recombinant cell, method for preparing a polypeptide, composition, method for treating a substrate c

专利摘要:
POLYPEPTIDE HAVING OR AIDING THE DEGRADATION ACTIVITY OF CARBOHYDRATE MATERIAL AND USES THEREOF. The invention relates to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polynucleotide of a polypeptide or a variant thereof, in that the variant polypeptide has sequence identity of at least 76% with the sequence set forth in SEQ ID NO: 2 or the polynucleotide encodes a variant polypeptide that has at least 76% sequence identity with the sequence set forth in SEQ ID NO: 2. The invention provides the full-length coding sequence of the novel gene, as well as the full-length amino acid sequence of the polypeptide functional and functional equivalents of the gene or amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the present invention are cells transformed with a polynucleotide according to the invention suitable for the production of these proteins.
公开号:BR112012033404B1
申请号:R112012033404-2
申请日:2011-06-23
公开日:2021-05-18
发明作者:Margot Elisabeth Francoise Schooneveld-Bergmans；Wilbert Herman Marie Heijne；Alrik Pieter Los
申请人:Dsm Ip Assets B.V.；
IPC主号:

专利说明:

Research and Development Statement Sponsored by the Federal Government
[0001] This invention was made with the support of the United States Government under grant no. DE-FC36-08G018079, issued by the Department of Energy. The Government of the United States Government may have certain rights in the present invention. field of invention
[0002] The invention relates to sequences comprising genes encoding polypeptides that have or assist in the activity of degradation of carbohydrate material. The invention features the full-length coding sequence of the new gene, as well as the functional full-length protein amino acid sequence, and the gene variants and fragments or amino acid sequence. The invention also relates to methods for using these proteins in industrial processes. Also included in the present invention are cells transformed with a polynucleotide according to the invention, suitable for the production of these proteins. Furthermore, the invention relates to the successful expression of genes encoding polypeptides having carbohydrate material degradation activity in a host. The host may be any suitable host, for example Aspergillus, for example Aspergillus niger or Talaromyces, for example Talaromyces emersonii. Background of the invention
[0003] Carbohydrates constitute the most abundant organic compounds on Earth. However, much of this carbohydrate is sequestered into complex polymers, including starch (the main storage carbohydrate in seeds and grains), and a collection of carbohydrates and lignin known as lignocellulose. The main components of lignocellulose carbohydrates are cellulose, hemicellulose, and pectins. These complex polymers are often collectively referred to as lignocellulose.
[0004] The bioconversion of renewable lignocellulosic biomass to a fermentable sugar that is subsequently fermented to produce alcohol (eg ethanol) as an alternative to liquid fuels has attracted intense attention from researchers since the 1970s, when the oil crisis erupted, due to the decrease in oil production by OPEC. Ethanol has been widely used as a 10% blend for gasoline in the US or as a clean fuel for vehicles in Brazil for the past two decades. More recently, the use of E85, an 85% ethanol blend, has been applied especially for clean municipal applications. The importance of bioethanol fuel production will increase in parallel with the rise in oil prices and the gradual depletion of its sources. In addition, fermentable sugars are used to produce plastic polymers, and other bio-based products and this industry is expected to grow substantially, therefore increasing the demand for plentiful low cost fermentable sugars that can be used as a feed material, in instead of petroleum-based raw materials.
[0005] The sequestration of such large amounts of carbohydrates in plant biomass provides an abundant source of potential energy in the form of sugars, both five-carbon and six-carbon sugars that can be used for numerous industrial and agricultural processes. However, the enormous energy potential of these carbohydrates is currently underutilized, as sugars are locked in complex polymers and, therefore, are not easily accessible for fermentation. Methods that generate sugars from plant biomass can provide abundant, economically competitive raw materials for fermentation into chemicals, plastics, such as, for example, succinic acid and (bio)fuels, including ethanol, methanol, butanol liquid fuels synthetics and biogas.
[0006] Regardless of the type of cellulosic raw material, the cost and efficiency of hydrolytic enzymes are main factors that limit the commercialization of biomass bioconversion processes. The production costs of microbially produced enzymes are closely related to the productivity of the enzyme-producing strain and the yield of the final activity in the fermentation broth.
[0007] Despite continuous research in recent decades to understand the degradation of enzymatic lignocellulosic biomass and cellulase production, it remains desirable to identify or construct new highly active cellulases and hemicellulases. It would also be highly desirable to construct highly efficient enzyme compositions capable of effecting rapid and efficient biodegradation of lignocellulosic materials, in particular, cellulases and hemicellulases which have increased thermostability.
[0008] Such enzymes can be used for the production of sugars for fermentation into chemicals, plastics, such as, for example, succinic acid and (bio)fuels, including ethanol, methanol, butanol, synthetic liquid fuels and biogas production , for silage, and also as an industrial enzyme in other processes, for example, in the food or animal feed, textile, paper or paper pulp or detergent and other industries. Invention Summary
[0009] The present invention provides polynucleotides that encode polypeptides that have the ability to degrade (i.e., assist in the degradation of), a carbohydrate (for example, polysaccharides), in lignocellulose, in particular. The polynucleotides of the present invention typically encode a polypeptide or aid in carbohydrate degrading activity.
[0010] The invention also provides naturally and recombinantly the polypeptides produced with such activity, as well as recombinant cell lines that produce such enzymes. In addition, methods of making and using the polynucleotides and polypeptides of the present invention are provided.
[0011] According to the invention, there is provided a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polynucleotide or variant polypeptide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set forth in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide having at least 76% sequence identity with the sequence set forth in SEQ ID NO: 2.
[0012] The polypeptides according to the invention have advantageous properties, in particular the property of having or assisting in the activity of degradation of carbohydrate material. In one embodiment, the polypeptide according to the invention has oxidohydrolase activity. In one embodiment, the polypeptide has GH61 activity.
In one embodiment the variant polypeptide has a His27 residue (positioned as in SEQ ID NO: 2). It is here assumed that His27 plays a role in binding metal ions (Ni2+ and Mg2+ and potentially other cations. In one embodiment the variant polypeptide has at least 76% sequence identity with the sequence set forth in SEQ ID NO: 2 , a His27 catalytic residue and oxidohydrolase and/or GH61 activity.
[0014] Furthermore, the polypeptides have a high thermostability. The polypeptides, according to the invention, maintain a high relative activity (% of initial activity) as a function of the incubation time (h), for example, 2 hours, 3 hours, 4 hours, five hours, six hours, eight hours , nine hours, 10 hours or more, 20 hours or more, 30 hours or more, especially at elevated temperatures, for example, at 60°C or more, at 65°C or more, or at 70°C or more, for example, 8 hours at 65°C or 72 hours at 60°C.
[0015] The invention also provides a polynucleotide comprising: (a) the nucleotide sequence set forth in SEQ ID NO: 1, or (b) a nucleotide sequence that selectively hybridizes to a polynucleotide being the reverse complement of SEQ ID NO: 1, or (c) a nucleotide sequence having at least about 80% sequence identity with the nucleotide sequence of SEQ ID NO: 1, or (d) a fragment of a nucleotide sequence as defined in (a), (b) or (c), which is at least about 100 nucleotides in length, or (e) a sequence which is degenerate as a result of the genetic code to a sequence as defined in any one of (a ), (b), (c) or (d), or (f) a nucleotide sequence that is the reverse complement of a nucleotide sequence as defined in (a), (b), (c), (d) or (e).
[0016] Also provided is a vector according to the invention, such as an expression vector, which incorporates a polynucleotide sequence of the present invention and a cell comprising a polypeptide, a polynucleotide or a vector of the invention.
[0017] The invention also provides:
[0018] a method for preparing a polypeptide having or aiding the activity of carbohydrate degradation, which method comprises culturing a cell of the invention under conditions that allow the expression of said polypeptide and, optionally, the recovery of the expressed polypeptide;
[0019] a polypeptide obtained by such a method; and
[0020] a composition comprising: (i) a polypeptide of the present invention, and (ii) a cellulase and/or a hemicellulase and/or a pectinase;
[0021] The polypeptides of the present invention having or aiding the activity of carbohydrate degradation can be used in industrial processes. Thus, the invention provides a method for treating a substrate comprising a carbohydrate material wherein the method comprises contacting the substrate with a polypeptide or a composition of the present invention.
[0022] In particular, the invention provides a method for producing a sugar or sugars from lignocellulosic material wherein the method comprises contacting the lignocellulosic material with a polypeptide or a composition of the present invention.
[0023] Sugars produced in this way can be used in a fermentation process. Therefore, the present invention provides a method of producing a fermentation product, which method comprises: producing a fermentable sugar using the above; and the fermentation of the resulting fermentable sugar, thus producing a fermentation product.
[0024] A composition or a polypeptide of the invention can also be used, for example, in the preparation of a food product, in the preparation of a detergent, in the preparation of an animal feed, in the treatment of paper pulp or in the manufacture of a paper or in preparing a fabric or textile fabric or in cleaning it.
[0025] The invention also provides:
[0026] a processed material obtained by contacting a plant material or lignocellulosic material with a polypeptide or a composition of the invention;
[0027] a food or feed for animals comprising a polypeptide or a composition of the invention; and a plant or a part thereof, which comprises a polynucleotide, a polypeptide, a vector or a cell according to the invention. Brief description of the drawings
[0028] Fig. 1: Map of pGBTOP for A. niger gene expression. Represented are the gene of interest (GOI) expressed from the glucoamylase promoter (PglaA). In addition, the glucoamylase (3'glaA) flank of the expression cassette is described. In this application, a gene of interest is the coding sequence of TEMER07589 as defined below.
[0029] Fig. 2: Graph of glucose release, expressed in mmol/L, with time, from 2% dm of pretreated wheat straw, incubated with 15 mg of protein, as equivalent BSA per gram of wheat straw pretreated dry, by 4 enzyme mixtures, which contains 1 BG , 1 CBHI, 1 CBHII 1 and 1 EG or, assist in the carbohydrate degradation activity (-- • -- = CEA;. -.. x-.. = CEB; -- ■ -- = TEMER07589) or by the classical cellulase product (--▲-- = Filtrase® NL). Brief Description of Sequence Listing
[0030] SEQ ID NO 1 sets forth the coding sequence of TEMER07589;
[0031] SEQ ID NO 2 establishes the amino acid sequence of TEMER07589;
[0032] SEQ ID NO 3 sets forth the signal sequence of TEMER07589; Detailed description of the invention
[0033] Throughout this specification and the appended claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" shall be interpreted inclusively . That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
[0034] The articles "a," and "an" are used here to refer to one or more than one (ie, one or at least one) grammatical object of the article. By way of example, "an element" can mean one element or more than one element.
[0035] The present invention provides polynucleotides that encode polypeptides, for example, enzymes that have the ability to modify, for example, degrade, a carbohydrate material. A carbohydrate material is a material that comprises, consists of, or consists essentially of one or more carbohydrates. Enzymes here are a subclass of polypeptides.
[0036] The substrate here (also called raw material) is used to refer to a substance comprising the carbohydrate material, which can be treated with enzymes according to the invention, so that the carbohydrate material therein is modified . In addition, the substrate carbohydrate material may contain any other component, including, but not limited to, non-carbohydrate materials and starches.
[0037] The present invention provides polynucleotides that encode polypeptides, for example, enzymes that have the ability to modify, for example, degrade, a carbohydrate material. A carbohydrate material is a material that comprises, consists of, or consists essentially of one or more carbohydrates. Enzymes here are a subclass of polypeptides.
[0038] Typically, a polypeptide of the present invention encodes a polypeptide having at least or aiding a carbohydrate degradation activity, provisionally called TEMER07589, having an amino acid sequence according to SEQ ID NO: 2, or a sequence that is a variant thereof, typically functionally equivalent to the polypeptide having the sequence of SEQ ID NO: 2, or a sequence that is a fragment of any of these.
[0039] "Having or assisting in carbohydrate degrading activity" is defined herein as the polypeptide that has carbohydrate degrading activity, or which polypeptides assist in carbohydrate degradation, or both. In one embodiment, the polypeptide increases the activity of at least one cellulase. In this modality, when the polypeptide of the present invention is present in a mixture with one or more cellulases, for example, in a mixture with cellobiohydrolase (CBH) and beta-glucosidase (BG), this will increase the activity of these cellulases, which will result in an increase in the activity of the mixture for cellulose degradation. TEMER07589 is believed to belong to the GH61 family of enzymes. The GH61 enzymes in this family were originally classified as a glycoside hydrolase family based on the measurement of very weak endo-1,4-b-D-glucanase activity of a family member (endoglucanase (EC 3.2.1.4)). The structure and mode of action of these enzymes are, of course, non-canonical and may not be considered bona fide glycosidases. However, they are kept in the CAZy rating based on their ability to increase lignocellulose breakdown when used in conjunction with a cellulase or a mixture of cellulases. An overview of known members of the GH61 family is given in Figure 5 of Harris, PV et al., Biochemistry 2010, 49, 3305-3316.
[0040] In one embodiment, in addition to the enhanced cellulase activity, the cellulase enhancer protein according to the invention has endoglucanase (EG) activity. In this embodiment, the addition of endoglucanase (other than the cellulase enhancing protein according to the invention) to a cellulase mixture, which is generally essential for the effective degradation of cellulose, can be avoided.
[0041] Thus, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,4-β-D-glucosidic bonds in cellulose, lichenin or β-D -cereal glucans. Such a polypeptide may also be capable of hydrolyzing 1,4-linkages in β-D-glucans also containing 1,3-linkages. This enzyme may also be referred to as cellsase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase, carboxymethyl cellulose, celludextrinase, endo-1,4-β-D-glucanase, endo-1,4 - β-D-glucanohydrolase, endo-1,4-β-D-glucanase or endoglucanase.
[0042] In one embodiment, a polypeptide of the present invention may have one or more alternative and/or complementary activities other than increasing cellulase activity and endoglucanase activity as mentioned above, for example, one of the other hydrolysis activities of carbohydrates and/or carbohydrate degradation mentioned here.
[0043] "Carbohydrates" in this context includes all saccharides, for example, polysaccharides, oligosaccharides, disaccharides or monosaccharides.
[0044] A polypeptide according to the invention can modify and/or degrade a carbohydrate material by chemically degrading or physically degrading such material or hydrolyzing the carbohydrate. Physical includes, for example, interruption of the interaction between the cellulose microfibrils and/or the opening of the structure of the cellulose fibers. Chemical modification of the carbohydrate material can result in the degradation of materials such as, for example, by hydrolysis, oxidation or chemical modification, such as through the action of a lyase. Physical modification may or may not be accompanied by chemical modification. Suitable carbohydrate materials
[0045] A non-starch carbohydrate suitable for the modification of a polypeptide of the present invention is lignocellulose. The main polysaccharides comprising different lignocellulosic residues, which can be considered as a potential renewable raw material are cellulose (glucans), hemicellulose (xylans, heteroxylans and xyloglucans). Furthermore, some hemicelluloses may be present as glucomannans, for example, in wood raw material derivatives. The enzymatic hydrolysis of these polysaccharides to soluble sugars, eg glucose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, D-galacturonic and other hexoses and pentoses takes place under the action of different enzymes that act in combination.
[0046] In addition, pectins and other pectic substances such as arabinans can make up considerably dry mass ratio typically of cell walls from non-woody plant tissues (about a quarter to half of the dry mass can be pectin ).
[0047] Cellulose is a linear polysaccharide consisting of glucose residues linked by β-1,4 bonds. The linear nature of cellulose fibers as well as the stoichiometry of bound β-glucose (relative to α) generates structures more prone to hydrogen bonding between the strands than the highly branched α-bonded structures of starch. Thus, cellulose polymers are generally less soluble, and form more tightly bound fibers than those found in starch fibers.
[0048] Hemicellulose is a complex polymer, and its composition varies very often from organism to organism, and from one type of tissue to another. Generally speaking, a major component of hemicellulose is β-1,4-linked xylose, a five-carbon sugar. However, this xylose is often branched into O-3 and/or O-2 and can be replaced with linkages to arabinose, galactose, mannose, glucuronic acid, galacturonic acid, or by acetic acid esterification (and ferulic acid esterification with arabinose). Hemicellulose may also contain glucans, which is a general term for β-linked six-carbon sugars (such as the β-(1,3)(1,4) glucans and heteroglucans mentioned above) and, additionally, glucomannans (wherein glucose and mannose are present in the linear skeleton, linked together by e-bonds).
[0049] The composition, nature of the replacement, and degree of branching of hemicellulose is very different in dicot plants (dicots, for example, plants whose seeds have two cotyledons or seed leaves, such as beans, peanuts, almonds, peas, pods ), compared to monocotyledonous plants (monocotyledonous, ie plants that have a single cotyledon or seed leaf, such as corn, wheat, rice, turf, barley). In dicots, hemicellulose is mainly composed of xyloglucans which are 1,4-β-linked glucose chains with 1,6-β-linked xylosyl side chains. In monocots, including most grain crops, the main components of hemicellulose are heteroxylans. These are mainly composed of 1,4-β-xylose backbone polymers linked with 1,3-α bonds with arabinose, galactose, mannose and glucuronic acid or 4-O-methyl-glucuronic acid, as well as acid-modified xylose acetic esters linked to esters. β-glucans composed of 1,3- and 1,4-β linked glucose chains are also present. In monocots, cellulose, heteroxylans and β-glucans can be present in approximately equal amounts, each comprising about 1525% cell wall dry matter. Furthermore, different plants may comprise different amounts of, and different compositions of, pectic substances. For example, sugar beet contains about 19% pectin and about 21% arabinan, on a dry weight basis.
[0050] Therefore, a composition of the present invention can be tailored in view of the particular raw material (also called substrate) that is to be used. That is, the spectrum of activities in a composition of the present invention can vary depending on the raw material in question.
[0051] Combinations of enzymes or physical treatments can be administered concurrently or sequentially. Enzymes can be produced exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added to lignocellulosic raw material. Alternatively, enzymes are produced, but not isolated, and fermentation broth from the crude cell mass, or plant material (eg, corn husk), and the like, is added to the raw material. Alternatively, the crude cell mass or enzyme production medium or plant material can be treated to prevent further microbial growth (eg by heating or by adding antimicrobial agents) then added to the raw material. Such raw enzyme mixtures can include the organism producing the enzyme. Alternatively, the enzyme can be produced in a fermentation that uses the raw material (eg, as corn husks) to provide nutrition to an organism that produces an enzyme(s). In this way, the enzyme-producing plants can serve as the lignocellulosic feedstock and be added to the lignocellulosic feedstock. Enzymatic activity
[0052] Endo-1,4-β-glucanases (EG) and exocellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to cello-oligosaccharides (cellobiose as the main product), while β-glucosidases (BGL) convert oligosaccharides , mainly cellobiose and glucose cellotriosis.
[0053] Xylanases together with accessory enzymes, for example, α-L-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and β-xylosidases, catalyze the hydrolysis of part of the hemicelluloses.
Pectic substances include pectins, arabinans, galactans and arabinogalactans. Pectins are the most complex polysaccharides in the plant cell wall. They are built around a core of a chain of α(1,4)-D-linked galacturonic acid units interspersed to some degree with L-rhamnose. In any one cell wall, there are a number of structural units that match this description and it has generally been assumed that, in a single pectic molecule, the core chains of different structural units are continuous with one another.
Pectinases include, for example, an endo polygalacturonase, a pectin methyl esterase, an endo-galactanase, a beta-galactosidase, an acetyl esterase pectin, an endo-pectin lyase, pectin lyase, alpha rhamnosidase, an exo -galacturonase, an expolygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, an acetyl esterase rhamnogalacturonan, a rhamnogalacturonan galacturonase, a xylogalacturonase, an α-arabinofurans.
