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    • 专利摘要:
    A method for rational mining for induced enzymes in microbial communities is described. The method is characterized in that a community of microorganisms is provided and that the microbial populations of the community are cultivated in a container under conditions of choice, where the microorganisms are given a defined culturing medium, to eliminate matter deriving from the natural habitat and to allow the microbial community to reach a metabolic steady-state. The method is further characterized in that at least one fraction of microorganisms is taken from the container and transferred into at least two separate containers, where at least one of the fractions of microorganisms is provided with a defined medium which includes an inducing substance and/or a substance against which enzyme/enzymes is/are desired, to induce regulation of expression of the desired enzyme/enzymes, and at least one fraction is provided with a defined medium without said inducing substance and without said substance against which enzyme/enzymes is/are desired, for the purpose of comparison, and the fractions are maintained and/or cultivated. Samples of the two fractions are then withdrawn and analyzed for identification of the induced enzyme/enzymes.
    公开号:SE1051254A1
    申请号:SE1051254
    申请日:2010-11-29
    公开日:2012-05-30
    发明作者:Martin Karlsson；Bengt-Harald Jonsson；Uno Carlsson
    申请人:Rational Enzyme Mining Rem Ab；
    IPC主号:
    专利说明:

    RATIONAL ENZYME MINING Technical Field of the lnvention The present invention relates to a method for rational mining forinduced enzymes in microbial communities and to a method for mining andproduction of enzymes. The present invention further relates to an enzymeproduced using the methods according to the invention.
    Background Art Enzymes are protein biomolecules that are able to function as highlyeffective, high-performing biological catalysts and are fundamental for allbiological life. They are substances that accelerate the chemical reactions oflife without being consumed themselves. lsolated enzymes are important inmany industrial processes for treating biological substrates. Examples ofindustries that benefit from the use of enzymes are food and feed industry,detergent industry, leather industry and the increasing application of enzymesin bioenergy industry as exemplified by the production of bioethanol. All theabove industries employ various hydrolytic enzymes such as amylases,cellulases, proteases, lipases etc. for increasing production rate and yield ofproducts from biobased feed stock m. There are numerous other conceivableapplications in related fields e.g. for production of biogas or break down ofharmful compounds in process water. Thus, enzymes for industrial andenvironmental applications have a large and increasing economic andecological value.
    One bottleneck in the application of enzymes in industrial processes isthat in order to be active, enzymes and other proteins must keep a highlyordered structure. However, the highly ordered structure of proteins is onlymaintained if the proteins are stable at the prevailing conditions, i.e. pH, ionicstrength, temperature, etc., within certain limits that are specific for each typeof protein. ln terms of natural selection of proteins during evolution, this notionstresses the fact that a protein molecule only makes structural sense when itexists under conditions similar to those for which it was selected, or the socalled, native state. That is, the enzymes for treating biological substratesshould ideally be evolutionarily adapted to the environment in which they areto be used, to assure that the enzymes have a high activity and longevity in 2 that unique environment. lf however the protein is not stable enough for theapplication in mind the stability of the enzyme needs to be altered by variousprotein engineering methods m, which is a very complex and expensiveprocess with an often uncertain outcome.
    Microorganisms are a valuable source of industrially importantenzymes. Microorganisms are the sma|est form of life, nevertheless theycollectively constitute the largest mass of living material on earth. Not only arethey the most abundant, but the diversity of microbial life by far exceeds thatof the plants and animals, which is a result of microorganisms' ability to live atplaces unsuitable for other organisms. An ability that comes from microorga-nisms' highly specialized physiological capabilities. Part of these capabilitiescomes from having enzymes that are stable, active and adapted to differenthabitats so that different microorganisms can feed and make use of thenutrients available in different environments.
    To feed and utilize nutrition in their environment, microorganisms aregenerally not able to break down more complex compounds like polysaccha-rides and proteins within the cell. Their only way to digest them is via extra-cellular enzymes. For example, to break down starch into glucose the cellssecrete amylases into their environment in what could be described as anexternal digestion process. ln the environment the amylases break downstarch until the microbes are surrounded by small glucose molecules whichcan then easily be ingested.
    Microorganisms can secrete both cell-bound and free extracellularenzymes. Cell-bound enzymes are important for the microorganisms sincethey exert their activities close to the microbial cell, which assures that e.g.the glucose produced from the starch can be ingested by the very organismthat produced the amylases. For industrial applications however extracellularfree enzymes are of larger interest than cell-bound enzymes as they areadapted to be stable independently of the cell and furthermore since theefficiency in degrading complex compounds is far higher for an excreted freeenzyme than for a cell-bound one. All microorganisms, both prokaryotic andeukaryotic, have the ability to produce extracellular enzymes. However, forthe large scale production of enzymes from isolated genes, prokaryotic 3 enzymes are most often preferred as they lack posttranslational modificationsand are thereby easier and cheaper to produce in heterologous expressionsystems.
    Thus, as described above, the large diversity of microorganisms thathas evolved in different environments offers an almost inexhaustible bio-bankof organisms with the ability to produce enzymes with different functions indifferent environments, suitable for various industrial processes. ln nature,microorganisms live in association with other microorganisms in populations.The environment in which a population lives is called a habitat in whichdifferent populations interact in assemblages called microbial communities.The diversity and abundance of microorganisms in a microbial community isdependent on the resources, i.e. food, and conditions such as temperature,pH, oxygen content etc., that exist in that environment. Microbial habitats canbe found in all environments that can sustain life. These includes familiar oxicand anoxic habitats like soil, water, animals and plants but also extremeenvironments with high or low pH, high or low temperature, high pressure,high salt concentration etc. Following the above reasoning, microorganismsthat have evolved to live in any of the above habitats naturally also secreteenzymes that are stable in that very environment.
    Microbial populations in a community interact and cooperate in variousways, some of which are beneficial to the whole community. For example, thewaste products of metabolic activities of some microorganisms can be nutri-ents for others. A special case of interaction is syntrophy (literarily meaning“eating together”), a situation in which tvvo or more microorganisms team upto degrade a substance that neither microorganism can degrade individually.
    Due to technical limitations, historically and traditionally, enzymes havebeen discovered and isolated from pure cultures of microorganisms. Theprocess of obtaining a pure culture of a microorganism is very time consum-ing, if at all possible. A microorganism of course needs to be known toproduce the product or enzyme of interest, preferably at the conditions ofinterest. This microorganism then needs to be purified from all the othermicroorganisms in its habitat by differential and selective media in hope to getthe microorganism in a pure culture. However, recent findings by meta- 4 genomics, where the whole microbial community is considered to represent ameta-organism, have made it clear that the interactions and co-operationsbetween populations of microorganism in a microbial community are so vitalthat only a fraction (1-10%) of all microorganisms can be obtained in purecultures. Consequently, the enzymes that have been found so far and are inuse in industrial applications today are very much biased towards geneproducts of the very few microorganisms that can be obtained in purecultures. Thus, there are some 90-99 % of the microorganisms that are notaccessible for screening for valuable enzymes with methods that rely on purecultures of microorganisms m. ln view of the limitations of pure culturing described above, othermethods to enable screening for novel enzymes in full microbial communitiesare desirable. One way of doing this is to take the route via DNA, using meta-genomics and produce a meta-genomic library of microbial communities innatural habitats W. However, one drawback of this approach is that all genesneed to be cloned, even those that might not be of interest for industrialbiotechnology, such as genes coding for e.g. structural proteins. Anotherdrawback of this approach is that the huge number of clones that areproduced, all carrying different gene segments, have to be expressed andsubmitted to either sequence or activity based screening for identification ofthe correct enzyme.
