Gene

专利摘要:
An isolated gene having a DNA sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity, or a polypeptide having a function functionally equivalent to the activity.
公开号:KR20010023509A
申请号:KR1020007002155
申请日:1998-05-26
公开日:2001-03-26
发明作者:다카야마마사노리；고야마노부토；가토이쿠노신；사카이다케시
申请人:오미야 히사시；다카라츠죠 가부시키가이샤；
IPC主号:

专利说明:

Gene
Fucose sulfate-containing polysaccharide derived from seaweed is a sulfated polysaccharide mainly composed of fucose, commonly referred to as fucoidan, and known to include galactose, glucuronic acid, xylose, mannose, glucose and the like. The type and amount of these constituent sugars vary depending on the type of seaweed from which it is derived. For example, there are reports that commercially available Sigma-made fucoidan is divided into about 13 molecular species (Carbohydrate Research, Vol. 255, pp. 213-224 (1994)).
When these are largely divided, it is divided into two types, a substance which comprises fucose substantially without containing uronic acid, and a thing containing fucose and mannose in a component sugar, including uronic acid.
The biological activity of fucose sulfate-containing polysaccharides has been reported in several ways, such as enhancing macrophage activity, inhibiting cancer metastasis, and anticoagulant.However, there are molecular species in fucose sulfate-containing polysaccharides. In order to do so, it was necessary to separate and purify the fucose sulfate-containing polysaccharide. However, in the previous method, the separation was insufficient, making it difficult to prepare a large amount as a drug. Furthermore, fucose sulfate-containing polysaccharide is a sulfated polysaccharide having a very high molecular weight, and if used as it is as a medicine, there are problems such as antigenicity, uniformity, anticoagulant activity, etc. There was.
The method of preparing low molecular weight by enzymatically decomposing the fucose sulfate-containing polysaccharide is an advantageous method that can react under mild conditions and obtain a uniform low molecular weight from the substrate specificity of the enzyme. Background Art Conventionally, abalone, scallops, sea urchins, marine microorganisms and the like have been reported to produce enzymes that degrade fucose sulfate-containing polysaccharides. However, since these enzymes are generally contained in a small amount in the living body and have a plurality of fucose sulfate-containing polysaccharide degrading enzymes, various purification steps are required to obtain a single enzyme. In addition, the amino acid sequence and gene structure of these enzymes were not clearly identified.
An object of the present invention is to use a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity useful for the preparation or structural analysis of a fucose sulfate-containing polysaccharide, and for preparing a low molecular weighted fucose sulfate-containing polysaccharide, and genetic engineering using the gene. The present invention provides a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity that can be obtained.
The present invention provides a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity useful for structural analysis of a fucose sulfate-containing polysaccharide or preparation of a low molecular weight polysaccharide, a method for producing the polypeptide, and a polypeptide obtained by the method. It is about.
1 is a diagram illustrating positions of ORF-1 and ORF-2.
2 is a view showing the precipitation formation rate of the fucose sulfuric acid-containing polysaccharide.
3 is a diagram illustrating the position of fdlA.
4 shows the position of fdlB.
5 is a chromatogram using DEAE-Sepharose FF.
Embodiment of the invention
Hereinafter, the present invention will be described in detail.
The present invention relates to a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity. As an example of the polypeptide encoded by this gene, the polypeptide which has the endo type fucose sulfate-containing polysaccharide degradation activity of following (1) derived from the genus Alteromonas is mentioned.
(1) It acts on the fucose sulfuric acid containing polysaccharide (henceforth fucose sulfuric acid containing polysaccharide-F) which has the following physicochemical property, and makes this fucose sulfuric acid containing polysaccharide low molecular weight.
(a) Constitution sugars: It contains substantially no uronic acid.
(b) Flavobacterium sp. It is not substantially low molecular weight by the fucoidan degrading enzyme produced by SA-0082 (FERM BP-5402).
Polypeptides having a fucose sulfate-containing polysaccharide-degrading activity include Alteromonas sp. There is an endo type fucose sulfate-containing polysaccharide degrading enzyme produced by SN-1009, and the enzyme can be prepared as described in Reference Example 1- (3).
The fucose sulfuric acid-containing polysaccharide-F can be prepared as described in Reference Example 1.
Flavobacterium sp. The fucoidan degrading enzyme produced by SA-0082 (FERM BP-5402) can be prepared as described in Reference Example 5.
As another polypeptide having a fucose sulfate-containing polysaccharide decomposition activity, a polypeptide having a fucose sulfate-containing polysaccharide degradation activity of the following (2) derived from the genus Flavobacterium bacteria can be exemplified.
(2) acting on fucose sulfuric acid-containing polysaccharide (hereinafter referred to as fucose sulfuric acid-containing polysaccharide-U) having the following physicochemical properties to lower the molecular weight of the fucose sulfuric acid-containing polysaccharide, and the following formulas (I), (II) and (III) At least one compound selected from IV is liberated.
(c) Constitution sugars: contains uronic acid.
(d) Flavobacterium sp. It is degraded by the fucoidan degrading enzyme produced by SA-0082 (FERM BP-5402).