[0056] The main types of structural unit are: galacturonan (homogalaturonane), which can be substituted with methanol in the carboxyl group and acetate in O-2 and O-3; rhamnogalacturonan I (RGI), in which the galacturonic acid units alternate with rhamnose units that carry (1,4)-linked galactan and (1,5)-linked arabinan side chains. Arabinan side chains can be linked directly to rhamnose or indirectly through galactan chains; xylogalacturonane, with a single xylosyl unit in galacturonic acid O-3 (intimately associated with RGI); and rhamnogalacturonan II (RGI I), a particularly smaller complex unit that contains the unusual sugars, eg, apiose. An RGII unit can contain two apiosyl residues which, under suitable ionic conditions, can reversibly form borate esters.
As set out above, a polypeptide of the present invention will typically have enhanced cellulase activity. However, a polypeptide of the present invention may have one or more of the above activities, in addition to or alternative to that activity. Furthermore, a composition of the present invention, as described herein, may have one or more of the above activities, in addition to what is provided by a polypeptide of the present invention having enhanced cellulase activity. Polynucleotide sequence
[0058] The invention provides genomic polynucleotide sequences comprising the gene encoding TEMER07589 as well as its coding sequence. Accordingly, the invention relates to an isolated polynucleotide comprising the nucleotide sequence of the genome according to the nucleotide coding sequence according to SEQ ID NO: 1 and variants, such as functional equivalents, of one or the other of these.
In particular, the invention relates to an isolated polynucleotide that is capable of selectively hybridizing, for example, under stringent conditions, preferably under highly stringent conditions, with the reverse complement of a polynucleotide comprising the sequence set forth in SEQ ID NO : 1.
[0060] More specifically, the invention relates to a polynucleotide comprising or consisting essentially of a nucleotide sequence according to SEQ ID NO: 1.
[0061] The invention also relates to an isolated polynucleotide comprising or consisting essentially of a sequence encoding at least one functional domain of a polypeptide according to SEQ ID NO: 2 or a variant thereof, such as a functional equivalent or a fragment of any of these.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that can be isolated from chromosomal DNA, which include an open reading frame encoding a protein, for example , the cellulase enhancer protein according to the present invention.
[0063] A gene may include coding sequences, non-coding sequences, introns, and/or regulatory sequences. Furthermore, the term "gene" can refer to an isolated nucleic acid molecule as defined herein.
A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or a variant thereof, such as a functional equivalent, can be isolated using standard biological techniques molecular structure and the sequence information provided herein. For example, using all or a portion of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe, the nucleic acid molecules according to the invention can be isolated by means of standard hybridization and cloning techniques ( for example, as described in Sambrook, J., Fritsh, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
[0065] Furthermore, a nucleic acid molecule comprising all or a portion of SEQ ID NO: 1, can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in SEQ ID NO: 1.
A nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
[0067] Furthermore, oligonucleotides which correspond to or hybridize to a nucleotide sequence according to the invention can be prepared by standard synthetic techniques, for example using an automated DNA synthesizer.
[0068] In a preferred embodiment, an isolated nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 1.
[0069] In another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule that is the reverse complement of the nucleotide sequence shown in SEQ ID NO: 1 or a variant thereof, such as an equivalent functional, of any of these nucleotide sequences.
A nucleic acid molecule that is complementary to another nucleotide sequence is one that is sufficiently complementary to the other nucleotide sequence such that it can hybridize with the other nucleotide sequence, thus forming a stable duplex.
[0071] One aspect of the invention concerns isolated nucleic acid molecules encoding a polypeptide of the invention or a variant thereof, such as a functional equivalent thereof, e.g., a biologically active fragment or domain, as well as acid molecules sufficient nucleic acid for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
[0072] A polynucleotide according to the invention can be "isolated". In the context of the present invention, an "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous to one or both of the coding sequences with which it is immediately contiguous (one at the 5' end and one over the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding sequences (eg, promoters) that are immediately contiguous to the coding sequence. The term thus includes, for example, a recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (eg, a cDNA or a genomic DNA fragment produced by PCR or by restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide that is substantially free of cellular material, viral material or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when synthesized chemically). Furthermore, an "isolated nucleic acid fragment" is a nucleic acid fragment that does not naturally occur as a fragment and that would not be found in the natural state.
[0073] As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg mRNA) and DNA analogues or RNA generated using nucleotide analogues. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. Nucleic acid can be synthesized using oligonucleotide analogs or derivatives thereof (for example, phosphorothioate or inosine nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have had altered base pairing ability or increased nuclease resistance.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a TEMER07589 nucleic acid molecule, for example the coding strand of a TEMER07589 nucleic acid molecule. Also included within the scope of the present invention are the complementary strands of nucleic acid molecules described herein.
[0075] Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of the polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. The nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
[0076] The actual sequence can be more accurately determined by other approaches, including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or removal of a given nucleotide sequence relative to the actual sequence will cause a translational structure change of the nucleotide sequence such that the predicted amino acid sequence encoded by a nucleotide sequence determined will be completely different from the actual amino acid sequence encoded by the sequenced DNA molecule, starting at the point of such insertion or deletion.
[0077] The person skilled in the art is able to identify these erroneously identified bases and know how to correct these errors.
A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in SEQ ID NO: 1 (or a variant of any of these), for example a fragment which may be used as a probe or primer or fragment encoding a portion of a TEMER07589 polypeptide.
[0079] The nucleotide sequence determined from the cloning of the TEMER07589 cDNA gene allows the generation of probes and primers designed for use in the identification and/or cloning of other members of the TEMER07589 family, as well as TEMER07589 homologues from other species.
The probe/primer typically comprises a substantially purified oligonucleotide which typically comprises a region of nucleotide sequence which preferably hybridizes under highly stringent conditions with at least about 12 to about 15, preferably from about 18 to about 20, preferably from about 22 to about 25, more preferably from about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO: 1 or of a variant, such as a functional equivalent, or any of the same.
[0081] Probes based on TEMER07589 nucleotide sequences can be used to detect transcribed or genomic TEMER07589 sequences encoding the same polypeptides or homologous polypeptides, for example, in other organisms. In preferred embodiments, the probe further comprises a label group attached thereto, for example, the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. These probes can also be used as part of a diagnostic test kit for identifying cells that express a TEMER07589 polypeptide.
[0082] The polynucleotides described herein may be synthetic polynucleotides. Synthetic polynucleotides can be optimized in the usage codon, preferably, according to the methods described in WO2006/077258 and/or PCT/EP2007/055943, which are hereby incorporated by reference. The document PCT/EP2007/055943 addresses the codon pair optimization. Codon pair optimization is a method in which the nucleotide sequences encoding a polypeptide have been modified in relation to their codon usage, in particular the codon pairs that are used, to obtain a better expression of the nucleotide sequence that encodes the polypeptide and/or better produce the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) of a coding sequence.
The invention further relates to a nucleic acid construct comprising the polynucleotide as described above. "Nucleic acid construct" is defined herein as a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain nucleic acid segments that are combined and juxtaposed so that they would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains all of the control sequences necessary for the expression of a coding sequence. The term "coding sequence" as defined herein is a sequence that is transcribed into mRNA and translated into a transcriptional activator of a protease promoter of the invention. The boundaries of the coding sequence are generally determined by the ATG initiation codon at the 5' end of the mRNA and an open reading frame termination sequence translation stop codon at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. Preferably, the nucleic acid has a high GC content. The GC content here indicates the number of G and C nucleotides in the construct, divided by the total number of nucleotides, expressed in %. The GC content is preferably 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, or in the range of 56-70%, or in the range of 58-65% .
[0084] Preferably, the DNA construct comprises a promoter DNA sequence, a coding sequence in operative association with said promoter DNA sequence and control sequences, such as:
[0085] - a 5' to 3' oriented translational termination sequence selected from the following list of sequences: TAAG, TAGA and ATA, preferably, AATA, and/or
[0086] - a 5' to 3' oriented translational primer coding sequence selected from the following sequence list: GCTACCCCC; GCTACCTCC; GCTACCTC; GCTACCTTC; GCTCCCCCC; GCTCCCTCC; GCTCCCCTC; GCTCCCTTC; GCTGCCCCC; GCTGCCTCC; GCTGCCCTC; GCTGCCTTC; GCTTCCCCC; GCTTCCTCC; GCTTCCCTC and GCTTCCTTC, preferably TGC TTC TCC, and/or
[0087] - a translational primer selected from the following list of sequences: 5'-mwChkyCAAA-3'; 5'-mwChkyCACA-3' or 5'-mwChkyCAAG-3', using ambiguity codes for nucleotides: m (A/C); w (A/T), y (C/T), k (G/T) h (A/C/T), preferably 5'-CACCGTCAAA-3' or 5'-CGCAGTCAAG-3'.
In the context of the present invention, the term "translational primer coding sequence" is defined as the nine nucleotides immediately downstream of the primer initiation codon or open reading frame of a DNA coding sequence. The initiator or start codon encodes the AA methionine. The initiator codon is usually ATG, but it can also be any functional start codon such as GTG.
[0089] In the context of the present invention, the term "translational stop sequence" is defined as the four nucleotides from the translational stop codon at the 3' end of the open reading frame or coding sequence of 5' oriented and nucleotides for 3'.
[0090] In the context of the present invention, the term "translational primer" is defined as the ten nucleotides immediately upstream of the start or start codon of the open reading frame of a DNA sequence encoding a polypeptide. The initiator or start codon encodes methionine AA. The initiator codon is usually ATG, but it can also be any functional start codon such as GTG. It is well known in the art that uracil, U, replaces the deoxynucleotide thymine, T, in RNA. homology and identity
[0091] Amino acids or nucleotide sequences are considered homologous when they exhibit a certain degree of similarity. Two sequences that are homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "percent identity" or "percent similarity", which is high or low, respectively. Despite the argument to indicate "percent identity" or "percent similarity", the "level of homology" or "percent homology" is often used synonymously.
[0092] The terms "homology", "percent homology", "percent identity" or "percent similarity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that, in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the complete sequences are aligned for purposes of optimal comparison. In order to optimize the alignment between the two sequences gaps can be introduced into either of the two sequences that are compared. Such alignment is carried out over the full length of the sequences being compared. Alternatively, the alignment can be carried out over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. Identity is the percentage of identical matches between the two sequences over the reported aligned region.
[0093] A comparison of sequences and determination of percentage identity between two sequences can be performed using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine homology between the two sequences (Kruskal, JB (1983) An overview of Sequence Comparison in D. Sankoff and Kruskal JB, (ed. .), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch for the alignment of two sequences. (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453). The algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm was implemented in the NEEDLE computer program. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Software Suite Open (2000) Rice, P. LongdenJ and Bleasby A. Trends in Genetics 16 , (6) pp276-277, http://emboss.bioinformatics.nl/). For polypeptide sequences, EBLOSUM62 is used for the replacement matrix. For nucleotide sequences, EDNAFULL is used. Other matrices can be specified. Optional parameters used for amino acid sequence alignment are a gap opening penalty of 10 and a gap extension penalty of 0.5. The versed will appreciate that all of these different parameters will yield slightly different results, but that the overall percentage identity of two sequences does not change significantly when using different algorithms. Definition of World Homology
[0094] Homology or identity is the percentage of identical matches between the two complete sequences over the entire aligned region including any gaps or extensions. The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment, including gaps. The identity as defined here can be obtained from NEDDLE and is marked in the program exit as "IDENTITY". Definition of Longer Identity
The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences, divided by the total length of the alignment, after subtracting the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked in the program output as "the longest identity". For the purposes of the invention the level of identity (homology) between two amino acid (or nucleotide) sequences is calculated according to the definition of "longer identity", as can be performed using the NEEDLE program.
[0096] The polypeptide sequences of the present invention can further be used as a "search sequence" to perform a search against sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using BLAST programs. Software for performing BLAST analyzes is publicly available through the National Biotechnology Information Center (http://www.ncbi.nlm.nih.gov). BLASTP is used for the BLASTN amino acid and nucleotide sequences for sequences. The BLAST program defaults to:
[0097] Cost for gap opening: default = 5 for nucleotides/11 for polypeptides
[0098] Cost for gap extension: default = 2 for nucleotides/1 for polypeptides - Penalty for mismatched nucleotides: default = -3 - Reward for matching nucleotide: default = 1 - Wait Value: default = 10 -Word length: default = 11 for nucleotides / 28 for Megablast / 3 for polypeptides
[0099] Furthermore, the degree of local identity (homology) between the amino acid search sequence or nucleic acid search sequence and the retrieved homologous sequences is determined by the BLAST program. However, only the segments of sequences that are compared generate a match above a certain threshold. Therefore, the program calculates the identity only for these matching segments. Thus, the identity calculated in this way is referred to as the local identity. Hybridization
[0100] As used herein, the term "selectively hybridize", "selectively hybridize" and similar terms are used to describe conditions for hybridization and washing under which nucleotide sequences are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, more preferably at least about 85%, still more preferably at least about 90%, preferably at least 95%, more preferably at least about 98% or more, preferably at least about 99% homologous to each other typically remain hybridized to each other. That is, such hybridizing sequences can share at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, more preferably at least 95%, more preferably at least 98% or more, preferably at least about 99% of sequence identity.
[0101] A preferred non-limiting example of such hybridization conditions is hybridization in 6X sodium citrate/sodium chloride (SSC) at about 45°C, followed by one or more washes in 1X SSC, 0.1 SDS % at about 50°C, preferably at about 55°C, preferably at about 60°C and even more preferably at about 65°C.
[0102] Highly stringent conditions include, for example, hybridization at about 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS and washing in 0.2x SSC/0.1% SDS at temperature environment. Alternatively, washing can be carried out at 42°C.
[0103] The person skilled in the art will know which conditions to apply to stringent and very stringent hybridization conditions. Additional guidance regarding these conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (Eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
[0104] Naturally, a polynucleotide that hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract mRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the present invention, since such polynucleotide hybridizes to any nucleic acid molecule containing a poly(A) stretch or its complement (e.g., virtually any double-stranded cDNA clone).
[0105] In a typical approach, cDNA libraries built from other organisms, for example, filamentous fungi, in particular, from the family of microorganisms Trichomaceae, for example, from the genus Penicillium can be traced, as Penicillium decumbens.
[0106] For example, Penicillium strains can be screened for homologous TEMER07589 polynucleotides by means of Northern blot analysis. After detecting transcripts homologous to polynucleotides according to the invention, cDNA libraries can be constructed from RNA isolated from the appropriate strain, using standard techniques well known to those skilled in the art. Alternatively, a total genomic DNA library can be screened using a probe capable of hybridizing to a TEMER07589 polynucleotide according to the invention.
[0107] Homologous gene sequences can be isolated, for example, by performing PCR using two pools of degenerate oligonucleotide primers designed based on the nucleotide sequences, as taught here.
[0108] The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new TEMER07589 nucleic acid sequence, or a functional equivalent thereof.
[0109] The PCR fragment can then be used to isolate a full-length cDNA clone by a variety of known methods. For example, the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library. Alternatively, the tagged fragment can be used to search a genomic library.
[0110] PCR technology can also be used to isolate full length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. The reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the larger 5' end of the amplified fragment for the initiation of first strand synthesis.
[0111] The resulting RNA/DNA hybrid can then be "finished" (eg with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H and the second strand synthesis can then be prepared ( for example, with a poly-C initiator). Thus, cDNA sequences upstream of the amplified fragment can easily be isolated. For a review of useful cloning strategies see, for example, Sambrook et al., supra,. And Ausubel et al., supra. Vectors
[0112] Another aspect of the invention relates to vectors, including cloning and expression vectors comprising a polynucleotide of the invention encoding a TEMER07589 polypeptide or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors into a host cell suitable, for example, under conditions where expression of a polypeptide of the present invention occurs. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
[0113] The polynucleotides of the present invention can be incorporated into a recombinant replicable vector, for example, a cloning or expression vector. The vector can be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention provides a method for making the polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that cause replication vector icon. The vector can be retrieved from the host cell. Suitable host cells are described below.
[0114] The vector into which the expression cassette or polynucleotide of the invention is inserted can be any vector that can conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
[0115] A vector according to the invention can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell's genome and replicated along with the chromosome(s) into which it has been integrated.
[0116] One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. These vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably throughout this document as plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as cosmids, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors, which serve equivalent functions.
[0117] For most filamentous fungi and yeasts, the expression vector construct is preferably either integrated into the host cell genome in order to obtain stable transformants. However, for certain yeasts also suitable episomal vectors that are available in the expression construct can be incorporated for stable and high level expression, examples of these include vectors derived from 2 µ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (for example, AMA1 from Aspergillus). In case the expression constructs are integrated into the host cell genome, the constructs are either integrated at random loci in the genome or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.
[0118] Consequently, expression vectors useful in the present invention include chromosomal, episomal and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosome elements, viruses such as baculoviruses, Papova viruses, vaccinia virus, adenovirus, bird pox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as those derived from plasmids and bacteriophage genetic elements, such as cosmids and phagemids.