    Recent progress in proteomics, i.e. the large-scale study of proteinsexpressed by an organism, has also lead to the development of metaproteo-mics, i.e. the large-scale characterization of the entire protein complement ofenvironmental microbiota at a given point in time[5]. However, up till nowmetaproteomics has only been used to collect intracellular proteins in order tounderstand metabolic pathways and interactions among the populations thatmakes up a certain microbial community[6'8]. Even if it would be desirable toscreen for secreted proteins/enzymes in environmental samples this wouldnot be possible since the sample would be too contaminated with interferingsubstances and the enzymes would be too diluted. For the purpose ofscreening for secreted proteins (known as secretomics) by microorganisms, 5 so far only microorganisms in pure cultures have been used, often with theaim of finding virulence factors for pathogenic microorganisms[9' 101.
    The growing worldwide interest to increase the production of biogasfrom organic residues, to be used as an alternative fuel, can serve as animportant example of an industrial process that clearly would benefit fromemploying hydrolytic enzymes. However, this will only be possible if enzymesthat are effective and have a long lifetime in the prevailing conditions of ananaerobic digester can be found. Much of the organic matter used assubstrate in biogas production has a low biological availability, due to beingphysically and chemically stable, which results in that some substratesdisplay a low degree of degradation. Most suggested pretreatment methodsfor increasing biogas production rely on energy intensive thermal and/orphysical disintegration of various cell walls, with the intention to release thecontent of the cells. Examples are steam explosion, ultrasonication, electro-poration, bead-milling etc. However, even if successful and economicallyviable, these methods alone will only marginally influence the rate and degreeof degradation of the actual cell walls and other structural components thatmake up a significant part of most organic material.
    Although established in many other industrial biotechnological appli-cations, the addition of hydrolytic enzymes in order to increase both the rateand yield ofdigestion in the biogas production is fairly new. The use ofenzymes for this purpose is applicable in processes where the first andsecond step in methanogenesis, i.e. disintegration and hydrolysis, are ratelimiting (see fig. 1). The idea is that enzymes with specific activity towardsvarious biopolymers such as proteins and polysaccharides will hydrolyze theorganic matter and that the addition of hydrolytic enzymes to the processleads to a more effective use of substrates that are difficult to degrade, and tothe possible use of various new substrates. Experiments have been made byadding various commercially available hydrolytic enzymes to anaerobicdigesters. However, these enzymes have originated from various microorga-nisms that are not part of microbial communities in methanogenic habitats.Thus, those enzymes are not evolutionarily adapted to the prevailing condi- 6 tions in an anaerobic digester and the intended use. Consequently,experiments made so far have generally not shown any high success rate.
    However, what is clear is that upon addition of recalcitrant organicmaterial, such as cellulose, to an anaerobic digester there is an increase ingas production although this process is very slow. Thus, within the microbialcommunity there are microorganisms present that are able to synthesize andsecrete enzymes that are active against e.g. cellulose at the prevailingconditions, but in too small amounts. Such an enzyme or enzymes are ofcourse of a considerable value in increasing biogas production if it would bepossible to identify, produce and add them to anaerobic digesters.
    Summary of the lnvention The aim of the present invention is therefore to solve the problems anddisadvantages described above by providing a method that is useful for bothinducing and identifying enzymes of choice that are optimally adapted to theconditions and substrates of choice, whilst at the same time being indepen-dent of pure culturing.
    This is achieved according to the present invention by means of amethod for rational mining for induced enzyme/enzymes in microbialcommunities, comprising the steps of: (a) providing a community of microorganisms, (b) cultivating the microbial populations of the community in a containerunder conditions that mimic the conditions of the microbialcommunity's natural habitat, where the microorganisms are given adefined culturing medium to eliminate matter deriving from thenatural habitat and to allow the microbial community to reach ametabolic steady-state, (c) removal from the container of step (b) of two fractions of microorga-nisms, and transfer of said two fractions into two separatecontainers, (d) providing one of the fractions of microorganisms from step (c) with adefined medium which includes a substance against whichenzyme/enzymes is/are desired, to induce a regulation of expressionof the desired enzyme/enzymes, and the other fraction from step (c) 7 with a defined medium without said Substance for the purpose ofcomparison, and maintaining and/or cultivating the two fractions ofmicroorganisms, and (e) withdrawing and analyzing samples of the two fractions of step (d) foridentification of the induced enzyme/enzymes.
    According to one embodiment the container of step (b) is a chemostator an anaerobic digester.
    According to a further embodiment the microbial community originatesfrom animals, p|ants, insects, soil or aqueous environments. ln one embodiment the defined medium is a medium comprisingdecomposed organic material, fermentation products and other essentialsubstances, such as salts, vitamins, metals and trace elements. ln another embodiment of the present invention the contents of thesamples taken in step (e) are separated into fractions before analyses andidentification. ln one embodiment the samples taken in step (e) are subject topunficaflon. ln a further embodiment the samples taken in step (e) are subject toconcentration. ln a further embodiment the purification and/or concentration is doneby separation methods based on the varying physicochemical properties ofproteins. ln another embodiment the analyses and identification of step (e) isdone by 1 or 2 dimensional gel electrophoresis or chromatography followedby comparison of the results for the two fractions.
    The aim of the present invention is further achieved by means of amethod for rational mining for induced enzyme/enzymes and productionthereof, comprising the steps of: (a) providing a community of microorganisms, (b) cultivating the microbial populations of the community in a containerunder conditions that mimic the conditions of the microbialcommunity's natural habitat, where the microorganisms are given adefined culturing medium to eliminate matter deriving from the 8 natural habitat and to allow the microbial community to reach ametabolic steady-state, (c) removal from the container of step (b) of two fractions ofmicroorganisms, and transfer of said two fractions into two separatecontainers, (d) providing one of the fractions of microorganisms from step (c) witha defined medium which includes a substance against which enzyme/enzymes is/are desired, to induce a regulation of expressionof the desired enzyme/enzymes, and the other fraction from step (c)with a defined medium without said substance for the purpose ofcomparison, and maintaining and/or cultivating the two fractions ofmicroorganisms, and (e) withdrawing and analyzing samples of the two fractions of step (d)for identification of the induced enzyme/enzymes. (f) analyzing the identified enzyme/enzymes, deriving the DNAsequence/sequences that codes for the enzyme/enzymes, and (g) producing the identified enzyme/enzymes by use of a hostorganism or the original parental organism.