As a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity, Flavobacterium sp. There is a fucoidan decomposing enzyme produced by SA-0082, and the enzyme can be prepared as described in Reference Example 5.
In addition, the mixture of fucose sulfuric acid containing polysaccharide-F and fucose sulfuric acid containing polysaccharide-U is described below as a fucose sulfuric acid containing polysaccharide mixture.
In the present invention, a polypeptide having a fucose sulfuric acid-containing polysaccharide degrading activity means a deletion of an amino acid in a natural amino acid sequence as long as it has not only a natural fucose sulfuric acid-containing polysaccharide degrading enzyme but also a fucose sulfuric acid-containing polysaccharide degrading activity ( It is also meant that the polypeptide whose amino acid sequence has been modified by vi), substitution, insertion, addition, or the like is also included in the present invention.
The natural fucose sulfate-containing polysaccharide degrading enzyme referred to herein includes, for example, those derived from the genus Alteromonas bacterium and Flavobacterium bacterium, but the present invention is not limited thereto. Of course, those derived from microorganisms, such as yeasts, filamentous fungi, asymptomatic fungi and basidiomycetes, or those derived from living things such as plants and animals are included.
In the present specification, a polypeptide having a functionally equivalent activity refers to the following.
In addition to the polymorphisms or mutations of the gene encoding the protein present in nature, deletion, addition, insertion, or insertion of amino acid residues in the amino acid sequence may be caused by modification reaction during in vivo and purification of the protein after production. Although variations such as substitution may occur, it is known that there is a physiological and biological activity that is substantially equivalent to a protein having no mutation regardless. Even though these structural differences are different, a large difference in its function is not recognized as a polypeptide having functionally equivalent activity.
The same is true when the above-described mutations are artificially introduced into the amino acid sequence of the protein. In this case, it is possible to produce a wider variety of variants, but as long as they exhibit substantially the same physiological activity as those having no mutation, It is interpreted as a polypeptide having functionally equivalent activity.
For example, methionine residues present at the N terminus of a protein expressed in E. coli are considered to be removed in most cases by the action of methionine aminopeptidase, but depending on the type of protein, both methionine residues are produced and those which do not have them. . However, the presence or absence of this methionine residue often does not affect the activity of the protein. In addition, it is known that a polypeptide in which a cysteine residue is substituted with serine in the amino acid sequence of human interleukin 2 (IL-2) maintains interleukin 2 activity (Science, Vol. 224, p. 1431 (1984)). .
Moreover, when producing proteins genetically, expression as a fusion protein is often done. For example, in order to increase the amount of expression of the target protein, an N-terminal peptide ring derived from another protein is added to the N-terminal of the target protein, or an appropriate peptide ring is added to the N-terminal or C-terminal of the target protein and expressed. By using a carrier having an affinity for the added peptide ring, a method for facilitating purification of the target protein is performed.
In addition, polypeptides that have at least one of deleted, added, inserted, or substituted one or more amino acid residues in the amino acid sequence of the target protein rarely have activities equivalent to those of the target protein. The gene coding for is also included in the present invention, whether it is derived from nature or artificially produced.
In general, it is known that codons (combinations of three bases) that designate amino acids on genes are present in one to six kinds of amino acids. Thus, a gene encoding an amino acid sequence may exist in a large number depending on the amino acid sequence. Genes are never reliably present in nature, and mutations in these nucleic acids are rare. In some cases, an amino acid sequence encoded by a mutation on a gene does not change (called a silent mutation), and in this case, another gene encoding the same amino acid sequence is generated. Therefore, even if a gene encoding a particular amino acid sequence is isolated, the possibility of generating many kinds of genes encoding the same amino acid sequence cannot be denied while the organism containing it is passaged.
In addition, artificially producing many kinds of genes encoding the same amino acid sequence is not difficult using various genetic engineering techniques.
For example, in gene engineering protein production, when the frequency of use is low among the hosts used by the codon used on the original gene which codes the target protein, the expression amount of a protein may be low. In such a case, high expression of the target protein is aimed at by artificially converting the codon to one frequently used in the host without changing the encoded amino acid sequence. As a matter of course, it is possible to artificially produce many kinds of genes encoding a specific amino acid sequence. Therefore, even these artificially produced different polynucleotides are included in the present invention as long as the amino acid sequence disclosed in the present invention is encoded.
Moreover, in the polypeptide which has functionally equivalent activity, the gene which codes it is often homology. Therefore, the gene which can hybridize with the gene used for this invention in strict conditions, and which encodes the polypeptide which has a fucose sulfate-containing polysaccharide degradation activity is also included in this invention.
Hereafter, Alteromonas sp. SN-1009 and Flavobacterium sp. The present invention will be specifically described by taking SA-0082 as an example.
This alteromonas sp. SN-1009 is modified by Alteromonas sp. It is displayed as SN-1009, and from February 13, 1996 (original deposit day), Ministry of Trade, Industry and Technology Institute biotechnology industrial technology research institute [Higashi 1Chome 1Chome 1 Ibaakiken Tsukuba-shi Japan 3 (postal code 305-) 8566), has been deposited internationally as FERM BP-5747. Flavobacterium sp. SA-0082 is Flavobacterium sp. It is designated as SA-0082 and has been deposited internationally as the FERM BP-5402 at the Institute of Biotechnology and Industrial Technology at the Ministry of Trade, Industry and Energy.
Alteromonas sp. SN-1009 or Flavobacterium sp. In order to obtain a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity produced by SA-0082, for example, a hybridization method, a PCR method, or a combination thereof can be used. These methods require probes capable of hybridizing to the gene or primers capable of amplifying the gene or a part thereof by PCR, but the amino acid sequence and gene of the polypeptide having the fucose sulfate-containing polysaccharide degradation activity produced by these strains. Since the structure is not known at all, synthetic oligonucleotides that can be used as probes or primers cannot be produced. Therefore, the partial amino acid sequence of the fucose sulfate-containing polysaccharide degrading enzyme produced by the microorganism is first determined, and the preparation of synthetic oligonucleotides usable as probes or primers is examined.
First, for example, Alteromonas sp. SN-1009 or Flavobacterium sp. SA-0082 is cultured, and then the resulting fucose sulfuric acid-containing polysaccharide degrading enzyme is isolated and purified from the culture, respectively.
Subsequently, information regarding the partial amino acid sequence of each of the purified fucose sulfate-containing polysaccharide degrading enzymes is obtained. In order to determine the partial amino acid sequence, for example, fucose sulfate-containing polysaccharide degrading enzyme can be directly sequenced by edman digestion according to a conventional method (for example, protein sequencer 476A (manufactured by Loaded Biosystems Co., Ltd.) can be used). By providing to the N-terminal amino acid sequence of the fucose sulfate-containing polysaccharide degrading enzyme is determined. Alternatively, a highly specific proteolytic enzyme, such as Achromobacter protease I, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) -trypsin, or the like is applied to purified fucose sulfate-containing polysaccharide degrading enzyme. The amino acid sequence of the purified peptide fragment can be obtained by separating and purifying the obtained peptide fragment using reversed phase HPLC, and most amino acid sequence information can be obtained.
The information on the partial amino acid sequence specific to the fucose sulfate-containing polysaccharide degrading enzyme thus obtained is selected, and based on the information, an oligonucleotide obtained by degenerating the nucleotide sequence is designed and synthesized. At this time, it is necessary to synthesize oligonucleotides having a low degree of accumulation and long oligonucleotides, in other words, oligonucleotides having high specificity to genes encoding polypeptides having a fucose sulfate-containing polysaccharide-degrading activity. It is an important factor in the cloning of genes encoding polypeptides having polysaccharide degradation activity.
Subsequently, it is necessary to examine the conditions of specific hybridization between the synthetic oligonucleotide and the gene encoding the polypeptide having the polysaccharide decomposition activity containing fucose sulfate by Southern hybridization.
For example, Alteromonas sp. SN-1009 or Flavobacterium sp. The genomic DNA of SA-0082 is completely digested with a suitable restriction enzyme, separated by agarose gel electrophoresis, and blotted to a nylon membrane or the like according to a conventional method. Hybridization first involves, for example, 6 × SSC (1 × SSC is 8.77 g sodium chloride and 4.41 g sodium citrate dissolved in 1 liter of water), 1% sodium lauryl sulfate (SDS), 100 μg / ml salmon sperm DNA, 5 After blocking the nylon membrane by incubating at 65 DEG C for several hours in a preliminary hybridization solution containing x Denharz (containing bovine serum albumin, polyvinylpyrrolidone, and picol at a concentration of 0.1% each), for example, ³² Synthetic oligonucleotides labeled with P are added and incubated overnight at 42 ° C. After washing this nylon membrane with 1 * SSC containing 0.1% SDS for 42 degreeC for 30 minute (s), autoradiography is performed and the DNA fragment which hybridizes with a synthetic oligonucleotide probe is detected. At this time, depending on the length of the synthetic oligonucleotide used and complementarity with the gene encoding the polypeptide having a polysaccharide-degrading activity containing fucose sulfate, it is recommended to select the optimum conditions by examining the incubation temperature, the salt concentration of the washing solution, and the like. effective.
As a method of obtaining a DNA fragment containing a gene encoding a polypeptide having a detected fucose sulfate-containing polysaccharide decomposition activity, a DNA fragment corresponding to the position of a directly detected band is extracted and purified from a gel, and then A library assembled to a host-vector vector to be used may be prepared, and colony hybridization or plaque hybridization may be performed under the same conditions as in the Southern hybridization method to screen and isolate a clone containing the DNA fragment of interest. Or directly atteromonas sp. SN-1009 or Flavobacterium sp. After digesting the genomic DNA of SA-0082 with a suitable restriction enzyme, a library assembled to a commonly used host-vector vector may be produced. Similarly, a hybridization method may be used to screen and isolate clones containing the DNA fragment of interest. good.
As the host-vector system to be used, known ones can be used, and for example, plasmid vectors such as pUC18 and pUC19 having E. coli, or phage vectors such as lambda phage, etc. may be mentioned, but are not particularly limited thereto.
The type and method of handling these host-vector systems may be generally used. For example, the Molecular Cloning, A Laboratory Manual, Second Edition, J. Sambrook et al. Cold Spring Harbor Laboratories, published in 1989).
If the vector containing the DNA fragment of interest can be selected, the base sequence of the DNA fragment of interest inserted into the vector may be determined by a conventional method such as dideoxy method [Procedings of the National Academy of Sciences of the USA. (Proc. Nat. Acad. Sci, USA), Vol. 74, pp. 5645 (1977). By comparing the determined nucleotide sequence with the N-terminal analysis, partial amino acid sequence, molecular weight and the like of the fucose sulfate-containing polysaccharide degrading enzyme, the gene structure in the obtained DNA fragment and the amino acid sequence of the polypeptide encoded by the gene can be known.
Further, the PCR method can be used as a method of obtaining a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide degrading activity using an oligonucleotide obtained based on the partial amino acid sequence of the fucose sulfate-containing polysaccharide degrading enzyme. Especially, the PCR method using cassette DNA is a method of obtaining the fragment of the target gene which can be used for the hybridization method from the short amino acid sequence information.
For example, Flavobacterium sp. Genomic DNA extracted from the cultured cells of SA-0082 according to a conventional method is digested with a suitable restriction enzyme, and then synthetic DNA (cassette DNA) having a known sequence is linked. Using this mixture as a template, a PCR reaction is carried out using the gene-specific oligonucleotide primer designed based on the information of the partial amino acid sequence and the oligonucleotide primer (cassette primer) complementary to the cassette DNA, to thereby prepare a DNA fragment of interest. It is possible to amplify. As cassette DNA or a cassette primer, the thing manufactured by Takarazo Corporation can be used, for example. The cassette DNA preferably contains a sequence corresponding to two kinds of cassette primers. First, a first PCR reaction is carried out using a primer farther from the restriction enzyme site to which it is linked, and then a part of the reaction solution is templated. The second PCR reaction is effective by using the inner primer. In addition, two kinds of the gene-specific oligonucleotide primers can be designed and synthesized side by side, using upstream primers for the first PCR reaction and downstream primers for the second PCR reaction. This increases the possibility of specific amplification of the target DNA fragment.
However, since the nucleotide sequence of the target gene is not clear, the restriction enzyme site used for linking the cassette DNA cannot be said to be in a suitable position for amplification reaction by PCR from the region encoding the partial amino acid sequence. Therefore, it is necessary to use cassette DNA of many kinds of restriction enzyme sites. In addition, PCR can be carried out under generally used conditions, such as those described in PCR Technology (PCR Technology, Erlich HA Edit, Stockton Press, 1989, for example). The annealing temperature, cycle number, magnesium concentration, heat resistant polymerase concentration, etc. are examined according to the complementarity with the gene encoding the polypeptide having the polysaccharide-degrading activity containing fucose sulfate. You need to choose a condition.
The PCR reaction solution is subjected to electrophoresis such as agarose gel to confirm amplified DNA fragments. These fragments can be extracted and purified according to a conventional method, inserted into a cloning vector such as pUC18, pUC19, or the like which is commonly used, and the base sequence can be analyzed, for example, by dideoxy. Alternatively, the recovered amplified DNA fragment may be analyzed directly by using a cassette primer used in a PCR reaction. As a result, if the partial amino acid sequence of the fucose sulfate-containing polysaccharide degrading enzyme determined in addition to the primer sequence can be obtained, a gene encoding the enzyme or a fragment of a gene showing homology to the enzyme can be obtained.
In this way, when the DNA fragment obtained by Southern hybridization or PCR is part of a gene encoding a target enzyme, the genomic library is screened by hybridization using the DNA fragment as a probe or the DNA By PCR using oligonucleotides prepared based on the nucleotide sequences of the fragments as primers, a DNA fragment containing the full length of the gene encoding the target enzyme can be obtained.
Further, the fucose sulfate-containing polysaccharide degrading enzyme gene or a part thereof obtained as described above was used as a probe to altereromonas sp. SN-1009 or Flavobacterium sp. When the genomic DNA of SA-0082 was analyzed by Southern hybridization, Alteromonas sp. Containing a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide degrading activity from the position of the detected band. SN-1009 or Flavobacterium sp. The genomic DNA restriction enzyme fragment size of SA-0082 can be obtained, and according to the number of bands detected, the gene encoding the polypeptide having the fucose sulfate-containing polysaccharide decomposition activity and the number of genes homologous thereto can be obtained. As can be expected, DNA fragments comprising these genes can be isolated by methods as described above.
Whether or not the DNA fragment thus obtained contains a gene encoding the enzyme of interest is prepared by using an expression vector containing the DNA fragment finally isolated, and transforming the host using the vector. It is confirmed by culturing the transformant and measuring the fucose sulfate-containing polysaccharide degradation activity of the expressed polypeptide.
In the present invention, Alteromonas sp. From SN-1009, a gene having an amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and having a base sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity was isolated. Examples of nucleotide sequences encoding polypeptides having an amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the Sequence Listing, respectively.
In addition, Flavobacterium sp. From SA-0082, a gene having an amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 of the Sequence Listing, respectively, and having a base sequence encoding a polypeptide having a fucose sulfuric acid containing polysaccharide decomposition activity was isolated. Examples of base sequences encoding polypeptides having an amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, in the sequence listing.
As a method for obtaining a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity or a functionally equivalent activity by hybridization using the base sequence of the gene of the present invention, for example, the following method can be applied.
First, cDNA produced by reverse transcriptase from chromosomal DNA or mRNA obtained from the target gene source is connected to a plasmid or phage vector according to a conventional method and introduced into a host to prepare a library. The library is cultured on a plate, the grown colonies or plaques are transferred to a membrane of nitrocellulose or nylon, and the DNA is fixed to the membrane by denaturation. A probe labeled with this membrane, for example, ³² P or the like (the probe to be used may be a nucleotide sequence encoding an amino acid sequence represented by any one of SEQ ID NOs 1 to 4 in the sequence listing or a part thereof, for example, SEQ ID NO: 5 in the sequence listing). Incubated in a solution containing the base sequence represented by any of -8 or a part thereof) to form a hybrid between the DNA on the membrane and the probe. For example, the membrane immobilized with DNA is hybridized with the probe for 20 hours at 65 ° C. in a solution containing 6 × SSC, 1% SDS, 100 μg / ml salmon sperm DNA, and 5 × Denharz. After hybridization, the non-specifically adsorbed probe is washed away, and then clones hybridized with the probe are identified by autoradiography or the like. This operation is repeated until the hybrid-formed clone becomes single. In the clone thus obtained, a gene encoding the target polypeptide is inserted.
The obtained gene is determined as follows, for example, to confirm that the obtained gene is a gene encoding a polypeptide having a desired fucose sulfate-containing polysaccharide decomposition activity or a functionally equivalent activity.
In the determination of the nucleotide sequence, if the transformant is E. coli transformed with the plasmid, it is cultured in a test tube or the like, and the plasmid is extracted according to a conventional method. This is digested with a restriction enzyme, the inserted fragment is taken out, subcloned in an M13 phage vector or the like, and the nucleotide sequence is determined by the dideoxy method. Even when the recombinant uses a phage vector, the base sequence can be basically determined by the same process. Regarding the basic experiment method from these cultures to sequencing, for example, the Molecular Cloning A Laboratory Manual 2nd Edition [J. J. Sambrook et al., Cold Spring Harbor Laboratories, 1989).
In order to confirm whether the obtained gene is a gene encoding a polypeptide having a desired fucose sulfate-containing polysaccharide degradation activity or a functionally equivalent activity, the determined nucleotide sequence or the amino acid sequence to be encoded is determined by SEQ ID NO: 5 in the sequence list of the present invention. It compares with the nucleotide sequence shown by any of -8, or the amino acid sequence shown by any of sequence numbers 1-4 of a sequence list.
If the obtained gene does not contain all of the regions encoding the polypeptide having the fucose sulfate-containing polysaccharide degrading activity or functionally equivalent activity, a synthetic DNA primer is prepared based on the obtained gene, and the region lacked by PCR. By screening a DNA library or cDNA library using a fragment of the obtained gene as a probe, and further screening a DNA library or a cDNA library, thereby reducing the total coding region of a polypeptide having a fucose sulfate-containing polysaccharide degradation activity or a functionally equivalent activity. The base sequence can be determined.
On the other hand, the primer for PCR reaction can be designed from the base sequence of the gene of this invention. By carrying out a PCR reaction using this primer, the gene fragment with high homology with the gene of this invention can be detected, or the whole gene can also be obtained.
Next, the obtained gene is expressed, the fucose sulfate-containing polysaccharide decomposition activity is measured, and the function of the obtained gene is confirmed.
The following method is convenient for producing a polypeptide having a fucose sulfate-containing polysaccharide degrading activity using a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide degrading activity of the present invention.
First, the host is transformed using a vector containing a gene encoding a polypeptide having the desired fucose sulfate-containing polysaccharide decomposition activity, and then the culture of the transformant is carried out under the conditions normally used. Polypeptides having sulfuric acid containing polysaccharide degradation activity can be produced. In this case, the polypeptide may be produced in the form of an inclusion body. As the host, cultured cells such as microorganisms, animal cells, and plant cells can be used.
It is convenient to confirm the expression by, for example, measuring the fucose sulfate-containing polysaccharide decomposition activity. Activity measurement can be measured, for example, using the cell extract of recombinant E. coli as an enzyme solution.
When expression of a polypeptide having a desired fucose sulfate-containing polysaccharide decomposition activity is recognized, for example, if the transformant is Escherichia coli, the composition of the medium, the pH of the medium, the culture temperature, the amount and time of use of the inducer, the incubation time, etc. By determining optimum conditions, polypeptides having a fucose sulfate-containing polysaccharide decomposition activity can be produced efficiently.
Conventional methods are used to purify a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity from the culture of the transformant. When the transformants accumulate polypeptides having a fucose sulfate-containing polysaccharide-degrading activity in cells as in the case of Escherichia coli, the transformants are collected by centrifugation after the end of culture, and then disrupted by sonication, followed by centrifugation. Obtain a cell free extract. From this, the target polypeptide can be purified using general protein purification methods such as salting out, ion exchange, gel filtration, various chromatography such as hydrophobicity and affinity. Depending on the host-vector system used, the expression product may be secreted out of the transformant. In this case, purification may be performed in the same manner from the culture supernatant.
Polypeptides having a fucose sulfate-containing polysaccharide-degrading activity produced by the transformant coexist with various enzymes in the cells when they are produced in the cells, but they are only a small amount compared to the amount of polypeptides having a fucose sulfate-containing polysaccharide-degrading activity. , Its purification is very easy. Moreover, when the cell used as a host is selected, the enzyme derived from the host which acts on a fucose sulfate-containing polysaccharide is greatly reduced. In addition, when a polypeptide having a fucose sulfate-containing polysaccharide degrading activity is secreted out of the cells, media components and the like coexist, but these can be easily separated from a polypeptide having a fucose sulfate-containing polysaccharide degrading activity.
For example, when the host is Escherichia coli, the expression product may be formed as an insoluble inclusion body. In this case, after incubation, the cells are collected by centrifugation, crushed by sonication, and then centrifuged to collect insoluble fractions containing inclusion bodies. After washing the inclusion body, it is solubilized with a commonly used protein solubilizer such as urea, guanidine hydrochloride, and the like, and purified by various chromatography such as ion exchange, gel filtration, hydrophobicity, affinity, if necessary, and then dialyzed. By carrying out the reholding operation using a method or a dilution method, a polypeptide having a fucose sulfate-containing polysaccharide degrading activity for maintaining the activity can be obtained. If necessary, the sample can be further purified by various chromatography to obtain a polypeptide having high purity fucose sulfate-containing polysaccharide decomposition activity.
The same production method and purification method may also be used when producing a polypeptide having a functionally equivalent activity with a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity.
As described above, according to the present invention, it is possible to provide a primary structure and a genetic structure of a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity. In addition, genetic engineering of a polypeptide having a fucose sulfuric acid-containing polysaccharide decomposition activity or a polypeptide functionally equivalent to the activity thereof becomes possible.
By using the genetic engineering method of the present invention, it is possible to inexpensively obtain a polypeptide having a high purity fucose sulfate-containing polysaccharide decomposition activity or a polypeptide having a functionally equivalent activity.
The method for producing fucose sulfate-containing polysaccharide degrading enzymes by culturing the bacteria of the genus alteromonas or flabobacterium which produces the polysaccharide degrading enzyme containing fucose sulfate, at the same time protease or other polysaccharide degrading enzymes are produced Therefore, in order to isolate the desired fucose sulfate-containing polysaccharide degrading enzyme, it is necessary to separate and purify these enzymes which are very cumbersome, and to induce the production of the enzyme, an expensive fucose sulfate-containing polysaccharide is added to the medium at the time of culture. Although it was necessary to induce a fucose sulfuric acid-containing polysaccharide degrading enzyme, the present invention made it possible to provide a polypeptide having a high purity fucose sulfuric acid-containing polysaccharide decomposition activity at low cost.
The present inventors conducted intensive studies on the genes of microorganisms producing fucose sulfate-containing polysaccharide degrading enzymes in order to identify the amino acid sequence and the nucleotide sequence of a polypeptide having a fucose sulfate-containing polysaccharide degrading activity. Revealed that each of two genes encoding a polypeptide having a fucose sulfate-containing polysaccharide degrading activity derived from the Flavobacterium bacterium was present, and the entire nucleotide sequence thereof was determined to determine the amino acid sequence of the polypeptide for the first time. The present invention was also successful in developing a method for industrially advantageously producing a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity using the gene, thereby completing the present invention.
Briefly describing the present invention, a first invention of the present invention relates to an isolated gene having a DNA sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity or a polypeptide having a functionally equivalent activity.
The second invention of the present invention relates to a recombinant DNA comprising the gene of the first invention of the present invention.
The third invention of the present invention relates to an expression vector in which the recombinant DNA of the second invention of the present invention is inserted and which contains a microorganism, an animal cell or a plant cell as a host cell.
The fourth invention of the present invention relates to a transformant transformed with the expression vector which is the third invention of the present invention.
According to a fifth aspect of the present invention, culturing the transformant of the fourth aspect of the present invention, extracting a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity or a polypeptide having a functionally equivalent activity from the culture The present invention relates to a polypeptide having a fucose sulfate-containing polysaccharide degrading activity or a polypeptide having a functionally equivalent activity.
The sixth invention of the present invention relates to a polypeptide having an amino acid sequence represented by any of SEQ ID NOs: 1 to 4 in the Sequence Listing, and also having a polypeptide having a fucosulfuric acid-containing polysaccharide decomposition activity or a functionally equivalent activity thereof. will be.
Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to a following example.
Reference Example 1
(1) 2 kg of dried kagome kelp (Kjellmaniella crassifolia) was pulverized with a free grinder M-2 type (manufactured by Naraki Kasei Co., Ltd.), and treated after processing at 80 ° C. for 2 hours in 4.5 times of 80% ethanol, followed by filtration. With respect to the residue, the process of 80% ethanol extraction and filtration was repeated three more times to obtain 1870 g of ethanol washing residue. 36 liters of water was added to the residue, and the mixture was treated at 100 ° C for 2 hours to obtain an extract by filtration. The salt concentration of the extract was made equal to 400 mM sodium chloride solution, then 5% cetylpyridinium chloride was added until no further precipitation occurred and centrifuged. The precipitate was washed repeatedly with 80% ethanol to completely remove cetylpyridinium chloride, dissolved in 3 liters of 2M sodium chloride, insolubles were removed by centrifugation, and equilibrated with 2M sodium chloride, 100 ml of DEAE- Celerine A-800 was suspended, stirred and filtered to remove the resin. The filtrate was hung on a column of 100 mL of DEAE-Selepine A-800 equilibrated with 2 M sodium chloride, and the excess fraction was desalted and removed by ultrafiltration (100,000 molecular weight exclusion of the membrane). The precipitate formed was removed by centrifugation. The supernatant was freeze-dried to obtain 82.2 g of a purified Kagome Kelp Fucose sulfuric acid-containing polysaccharide mixture.