[0119] Vectors according to the invention can be used in vitro, for example, for the production of RNA or used to transfect or transform a host cell. The recombinant expression vector can be transcribed and translated in vitro, for example, using regulatory sequences from the T7 promoter and T7 polymerase.
[0120] A vector of the invention may comprise two or more, for example, three, four or five polynucleotides of the present invention, for example, for overexpression.
[0121] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected on the basis of the cells host to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
[0122] Within a vector, such as an expression vector "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a way that allows expression of the sequence of nucleotides (eg, in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), ie the term "operably linked" refers to a juxtaposition in which the described components they are in a relationship that allows them to function in their intended way. A regulatory sequence, such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned such that expression of the coding sequence is achieved under conditions compatible with the control sequences or the sequences are arranged so that they work together for their intended purpose, for example, transcription starts at the promoter and proceeds through the DNA sequence encoding the polypeptide.
[0123] An expression vector or construct for a given host cell may thus comprise the following elements operably linked together in a consecutive order from the 5' end to the 3' end with respect to the coding strand of the sequence encoding the polypeptide of the first invention: (1) a promoter sequence capable of directing the transcription of the nucleotide sequence encoding the polypeptide in the given host cell, and (2) optionally, a signal sequence capable of directing the secretion of the polypeptide from the given host cell in a culture medium; (3) a DNA sequence of the invention encodes a mature and preferably active form of the polypeptide with booster cellulase activity and, preferably, also (4) a transcription termination region (terminator) capable of terminating the downstream transcription of the sequence of nucleotides that encodes the polypeptide.
[0124] Downstream of the nucleotide sequence according to the invention, this may be the 3' untranslated region containing one or more transcription termination sites (for example, a terminator). Terminator origin is less critical. The terminator can, for example, be native to the DNA sequence encoding the polypeptide. However, preferably, a yeast terminator is used in yeast host cells and a filamentous fungus terminator is used in filamentous fungus host cells. Most preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a translation starting with AUG at the beginning and a stop codon appropriately positioned at the end of the polypeptide to be translated.
[0125] Enhanced expression of the polynucleotide of the invention can also be achieved through selection of heterologous regulatory regions, for example, promoter, secretion leader and/or terminator regions that can serve to increase expression and, if desired, levels to secrete the polypeptide of interest from the expression host and/or to provide inducible control of expression of a polypeptide of the present invention.
[0126] It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the expression level of the desired polypeptide, etc.. expression, of the present invention can be introduced into host cells to thereby produce proteins or peptides encoded by nucleic acids as described herein (e.g., TEMER07589 polypeptides, mutant forms of TEMER07589 polypeptides, fragments, variants or functional equivalents thereof. The vectors. , such as recombinant expression vectors, of the present invention can be designed for expression of TEMER07589 polypeptides in prokaryotic or eukaryotic cells.
[0127] For example, TEMER07589 polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of suitable hosts are described below.
[0128] Appropriate culture media and conditions for the host cells described above are known in the art.
[0129] The term "control sequences" or "regulatory sequences" is defined herein to include at least any component that may be necessary and/or advantageous for the expression of a polypeptide. Any control sequence can be native or foreign to the nucleic acid sequence of the present invention that encodes a polypeptide. Such control sequences may include, but are not limited to, a promoter, a leader, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), a signal sequence of secretion, a pro-peptide sequence, a polyadenylation sequence, a transcription terminator. At a minimum, control sequences typically include a promoter, and transcription and translation stop signals. As set out above, the term "operatively linked" is defined herein as a configuration in which a control sequence is suitably placed in a position relative to the coding sequence of the DNA sequence such that the control sequence directs production of a polypeptide.
[0130] Control sequences can be provided with linkers for the purpose of introducing specific restriction sites that facilitate the linkage of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term "operatively linked" is defined herein as a configuration in which the control sequence is suitably placed in a position relative to the coding sequence of the DNA sequence in such a way that the control sequence directs the production of a polypeptide.
[0131] The control sequence may be a suitable promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcription control sequences, which mediate expression of the polypeptide. The promoter can be any nucleic acid sequence, which shows transcriptional activity in the cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides or homologous or heterologous to the cell.
[0132] The control sequence may also be a suitable transcription termination sequence, a sequence recognized by a fungal filamentous cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the cell can be used in the present invention.
[0133] The control sequence can also be a terminator. Preferred terminators for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC gene and Fusarium trypsin-like protease oxysporum.
[0134] The control sequence may also be an appropriate leader sequence, an untranslated region of an mRNA that is important for translation by the fungal filamentous cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the cell can be used in the present invention. Preferred leaders for filamentous fungal cells are derived from the genes encoding amylase from A. oryzae TAKA and A. nidulans triose phosphate isomerase and A. niger glaA.
[0135] Other control sequences can be isolated from the Penicillium IPNS gene, or pcbC gene, the beta-tubulin gene. All control sequences cited in WO 01/21779 are incorporated herein by reference.
[0136] The control sequence may also be a polyadenylation sequence, a sequence that is operably linked to the 3' terminus of the nucleic acid sequence, and which, when transcribed, is recognized by the filamentous fungus cell as a signal to add residues from polyadenosine to transcribed mRNA. Any polyadenylation sequence, which is functional in the cell, can be used in the present invention. Preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger, A. nidulans anthranilate synthase glucoamylase, Fusarium oxysporum trypsin-like protease and A. niger alpha-glucosidase.
[0137] When the polypeptide according to the invention must be secreted from the host cell into the culture medium, an appropriate signal sequence can be added to the polypeptide in order to direct the newly synthesized polypeptide to the secretion pathway of the host cell. The person skilled in the art knows how to choose an appropriate signal sequence for the specific host. The signal sequence can be natural to the host cell, or it can be foreign to the host cell. As an example, a signal sequence from a polypeptide native to the host cell can be used. Preferably, said native polypeptide is a highly secreted polypeptide, that is, a polypeptide that is secreted in amounts greater than 10% of the total amount of polypeptide to be secreted. The signal sequences preferably used according to the invention are, for example: pmeA.
[0138] As an alternative to a signal sequence, the polypeptide of the present invention can be fused to a secreted carrier polypeptide, or part thereof. This chimeric construct is directed to the secretion pathway through the signal sequence of the transporter polypeptide, or part of it. Furthermore, the carrier polypeptide will provide a stabilizing effect for the polypeptide according to the invention and/or may increase solubility. Such carrier polypeptide can be any polypeptide. Preferably, a highly secreted polypeptide is used as a carrier polypeptide. The carrier polypeptide can be natural or foreign to the polypeptide according to the invention. The transporter polypeptide can be native or it can be foreign to the host cell. Examples of transport polypeptides are glucoamylase, alpha-mating factor pre-pro sequence, cellulose-binding domain of Clostridium cellulovorans cellulose-binding protein A, glutathione S-transferase, a chitin-binding domain of chitinase A1 from Bacillus circulans, maltose-binding domain encoded by the E. coli K12 malE gene, beta-galactosidase and alkaline phosphatase. A preferred carrier polypeptide for expressing such a chimeric construct in Aspergillus cells is glucoamylase. The carrier protein and polypeptide may contain a specific amino acid motif to facilitate isolation of the polypeptide; the polypeptide according to the invention can be released by a special release agent. The release agent can be a proteolytic enzyme or a chemical agent. An example of such an amino acid motif is the KEX protease cleavage site, which is well known to the person skilled in the art.
[0139] A signal sequence can be used to facilitate the secretion and isolation of a protein or polypeptide of the present invention. Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage winds. Such signal processing peptides contain sites that allow for the cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is cleaved subsequently or simultaneously. The protein can then be readily purified from the extracellular medium by known methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence, which facilitates purification, such as with a GST domain. Thus, for example, the sequence encoding the polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag (identifier) provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Academic Sci. USA 86:821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. The HA tag is another peptide useful for purification, which corresponds to an epitope derived from the influenza hemagglutinin protein, which was described by Wilson et al., Cell 37:767 (1984), for example.
[0140] Preferably, a TEMER07589 fusion protein of the invention is produced by recombinant DNA techniques. For example, the DNA fragments encoding the different polypeptide sequences are linked together in structure according to conventional techniques, for example by employing blunt-ended or staggered-end ends for ligation, digestion with restriction enzymes to provide proper ends, fill in cohesive ends as appropriate, alkaline phosphatase treatment to avoid unwanted joining, and enzymatic binding. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that give rise to complementary protrusions between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds John Wiley & Sons:1992). In addition, many expression vectors are commercially available as they encode a fraction of the fusion (eg, a GST polypeptide). A nucleic acid encoding TEMER07589 can be cloned into such an expression vector such that the fusion moiety is linked in-frame with the TEMER07589 protein.
[0141] Preferably, the efficiency of target integration into the host cell genome, i.e. integration into the predetermined target locus, is increased by the increased capacity of homologous recombination in the host cell. Such a cell phenotype preferably involves a defective hdfA or hdfB gene, as described in WO2005/095624. WO2005/095624 describes a preferred method for obtaining a fungal filamentous cell comprising a greater target integration efficiency.
[0142] Optionally, the host cell comprises a high unfolded protein (UPR) response compared to the wild-type cell to enhance the ability to produce a polypeptide of interest. UPR can be increased by techniques described in US2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2 and/or WO2005/123763. More specifically, the protein level of HAC1 and/or IRE1 and/or PTC2 was modulated, and/or the SEC61 protein was designed to obtain a host cell having a high UPR.
[0143] Alternatively, or in combination with an elevated UPR, the host cell is genetically modified to obtain a phenotype exhibiting lower protease expression and/or enzyme secretion compared to the wild-type cell, in order to increase the ability to produce a polypeptide of interest. The phenotype can be obtained by deleting and/or modifying and/or inactivating a transcriptional regulator of protease expression. This transcriptional regulator is, for example, prtT. Lowering the expression of proteases by modulating prtT can be accomplished by techniques described in US2004/0191864A1.
[0144] Alternatively, or in combination with a high UPR and/or a phenotype exhibiting low protease expression and/or protease secretion, the host cell exhibits an oxalate deficient phenotype in order to increase the yield of production of a polypeptide of interest. An oxalate deficient phenotype can be obtained by techniques described in WO2004/070022A2.
[0145] Alternatively, or in combination with an elevated UPR and/or a phenotype exhibiting lower protease expression and/or protease secretion and/or oxalate deficiency, the host cell exhibits a combination of phenotypic differences compared to the cell to increase the yield of production of the polypeptide of interest. These differences may include, but are not limited to, low expression of glucoamylase and/or neutral alpha-amylase A and/or neutral alpha-amylase B, protease, and oxalic acid hydrolase. Said phenotypic differences presented by the host cell can be obtained by genetic modification according to the techniques described in document US2004/0191864A1.
[0146] Alternatively, or in combination with an elevated UPR and/or a phenotype exhibiting lower protease expression and/or protease secretion and/or oxalate deficiency and a combination of phenotypic differences compared to the wild-type cell to increase the Through the production of the polypeptide of interest, the host cell exhibits a deficiency in toxin genes, disabling the capacity of the filamentous fungal host cell to express toxins. These toxins include, but are not limited to, ochratoxins, fumonisins, cyclapiazonic acid, 3-nitropropionic acid, emodin, malformin, aflatoxins, and secalonic acids. Such deficiency is preferably as described in WO2000/039322. (super) expression
[0147] In a preferred embodiment, the polynucleotides of the present invention, as described herein, may be overexpressed in a microbial strain of the invention compared to the microbial strain of origin in which said gene is not overexpressed. Overexpression of a polynucleotide sequence is defined herein as the expression of the gene of said sequence that results in an enzyme activity encoded by said sequence in a microbial strain having at least about 1.5 times the enzyme activity in the microbe of origin, preferably, the activity of said enzyme is at least about 2 times, more preferably, at least about 3 times, more preferably, at least about 4 times, more preferably, at least about 5 times, even more preferably, at least about 10 times and more preferably at least about 20 times the activity of the parent microbial enzyme.
[0148] The vector can also include sequences that flank the polynucleotide giving rise to RNA that comprises sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell.
[0149] An integrative cloning vector can integrate randomly or to a predetermined target locus in the chromosome(s) of the host cell in which it will be integrated. In a preferred embodiment of the present invention, an integrative cloning vector may comprise a DNA fragment that is homologous to a DNA sequence at a predetermined target locus in the host cell genome to direct the integration of the cloning vector to that locus predetermined. In order to promote targeted integration, the cloning vector can preferably be linearized prior to transformation of the host cell. Linearization can preferably be carried out in such a way that at least one, but preferably both ends of the cloning vector are flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least about 0.1 kb, such as about at least 0.2kb, more preferably at least about 0.5 kb, even more preferably at least about of 1 kb, more preferably at least about 2 kb. Preferably, the source host strains can be modified for a better frequency of target DNA integration, as described in WO05/095624 and/or WO2007/115886.
[0150] The deletion example provided in the present invention uses the gene promoter as the 5' flank and the gene as the 3' flank to insert a selection marker between the promoter and the gene, thus breaking (i.e., functionally inactivating) the transcription gene. The above gene sequences can be used to make functionally similar inactivated genes. The genes can be split in two, providing a 5' flank and a 3' flank, but the gene can also be used to clone a larger piece of genomic DNA that contains the gene terminator and promoter regions, which cannot function as a -5' flank and a 3' flank.
[0151] The vector system can be a vector, such as a single plasmid, or two or more vectors, such as two or more plasmids, which together contain the total DNA to be introduced into the genome of the host cell.
[0152] The vector may contain a polynucleotide of the invention oriented in an antisense direction to provide antisense RNA production.
[0153] The vector DNA can be introduced into prokaryotic or eukaryotic cells through conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (eg, DNA) into a host cell, including calcium phosphate co-precipitation or calcium chloride, DEAE-dextran mediated transfection, transduction, infection, lipofection, cationic lipid mediated transfection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al, Basic Methods in Molecular Biology (1986) and other laboratory manuals.
[0154] For stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene encoding a selectable marker (eg, antibiotic resistance) is usually introduced into host cells, along with the gene of interest. Preferred selectable markers include, but are not limited to, those that confer drug resistance or that complement a defect in the host cell. They include, for example, the versatile marker genes that can be used for the transformation of most filamentous fungi and yeasts, such as acetamidase genes or cDNAs (the amdS, niaD, facD or cDNAs genes from A. nidulans, A. oryzae or A. niger) or genes that provide antibiotic resistance such as resistance to G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomila (benA). Alternatively, specific selection markers can be used as auxotrophic markers that require corresponding mutant host strains: for example, URA3 (from S. cerevisiae or other yeast analogue genes), pyrG or pyrA (from A. nidulans or A. niger) , argB (from A. nidulans or A. niger) or trpC. In a preferred embodiment, the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide, which are free of selection marker genes.
[0155] Other markers include ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate decarboxylase (pvrA), the bacterial resistance gene G418 (this can also be used in yeast but not fungi), the resistance gene ampicillin (E. coli ), the neomycin resistance gene (Bacillus), the nourseotricin nat1 resistance gene from Streptomyces nursei, the pyrthiamine PTRA resistance gene from Aspergillus oryzae, and the E. coli uidA gene, which encodes β-glucuronidase (GUS). Vectors can be used in vitro, for example, for the production of RNA or used to transfect or transform a host cell.
[0156] Protein expression in prokaryotes is generally performed in E. coli with vectors containing constitutive or inducible promoters directing the expression of fusion or non-fusion proteins. Fusion vectors add a number of amino acids from a protein encoded therein, for example, to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein, 2) to increase the solubility of the recombinant protein, and 3) to aid in the purification of the recombinant protein by acting as a binder in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and recombinant protein to allow separation of the recombinant protein from the later fusion moiety for purification of the fusion protein.
[0157] As indicated, expression vectors preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for culturing eukaryotic cells and tetracycline or ampicillin resistance for culturing in E. coli and other bacteria.
[0158] Preferred vectors for use in bacteria, for example, are described in WO-A1-2004/074468, which are incorporated herein by reference. Other suitable vectors will be readily apparent to a person skilled in the art.
[0159] For secretion of translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular medium, the appropriate secretion signal can be incorporated into the expressed polypeptide. Signals can be endogenous to the polypeptide or they can be heterologous signals.
[0160] The TEMER07589 polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. Thus, for example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Additionally, peptide fractions can be added to the polypeptide to facilitate purification.
[0161] The invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO: 2, and an amino acid sequence obtained by expression of the polynucleotide of SEQ ID NO: 1, in an appropriate host. Also, a peptide or polypeptide comprising a variant of the above polypeptides, such as a functional equivalent, is encompassed by the present invention. The above polypeptides are collectively encompassed within the term "polypeptides according to the invention".
[0162] The term "variant peptide" or "variant polypeptide" is defined herein as a peptide or polypeptide, respectively, comprising one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more specific amino acid residues at one or more specific positions on the peptide or polypeptide, respectively. Thus, a variant signal peptide is a signal peptide consisting of one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more specific amino acid residues at one or more specific positions of the signal peptide.
[0163] The term "polynucleotide" is identical to the term "nucleic acid molecule" and may be read interchangeably herein. The term refers to a polynucleotide molecule, which is either a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), either single-stranded or double-stranded. A polynucleotide can either be present in its isolated form, be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell.
[0164] The "variant polynucleotide" is defined herein as a polynucleotide that comprises one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more nucleotides at one or more specific positions in the polynucleotide.