    According to one embodiment the container of step (b) is a chemostat or an anaerobic digester.
    According to a further embodiment the microbial community originates from animals, plants, insects, soil or aqueous environments. ln one embodiment the defined medium is a medium comprising decomposed organic material, fermentation products and other essentialsubstances, such as salts, vitamins, metals and trace elements. ln another embodiment of the present invention the content of the samples taken in step (e) are separated into fractions before analyses andidentification. ln one embodiment the samples taken in step (e) are subject to punficaflon. ln a further embodiment the samples taken in step (e) are subject to concentration. 9 ln a further embodiment the purification and/or concentration is doneby separation methods based on the varying physicochemical properties ofproteins. ln another embodiment the analyses and identification of step (e) isdone by 1 or 2 dimensional gel electrophoresis or chromatography followedby comparison of the results for the two fractions. ln a further embodiment the enzyme/enzymes that is/are identifiedis/are analyzed and classified by e.g. mass spectrometry and de-novosequencing.
    The present invention also relates to enzymes that are produced usingthe methods as described above.
    Brief Description of the Drawinqs The present invention will be further described with reference to theenclosed drawings.
    Fig. 1 is a schematic outline of the major steps of anaerobic digestionand biogas production, including main organisms and metabolites.
    Fig. 2 is a schematic view of a method according to the presentinvention where the microbial community, that originates from a habitat ofchoice (e.g. an anaerobic digester), is cultured in a chemostat with a definedmedium and under defined conditions to obtain a nullified reactor.
    Fig. 3A-3D show registred data and time points for main events(switch from slaughter house waste to defined medium and sample collectionfor batch cultures) for a chemostat started with a microbial community from ananerobic digester.
    Fig. 4A. shows two 2-Dimensional Electrophoresis (2-DE) gels with alarge number of protein spots at high resolution, representing the meta-secretome of the methanogenic microbial community maintained in thechemostat and collected from the liquid fraction of the two batch cultures.One culture was supplemented with defined medium and cellulose asinducing substance of interest (MC) and the other supplemented with definedmedium only (M) as a reference.
    Fig.4B shows the same gels as in 4A, after a simple image analysis(increasing brightness and contrast to the same degree). Arrows indicate proteins that have been significantly up-regulated in the culture supplementedwith an inducing Substance.
    Detailed Description of Preferred Embodiments of the lnvention Definitions “Defined medium” refers to a chemically defined medium for culturegrowth wherein those nutrients and other components required for growth ofthe microbial community are supplied. Compounds which the organismsthemselves are capable of synthesizing using their own metabolic pathwaysare generally not included in the medium. The defined medium also containscomponents that adjust the properties of the medium to mimic the conditionsprevailing in the natural habitat of the microbial community. As a result, thecontent of a particular defined medium depends on the nutritional needs andconditions of the natural habitat of the microbial community of interest.Standard literature references describing the needs of the microbial commu-nity can be consulted for finding the composition of the correctly definedmedium for various microbial communities.
    ”“Conditions”” mean the physical and chemical state of the environmentin which the microbial community exists. Examples of different conditionsrelate to e.g. temperature, pH, water availability, oxygen levels and ionicstrength.
    “Chemostat” means a device for continuous culturing of microorga-nisms. “lnduce enzyme” means to affect the regulation of enzyme expression,both upwards and downwards. “lnducing substance” means a substance that is added to the microbialcommunity with the aim to affect the regulation of enzyme expression.
    “Metabolic steady-state” means a stable condition of metabolic activitythat does not change over time.
    “Nullified reactor” means a chemostat in which the microbial commu-nity has been kept under constant conditions, supplied with a definedmedium, so that residuals from the natural habitat has been washed outand/or consumed and the microbial community has reached a metabolicsteady-state in an environment of known composition. 11 “Anaerobic digester” means an artificial biodegradation facility for theanaerobic digestion of organic substrate to produce biogas.
    “Batch culture” means a closed-system microbial culture.
    “Meta-proteomics” is the study of all protein samples recovered directlyfrom environmental samples. Metaproteomics should be used to classifyexperiments that deal with all the proteins identified from complex communi-ties, where individuals cannot be binned into species or organism types.
    “Secretomics” relates to the subset of the proteome consisting ofproteins actively exported from a cell type.
    “Meta-secretomics” is the study of samples of all the actively exportedproteins from complex microbial communities, where individuals cannot bebinned into species or organism types.
    The method according to the present invention is not limited to the useof enzymes that catalyze any specific pathway. The method is general andcan be used to induce and identify any industrially important enzyme frome.g. a biogas process, a hot spring or any other microbial habitat whoseenvironment can be cleaned whilst keeping the microbial community viableusing a defined medium, regardless if it is an enzyme that affects differenttypes of fats, proteins, polysaccharides or any other organic molecules.
    The population of microorganisms according to the present inventioncan e.g. originate from animals, insects, plants, soil, aqueous environments,extreme environments with high or low oxygen content, high or low pH, highor low temperature, high pressure or high salt concentration. The populationsof microorganisms may further originate from methanogenic habitats such asan anaerobic digester, the rumen of ruminants, hindgut of cellulolytic insects,anoxic sediments or cecum of cecal animals.
    The enzyme/enzymes that are identified and/or produced according tothe present invention are preferably suitable for applications in industrial andenvironmental biotechnology, production of renewable energy from biobasedfeedstocks, production of biogas, production of bioethanol, food/feed industry,dairy industry, baking industry, wine and fruitjuice industry, brewing industry,textile industry, leather industry, applications in detergents, pulp and paperindustry, processing of fats and oils, application as digestive aid, chemical 12 Synthesis and for degradation of harmful compounds at contaminated sites orat waste water treatment plants, but may of course be suitable for many othertypes of applications.
    The present invention can be envisaged to be used to find enzymes fornumerous of other applications, which is limited only by imagination. Forexample, one possible use of the present invention is to use a bacterialpopulation taken from a waste water treatment plant to find and produceenzymes that decompose residues of medicaments in waste water. This is avery important aspect, since these residues can be very detrimental to bothhumans and animals, e.g. making fish sterile and affecting human fetus.
    Another example is for finding enzymes for the production of biogas inanaerobic digesters, in which the microbial populations of the communitywork together to break down organic matter by anaerobic digestion. lf it waspossible to find enzymes from within the microbial community of methano-genic habitats, as with the present invention, and then produce and add theseenzymes to anaerobic digesters, this could very well prove to be a successfulroute.