(2) After dissolving 7 g of the fucose sulfate-containing polysaccharide mixture derived from kagome kelp in 20 mM sodium acetate (pH 6.) containing 700 ml of 0.2 M calcium chloride, 20 mM acetic acid containing 0.2 M calcium chloride in advance The column was washed with 4000 ml of DEAE-Sepharose FF equilibrated with sodium (pH 6.0) and washed thoroughly with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride. Eluted with a sodium chloride gradient. Fractions eluting at a sodium chloride concentration of 0.9 to 1.5 M were collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane with an exclusion molecular weight of 100,000, and lyophilized to obtain 4.7 g of a freeze-dried sample of polysaccharide-F containing fucose sulfate.
In addition, the fractions eluted at a sodium chloride concentration of 0.05 to 0.8 M were collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane with an exclusion molecular weight of 100,000, and lyophilized to obtain 2.1 g of a freeze-dried sample of polysaccharide-U containing fucose sulfate. .
(3) alteromonas sp. Sterilized SN-1009 (FERM BP-5747) by dividing 600 ml of a medium consisting of pH8.2 of artificial seawater (manufactured by Jamarin Reboratori) containing glucose 0.25%, peptone 1.0%, and yeast extract 0.05% (120 C, 20 minutes) inoculated into a 2-liter Erlenmeyer flask and incubated at 25 ° C for 25 hours to obtain a seed culture solution. 18 liters of an artificial seawater pH8.0 containing 200 g of peptone, 4 g of yeast extract and 4 ml of an antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) was placed in a 30 liter fermenter and sterilized at 120 ° C for 20 minutes. After cooling, fucosulfuric acid-containing polysaccharide-F derived from Kagome kelp prepared by using the method of 20 g of Reference Example 1- (1) dissolved in 2 liters of artificial seawater sterilized separately at 120 ° C. for 15 minutes was added. Further, 600 ml of the seed culture solution was inoculated and incubated at 24 ° C. for 20 hours under conditions of aeration rate of 10 liters per minute and stirring speed of 250 revolutions per minute. After the completion of the culture, the culture solution was centrifuged to obtain cells and culture supernatant.
The activity of the endo type fucose sulfuric acid-containing polysaccharide degrading enzyme in the culture supernatant was measured by the method described in Reference Example 2 using fucose sulfuric acid-containing polysaccharide-F as a substrate, and the result was 10 mU / ml culture medium.
The obtained culture supernatant was concentrated by ultrafiltration with a fractional molecular weight of 10,000, and then the precipitate formed was removed by centrifugation, followed by 85% saturated ammonium sulfate salt, and the precipitate formed was collected by centrifugation. It was sufficiently dialyzed against 20 mM Tris-hydrochloric acid buffer solution (pH8.2) containing artificial seawater (jamarin S) to obtain 400 ml of coenzyme.
The obtained coenzyme solution was previously equilibrated with 20 mM tris-hydrochloric acid buffer (pH8.2) containing 5 mM sodium azide and artificial seawater (jamarin S) at a concentration of 10/10 (DEAE-Selepine A-800 ( After adsorbing to a column of Seigagaku Kogyo Co., Ltd., the adsorbate was sufficiently washed with copper buffer, eluted with a solution containing 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM sodium chloride in the buffer to collect the active fractions. .
The enzyme activity of the obtained active fraction was measured by the method described in Reference Example 2, which was 20400 mU (20.4 U).
The resulting active fractions were concentrated by ultrafiltration with a fractional molecular weight of 10,000, followed by ultrafiltration with addition of 20 mM tris-hydrochloric acid buffer (pH8.2) containing 10 mM calcium chloride and 50 mM sodium chloride to completely replace the buffer.
The obtained enzyme solution was adsorbed onto a column of DEAE-Sepharose FF previously equilibrated with copper buffer, the adsorbate was sufficiently washed with copper buffer, further washed with copper buffer having a sodium chloride concentration of 150 mM, and thereafter, 150 mM to 400 mM. A sodium chloride gradient was eluted with the same buffer containing sodium chloride.
The obtained active fractions were collected and concentrated by ultrafiltration, followed by gel filtration with Sephacryl S-200. As the eluate, 10 mM Tris-HCl buffer (pH 8.0) containing 5 mM sodium azide and 10/10 concentration of jamarin S was used. Moreover, when molecular weight was calculated | required by the said chromatography, it was about 100,000.
The obtained active fractions were collected, sufficiently dialyzed against 20 mM Tris-HCl buffer (pH8.2) containing 10 mM calcium chloride, 10 mM potassium chloride and 4.2 M sodium chloride, and the phenyl pre-equilibrated with a copper buffer having a sodium chloride concentration of 4M. Hang on a column of Sepharose CL-4B and elute with copper buffer containing 4M, 3M, 2M, 1M, 0.5M and 0.15M sodium chloride.
The obtained active fractions were collected and concentrated by ultrafiltration, followed by ultrafiltration with addition of 20 mM tris-hydrochloric acid buffer (pH8.2) containing 10 mM calcium chloride, 10 mM potassium chloride and 150 mM sodium chloride, thereby completely buffering the buffer solution. Substituted. The enzyme solution was suspended in DEAE-Selepine A-800, previously equilibrated with the same buffer solution, washed with the same buffer solution, and gradient elution of 150 mM to 350 mM sodium chloride was performed.
The obtained active fractions were collected, sufficiently dialyzed with a copper buffer containing 50 mM sodium chloride, and then adsorbed to DEAE-Selepine A-800, which had previously been equilibrated with a copper buffer containing 50 mM sodium chloride, and washed with the same buffer. A sodium chloride gradient elution of 50 mM to 150 mM was performed. The obtained active fractions were combined to obtain purified enzyme.
The molecular weight of the purified enzyme was determined to be about 90,000 by SDS (sodium dodecyl sulfate) -polyacrylamide electrophoresis.
Reference Example 2
Using the fucose sulfuric acid containing polysaccharide-F obtained by the process of Reference Example 1- (2), the endo type fucose sulfuric acid containing polysaccharide decomposition activity was measured as follows.
12 μl of a 2.5% fucose sulfate-containing polysaccharide-F solution, 6 μl of 1M calcium chloride solution and 9 μl of 4M sodium chloride solution, 60 μl of 50 mM acetic acid, imidazole, and tris-hydrochloric acid ( pH7.5), 21 μl of water, and 12 μl of the test solution for measuring the degradation activity were mixed and reacted at 30 ° C. for 3 hours. Was analyzed by HPLC to determine the degree of low molecular weight.
As a control, a reaction prepared under the same conditions using the buffer used for the test solution instead of the test solution for measuring the degradation activity and a reaction using water instead of the polysaccharide-F solution containing fucose sulfate were prepared. , Respectively, by HPLC in the same manner.
One unit of enzyme is defined as the amount of enzyme that cleaves 1 μmol of the fucosyl bond of fucose sulfate-containing polysaccharide-F in 1 minute in the reaction system. Quantification of cleaved prokosyl bonds was obtained by the following formula.
{(12 × 2.5) / (100 × MF)} × {(MF / M) -1} × {1 / (180 × 0.01)} × 1000 = U / ml
(12 x 2.5) / 100: Fucose sulfuric acid-containing polysaccharide-F (mg) added in the reaction system
MF: Average molecular weight of substrate fucose sulfate-containing polysaccharide-F
M: average molecular weight of the reaction product
(MF / M) -1: A number of molecules in which one molecule of fucose sulfate-containing polysaccharide-F was cleaved by an enzyme
180: reaction time (minutes)
0.01: enzyme liquid amount (ml)
In addition, the conditions of HPLC are as follows.
Equipment: L-6200 type (manufactured by Hitachi Seisakusho)
Column: OHpak SB-806 (8 mm x 300 mm) (Showa Denko Corporation)
Eluent: 25 mM imidazole buffer (pH 8) containing 5 mM sodium azide, 25 mM calcium chloride and 50 mM sodium chloride
Detection: Differential refractive index detector (Shodex RI-71, Showa Denko Corporation)
Flow rate: 1 ml / min
Column temperature: 25 ℃
In order to determine the average molecular weight of the reaction product, a commercially known molecular weight pullane (STANDARD P-82, manufactured by Showa Denko) was analyzed under the same conditions as the HPLC analysis to maintain the molecular weight of the pulley and OHpak SB-806. The relationship with time is shown by the curve and used as the standard curve for the molecular weight measurement of the said enzyme reaction product.
Reference Example 3
The fucose sulfuric acid containing polysaccharide decomposition activity was measured using the fucose sulfuric acid containing polysaccharide-U obtained by the process of Reference Example 1- (2) as follows.
50 [mu] l of a 2.5% fucose sulfate-containing polysaccharide-U solution, 10 [mu] l of the test solution for measuring the degradation activity, and 83 [mu] m phosphate buffer (pH7.5) containing 60 [mu] l of 667 mM sodium chloride were mixed. After making it react for time, 105 microliters of reaction liquids and 2 ml of water are mixed and stirred, and the absorbance (AT) in the 230 nm is measured. As a control, instead of the test solution for measuring the degradation activity, the reaction was carried out under the same conditions using only the buffer used for the test solution, and the reaction was carried out using only water instead of the polysaccharide-U solution containing fucose sulfate. , The absorbance is measured in the same manner (AB1 and AB2), respectively.
One unit of enzyme is defined as the amount of enzyme that cleaves glycosidic bonds between 1 μmo1 of mannose and uronic acid in one minute in the reaction system. Quantification of cleaved bonds is carried out by calculating the millimolar molecular extinction coefficient of unsaturated uronic acid generated during the detachment reaction as 5.5. In addition, the activity of the enzyme was calculated according to the following formula.
(AT-AB1-AB2) × 2.105 × 120 / 5.5 × 105 × 0.01 × 180 = U / mL
In the formula,
2.105 is the liquid amount (ml) of the sample for measuring absorbance,
120 is the amount of the enzyme reaction solution (μl),
5.5 is the millimolar molecular extinction coefficient (/ mM) at 230 nm of unsaturated uronic acid,
105 is the liquid amount (µl) of the reaction solution used for dilution,
0.01 is the amount of enzyme solution (ml),
180 is the reaction time in minutes.
Reference Example 4
(1) Molecular weight distribution of fucosulfuric acid-containing polysaccharide-F and fucose sulfuric acid-containing polysaccharide-U was determined by gel filtration using Sephacryl S-500.
(2) Precipitation formation properties in the presence of excess cetylpyridinium chloride in the concentration of sodium chloride of fucose sulfate-containing polysaccharide-U and fucose sulfate-containing polysaccharide-F are shown in FIG. 2.
2 represents the precipitation formation rate (%), and the horizontal axis represents sodium chloride concentration (M). In the figure, the solid line and the empty circle represent the precipitation formation rate at each sodium chloride concentration of the fucose sulfate-containing polysaccharide-U, and in the figure, the dotted line and the empty triangle represent the sodium chloride concentration (M) of the fucose sulfate-containing polysaccharide-F. Shows the precipitation formation rate.
The measurement of the precipitation formation rate was performed at the solution temperature of 37 ° C. as follows.
Fucose sulfuric acid-containing polysaccharide-U and fucose sulfuric acid-containing polysaccharide-F were dissolved in water and 4M sodium chloride at a concentration of 2%, respectively, and mixed in various ratios to dissolve fucose sulfate-containing polysaccharide in various concentrations of sodium chloride. 125 µl of -U and fucose sulfate-containing polysaccharide-F solutions were prepared, respectively. Next, cetylpyridinium chloride was dissolved in water and 4M sodium chloride at a concentration of 2.5%, and mixed to prepare a 1.25% cetylpyridinium chloride solution dissolved in various concentrations of sodium chloride.
The 2% fucose sulfuric acid-containing polysaccharide-U and the fucose sulfuric acid-containing polysaccharide-F dissolved in water were required to be 3.2 times in capacity to completely precipitate with 1.25% cetylpyridinium chloride. Thus, for each 125 μl of 2% fucose sulfate-containing polysaccharide-U and fucose sulfate-containing polysaccharide-F dissolved in sodium chloride at each concentration, 400 μl of the cetylpyridinium chloride solution dissolved in sodium chloride at each concentration was added, followed by stirring sufficiently. The mixture was allowed to stand for 30 minutes, and then centrifuged to determine the sugar content in the supernatant by the phenol-sulfuric acid method (Analyical Chemistry, Vol. 28, pp. 350 (1956)). The precipitate formation rate of the fucose sulfate-containing polysaccharide was calculated.
(3) The component of fucose sulfuric acid containing polysaccharide-F was analyzed by the method shown below.
First, the amount of fucose was quantified according to the description of the Journal of Biological Chemistry, Vol. 175, p. 595 (1948).
A dried sample of fucose sulfuric acid-containing polysaccharide-F was dissolved in 1 N hydrochloric acid at a concentration of 0.5%, treated at 110 ° C. for 2 hours, and hydrolyzed to constituent monosaccharides. Pyridyl- ( ² ) -aminoation (PAylation) of the reducing end of the monosaccharide obtained by hydrolysis using GlycoTAG ^{™ and} GlycoTAG ^™ Reagent Kit (both manufactured by Takarazo Corporation). The proportion of constituent sugars was examined by HPLC.
Subsequently, the amount of uronic acid was quantified according to the description of Analytical Biochemistry, Vol. 4, page 330 (1962).
In addition, sulfuric acid content was quantified according to the description in Biochemical Journal, Vol. 84, 106 (1962).
The constituent sugars of this fucose sulfate-containing polysaccharide-F are fucose and galactose, and their molar ratio was about 10: 1. Uronic acid and other neutral sugars were substantially free. In addition, the molar ratio of fucose and sulfuric acid group was about 1: 2.
The component of the fucose sulfate-containing polysaccharide-U was measured according to the above method. As a result, the constituent sugars of the fucose sulfate-containing polysaccharide-U were fucose, mannose, galactose, glucose, rhamnose, xylose and uronic acid. No other neutral sugars were found. In addition, the main component of the fucose: mannose: galactose: uronic acid: sulfuric acid group was about 10: 7: 4: 5: 20 in molar ratio.
(4) The specific optical density of the freeze-dried product of this fucose sulfate-containing polysaccharide-F was -135 degrees when measured by a high-speed, highly sensitive photometer SEPA-300 (manufactured by Horiba Seisakusho).
In addition, the specific light intensity of the fucose sulfate-containing polysaccharide-U was -53.6 degrees.
(5) Endo-type fucoidan described in Reference Example 5 below, 16 ml of 1% fucose sulfate-containing polysaccharide-F solution, 12 ml of 50 mM phosphate buffer (pH8.0), 4 ml of 4M sodium chloride, and 32 mU / ml 8 ml of the degrading enzyme solution was mixed and reacted at 25 ° C. for 48 hours. The formation of decomposition products by the reaction was not confirmed.
Under the above conditions, the fucosulfuric acid-containing polysaccharide-U and the fucoidan degrading enzyme described in Reference Example 5 were reacted at 25 ° C for 48 hours. It was confirmed that the absorbance at 230 nm increased with the progress of the reaction, and it was found that the fucose sulfate-containing polysaccharide-U was decomposed by the present enzyme.
Reference Example 5
The fucoidan degrading enzyme described in Reference Example 4 is prepared by the following method. As the strain used for the production of the fucoidan decomposing enzyme, any strain may be used as long as it has the enzyme producing ability. Specific examples thereof include Flavobacterium sp. SA-0082 strain (FERM BP-5402) is mentioned.
This strain was newly obtained from the seawater of Aomoriken, and this strain was Flavobacterium sp. It is designated as SA-0082 and has been deposited internationally as an accession number of FERM BP-5402 to the Institute of Biotechnology and Industrial Technology, Ministry of Commerce, Industry and Technology since March 29, 1995 (original deposit date).
The nutrient source to be added to the medium of the strain may be used by the strain to be used to produce fucoidan degrading enzymes. Examples of carbon sources include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, maltose and lactose. Starch and the like can be used, and as the nitrogen source, yeast extract, peptone, crab acid, cornslicker, gravy, skim soybean, ammonium sulfate, ammonium chloride and the like are suitable. In addition, inorganic and metal salts such as sodium salt, phosphate, potassium salt, magnesium salt and zinc salt may be added.
In culturing the production bacteria of this fucoidan decomposing enzyme, the production amount varies depending on the culture conditions, but in general, the culture temperature is 15 ° C to 30 ° C, the pH of the medium is 5 to 9, and the aeration and agitation for 5 to 72 hours are performed. As seen, the production of fucoidan degrading enzyme reaches the highest. It is a matter of course that the culture conditions are set so that the production amount of this fucoidan decomposing enzyme is maximized depending on the strain used, the medium composition, and the like.
The fucoidan degrading enzyme is present in the cells or in the culture supernatant.
Flavobacterium sp. Cell-free extracts can be obtained by culturing SA-0082 strains in a suitable medium, collecting the cells, and crushing the cells by commonly used cell disruption means such as ultrasonication.
Subsequently, a purified enzyme sample can be obtained from this extract by the purification means usually used. For example, the purified present fucoidan degrading enzyme can be obtained by purification by salting out, ion exchange column chromatography, hydrophobic bond column chromatography, gel filtration or the like.
In addition, since the present enzyme (extracellular enzyme) is present in a large amount in the culture supernatant from which the cells are removed from the culture medium, it can be purified by a purification means such as an intracellular enzyme.
The purification example of a fucoidan decomposing enzyme is shown.
Flavobacterium sp. SA-0082 (FERM BP-5402) was sterilized by dividing 600 ml of a medium consisting of pH7.5 of artificial seawater (made by Jamarin Retortori) containing glucose 0.25%, peptone 1.0%, and yeast extract 0.05%. (C, 20 minutes) 2 liters of Erlenmeyer flasks were inoculated and incubated at 24 캜 for 24 hours to obtain a seed culture solution. 20 liters of a medium consisting of artificial seawater (made by Jamarin Laboratories) pH 7.5 containing 0.25% glucose, 1.0% peptone, 0.05% yeast extract, and 0.01% antifoaming agent (manufactured by Shin-Etsu Chemical Co., Ltd., KM70) The liters were put into a small fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the seed culture solution was inoculated and incubated at 24 ° C for 24 hours under conditions of aeration rate of 10 liters per minute and stirring speed of 125 revolutions per minute. After incubation, the culture medium was centrifuged to obtain cells.
The cells were suspended in 20 mM acetic acid-phosphate buffer (pH7.5) containing 200 mM sodium chloride, crushed by ultrasonication, and centrifuged to obtain cell extracts. When the activity of the fucoidan degrading enzyme in the cell extract was measured by the method described in Reference Example 3, 5 mU of activity was detected in 1 ml of medium. In addition, the activity measurement is described later.
To the extract, ammonium sulfate was added to the final concentration at 90% saturation, stirred and dissolved, followed by centrifugation. The precipitate was suspended in the same buffer as the cell extract, and 20 mM acetic acid-phosphate buffer containing 50 mM sodium chloride ( pH 7.5) was sufficiently dialyzed. The precipitate formed by dialysis was removed by centrifugation, and then adsorbed onto a column of DEAE-Sepharose FF previously equilibrated with 20 mM acetic acid-phosphate buffer (pH7.5) containing 50 mM sodium chloride, and the adsorbate was buffered with copper buffer. After washing sufficiently with, the mixture was eluted by a linear concentration gradient of 50 mM to 600 mM sodium chloride, and the active fractions were collected. Subsequently, sodium chloride was added to this active fraction to a final concentration of 4M, and adsorbed onto a column of phenylsepharose CL-4B (manufactured by Pharmacia Co., Ltd.) previously equilibrated with 20 mM phosphate buffer (pH8.0) containing 4M sodium chloride. The adsorbate was washed sufficiently with the same buffer, and then eluted by a linear concentration gradient of 4M to 1M sodium chloride to collect the active fractions. Subsequently, the active fractions were concentrated by an ultrafilter, and the active fractions were collected by gel filtration with Sephacryl S-300 (manufactured by Pharmacia) previously equilibrated with 10 mM phosphate buffer containing 50 mM sodium chloride. The molecular weight of this enzyme was found to be about 460,000 based on the retention time of Sephacryl S-300. This active fraction was then dialyzed with 10 mM phosphate buffer (pH7) containing 25 mM sodium chloride. The enzyme solution was adsorbed onto a column of Mono Q HR5 / 5 (Pharmacia Co., Ltd.), previously equilibrated with 10 mM phosphate buffer (pH7) containing 250 mM sodium chloride, and the adsorbate was sufficiently washed with the same buffer. , Eluted with a linear concentration gradient of 450 mM sodium chloride at 250 mM, and the active fractions were collected to obtain a purified enzyme. The above purification process is shown in Table 1. The protein is quantified by measuring the absorbance at 280 nm of the enzyme solution. At that time, the absorbance of the 1 mg / mL protein solution is calculated to be 1.0.
fairTotal protein amount (mg)Total activity (mU)Inactive (mU / mg)yield(%) Cell Extract61,900101,0001.63100 Ammonium Sulfate Salting33,80088,6002.6287.7 DEAE-Sepharose FF2,19040,40018.440.0 Phenyl Sepharose48.229,00060128.7 CL-4B7.2419,6002,71019.4 Sephacryl S-3000.82415,00018,20014.9
Moreover, fucoidan decomposing enzyme can also be refine | purified by the following method.
Flavobacterium sp. SA-0082 (FERM BP-5402) was sterilized by dividing 600 ml of a medium consisting of pH 7.5 of artificial seawater (made by Jamarin Retortory) containing 0.1% glucose, 1.0% peptone, and 0.05% yeast extract (120). 20 minutes) and inoculated into a 2-liter Erlenmeyer flask and incubated at 24 ° C for 20 hours to obtain a seed culture solution. Medium consisting of artificial seawater (made by Jamarin Retortori) pH7.5 containing 0.3% of fucoidan derived from Kagome Kelp, 0.5% of peptone, 0.01% of yeast extract, and 0.01% of antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) 20 liters were placed in a 30 liter small fermenter and sterilized for 20 minutes at 120 ° C. After cooling, 600 ml of the seed culture solution was inoculated and incubated at 24 ° C. for 20 hours under conditions of aeration rate of 10 liters per minute and stirring speed of 125 revolutions per minute. After the completion of the culture, the culture solution was centrifuged to obtain the cells and the culture supernatant. The cells obtained in this culture were suspended in 20 mM acetic acid-phosphate buffer (pH7.5) containing 200 mM saline, crushed by ultrasonication, and centrifuged to obtain cell extracts. When the activity of the fucoidan decomposing enzyme in this cell extract was measured, the activity of 20 mU was detected in 1 ml of medium.
On the other hand, the culture supernatant was concentrated by ultrafiltration (10,000 molecular weight exclusion of the filtration membrane) (manufactured by Amicon), and when the fucoidan degrading enzyme was measured, 6mU of activity per ml of the culture was detected.
To the concentrated solution of the culture supernatant, ammonium sulfate was added so that the final concentration was saturated 90%, stirred, dissolved, and centrifuged. The precipitate was suspended in the same buffer as the cell extract, and 20 mM acetic acid-phosphate containing 50 mM salt was used. Dialysis was sufficiently with buffer pH 7.5. The precipitate formed by dialysis was removed by centrifugation, and then adsorbed onto a column of DEAE-Sepharose FF previously equilibrated with 20 mM acetic acid-phosphate buffer (pH 7.5) containing 50 mM salt, and the adsorbate was absorbed into the same buffer. After washing sufficiently with, the mixture was eluted by a salt gradient of 50 mM to 600 mM, and the active fractions were collected. Then, salt was added to the active fraction to a final concentration of 4M, and the adsorbate was adsorbed onto a column of phenylsepharose CL-4B previously equilibrated with 20 mM phosphate buffer (pH8.0) containing 4M salt. After sufficient washing with the same buffer, the mixture was eluted with a salt gradient of 4M to 1M to collect the active fractions. Subsequently, the active fractions were concentrated by an ultrafilter (manufactured by Amicon), and the active fractions were collected by gel filtration with Sephacryl S-200 previously equilibrated with 10 mM phosphate buffer containing 50 mM saline. Salt was then added to this active fraction to a final concentration of 3.5 M, adsorbed onto a column of phenylsepharose HP previously equilibrated with 10 mM phosphate buffer (pH 8) containing 3.5 M of salt, and the adsorbate with copper buffer. After washing, the mixture was eluted by a salt gradient of 3.5M to 1.5M, and the active fractions were collected to obtain a purified enzyme. The molecular weight of this enzyme was found to be about 70,000 based on the holding time of Sephacryl S-200.
Example 1
(1) Alteromonas sp., A production strain of an endo type fucose sulfate-containing polysaccharide degrading enzyme. Sterilized SN-1009 (FERM BP-5747) by dividing and disinfecting 500 ml of a medium consisting of pH 8.0 of artificial seawater (manufactured by Jamarin Reboratori) containing glucose 0.25%, peptone 1.0%, yeast extract 0.05% 20 minutes) and inoculated into a 2 liter Erlenmeyer flask and incubated at 25 ℃ for 23 hours. After completion of the culture, the culture solution was centrifuged to collect the cells, and half of the cells were suspended in 10 ml of extraction buffer [50 mM Tris hydrochloric acid buffer (pH8.0), 100 mM ethylenediaminetetraacetic acid (EDTA), and then 1 ml. 20 mg / mL lysozyme solution dissolved in the extraction buffer was added and incubated for 30 minutes on an ice bath. Then 10 ml proteinase K solution [1 mg / ml proteinase K, 50 mM Tris hydrochloric acid buffer (pH8.0), 100 mM EDTA, 1% SDS] was added, followed by constant temperature at 50 ° C. for 2 hours. Treated. After returning to room temperature, TE buffer [10 mM Tris hydrochloric acid buffer (pH8.0), 1 mM EDTA] saturated phenol of equal capacity was added thereto, the mixture was stirred gently for 1 hour, centrifuged at 10000 rpm for 20 minutes, and the upper layer was recovered thereafter. Manipulation is called phenol extraction).
Equivalent volume of TE buffer saturated phenol / chloroform 1: 1 was added to the upper layer, followed by gentle stirring, centrifugation at 10000 rpm for 20 minutes, and then the upper layer was recovered (hereinafter this operation was called phenol / chloroform extraction). After extracting phenol / chloroform again, sodium chloride was added to the aqueous layer to 0.1 M, and twice the amount of ethanol was added to precipitate DNA, which was wound up with a glass rod, washed with 80% ethanol, and air dried. The genomic DNA was dissolved in a TE buffer in which 20 ml of 20 µg / ml ribonuclease A was dissolved and incubated at 37 ° C for 5 hours to degrade RNA. After phenol extraction and phenol / chloroform extraction, ethanol was added in the same manner as above, DNA was recovered and suspended in 5 ml of TE buffer. By the above operation, about 20 mg of genomic DNA was obtained.
(2) 100 μg of genomic DNA prepared in Example 1- (1) was partially digested with 10 units of restriction enzyme Sau3AI at 37 ° C. for 1 minute 40 seconds, and then phenol / chloroform extracted to recover the supernatant. DNA was precipitated by adding 1 / 10-fold 3M aqueous sodium acetate solution (pH5.0) and 2.5-fold ethanol to the upper layer, and the precipitate was recovered by centrifugation, washed with 80% ethanol and dried in the wind ( This operation is then called ethanol precipitation). The obtained partial digested product was fractionated by size by 1.25 to 5 M sodium chloride density gradient ultracentrifugation, and DNA was recovered by ethanol precipitation from a fraction containing a size of 10 to 20 kbp. 0.18 µg of the obtained genomic DNA partial digest and 0.6 µg of lambda Blue STAR BamHI arm manufactured by Novagene were mixed and linked using a DNA ligation kit manufactured by Takarazo Corporation. The lambda phage was packaged using a GigaPack II Gold kit manufactured by Stratagene, and then Alteromonas sp. A genomic DNA library of SN-1009 was constructed.
(3) Alteromonas sp. Obtained in Reference Example 1- (3). 200 pmol of purified endo type fucose sulfate-containing polysaccharide degrading enzyme protein of SN-1009 was charged to a desalting column (first desorption column PC3.2 / 10, manufactured by Pharmacia) equilibrated with 20 mM ammonium bicarbonate. Elution with buffer was used to replace the buffer. The eluate was collected in a glass vial, concentrated to dryness, and then the glass vial was placed in a larger sized glass test tube loaded with 10 μl pyridine, 2 μl 4-vinylpyridine, 2 μl tri-N-butylphosphine and 10 μl water. The glass test tube was sealed, and it reacted at 95 degreeC for 10 minutes, and pyridyl ethylation was carried out. After completion of the reaction, the glass vial was taken out and azeotropically with water several times to remove volatile components.
The obtained pyridylethylated fucose-containing polysaccharide degrading enzyme protein was purified by 10 mM Tris hydrochloric acid buffer (pH9.0) containing 40 µl of 8 M urea, 90 µl of 10 mM Tris hydrochloric acid buffer (pH9.0) and 0.5 pmo1 of acromo. Achromobacter protease I (manufactured by Takarazo Corp.) was added thereto, and the peptide fragment was digested overnight at 30 ° C., and the peptide fragment was purified by an HPLC system (Smart Systems, Pharmacia). The column was run at a flow rate of 100 μl / min using μRPC C2 / C18 SC2 · 1/10 (manufactured by Pharmacia). Elution was carried out using acetonitrile (eluent B) containing 0.12% trifluoroacetic acid aqueous solution (eluent A) and 0.1% trifluoroacetic acid as eluent, and loading the sample when the ratio of eluent B was 0%. The eluate B was eluted by a linear concentration gradient method up to 55% in minutes, and separated and purified. Each peptide fraction was subjected to amino acid sequence analysis to determine partial amino acid sequences F27 (SEQ ID NO: 9), F34 (SEQ ID NO: 10), F47 (SEQ ID NO: 11), and F52 (SEQ ID NO: 12).
(4) 20 μg of genomic DNA prepared in Example 1- (1) was digested with 100 units each of restriction enzymes BamHI, EcoRI, HindIII, PstI, SacI, SalI, SphI and XbaI at 37 ° C for 4 hours, and then phenol / Chloroform extraction. The digested product was recovered by ethanol precipitation, and each 10 µg was further digested with 50 units of the same restriction enzyme for 16 hours at 37 ° C, followed by phenol / chloroform extraction, and the digested product was recovered by ethanol precipitation. Each 5 µg of this digested product was subjected to 0.8% agarose gel electrophoresis and subjected to Southern blotting (Genetic Method II, pp. 218-221, Tokyo Chemical Co., Ltd.). N + (Hybond-N +)] was transcribed DNA.
As a hybridization probe, a mixed oligonucleotide pFDA27 (SEQ ID NO: 13) was synthesized from the partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3). The synthetic oligonucleotide 20pmo1 was labeled with ³² P using a Megalabel kit (MEGALABEL KIT, manufactured by Takarazojo Corporation).