[0165] The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably, as the context requires to indicate a chain of at least two amino acids coupled through peptide bonds. The word "polypeptide" is used here for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences here are written from left to right and in the direction from amino terminus to carboxyl terminus. The one-letter amino acid code used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
[0166] By "isolated" polypeptide or protein is meant a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides that have been substantially purified by any suitable technique, such as, for example, the method of single-step purification described in Smith and Johnson, Gene 67:31-40 (1988).
[0167] The cellulase enhancing polypeptide TEMER07589 according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.
[0168] The polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, yeasts, bacteria, higher plants, insect and insect cells. mammals. Dependent on the host employed in a recombinant production process, the polypeptides of the present invention can be glycosylated or they can be non-glycosylated. Furthermore, the polypeptides of the present invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
[0169] The invention also includes biologically active fragments of the polypeptides according to the invention.
[0170] Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the TEMER07589 polypeptide (for example, the amino acid sequence of SEQ ID NO: 2), which include fewer amino acids than the full-length polypeptide, but which exhibit at least one biological activity of the corresponding full-length polypeptide. Typically, biologically active fragments comprise a domain or motif with at least one activity of the TEMER07589 polypeptide.
[0171] A biologically active fragment of a polypeptide of the present invention can be a polypeptide that is, for example, about 10, about 25, about 50, about 100 or more amino acids in length, or at least about 100 amino acids at least 150, 200, 250, 300, 350, 400 amino acids in length, or a length up to the total number of amino acids of the polypeptide of the present invention.
[0172] In addition, other biologically active portions, in which other regions of the polypeptide are excluded, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the present invention.
[0173] The invention also includes nucleic acid fragments encoding the above biologically active fragments of the TEMER07589 polypeptide. Polypeptides
[0174] In another aspect of the present invention, improved TEMER07589 polypeptides are provided. Improved TEMER07589 polypeptides are polypeptides in which at least one biological activity is enhanced. Such polypeptides can be obtained by randomly introducing mutations along all or part of the TEMER07589 coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity.
[0175] Improved variants of the amino acid sequences of the present invention leading to an improved booster function of cellulase can be obtained by the corresponding genes of the present invention. Among such modifications are included: 1. PCR Error prone to introduce random mutations, followed by a screening of the obtained variants and isolation of improved variants with kinetic properties. 2. A shuffling family of variants related to genes encoding the cellulase booster enzyme, followed by a screening of the obtained variants and isolation of variants with improved kinetic properties.
[0176] Variants of the genes of the present invention, leading to an increase in the level of mRNA and/or polypeptide, resulting in cellulase reinforcement activity can be obtained by the polynucleotide sequences of said genes. Among such modifications include: 1. Improving codon usage such that codons are (optimally) tailored to the primary microbial host. 2. Improve codon pair usage such that codons are (optimally) tailored to the primary microbial host. 3. Addition of stabilization sequences to the genomic information encoding the cellulase booster polypeptide resulting in mRNA molecules with an increased half-life.
[0177] Preferred methods for isolating variants with improved catalytic properties or increased levels of mRNA or polypeptide are described in WO03/010183 and WO03/0131 1. Preferred methods for improving codon usage in major microbial strains are described in PCT/EP2007/05594. Preferred methods for adding stabilizing elements to the genes encoding the cellulase enhancing polypeptide of the invention are described in WO2005/059149.
[0178] In a preferred embodiment, the TEMER07589 polypeptide has an amino acid sequence according to SEQ ID NO: 2. In another embodiment, the TEMER07589 polypeptide is substantially homologous to the amino acid sequence according to SEQ ID NO: 2 and retains by less one biological activity of a polypeptide according to SEQ ID NO: 2, but differs in amino acid sequence due to natural variation or mutagenesis as described.
[0179] In another preferred embodiment, the TEMER07589 polypeptide has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridizing with a nucleic acid according to SEQ ID NO: 1, preferably under highly stringent hybridization conditions.
[0180] Accordingly, the TEMER07589 polypeptide is preferably a polypeptide comprising an amino acid sequence of at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92% , 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 2, and normally at least retains a functional activity of the polypeptide according to SEQ ID NO:2.
[0181] Functional equivalents of a polypeptide according to the invention can also be identified, for example, by screening combinatorial libraries of mutants, for example, truncation mutants, of the polypeptide of the present invention to increase cellulase activity. In one embodiment, a diverse library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A varied library of variants can be produced, for example, by enzymatically linking a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential polypeptide sequences is expressed as individual polypeptides, or alternatively, as a set of fusion proteins larger (eg for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the present invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev. Biochem 53:323; Itakura et al. (1984) Science 198 :1056; Ike et al. (1983) Nucleic Acid Res. 11:477).
[0182] Furthermore, libraries of fragments of the coding sequence of a polypeptide of the present invention can be used to generate a varied population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of the coding sequence of interest with a nuclease under conditions where cleavage occurs only about once per molecule, denaturation of double-stranded DNA, the renaturation of DNA to form double-stranded DNA that can include sense/antisense pairs from different pairs of cut products, removing single-stranded portions of reformed double-stranded, by treatment with S1 nuclease, and linking the resulting fragment library into an expression vector. By this method, an expression library can be derived that encodes N-terminal and internal fragments of various sizes of the protein of interest.
[0183] Several techniques are known in the art for screening gene products from combinatorial libraries produced by point truncation mutations, and for screening cDNA libraries for gene products that possess a chosen property. The most widely used techniques, which are amenable to high-throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions where detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in libraries, can be used in combination with screening assays to identify variants of a polypeptide of the present invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
[0184] In addition to the TEMER07589 gene sequence shown in SEQ ID NO:1, it will be apparent to the person skilled in the art that DNA sequence polymorphisms may exist within a given population, which may lead to changes in the amino acid sequence of TEMER07589 polypeptide. Such genetic polymorphisms can exist in cells from different populations or within a population due to natural allelic variation. Allelic variants can also include functional equivalents.
[0185] Fragments of a polynucleotide according to the invention may also comprise polynucleotides encoding non-functional polypeptides. Such polynucleotides can function as probes or primers for the PCR reaction.
[0186] The nucleic acids according to the invention, regardless of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a TEMER07589 activity include, inter alia, (1) isolation of the gene encoding TEMER07589 polypeptide variants or allelic variants from a cDNA library, for example, from of appropriate microorganisms, (2) in situ hybridization (eg FISH) to chromosomal metaphase spreads to provide the exact chromosomal location of the TEMER07589 gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for the detection of TEMER07589 mRNA expression in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyze for the presence of a hybridizable nucleic acid to the TEMER07589 probe in a given biological sample (eg tissue).
[0187] A method of obtaining a functional equivalent of a TEMER07589 gene is also encompassed by the present invention. Such a method entails obtaining a labeled probe which includes an isolated nucleic acid encoding all or a portion of the polypeptide sequence according to SEQ ID NO:2 or a variant thereof; screening a nucleic acid fragment library with the labeled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence to from the nucleic acid fragments in any labeled double strand to obtain a gene related to the TEMER07589 gene.
[0188] In one embodiment, a TEMER07589 nucleic acid of the invention is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least minus 97%, at least 98%, at least 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO:1 or the complement thereof.
[0189] Further provided are: host cells comprising a polynucleotide or vector of the invention. The polynucleotide can be heterologous to the host cell genome. The term "heterologous", usually in relation to the host cell, means that the polynucleotide does not naturally occur in the host cell's genome or that the polypeptide is not naturally produced by that cell.
[0190] In another embodiment, the present invention features, for example, cells, transformed host cells, or recombinant host cells containing a nucleic acid encompassed by the invention. A "transformed cell" or "recombinant cell" is a cell into which (or an ancestor into which) a nucleic acid according to the invention has been introduced, by means of recombinant DNA techniques. Both prokaryotic and eukaryotic cells are included, for example bacteria, fungi, yeasts and the like, especially preferred are filamentous fungal cells such as Aspergillus niger or Talaromyces emersonii.
[0191] A host cell can be chosen that modulates the expression of the inserted sequences or modifies and processes the gene product in a specific and desired way. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can facilitate optimal protein functioning.
[0192] Several host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems known to those skilled in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that have the cellular machinery for proper processing of primary transcription, glycosylation and phosphorylation of the gene product can be used. Such host cells are well known in the art.
[0193] If desired, a cell as described above can be used for the preparation of a polypeptide according to the invention. Such a method typically comprises culturing a host cell (e.g., transformed or transfected with an expression vector, as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and, optionally, recovering the expressed polypeptide. The polynucleotides of the present invention can be incorporated into a recombinant replicable vector, e.g., an expression vector. The vector can be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention provides a method of manufacturing a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that elicit vector replication. The vector can be retrieved from the host cell.
[0194] Preferably, the polypeptide is produced as a secreted protein, in which case the nucleotide sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a nucleotide sequence encoding a signal sequence. Preferably, the signal sequence is native (homologous) to the nucleotide sequence encoding the polypeptide. Alternatively, the signal sequence is foreign (heterologous) to the nucleotide sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence according to the invention is expressed. Examples of suitable signal sequences for yeast host cells are signal sequences derived from yeast a-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is, for example, a signal sequence derived from a filamentous fungus amyloglucosidase (AG) gene, e.g., the A. niger glaA gene. This can be used in combination with the amyloglucosidase (also called (gluco)amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences can also be used within the context of the present invention.
[0195] Preferred heterologous secretion guide sequences are those that originate from the fungal amyloglucosidase (GA) gene (glaA-both 18 and 24 amino acid versions, eg from Aspergillus), the α-factor gene (eg , Saccharomyces and Kluyveromyces yeasts) or the α-amylase gene (Bacillus).
[0196] Vectors can be transformed or transfected into a suitable host cell, as described above, to provide expression of a polypeptide of the present invention. This process may comprise culturing a host cell transformed with an expression vector, as described above, under conditions to provide for expression by the vector of a coding sequence that encodes the polypeptide. host cells
[0197] Thus, the invention provides host cells transformed or transfected with or comprising a polynucleotide or vector of the invention. Preferably, the polynucleotide is carried in a vector for the replication and expression of the polynucleotide. Cells will be chosen to be compatible with said vector and may, for example, be prokaryotic (e.g. bacterial), fungal, yeast, or plant cells.
[0198] A heterologous host can also be chosen in which the polypeptide of the present invention is produced in a manner that is substantially free of other enzymes that degrade cellulose or degrade hemicellulose. This can be achieved by choosing a host, which normally does not produce such enzymes.
[0199] The present invention encompasses processes for the production of the polypeptide of the present invention through recombinant expression of a DNA sequence encoding the polypeptide. For this purpose, the DNA sequence of the present invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow the economical production of the polypeptide in a homologous host cell or suitable heterologous. A homologous host cell is a host cell that is of the same species or that is a variant within the same species as the species from which the DNA sequence is derived.
[0200] Suitable host cells are preferably prokaryotic microorganisms such as bacteria or more preferably eukaryotic organisms, for example fungi such as yeast or filamentous fungi, or plant cells. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are poorly secreted from yeast, or in some cases are not processed correctly (eg hyperglycosylation in yeast). In such cases, a fungal host organism must be selected.
[0201] The host cell can overexpress the polypeptide and techniques for overexpression engineering are well known. The host may thus have two or more copies of the polynucleotide encoding (and the vector may thus have two or more copies accordingly).
[0202] Bacillus genus bacteria are very suitable as heterologous hosts due to their ability to secrete proteins into the culture medium. Other suitable bacteria as hosts are those of the genus Streptomyces and Pseudomonas. A preferred yeast host cell for expressing the DNA sequence encoding the polypeptide is from the genera Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharomyces.
More preferably, a yeast host cell is selected from the group consisting of species of Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var.lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica and Schizosaccharomyces pombe.
[0204] Most preferred are, however, fungal host cells (eg, filamentous). Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma/Hypocrea, Fusarium, Disporotrichum, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, Chryosporium, Neuromycospora, and Husporium.
[0205] More preferably, a filamentous fungal host cell is one of the species including, but not limited to, Aspergillus niger, Aspergillus awamori, Aspergillus tubingensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, and Aspergillus opergillus , Trichoderma reesei/Hypocrea jecorina, Fusarium graminearum, Talaromyces emersonii, Penicillium decumbens, Acremonium alabamense, Neurospora crassa, Myceliophtora thernaophilurri, Sporotrichum cellulophilum, Disporotrichum dimorphosmyela teres et al.
[0206] Host cells according to the invention include plant cells and the invention therefore extends to transgenic organisms, such as plants and parts thereof, which contain one or more cells of the invention. Cells can heterologously express the polypeptide of the invention or can heterologously contain one or more of the polynucleotides of the invention. The transgenic (or genetically modified) plant may therefore have (for example, stably) inserted into its genome a sequence encoding one or more of the polypeptides of the invention. Transformation of plant cells can be carried out using known techniques, for example using a Ti or Ri plasmid from Agrobacterium tumefaciens. The plasmid (or vector) can thus contain sequences necessary to infect a plant, and derivatives of the Ti and/or Ri plasmids can be employed.
[0207] Alternatively, direct infection of a part of a plant such as a leaf, root or stem can be effected. In this technique, the plant to be infected can be wrapped, for example, by cutting the plant with a razor or piercing the plant with a needle or rubbing the plant with an abrasive. The wrapped plant is then inoculated with Agrobacterium. The plant or plant part can then be grown in a suitable culture medium and allowed to develop into a mature plant. Regeneration of transformed cells into genetically modified plants can be accomplished by known techniques, for example, by selecting transformed shoots using an antibiotic and by subculture the shoots in a medium containing the appropriate nutrients, plant hormones and the like.
[0208] The invention also includes cells that have been modified to express the cellulase enhancing polypeptide of the invention or a variant thereof. Such cells include transient, or preferably more stable, eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as fungal (e.g., filamentous) cells, and yeast or prokaryotic cells, such as bacterial cells.
[0209] Furthermore, it is possible that the polypeptides of the present invention are transiently expressed in a cell lineage or in a membrane, such as, for example, in a baculovirus expression system. Such systems, which are adapted to express the polypeptides according to the invention, are also included within the scope of the present invention.
[0210] According to the present invention, the production of the polypeptide of the present invention can be carried out by culturing microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium . Polypeptide/enzyme production
[0211] Recombinant host cells according to the invention can be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available that are conducive to expression of the DNA sequence encoding the polypeptide. After reaching the desired cell density or titration of the polypeptide, the culture is stopped and the polypeptide is recovered using known procedures.
[0212] The fermentation medium may comprise a known culture medium containing a carbon source (glucose, for example, maltose, molasses, starch, cellulose, xylan, pectin, a lignocellolytic biomass hydrolyzate, etc.), a source of nitrogen (eg ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (eg yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (per example, phosphate, magnesium, potassium, zinc, iron, etc.) Optionally, an inducer (eg, cellulose, pectin, xylan, maltose, maltodextrin or xylogalacturonane) can be included.
[0213] The selection of the appropriate medium can be based on the choice of the expression host and/or based on the regulatory requirements of the expression construct. Such means are well known to those skilled in the art. The medium can, if desired, contain additional components that favor the transformed expression hosts over other potentially contaminating microorganisms.
[0214] The production of the polypeptide by the transformed host (fermentation) can be performed according to any known procedure. The production time can be extended over a period of about 0.5 to about 30 days. It can be a continuous batch or fed-batch process, suitably at a temperature in the range of 0 to 100°C or 0 to 80°C, for example from about 0 to about 60°C and/or at a pH value, for example, from about 2 to about 10, or from about 3 to about 9. Preferred fermentation conditions are a temperature in the range of about 20 to about 55°C and/or at a pH of about 3 to about 5. Appropriate conditions are usually selected based on the choice of expression host and the polypeptide to be expressed.
[0215] After fermentation, if necessary, cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after cell removal, the polypeptide of the invention can then be recovered and, if desired, purified and isolated by conventional means. Polypeptide/Enzyme Compositions
[0216] The present invention provides a composition comprising a polypeptide of the present invention and a cellulase and/or a hemicellulase and/or a pectinase.
[0217] When the polypeptide of the present invention is a cellulase, a composition of the present invention will normally comprise a hemicellulase and/or a pectinase in addition to the polypeptide of the invention.
[0218] When the polypeptide of the present invention is a hemicellulase, a composition of the present invention will normally comprise a cellulase and/or a pectinase in addition to the polypeptide of the invention.
[0219] When the polypeptide of the present invention is a pectinase, a composition of the present invention will normally comprise a cellulase and/or a hemicellulase, in addition to the polypeptide of the invention.
[0220] A composition of the invention may comprise one, two or three or more classes of cellulase, for example, one, two or all of an endo-1,4-β-glucanase (EG), an exocellobiohydrolase ( CBH) and β-glucosidase (BGL).
[0221] A composition of the invention may comprise a polypeptide that has the same enzymatic activity, for example, the same type of cellulase, hemicellulase and/or pectinase activity as provided by a polypeptide of the present invention.
[0222] A composition of the invention may comprise a polypeptide, which has a different type of cellulase activity and/or hemicellulase activity and/or pectinase activity than that provided by a polypeptide of the present invention. For example, a composition of the invention may comprise one type of cellulase and/or hemicellulase activity and/or pectinase activity provided by a polypeptide of the present invention and a second type of cellulase and/or hemicellulase activity and/or pectinase activity is provided by an additional hemicellulase/pectinase.