    The microbial process of anaerobic digestion of organic material tocarbon dioxide and methane (i.e. biogas) is also an important example of thepracticability of the present invention as this is possibly one of the mostdifficult processes to work with. The microbial community is made up ofseveral populations with very different generation times ranging from 1-2 daysfor primary fermentative bacteria to more than a week for some methanogenicarchaea. Further, the microbial community is made up of primary fermenta-tive, secondary fermentative, homoacetogenic, sulfate reducing and hydrogenproducing fatty acid oxidizing bacteria in addition to hydrogenotrophic andaceticlastic archaea. Thus, the microbial community in anaerobic digestersrepresents a highly complex, cooperative and syntrophic community andthere will therefore be present a high number of metabolites in the mediumfrom which enzymes need to be purified (see figure 1 for a schematic outlineof the process of anaerobic digestion, including main organisms andmetabolites). lt is further an anoxic process supporting the life of methano- 13 genic archaea, of which some are the most oxygen sensitive organisms oneanh.
    However, although one of the most difficult microbial communities towork with, the many steps involved in methanogenesis are a good indicationof the feasibility of the present invention. Thus, if for some reason the definedmedium or the conditions are not able to support life for one or more of thepopulations there will be an imbalance in the microbial population which willlead to accumulation of metabolites and/or that the process will not lead allthe way to methane. On the other hand, if the process leads all the way tomethane production this is in itself proofs that all the necessary populationsare viable and that the full microbial community is screened for induced andexpressed enzymes at the desired condition.
    A method that enables a rational way of inducing, finding and identi-fying enzymes for diverse areas is enzyme mining in microbial communitiesaccording to the present invention. ln one example of the present invention a microbial community is takenfrom an anaerobic digester, as can bee seen in Fig. 2. According to anotherexample, the microbial community can be taken from any other microbialhabitat. The microbial population is then transferred to a chemostat, operatedto mimic the natural habitat of the microbial community, where the microorga-nisms are fed with a nutrition solution of known composition, i.e. a chemicallydefined medium. The composition of the defined medium and/or conditionsthat is used depends on the natural habitat of the microbial community. Bycultivation of the microbial community with a defined medium the content ofthe chemostat becomes clean, since particulate matter and other contami-nants of the natural habitat no longer will be present and the populations ofthe microbial community are kept alive and viable. This results in that anullified reactor at steady-state is established, which does not have anymemory of the original substrate comprising a lot of particulate matter andother contaminants. Furthermore, the microorganisms of the nullified reactordo not have to express large amounts of hydrolytic enzymes to obtain aminoacids, sugars etc that are needed, since they are fed with decomposedsubstrates, which results in a steady-state expression of enzymes. 14 The nullified reactor according to the present invention is veryimportant for a multitude of reasons. To search for enzymes produced bymicrobial communities in natural habitats without induction would not berational since even if it was possible to purify and obtain the enzymes in highenough concentration their function would still be unknown. Thus cloning andscreening for sequence or activity for all proteins would still be necessary, i.e.it is a non-rational, non-feasible route (see A in fig. 2). There would further beno point in trying to induce regulation of an enzyme directly in the originalhabitat ofa microbial community, since it is too contaminated and complex,and the concentration of microorganisms and enzymes is too low. Thus,fractionation, purification and concentration of the enzymes or the microorga-nisms of interest are thereby almost impossible, i.e. it is a non-feasible route(see B in fig. 2). Moreover, it would be very difficult to identify the inducedenzymes of choice in samples collected directly from the natural habitat sincethere is an already ongoing and fluctuating active production of variousenzymes by the microbial community.
    From the clean microbial community of the nullified reactor twofractions are taken and the cells are preferably concentrated by for examplecentrifugation and washed with a defined medium once or several times andthereafter transferred into two separate containers for batch culturing.
    Examples of suitable containers for batch culturing are flasks, bottlesor any type of vessel. However, the cultivation of these two fractions can bedone either in a chemostat or in batch cultures. The purpose of the batchculturing is not neccesarily to culture the microorganisms to a higher celldensity, as in the regular use of batch cultures, but to find what enzymes areproduced by the microbial community in response to a certain substance. Theregular way to grow microbial cells in a batch culture normally encompassesinoculation of a small volume of pure culture into a larger volume of medium(normally at an approximate 1:100 ratio). Contrary to batch cultures of purecultured microorganisms, a batch experiment with the normally smallinoculum would however not work for rational enzyme mining since, accor-ding to the present invention, a microbial community is maintained in thenullified reactor. ln a microbial community different populations of microorga- nisms have different generation times, which can range from hours to evenweeks. Thus, if a small inoculum was used, different growth rates in the batchculture would lead to a microbial imbalance. Therefore it is necessary tosample a larger volume of microorganisms from the nullified reactor, which isconcentrated by centrifugation, so what is added to the batch cultures is acomplete microbial community that is already from the start in balance.
    Thus, importantly, the nullified continuous reactor serves severalpurposes that are instrumental for the present invention of rational enzymemining from microbial communities as (i) it provides a “clean” starting materialthat has no “memory” of the original substrate (ii) it provides a microbialcommunity that is at steady-state and viable under the chosen conditions andthus have the ability to produce enzymes that are active and stable under thesaid conditions (iii) it provides a microbial community that is in balance insuch a way that each type of microorganism is present in a correct numberrelative to other microorganisms so that the microbial community is able tometabolize all parts of the substrate or the produced metabolites at the samerate and (iv) it provides a starting material with a high absolute number ofeach and all microorganisms so that there is a high probability that anyexpressed protein/enzyme that is present after induction is present in suchlarge amount that it can be detected.
    One of the fractions of the microbial community is provided with adefined medium, including a substance against which one or more enzymesare desired. This will disturb the steady-state and at some point result in aresponse in enzyme expression regulation. The other fraction is provided onlywith a defined medium. The reason to this arrangement is that the content ofthe container that is given only the defined medium is used for comparison tobe able to identify the enzyme/enzymes that have been induced in the othercontainer, i.e. the content of the container given only defined medium is usedas a reference showing what proteins/enzymes that are present naturally inthe microbial community at steady-state.
    The addition of exactly the substance or substances against whichenzyme/enzymes are desired results in that only the microorganisms that areable to produce these enzyme/enzymes will react. Thus, contrary to e.g. 16 metagenomics, where the DNA of the full microbial community is cloned andscreened, according to the present invention the addition of a targetSubstance results in that the microbial community reacts and regulates theexpression of the requested enzyme/enzymes. Thus, by adding a singleknown substance to a microbial community, viable under certain conditions,several goals are achieved since (i) the activity of the identified enzyme isknown, for example if adding cellulose this will induce the regulation ofexpression of cellulases or if adding keratin this will induce the regulation ofexpression of keratinases, and (ii) the expressed and identified enzyme willbe stable under the conditions that the microbial community was selected forand no further screening is necessary.