The prepared filter was subjected to prehybridization at 65 ° C. for 3 hours in a solution containing 6 × SSC, 1% SDS, 100 μg / ml salmon sperm DNA, and 5 × Denharz, and then labeled label probe was 0.5 pmol / The solution was added at a concentration of ml, and hybridization was performed overnight at 42 ° C. After completion of hybridization, first wash at room temperature in 6 × SSC for 10 minutes, at room temperature in 1 × SSC, 0.1% SDS, for 10 minutes at 42 ° C in 1 × SSC, 0.1% SDS, and remove excess water. After exposing to a FUJIFILM imaging plate for 30 minutes, it detected by the FUJIFILM BAS2000 imaging analyzer.
As a result, at least 23 kbp with BamHI, EcoRI and SalI digests, about 4.8, 1.4 and 0.3 kbp with HindIII digests, at least 23 kbp and 3.6 kbp with PstI digests, at least 23 kbp and 9.8 kbp with SacI digests, and SphI The bands hybridizing with the probes were identified at positions of at least 23 kbp, 4.9 kbp and 3.0 kbp as digests, and at positions of about 12, 5.2 and 3.5 kbp as XbaI digests, respectively.
Alteromonas sp. Prepared in Example 1- (2). Clones containing the fucose sulfate-containing polysaccharide degrading enzyme gene from the genomic DNA library of SN-1009 were screened by the plaque hybridization method according to the Lambda Blue Star instruction manual of Novagen. First, the phage library was infected with E. coli ER1647, and about 500 plaques per sheet were formed on four L medium plates having a diameter of 8.5 cm. A nylon membrane (trade name High Bond N +) manufactured by Amarsham Co. was contacted with the plate for about 30 seconds for the first sheet and for about 2 minutes for the second sheet, and two sheets of each plate were transferred. The nylon membrane was denatured on a filter paper immersed in a solution of 0.5M sodium hydroxide and 1.5M sodium chloride for 5 minutes, and then neutralized on a filter paper immersed in a solution of 0.5M Tris hydrochloric acid buffer (pH7.0) and 3M sodium chloride for 5 minutes. After that, it was washed with 2 x SSC. When the nylon membrane and the synthetic oligonucleotide pFDA27 (SEQ ID NO: 13) were hybridized, washed and detected under the same conditions as those of the Southern hybridization described above, 18 positive signals were obtained. Plaques near the positive signal were taken from the original plate, suspended in SM buffer, plaques were formed on the new plate, and the same operation was repeated to isolate phages giving 14 positive signals.
According to the Novagen Lambda Blue Star Instruction Manual, each obtained phage was infected with E. coli BM25.8, and then ampicillin-resistant colonies were selected and the phages were converted into plasmid form. Colonies of the obtained clones were inoculated in L medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) containing 100 μg / ml of ampicillin, and then plasmid was obtained by alkaline lysis method from the culture medium cultured at 37 ° C. overnight. DNA was prepared. Using this plasmid DNA, E. coli JM109 (manufactured by Takarazo Corporation) was transformed, and the colonies of each clone obtained were inoculated in L medium containing 100 µg / ml of ampicillin, and alkalis were obtained from the culture medium at 37 ° C overnight. Plasmid DNA was prepared again by the lysis method. The plasmids of each clone obtained were named pSFDA1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16 and pSFDA17, respectively.
Each plasmid was digested with restriction enzyme NotI at both ends of the cloning region of the lambda blue star vector, and analyzed by 0.3% agarose gel electrophoresis, and it became clear that 8.4 to 17.4 kbp fragment was inserted. . Furthermore, each plasmid was digested with a restriction enzyme HindIII, and after Southern blotting as described above, hybridization was performed by synthetic oligonucleotide pFDA27 (SEQ ID NO: 13), and an analysis was performed to give a band of about 4.8 kbp. (pSFDA1 and pSFDA17), giving bands of about 4.8 and 0.3 kbp (pSFDA5, 6, 7, 11 and pSFDA13), giving bands of about 1.4 kbp (pSFDA10), bands of about 0.3 kbp and others Of giving a band of size (pSFDA2 and pSFDA14), of giving a band of about 4.8 kbp and other bands (pSFDA16), of giving a band of 5.0 kbp or more (pSFDA4,8 and pSFDA12). It was divided into six groups. However, except that pSFDA10, which gives a band of about 1.4 kbp, a plurality of bands of very similar size were detected by ethidium bromide staining of agarose gel electrophoresis, so that each plasmid had about 4.8 in genomic DNA of approximately the same position. And 0.3 kbp HindIII fragment or one or a part thereof was estimated. In addition, it was estimated that about 4.8 and 0.3 kbp HindIII fragments hybridizing with this pFDA27 are in close proximity to the genome. Thus, the 14 plasmids thus obtained were largely divided into pSFDA10 and other plasmids giving a band of about 1.4 kbp, and any of about 4.8, 1.4 and 0.3 kbp detected by Southern hybridization of genomic DNA HindIII digests, or about It could be assumed to have both 4.8 and 0.3 kbp. Among the resulting plasmids, pSFDA7 having an insertion fragment of about 10.2 kbp, having an insertion fragment of about 8.4 kbp and pSFDA7 comprising both HindIII fragments of about 4.8 and 0.3 kbp, and containing about 1.4 kbp HindIII fragment, After digestion with different kinds of restriction enzymes, analysis was performed by agarose gel electrophoresis to prepare restriction enzyme maps. Subsequently, the region hybridized with the synthetic oligonucleotide pFDA27 was subcloned into a plasmid pUC119 or the like, and the base sequence was determined by the dideoxy method. Interpreted In the HindIII fragment of about 1.4kbp of pSFDA10, a sequence of 14 bases out of 17 bases of pFDA27 was found, but the fragment hybridized with pFDA27 in that it did not match the sequence encoding the amino acid sequence of F27. It was thought to be a fragment independent of the type fucose sulfate-containing polysaccharide degrading enzyme gene. On the other hand, among the HindIII fragments of both about 4.8 and 0.3kbp of pSFDA7, the partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3) by completely matching one sequence of pFDA27 and including the surrounding sequence was also included. A sequence encoding the matching amino acid was found.
In addition, the HindIII fragments of about 4.8kbp and 0.3kbp were separated by at least about 3kbp on the restriction enzyme map, and the Alteromonas sp. Described in Reference Example 1- (3). At least two similar genes can be expected in that they are larger than the size of the enzyme gene expected from a molecular weight of about 100,000 as determined by gel filtration of the endotype fucose sulfate-containing polysaccharide degrading enzyme purified from SN-1009. there was.
E. coli JM109 strain incorporating pSFDA7 is designated as Escherichia coli JM109 / pSFDA7. In addition, E. coli JM109 strain introduced pSFDA7 is indicated as Escherichia coli JM109 / pSFDA7, and deposited as FERM P-16362 from the August 1, 1997 to the Institute of Biotechnology Industrial Technology, Ministry of Commerce, Industry and Technology Internationally deposited as FERM BP-6340 (Request for Escalation to International Deposit: 6 May 1998) to the Technical Laboratory.
The base sequence of pSFDA7 was analyzed in detail by the dideoxy method using primer extension, and the base sequence of 8.3 kbp was determined from the insertion fragment of 10.2 kbp of pSFDA7. Two reading frames were found, read frame 1 (hereinafter referred to as ORF-1) and read frame 2 (hereinafter referred to as ORF-2) of 2445 bases (including the stop codon). About 0.3 and 4.8 kbp HindIII fragments detected by Southern hybridization of genomic DNA HindIII digests were precisely 140 and 4549 base pairs in length, each containing some or the entire length of ORF-1 and ORF-2, respectively. In the amino acid sequence encoded by ORF-2, the partial amino acid sequence F34 (SEQ ID NO: 1), in addition to the sequence corresponding to the endo-type fucose sulfate-containing polysaccharide degrading enzyme partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3) 10), sequences highly homologous to F47 (SEQ ID NO: 11) and F52 (SEQ ID NO: 12) were found. Thus, ORF-2 is composed of Alteromonas sp. It is thought that the endotype fucose sulfate-containing polysaccharide degrading enzyme of SN-1009 is substantially encoded. On the other hand, among the amino acid sequences encoded by ORF-1, the partial amino acid sequence F34 (except for the sequence corresponding to the endo type fucose sulfate-containing polysaccharide degrading enzyme partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3)) Sequences with high homology to SEQ ID NO: 10) and F52 (SEQ ID NO: 12) were found, but no sequences with high homology to partial amino acid sequence F47 (SEQ ID NO: 11) were found. Comparing the amino acid sequences encoded by ORF-1 and ORF-2, there is an insertion sequence of 67 amino acid residues that are not present in ORF-2 near the N terminus of ORF-1, and the amino acid sequence after the insertion sequence is more than 70%. In view of high homology, it was thought that ORF-1 also encodes a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity. In the above manner, the gene (ORF-2) which is considered to encode a polypeptide having a fucose sulfate-containing polysaccharide degrading activity and the fucose sulfate-containing polysaccharide degrading activity, which exhibits very high homology with the gene, are considered. The entire nucleotide sequence of the gene (ORF-1) encoding the new polypeptide was determined.
The result is shown in FIG. 1 illustrates the positions of ORF-1 and ORF-2. Black arrows in the figure indicate the encryption region and direction of ORF-1, and diagonal arrows in the figure indicate the encryption region and direction of ORF-2, respectively. The nucleotide sequence of ORF-1 is shown in SEQ ID NO: 6 in the sequence listing, and the amino acid sequence encoded by ORF-1 is shown in SEQ ID NO: 2 in the sequence listing. Furthermore, the base sequence of ORF-2 is shown in SEQ ID NO: 5 in the Sequence Listing, and the amino acid sequence encoded by ORF-2 is shown in SEQ ID NO: 1 in the Sequence Listing.
As mentioned above, the gene (ORF-2) considered to encode substantially an endo type fucose sulfate-containing polysaccharide degrading enzyme by this invention, and the novel which show homology to the gene, and are considered to have a fucose sulfate-containing polysaccharide decomposition activity The gene (ORF-1) which is supposed to encode a polypeptide was isolated and purified.
Example 2
In order to construct a direct expression vector of gene (ORF-2) that is thought to encode substantially the endo type fucose sulfate-containing polysaccharide degrading enzyme obtained in Example 1, the initiation codon of ORF-2 was optimized on the expression vector. Consistent with the codon, the plasmid pEFDA-N was constructed by inserting a portion of the 5 'region of ORF-2.
First, synthetic DNA, FDA-N1 (SEQ ID NO: 14) and FDA-N2 (SEQ ID NO: 15) were synthesized. FDA-N1 is 15 mer synthetic DNA comprising the sequences of SEQ ID NOs 1-13 of SEQ ID NO: 5 in the Sequence Listing, and FDA-N2 is complementary to the sequences of SEQ ID NOs 4-13 of SEQ ID NO: 5 in the Sequence Listing 15 mer of synthetic DNA containing the sequence of interest.
These synthetic DNAs were incubated in a solution containing 0.2M Tris hydrochloric acid buffer (pH7.5) and 0.3M sodium chloride at 70 ° C for 10 minutes, and then gradually cooled to room temperature to form a double stranded synthetic DNA linker FDA-N. Was prepared. The resulting FDA-N is a synthetic DNA linker comprising the SnaBI site in SEQ ID NOs: 8-13 of SEQ ID NO: 5 in the Sequence Listing and linkable to the NcoI site in the initiation codon portion and the BamHI site immediately downstream of the SnaBI site.
On the other hand, pET21d (manufactured by Novagen), an expression vector using the T7 promoter, was cleaved at the NcoI site containing the start codon optimized for expression downstream from the T7 promoter and the BamHI site within the multicloning site. The digested product and the synthetic DNA linker FDA-N prepared first were linked using a DNA ligation kit (manufactured by Takaratsujo Co., Ltd.), and then transformed into E. coli JM109, on an L medium plate containing 100 μg / ml of ampicillin. Growing colonies were selected. Each transformant was inoculated in L medium containing 100 μg / ml of ampicillin, incubated overnight at 37 ° C., and plasmid DNA was prepared from the cultured cells by alkaline lysis. SnaBI digestion of the plasmid DNA was followed by 1% agarose gel electrophoresis to select a plasmid cleavable with SnaBI, and further confirmed the nucleotide sequence of the insertion fragment by the dideoxy method, and ORF between the NcoI-BamHI sites of pET21d. The plasmid pEFDA-N with the region from the start codon of -2 to the SnaBI site was obtained.
Subsequently, about 5 μg of the plasmid pSFDA7 obtained in Example 1 was decomposed with 30 units of SnaBI at 37 ° C. for 2 hours, and then separated by 1% agarose gel electrophoresis to include an almost full-length region of ORF-2. Approximately 2.5kbp of SnaBI fragment was cut and extracted and purified. This SnaBI fragment was mixed with the SnaBI digest of the plasmid pEFDA-N previously constructed, ligated using a DNA ligation kit, and transformed into E. coli JMl09 and grown on an L medium plate containing 100 µg / ml ampicillin. Colonies were chosen. Plasmid DNA was prepared in the same manner as above, 1% agarose gel electrophoresis was performed after SnaBI digestion to select a plasmid free of 2.5kbp SnaBI fragment. Furthermore, the orientation of the insertion fragment was confirmed by the dideoxy method, and the plasmid in which ORF-2 was inserted in the same direction as the T7 promoter was selected. The expression plasmid in which the full length of ORF-2 was inserted from the start codon in the NcoI site | part of pET21d obtained in this way was named pEFDAII103.
E. coli BL21 (DE3) strain (manufactured by Novagen) was transformed using pEFDAII103 thus obtained. E. coli BL21 (DE3) / pEFDAII103 obtained was inoculated in 5 ml of L medium containing 100 µg / ml of ampicillin and 5 mM calcium chloride and shake-cultured at 37 DEG C., and the turbidity was OD600 = 0.8 at a final concentration of 1 mM. After IPTG was added to this, shaking culture was again performed overnight with the culture temperature at 15 ° C. After completion of the culture, the culture solution was centrifuged to collect the cells, and suspended in 1 ml of cell disruption buffer [20 mM Tris hydrochloric acid buffer (pH7.5), 10 mM calcium chloride, 10 mM potassium chloride, 0.3 M sodium chloride], and the cells were sonicated. Crushed. The supernatant was collected by centrifugation to obtain E. coli extract.
As a control, E. coli BL21 (DE3) / pET21d transformed with pET21d was cultured under the same conditions at the same time to prepare an E. coli extract and used for the following analysis.
First, when the E. coli extract was analyzed by SDS polyacrylamide gel electrophoresis, in the extract of E. coli BL21 (DE3) / pEFDAII103, a band of about 90,000 molecular weights not seen in the extract of E. coli BL21 (DE3) / pET21d were observed. This molecular weight agrees well with the molecular weight 88210 of the polypeptide calculated from the amino acid sequence that ORF-2 shown in SEQ ID NO: 1 in the Sequence Listing can encode. The molecular weight of approximately 90,000 was analyzed by SDS polyacrylamide gel electrophoresis of the endo type fucose sulfate-containing polysaccharide degrading enzyme purified from SN-1009. As mentioned above, it was confirmed that E. coli BL21 (DE3) / pEFDAII103 expresses the polypeptide encoded by ORF-2.
Subsequently, the endotype fucose sulfuric acid-containing polysaccharide decomposition activity of the E. coli extract was measured by the method described in Reference Example 2.
As a result, in the extract of E. coli BL21 (DE3) / pEFDAII103, 520 mU / ml endo type fucose sulfate-containing polysaccharide decomposition activity was detected. That is, the polypeptide encoded by ORF-2 retains the fucose sulfate-containing polysaccharide degradation activity, and exhibits about 104 mU of the fucose sulfate-containing polysaccharide degradation activity in 1 ml of the culture solution of E. coli BL21 (DE3) / pEFDAII103 having the gene of the present invention. It was found that the polypeptide encoded by the having ORF-2 was produced.
On the other hand, the endo type fucose sulfate-containing polysaccharide decomposition activity was not detected at all from the extract of E. coli BL21 (DE3) / pET21d.
Example 3
The sequences up to 15 bases from the initiation codons of ORF-1 and ORF-2 are completely identical and there is a restriction enzyme SnaBI site in that region. That is, since the insertion sequence in the plasmid pEFDA-N constructed in Example 2 is completely identical to ORF-1 and ORF-2, this pEFDA-N can be used for constructing the expression vector of ORF-1.
First, about 5 μg of the plasmid pSFDA7 obtained in Example 1 was decomposed by 30 units of SnaBI at 37 ° C. for 2 hours, and then separated by 1% agarose gel electrophoresis to include an almost full-length region of ORF-1. The SnaBI fragment of about 3.2 kbp was cut out and purified. This SnaBI fragment was mixed with the SnaBI digest of plasmid pEFDA-N, ligated using a DNA ligation kit, and transformed into E. coli JM109 to select colonies growing on L medium plates containing 100 μg / ml ampicillin. It was.
Plasmid DNA was prepared in the same manner as in Example 2, SnaBI digested, and then subjected to 1% agarose gel electrophoresis to select a plasmid free of 3.2 kbp SnaBI fragment. Furthermore, the orientation of the insertion fragment by the dideoxy method was confirmed, and the plasmid in which ORF-1 was inserted in the same direction as the T7 promoter was selected. The expression plasmid in which the full length of ORF-1 was inserted from the start codon in the NcoI site | part of pET21d obtained in this way was named pEFDAI103.
Using pEFDAI103 thus obtained, expression of the polypeptide encoded by ORF-1 and the polysaccharide degradation activity containing fucose sulfate were confirmed in the same manner as in Example 2.
That is, E. coli BL21 (DE3) strain was first transformed using pEFDAI103. E. coli BL21 (DE3) / pEFDAI103 obtained was inoculated in 5 ml of L medium containing 100 µg / ml of ampicillin and 5 mM calcium chloride and shake-cultured at 37 DEG C., in which the final concentration was OD600 = 0.8. After IPTG was added to 1 mM, the culture was shaken again overnight at 15 ° C. After completion of the culture, the culture solution was centrifuged to collect the cells, suspended in 1 ml of the cell disruption buffer, and the cells were disrupted by sonication. The supernatant was collected by centrifugation to obtain E. coli extract.
As a result of analyzing the E. coli extract by SDS polyacrylamide gel electrophoresis, in the extract of E. coli BL21 (DE3) / pEFDAI103, a band of about 100,000 molecular weights not seen in the extract of E. coli BL21 (DE3) / pET21d were observed. This molecular weight is in good agreement with the molecular weight 94910 of the polypeptide calculated from the amino acid sequence capable of encoding the ORF-1 shown in SEQ ID NO: 2 of the Sequence Listing, and E. coli BL21 (DE3) / pEFDAI103 expresses the polypeptide encoded by the ORF-1 I could confirm that I was doing.
Subsequently, the endotype fucose sulfuric acid-containing polysaccharide decomposition activity of the E. coli extract was measured by the method described in Reference Example 2.
As a result, 35.8 mU / mL endo type fucose sulfuric acid containing polysaccharide degradation activity was detected from the extract of E. coli BL21 (DE3) / pEFDAI103. That is, the polypeptide encoded by ORF-1 has a fucose sulfate-containing polysaccharide degradation activity, and about 7.2 mU of fucose sulfate-containing polysaccharide degradation activity in 1 ml of the culture solution of E. coli BL21 (DE3) / pEFDAI103 having the gene of the present invention. It can be seen that a polypeptide encoding ORF-1 having
On the other hand, the endo type fucose sulfate-containing polysaccharide decomposition activity was not detected at all from the extract of E. coli BL21 (DE3) / pET21d.
Example 4
(1) Flavobacterium sp. Sterilized SA-0082 (FERM BP-5402) by dividing 500 ml of a medium consisting of pH 8.0 of artificial seawater (made by Jamarin Reboratori) containing glucose 0.25%, peptone 1.0%, yeast extract 0.05% 20 minutes) and inoculated into a 2 liter Erlenmeyer flask and incubated at 25 ℃ for 23 hours. After completion of the culture, the culture solution was centrifuged to collect the cells, and half of the cells were suspended in 10 ml of extraction buffer [50 mM Tris hydrochloric acid buffer (pH8.0), 100 mM ethylenediaminetetraacetic acid (EDTA), and then 1 ml. 20 mg / ml lysozyme solution dissolved in the extraction buffer of was added and incubated for 30 minutes on an ice bath. Subsequently, 10 ml of proteinase K solution [1 mg / ml proteinase K, 50 mM Tris hydrochloric acid buffer (pH8.0), 100 mM EDTA, 1% SDS] was added, followed by constant temperature at 50 ° C for 2 hours. Treated. After returning to room temperature, an equal volume of TE buffer [10 mM Tris hydrochloric acid buffer (pH8.0), 1 mM EDTA] saturated phenol was added thereto, stirred gently for 1 hour, centrifuged at 10000 rpm for 20 minutes, and the upper layer was recovered. Equivalent volume of TE buffer saturated phenol / chloroform 1: 1 was added to the upper layer, followed by gentle stirring, centrifugation at 10000 rpm for 20 minutes, and the upper layer was recovered. After extracting phenol / chloroform again, sodium chloride was added to the aqueous layer to make 0.1 M, and twice the amount of ethanol was added to precipitate DNA, which was wound up with a glass rod, washed with 80% ethanol, and dried lightly in the wind. The genomic DNA was dissolved in TE buffer in which 20 ml of 20 μg / ml ribonuclease A was dissolved, and incubated at 37 ° C. for 5 hours to degrade RNA. After phenol extraction and phenol / chloroform extraction, ethanol was added in the same manner as above, DNA was recovered and suspended in 5 ml of TE buffer. By the above operation, about 20 mg of genomic DNA was obtained.
(2) 100 µg of the genomic DNA prepared in Example 4- (1) was partially digested with 10 units of restriction enzyme Sau3AI for 1 minute and 40 seconds at 37 ° C, and the upper layer was recovered by phenol / chloroform extraction. DNA was precipitated by adding 1 / 10-fold 3M aqueous sodium acetate solution (pH5.0) and 2.5-fold ethanol to the upper layer, and the precipitate was recovered by centrifugation, washed with 80% ethanol and dried in the wind. The obtained partial digested product was fractionated in size by 1.25 to 5 M sodium chloride density gradient ultracentrifugation, and DNA was recovered by ethanol precipitation from a fraction containing a size of 10 to 20 kbp. 0.2 µg of the obtained genomic DNA partial digest and 0.6 µg of Novagen Lambda Blue Star BamHI arm were mixed and linked using a DNA ligation kit manufactured by Takarazo Corporation, followed by lambda phage using Gigapack II Gold Kit manufactured by Stratagene. Packaging was performed, Flavobacterium sp. A genomic DNA library of SA-0082 was constructed.
(3) Flavobacterium sp. Obtained in the same manner as in Reference Example 5. Purified Fucoidan Degrading Enzyme Protein 200pmo1 of SA-0082 was charged into a desalting column (First Desorption Column PC3.2 / 10, manufactured by Pharmacia) equilibrated with 20 mM ammonium bicarbonate, eluted with the same buffer solution, and buffered. Was substituted. The eluate was concentrated in a glass vial, concentrated to dryness, and then loaded into a glass vial of 1 size larger glass containing 10 μl of pyridine, 2 μl of 4-vinylpyridine, 2 μl of tri-N-butylphosphine, and 10 μl of water. Was added, the glass test tube was sealed, and it reacted at 95 degreeC for 10 minutes, and pyridyl ethylation was carried out. After completion of the reaction, the glass vial was taken out and azeotropically with water several times to remove volatile components.
The resulting pyridylethylated fucoidan degrading enzyme protein was purified by 10 mM Tris hydrochloric acid buffer (pH9.0) containing 40 µl of 8M urea, 90 µl of 10 mM Tris hydrochloric acid buffer (pH9.0), and 0.5 pmo1 of Acromobacter protease I. (Takaratsujo Co., Ltd.) was added, and the peptide fragment was digested overnight at 30 ° C, and the peptide fragment was purified by an HPLC system (Smart Systems, Pharmacia Co., Ltd.). The column was run at a flow rate of 100 μl / min using μRPC C2 / C18 SC2.1 / 10 (manufactured by Pharmacia). Elution was carried out using an acetonitrile (eluent B) containing 0.1% trifluoroacetic acid aqueous solution (eluent A) and 0.1% trifluoroacetic acid as eluent. And eluting fractions L27, L31, L36 and the like were obtained as peaks eluted when the proportion of the eluent B was raised to 55% within 80 minutes, and the ratio of the eluent B was 27% or more. In addition, fractions containing 17-27% of the eluent B, which was not well separated, were collected, concentrated, and added to the same HPLC system, and the elution pattern was increased in a straight line to raise the eluent B to 15% to 40% within 87 minutes. Elution was carried out in a concentration gradient to obtain the elution fractions LR8, LR9, LR14, LR16 and the like. Amino acid sequence analysis was performed on the obtained peptide fractions to determine partial amino acid sequences L27 (SEQ ID NO: 16), L36 (SEQ ID NO: 17), LR9 (SEQ ID NO: 18), LR14 (SEQ ID NO: 19), and LR16 (SEQ ID NO: 20). Decided.
(4) 2.4 μg of genomic DNA prepared in Example 4- (1) was digested with 37 units of restriction enzymes EcoRI, HindIII, MunI, SpeI, XbaI, and Sau3AI at 37 ° C. for 3 hours, and then phenol / chloroform extracted. The digest was recovered by ethanol precipitation. About 0.5 μg of each digest, 20 ng of EcoRI cassette for EcoRI and MunI digest, HindIII cassette for HindIII digest, XbaI cassette for SpeI and XbaI digest and Sau3AI cassette for Sau3AI digest (each cassette DNA manufactured by Takarazojo Co., Ltd.) The cassette was mixed with 200 ng), and then linked using a DNA ligation kit manufactured by Takarazozu Corporation. The reaction was recovered by ethanol precipitation, dissolved in 10 µl of water, and used as template DNA of PCR using cassette DNA.
On the other hand, a mixture of pL14F17 (SEQ ID NO: 21) from the amino acid sequences 1-6 of partial amino acid sequence LR14 (SEQ ID NO: 19) determined in Example 4- (3) and pL14F26 (SEQ ID NO: 22) from the amino acid sequences of 3-11; Oligonucleotides were each synthesized in the same direction as the amino acid sequence.
1 μl of each template DNA prepared above, 20 pmo1 of cassette primer C1 (manufactured by Takarazo Corporation), 100 pmo1 of mixed oligonucleotide pL14F17 (SEQ ID NO: 21), and sterile water were added thereto to make 22 μl, and the mixture was quenched after heating at 94 ° C. for 10 minutes. 10 μl of 10-fold concentration of Ex Taq amplification buffer (manufactured by Takaratsujo Co., Ltd.), 16 μl of 1.25mM dNTP mixture solution, 2.5 units of Takara Ex Taq (Takara Ex Taq, manufactured by Takarazojo Co.) and sterile water were added thereto. It was made into microliter and the mineral oil was layered. This mixed solution was subjected to an amplification reaction by an automatic gene amplification apparatus thermal cycler (DNA Thermal Cycler 480, manufactured by Takarazo Corporation). In the PCR reaction, 25 cycles of denaturation at 94 ° C. for 0.5 minutes, annealing at 45 ° C. for 2 minutes, and synthesis reaction at 72 ° C. for 3 minutes were performed, followed by incubation at 72 ° C. for 7 minutes to complete synthesis.
Subsequently, the first PCR reaction solution diluted 10-fold with sterile water was heated at 94 DEG C for 10 minutes, and then quenched to give a template DNA for a second PCR reaction. That is, 10 μl of the first PCR reaction solution, 20 pmo1 of cassette primer C2 (manufactured by Takarazo Corporation), 100 pmol of mixed oligonucleotide pL14F26 (SEQ ID NO: 22), 10 μl of 10-fold concentration Ex Taq amplification buffer, 16 μl 1.25 mM dNTP mixed solution, 2.5 units of Takara Ex Taq and sterile water were added to make 100 µl, and the mineral oil was further layered. The mixed solution was subjected to 25 cycles of denaturation at 94 ° C. for 0.5 minutes, annealing at 55 ° C. for 2 minutes, and synthesis reaction at 72 ° C. for 3 minutes, and then incubated at 72 ° C. for 7 minutes to complete synthesis. At the same time, the reaction solution in which only the cassette primer C2 or only the mixed oligonucleotide pL14F26 was added to each reaction solution was also subjected to PCR reaction and used as a control for the nonspecific amplification product of each primer.
The reaction solution was analyzed by agarose gel electrophoresis and compared to the control of the non-specific amplification product, a plurality of amplification bands were detected in many reaction solutions. Among them, a band of about 0.7 kbp was extracted and purified from the second PCR reaction solution of the MunI digest product-EcoRI cassette having a relatively low background and strongly amplifying one band. The DNA fragment and the pT7 blue T-Vector (pT7blue T-Vector, manufactured by Novagen) were mixed and linked using a DNA ligation kit, followed by transformation of E. coli JM109, and 100 µg / ml Ampicillin, 0.004%. White colonies growing on L medium plates containing X-Gal and 1 mM IPTG were selected. Each transformant was inoculated in L medium containing 100 μg / ml of ampicillin, incubated overnight at 37 ° C., and then plasmid DNA was prepared from the cultured cells by alkaline lysis, and a plasmid having a band of about 0.7 kbp was inserted. Was chosen and named pT7-Mun. When the insertion sequence of this pT7-Mun was subjected to sequencing by the dideoxy method, the amino acid sequence of the 12th or later amino acid of the partial amino acid sequence LR14 (SEQ ID NO: 19) was encoded following the sequence of pL14F26 used for the 2nd PCR reaction. Area was found. Also downstream was found a region coding for an amino acid sequence showing very high homology with the partial amino acid sequence L27 (SEQ ID NO: 16). From these, it became clear that the DNA fragment of about 0.7 kbp was part of the fucoidan degrading enzyme gene. The sequence from the primer pL14F26 of this about 0.7 kbp DNA fragment to the junction with the EcoRI cassette is shown in SEQ ID NO: 23.
(5) Each 20 µg of genomic DNA prepared in Example 4- (1) was digested with each of 100 units of restriction enzymes BamHI, EcoRI, HindIII, PstI, SacI, SalI, SphI and XbaI at 37 ° C for 4 hours, and then phenol / Chloroform extraction. The digested product was recovered by ethanol precipitation, and each 10 µg was further decomposed with 50 units of the same restriction enzyme for 16 hours at 37 ° C, followed by phenol / chloroform extraction to recover the digested product by ethanol precipitation. Each 5 µg of this digested product was used for 0.8% agarose gel electrophoresis, and DNA was transferred to nylon membrane high bond N + manufactured by Amarsham Co. by Southern blotting.
On the other hand, about 4 µg of pT7-Mun obtained in Example 4- (4) was digested with BamHI and SphI derived from a vector, and a fragment containing a part of the free 0.7 kbp fucoidan degrading enzyme gene was extracted and purified. This DNA fragment was labeled with ³² P using a BcaBEST Labeling Kit (manufactured by Takarazo Corporation) to prepare probe DNA for hybridization.
The prepared filter was subjected to preliminary hybridization at 65 ° C. for 1 hour in a solution containing 6 × SSC, 1% SDS, 100 μg / ml salmon sperm DNA, and 5 × Denharz, and then the labeling probe was about 1 million. The solution was added at a concentration of cpm / ml, and hybridization was performed overnight at 60 ° C. After completion of hybridization, first at room temperature in 6 × SSC, 10 minutes at room temperature in 2 × SSC, 0.1% SDS, 5 minutes at room temperature in 0.2 × SSC, 0.1% SDS, and further at 0.2 × SSC, 0.1% SDS After 30 minutes of washing at 45 ° C. to remove excess water, the plate was exposed to an imaging plate manufactured by Fujifilm for 30 minutes, and then detected by a BAS2000 imaging analyzer manufactured by Fujifilm.
As a result, one BamHI, SalI and SphI, SalI decomposes at a position of 23 kbp or more, two at Eco 8 digests of about 8 and 3 kbp, two HindIII digests of about 11 and 4 kbp, and PstI digests of about 12 And two bands at a position of 2.5 kbp, two at a position of about 10 and 9.5 kbp with SacI digest, and one at a position of about 6 kbp with XbaI digest, and a band that hybridized strongly to each probe. From this, Flavobacterium sp. It was strongly suggested that there are two kinds of fucoidan degrading enzyme genes on the genomic DNA of SA-0082.
(6) Flavobacterium sp. Prepared in Example 4- (2). Clones containing the fucoidan degrading enzyme gene from the genomic DNA library of SA-0082 were screened by plaque hybridization according to the Lambda Blue Star instruction manual of Novagene.