[0223] Here, a cellulase is any polypeptide that is capable of degrading or degrading cellulose. A polypeptide that is capable of degrading cellulose is one that is capable of catalyzing the process of breaking down cellulose into smaller units, either partially, for example, into cellodextrins, or completely into glucose monomers. The cellulase according to the present invention can give rise to a mixed population of cellodextrins and glucose monomers when in contact with cellulase. Such degradation will normally take place through a hydrolysis reaction.
[0224] Here, a hemicellulase is any polypeptide that is capable of degrading or of hemicellulose. That is, a hemicellulase may be capable of degrading either one or more of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan. A polypeptide that is capable of degrading a hemicellulose is one that is capable of catalyzing the process of hemicellulose decomposition into smaller polysaccharides, either partially, for example, into oligosaccharides, or completely into sugar monomers, for example, hexose sugar monomers or pentose. The hemicellulase according to the present invention can give rise to a mixed population of oligosaccharides and sugar monomers when in contact with the hemicellulase. Such degradation will normally take place through a hydrolysis reaction.
[0225] Here, a pectinase is any polypeptide that is capable of degrading pectin. A polypeptide that is capable of degrading pectin is one that is capable of catalyzing the process of breaking down pectin into smaller units, either partially, for example, into oligosaccharides, or completely into sugar monomers. The pectinase according to the present invention can give rise to a mixed population of oligosaccharides and sugar monomers when in contact with pectinase. Such degradation will normally take place through a hydrolysis reaction.
[0226] Consequently, a composition of the invention may comprise any cellulase, for example, a cellobiohydrolase, an endo-β-1,4-glucanase, a β-glucosidase or a β-(1,3)(1,4 )-glucanase.
[0227] Here, a cellobiohydrolase is any polypeptide that is capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic bonds in cellulose or cellotetraose, releasing cellobiose from the ends of the chains. This enzyme may also be referred to as cellulase 1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucan cellobiohydrolase, avicelase, exo-1,4-β-D -glucanase, exocellulase booster or exoglucanase. This may have the code EC EC 3.2.1.91. Cellobiohydrolases can be subdivided into cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II). CBH I is defined as cellobiohydrolase that hydrolyzes cellulose predominantly from the reducing ends, the cellobiose division. CBH II is defined as cellobiohydrolase that hydrolyzes cellulose from predominantly non-reducing ends, the cellobiose breakdown.
[0228] Here, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,4-β-D-glucosidic bonds in cellulose, or β- Cereal D-glucans. Such a polypeptide may also be capable of hydrolyzing 1,4-links to β-D-glucans also containing 1,3-linkages. This enzyme may also be referred to as cellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase, cellulase, carboxymethylcellulose, celludextrinase, endo-1,4-β-D-glucanase, endo-1, 4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase. CEA is here an EC 3.2.1.4 endoglucanase which, based on its 3D structure is classified under the Glycosyl Hydrolase 5 (GH5) family. Activities known to members of the GH5 family include chitosanase (EC 3.2.1.132); β-mannosidase (EC 3.2.1.25); cellulase (EC 3.2.1.4); 1,3-β-glucosidase glucan (EC 3.2.1.58); licheninase (EC 3.2.1.73); glucan endo-1,6-β-glucosidase (EC 3.2.1.75); endo-β-1,4-mannosidase mannan (EC 3.2.1.78); endo-β-1,4-xylanase (EC 3.2.1.8); cellulose β-1,4-cellobiosidase (EC 3.2.1.91); endo-β-1,6-galactanase (EC 3.2.1.-); β-1,3-mannanase (EC 3.2.1.-); endo-β-1,4-xyloglucan-specific glucanase (EC 3.2.1.151); mannan transglycosylase (EC 2.4.1.-). CEB is here an EC 3.2.1.4 endoglucanase which, based on its 3D structure is classified under the Glycosyl Hydrolase 7 (GH7) family. Activities known to members of the GH7 family include endo-β-1,4-glucanase (EC 3.2.1.4); [reducing end action] cellobiohydrolase (EC 3.2.1.-); chitosanase (CE 3.2.1.132); endo-β-1,3-1,4-glucanase (EC 3.2.1.73).
[0229] Here, a β-glucosidase (abbreviated BG) (EC 3.2.1.21) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing β-D-glucose residues with the release of β-D-glucose . Such a polypeptide may have broad specificity for β-D-glucosides and may also hydrolyze one or more of the following: a β-D-galactoside, an α-L-arabinoside, a β-D-xyloside, or a β-D- fucoside. This enzyme may also be referred to as amidalase, β-D-glucoside glucohydrolase, cellobiase or gentiobiase.
[0230] Here a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide that is capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic bonds into β-D -glucans containing 1,3 and 1,4 linkages. Such a polypeptide can act on lechitin and cereal β-D-glucans, but not on β-D-glucans containing only 1,3- or 1,4-links. This enzyme may also be referred to as licheninase, 1,3-1,4-β-D-glucan 4-glucan hydrolase, β-glucanase, endo-β-1,3-1,4 glucanase, lichenase, or mixed linkage of β-glucanase. An alternative for this type of enzyme is EC 3.2.1.6, which is described as endo-1,3(4)-beta-glucanase. This type of enzyme hydrolyzes 1,3- or 1,4- bonds in beta-D-glucans when the glucose residue whose reducing group is involved in the bond to be hydrolyzed is itself replaced at C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase, 1,3-(1,3,1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates include laminarin, cereal lichen, and beta-D-glucans.
[0231] A composition of the invention may comprise any hemicellulase, for example, an endoxylanase, a β-xylosidase, an α-L-arabionofuranosidase, an α-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanase or a β-mannosidase.
[0232] Here, an endoxylanase (EC 3.2.1.8) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,4-β-D-xylosidic bonds in xylans. This enzyme may also be referred to as endo-1,4-β-xylanase or 1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is capable of hydrolyzing 1,4-xylosidic bonds in glucuronoarabinoxylans.
[0233] Here, a β-xylosidase (EC 3.2.1.37) is any polypeptide that is capable of catalyzing the hydrolysis of 1,4- β-D-xylans, to remove successive D-xylose residues from residues from of non-reducing terminals. Such enzymes can also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase or xylobiase.
[0234] Here, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptide that is capable of acting on α-L-arabinofuranosides, α-L-arabinans containing (1,2) and/or (1, 3)- and/or (1.5), arabinoxylans and arabinogalactans. This enzyme may also be referred to as α-N-arabinofuranosidase, arabinofuranosidase, or arabinosidase.
[0235] Here, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide that is capable of catalyzing a reaction as follows: alpha-D-glucuronoside + H(2)O = an alcohol + D-glucuronate. This enzyme may also be referred to as alpha-glucuronidase or alpha-glucosiduronase. These enzymes can also hydrolyze 4-O-methylated glucuronic acid, which may also be present as a substituent in xylans. Alternative is EC 3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyzes the hydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl bonds.
[0236] Here, an acetyl xylan esterase from (EC 3.1.1.72) is any polypeptide that is capable of catalyzing the deacetylation of xylans and xylo-oligosaccharides. Such a polypeptide can catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-naphthyl acetate or p-nitrophenyl acetate, but normally not from triacylglycerol. Such a polypeptide normally does not act on acetylated mannan or pectin.
[0237] Here, a feruloyl esterase (EC 3.1.1.73) is any polypeptide that is capable of catalyzing a reaction in the form: feruloyl-saccharide + H(2)O = ferulate + saccharide. The saccharide can be, for example, an oligosaccharide or a polysaccharide. It can normally catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is generally in arabinose on "natural" substrates, p-nitrophenol acetate and methylferulate are usually poor substrates. This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, as this can help xylanases and pectinases break down plant cell wall hemicellulose and pectin.
[0238] Here, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide that is capable of catalyzing a reaction in the form: coumaroyl-saccharide + H(2)O = coumarate + saccharide. The saccharide can be, for example, an oligosaccharide or a polysaccharide. This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme is also encompassed in EC 3.1.1.73, it may also be referred to as a feruloyl esterase.
[0239] Here, an α-galactosidase (EC 3.2.1.22) is any polypeptide that is capable of catalyzing the hydrolysis of non-reducing, terminal α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing α-D-fucosides. This enzyme can also be referred to as melibiase.
[0240] Here, a β-galactosidase (EC 3.2.1.23) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing β-D-galactose residues into β-D-galactosides. Such a polypeptide may also be capable of hydrolyzing α-L-arabinosides. This enzyme may also be referred to as exo-(1^4)-3-D-galactanase or lactase.
[0241] Here, a β-mannanase (EC 3.2.1.78) is any polypeptide that is capable of catalyzing the random hydrolysis of 1,4-β-D-mannosidic bonds in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.
[0242] Here, a β-mannosidase (EC 3.2.1.25) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing β-D-mannose residues into β-D-mannosides. This enzyme can also be referred to as mannanase or mannase.
[0243] A composition of the invention may comprise any pectinase, for example an endopolygalacturonase, a methyl pectin esterase, an endo-galactanase, a beta-galactosidase, an acetyl pectin esterase, an endo-pectin lyase, a lyase of pectate, alpha-rhamnosidase, an exo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, an acetyl rhamnogalacturonase esterase, a rhamnogalacturonan, a galacturonohydrolase, or a rhamnogalacturonan.
[0244] Here, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide that is capable of catalyzing the random hydrolysis of 1,4-a-D-galactosiduronic bonds in pectate and other galacturonanes. This enzyme may also be referred to as pectin polygalacturonase depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-α-1,4-galacturonide glycanhydrolase, endogalacturonase; endo-D-galacturonase or poly(1,4-α-D-galacturonide glycanohydrolase).
[0245] Here, a pectin methyl esterase (EC 3.1.1.11) is any enzyme that is capable of catalyzing the reaction of: pectin + n H2O = n methanol + pectate. The enzyme may also be known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinesterase or pectin pectin hydrolase.
[0246] Here, an endo-galactanase (EC 3.2.1.89) is any enzyme capable of catalyzing the endohydrolysis of 1,4-β-D-galactosidic bonds in arabinogalactans. The enzyme may also be known as endo-1,4-β-galactosidase arabinogalactan, endo-1,4-β-galactanase, galactanase, arabinogalactanase or 4-β-D-galactan arabinogalactan hydrolase.
[0247] Here, an acetyl pectin esterase is defined herein as any enzyme that has an acetyl esterase activity that catalyzes the deacetylation of the acetyl groups on the hydroxyl groups of the GaIU residues of pectin.
[0248] Here, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminative cleavage of (1^4)-α-D-galacturonan methyl ester to provide the oligosaccharides with 4-deoxy groups -6-O-methyl-α-D-galact-4-enuronosyl at their non-reducing ends. The enzyme may also be known as pectin lyase, pectin transeliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1^4)-6-O-methyl-α-D-galacturonan lyase.
[0249] Here, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminative cleavage of (1^4)-α-D-galacturonan to provide oligosaccharides with 4-deoxy-aD-4-galact groups -enuronosyl at its non-reducing ends. The enzyme may also be known as polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, α-1,4-D-acid lyase endopolygalacturonic, PGA lyase, PPase-N, endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin transeliminase, polygalacturonic acid transeliminase or (1^4)-α-D-galacturonan lyase .
[0250] Here, an alpha-rhamnosidase (EC 3.2.1.40) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing α-L-rhamnose residues into α-L-rhamnosides or alternatively into rhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T, α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.
[0251] Here, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate. The enzyme may also be known as exo-poly-α-galacturonosidase, exopolygalacturonosidase, or exopolygalacturonosidase.
[0252] Here, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable of catalyzing: (1,4-aD-galacturonide)n + H2O = (1,4-aD-galacturonide)n-1 + D-galacturonate . The enzyme may also be known as galacturan 1,4-α-galacturonidase, exopolygalacturonase, poly(galacturonate hydrolase), exo-D-galacturonase, exo-D-galacturonase, exopoly-D-galacturonase, or poly(galacturonate hydrolase). 1,4-aD-galacturonide).
[0253] Here, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing the eliminative cleavage of 4-(4-desoxy-aD-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate , that is, the deesterified pectin. This enzyme may be known as pectate disaccharide lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid transeliminase, PATE, exo-PATE, exo-PGL, or end-reducing end disaccharide lyase (1^4)-α-D-galacturonan.
[0254] Here, rhamnogalacturonan hydrolase is any polypeptide that is capable of hydrolyzing the bond between galactosyluronic acid and rhamnopyranosyl in an endo-like form in strictly alternating rhamnogalacturonan structures consisting of disaccharide [(1,2-di-alpha-L - rhamnoyl-(1,4)-alpha-galactosyluronic acid)]. (a) Here, rhamnogalacturonan lyase is any polypeptide that is any polypeptide that is capable of cleaving α-L-Rhap-(1^4)-α-D-GalpA bonds in an endo-like form in rhamnogalacturonan by beta-elimination.
[0255] Here, rhamnogalacturonan acetyl esterase is any polypeptide that catalyzes the deacetylation of the alternating backbone of rhamnose and galacturonic acid residues in rhamnogalacturonan.
[0256] Here, rhamnogalacturonan hydrolase is any polypeptide that is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-form.
[0257] Here, xylogalacturonase is any polypeptide that acts on xylolacturonan by cleaving the backbone of β-xylose-substituted galacturonic acid in an endo form. This enzyme may also be known as xylogalacturonane hydrolase.
[0258] Here, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptide that is capable of acting on α-L-arabinofuranosides, (1,2) and/or (1,3)- and/or linkages (1,5) containing α-L-arabinans, arabinoxylans and arabinogalactans. This enzyme may also be referred to as α-N-arabinofuranosidase, arabinofuranosidase, or arabinosidase.
[0259] Here, endo-arabinanase (EC 3.2.1.99) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,5-a-arabinofuranosidic bonds into 1,5-arabinanes. The enzyme may also be known as endo-arabinase, endo-1,5-α-L-arabinosidase arabinan, endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or 1,5-α-L-arabinane hydrolase from 1,5-α-L-arabinan.
[0260] A composition of the present invention will normally comprise at least one cellulase and/or at least one hemicellulase and/or at least one pectinase (one of which is a polypeptide according to the invention). A composition of the invention may comprise a cellobiohydrolase, an endoglucanase and/or a β-glucosidase. Such a composition may also comprise one or more hemicellulases and/or one or more pectinases.
[0261] One or more (eg two, three, four or all) of an amylase, a protease, a lipase, a ligninase, a hexosyltransferase or a glucuronidase may be present in a composition of the invention.
[0262] "Protease" includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention incorporated herein by reference. Some specific types of proteases include cysteine proteases, including pepsin, papain, and serine proteases, including chymotrypsins, carboxypeptidases and metalloendopeptidases.
[0263] "Lipase" includes enzymes that hydrolyze lipids, fatty acids, and particularly acylglycerides, including phosphoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogenic infection. These lipids include waxes derived from fatty acids as well as cutin and suberin.
[0264] "Ligninase" includes enzymes that hydrolyze or can break down the structure of lignin polymers. Enzymes that degrade lignin can include lignin peroxidases, manganese peroxidases, laccases and feruloyl esterases, and other enzymes described in the known art for depolymerizing or otherwise breaking down lignin polymers. Enzymes capable of hydrolyzing bonds formed between hemicellulose sugars (particularly arabinose) and lignin are also included. Ligninases include, but are not limited to the following group of enzymes: lignin peroxidases (EC 1.11.14), manganese peroxidases (EC 1.11.1.13), the laccases (EC 1.10.3.2) and feruloyl esterases (EC 3.1.1.73) ).
[0265] "Hexosyltransferase" (2.4.1-) includes enzymes that are capable of transferring glycosyl groups, more specifically hexosyl groups. In addition, transferring a glycosyl group from a glycosyl-containing donor to another glycosyl-containing compound, the recipient, enzymes can also transfer the glycosyl group to water as a recipient. This reaction is also known as a hydrolysis reaction rather than a transfer reaction. An example of a hexosyltransferase that can be used in the present invention is β-glucanosyltransferase. Such an enzyme may be capable of catalyzing the degradation of (1,3)(1,4)glucan and/or cellulose and/or a cellulose degradation product.
[0266] "Glucoronidase" includes enzymes that catalyze the hydrolysis of a glucuronoside, eg, β-glucuronoside to obtain an alcohol. Many glucuronidases have been characterized and may be suitable for use in the present invention, for example, β-glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase (3.2.1.56), β-glucuronidase (3.2.1.56), β- glycyrrhizinate glucuronidase (3.2.1.128) or α-D-glucuronidase (EC 3.2.1.139).
[0267] A composition of the invention may comprise an expansin or expansin-like protein, such as a swolenin (see Salheimo et al., Eur. J. Biohem. 269, 4202-4211, 2002) or a swolenin-like protein.
[0268] Expansins are implicated in loosening the cell wall structure during plant cell growth. Expansins have been proposed to break hydrogen bonds between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of the cellulose fibers and the widening of the cell wall. Swolenin, an expansin-like protein contains an N-terminal 1 Carbohydrate Binding Module Family (CBD) domain and a C-terminal expansin-like domain. For the purposes of the present invention, an expansin-like protein or a swolenin-like protein similar can comprise one or both of these domains and/or can disturb the structure of the cell walls (such as disturb the structure of cellulose), optionally, without producing detectable amounts of reducing sugars.
[0269] The composition of the invention may comprise the polypeptide product of a cellulose-integrating protein, a structure or a structured-like protein, such as, for example, CipA or CipC from Clostridium thermocellum or Clostridium cellulolyticum respectively.
[0270] Cellulose or structured integration proteins are multifunctional that integrate subunits that can organize cellulolytic subunits into a multienzyme complex. This is achieved through the interaction of two complementary domain classes, that is, an adhesion domain on the structure and a dokerin domain in each enzymatic unit. The structure subunit also has a cellulose binding module (CBM), which mediates the binding of the cellulosome to its substrate. A cellulose-integrating or structured protein for the purposes of the present invention may comprise one or both of these domains.
[0271] A composition of the invention may comprise a cellulose-induced protein or modulating protein, for example, as encoded by the cipl or cip2 gene or similar genes from Trichoderma reesei/Hypocrea jecorina (see Foreman et al., J. Biol Chem. 278(34), 31988-31997, 2003). The polypeptide product of these genes are bimodular proteins, which contain a cellulose binding module and a domain that functions or activity cannot be related to the known glycosyl hydrolase families. However, the presence of a cellulose binding module and the co-regulation of the expression of these genes with cellulase components previously indicates unrecognized activities with a potential role in biomass degradation.
[0272] A composition of the invention may comprise an element of each of the classes of polypeptides mentioned above, several elements of a class of polypeptide, or any combination of these classes of polypeptides.
[0273] A composition of the present invention may be composed of polypeptides, eg enzymes, from (1) commercial suppliers, (2) polypeptides expressing cloned genes, eg enzymes, (3) complex broth (such as resulting from the growth of a microbial strain in the medium, in which the strains secrete proteins and enzymes in the medium, (4) cell lysates from strains cultivated as in (3); and/or (5) polypeptides expressing plant material, for example, enzymes Different polypeptides, such as, for example, enzymes, in a composition of the present invention can be obtained from different sources. Use of Polypeptides
[0274] Polypeptides and polypeptide compositions according to the invention can be used in many different applications. For example, they can be used to produce fermentable sugars. Fermentable sugars can then, as part of a biofuel process, be converted to biogas or ethanol, butanol, isobutanol, 2-butanol or other suitable substances. Alternatively, polypeptides and their compositions can be used as enzymes, for example, in the production of food products, in detergent compositions, in the pulp and paper industry and in antibacterial formulations, in pharmaceuticals such as throat lozenges, toothpaste, and mouthwash. Some of the applications will be illustrated in more detail below.
[0275] In the uses and methods described below, the components of the compositions described above may be provided simultaneously (that is, as a single composition per se), or separately or sequentially.
[0276] The present invention also relates to the use of the cellulase enhancing polypeptide according to the invention and to compositions comprising such an enzyme in industrial processes.
[0277] Despite the long experience obtained with these processes, the cellulase enhancing polypeptide according to the invention may present a number of significant advantages over currently used enzymes. Depending on the specific application, these advantages can include aspects such as lower production costs, greater substrate specificity, reduced antigenicity, fewer undesirable side actions, higher yields when produced in a suitable microorganism, pH and ranges more suitable temperature, non-inhibition by products derived from lignin, hydrophobic or less product inhibition or, in the case of the food industry, a better taste or texture of the final product, as well as food quality and kosher aspects.