    After leaving the induced microbial population to express enzymes,while maintaining and or cultivating the fractions of microorganisms, samplesare taken from the container with the induced population and also from theother container used as a reference. At this point the samples can befractionated to find different proteins/enzymes in different fractions forexample (i) the liquid phase could be collected to identify free extra-cellularproteins/enzymes (ii) the substrate could be collected to search for substrateadsorbed proteins/enzymes (iii) the microorganisms could be collected toidentify cell-bound extracellular proteins/enzymes or (iv) the microorganismscould be collected to identify intracellular proteins/enzymes. Theproteins/enzymes that are collected are then purified and concentrated.Since, the samples are taken from a rather clean environment of knowncomposition, due to the cultivation in a chemically defined medium,fractionation, purification and concentration of the proteins/enzymes can bedone with a number of established techniques based on the physicochemicalproperties of proteins, such as centrifugation, ultra filtration, dialysis, electro-phoresis, iso-electric focusing, specific precipitation etc.
    After purification of the protein/enzyme samples they are analyzed bye.g. gel electrophoresis or chromatography. The purified samples, when usinggel electrophoresis, are separated on the gels with regard to size or, alterna-tively, size combined with charge. One gel is used for the sample that hasbeen induced and one gel for the reference sample. These gels will give 17 patterns reflecting the enzyme expression from the microbial community atsteady-state that has not been induced with any Substance and also theenzyme expression from the induced sample. By analysis and comparison ofthese two gels, e.g. by overlapping, it is possible to identify protein spots thatdiffer between the gel from the induced sample from the gel with thereference sample. These spots represent potential enzymes with activity ofchoice that have been rationally induced using a substance of interest andidentified from the microbial community, viable under predeterminedconditions, according to the present invention.
    After this comparison, interesting enzyme spots may be cut out fromthe gels and analyzed with established methods, such as mass-spectrometryand de-novo sequencing in order to classify the enzyme, find the likelyfunction of the enzyme, what the amino acid sequence of the enzyme is, etc.
    Using the information from this analysis it is possible to derive the DNAsequence and to clone or construct the gene in order to produce the enzymefor further tests regarding activity, stability, pH- and temperature optimum etc.lt is further possible to use the DNA sequence for production of theenzyme/enzymes in a host organism or the original organism on an industrialscale, e.g. for use in any of the earlier described processes.
    Thus, by using the method according to the present invention it is notonly possible to find and produce new enzymes with properties superior tothose of already known enzymes but also to identify new unique enzymesnever used industrially by man since the whole microbial population is usedas a source.
    Examples The following examples illustrate only preferred embodiments of thepresent invention. The examples are only illustrative and should not beconstrued as limiting the invention in any way. The examples further describehow the present invention enables rational enzyme mining of enzymessecreted into the medium, which is the most complex problem to solve.Nullified Reactor The anaerobic microbial community was collected in the form of adigestate from a full-scale anaerobic digester treating slaughter house and 18 food waste. The digestate was transferred under oxygen exclusion to achemostat operated under anaerobic conditions as an anaerobic digester witha total volume of 15 litres and an active volume of 9 litres. The reactor wasduring a start up phase fed with the same substrate as the full-scale digesterand run under mesophilic conditions (38 °C) with an average retention time of35 days and an organic loading rate (OLR) of 2.5 g volatile solids (VS) / L andday. During the experiment several parameters were monitored in order toverify that the bacterial population was stable and at steady state, including:digestate total dry solids (DS) and volatile solids (VS), volatile fatty acids(VFA), nitrogen, pH and alkalinity. Gas production and methane content weremeasured online with a milli gas counter (MGC-10, Ritter, Germany) and anIR gas sensor (Bluesense Gas sensor GmbH, Germany) and through gassampling. VFA was measured with a GC-FID (Clarus 500, Perkin-Elmer),column: Perkin Elmer Elite-FFAP (Perkin-Elmer, USA). Ammonium-nitrogenwas analyzed according to FOSS Tecators application sub note 3502 with aKjeltec 8200 (FOSS in Scandinavia, Sweden). The total organic nitrogen,Kjeldhal-nitrogen, was analyzed according to Tecators AN 300 Sv 1999-04-09v. 2 with a Kjeltec 8200 (FOSS in Scandinavia, Sweden). pH was measuredaccording to Swedish standard EN 12176 with the pH electrode WTW lnolabpH Level 2 (Weilheim, Germany). Alkalinity was measured with a hydro-chloride acid titration method according to guidelines in Swedish standard ENlSO9963. Total solids and volatile solids were analyzed according to SwedishStandard SS 028113.
    When all monitored parameters were stable the substrate wasreplaced with a defined medium designed to provide all nutritional compo-nents and conditions that are required by the anaerobic microbial community.The medium was designed based on several anaerobic media used forgrowth of methanogenic communitiesmß] and consisted of the following: Buffer and minerals: 10 mM KH2PO4, 10mM Na2HPO4_61 mMNaHCOg, 5.6 mM NH4Cl, 3.5 mM Na2SO4_ 0.5 mM MgClg - 6H2O, 0.3 mMFeCl2- 4H2O, Vitamins: 1 uM pyridoxamine-2HCl (Vit. BG), 0.81 uM nicotinic acid(Vit, Bg), 0.57 uM L-ascorbic acid (Vit. C), 0.36 uM p-aminobenzoic acid, 0.24 19 (JM lipoic acid, 0,22 (JM thiamin hydrochloride (Vit. B1), 0.13 (JM riboflavin (Vit.Bg), 0.11 (JM calcium D(+) pantothenate (Vit. B5), 0.08 (JM biotin (Vit. Bg), 0.05(JM folic acid (Vit. Bg), 0.04 (JM vitamin B12, Trace elements: 0.65 (JM H3BO3, 0.37 (JM AlClß, 0.29 (JM ZnClg, 0.21(JM CoClg - 6H2O, 0.18 (JM CuClg - 2H2O, 0.15 (JM MnCl2- 2H2O, 0.105 (JMNiClg - 6H2O, 0.06 (JM NagSeOß - 5H2O, 0.06 (JM Na2WO4 - 5H2O, 0.04 (JM(NH4)6Mo7O24 - 4 H20, Carbon and nitrogen sources: 170 mM glucose, 170 mM amino acids(acid hydrolyzed casein), 22 mM saccharose and 22 mM Ca-Iactate, Fermentation products: 15 mM ethanol, 15 mM methanol, 9 mM aceticacid, 3 mM propionic acid, 1.5 mM butyric acid and 1.5 mM formic acid.