First, the phage library was infected with E. coli ER1647, and about 300 plaques per sheet were formed on five L medium plates having a diameter of 8.5 cm. The phage was transferred by contacting the plate with Amaksham nylon membrane high bond N + for about 30 seconds. The nylon membrane was denatured in a solution of 0.5 M sodium hydroxide and 1.5 M sodium chloride for 5 minutes, and then neutralized for 5 minutes in a solution of 0.5 M Tris hydrochloric acid buffer (pH 7.0) and 3 M sodium chloride, and washed with 2 x SSC. Dried in the wind After the nylon membrane was immobilized by UV irradiation to fix the DNA, 36 positive signals were obtained as a result of hybridization, washing and detection under the same conditions as Southern hybridization of Example 4- (5). Plaques near the positive signal were taken from the original plate and suspended in SM buffer to recover phage. About how many phage liquids were obtained, the plaque was formed again on a new plate, and the phage which gives nine positive signals was isolated by repeating the same operation.
In accordance with Novagen's Lambda Blue Star Instruction Manual, each obtained phage was infected with E. coli BM25.8 and plated with an L medium plate containing 100 μg / ml of ampicillin to select ampicillin-resistant colonies for plasmid Converted to form. Colonies of the obtained clones were inoculated in L medium containing 100 µg / ml of ampicillin, and plasmid DNA was prepared by alkaline lysis from a culture medium cultured overnight at 37 ° C. Using this plasmid DNA, E. coli JM109 (manufactured by Takarazo Corporation) was transformed, and the colonies of each clone obtained were inoculated in L medium containing 100 µg / ml of ampicillin, and alkalis were obtained from the culture medium at 37 ° C overnight. Plasmid DNA was prepared again by the lysis method. The plasmids of each clone obtained were named pSFLA1, 5, 10, 11, 12, 13, 15, 17 and pSFLA18, respectively.
Each plasmid was digested by appropriate combination of restriction enzymes KpnI, SacI, and Xbal, and then subjected to agarose gel electrophoresis, followed by Southern hybridization as described above to prepare and interpret restriction enzyme maps of each inserted fragment. . As a result, by ethidium bromide staining of agarose gel electrophoresis, a plurality of bands of very similar size were detected, and at the same time, two bands hybridized with about 0.7 kbp insertion fragment of pT7-Mun by SacI decomposition, It is clear that the pairs of 10, 12, 15 and pSFLA18 are likewise divided into two groups, pairs of pSFLA5, 11, 13 and pSFLA17 which have two bands which hybridize with about 0.7 kbp insert fragment of pT7-Mun by KpnI digestion. Was done. In addition, it is evident that the three plasmids of pSFLA5, pSFLA10 and pSFLA17 liberated fragments of about 6 kbp by XbaI digestion, and a band of about 6 kbp XbaI digests detected by Southern hybridization of Example 4- (5) It is suggested that two bands overlap each other, and Flavobacterium sp. It has become clear that there are at least two fucoidan degrading enzyme genes on the genomic DNA of SA-0082.
Therefore, pSFLA10 and pSFLA17 were selected from each group of plasmids, and the insertion fragments were analyzed again. That is, about 3 µg of these plasmids were digested by XbaI to extract and purify about 6 kbp of free fragments. Each of these KbaI fragments was mixed with an XbaI digest of pHSG399 (manufactured by Takarazo Corporation), a chloramphenicol resistance vector, linked using a DNA ligation kit (manufactured by Takarazujo Corporation), and then transformed into E. coli JM109, 30 µg / ml. White colonies growing on L medium plates containing chloramphenicol, 0.004% X-Ga1 and 1 mM IPTG were selected. Each transformant was inoculated in L medium containing 30 µg / ml chloramphenicol, incubated overnight at 37 ° C, and then plasmid DNA was prepared from the cultured cells by alkaline lysis, followed by restriction enzyme digestion followed by agarose gel electrolysis. Youngdong was interpreted. As a result, pH10X6-1 and pH10X6-2 in which about 6 kbp DNA derived from pSFLA10 were inserted in the reverse direction with respect to the vector DNA were obtained, respectively. Similarly, pH17X6-7 and pH17X6-11 containing about 6 kbp DNA derived from pSFLA17 were obtained. E. coli JM-109 strain, introduced with pHlOX6-1, is designated as Escherichia coli JM109 / pH10X6-1 and is commonly referred to as the FERM P-16659, dated February 24, 1998, at the Institute of Biotechnology and Technology, It has been deposited internationally as FERM BP-6341 (date of transfer of international deposit: May 6, 1998) to the Institute of Biotechnology and Industrial Technology, Institute of Commerce and Industry. Escherichia coli JM-109 strain, introduced with pH17X6-7, is expressed as Escherichia coli JM109 / pH17X6-7, and deposited as FERM P-16660 on February 24, 1998, at the Institute of Biotechnology and Technology, Ministry of Commerce, Industry and Technology. Internationally deposited as FERM BP-6342 (International Deposit Request Date: May 6, 1998) to the Institute of Biotechnology and Industrial Technology, Industrial and Industrial Technology Institute. These plasmids were subjected to direct primer extension or subcloning after restriction enzyme digestion, and then analyzed in detail by the dideoxy method. As a result, 2094 bases (final codons) were obtained from a DNA fragment of about 6 kbp derived from pSFLA10. Read frame of 2115 bases (including stop codons) from a DNA frame of about 6kbp derived from pSFLA17, and the amino acid sequence encoded by these read frames A region showing very high homology with the partial amino acid sequence determined at.
Read frames derived from pSFLA10 were named fdlA and read frames derived from pSFLA17 were named fdlB.
The results are shown in FIGS. 3 and 4. That is, FIG. 3 is a diagram showing the position of fdlA in pSFLA10, and FIG. 4 is a diagram showing the position of fdlB in pSFLA10. The black arrows in FIG. 3 indicate the cipher regions and directions of fdlA, respectively, and the black arrows in FIG. 4 indicate the cipher regions and directions of fdlB.
Among the amino acid sequences coded by fdlA, the sequence and partial amino acid sequence L27 (SEQ ID NO: 16) consistent with the fucoidan degrading enzyme partial amino acid sequence L36 (SEQ ID NO: 17) and LR14 (SEQ ID NO: 19) determined in Example 4- (3) , Sequences highly homologous to LR9 (SEQ ID NO: 18) and LR16 (SEQ ID NO: 20) were found. Further, in the peptide fraction named LR8 in Example 4- (3), two amino acid derivatives were detected by amino acid sequence analysis for each cycle, and two amino acid sequences encoded by fdlA were not determined uniformly. By applying a sequence of sequences, the amino acid derivatives detected in each cycle can be described, and it became clear that this fraction contained a peptide containing these two sequences. Each sequence was named LR8-1 (SEQ ID NO: 24) and LR8-2 (SEQ ID NO: 25). Thus, the amino acid sequence coded by fdlA is consistent with or high homology to all amino acid sequences identified by the fucoidan decomposing enzyme partial amino acid sequence analysis of Example 4- (3). It is considered that the fucoidan degrading enzyme of SA-0082 is substantially encoded.
On the other hand, among the amino acid sequences coded by fdlB, sequences corresponding to the fucoidan decomposing enzyme partial amino acid sequences LR14 (SEQ ID NO: 19) and LR8-1 (SEQ ID NO: 24), and partial amino acid sequences L27 (SEQ ID NO: 16) and LR16 ( Sequences with high homology with SEQ ID NO: 20) and LR8-2 (SEQ ID NO: 25) were found, but no sequences with high homology to the partial amino acid sequences L36 (SEQ ID NO: 17) and LR9 (SEQ ID NO: 18) were found. . However, when fdlA and fdlB are compared, the homology between the base sequence and the amino acid sequence is higher than about 67% and about 56%, respectively. Therefore, it was considered that fdlB also encodes a polypeptide having fucoidan degrading activity. In the above manner, the entire nucleotide sequence of the gene (fdlA), which is thought to encode a polypeptide having fucoidan-degrading activity, and the gene (fdlB) showing very high homology to the gene, were determined. The nucleotide sequence of fdlA is shown in SEQ ID NO: 7 of the sequence list, and the amino acid sequence coded by fdlA is shown in SEQ ID NO: 3 of the sequence list. The nucleotide sequence of fdlB is shown in SEQ ID NO: 8 of the sequence listing list, and the amino acid sequence coded by fdlB is shown in SEQ ID NO: 4 of the sequence listing.
In the above, the gene (fdlA) which is substantially coded by the present invention, which is considered to encode a fucoidan degrading enzyme, and the gene which is homologous to the gene and is considered to encode a novel polypeptide which is thought to have a fucose sulfate-containing polysaccharide decomposition activity ( fdlB) was isolated and purified.
Example 5
First, the direct expression vector of the gene (fdlA) which is considered to encode substantially the fucoidan decomposing enzyme obtained in Example 4 was constructed.
In the plasmid pH10X6-1 obtained in Example 4, an about 6 kbp XbaI fragment derived from pSFLA10 was inserted in a direction facing the lac promoter on the fdlA gene and the vector. This pHl0X6-1 was digested at the SacI site inside the fdlA gene, further at BspflI at the start codon of the fdlA gene, separated by 1% agarose gel electrophoresis, and the N-terminal region of fdlA was encoded. The DNA fragment of about 400bp was cut out and purified.
Meanwhile, pET21d (manufactured by Novagen), an expression vector using the T7 promoter, was cleaved at the NcoI site containing the start codon optimized for expression downstream of the T7 promoter, and at the SacI site at the multiple cloning site, and then prepared first. About 400 bp of BspHI-SacI fragment encoding the N-terminal region of fdlA was inserted to construct plasmid pEFLA10-N.
Subsequently, HindIII digestion of the plasmid pH10X6-1 was cleaved at the HindIII site derived from the fdlA gene and the vector, and a DNA fragment of about 2 kbp encoding the C-terminal region containing the stop codon of the fdlA gene was cut out and purified. The fragment was linked to the HindIII digest of the plasmid pEFLA10-N, which was constructed first, to select the plasmid into which the 2kbp HindIII fragment was inserted in the direction in which the entire length of the fdlA gene was reproduced. The expression plasmid in which the entire length of fdlA was inserted from the start codon in the NcoI site | part of pET21d obtained in this way was named pEFLA10.
E. coli BL21 (DE3) strain (manufactured by Novagen) was transformed using pEFLA10 obtained in this way. E. coli BL21 (DE3) / pEFLA10 obtained was inoculated in 5 ml of L medium containing 100 µg / ml of ampicillin and shake-cultured at 37 ° C. At the stage of turbidity of OD600 = 0.8, IPTG so that the final concentration was 1 mM. After addition, shaking culture was further performed overnight at a culture temperature of 15 ° C. After completion of the culture, the culture medium was centrifuged to collect the cells, suspended in 0.5 ml of cell disruption buffer [20 mM sodium phosphate buffer (pH7.0), 0.3 M sodium chloride], and the cells were disrupted by sonication. A part of this suspension was taken as E. coli crushing liquid. Furthermore, this was centrifuged to remove insoluble matter, and the E. coli extract was obtained.
As a control, Escherichia coli BL21 (DE3) / pET21d transformed with pET21d were cultured under the same conditions at the same time to prepare an E. coli lysate and extract, which were used for the following analysis.
First, when E. coli crushing solution was analyzed by SDS polyacrylamide gel electrophoresis, in the lysing solution of E. coli BL21 (DE3) / pEFLA10, a band of about 7.6 million molecular weight was not observed in the lysate of E. coli BL21 (DE3) / pET21d. It became. This molecular weight is in good agreement with the molecular weight 75,740 of the polypeptide calculated from the amino acid sequence which can be encoded by fdlA shown in SEQ ID NO: 3 of the Sequence Listing, and the Flavobacterium sp. It is in good agreement with the molecular weight of about 70,000 analyzed by gel filtration of the more purified fucoidan degrading enzyme of SA-0082. As mentioned above, it was confirmed that E. coli BL21 (DE3) / pEFLA10 expresses the polypeptide encoded by fdlA.
Subsequently, the fucose sulfate-containing polysaccharide decomposition activity of the E. coli extract was measured by the method described in Reference Example 3.
As a result, it was found that the fucoidan decomposition activity in the extract of E. coli BL21 (DE3) / pEFLA10 was 2,300 mU / ml. In other words, the polypeptide encoded by fdlA has fucoidan-degrading activity, and the polypeptide encoded by fdlA having a fucose sulfate-containing polysaccharide-degrading activity of about 230 mU in 1 ml of the culture medium of E. coli BL21 (DE3) / pEFLA10 having the gene of the present invention You can see that it was produced.
On the other hand, no fucoidan decomposing activity was detected from the extract of E. coli BL21 (DE3) / pET21d.
Example 6
A direct expression vector of fdlB, a gene encoding a novel polypeptide that is thought to have fucose sulfate-containing polysaccharide degradation activity obtained in Example 4, was constructed. Although the recognition sequence of BspHI exists in the position of the start codon of this fdlB gene, since it is methylated by the influence of the upstream sequence, the plasmid collect | recovered from E. coli JM109 used as a host could not be directly degraded by BspHI. Thus, constructing was performed using DNA fragments amplified by PCR.
In the plasmid pH17X6-7 obtained in Example 4, an about 6 kbp XbaI fragment derived from pSFLA17 was inserted in the same direction as the lac promoter on the fdlB gene and the vector. First, pH17X6-7 was cleaved at the KpnI site derived from the fdlB gene and the vector, and then self-ligated to construct plasmid pH17X6-7K. Using a pH17X6-7K as a template and a pair of synthetic DNA primer 17X6F4 (SEQ ID NO: 26) in the same direction as fdlB and an M13 primer M4 (manufactured by Takarazo Corporation) that is upstream from the coding region of fdlB. PCR reaction was performed. In the PCR reaction, the same reaction solution composition as in Example 4- (4) was subjected to 25 cycles of denaturation at 94 ° C for 0.5 minute, primer annealing at 55 ° C for 0.5 minute, and synthesis reaction at 72 ° C for 1 minute. The synthesis was completed by incubation at 72 ° C. for 7 minutes. After the reaction solution was extracted with phenol / chloroform, the amplified DNA fragment of about 1 kbp was recovered by ethanol precipitation. This fragment was cleaved at the BspHI at the start codon and the MflI site within the coding region of the fdlB gene and then separated by 3% agarose gel electrophoresis to obtain an approximately 450 bp BspHI- coding for the N-terminal region of fdlB. The MflI fragment was cut and extracted and purified.
Meanwhile, as in Example 5, pET21d, which is an expression vector using a T7 promoter, was cleaved at the BamHI site within the NcoI site and the multiple cloning site, and then inserted a BspHI-MflI fragment of about 450 bp encoding the N-terminal region of fdlB prepared above. Thus, plasmid pEFLA17-N was constructed.
The plasmid pH17X6-7 was then cleaved at the NspV site within the fdlB gene and at the EcoRI site downstream of the fdlB gene to cut and extract an approximately 2 kbp NspV-EcoRI fragment encoding the C-terminal region containing the stop codon of the fdlB gene. Purified. This fragment was inserted between NspV-EcoRI of plasmid pEFLA17-N constructed earlier. The expression plasmid in which the entire length of fdlB was inserted from the start codon in the NcoI site | part of pET21d obtained in this way was named pEFLA17.
Using pEFLA17 thus obtained, expression of the polypeptide encoded by fdlB and fucoidan degradation activity were confirmed in the same manner as in Example 3.
That is, E. coli BL21 (DE3) strain was first transformed using pEFLA17. E. coli BL21 (DE3) / pEFLA17 obtained was inoculated in 5 ml of L medium containing 100 µg / ml of ampicillin, shake-cultured at 37 ° C, and at a stage of turbidity of OD600 = 0.8, IPTG so that the final concentration was 1 mM. After the addition, the culture temperature was 15 ° C., followed by further shaking culture. After completion of the culture, the culture solution was centrifuged to collect the cells, suspended in 0.5 ml of cell disruption buffer, and the cells were disrupted by sonication. A part of this suspension was taken as E. coli crushing liquid. Furthermore, this was centrifuged to remove insoluble matter, and the E. coli extract was obtained.
As a result of analyzing the E. coli crushing solution by SDS polyacrylamide gel electrophoresis, in the lysate of E. coli BL21 (DE3) / pEFLA17, a band of about 7.7 million molecular weight was not observed in the lysate of E. coli BL21 (DE3) / pET21d. This molecular weight agrees well with the molecular weight 76,929 of the polypeptide calculated from the amino acid sequence coded by fdlB shown in SEQ ID NO: 4 in the Sequence Listing, confirming that E. coli BL21 (DE3) / pEFLA17 expresses a polypeptide encoding fdlB. Could.
Subsequently, the fucoidan degrading activity of the E. coli extract was measured by the method described in Example 3.
As a result, 480 mU / ml of fucoidan degradation activity was detected from the extract of E. coli BL21 (DE3) / pEFLA17. That is, the polypeptide encoded by fdlB has a fucose sulfate-containing polysaccharide degradation activity, and fdlB having a fucose sulfate-containing polysaccharide degradation activity of about 48 mU in 1 ml of the culture medium of E. coli BL21 (DE3) / pEFLA17 having the gene of the present invention. It turns out that the polypeptide which coding for is produced.
On the other hand, no fucoidan decomposing activity was detected from the extract of E. coli BL21 (DE3) / pET21d.
Example 7
The action of fucose sulfate-containing polysaccharide-U on the polypeptides obtained in Examples 5 and 6 with fucose sulfate-containing polysaccharide degradation activity was investigated.
That is, in a mixture of 50 μl of 100 mM phosphate buffer (pH7.5), 50 μl of 2.5% fucose sulfate-containing polysaccharide-U, and 10 μl of 4M sodium chloride, 10 μl of the Fu obtained in Example 5 or Example 6 was used. The polypeptide solution having the coarse sulfuric acid containing polysaccharide decomposition activity was added and maintained at 25 degreeC. The reaction solution was aliquoted at 16, 40 and 65 hours from the start of the reaction and the reaction product was analyzed by HPLC. The conditions of HPLC were as follows.
Column: Shodex SB802.5 (manufactured by Showa Denko Co., Ltd.)
Column temperature: 25 ℃
Eluent: 50 mM sodium chloride solution containing 5 mM sodium azide
Flow rate: 1 ml / min
Detector: Differential Refractive Index Detector (manufactured by Shodex RI-71 Showa Denko Corporation)
Moreover, since the thing of the following structure is known so far as a reaction product, the following substance was used as a standard substance. In addition, the substance manufactured by the following method was used for the substance of following formula (I)-(IV).
The dried Kagome kelp was pulverized with a free grinder M-2 (manufactured by Narakai Chemical Co., Ltd.), and after filtration at 70 ° C. for 2 hours in 85% of 10% methanol. 20 times of water was added to the residue, and the mixture was treated at 100 ° C. for 3 hours to obtain an extract by filtration. The salt concentration of the extract was made equal to 400 mM sodium chloride solution, and then 5% cetylpyridinium chloride was added until no further precipitation occurred and centrifuged. The precipitate was sufficiently washed with ethanol to completely remove the cetylpyridinium chloride, and then desalted and removed with an ultrafiltration (excluded molecular weight of the filtration membrane of 100,000) (manufactured by Amicon) to centrifuge the resulting precipitate. Removed by. This supernatant was lyophilized to obtain purified Kagome Kelp Fucoidan. The yield was about 4% by weight of dry Kagome Kelp powder.
600 ml of the 5% purified Kagome Kelp Fucoidan solution, 750 ml of 100 mM phosphate buffer (pH8.0), 150 ml of 4M sodium chloride and 3.43 ml of the fucoidan degrading enzyme solution described in Reference Example 5 of 1750 mU / ml It mixed and reacted at 25 degreeC for 144 hours. The reaction solution was dialyzed using a dialysis membrane having a pore size of 3500 to collect fractions having a molecular weight of 3500 or less. This fraction was desalted by Micro Acylizer G3 (manufactured by Asahi Kasei Co., Ltd.) and then subjected to ion exchange chromatography using a DEAE-Sepharose FF column (4 cm × 25 cm) equilibrated with 10 mM ammonium acetate. Elution was carried out by a concentration gradient with ammonium acetate, and the eluate was aliquoted 50 ml each. Fig. 5 shows the elution of the chromatography. Nine fractions (a) to (i) were obtained by the above chromatography, and the fractions (a), (b), (c) and (f) were used for structural analysis.
Next, the (a), (b), (c) and (f) fractions were subjected to structural analysis according to a conventional method. After confirming that the fractions are of formula IV, and (f) the compounds of formula II, each fraction was used as a standard sample for the reaction product analysis.
In the ion exchange chromatography using the DEAE-Sepharose FF column, the numbers of the fractions were (a): 42-43, (b): 84-91, (c): 51-52, (f): 62-63.
Formula I
Formula II
Formula III
Formula IV
As a result of analysis of the reaction product, when the polypeptide having the fucose sulfate-containing polysaccharide decomposition activity obtained in Example 5 was used, the reaction products were only I, II and III, but the fucose sulfate-containing polysaccharide decomposition activity obtained in Example 6 In the case of using the polypeptide having the reaction product, in addition to the above-mentioned I, II and III, IV is contained in large quantities, and the IV was not decomposed to I even after the reaction was continued.
That is, the polypeptide encoded by fdlA obtained in the same manner as in Example 5 acts on the fucose sulfate-containing polysaccharide-U to liberate trisaccharides such as I, II, and III as the final cleavage unit, The polypeptide encoded by fdlB obtained in the above formula has been found to act on fucose sulfate-containing polysaccharide-U, thereby releasing six sugars such as IV as the final cleavage unit.
Example 8
In Examples 5 and 6, polypeptides encoded by each gene were produced by the respective recombinant Escherichia coli including the fdlA and fdlB genes, and the fucose sulfate-containing polysaccharide degradation activity was detected in these E. coli extracts, and fdlA was detected. And the polypeptide encoded by fdlB was confirmed to have a fucose sulfate-containing polysaccharide decomposition activity. However, analysis of the E. coli extract by SDS polyacrylamide gel electrophoresis revealed that only a small amount of recombinant polypeptide was detected as compared to E. coli crushing solution, and only a part of the produced polypeptide was expressed as an active soluble polypeptide. It became.
Flavobacterium sp. The fucoidan degrading enzyme of SA-0082 is an extracellular secretory enzyme, and it is expected that the secretion signal for membrane permeation is expressed in the form of a precursor added to the N terminus. Therefore, when the N-terminal amino acid sequences encoded by fdlA and fdlB were examined, regions from the starting methionine to the 25th and 24th alanine residues were expected to be secreted signals. Since the presence of this hydrophobic secretion signal was considered to be related to the solubility of each of the recombinant polypeptides described in Examples 5 and 6, an expression plasmid was constructed except for the secretion signals of fdlA and fdlB.
(1) First, primers FDL-Q-Bam (SEQ ID NO: 27) and primer 10X6R4 (SEQ ID NO: 28) were designed and synthesized to introduce a restriction enzyme BamHI site immediately upstream of the 26th glutamine residue from the starting methionine of fdlA. It was. FDL-Q-Bam is 28 mer of synthetic DNA with a BamHI site located upstream of the sequences SEQ ID NOs: 76-95 of SEQ ID NO: 7 in the Sequence Listing. Also, 10X6R4 is 21 mer of synthetic DNA complementary to the sequence of SEQ ID NOs: 1471-1491 of SEQ ID NO: 7 in the Sequence Listing.
Using pEFLA10 constructed in Example 5 as a template, a PCR reaction was carried out with a pair of primers FDL-Q-Bam and 10X6R4. In the PCR reaction, the same reaction solution composition as in Example 4- (4) was subjected to 25 cycles of denaturation at 94 ° C. for 0.5 minutes, primer annealing at 50 ° C. for 1 minute, and synthesis reaction at 72 ° C. for 2 minutes. The synthesis was completed by incubation at 72 ° C. for 7 minutes. After the reaction solution was separated by agarose gel electrophoresis, the amplified DNA fragment of about 1.4 kbp was extracted and purified. In the same manner as in Example 4- (4), the fragment was prepared by plasmid DNA inserted into a pT7 blue T-vector (manufactured by Novagen) to confirm the nucleotide sequence. The resulting plasmid was cleaved at the BamHI site and the SnaBI site within the fdlA coding region, placed in the primer FDL-Q-Bam, separated by 5% polyacrylamide gel electrophoresis, after the 26th glutamine residue from the starting methionine of fdlA. About 480 bp BamHI-SnaBI fragment encoding the N-terminal region of was cut out and purified.
On the other hand, pET21a (manufactured by Novagen), which is an expression vector using the T7 promoter of the same formula as Example 5, was cleaved at the HindIII site in the multiple cloning site, and the C-terminal region containing the stop codon of the fdlA gene prepared in Example 5 A plasmid pEFDLA-C was constructed by inserting a coding sequence of about 2 kbp HindIII DNA so that the T7 promoter and the fdlA gene were in the same direction. This pEFDLA-C was cleaved at the BamHI site derived from the multiple cloning site and the SnaBI site within the fdlA coding region to encode an N terminal region after the 26th glutamine residue from the starting methionine of fdlA prepared above, and about 480 bp BamHI-SnaBI. The fragment was inserted to obtain plasmid pEFDLA101.
This plasmid pEFDLA101 encodes a polypeptide linked to the N-terminal leader sequence (SEQ ID NO: 29) of 14 residues derived from pET21a downstream of the T7 promoter, followed by the 26th and subsequent sequences of SEQ ID NO: 3 in the Sequence Listing.
(2) The sequence of 20 bases after the 26th glutamine residue of fdlA used in the design of primer FDL-Q-Bam is similar to the sequence after the 25th glutamine residue of fdlB (SEQ ID NO: 73 of SEQ ID NO: 8 of Sequence Listing). -92). Therefore, the primer FDL-Q-Bam used in Example 8- (1) can be used for constructing an fdlB expression vector as it is, and basically constructing an fdlB expression vector pEFDLB101 using the same method as Example 8- (1). It was.
First, in order to perform PCR reaction using FDL-Q-Bam, 20 mer of synthetic DNA, 17X6R1 (SEQ ID NO: 30), complementary to the sequence of SEQ ID NO: 1489-1508 of SEQ ID NO: 8 of Sequence Listing was produced. . Using pEFLB17 constructed in Example 6 as a template, PCR reaction was carried out using a pair of primers FDL-Q-Bam and 17X6R1 to extract and purify the amplified DNA fragment of about 1.4 kbp. This fragment was inserted into the pT7 blue T-vector to prepare plasmid DNA and to confirm the nucleotide sequence.
Subsequently, the obtained plasmid was cleaved at the BamHI site and the NspV site located in the fdlB coding region placed in the primer FDL-Q-Bam, separated by 5% polyacrylamide gel electrophoresis, and separated from the starting methionine of fdlB. An approximately 210 bp BamHI-NspV fragment encoding the N terminal region after the glutamine residue was cut out and purified.
On the other hand, the plasmid pH17X6-7 obtained in Example 4 was cut at the EcoRI site, and an approximately 2.6 kbp EcoRI fragment including the full length of the fdlB gene was cut out and purified. This fragment was inserted into the EcoRI site in the multiple cloning site of pET21a so that the T7 promoter and the fdlB gene were in the same direction to construct the plasmid pEFDLB-W. This pEFDLB-W was cleaved at the BamHI site and the NspV site within the fdlB coding region derived from the multiple cloning site to encode an N terminal region after the 25th glutamine residue from the starting methionine of fdlB prepared above, about 210 bp of BamHI- The NspV fragment was inserted to obtain plasmid pEFDLB101.
This plasmid pEFDLB101 has a 14-terminal N-terminal leader sequence (SEQ ID NO: 29) that is exactly the same as the plasmid pEFDLA101 obtained in Example 8- (1) downstream of the T7 promoter, followed by the 25th or later sequence of SEQ ID NO: 4 in the Sequence Listing. The subsequent polypeptide is encoded.
(3) Using pEFDLA101 and pEFDLB101 obtained in Example 8- (1) and (2), culture of recombinants and preparation of cell extracts were carried out in the same manner as in Example 5, and in Example 5 and Example 6 The expression level and fucoidan degrading activity of each recombinant polypeptide by pEFLA10 and pEFLA17 thus constructed were compared.
That is, E. coli BL21 (DE3) strain was transformed using pEFLA10, pEFLA17, pEFDLA101 or pEFDLB101. The obtained transformants were inoculated in 5 ml of L medium containing 100 µg / ml of ampicillin, respectively, and shaken at 37 ° C. After the culture solution had a turbidity of O.D. 600 = 0.8, IPTG was added so that the final concentration was 1 mM, and the culture was further shaken overnight at 15 ° C. After completion of the culture, the culture solution was centrifuged to collect the cells, suspended in 0.5 ml of cell disruption buffer, and the cells were disrupted by sonication. A part of this suspension was taken as E. coli crushing liquid. In addition, this was centrifuged to remove insoluble matter, to obtain an E. coli extract.
Each of the E. coli lysate and extract was separated by SDS polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining and interpreted according to a conventional method. Was observed. At the same time, the expression level was approximated by comparison with the bovine serum albumin amount electrophoresis, and fucoidan decomposition activity in each extract was measured by the method described in Reference Example 3. These results are summarized in Table 2.
PlasmidgeneAmount of expressed protein per 1 L of culture medium (mg / L)Active amount (mU / mL) extract per 1ml of culture pEFLA10fdlA5One200 pEFDLA101fdlA10101300 pEFLA17fdlB35*34 pEFDLB101fdlB75755700 * Since it is below the detection limit, it cannot be estimated.
As shown in Table 2, pEFDLA101 (fdlA) and pEFDLB101 (fdlB) constructed to add an N-terminal leader sequence instead of a sequence expected to be a secretion signal are expressed in the amount of the target polypeptide contained in the lysate. Compared with the direct expression plasmids pEFLA10 (fdlA) and EFLA17 (fdlB), the genes showed about twice as high yield.
In addition, in the case of having a secretion signal, the amount of the target polypeptide present in the extract solution is extremely small compared to the lysate solution, whereas most of the expressed polypeptides are insoluble, whereas when the secretion signal is removed, Nearly equivalent amounts of the desired polypeptide were detected in the extract, and it became clear that the expressed polypeptide was present almost as soluble. In addition, the amount of activity in the extract was also significantly increased corresponding to the amount of the target polypeptide present.
Example 9
The action of fucose sulfate-containing polysaccharide-F of polypeptides obtained in Examples 2 and 3 with fucose sulfuric acid-containing polysaccharide decomposition activity was investigated.
That is, 500 µl of 50 mM imidazole-HCl buffer (pH7.5), 50 µl of fucose sulfate-containing polysaccharide-F 2.5% solution described in Reference Example 1- (2), 50 µl of 1M calcium chloride and 75 µl of To the mixed solution of 4M sodium chloride, 100 µl of a polypeptide solution having 9.7 mU / ml of fucose sulfuric acid-containing polysaccharide decomposition activity obtained in Example 2 or Example 3 was added, and distilled water was added to make the whole amount 1 ml. After 18 hours of reaction at 25 ° C., the reaction product was analyzed by HPLC. The conditions of HPLC were as follows.
Column: Shodex SB804 (manufactured by Showa Denko Co., Ltd.)
Column temperature: 25 ℃
Eluate: 50 mM sodium chloride solution containing 5 mM sodium azide
Flow rate: 1 ml / min