[0278] In principle, a cellulase enhancing polypeptide or composition of the invention can be used in any process that requires the treatment of a material comprising polysaccharide. Thus, a polypeptide or composition of the present invention can be used in treating polysaccharide material. Thus, a polysaccharide material is a material that comprises or consists essentially of one or, more usually, more than one polysaccharide. Typically, plants and derived materials comprise significant amounts of non-starch polysaccharide material. Thereby, a polypeptide of the invention can be used in the treatment of a plant material or fungi or a material derived therefrom. Lignocellulose
[0279] An important component of plant non-starch polysaccharide material is lignocellulose (also referred to herein as lignocellulolytic biomass). Lignocellulose is the plant material comprising cellulose and hemicellulose and lignin. Carbohydrate polymers (cellulose and hemicellulose) are strongly bonded to lignin by hydrogen and covalent bonds. Thus, a polypeptide of the invention can be used in the treatment of lignocellulolytic material. Here, the lignocellulolytic material is a material that comprises or consists essentially of lignocellulose. Thus, in a method of the present invention for treating a non-starch polysaccharide, the non-starch polysaccharide may be a lignocellulosic material/biomass.
[0280] Accordingly, the invention provides a method of treating a substrate comprising the non-starch polysaccharide wherein the treatment comprises the degradation and/or hydrolysis and/or the modification of cellulose and/or hemicellulose and/or a pectic substance.
[0281] Degradation in this context indicates that the treatment results in the generation of hydrolysis products of cellulose and/or hemicellulose and/or a pectic substance, ie saccharides of shorter length are present as a result of the treatment than are present in a similar untreated non-starch polysaccharide. Thus, degradation, in this context, can result in the release of oligosaccharides and/or sugar monomers.
[0282] All plants contain non-starch polysaccharide as do virtually all plant-derived polysaccharide materials. Accordingly, in a method of the present invention for treating the substrate comprising a non-starch polysaccharide, said substrate may be provided in the form of a plant or plant-derived material or a material comprising plant or plant-derived material. , for example, a plant pulp, a plant extract, a food or ingredient, therefore a fabric, a textile product or an article of clothing.
[0283] Lignocellulolytic biomass suitable for use in the present invention includes biomass, which may include virgin biomass and/or non-virgin biomass, such as agricultural biomass, commercial organics, construction and demolition of debris, municipal solid waste, paper waste and garden waste. The most common forms of biomass include trees, shrubs and grasses, wheat, wheat straw, sugarcane bagasse, corn, corn husks, corn cobs, corn grain, including grain fibers, grain milling products and by-products, such as corn, wheat and barley (including wet milling and dry milling), often called "bran or fiber", as well as municipal solid waste, paper waste and garden waste. Biomass can also be, but is not limited to, herbaceous material, agricultural residues, forest residues, municipal solid residues, paper residues and pulp and paper mill residues.
[0284] "Agricultural biomass" includes twigs, shrubs, reeds, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous plants, leaves, bark, needles, trunks, roots, seedlings, woody crops short rotation, shrubs, panicum, trees, plants, fruit peels, vines, beet pulp, wheat, oat husks, and hard and soft woods (not including forest with harmful materials). In addition, agricultural biomass includes organic waste materials generated from agricultural processes, including forestry and agricultural activities, specifically including forest wood residues. Agricultural biomass can be any one of those singularly mentioned above or in any combination or a mixture thereof. Additional examples of suitable biomass are orchard starters, chaparral, municipal waste, wood waste mill, municipal waste, log waste, forest thinning, short rotation woody crops, industrial waste, wheat straw, oat straw, straw of rice, barley straw, rye straw, flax straw, soybean husk, rice husk, rice straw, corn gluten feed, oat husk, sugar cane, corn husk, corn stalk , corncobs, corn husks, grazing pasture, pasture, foxtail; beet pulp, citrus pulp, seed husks, cellulosic animal waste, grass clippings, cotton, algae, trees, shrubs, grasses, wheat, wheat straw, sugarcane bagasse, corn, corn husks, corn slabs , corn grain, grain fibers, wet or dry grain milling products and by-products, municipal solid waste, paper waste, garden waste, herbaceous material, agricultural waste, forestry waste, municipal solid waste, paper waste, cellulose , pulp mill waste, twigs, shrubs, reeds, corn, corn husks, energy yield, forest, fruit, flower, grain, grass, herbaceous crop, leaf, bark, needle, log, root, seedling, shrub, grass, tree, plant, fruit peel, vine, sugar beet pulp, wheat, oat husks, hard or soft wood, organic waste material produced from an agricultural process, wood forestry waste, or a combination of which er two or more of the same.
[0285] In addition to virgin biomass or raw materials that have already been processed in food and feed, or pulp and pulping industries, biomass/raw material can still be pretreated with heat, mechanical and/or chemical modification , or any combination of these methods, for the purpose of increasing enzymatic degradation. Pre-treatment
[0286] Prior to enzymatic treatment, the raw material may optionally be pretreated with heat, mechanical and/or chemical modification, or any combination of these methods, in order to improve substrate accessibility for enzymatic and/or hydrolysis hydrolyzing the hemicellulose and/or solubilizing the hemicelluloses and/or cellulose and/or lignin, in any manner known in the art. The pre-treatment may comprise exposing the lignocellulosic material to water (hot), steam (steam explosion), an acid, a base, a heat, a solvent, a peroxide, ozone, mechanical fragmentation, milling, grinding or depressurization fast, or a combination of any two or more of them. A chemical pretreatment is often combined with a thermal pretreatment, for example at 150 to 220°C for 1 to 30 minutes. Pre-saccharification
[0287] After the pretreatment step, a liquefaction/hydrolysis or pre-saccharification step involving incubation with an enzyme or enzyme mixture can be used. The presaccharification step can be carried out at many different temperatures, but it is preferable that the presaccharification step takes place at a temperature most suitable for the enzyme mixture to be applied, or the optimal predicted enzyme of the enzymes to be applied. The temperature of the presaccharification step can range from about 10°C to about 95°C, about 20°C to about 85°C, about 30°C to about 70°C, about 40° C to about 60°C, about 37°C to about 50°C, preferably from about 37°C to about 80°C, more preferably about 60 to 70°C still preferably about 65° Ç. The pH of the pre-saccharification mixture can range from about 2.0 to about 10.0, but is preferably about 3.0 to about 7.0, preferably about 4.0 to about 6.0. preferably about 4.0 to about 5.0. Again, pH can be adjusted to maximize enzyme activity and can be adjusted with the addition of enzyme.
[0288] The reaction of the liquefaction/hydrolysis or pre-saccharification step can take place from several minutes to several hours, such as from about 1 hour to about 120 hours, preferably from about 2 hours to about 48 hours , preferably from about 2 to about 24 hours, preferably for about 2 to about 6 hours. Cellulase treatment can take place from several minutes to several hours, such as from about 6 hours to about 120 hours, preferably about 12 hours to about 72 hours, preferably about 24 to 48 hours. saccharification
[0289] The present invention provides a method for producing a sugar from a lignocellulosic material which method comprises contacting a polypeptide of the present invention to a composition of the present invention with the lignocellulosic material.
[0290] Such a method allows free sugars (monomers) and/or oligosaccharides to be generated from lignocellulosic biomass. These methods involve the conversion of lignocellulosic biomass to free sugars and small oligosaccharides with a polypeptide or composition of the invention.
[0291] The process of converting a complex carbohydrate such as lignocellulose into sugars preferentially allows the conversion to fermentable sugars. Such a process may be referred to as "saccharification". Thus, a method of the invention can cause the release of one or more hexose and/or pentose sugars, such as one or more of glucose, xylose, arabinose, galactose, galacturonic acid, glucuronic acid, mannose, rhamnose, ribose and fructose .
[0292] Consequently, another aspect of the present invention includes methods using the polypeptide of the composition of the invention, described above, in conjunction with other enzymes or physical treatments such as temperature and pH to convert lignocellulosic biomass into sugars and oligosaccharides.
[0293] Although the composition has been discussed as a single mixture, it is recognized that enzymes can be added sequentially, in which temperature, pH, and other conditions can be changed to increase the activity of each individual enzyme. Alternatively, an optimal pH and temperature can be determined for the enzyme mixture.
[0294] Enzymes are reacted with the substrate under the appropriate conditions. For example, enzymes can be incubated at about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C or more . That is, they can be incubated at a temperature of between about 20°C to about 95°C, for example, in low to medium ionic strength buffers and/or from low to neutral pH. By "average ionic strength" the buffer is intended to have an ion concentration of about 200 millimolar (mM) or less, for any single ion component. The pH can range from about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, to about pH 5 .5, about pH 6, to about pH 6.5, about pH 7, to about pH 7.5, about pH 8.0, to about pH 8.5. Generally, the pH range will be from about pH 3.0 to about pH 7. For the production of ethanol in an acidic medium it is preferable, for example, pH = 4, while for the production of neutral pH biogas, by example, pH = 7 is desirable. Incubating enzyme combinations under these conditions results in the release or release of substantial amounts of sugar from the lignocellulose. By substantial amount at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of available sugar is intended.
[0295] Polypeptides such as enzymes, can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added, for example, to lignocellulosic raw material. Alternatively, enzymes are produced, but not isolated, and the fermentation broth from crude cell mass, or plant material (eg corn husk), and this can be added as, for example, the raw material. Alternatively, the crude cell mass or enzyme production medium or plant material can be treated to prevent further microbial growth (eg by heating or by adding antimicrobial agents), then added, for example, to a material. -cousin. These crude enzyme mixtures can include the organism producing the enzyme. Alternatively, the enzyme can be produced in a fermentation that uses a raw material (eg, corn husk) to provide nutrition to an organism that produces the enzyme(s). In this way, the plants that produce the enzymes can themselves serve as a lignocellulosic feedstock and be added to the lignocellulosic feedstock. Sugar fermentation
[0296] Fermentable sugars can be converted into useful value-added fermentation products, non-limiting examples of which include amino acids, vitamins, pharmaceuticals, feed supplements, specialty chemicals, chemical raw materials, plastics, solvents, fuels , or other organic polymers, lactic acid, and ethanol, including ethanol fuel. In particular, sugars can be used as raw materials for fermentation in chemicals, plastics such as, for example, succinic acid and (bio)fuels, including synthetic liquid fuels of ethanol, methanol, butanol and biogas.
[0297] For example, in the method of the present invention, an enzyme or combination of enzymes acts on a lignocellulosic substrate or plant biomass, which serves as a food product, in order to convert this complex substrate into simple sugars and oligosaccharides for production of ethanol or other useful fermentation products.
[0298] Sugars released from biomass can be converted into useful fermentation products, one of those including, but not limited to, amino acids, vitamins, pharmaceuticals, feed supplements, specialty chemicals, chemical raw materials, plastics , and ethanol, including ethanol fuel. Accordingly, the invention provides a method for preparing a fermentation product, which method comprises: a. degradation of lignocellulose using a method as described herein, and b. fermentation of the resulting material, to thereby prepare a fermentation product.
[0299] Fermentation can be carried out under aerobic or anaerobic conditions. Preferably, the process is carried out under oxygen-limited or microaerophilic conditions.
[0300] An anaerobic fermentation process is defined herein as a fermentation process carried out in the absence of oxygen or in which substantially no oxygen is consumed, preferably about 5 or less, about 2.5 or less, or about 1 mmol /L/h or less, and in which organic molecules serve as both electron donor and electron taker.
[0301] An oxygen-limited fermentation process is a process in which oxygen consumption is limited by the transfer of oxygen from gas to liquid. The degree of oxygen limitation is determined by the amount and composition of the continuous gas stream as well as the actual mix/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the oxygen consumption rate is at least about 5.5, preferably at least about 6, and most preferably at least about 7 mmol/L/h.
[0302] A method for preparing a fermentation product may optionally comprise recovery of the fermentation product. SSF
[0303] Fermentation and saccharification can also be carried out in simultaneous saccharification and fermentation (SSF) mode. One of the advantages of this mode is the reduction of sugar inhibition of enzymatic hydrolysis (sugar inhibition in cellulases is described by Caminal B&B Vol. XXVII Pp 12821290). Fermentation Products
[0304] Fermentation products that can be produced in accordance with the present invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical raw materials, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including ethanol fuel (the term "alcohol" is to be understood to include ethyl alcohol or mixtures of ethyl alcohol and water).
[0305] Specific value-added products that can be produced by the methods of the present invention include, but are not limited to, biofuels (including ethanol and butanol and a biogas), lactic acid, a plastic, a specialty chemical; an organic acid, including citric acid, succinic acid, fumaric acid, itaconic acid, and maleic acid; 3-hydroxy-propionic acid, acrylic acid, acetic acid, 1,3-propane-diol; ethylene, glycerol, a solvent, an animal food supplement, a pharmaceutical such as a β-lactam antibiotic or a cephalosporin; vitamins; an amino acid such as lysine, methionine, tryptophan, threonine and aspartic acid; an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, a transferase or a xylanase, and a chemical starting material. Biogas
[0306] The present invention, furthermore, provides the use of a polypeptide or a composition described herein in a method for the preparation of biogas. Biogas usually refers to a gas produced by the biological decomposition of organic matter, eg material containing non-starch carbohydrate, in the absence of oxygen. Biogas originates from biogenic material and is a type of biofuel. One type of biogas is produced by the anaerobic digestion or fermentation of biodegradable materials such as biomass, manure or sewage, municipal waste, and energy yields. This type of biogas is mainly composed of methane and carbon dioxide. Methane gas can be flared or oxidized with oxygen. Air contains 21% oxygen. This allows the release of biogas energy to be used as fuel. Biogas can be used as a low-cost fuel across the country for any heating purpose, such as cooking. It can also be used in modern waste management facilities, where it can be used to run any type of engine heating, to generate both mechanical and electrical energy.
[0307] The first step in the production of microbial biogas consists of the enzymatic degradation of polymers and complex substrates (eg non-starch carbohydrate). Consequently, the invention provides a method for the preparation of a biogas in which a substrate comprising non-starch carbohydrate is contacted with a polypeptide or composition of the present invention, thus to obtain fermentable material that can be converted to a biogas by a organism such as a microorganism. In such a method, a polypeptide of the present invention may be provided in the form of an organism, e.g., a micro-organism that expresses such a polypeptide. Use of enzymes in food products
[0308] The polypeptides and compositions of the present invention can be used in a method for processing plant material to degrade or modify cellulose or hemicellulose or pectic substance constituents of the cell walls of the plant or fungal material. Such methods can be useful for preparing a food product. Accordingly, the present invention provides a method for preparing a food product which method comprises incorporating a polypeptide or composition of the present invention during the preparation of the food product.
[0309] The invention also provides a method of treating a plant material which method comprises contacting the plant material with a polypeptide or composition of the present invention to degrade or modify the cellulose in the (vegetable) material. Preferably, the plant material is a plant cellulose or plant extract such as juice.
[0310] Pectic substance/hemicellulose/cellulose and plant-containing materials include plant cellulose, plant parts and plant extracts. In the context of this invention, an extract of a plant material is any substance that can be derived from plant material by extraction (mechanical and/or chemical), processing or by other separation techniques. The extract can be juice, nectar, base, or concentrates made from them. The plant material may comprise or be derived from plants, for example, carrots, celery, onion, vegetables or legumes (soybeans, soybeans, peas) or fruits, for example, stone fruits or seeds (apples, pears, quince, etc.), grapes, tomatoes, citrus fruits (orange, lemon, lime, tangerine), melons, plums, cherries, currants, raspberries, strawberries, blackberries, pineapple and other tropical fruits, trees and their parts (eg pollen, of pine trees), or cereals (oats, barley, wheat, corn, rice). The material (to be hydrolyzed) can also be agricultural waste, such as sugar beet pulp, corn slab, wheat straw, nut shells (from soil), or recyclable materials, for example, paper (waste).
[0311] The polypeptides of the present invention can thus be used to treat plant material including plant cellulose and plant extracts. These can also be used to treat liquid or solid foodstuffs or edible food ingredients, or be used for extracting vegetable oils such as starch or a thickening agent in foods.
[0312] Typically, the polypeptides of the present invention are used as a composition/enzyme preparation as described above. The composition will generally be added to plant pulp obtained through, for example, mechanical processing such as crushing or milling plant material. Incubation of the composition with the plant will normally be carried out for a period of between 10 minutes and 5 hours, such as 30 minutes to 2 hours, preferably for about 1 hour. The processing temperature is preferably from about 10°C to about 55°C, e.g. from about 15°C to about 25°C, optimally about 20°C and between about 10°C can be used. g about 300 g, preferably from about 30 g to about 70 g, optimally about 50 g of enzyme per ton of material to be treated.
[0313] All enzymes or their compositions used can be added sequentially or simultaneously to the pulp of the plant. Depending on the composition of the enzyme preparation, the plant material may first be macerated (eg pure) or liquefied. Using the polypeptides of the invention processing parameters such as extraction yield, extract viscosity and/or extract quality can be improved.
[0314] Alternatively, or in addition to the above, a polypeptide of the present invention can be added to the raw juice obtained from pressing, or liquefaction of the vegetable pulp. The treatment of raw juice will be carried out in a similar way to plant pulping in terms of dosage, temperature and storage time. Again, other enzymes such as those discussed above may be included. Typical incubation conditions are as described in the previous paragraph.
[0315] Once the raw juice has been incubated with the polypeptides of the present invention, the broth is then centrifuged or (ultra)filtered to produce the final product.
[0316] After treatment with the polypeptide of the present invention, the (final) product can be heat treated, for example, at about 100°C for a time of about 1 minute to about 1 hour, under conditions for partially or fully inactivate the polypeptides of the invention.
[0317] A composition containing a polypeptide of the present invention can also be used during the preparation of fruit or vegetable purees.
[0318] Baking the polypeptide can improve the structure of the dough, modify its stickiness or flexibility, improve the bread volume and/or crumb structure or provide better texture characteristics, such as the quality of the bread, crumb, or crumb.
[0319] The present invention thus relates to methods for the preparation of a cereal-based pasta or food product comprising incorporating in the pasta a polypeptide or composition of the present invention. This can improve one or more properties of the pasta or cereal-based food product obtained from the pasta relative to a pasta or cereal-based food product in which the polypeptide is not incorporated.
[0320] The preparation of the cereal-based food product according to the invention may also comprise steps known in the art, such as boiling, drying, frying, baking or steaming the obtained mass. Products that are made from a dough that is boiled are, for example, noodles, baked dumplings, products that are made from fried dough are, for example, donuts, beignets, fried noodles, products that are made for steam dough they are, for example, steamed buns and pasta, examples of products made from dry pasta are pasta and dry pasta, and examples of products made from cooked pasta are bread, biscuits, cake.
[0321] The term "improved property" is defined herein as any property of a dough and/or a product obtained from the dough, in particular a cereal-based food product, which is improved by the action of the polypeptide according to invention in relation to a mass or a product into which the polypeptide according to the invention is not incorporated. The improved property may include, but is not limited to, increased dough strength, increased dough elasticity, increased dough stability, improved dough machinability, improved dough proof strength, reduced dough viscosity, improved dough extensibility, volume of cereal food product increased, cereal food product blistering reduced, baked product crumb structure improved, cereal food product softness improved, cereal food product taste improved, anti-setting of improved cereal-based food product. Improved properties related to pasta and pasta-like cereal food products are, for example, improved firmness, reduced viscosity, better tackiness and reduced cooking loss.