    Depending on process stability as evaluated by VFA concentration theOLR of the defined media was between 0 and 2 g VS/L and day.Batch lnduction A sample of 200 mL of digestate was co|ected from the chemostat 19 months (see arrow in fig. 3A - D) after the switch to the defined medium. Thesample was sp|it into two aiiquots of 100 mL and transferred to two centrifugetubes which were thereafter centrifuged at 250 g for 5 minutes to allow for anyparticulate matter to sediment. The remaining supernatant was then centri-fuged at 9000 g for 20 min. The supernatant was poured off and the pelletwas re-dissolved in a de-gassed washing buffer consisting of the definedmedium minus the carbon source and fermentation products. The sampleswere once again centrifuged and the supernatant was poured off. Thebacterial cell pellet was thereafter used to start new bacterial populationcultures in batch flasks (544 ml flasks with rubber septum). Both flaskscontained 250 mL of the defined medium described above but with an organicloading of 0.75 g VS/L. One of the flasks was also supplemented with thenew, polymeric, carbon source cellulose in the form of Whatman paper (nr597) to an additional organic load of 0.75 g VS/L. Gas production wasdetermined by pressure measuremnets (Testo 312-3, Germany) and methanecontent was measured in withdrawn gas-samples by GC-FID (Clarus 500,Perkin-Elmer), column: packed Porapak T 80/100 Mesh (Perkin-Elmer, USA). 2-Dimensional Electrophoresis (2-DE). Sample preparation and qelsSampling for 2-DEAfter 4 days 80 ml of sample from each batch culture was transferred to 2 x 50 ml centrifugation tubes (Sarstedt). The samples were centrifuged for45 min at 9 000 g at 4°C in a Hettich R35 centrifuge. The supernatant wascollected for sampling of free extra-cellular proteins/enzymes and the pelletswere discarded. The samples were frozen in liquid nitrogen and freeze-driedfor two days to reduce the sample volume to approximately 4 ml.
    Protein Precipitation The proteins were precipitated using a 20 % trichloroacetic acid (TCA)/ acetone mixture (Sigma-Aldrich) containing 0.2% dithiothreitol (DTT). Thesamples of 4 ml were mixed with 20 ml precipitation solution and the proteinswere precipitated overnight at -20 °C and thereafter pelleted by centrifugationat 9 000 g for 45 min at -15°C. The pellets were washed twice with 2 ml ofacetone at -20 °C. Subsequently the pellets were resuspended and allowed tostand in 2 ml acetone for 1 h at -20°C. The acetone was removed after anadditional centrifugation and the white pellets were allowed to air-dry for 10min.
    Gel Filtration and Sample concentration The protein pellets were dissolved in 2 ml of 8 M urea and each sample was applied to a PD-10 desalting coulomn (GE-Healthcare)equilibrated with 8 M urea. The samples were eluted with 3 ml 8 M urea. Thefirst and last 0.75 ml of liquid was descarded and the middle fraction of 1,5 mlwere collected for each sample. The collected fractions were concentratedusing an Amicon Ultra-2 ml pre-launch centrifugal filter (Millipore) with amolecular weight cut-off of 10 kDa, reducing the sample volume from 1,5 mlto approximately 100 ul.
    Protein guantification The protein concentration of the samples was determined with a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies lnc.) usingthe Protein A280 method. The protein concentration was calculated bycomparison to a bovine serum albumin standard curve in 8 M urea. 21 2-DE. 1st Dimension (lsoelectric Focusinq) An aliquot of sample containing 120 ug protein was diluted to a finalvolume of 140 ul in lysis buffer (8 M urea, 1% (w/v) CHAPS, 0.4% DTT and0.5% carrier ampholytes, Bio-Rad). The mixture was allowed to stand for 30min at room temperature. The samples were centrifuged at 22 000 g for 30min and the supernatant was loaded via anodic cup loading to a pre-swollen11 cm IPG strip, pH 4-7 (Bio-Rad). A voltage of 200 V was applied for 6 h toallow the proteins to enter the gel.
    The following voltage ramp protocol on the Protean IEF cell (Bio-Rad)was used: 30 min 250 V rapid voltage ramping, 1 h 8000 V slow voltageramping, rapid voltage ramping to 20 kVh, 50 V rapid ramping for 20 h(holding step). The IPG strips were re-focussed for 15 min at 8000V prior tothe 2“d dimension analysis.
    The IPG strips were equilibrated under gentle agitation for 15 min inequilibration base buffer (6 M urea, 30% (w/v) glycerol, 2 % (w/v) SDS in 0.05M Tris-HCl buffer (pH 8.8)) and 50 mg DTT (Sigma-Aldrich) per strip.Subsequently the strips were equilibrated for 15 min in equilibration basebuffer containing 200 mg lodoacetamide (Bio-Rad) per strip.2-DE. 2“d Dimension (electrophoresis).
    The second dimension was run on the Criterion Cell System (Bio-Rad).Bio-Rad 's Criterion 10,5-14% Tris-HCl gels were run in the TGS buffersystem at 50 V for 30 min and followed by 200 V for 50 min. 5 ul FermentasPageruler plus prestained Protein Ladder was used as molecular weightstandard. After electrophoresis the gels were stained with SYPRO rubyfluorescent stain (lnvitrogen) according to the manufacturer's protocol.
    The gels were scanned and documented with A FLA-5100 imagingsystem (Fuji-Film) using a SHG blue laser at 473 nm.
    Results and DiscussionNullified Reactor Except for a short period (approx. 9 months after the switch to thedefined medium) when technical problems occurred (leakage to air), thechemostat could be stably operated during the full experiment to reach anullified reactor. 22 lmportantly, the bacterial community in the chemostat maintainedmethane gas production during the full experiment. ln fig. 3A the relationshipbetween organic loading rate and gas production is illustrated. After anappropriate OLR for the defined medium of 1 g VS/L and day was found, thegas production maintained stable (the decrease in organic loading ratemidterm of the experiment was a necessary response to the increasing acidsduring the technical problems). The stable production of biogas is importantbecause it shows that the microbial community is stable in the sence that alltypes of microorganisms that are necessary to metabolize the carbon sourcesall the way from monomers via fermentation products to methane are presentalso after feeding with the defined culturing medium.
    As illustrated in figure 3B the total solids dropped from 5.5 % to 1 %after approximately 5 months after the onset of feeding with defined medium.Thus, the residues of the original substrate in the form of slaughter housewaste have been either degraded or washed out depending on whether thematerial is biologically accessible or not. Arithmetically, 95 % of an inertsubstance would be washed out after 3 retention times (105 days). This isimportant because once the dry substance (DS) is stable at very low values itshows that there are no residual components from the original substrate left inthe reactor. Thus, the remaining volatile solids (VS) of the dry substance (DS)is made up of the bacterial community solely. Furthermore, after the switch insubstrate the degree of degradation increased from 55 % to 85 %. This isexpected since the defined medium only contains carbon sources in the formof monomeric sugars and amino acids that are microbially accessible. Thus,more or less all the carbon is converted to gas in the process. lt is alsointeresting to note that this increase of 30% in degree of degradation is whatcould possibly be reached if all organic material in e.g. slaughter house wastecould be hydrolyzed to monomers. A 100 % degree of degradation is notpossible as some of the carbon is neccessary to produce components of newmicroorganisms. ln order to provide substrate for the primary fermentative bacteria thedefined medium contained the carbon sources glucose, saccharose andamino acids. This is however not the substrate for secondary fermentative 23 bacteria or methanogens, so in order to satisfy the substrate needs of all themicroorganisms the defined medium also comprises low concentrations offermentation products (alcohols and fatty acids). As is illustrated in Figure 3Cthere is a sudden increase in acids at the switch from the original substrate tothe defined medium which is due to that acids are both produced by theresidual original substrate, the added highly microbially accessible carbonsources and the added fatty acids. However, it also shows that the meta-bolically downstream microorganisms are able to cope with the increasingVFA content as the concentration falls after a few weeks and is thereafterfairly stable (below 30 mM total VFA) until problems with the chemostatoccured. However, even after the technical problems leading to fatty acidconcentrations of above 60 mM, the microbial community could be main-tained, and after the technical problem was solved fatty acid concentrationremained very low.