Detector: Differential refractive index detector (manufactured by Shodex RI-71 Showa Denko)
As a result of the analysis of the reaction product produced by the action of the polypeptide encoded by the gene of the present invention, all of the reaction products in the case of using the polypeptide having the fucose sulfate-containing polysaccharide decomposition activity obtained in Examples 2 and 3 were used as HPLC. Retention time detected two peaks, 8.62 minutes and 9.30 minutes.
When the polypeptide having the fucose sulfate-containing polysaccharide degrading activity obtained in Example 2 was used and the polypeptide having the fucose sulfate-containing polysaccharide degrading activity obtained in Example 3 was used, It was producing a lot of material.
According to the present invention, the amino acid sequence and the nucleotide sequence of a polypeptide having a fucose sulfuric acid-containing polysaccharide decomposition activity are finally clarified, thereby making it possible to provide a polypeptide having a fucose sulfuric acid-containing polysaccharide decomposition activity. Provided are industrially advantageous methods for producing genetically engineered polypeptides having polysaccharide degradation activity.
According to the present invention, it is not necessary to add the fucose sulfate-containing polysaccharide to the medium for the induction production of the fucose sulfate-containing polysaccharide degrading enzyme, and the productivity is high. In addition, enzymes such as proteases and other polysaccharide degrading enzymes are not produced at the same time, and purification is easy. By providing the amino acid sequence and the nucleotide sequence of the polypeptide having a fucose sulfate-containing polysaccharide decomposition activity, to produce a polypeptide antibody having anti-fucose sulfate-containing polysaccharide degradation activity based on the amino acid sequence, or containing a fucose sulfate based on the nucleotide sequence Probes and primers specific for the nucleotide sequence of a polypeptide having polysaccharide degradation activity can be produced.
SEQUENCE LISTING
<110> Takara Shuzo Co., Ltd.
〈120〉 GENES
<130> F-3528
〈140〉 PCT / JP98 / 02310
<141> 1998-05-26
〈150〉 252624/97
<151> 1997-09-03
〈160〉 30
<170> Patent In Ver. 2.0
〈210〉 1
<211> 814
<212> PRT
213 Alteromonas sp.
<400> 1
Met Lys Ile Arg Asn Val Cys Arg Ser Ala Val Leu Leu Gly Leu Met
1 5 10 15
Ser Leu Asn Thr Tyr Ala Glu Thr Lys Ala Asp Trp Met Gln Gly Asn
20 25 30
Trp Gly Ile Ser Tyr Arg Ile Pro Gly Gly Asp Ile Asn Tyr Ser Gly
35 40 45
Ser His Val Ala Glu Tyr Asn Val Arg Ala Ala Val Glu Gln Ile Ser
50 55 60
Ala Ile Pro Gly Leu Lys Trp Val Gln Ile Asn Leu Thr Asn Gly Ala
65 70 75 80
Ser Gly Asp Arg Phe Ile Val Pro Val Thr Glu Val Glu Ala Ile Asn
85 90 95
Pro Leu Ser Ala Pro Asn Ser Ile Asn Asp Leu Tyr Asp Pro Thr Leu
100 105 110
Pro Gly Arg Asp Leu Phe Glu Gln Leu Ala Leu Ala Phe Lys Ala Lys
115 120 125
Gly Ile Arg Val Val Ala Tyr Ile Ala Thr Gln Gly Pro Gly Met Leu
130 135 140
Lys His Gly Ala Glu Asn Ser Met Asp Glu Asp Asp Ser Ile Thr Asp
145 150 155 160
Cys Lys Ser Ser Lys Pro Leu Val Thr Asp Leu Asp Thr Gln Val Tyr
165 170 175
Cys Ser Ala Asn Met Asn Arg Trp Arg Asp Tyr Val Leu Glu Gln Tyr
180 185 190
Pro Ser Thr Ser Leu Tyr Arg Ser Phe Glu Leu Ala Met Val Asn Ile
195 200 205
Val Glu Thr Leu Ser Leu Arg Tyr Gly Ser Thr Ile Asp Gly Trp Trp
210 215 220
Phe Asp His Ser Gly Phe Gly Asp Ser Glu Leu Leu His Ala Ala Ala
225 230 235 240
Leu Ala Gly Asn Asn Asp Ala Ala Val Ala Phe Asn Glu Gly Asp Lys
245 250 255
Val Pro Leu Val Asn Asn Pro Glu Thr Leu Asp Asp Tyr Thr Phe Gly
260 265 270
His Pro Thr Pro Ile Gly Ser Glu Val Ser Ser Asp Asp Lys Asn Leu
275 280 285
Pro Met Leu Thr Ser Ile Glu Ala Thr Leu Asp Gly Ile Leu Thr Gly
290 295 300
Ser Gly Asp Asp Val Gly Ser Val Gly His Met Phe Met Pro Leu Gln
305 310 315 320
Glu Ser Trp Asn Gly Gly Thr Val Val Phe Ser Glu Ala Lys Gly Ser
325 330 335
Asp Trp Leu Asn Arg Ala Leu Lys Ala Gly Gly Ala Phe Thr Trp Ala
340 345 350
Leu Ser Gln Asp Ser Asn Asp Glu Leu Gly Gly Gly Gly Ala Arg Leu
355 360 365
Ile Ser Glu Pro Gln Val Lys Met Leu Glu Arg Met Ser Phe Asn Ile
370 375 380
Gly Lys Gln Leu His Met Asn Leu Asp Gly Ser Asp Gly Asp Thr Ala
385 390 395 400
Tyr Asp Asp Ser Val Asn Gln Tyr Thr Ala Thr Val Asn Gly Ala Asn
405 410 415
Phe Val Asp Asp Val Thr Arg Gly Lys Val Ala Ser Phe Thr Glu Asp
420 425 430
Asp Gln Leu Glu Leu Asp Asn Tyr Gln Gly Ile Ser Gly Gly Asn Ala
435 440 445
Arg Thr Thr Met Ala Trp Ile Lys Thr Ser Asp Ser Lys Gly Asp Ile
450 455 460
Ile Asp Trp Gly Asn Asn Thr Thr Ser Ser Glu Arg Trp Trp Leu Arg Leu
465 470 475 480
Val Asp Gly Lys Phe Lys Leu Ile Leu Lys Gly Pro Asn Leu Thr Gly
485 490 495
Thr Thr Thr Leu Asn Asp Asp Gln Trp His His Ile Ala Val Val Ala
500 505 510
Ser Asp Asn Val Val Ala Asn Ile Lys Val Tyr Ile Asp Gly Val Leu
515 520 525
Glu Thr Val Ala Val Asn Asp Asn Ala Ser Thr Thr Phe Asp Thr Thr
530 535 540
Leu Gly Gly Asn Ile Gln Ile Gly Gly Ala Tyr Thr Gly Leu Ile Asp
545 550 555 560
Lys Val Leu Val His Asp Arg Ala Leu Asp Glu Ser Glu Ile Glu Tyr
565 570 575
Val Val Asn Ser Ser Asn Ala Asp Leu Asp Leu Glu Val Ala Leu Asp
580 585 590
Val Arg Phe Glu Glu Ser Ala Asn Ser Thr Lys Val Thr Asp Asn Ser
595 600 605
Ile Tyr Gly Arg His Gly Thr Asn Arg Gly Ala Ile Thr Gly Val Phe
610 615 620
Asp Ala Glu Arg Asn Ser Asn Val Tyr Ser Leu Asp Gly Val Asp Ser
625 630 635 640
Gly Glu Asp Ile Asn Asp Leu Lys Asp Ser Asp Tyr Glu His Glu Val
645 650 655
Val Met Thr Thr Asp Asn Ser Lys Asp Ser Lys Gly Tyr Ser Gly Val
660 665 670
Asn Gly Ala Gly Pro Arg Thr Val Met Ala Trp Ile Lys Thr Thr Phe
675 680 685
Gly Gly Ala Val Ile Ala Gln Trp Gly Asn Lys Asn Ser Val Asp Gly
690 695 700
Glu Gln Tyr Glu Val Arg Leu Lys Asn Gly Ala Leu Arg Leu Asp Ile
705 710 715 720
Thr Gly Gly Ile Ile Lys Gly Thr Thr Ser Ser Ile Asn Asp Gly Glu Trp
725 730 735
His His Ile Ala Val Val Ser Pro Asp Glu Gln Leu Ala Asn Thr Lys
740 745 750
Leu Tyr Val Asp Gly Val Leu Glu Thr Ala Thr Thr Ser Gly Ser Gln
755 760 765
Ala Thr Ile Asp Thr Lys Thr Leu Asn Gly Asp Ser Lys Asp Val Ile
770 775 780
Ile Gly Ser Thr Phe Val Gly Glu Met Asp Asp Phe Ile Ile His Gln
785 790 795 800
Arg Ala Leu Arg Gln Phe Glu Val Lys Asn Ser Ala Gly Leu
805 810
〈210〉 2
<211> 881
<212> PRT
213 Alteromonas sp.
<400> 2
Met Lys Ile Arg Asn Met Cys Cys Thr Ala Leu Ile Val Ser Leu Met
1 5 10 15
Gly Cys Gly Gly Ser Gly Ser Glu Ala Ser Ser Pro Glu Val Glu Val
20 25 30
Asp Asn Gly Val Glu Ile Gln Pro Glu Pro Glu Val Glu Pro Glu Pro
35 40 45
Glu Val Glu Pro Glu Pro Glu Val Glu Pro Glu Pro Glu Val Glu Pro
50 55 60
Glu Pro Glu Val Glu Pro Glu Pro Glu Val Glu Pro Glu Pro Glu Val
65 70 75 80
Glu Pro Glu Pro Glu Asp Ile Arg Ala Ser Trp Met Gln Gly Asn Trp
85 90 95
Gly Ile Ser Phe Arg Ile Ser Gly Gly Asp Ile Ser Gln Asn Glu Ser
100 105 110
His Val Asn Glu Tyr Gln Val Ala Pro Ala Val Glu Gln Ile Ala Ala
115 120 125
Ile Pro Gly Leu Lys Trp Leu Gln Val Asn Leu Ser Asn Gly Ala Phe
130 135 140
Gly Asp Arg Phe Ile Val Pro Val Pro Glu Val Glu Ala Ile Asn Pro
145 150 155 160
Asn Ser Ala Pro Asn Ser Ser Ala Asp Leu Phe Asp Pro Ala Leu Pro
165 170 175
Gly Asp Asp Leu Phe Glu Gln Ile Ala Leu Gly Leu Gln Ala Lys Gly
180 185 190
Ile Lys Val Val Ala Tyr Ile Ala Thr Gln Gly Pro Ala Met Leu Lys
195 200 205
His Gly Ala Glu Arg Ser Met Asp Phe Asp Asp Ser Ile Val Asp Glu
210 215 220
Ser Asp Gly Ser Ala Cys Lys Ser Ser Arg Pro Val Val Ser Asp Pro
225 230 235 240
Asp Thr Gln Val Tyr Cys Ser Ala Asn Met Asn Arg Trp Arg Asp Tyr
245 250 255
Val Leu Gln Gln Tyr Pro Ser Thr Ser Leu His His Ser Phe Gln Leu
260 265 270
Gly Leu Val Asn Ile Val Glu Thr Leu Ser Leu Arg Tyr Gly Thr Leu
275 280 285
Ile Asp Gly Trp Trp Phe Asp His Ser Ile Tyr Gly Asp Tyr Asn Leu
290 295 300
Leu Pro Asp Ala Ala Arg Ala Gly Asn Ser Asn Ala Ala Val Ser Leu
305 310 315 320
Asn Leu Glu Gly Asp Ile Phe Leu Ser Asn Asn Pro Glu Val Met Glu
325 330 335
Asp Phe Thr Gly Gly His Pro Thr Pro Ile Ala Arg Val Val Ser Ser
340 345 350
Asp Asp Thr Asn Leu Pro Met Leu Thr Ala Ile Glu Asp Ala Pro Asn
355 360 365
Gly Ile Phe Thr Gly Thr Gly Asp Asp Val Asp Ala Leu Gly His Met
370 375 380
Phe Leu Pro Leu Gln Glu Thr Trp Asn Gly Gly Thr Val Val Phe Ser
385 390 395 400
Glu Ala Lys Gly Thr Glu Trp Leu Asn Arg Val Thr Arg Ala Gly Gly
405 410 415
Ala Leu Thr Trp Ala Leu Ser His Glu Gly Ser Val Ser Gly Gly Glu
420 425 430
Ala Met Leu Ile Ser Ala Pro Gln Ala Lys Met Leu Ala Arg Met Gln
435 440 445
Leu Asn Ile Gly Lys Gln Leu Asp Met Asp Leu Asp Gly Ala Asp Gly
450 455 460
Ala Thr Ala Tyr Asp Asp Ser Val Asn Gln His Thr Ala Thr Val Thr
465 470 475 480
Gly Ala Thr Phe Ile Asp Asp Val Thr Arg Glu Lys Val Ala Ser Phe
485 490 495
Thr Glu Thr Asp Leu Ile Thr Leu Asn Asn Phe Thr Gly Ile Leu Gly
500 505 510
Glu Ser Ala Arg Thr Thr Met Ala Trp Ile Lys Thr Ser Asp Ser Asn
515 520 525
Ala Asp Val Ile Gln Trp Gly Lys Gln Glu Thr Ser Glu Ala Trp Tyr
530 535 540
Val Gly Leu Asp Asn Gly Ile Leu Gln Leu Asn Ile Gln Gly Ser Thr
545 550 555 560
Val Ile Gly Ala Ser Val Leu Asn Asp Asp Ser Trp His His Ile Ala
565 570 575
Val Ile Ala Pro Asp Asn Ser Ile Ala Asn Thr Gln Val Tyr Ile Asp
580 585 590
Gly Val Leu Glu Thr Leu Thr Val Asn Asp Gly Gly Ser Ser Thr Phe
595 600 605
Asn Thr Val Ala Asp Thr Asn Val Val Ile Gly Gly Glu Phe Thr Gly
610 615 620
Leu Ile Asp Lys Thr Val Val Tyr Asn Arg Ala Leu Glu Glu Ser Glu
625 630 635 640
Ile Asp Tyr Ile Val Asn Ser Ala Asp Ala Asp Ile Asp Leu Gly Ile
645 650 655
Ser Leu Asp Val Arg Phe Asp Glu Asp Ala Asn Ala Thr Thr Val Ala
660 665 670
Asp Asn Ser Ala Tyr Glu Arg Ser Gly Ile Asn Arg Gly Ala Ile Thr
675 680 685
Gly Val Phe Asp Ala Thr Arg Asn Ser Asn Val Tyr Ser Leu Asp Gly
690 695 700
Val Asp Ser Gly Glu Asp Leu Asp Asp Leu Ile Asp Ser Asp Tyr Glu
705 710 715 720
His Gln Ile Val Met Thr Thr Asn Asn Lys Arg Asp Asn Lys Gly Tyr
725 730 735
Ser Gly Val Asn Gly Gly Asp Pro Arg Thr Val Met Ala Trp Ile Lys
740 745 750
Thr Thr Phe Gly Gly Ala Val Ile Ala Gln Trp Gly Asn Lys Asp Ser
755 760 765
Val Asp Gly Glu Gln Tyr Glu Val Arg Leu Lys Asn Gly Glu Leu Arg
770 775 780
Val Asp Ile Thr Gly Gly Leu Ile Lys Gly Thr Thr Leu Ile Asn Asp
785 790 795 800
Gly Glu Trp His His Ile Ala Val Val Ser Pro Asp Asp Gln Leu Ala
805 810 815
Asn Thr Lys Leu Tyr Val Asp Gly Val Leu Glu Thr Thr Thr Thr Ser
820 825 830
Gly Ser Gln Thr Thr Ile Asp Thr Leu Thr Leu Asn Gly Asp Ser Lys
835 840 845
Asp Val Ile Ile Gly Ser Thr Phe Val Gly Glu Met Asp Asn Phe Val
850 855 860
Ile His Gln Arg Ala Leu Lys Gln Phe Glu Val Lys Val Ala Ala Gly
865 870 875 880
Ile
〈210〉 3
<211> 697
<212> PRT
213 Flavobacterium sp.
<400> 3
Met Ile Lys Lys Tyr Asn Leu Ile Lys Thr Gly Val Ile Thr Phe Leu
1 5 10 15
Val Leu Phe Phe Gln Gln Thr Tyr Ala Gln Thr Thr Thr Val Tyr Ser
20 25 30
Leu Glu Asp Leu Leu Pro Tyr Leu Lys Gln Asp Asn Val Asp Val Lys
35 40 45
Leu Ala Pro Gly Thr Tyr Asn Val Asn Gly Phe Asp Val Gly Glu Asp
50 55 60
Arg Leu Phe Ser Thr Thr Pro Leu Phe Leu Phe Glu Gly Ser Asn Ser
65 70 75 80
Thr Tyr Asp Phe Thr Asp Val Lys Leu Asn Ile Asn Thr Val Val Leu
85 90 95
Thr Lys Phe Gly Asn Asn Glu Val Asn Glu Ile Gln Ile Leu Gly Asn
100 105 110
Asn Asn Val Leu Lys Asn Leu Lys Leu Glu Asp Ile Gly Thr Thr Ala
115 120 125
Pro Ser Asn Arg Ala Gln Ser Ile Val Ile Asp Gly Arg Asp Asn Arg
130 135 140
Ile Glu Gly Phe His Leu Thr Ile Arg Gly Ser Tyr Pro Tyr Gly Tyr
145 150 155 160
Gly Asp Ala Phe Gly Lys Gly Gly Gly Ser Val Ile Asn His Arg Lys
165 170 175
His Ser Gly Val Leu Ile Arg Gly Leu Arg Asn His Leu Lys Asp Cys
180 185 190
Thr Ile Ile Ser Arg Ser Tyr Gly His Ile Val Phe Met Gln Ala Ala
195 200 205
Ser Tyr Pro Thr Val Glu Gly Cys Tyr Ile Glu Gly Glu Met Arg Ser
210 215 220
Thr Asp Asp Met Leu Ala Glu Glu Gly Thr Gly Ser Pro Ala Asp Lys
225 230 235 240
Val Asp Phe Met Thr Val Trp Gly Tyr Lys Leu Pro Ala Gly Tyr Met
245 250 255
Met Ser Leu Gln Glu Gly Gly Ile Arg Ala Tyr Asn Ala Gly Thr Thr
260 265 270
Tyr Ile Asp Gly Val Glu Ile Gln Arg Ala Thr Asp Asn Pro Thr Val
275 280 285
Leu Asn Cys Thr Ile Lys Asn Ala Arg Thr Gly Val Thr Leu Ala His
290 295 300
Ala Asn Gly Thr Lys Tyr Val Glu Gly Cys Thr Val Leu Gly Cys Glu
305 310 315 320
Asn Gly Tyr Ser Ile Gly Ser Gly Thr Val Val Asn Cys Gly Ala Asp
325 330 335
Ala Ile Tyr Gly Pro Val Phe Lys Asn Thr Tyr Gly Ser Asp Lys Gly
340 345 350
Tyr Asn Ala Asp Ile Thr Ile Leu Pro Pro Ser Asp Ala Tyr Tyr Asn
355 360 365
Gly His Asp Ala Val Ala Tyr Ile Gly Gly Ser Asn His Asn Leu Thr
370 375 380
Phe Arg Ser Glu Ile Thr Glu Ile Pro Ser Asn Leu Lys Ile Met Val
385 390 395 400
Ser Gly Asp Leu Gln Gly Leu Arg Val Leu His Gly Ser Asn Pro Ser
405 410 415
Gln Asn Asn Phe Ala Gly Thr Asn Ile Val Leu Arg Asn Leu Thr Asn
420 425 430
Phe Pro Val Asp Leu His Ser Asp Ser Ser Asn Ile Thr Val Thr Ser
435 440 445
Cys Asp Thr Asp Asn Ile Thr Asp Asn Gly Thr Asn Asn Ser Ile Glu
450 455 460
Ala Ile Asp Cys Asp Ser Asp Asn Leu Ala Leu Lys Gly Glu Ala Ser
465 470 475 480
Gln Ser Ser Ser Arg Pro Ser Asp Gly Phe Ala Ala Asn Ala Ile Asp
485 490 495
Gly Asn Thr Asn Gly Ala Trp Ser Asn Asn Ser Val Ser His Thr Gly
500 505 510
Thr Glu Glu Asn Pro Trp Trp Gln Val Asp Leu Gly Thr Asp Ala Ile
515 520 525
Ile Gly Ser Ile Asn Ile Phe Asn Arg Thr Asp Gly Cys Cys Lys Gly
530 535 540
Arg Leu Asp Asn Phe Thr Val Tyr Val Ile Asp Lys Asp Asp Lys Val
545 550 555 560
Thr Phe Ser Lys Thr Tyr Val Thr Val Pro Asp Pro Ser Ile Thr Val
565 570 575
Asp Ala Gly Gly Val Asn Gly Lys Ile Val Lys Ile Val Leu Asn Asn
580 585 590
Ser Ser Gln Ala Leu Ala Leu Ala Glu Val Glu Val Tyr Gly Thr Ser
595 600 605
Leu Ser Asn Lys Glu Thr Ile Lys Asn Pro Ile His Phe Tyr Pro Asn
610 615 620
Pro Val Glu Asp Glu Val Thr Ile Ser Leu Glu Ser Ala Asp Leu Asn
625 630 635 640
Leu Asn Glu Thr Arg Val Val Ile Tyr Asn Ile Lys Gly Gln Lys Ile
645 650 655
Leu Glu Thr Thr Pro Ser Asn Ser Thr Glu Val Asn Leu Asn Leu Ser
660 665 670
His Leu Pro Thr Gly Val Tyr Leu Ile Arg Val Ser Asp Gln Asn Lys
675 680 685
Asn Ile Ile Asn Lys Ile Val Lys Leu
690 695
〈210〉 4
<211> 704
<212> PRT
213 Flavobacterium sp.
<400> 4
Met Lys Lys Tyr Ser Ile Leu Lys Ile Gly Ile Ile Ala Val Ile Met
1 5 10 15
Leu Phe Val Gln Gln Ser Tyr Ala Gln Thr Thr Thr Thr Tyr Ser Leu
20 25 30
Glu Asp Leu Leu Pro Tyr Leu Lys Gln Asp Asn Val Asp Val Lys Leu
35 40 45
Ala Pro Gly Thr Tyr Asn Ile Asn Ala Phe Asp Ile Thr Gln Gly Lys
50 55 60
Phe Ser Asn Pro Leu Phe Leu Phe Glu Gly Ser Asn Asn Thr Phe Asp
65 70 75 80
Phe Thr Asp Val Lys Ile Asn Ile Asn Thr Leu Val Leu Thr Lys Phe
85 90 95
Gly Asn Asn Glu Val Asn Glu Ile Gln Ile Leu Gly Asn Asn Asn Val
100 105 110
Leu Lys Asn Leu Lys Leu Glu Asp Ile Gly Thr Thr Ala Pro Ser Asn
115 120 125
Arg Ala Gln Ser Ile Ile Met Asp Gly Arg Asp Asn Arg Ile Glu Gly
130 135 140
Phe His Leu Thr Ile Arg Gly Ser Tyr Pro Tyr Gly Tyr Gly Asp Ala
145 150 155 160
Phe Gly Lys Gly Gly Gly Ser Val Ile Asn His Arg Lys His Ser Gly
165 170 175
Val Leu Ile Arg Gly Leu Arg Asn His Leu Lys Asp Cys Thr Ile Ile
180 185 190
Ser Arg Ser Tyr Gly His Ile Val Phe Met Gln Ala Ala Ser Tyr Pro
195 200 205
Thr Val Glu Gly Cys Tyr Ile Glu Gly Glu Met Arg Ser Thr Asp Asp
210 215 220
Met Leu Ala Glu Glu Gly Thr Gly Ser Pro Ala Asp Asn Val Asp Phe
225 230 235 240
Met Thr Val Trp Gly Tyr Lys Leu Pro Ala Gly Tyr Met Met Ser Leu
245 250 255
Gln Glu Gly Gly Ile Arg Ala Tyr Asp Ala Gly Thr Thr Tyr Ile Asp
260 265 270
Gly Glu Val Ile Gln Arg Ala Thr Asp Asn Pro Thr Val Leu Asn Cys
275 280 285
Thr Ile Lys Asn Ala Arg Thr Gly Val Thr Leu Ala His Ala Lys Gly
290 295 300
Thr Lys His Val Glu Asn Val Lys Ala Ile Gly Cys Glu Gln Gly Tyr
305 310 315 320
Ser Ile Gly Ser Gly Thr Val Ser Asn Cys Ser Gly Asp Ala Gln Tyr
325 330 335
Gly Pro Leu Leu Ser Phe Ala Tyr Ser Ser Asp Lys Asn Thr Asn Ile
340 345 350
Asp Ile Glu Val Leu Pro Ala Glu Asn Tyr Tyr Asn Gly Ser Glu Thr
355 360 365
Ala Ala Tyr Val Gly Gly His Ser His Asn Ile Thr Leu Arg Gly Gly
370 375 380
Asp Pro Asn Ala Asp Leu Arg Val Gln Val Gly Gly Glu Lys Asn Asn
385 390 395 400
Val Arg Leu Leu Gly Val Thr Ser Asn Gln Asn Pro Leu Ser Ala Ser
405 410 415
Asn Leu Glu Leu Asn Asn Leu Thr Asn Phe Pro Val Val Leu Asp Glu
420 425 430
Met Ser Ser Asn Ile Ile Val Glu Ser Cys Gly Glu Val Thr Asn Asn
435 440 445
Gly Ser Asn Asn Ser Ile Thr Asp Cys Pro Asp Gly Pro Ile Ser Phe
450 455 460
Pro Asp Ser Ser Lys Ala Tyr Arg Leu Gly Asn Asn Arg Phe Thr Phe
465 470 475 480
Trp Val Ala Ala Asn Gly Gly Asp His Ala Tyr Ser Ile Lys Tyr Asn
485 490 495
Asp Gly Ile Ser Gly Asn Ile Asn Asp Tyr Glu Asp Leu Phe Pro Glu
500 505 510
Gly Glu Glu Ser Phe Trp Val Phe Thr Pro Val Glu Gly Arg Asp Gly
515 520 525
Tyr Phe Phe Val Asp Cys Val Gly Gly Gly Asp Lys Gln Arg Leu Ser
530 535 540
Ala Thr Thr Asp Ser Gly Leu Pro Val Met Val Ser Lys Thr Ile Thr
545 550 555 560
Ser Ala Ser Val Gln Trp Ser Val Val Gln Pro Glu Gly Arg Asp Thr
565 570 575
Phe His Ile Thr Asn Asp Tyr Ala Arg Met Val Gly Ala Asn Thr Thr
580 585 590
Thr Asn Gln Thr Ile Leu Ser Thr Val Gly Asn Thr Ser Asn Gln Ser
595 600 605
Arg Phe Glu Val Leu Glu Val Ser Asn Tyr Ser Leu Ser Ile Lys Asn
610 615 620
Asp Ile Leu Asn Asn Asn Ile Thr Val Phe Pro Ile Pro Thr Ser Asp
625 630 635 640
Ile Leu Asn Ile Asn Leu Lys Asn Met Glu Ser Val Thr Val Glu Leu
645 650 655
Tyr Asn Ser Ile Gly Gln Lys Ile Leu Ser Lys Glu Ile Lys Gln Gly
660 665 670
Glu Asn Thr Leu Asn Leu Ser Gly Ile Tyr Thr Gly Val Tyr Leu Leu
675 680 685
Lys Leu Asn Asp Gly Gln Asn Ser Tyr Thr Lys Arg Ile Ile Met Lys
690 695 700
〈210〉 5
<211> 2442
<212> DNA
213 Alteromonas sp.
<400> 5
atgaaaatac gtaatgtttg tcgtagtgcg gtgcttttag gcttgatgtc tttaaataca 60
tacgcagaaa caaaagctga ttggatgcaa ggtaactggg ggatcagtta tcgaatacct 120
ggaggagata ttaattactc aggtagtcat gttgcagaat acaatgtaag agccgcagtt 180
gaacaaatct cagcaattcc tggtttgaag tgggtacaaa ttaatttaac caacggtgca 240
tctggtgatc gttttatagt ccctgtaaca gaagttgaag ccattaatcc tttatccgct 300
cctaacagta ttaatgactt atacgatcct actttacctg ggcgagatct ttttgagcaa 360
ctggcattag ccttcaaagc taaaggcata agagttgttg cttatattgc gactcaaggg 420
cctggcatgc tcaagcatgg tgctgaaaac tcgatggatg aagatgactc cattactgac 480
tgtaaatcgt ctaagccatt agtaaccgat cttgatacac aagtttactg ttcagcaaat 540
atgaatcgct ggagagatta cgttttagaa caatacccat caaccagtct ttatagaagt 600
tttgaattgg caatggtcaa tattgtagaa acattatcac tgcgttatgg aagtacaatt 660
gatggctggt ggtttgatca ttcaggtttt ggtgacagtg aattacttca tgctgcggct 720
ctagctggaa ataatgatgc ggcagtagcc tttaatgaag gcgataaagt tcctttggta 780
aataacccag agacattaga cgattacacc tttggtcatc caacacctat aggtagtgag 840
gtttcttctg atgataaaaa cctacctatg ttaacgtcta tagaagctac tttagatggt 900
attttaactg gttcaggtga tgatgtaggc tctgtgggac atatgtttat gccacttcaa 960
gaaagttgga atggtggcac tgttgtattt tctgaagcga aaggatctga ctggcttaat 1020
cgagcattaa aagccggagg tgcatttaca tgggcactaa gccaagacag taatgatgag 1080
ttaggtggtg gcggagcaag attaatttca gaaccgcagg taaaaatgct tgaacgtatg 1140
agttttaata taggtaaaca attacatatg aatctagatg gttcagatgg tgatactgct 1200
tatgatgact ccgtcaacca atataccgct actgtaaacg gtgctaattt tgttgatgat 1260
gttacaagag gaaaagttgc aagttttact gaagacgacc agttagaact agacaattat 1320
caaggtattt caggtggaaa tgcgcgtaca accatggctt ggataaaaac ttcagacagc 1380
aaaggcgata ttattgattg gggtaataac acaacaagcg aacgttggtg gttacgttta 1440
gttgacggta aatttaaact gatattaaaa ggtcctaatc ttacaggaac tacaacactt 1500
aatgacgacc aatggcacca tattgctgtt gtagcttctg ataacgtagt tgctaatatc 1560
aaagtataca ttgatggtgt tttagaaact gttgctgtaa atgacaatgc ttcaactacc 1620
ttcgatacaa ccttaggtgg caatatacaa ataggtgggg cctacaccgg acttatcgat 1680
aaagtgcttg tgcatgatag agcattagat gaaagcgaga ttgagtatgt tgttaattca 1740
tccaatgctg atcttgattt agaggttgca ttagatgtgc gttttgaaga gtcagcaaac 1800
tcaactaaag taaccgataa ttctatatat ggacgtcatg gcacaaatcg aggtgctatt 1860
actggcgtgt ttgatgcaga acgtaacagc aatgtgtact cacttgatgg tgttgatagt 1920
ggcgaagata taaatgattt aaaagatagc gactacgaac atgaagttgt aatgacaaca 1980
gataattcta aagactcaaa aggttatagt ggagttaatg gtgcaggtcc gcgtactgta 2040
atggcatgga taaaaacaac ttttggcggt gctgttattg cccaatgggg taataaaaat 2100
tcagttgatg gcgaacaata tgaagttcgt ttaaaaaatg gtgcactgag attagatatt 2160
acaggtggca ttattaaagg cacaacatca attaatgatg gcgagtggca tcatattgct 2220
gtggtttcac ctgatgaaca gttagctaat actaaattgt atgttgatgg tgtactagaa 2280
acagcaacca cttcgggttc tcaagcaacg attgatacta aaactcttaa tggcgatagc 2340
aaagacgtaa taattggtag tacgtttgtt ggcgagatgg acgattttat tattcatcaa 2400
cgcgctttaa gacagtttga agtgaaaaac tcagcaggac tc 2442
〈210〉 6
<211> 2643
<212> DNA
213 Alteromonas sp.
<400> 6
atgaaaatac gtaatatgtg ttgtactgct ttaatcgtaa gtttaatggg ctgcggtggt 60
tctggttcag aagctagttc tcctgaagta gaagttgata atggagtaga aattcaacct 120
gaaccagaag ttgaacctga gccagaagtt gaacctgaac cagaagttga acctgaacca 180
gaagttgaac ctgaaccaga agttgaacct gagccagaag ttgagcctga accagaagtt 240
gaacctgaac cagaagatat aagagcctca tggatgcaag gtaactgggg aatcagcttc 300
agaatttctg gtggtgacat cagtcaaaat gaaagtcatg taaatgaata ccaagtagca 360
ccagctgttg agcaaatagc cgcaattcct ggattaaagt ggttacaagt taatttaagt 420
aacggggctt ttggcgaccg ttttattgta cctgtacctg aagtagaagc tattaatcca 480
aattcagcgc caaacagctc ggcagattta tttgatcctg cattacctgg cgatgactta 540
tttgaacaaa tagcactagg acttcaagcc aaaggcataa aagtagtagc atatattgcg 600
actcaaggtc ctgcaatgct gaaacatggc gcagaaagat cgatggattt tgatgattct 660
attgttgatg aatcagatgg cagtgcttgt aaatcttcaa gacctgtcgt ttctgatcct 720
gatacgcaag tttattgttc agcaaatatg aatcgctgga gagattatgt gttacagcaa 780
tacccatcaa caagtttgca tcatagtttt caattgggac tcgtcaatat tgtagaaact 840
ttatcactac gttacggcac tctgattgat ggttggtggt ttgatcattc tatttacggt 900
gactacaact tacttcctga tgctgcaaga gcgggaaata gcaatgctgc ggtttctctt 960
aatttagaag gggatatttt cttaagtaat aacccagaag tgatggagga ttttaccggc 1020
ggacatccaa caccgattgc tcgagttgtt tcatctgatg ataccaattt acccatgtta 1080
acggctatag aagatgctcc aaacggtatt tttacaggaa caggtgatga tgtagatgct 1140
ttagggcaca tgtttttacc gctgcaagaa acctggaatg gcggaactgt agtattttca 1200
gaagccaaag gaactgagtg gcttaacaga gttactcgag ctggcggcgc attaacttgg 1260
gcattaagcc atgaaggcag tgtttctggt ggtgaggcta tgttgatttc tgcaccacaa 1320
gcaaaaatgc ttgcacgtat gcagctaaat attggtaaac aactcgatat ggatttagat 1380
ggtgccgatg gcgctacggc ttatgatgat tctgtcaatc aacatacagc tacggttaca 1440
ggtgcgacat ttatagatga tgttactcgt gaaaaagtgg caagctttac tgaaacagat 1500
ctgattacgt taaacaattt tactggtatt ttaggcgaaa gtgctcgtac aacaatggct 1560
tggataaaaa catcagacag taacgcagat gttattcaat ggggtaaaca agagacgagt 1620
gaagcttggt atgtgggctt agacaatgga atacttcaat taaatattca aggttctacg 1680
gttattggcg caagtgtact taacgatgat agttggcatc atattgctgt tatcgcgcct 1740
gataattcaa ttgccaatac tcaagtctat atcgatggtg ttttagaaac acttaccgtg 1800
aatgatggtg gttcatctac atttaataca gtggcagaca ccaacgttgt aataggagga 1860
gagtttactg gccttataga taaaaccgtt gtgtataaca gagcattaga agaaagcgag 1920
attgattata ttgttaattc agctgacgca gatattgatt taggtatttc acttgatgtg 1980
aggtttgatg aagatgctaa tgcaacaaca gtagctgata attctgccta tgaacgttca 2040
ggtataaatc gaggtgccat tacgggcgtt tttgatgcaa cacgtaacag caatgtttat 2100
tcacttgatg gtgttgatag cggcgaagat ctagatgatt taatagatag tgattatgag 2160
catcaaattg ttatgacaac caataacaaa agagataaca aaggttatag tggcgtgaat 2220
ggcggtgatc ctcgaactgt tatggcatgg ataaaaacaa cctttggtgg tgctgttatt 2280
gctcaatggg gtaataaaga ttcagtcgat ggcgaacaat atgaagtgcg cttgaaaaat 2340
ggcgaactta gagtcgatat cactggcggg cttattaaag gaacaacatt aataaacgat 2400
ggcgaatggc atcatattgc tgttgtatct cctgatgatc aattagctaa cactaaactt 2460
tatgttgatg gtgttctaga aacgaccacc acctccggct ctcaaacaac aatagatacg 2520
ttaaccctta acggtgacag caaagacgta atcattggaa gtacttttgt tggcgagatg 2580
gataactttg ttattcatca acgtgcttta aaacaatttg aagtaaaagt cgccgcaggt 2640
att 2643
〈210〉 7
<211> 2091
<212> DNA
213 Flavobacterium sp.
<400> 7
atgataaaaa aatacaattt aattaaaaca ggagttatta catttctagt tttgtttttt 60
cagcaaactt acgcacaaac aaccacagta tattctttag aagacttact accctattta 120
aaacaggata atgtagatgt taaattagcc ccaggaactt ataatgttaa tggttttgat 180
gtaggtgaag acaggttgtt ttccactact ccactttttt tgtttgaagg gtctaacagt 240
acttatgact ttacagatgt aaagcttaac atcaatacgg ttgtgttaac caagtttgga 300
aataatgagg ttaatgaaat tcagatttta ggaaataaca atgttcttaa aaacttaaaa 360
ctagaagata ttggaacaac agctccttct aacagagctc agtctattgt tatagatggg 420
cgagacaata gaatagaagg ttttcattta accattagag gatcttaccc ttatggatat 480
ggagatgctt ttggaaaagg aggaggttcc gtaattaatc accgaaaaca ttcaggtgtt 540
ttaataagag gattacgtaa tcacctaaaa gattgtacca ttatttctcg ttcttatggg 600
catatagtat tcatgcaagc agcaagttac ccaactgtgg aaggttgtta tattgaaggt 660
gaaatgcgtt caaccgatga tatgttggca gaagaaggaa caggttctcc agcagataaa 720
gtagatttta tgacggtttg gggatataag ttaccagctg gttatatgat gagtttacaa 780
gaaggaggaa ttagagcata taatgcagga accacttata ttgatggagt agagattcaa 840
cgagcaacag acaaccctac cgttctaaat tgtactatta aaaatgcaag aacaggagta 900
acattagcac atgcaaatgg aacaaaatat gttgagggtt gtactgtttt aggatgtgaa 960
aatggatact ccataggaag tggaactgta gtaaactgtg gagcagatgc tatttatgga 1020
cctgtattta aaaatacata cggaagcgat aaagggtaca atgcagacat taccattttg 1080
ccacctagtg atgcttacta caacggacat gatgctgtag catacattgg aggatcaaat 1140
cataacctta cttttagaag tgaaataaca gaaattccaa gcaatttaaa aattatggtc 1200
tctggagatt tacaaggatt aagagtattg catggaagta atcctagtca gaataatttt 1260
gctggaacca acattgtttt aagaaattta acaaactttc ctgtagactt acattcagac 1320
agttctaata taactgttac ttcttgtgat acggataata ttacagacaa tggtacaaat 1380
aatagtattg aagctataga ttgcgattcg gataatttag ctttaaaagg agaagctagt 1440
caatcatcct ctcgtccaag tgatggtttt gcagcaaatg ccattgatgg aaatacaaat 1500
ggggcatggt caaacaattc tgtttctcat acgggtacag aagaaaatcc atggtggcaa 1560
gtagatttag gaacagatgc tattataggt agcatcaata tttttaacag aacagatggt 1620
tgttgtaaag gtagattaga taattttact gtttacgtga tagataaaga tgataaggtt 1680
acattttcta aaacctatgt taccgttcca gatccgtcta taactgttga tgcaggtggt 1740
gtgaatggaa aaattgtaaa aattgttttg aataacagtt cacaggcttt ggctttagca 1800
gaggtagaag tgtacggaac gtctttgtct aataaagaaa ctataaagaa tcctattcat 1860
ttttatccta acccggtaga agatgaggta actatttctt tagagtcagc cgatttaaat 1920
ttaaacgaga ctcgagttgt tatttataat ataaaaggtc aaaaaatact agaaacaact 1980
ccaagtaatt ccacggaagt taatttaaac ttatctcact taccaacagg agtttattta 2040
ataagagtaa gcgatcaaaa taaaaatatc ataaataaaa ttgtaaaatt a 2091
〈210〉 8
<211> 2112
<212> DNA
213 Flavobacterium sp.
<400> 8
atgaaaaaat attctatcct aaaaatagga attatagctg ttataatgtt gtttgttcag 60
cagtcttacg cacaaacaac cacagtatat tctttagaag acttactacc ctatttaaaa 120
caggataatg tagatgttaa attagcccca ggaacttata atatcaatgc atttgacatt 180
actcaaggaa aattttcgaa ccccttattt ctttttgaag ggtctaataa tacttttgat 240
tttacagatg ttaaaataaa catcaatact ctggtgttaa caaagtttgg gaataatgaa 300
gtcaatgaaa ttcagatttt aggaaataac aatgttctta aaaacttaaa actagaagat 360
attggaacaa cagctccttc taacagagcc cagtcaatta taatggatgg gcgagacaat 420
agaatagaag gctttcattt aaccattaga ggatcttatc cttatggata tggagatgct 480
tttggaaaag gaggaggttc cgtaattaat caccgaaaac attcaggtgt tttaataaga 540
ggattacgta atcacctaaa agattgtact attatttctc gttcttatgg gcatatagta 600
tttatgcaag cagcaagtta cccaactgta gaaggttgtt atattgaagg tgaaatgcgt 660
tcaaccgatg atatgttggc agaagaagga acaggttctc cagcggataa tgtagatttt 720
atgacggttt ggggatataa gttaccagct ggttatatga tgagtttaca agaaggagga 780
attagagctt atgatgctgg taccacttat attgatggag aagtaatcca aagagcaaca 840
gataacccta ccgttctaaa ttgtaccatt aaaaatgcaa gaacaggagt gactttagca 900
catgctaaag gaacaaaaca cgtagaaaat gttaaggcta ttgggtgtga gcaaggatat 960
tcaattggta gtggtacagt gagtaattgt agtggtgatg ctcagtatgg tccgttgtta 1020
agttttgctt attctagtga taaaaatacg aatatagaca tagaagtttt gcctgcagaa 1080
aattattata acggtagtga aactgctgct tacgttggag gacattctca taatattaca 1140
ctaagaggag gtgatcctaa tgcggatctt agagttcagg tagggggaga aaaaaataac 1200
gttaggttgc ttggagttac ttctaatcaa aatccacttt ctgcttcaaa tttggaactg 1260
aataatttaa ctaattttcc tgtagtgtta gatgaaatga gttctaatat tattgtggag 1320
tcatgtgggg aggttaccaa taacggaagt aataatagta ttactgactg cccagatgga 1380
ccaattagct ttccagattc aagcaaagcg tatcgtttag gaaataatag atttacattt 1440
tgggttgcgg ccaatggagg agatcatgct tattctataa agtataatga tggtattagt 1500
ggtaacatta atgattatga ggatttgttt ccagaaggag aagagtcttt ttgggttttt 1560
actccagtag agggaagaga cggatacttt tttgttgatt gtgttggtgg tggtgataaa 1620
caaagattgt cagctactac agatagtggc ttgccagtaa tggtgtcaaa aaccattaca 1680
agtgcatctg ttcaatggtc tgtagtgcaa ccagaaggaa gagatacttt ccatataacg 1740
aatgattatg ctagaatggt aggagctaat acaactacta atcaaaccat tttgtctact 1800
gttgggaaca cctcaaacca atctcgtttt gaagttcttg aagtttctaa ctattcttta 1860
agtattaaaa acgacatctt aaacaataat attacggttt ttcctattcc aacatctgac 1920
attcttaata taaatttaaa aaatatggag tctgttactg ttgaattata caactcaata 1980
ggtcaaaaaa tattatcaaa agaaattaaa caaggtgaaa ataccctaaa cttgtctggt 2040
atttatacag gagtttattt gttaaaattg aacgatggac aaaattctta tacaaaaaga 2100
attattatga aa 2112
〈210〉 9
<211> 7
<212> PRT
213 Alteromonas sp.
〈400〉 9
Thr Thr Met Ala Trp Ile Lys
1 5
<210> 10
<211> 18
<212> PRT
213 Alteromonas sp.
<400> 10
Gly Thr Thr Ser Ile Asn Asp Gly Glu Glu His His His Ala Val Val
1 5 10 15
Ser Pro
<210> 11
<211> 15
<212> PRT
213 Alteromonas sp.
〈220〉
<221> UNSURE
222 (13)
<223> Undefined amino acid
<400> 11
Gly Pro Asn Leu Thr Gly Thr Thr Thr Leu Asn Asp Xaa Gln Thr
1 5 10 15
<210> 12
<211> 24
<212> PRT
213 Alteromonas sp.
〈220〉
<221> UNSURE
222 (5)
<223> Undefined amino acid
〈220〉
<221> UNSURE
222 (7)
<223> Undefined amino acid
〈220〉
<221> UNSURE
222 (8)
<223> Undefined amino acid
〈220〉
<221> UNSURE
222 (17)
<223> Undefined amino acid
<400> 12
Ala Asp Ile Met Xaa Gly Xaa Xaa Gly Ile Ser Tyr Arg Ile Pro Gly
1 5 10 15
Xaa Asp Ile Asn Tyr Ser Gly Ser
20
〈210〉 13
<211> 17
<212> DNA
〈213〉 Artificial Sequence
〈220〉
223 Description of Artificial Sequence: Synthetic
oligonucleotide pFDA27
〈220〉
〈221〉 unsure
222 (3)
<223> a or c or g or t
〈220〉
〈221〉 unsure
222 (9)
<223> a or c or g or t
<400> 13
acnatggcnt ggathaa 17
〈210〉 14
<211> 15
<212> DNA
〈213〉 Artificial Sequence
〈220〉
223 Description of Artificial Sequence: Synthetic DNA
FDA-N1
<400> 14
catgaaaata cgtag 15
<210> 15
<211> 15
<212> DNA
〈213〉 Artificial Sequence
〈220〉
223 Description of Artificial Sequence: Synthetic DNA
FDA-N2
<400> 15
gatcctacgt atttt 15
<210> 16
<211> 30
<212> PRT
213 Flavobacterium sp.
〈220〉
<221> UNSURE
222 (1)
<223> Undefined amino acid
〈220〉
<221> UNSURE
222 (25)
<223> Undefined amino acid
<400> 16
Xaa Pro Ala Gly Tyr Met Met Ser Leu Gln Glu Gly Gly Ile Arg Ala
1 5 10 15
Tyr Asn Ala Gly Thr Thr Tyr Ile Xaa Gly Val Glu Ile Gln
20 25 30
〈210〉 17
<211> 22
<212> PRT
213 Flavobacterium sp.
〈400〉 17
Gly Glu Ala Ser Gln Ser Ser Ser Arg Pro Ser Asp Gly Phe Ala Ala
1 5 10 15
Asn Ala Ile Asp Gly Asn
20
〈210〉 18
<211> 19
<212> PRT
213 Flavobacterium sp.
〈220〉
<221> UNSURE
222 (1)
<223> Undefined amino acid
〈400〉 18
Xaa Tyr Val Thr Val Pro Asp Pro Ser Ile Thr Val Asp Ala Gly Gly
1 5 10 15
Val asn gly
〈210〉 19
<211> 18
<212> PRT
213 Flavobacterium sp.
〈400〉 19
Phe Gly Asn Asn Glu Val Asn Glu Ile Gln Ile Leu Gly Asn Asn Asn
1 5 10 15
Val leu
<210> 20
<211> 17
<212> PRT
213 Flavobacterium sp.
〈220〉
<221> UNSURE
222 (11)
<223> Undefined amino acid
〈220〉
<221> UNSURE
222 (12)
<223> Undefined amino acid
<400> 20
Tyr Val Glu Gly Cys Thr Val Leu Gly Cys Xaa Xaa Gly Tyr Ser Ile
1 5 10 15
Gly
〈210〉 21
<211> 17
<212> DNA
〈213〉 Artificial Sequence
〈220〉
<223> Description of Artificial Sequence: Mixed
oligonucleotide pL14F17
〈220〉
〈221〉 unsure
222 (6)
<223> a or c or g or t
<400> 21
ttyggnaaya aygargt 17
<210> 22
<211> 26
<212> DNA
〈213〉 Artificial Sequence
〈220〉
<223> Description of Artificial Sequence: Mixed
oligonucleotide pL14F26
〈220〉
〈221〉 unsure
222 (12)
<223> a or c or g or t
<400> 22
aayaaygarg tnaaygarat hcarat 26
<210> 23
<211> 675
<212> DNA
213 Flavobacterium sp.
<400> 23
aataatgagg ttaatgaaat tcagatttta ggaaataaca atgttcttaa aaacttaaaa 60
ctagaagata ttggaacaac agctccttct aacagagccc agtcaattat aatggatggg 120
cgagacaata gaatagaagg ctttcattta accattagag gatcttatcc ttatggatat 180
ggagatgctt ttggaaaagg aggaggttcc gtaattaatc accgaaaaca ttcaggtgtt 240
ttaataagag gattacgtaa tcacctaaaa gattgtacta ttatttctcg ttcttatggg 300
catatagtat ttatgcaagc agcaagttac ccaactgtag aaggttgtta tattgaaggt 360
gaaatgcgtt caaccgatga tatgttggca gaagaaggaa caggttctcc agcggataat 420
gtagatttta tgacggtttg gggatataag ttaccagctg gttatatgat gagtttacaa 480
gaaggaggaa ttagagctta tgatgctggt accacttata ttgatggaga agtaatccaa 540
agagcaacag ataaccctac cgttctaaat tgtaccatta aaaatgcaag aacaggagtg 600
actttagcac atgctaaagg aacaaaacac gtagaaaatg ttaaggctat tgggtgtgag 660
caaggatatt caatt 675
<210> 24
<211> 14
<212> PRT
213 Flavobacterium sp.
<400> 24
His Ser Gly Val Leu Ile Arg Gly Leu Arg Asn His Leu Lys
1 5 10
<210> 25
<211> 10
<212> PRT
213 Flavobacterium sp.
<400> 25
Leu Asn Ile Asn Thr Val Val Leu Thr Lys
1 5 10
〈210〉 26
<211> 21
<212> DNA
〈213〉 Artificial Sequence
〈220〉
223 Description of Artificial Sequence: Synthetic DNA
primer 17X6F4
<400> 26
gttcaatagt aacagcaaac c 21
<210> 27
<211> 28
<212> DNA
〈213〉 Artificial Sequence
〈220〉
<223> Description of Artificial Sequence: Primer
FDL-Q-Bam
<400> 27
ccggatccca aacaaccaca gtatattc 28
<210> 28
<211> 21
<212> DNA
〈213〉 Artificial Sequence
〈220〉
<223> Description of Artificial Sequence: Primer 10X6R4
<400> 28
tccatcaatg gcatttgctg c 21
<210> 29
<211> 14
<212> PRT
〈213〉 Unknown
〈220〉
223 Description of Unknown Organism: N-terminal
sequence derived from plasmid pET21a
<400> 29
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser
1 5 10
<210> 30
<211> 20
<212> DNA
〈213〉 Artificial Sequence
〈220〉
223 Description of Artificial Sequence: Synthetic DNA
17X6R1
<400> 30
atgttaccac taataccatc 20