[0322] The improved property can be determined by comparing a pasta and/or a cereal-based food product prepared with and without the addition of a polypeptide of the present invention. Organoleptic qualities can be assessed using well-established procedures in the bakery industry, and may include, for example, the use of a panel of trained taste testers.
[0323] The term "dough" is defined herein as a mixture of cereal flour and other ingredients firm enough to knead or roll. Examples of cereal are wheat, rye, corn, grain, barley, rice, cereals, buckwheat and oats. Wheat is I and hereafter intended to encompass all known species of the genus Triticum, eg aestivum, durum and/or spelta. Examples of other components are: cellulase enhancing polypeptide according to the present invention, additional enzymes, chemical additives and/or processing aids. The dough can be fresh, frozen, pre-thined, or pre-cooked. The preparation of a dough from the above-described ingredients is well known in the art and comprises mixing said ingredients and processing aids and one or more molding and, optionally, fermentation steps. The preparation of frozen dough is described by Kulp and Lorenz in Frozen and Refrigerated Doughs and Batters.
[0324] The term "cereal-based food product" is defined herein as any product prepared from a dough, or of a soft or crumbly character. Examples of cereal-based food products, of a white, light or dark type, which can advantageously be produced by the present invention are bread (in particular white, wholemeal or rye bread flour), usually in the form of breads or rolls. , French baguette bread, pasta, pasta, donuts, biscuits, cake, pita bread, tortillas, tacos, cakes, pancakes, biscuits, biscuits, pie dough, steamed bread and toasted bread, and the like.
[0325] The term "baked product" is defined herein as any cereal-based food product prepared by cooking pasta.
[0326] Non-starch polysaccharides (NSP) can increase digestion viscosity which can, in turn, decrease nutrient availability and animal performance. The use of the cellulase enhancing polypeptide of the present invention can improve the utilization of phosphorus as well as cation minerals and polypeptide during animal digestion.
[0327] The addition of specific nutrients to feed improves animal digestion and thus reduces feed costs. A lot of feed additives are currently being used and new concepts are continually being developed. The use of specific enzymes such as non-starch carbohydrate-degrading enzymes could break down fiber to release energy, as well as increased protein digestibility due to better protein accessibility when fiber is broken down. In this way, the cost of food can be reduced as well as protein levels in the food can also be reduced.
[0328] Non-starch polysaccharides (NSPs) are also present in virtually all plant-based feed ingredients. NSPs are misused and can, when solubilized, have adverse effects on digestion. Exogenous enzymes can contribute to a better utilization of these NSPs and consequently reduce any anti-nutritive effects. The non-starch carbohydrate degradation enzymes of the present invention can be used for this purpose in cereal-based diets for poultry and, to a lesser extent, in swine and other species.
[0329] A non-starch carbohydrate degradation polypeptide/enzyme of the present invention (of a composition comprising the enzyme/polypeptide of the present invention) can be used in the detergent industry, for example, for the removal of carbohydrate-based dyes of the clothes. The detergent composition may comprise an enzyme/polypeptide of the present invention and, in addition, one or more of a cellulose, a hemicellulase, a pectinase, a protease, a lipase, a cutinase, an amylase or a carbohydrase. Use of enzymes in detergent compositions
[0330] A detergent composition comprising a polypeptide or a composition of the invention may be in any convenient form, for example, a paste, a gel, a powder or a liquid. A liquid detergent can be aqueous, typically containing up to about 70% water and between about 0 and about 30% organic or non-aqueous solvent material.
[0331] Such a detergent composition can, for example, be formulated as a hand or machine laundry detergent composition, including a laundry additive composition suitable for pre-treatment of stained fabrics and a fabric softening composition. fabric added to the rinse, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for machine or hand dishwashing operations.
[0332] In general, enzyme properties must be compatible with a selected detergent (eg optimum pH, compatibility with other enzymatic and/or non-enzymatic ingredients, etc.) and enzymes must be present in an effective amount .
[0333] The detergent composition can comprise a surfactant, for example, an anionic or nonionic surfactant, a detergent builder or complexing agent, one or more polymers, a bleaching system (eg, a source of H2O2) or an enzyme stabilizer. The detergent composition may also comprise any other ingredients of conventional detergents such as, for example, a conditioner including a clay, a suds activator, a suds suppressor, an anti-corrosion agent, a soil, a suspending agent, a suds suppressor. redeposition of a soil, a dye, a bactericide, an optical brightener, a hydrotrope, a stain inhibitor or a perfume. Use of enzymes in pulp and paper processing
[0334] A composition or a polypeptide of the present invention can be used in the paper and cellulose industry, particularly in the bleaching process to increase the brightness of bleached celluloses whereby the amount of chlorine used in the bleaching steps can be reduced, and to increase the degree of refining of celluloses in the recycled paper process (Eriksson, KEL, Wood Science and Technology 24 (1990):79-101; Paice, et al., Biotechnol. and Bioeng 32 (1988):235-239 and Pommier et al., Tappi Journal (1989):187-191). Furthermore, a composition or a polypeptide of the present invention can be used for the treatment of lignocellulosic cellulase in order to improve the whiteness of the same. In this way, the amount of chlorine needed to obtain a satisfactory bleaching of the cellulose can be reduced.
[0335] A composition or a polypeptide of the present invention can be used in a method to reduce the rate at which cellulose containing tissue becomes stiff or to reduce the stiffness of tissue containing cellulose, the method comprising treating cellulose containing contain tissues with a polypeptide or a composition as described above. The present invention further relates to a method for providing increased color clarity of colored cellulose-containing fabrics, the method comprising treating colored cellulose-containing fabrics with a polypeptide or composition as described above, and a method for providing a variation localized to the color of colored cellulose-containing fabrics, the method comprising treating colored cellulose-containing fabrics with a polypeptide or composition as described above. The methods of the present invention can be carried out by treating cellulose-containing fabrics during laundering. However, if desired, fabric treatment can also be carried out during soaking or rinsing, or simply by adding the composition or polypeptide, as described above, to the water in which the fabrics are or will be immersed. Other Enzyme Uses
[0336] Furthermore, a composition or a polypeptide of the present invention can also be used in the antibacterial formulation as well as in pharmaceuticals such as throat lozenges, toothpaste, and mouthwash.
[0337] The following examples illustrate the invention: EXAMPLES Materials and Methods DNA Procedures
[0338] Standard DNA procedures were performed as described elsewhere (Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), unless otherwise indicated. The DNA was amplified using the proofreading enzyme Physion polymerase (Finnzymes). Restriction enzymes were from Invitrogen or New England Biolabs. Preparation of cellulase samples
[0339] Cellulases originating from Talaromyces emersonii were expressed in Aspergillus niger. Enzyme concentrate filtrates were produced as described in WO2004/030468. After growth of Aspergillus niger, containing the appropriate expression, cell-free plasmid supernatants were prepared by centrifuging the fermentation broth at 5000 xg for 30 minutes at 4°C. The optional supernatant can be adjusted to pH = 5 with 4N KOH and sterile filtered through a 2 µm filter (top bottle) with suction to eliminate any fungal material. In addition, supernatants can be further filtered over a Whatmann GF/A glass microfiber filter (150mm 0) to remove any solids. Supernatants were ultrafiltered, concentrated, and stored until use at 4°C or frozen at -20°C. Method for determination of total protein
[0340] The method used was a combination of protein precipitation, using trichlorine acetic acid (TCA) to remove the disturbing substances and allow the determination of protein concentration, with the colorimetric Biuret reaction. During the biuret reaction, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. The violet color indicates the presence of proteins. Color intensity, and therefore absorption at 546 nm, is directly proportional to protein concentration, according to BeerLambert's law. Normalization was performed using BSA (Bovine Serum Albumin) and the protein content was expressed in g of proteins, as equivalent BSA/L or mg of protein, as equivalent BSA/ml. Protein content was calculated using standard calculation protocols known in the art, by graphical representation of OD546 as a function of the concentration of samples with known concentration, followed by calculation of the concentration of unknown samples using the equation generated from the calibration line.
[0341] Preparation of washed pretreated wheat straw substrate
[0342] Pretreated dilute acid wheat straw can be obtained as described in Linde, M. et al., Biomass and Bioenergy 32 (2008), 326-332 and the equipment described in Schell, DJ, Applied Biochemistry and Biotechnology (2003), vol. 105-108, pp 69-85, can be used. The pretreated wheat straw was washed with water until the wheat straw solution was pH 6.0 or higher. Wash water was removed by filtration. Screening for cellulose hydrolysis
[0343] Acid washed pretreated wheat straw (PWS) to 2% dry matter (dm) substrate solution is made in 50 mM sodium acetate buffer pH 4.5. For incubation a 96-well deep microtiter plate is used. In each well 1 ml of substrate is pipetted. Enzyme mixtures were prepared at various cellulase ratios, and the fixed protein dose per gram of substrate dry matter was added to the substrate solution.
[0344] The plate is incubated at 65°C for 20 hours. After incubation, the enzymatic reaction is stopped by adding 50 μl of a 1M sodium hydroxide solution. The plate is then centrifuged for 15 min at 3220 rcf and the supernatant is diluted to a glucose concentration between 40 and 100 µ.
[0345] 50 μl of the diluted sample is pipetted onto a PCR plate and 150 μl of BCA reagent is added. The BCA reagent is freshly made by mixing two 1:1 v/v stock solutions A and B. A solution A consisting of 54.3 g of Na2CO3, 24.2 g of NaHCO3 and 1.9 g of Na2BCA (Sigma D8284) for each liter of water (MQ). Solution B contained 31.24 ml of 4% CuSO4.5H2O solution (Pierce 185 9078) and 1.26 g of L-lysine (Sigma L5501) per liter of water (MQ). (Final concentration: 2.5 mM BCA, 2.5 mM Cu, 4.3 mM L-lysine, 400 mM carbonate). The plate is heated to 80°C for 60 minutes. After cooling down to 150 μl, they are pipetted into a second plate and the absorbance is measured at 560 nm.
[0346] In all plates the substrate background is analyzed, by substrate incubation, without adding enzymes. For each measurement a 55 µm glucose standard is analyzed against acetate buffer to calculate the glucose molar extinction coefficient.
[0347] Incubations are performed using a Peltier thermocycle PCR block (PTC200) and measurements are performed using a microtiter plate reader (TECAN Sunrise).
[0348] The glucose released from the substrate by the action of enzymes was calculated as follows:
As = sample absorbance at 560 nm Ablk = non-expressive substrate absorbance at 560 nm Densaio = dilution in the Vensaio assay = total assay volume (μL) Dincubated = dilution of incubated I = path length (with) c = = extraction coefficient molar glucose-BCA (mmol*L-1*with-1) Vsubstrate = total substrate volume (μL)
[0349] This value was further corrected for the reduction of protein/sugar content in the enzyme sample (analyzed by the BCA assay).
[0350] Extended Hydrolysis of Cellulose
[0351] Incubations of enzyme combinations from acid-washed pretreated wheat straw (PWS) to 2% dry matter (dm) substrate solution in 50 mM sodium acetate buffer pH 4.5, were performed on a 10 mL scale. Enzyme combinations were added to a fixed protein dose per gram of substrate dry matter. Samples were taken in time, up to 72 hours of incubation at 65°C. Reactions were terminated at the appointed time by turning the residue down, pipetting the supernatant and freezing the samples until analysis.
[0352] The analysis of the amount of glucose released was performed using an NMR flow. 1H NMR spectra were recorded on a Bruker AVANCE II best NMR system operating at a proton frequency of 500 MHz and probe temperature at 27°C. Example 1 1.1 Construction of expression plasmids
[0353] The sequence with SEQ ID NO:1 was cloned into pGBTOP vector (figure 1), using EcoRI and SnaBI sites, comprising the glucoamylase promoter and a terminator sequence. The E. coli part was removed by digestion with NotI prior to transformation of A. niger. CBS 513.88. 1.2 Transformation of A. niger A. niger WT-1: this A. niger strain is CBS513.88 comprising deletions of genes encoding glucoamylase (glaA), fungal amylase and acid amylase. A. niger WT 1 is constructed using the "GENE FREE-MARKER" method as described in EP 0 635 574 B1.
[0354] The expression constructs are co-transformed into the A. niger WT-1 strain according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479, with the following modifications:
[0355] - The spores are germinated and cultivated for 16 hours at 30 degrees Celsius in a shake flask placed on a rotary shaker at 300 rpm, in minimal Aspergillus medium (100 ml). The Aspergillus mineral medium contains, per liter: 6 g of NaNO3, 0.52 g of KCl, 0.52 g of KH2PO4, 1.12 ml of 4M KOH, 0.52 g of MgSO4.7H2O, 10 g of glucose, 1 g of casamino acids, 22 mg of ZnSO4.7H2O, 11 mg of H3BO3, 5 mg of FeSO4.7H2O, 1.7 mg of CoCl2.6H2O, 1.6 mg of CuSO4.5H2O, 5 mg of MnCl2.2H2O , 1.5 mg of Na2MoO4.2H2O, 50 mg of EDTA, 2 mg of riboflavin, 2 mg of thiamine-HCl, 2 mg of nicotinamide, 1 mg of pyridoxine-HCl, 0.2 mg of pantothenic acid, 4 g of biotin, 10 ml penicillin (5000 IU/ml) and streptomycin (5000 UG/ml) solution (Gibco).
[0356] - Novozym 234® (Novo Industries) instead of helicase is used for the preparation of protoplasts;
[0357] - After the formation of the protoplast (60 to 90 minutes), the KC buffer (0.8M KCl, 9.5 mM citric acid, pH 6.2) is added to a final volume of 45 ml, at Protoplast suspension was centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swing-bucket rotor. The protoplasts are resuspended in 20 ml of KC buffer and subsequently 25 ml of STC buffer (0.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ) is added. The protoplast suspension is centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swing-bucket rotor, washed in STC buffer and resuspended in STC buffer at a protoplast concentration 10E8/ml;
[0358] - At 200 microliters of the protoplast suspension, the DNA fragment was dissolved in 10 microliters of TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and 100 microliters of TE solution PEG (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ) are added;
[0359] - After incubating the protoplast-DNA suspension for 10 minutes at room temperature, a solution of 0.5 ml PEG (60% PEG 4000 (Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ) is added slowly, with repeated mixing of tubes. After incubation for 20 minutes at room temperature, the suspensions are diluted with 5 ml of 0.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. The protoplasts are gently resuspended in 1 ml of 1.2 M sorbitol and plated on solid selective regeneration medium consisting of one or another minimal Aspergillus medium without riboflavin, thiamine, HCL, nicotinamide, pyridoxine, pantothenic acid, biotin, casamino acids and glucose. In the case of acetamide selection the medium contains 10 mM acetamide as the sole nitrogen source and 1 M sucrose as the C-source and osmoticum. Alternatively, protoplasts are plated on PDA (Potato Dextrose Agar, Oxoid) supplemented with 1 to 50 micrograms/ml phleomycin and 1 M sucrose as osmosticum. Regeneration plates are solidified using 2% agar (Agar No.1, Oxoid L11). After incubation for 6 to 10 days at 30 degrees Celsius, transformants conidiospores are transferred to plates consisting of Aspergillus selective medium (minimum medium containing acetamide as the only nitrogen source in the case of selection of acetamide or PDA supplemented with 1 to 50 micrograms/ ml of phleomycin in the case of phleomycin selection) with 2% glucose and 1.5% agarose (Invitrogen) and incubated for 5 to 10 days at 30 degrees Celsius. Individual transformants are isolated and this selective purification step is repeated once the purified transformants are stored.
[0360] After transformation, transformants were selected in a medium containing acetamide as the only nitrogen source and purified by colony. Copy numbers were estimated by quantitative PCR and low and high copy numbers of transformants were selected. High copy transformants were cultured in shake flasks in 100 ml of the CSM-MES medium as described in EP 635 574 at 34°C at 170 rpm in an incubator shaker using a 500 ml shaker flask mixed in shake flask recesses in the background. After 3 and 4 days of fermentation, supernatant samples were collected to determine expression by SDS-PAGE. 1.3 Protein content
[0361] Concentrated and ultrafiltered supernatants from shake flask fermentations of transformants expressing cell-enhancing polypeptide (TEMER07589) were analyzed for protein content. The protein content was determined to be 24 mg protein, as BSA equivalent/ml. Example 2 2.1 Preparation of cellulase samples
[0362] Two endoglucanases from Talaromyces emersonii as described in Patent Application EP1621628 as CEA (SEQ 1) and CEB (SEQ 3) were prepared as described in Example 1 for TEMER07589. Furthermore, two exoglucanases, being CBHI as described in Patent Application EP09158739.4 and CBHII (as described in co-pending Case Patent Application DSM 27829, filed the same day as this application), were prepared in a similar manner. Finally, a beta-glucosidase from Talaromyces emersonii, as known from Murray et al., Protein Expression and Purification, 2004, 38, 248-257, was also overexpressed in Aspergillus niger. The protein content of the samples was determined and ranged from 20 to 60 mg of protein, as equivalent BSA/ml. 2.2 Hydrolysis of cellulose by enzyme mixtures in 20 h
[0363] Combinations of four cellulases were prepared. Each combination consisted of 1 BG, 1 CBHI, 1 CBHII, and 1 EG. However, for the boosting activity of cellulase containing the cellulase combination, EG was omitted and replaced by TEMER07589. In addition to different endoglucanase, or cellulase enhancing activity in the enzyme mixtures, also a little different ratio of the 4 enzymes was tested. All four of these enzyme combinations were added to 5 mg protein as BSA equivalent per g washed pretreated wheat straw, and screened for their ability to hydrolyze cellulose in 20 h incubations at 65°C as described above. In addition to these enzyme mixtures, a classic Talaromyces emersonii cellulase product, known as Filtrase® NL, was also incubated at a similar protein dose. Glucose release was determined and is shown in Table 1 for the 4 enzyme combinations. For Filtrase® NL, the glucose released was determined to be 30.1 mmol/L.
[0364] Table 1: Composition of four compound mixtures of 4 cellulase combinations, with different relative amounts of the four cellulases (given as a percentage of the total protein dose) as well as the glucose released (expressed in mmol/L) after 20 h incubation at pH 4.5 and 65°C, for each mixture, when containing CEA, CEB or TEMER07589 as EG.
*EG indicates the percentage of endoglucanase, in case of CEA and CEB, and indicates the percentage of cellulase boosting activity, in the case of TEMER07589, where the mixture of a real endoglucanase is omitted.
[0365] From these results, it is evident that for all four ratios of enzymes tested the cellulase boosting activity outperformed the two endoglucanases. However, in case the enzyme ratio is given in Mix 1 and Mix 4, the enzyme mix 4 containing the cellulase enhancing activity, TEMER07589, instead of endoglucanase, even outperformed Filtrase® NL, which contains several endoglucanases, beta-glucosidases and the two exoglucanases, CBHI and CBHII. Example 3 3.1 Enzyme Mix Ratio Optimization 4
[0366] The different 3 of 4 enzyme mixtures as used in Example 2 were used in a mixture design consisting of 10 vertices, and a total number of 55 different ratio combinations to find the optimal ratio of the four enzymes for each one of the three blends. The ranges of the different enzymes tested in this project were BG 4 to 12%, and each of the other three enzymes in ranges from 10 to 70%. In each series of 55 incubations, three duplicates were performed. Screening of these mixtures was performed on pretreated wheat straw washed in 2% DM in a 50 mM acetate buffer at pH 4.5. Incubation was carried out at a total protein dosage of 5 mg protein as BSA equivalent per g dry-washed pre-treated wheat straw for 20 h at 65°C in a 96-well microtiter plate. depth. Cellulose hydrolysis capacity was determined by the glucose released as determined by the BCA sugar reduction assay. The statistical evaluation of all data from each mixture resulted in the optimized index of the four enzymes in each mixture, as indicated in Table 2.
[0367] Table 2. Optimal composition of 3 different of 4 enzyme mixtures, each containing BG, 1 CBHI, CBHII 1 and 1 EG or a cellulase enhancing activity, expressed as a percentage of total protein, for each of the enzymes individual.
*EG indicates the percentage of endoglucanase, in case of CEA and CEB, and indicates the percentage of cellulase boosting activity, in the case of TEMER07589, where the mixture of a real endoglucanase is omitted. 3.2 Extended hydrolysis of 4 optimized enzyme mixtures
[0368] The 3 optimized mixtures were applied in prolonged hydrolysis on a 10 mL scale with wheat straw pretreated at 2% DM, at pH 4.5 and at 65°C. As a comparison, the aforementioned Filtrase® NL, a cellulase product from Talaromyces emersoniclassic, was also incubated at the same concentration, pH and temperature as the substrate. The total dose of protein in each of the incubations was 15 mg protein as BSA equivalent per gram of dry matter pretreated wheat straw. Incubations lasted 72 hours and at time intervals several samples were taken. Samples were removed by rotation, and the supernatant was frozen until NMR analysis. The release of glucose over time is shown in figure 2. From figure 2, it is evident that the mixture containing cellulase enhancing activity, TEMER07589, outperforms the two endoglucanase mixtures containing CEA or CEB. The mixture containing the cellulase boosting activity additionally has slightly higher glucose release at the end of the incubation than Filtrase® NL.