    The defined medium has a much lower pH buffering capacity thanmixed organic material, e.g. slaughter house waste, and the alkalinitydropped from above 20.000 to approx. 5.000 HCOg- equivalents per litre (fig.3D). This is however enough since acidic metabolites are consumed at a highrate by secondary fermenters and methanogens that are viable and active inthe reactor. Thus pH dropped only by approximately 0.7 units from the switchfrom slaughter house waste to defined medium (fig. 3D).
    To conclude it is shown that it is possible to compose and use adefined culturing medium under conditions that maintains the full microbialcommunity that is needed to anaerobically convert a carbon source all theway to carbon dioxide and methane via microorganisms performing primaryfermentation, secondary fermentation and methanogenesis. lf this was not thecase, there would at some point emerge a terminal accumulation of dry solids(DS), volatile solids(VS) or fermentation products such as various fatty acids,and gas production would have seized. Thus, the material from this reactor isa perfect starting point for the following induction steps as it has no “memory”of the original substrate. Furthermore, the reactor material does no longercontain any disturbing substances such as proteins, fats, sugars, DNA,particulate matter etc, other than what can be derived from the microbial 24 population, that can influence the subsequent steps in the rational enzymemining. That is, when the content in the container is stable and the productionof metabolites and end products of the microbial community are at a steady-state, a nullified reactor has been reached, which occurred after approx. 5months after switch to defined medium (see fig. 3A-D).
    Batch |nduction Both batch bottles started producing gas immediately upon mixing of microbial population from the nullified reactor with the two defined media. Thisis normally not the case in standard batch experiment proceedings where alag phase is most often noted, caused by the necessary hydrolysis ofsubstrate and/or microbial growth. lt was also found that the methaneproduction commenced concomitantly with the gas production and thatmethane concentration was as high as 50 % after 3 days. That is, the gasproduction was not only a result of fermentation but the number of methano-gens was high enough to convert fermentation products to methane.lmportantly, the bottle that had cellulose added to induce regulation ofexpression of cellulases showed a higher overall gas production(approximately 10 % more) relative to the batch culture that had only beensupplemented with the defined medium. Thus, this implies that the cellulosewas influenced and degraded to single sugars by the microbial community,and subsequently was fermented and further converted to methane. 2-DE Sample Preparation and Gels The resulting two gels are presented in fig. 4A. After sample preparation the extra-cellular proteins were concentrated by a factor 800(volume reduction of the medium from 80.000 to 100 ul). The protein sampleswere further pure enough and of high enough quality and concentration to besubject to 2-DE. What is important is the fact that the extra-cellular proteins,that are present in a very diluted form in the medium, can be fractionated,purified and concentrated, and run on 2-DE gels in such a way that the gelprovides protein spots of high resolution against a low background and arethus interpretable. What is further important is that both gels are similar intheir protein spot pattern even though samples were not collected until 4 daysafter induction. This is what should be anticipated if the two batches were treated identically (besides the added inducing substrate in one of thebottles). This result is further necessary for any small differences between thetwo samples to be detected that would be the result from induction of proteinexpression regulation from a single defined substrate.
    The methods of the present invention enables rational enzyme miningin microbial communities and the resulting gels represents the first example ofa meta-secretome from an active microbial community. The gels are of highenough quality to be subjected to image analysis and identification ofdifferences in protein expression pattern and/or expression strength betweenthe induced and non-induced culture. Even without advanced image analysistools it is possible to identify spots that differs in strength between the twogels (see arrows in figure 4B). ldentified spots is then subjected to furtheranalyses, whereas those spots that do not differ can be excluded, whichdramatically will reduce the time and effort associated with cloning andscreening of all the proteins.
    Obviously, since the extra-cellular proteins can be fractionated, purifiedand concentrated to give the desired result, so can also other fractions of thecultures. 26 REFERENCES 1) Kirk O, Borchert TV, Fuglsang CC. Industrial enzyme applications. Curr.Op. Biotech., vol. 13 (4), pp. 345-351, 2002. 2) Eijsink VGH, Bjork A, Gaseidnes S, Sirevag R, Synstad B, van den Burg B,Vriend G. Rational engineering of enzyme stability. J. Biotech., vol. 13 (1-3), pp. 105-120, 2004. 3) Torsvik V, Ovreas L, Thingstad TF. Prokaryotic diversity - Magnitude,dynamics, and controlling factors. Science, vol. 296 (5570), pp. 1064-1066, 2002. 4) Lorenz P, Eck J. Metagenomics and industrial biotechnology. Nature vol. 3,pp. 510-515, 2005. 5) Bond PL and Wilmes P. The application of two-dimensionalpolyacrylamide gel electrophoresis and downstream analyses to a mixedcommunity of prokaryotic microorganisms. Environmental Microbiology,vol. 6, pp. 911-920, 2004 6) Bond PL and Wilmes P. Metaproteomics: studying functional geneexpression in microbial ecosystems. TRENDS in Microbiology, vol. 14, pp.92-97, 2006. 7) Maron P-E, Ranjard L, Mougel C and Lemanceu P. Metaproteomics: Anew approach for studying functional microbial ecology. Microbial Ecology,vol. 53, pp. 486-493, 2007. 8) Maron P-A, Cristophe M, Séverine S, Houria A, Philippe L and Lionel R.Protein extraction and fingerprinting optimization of bacterial communitiesin natural environment. Microbial ecology, vol 53, pp. 426-434, 2007. 9) Mariappan V, Vellasamy KM, Thimma JS. Identification of immunogenicproteins from Burkholderia cepacia secretome using proteomicanalysis.VACClNE, vol. 28 (5), pp. 1318-1324, 2010. 10) Pocsfalvi G, Cacace G, Cuccurullo, M. Proteomic analysis of exoproteinsexpressed by enterotoxigenic Staphylococcus aureus strains.PROTEOMICS, vol. 8 (12), pp. 2462-2476, 2008. 27 11) Mould FL, Morgan R, Kliem KE and Krysttalidou E. A review andsimplification of the in vitro incubation medium. Animal feed science andTechnology, vol. 123-124, pp. 155-172, 2005. 12) Pavlostathis SG, Miller TL and Wolin MJ. Fermentation of insolublecellulose by continuous cultures of Ruminococcus albus. Applied andEnvironmental Microbiology, vol. 54, pp. 2655-2659, 1988. 13) Krause DO, Denman SE, Mackie RI, Morrison M, Rae AL, Attwood GTand McSweeney, C.S. Opportunities to improve fiber degradation in therumen: microbiology, ecology, and genomics. FEMS Microbiology Review,vol. 27, pp. 663-693, 2003. 14) Song H, Clarke WP and Blackall LL. Concurrent microscopic observationsand activity measurements of cellulose hydrolyzing and methanogenicpopulations during the batch anaerobic digestion of crystalline cellulose.Biotechnology and Bioengineering, vol. 93, pp. 369-378, 2005 15) Karlsson A, Ejlertsson J, Nezerevic D and Svensson BH. Degradation of phenol under meso- and thermophilic, anaerobic conditions. Anaerobe, vol. sn ), pp. 25-35, 1999.