权利要求:
Claims (15)
[1" claim-type="Currently amended] An isolated gene having a DNA sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide degradation activity, or a polypeptide having a function functionally equivalent to the activity.
[2" claim-type="Currently amended] The gene according to claim 1, wherein the polypeptide is derived from bacteria of the genus Alteromonas.
[3" claim-type="Currently amended] The gene according to claim 1 or 2, wherein the polypeptide has an activity of acting on fucose sulfuric acid-containing polysaccharide having the following physicochemical properties and low molecular weight of the fucose sulfuric acid-containing polysaccharide.
(a) Constituent sugar: substantially free of uronic acid.
(b) Flavobacterium sp. Not substantially low molecular weight by fucoidan degrading enzyme produced by SA-0082 (FERM BP-5402).
[4" claim-type="Currently amended] The gene according to claim 1, wherein the polypeptide is derived from the genus Flavoacterium.
[5" claim-type="Currently amended] The method according to claim 1 or 4, wherein the polypeptide acts on a fucose sulfuric acid-containing polysaccharide having the following physicochemical properties, thereby lowering the fucose sulfuric acid-containing polysaccharide to be selected from the following formulas A gene characterized in that it has an activity of releasing at least one or more compounds.
(c) Constitution sugars: contains uronic acid.
(d) Flavobacterium sp. Decomposed by fucoidan degrading enzyme produced by SA-0082 (FERM BP-5402).
Formula I