权利要求:
Claims (8)
[0001]
1. Recombinant cell characterized in that it comprises: (i) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (ii) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1, or (iii) a nucleic acid construct consisting of the polynucleotide of (ii) or a vector incorporating the polynucleotide of (ii), wherein the cell is a fungal cell selected from the group consisting of the genera Aspergillus, Trichoderma/Hypocrea, Fusarium, Disporotrichum, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, Chryosporium, Fusarium, Humicola and Neurospora which is different from fungal cells naturally produce the polypeptide of SEQ ID NO:2.
[0002]
2. Recombinant cell according to claim 1, characterized by the fact that the fungal cell is selected from the species Aspergillus oryzae, Aspergillus soybeane, Aspergillus nidulans, or Aspergillus niger.
[0003]
3. Method for the preparation of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, the method being characterized in that it consists in culturing a cell as defined in claim 1 or 2 under conditions allowing the expression of said polypeptide and, optionally, recovering the expressed polypeptide.
[0004]
4. Composition characterized in that it consists of: (i) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (ii) a cellobiohydrolase I, a cellobiohydrolase II and β-glucosidase.
[0005]
5. Method for the treatment of a substrate comprising carbohydrate material, optionally a plant material, the method being characterized by bringing the substrate into contact with a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, and /or a composition as defined in claim 4, and/or a recombinant cell as defined in claim 1 or 2.
[0006]
6. Method according to claim 5, characterized in that the substrate is a plant material and the plant material is supplied in the form of a plant, a plant pulp, a plant extract, a food product or derivative ingredient thereof, or a fabric, textile or item of clothing which comprises a plant material.
[0007]
7. Method according to claim 5 or 6, characterized in that the treatment comprises the degradation and/or modification of cellulose and/or hemicellulose and/or a pectic substance.
[0008]
8. Use of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, and/or a composition, as defined in claim 4, and/or a recombinant cell, as defined in claim 1 or 2, characterized in that be for the production of sugar from a lignocellulosic material.

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同族专利:

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公开号 | 公开日

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BR112012033404A2|2018-08-28|

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CA2803986A1|2012-01-05|

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MY160897A|2017-03-31|

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CA2803986C|2019-04-23|

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AU2011273690A1|2013-01-24|

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ES2628345T3|2017-08-02|

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DK2588603T3|2017-07-03|

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EA201300060A1|2013-05-30|

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EP2588603A1|2013-05-08|

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US20130095553A1|2013-04-18|

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AU2011273690B2|2014-09-04|

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CN102971419B|2015-02-11|

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CN102971419A|2013-03-13|

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PL2588603T3|2017-08-31|

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EP2588603B1|2017-03-22|

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MX2012015139A|2013-04-22|

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WO2012000892A1|2012-01-05|

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US9260704B2|2016-02-16|

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AU2011273690C1|2015-07-30|

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法律状态:
2019-07-16| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-06-23| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-22| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-04-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-18| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 23/06/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US35956310P| true| 2010-06-29|2010-06-29|

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EP10167771|2010-06-29|

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EP10167771.4|2010-06-29|

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US61/359,563|2010-06-29|

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PCT/EP2011/060577|WO2012000892A1|2010-06-29|2011-06-23|Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof|

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