    权利要求:
    Claims (20)
    [1] 1. A method for rational mining for induced enzyme/enzymes in microbialcommunities, comprising the steps of: (a) providing a community of microorganisms, (b) cultivating the microbial populations of the community in a containerunder conditions that mimic the conditions of the microbialcommunity's natural habitat, where the microorganisms are given adefined culturing medium to eliminate matter deriving from thenatural habitat and to allow the microbial community to reach ametabolic steady-state, (c) removal from the container of step (b) of two fractions ofmicroorganisms, and transfer of said two fractions into two separatecontainers, (d) providing one of the fractions of microorganisms from step (c) witha defined medium which includes a substance against whichenzyme/enzymes is/are desired, to induce a regulation ofexpression of the desired enzyme/enzymes, and the other fractionfrom step (c) with a defined medium without said substance for thepurpose of comparison, and maintaining and/or cultivating the twofractions of microorganisms, and (e) withdrawing and analyzing samples of the two fractions of step (d)for identification of the induced enzyme/enzymes.
    [2] 2. A method according to claim 1, wherein the container of step (b) is achemostat or an anaerobic digester.
    [3] 3. A method according to claim 1 or 2, wherein the community ofmicroorganisms originates from animals, plants, insects, soil or aqueous environments. 29
    [4] 4. A method according to any of the previous claims, wherein the definedmedium is a medium comprising decomposed organic material, fermentationproducts and other essential substances, such as salts, vitamins, metals andtrace elements.
    [5] 5. A method according to any one of the previous claims, wherein the contentof samples taken in step (e) are separated into fractions before analyses andidentification.
    [6] 6. A method according to any one of the previous claims, wherein thesamples taken in step (e) are subject to purification.
    [7] 7. A method according to any one of the previous claims, wherein thesamples taken in step (e) are subject to concentration.
    [8] 8. A method according to any one of claims 6-7, wherein the purificationand/or concentration is done by separation methods based on the varyingphysicochemical properties of proteins.
    [9] 9. A method according to any one of the previous claims, wherein theanalyses and identification of step (e) is done by 1 or 2 dimensional gelelectrophoresis or chromatography followed by comparison of the results forthe two fractions.
    [10] 10. A method for rational mining for induced enzyme/enzymes in microbial communities and production thereof, comprising the steps of:(a) providing a community of microorganisms,(b) cultivating the microbial populations of the community in a containerunder conditions that mimic the conditions of the microbial community'snatural habitat, where the microorganisms are given a defined culturingmedium to eliminate matter deriving from the natural habitat and toallow the microbial community to reach a metabolic steady-state, (c) removal from the container of step (b) of two fractions ofmicroorganisms, and transfer of said two fractions into two separatecontainers, (d) providing one of the fractions of microorganisms from step (c) with a defined medium which includes a substance against whichenzyme/enzymes is/are desired, to induce a regulation of expression ofthe desired enzyme/enzymes, and the other fraction from step (c) witha defined medium without said substance for the purpose ofcomparison, and maintaining and/or cultivating the two fractions ofmicroorganisms, and (e) withdrawing and analyzing samples of the two fractions of step (d)for identification of the induced enzyme/enzymes, (f) analyzing the identified enzyme/enzymes, deriving the DNA sequence/sequences that codes for the enzyme/enzymes, and (h) producing the identified enzyme/enzymes by use of a host organism or the original parental organism.
    [11] 11. A method according to claim 10, wherein the container of step (b) is achemostat or an anaerobic digester.
    [12] 12. A method according to claim 10 or 11, wherein the community ofmicroorganisms originates from animals, plants, insects, soil, or aqueousenvironments.
    [13] 13. A method according to any of claims 10-12, wherein the defined mediumis a medium comprising decomposed organic material, fermentation productsand other essential substances, such as salts, vitamins, metals and trace elements.
    [14] 14. A method according to any one any of claims 10-13, wherein the contentof samples taken in step (e) are separated into fractions before analyses andpunficaflon. 31
    [15] 15. A method according to any one of claims 10-14, wherein the samplestaken in step (e) are subject to purification.
    [16] 16. A method according to any one of claims 10-15, wherein the samplestaken in step (e) are subject to concentration.
    [17] 17. A method according to any one of claims 15-16, wherein the purificationand/or concentration is done by separation methods based on the varyingphysicochemicai properties of proteins.
    [18] 18. A method according to any one of claims 10-17, wherein the analyses andidentification of step (e) is done by 1 or 2 dimensional gel electrophoresis orchromatography followed by comparison of the results for the two fractions.
    [19] 19. A method according to claim 18, wherein the enzyme/enzymes that is/areidentified is/are analyzed and classified by e.g. mass spectrometry and de- novo sequencing.
    [20] 20. An enzyme mined and/or produced according to the method of any one of the previous claims.
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    同族专利:
    公开号 | 公开日
    EP2646547A4|2014-04-30|
    EP2646547B8|2017-10-25|
    US20130280731A1|2013-10-24|
    DK2646547T3|2017-11-13|
    WO2012074476A1|2012-06-07|
    CA2819222A1|2012-06-07|
    US9365887B2|2016-06-14|
    EP2646547B1|2017-08-16|
    SE535469C2|2012-08-21|
    EP2646547A1|2013-10-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2001012842A1|1999-08-19|2001-02-22|Omniscience Pharmaceuticals|Methods and targets of antibiotic resistance|
    EP1618183B1|2003-04-29|2014-11-19|Danisco US Inc.|Novel bacillus 029cel cellulase|
    法律状态:
    2021-06-29| NUG| Patent has lapsed|
    优先权:
    申请号 | 申请日 | 专利标题
    SE1051254A|SE535469C2|2010-11-29|2010-11-29|Rational enzyme washing|SE1051254A| SE535469C2|2010-11-29|2010-11-29|Rational enzyme washing|
    EP11844020.5A| EP2646547B8|2010-11-29|2011-11-29|Rational enzyme mining|
    DK11844020.5T| DK2646547T3|2010-11-29|2011-11-29|Rational Enzyme Extraction|
    CA2819222A| CA2819222A1|2010-11-29|2011-11-29|Rational enzyme mining|
    PCT/SE2011/051449| WO2012074476A1|2010-11-29|2011-11-29|Rational enzyme mining|
    US13/989,513| US9365887B2|2010-11-29|2011-11-29|Rational enzyme mining|
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