Formula II

Formula III

Formula IV

[6" claim-type="Currently amended] The polypeptide according to any one of claims 1 to 5, comprising a polypeptide having an amino acid sequence represented by any of SEQ ID NOs 1 to 4 in the Sequence Listing or a part of an amino acid sequence represented by any of SEQ ID NOs 1 to 4. And encoding a polypeptide having a fucose sulfate-containing polysaccharide decomposition activity or a polypeptide having a functionally equivalent activity with these activities.
[7" claim-type="Currently amended] The gene encoding the amino acid sequence according to claim 6, wherein the DNA encoding the amino acid sequence is a gene having a DNA sequence represented by any of SEQ ID NOs: 5 to 8 or a gene comprising a part of a DNA sequence represented by any of SEQ ID NOs: 5 to 8, A gene which encodes a polypeptide having a fucose sulfate-containing polysaccharide degrading activity or a polypeptide functionally equivalent to these activities.
[8" claim-type="Currently amended] The amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the sequence listing, wherein one or more amino acid sequences contains at least one of deletion, addition, insertion or substitution, and further contains fucose sulfate. A gene characterized by a gene encoding a polypeptide having polysaccharide degrading activity.
[9" claim-type="Currently amended] The gene according to claim 1, which encodes a polypeptide having a fucose sulfate-containing polysaccharide degrading activity capable of hybridization under strict conditions with the gene according to any one of claims 2 to 8.
[10" claim-type="Currently amended] A recombinant DNA comprising the gene according to any one of claims 1 to 9.
[11" claim-type="Currently amended] An expression vector in which the recombinant DNA according to claim 10 is inserted, wherein the microorganism, animal cell or plant cell is used as a host cell.
[12" claim-type="Currently amended] A transformant transformed with the expression vector according to claim 11.
[13" claim-type="Currently amended] The transformant according to claim 12 is cultured to obtain a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity or a polypeptide having a functionally equivalent activity with that activity from the culture. A method for producing a polypeptide having polysaccharide degrading activity or a polypeptide having a functionally equivalent activity.
[14" claim-type="Currently amended] A polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 in the sequence listing and having a fucose sulfate-containing polysaccharide decomposition activity or a polypeptide functionally equivalent to the activity.
[15" claim-type="Currently amended] 15. The amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the sequence listing, wherein one or more amino acid sequences contains at least one of deletion, addition, insertion, or substitution, and further contains fucose sulfate. A polypeptide having a polysaccharide degrading activity, or a polypeptide comprising a part of an amino acid sequence represented by any of SEQ ID NOs: 1 to 4, and having a fucose sulfate-containing polysaccharide degrading activity.

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同族专利:

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公开号 | 公开日

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AT380245T|2007-12-15|

---

RU2004133974A|2006-05-10|

---

KR100543051B1|2006-01-20|

---

KR20050042780A|2005-05-10|

---

CA2300797A1|1999-03-11|

---

TW570979B|2004-01-11|

---

AU757992B2|2003-03-13|

---

EP1010761A4|2005-01-05|

---

KR100479533B1|2005-03-31|

---

EP1826272A3|2008-12-03|

---

CN1271390A|2000-10-25|

---

US6489155B1|2002-12-03|

---

WO1999011797A1|1999-03-11|

---

DE69838810D1|2008-01-17|

---

RU2246539C2|2005-02-20|

---

CN1162544C|2004-08-18|

---

EP1010761A1|2000-06-21|

---

EP1826272A2|2007-08-29|

---

EP1010761B1|2007-12-05|

---

AU7451698A|1999-03-22|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-09-03|Priority to JP97-252624

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1997-09-03|Priority to JP25262497

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1998-05-26|Application filed by 오미야 히사시, 다카라츠죠 가부시키가이샤

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1998-05-26|Priority to PCT/JP1998/002310

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2001-03-26|Publication of KR20010023509A

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2005-03-31|Application granted

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2005-03-31|Publication of KR100479533B1

优先权:

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申请号 | 申请日 | 专利标题

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JP97-252624|1997-09-03|

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JP25262497|1997-09-03|

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PCT/JP1998/002310|WO1999011797A1|1997-09-03|1998-05-26|Gene|