JP2005312459A - Gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new fucose sulfate-containing polysaccharide hydrolase, and to obtain a gene therefor. <P>SOLUTION: An isolated gene contains a DNA sequence encoding a polypeptide comprising a specific amino acid sequence originating from Flavobacterium, or a polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, added, inserted or replaced and having an activity to convert a fucose sulfate-containing polysaccharide having the following physiochemical properties into a low molecule and releasing a fucose sulfate-containing oligosaccharide compound. (a) A constituting sugar: containing uronic acid. (b) The fucose sulfate-containing polysaccharide is hydrolyzed with a fucoidan hydrolase produced by Flavobacterium sp. SA-0082 (FERM BP-5402). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity useful for structural analysis of a sulfated-fucose-containing polysaccharide and preparation of a low molecular weight product of the polysaccharide, a method for producing the polypeptide by genetic engineering, and the It relates to the polypeptide obtained by the method.

  The seaweed-derived sulfated fucose-containing polysaccharide is a sulfated polysaccharide having fucose as a main component, which is generically called fucoidan, and is known to contain galactose, glucuronic acid, xylose, mannose, glucose and the like. The types and amounts of these constituent sugars vary depending on the type of seaweed from which they are derived. For example, there is a report that fucoidan manufactured by Sigma is divided into 13 molecular species. [Carbohydrate Research, Vol. 255, pp. 213-224 (1994)]

  If these are roughly classified, they can be classified into two types: those having substantially no uronic acid and containing fucose as the main component of the constituent sugar and those containing uronic acid and containing fucose and mannose.

  Various bioactivity of sulfated fucose-containing polysaccharides has been reported, including macrophage activity enhancement, suppression of cancer metastasis, and anticoagulation. In order to examine the presence, it was necessary to separate and purify the sulfated-fucose-containing polysaccharide. However, in the previous method, the separation was insufficient, so that it was difficult to prepare a large amount as a chemical. In addition, sulfated fucose-containing polysaccharides are sulfated polysaccharides with extremely large molecular weights, and there are problems such as antigenicity, homogeneity and anticoagulant activity when used as pharmaceuticals. Was needed.

  The method of enzymatically degrading a sulfated-fucose-containing polysaccharide to prepare a low molecular weight product is an advantageous method capable of performing a reaction under mild conditions and obtaining a uniform low molecular weight product from the substrate specificity of the enzyme. It has been reported that abalone, scallops, sea urchins, marine microorganisms and the like have produced enzymes that degrade sulfated fucose-containing polysaccharides. However, these enzymes are generally contained in a small amount in the living body and have a plurality of sulfated-fucose-containing polysaccharide-degrading enzymes, so that various purification steps are required to obtain a single enzyme. . Furthermore, the amino acid sequences and gene structures of these enzymes have not been clarified at all.

  An object of the present invention is to provide a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity useful for the preparation and structural analysis of a sulfated fucose-containing polysaccharide and the preparation of a low-molecular-weight fucose sulfate-containing polysaccharide, and a gene using the gene It is an object of the present invention to provide a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity, which can be obtained by engineering.

  In order to clarify the amino acid sequence and base sequence of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity, the present inventors have conducted extensive research on the genes of microorganisms that produce a fucose sulfate-containing polysaccharide-degrading enzyme. It is clarified that there are two kinds of genes encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity derived from bacteria, Flavobacterium genus bacteria, and the entire base sequence is determined, and the amino acid sequence of the polypeptide The present invention was clarified for the first time, and furthermore, a method for industrially producing a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity using the gene was also successfully developed, thereby completing the present invention.

  Briefly describing the present invention, the first invention of the present invention is an isolated DNA sequence encoding a polypeptide having sulfated fucose-containing polysaccharide-degrading activity or a polypeptide functionally equivalent to the activity thereof. Related to genes.

  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 having a microorganism, animal cell or plant cell as a host cell into which the recombinant DNA of the second invention of the present invention has been inserted.

  The fourth invention of the present invention relates to a transformant transformed with the expression vector of the third invention of the present invention.

  According to a fifth aspect of the present invention, a transformant according to the fourth aspect of the present invention is cultured, and the polypeptide having a fucose sulfate-containing polysaccharide-degrading activity or an activity functionally equivalent to the activity is cultured from the culture. The present invention relates to a method for producing a polypeptide having sulfated fucose-containing polysaccharide-degrading activity or a polypeptide functionally equivalent to the activity, characterized by collecting the polypeptide.

The sixth invention of the present invention is a polypeptide having any amino acid sequence represented by SEQ ID NO: 1 to 4 in the sequence listing and having a fucose sulfate-containing polysaccharide degrading activity, or a functionally equivalent polypeptide thereof. It relates to a polypeptide having activity.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the positions of ORF-1 and ORF-2.
FIG. 2 is a graph showing the rate of precipitation of sulfated fucose-containing polysaccharide.
FIG. 3 is a diagram showing the position of fdlA.
FIG. 4 is a diagram showing the position of fdlB.
FIG. 5 is a chromatogram using DEAE-Sepharose FF.

  The present invention will be specifically described below.

The present invention relates to a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity. An example of a polypeptide encoded by the gene is a polypeptide having an endo-fucose sulfate-containing polysaccharide-degrading activity of the following (1) derived from bacteria belonging to the genus Arteromonas.
(1) It acts on a sulfated-fucose-containing polysaccharide having the following physicochemical properties (hereinafter referred to as sulfated-fucose-containing polysaccharide-F) to lower the molecular weight of the sulfated-fucose-containing polysaccharide.
(A) Constituent sugar: substantially free of uronic acid.
(B) Flavobacterium sp. It is not substantially reduced in molecular weight by the fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402).

  Examples of the polypeptide having sulfated fucose-containing polysaccharide-degrading activity include Alteromonas sp. There is an endo-type fucose sulfate-containing polysaccharide-degrading enzyme produced by SN-1009, which can be prepared as described in Reference Example 1- (3).

  Said fucose sulfate-containing polysaccharide-F can be prepared as described in Reference Example 1.

  Flavobacterium sp. Fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402) can be prepared as described in Reference Example 5.

Examples of the other polypeptide having a fucose sulfate-containing polysaccharide-degrading activity include the following polypeptide (2) having a fucose sulfate-containing polysaccharide-degrading activity derived from a bacterium belonging to the genus Flavobacterium.
(2) Acting on a sulfated-fucose-containing polysaccharide having the following physicochemical properties (hereinafter referred to as sulfated-fucose-containing polysaccharide-U) to reduce the molecular weight of the sulfated-fucose-containing polysaccharide, the following formulas [I], [II], At least one compound selected from [III] and [IV] is liberated.
(C) Constituent sugar: contains uronic acid.
(D) Flavobacterium sp. It is degraded by fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402).

  Examples of the polypeptide having a fucose sulfate-containing polysaccharide-degrading activity include Flavobacterium sp. There is a fucoidan degrading enzyme produced by SA-0082, which can be prepared as described in Reference Example 5.

  The mixture of sulfated-fucose-containing polysaccharide-F and sulfated-fucose-containing polysaccharide-U is described below as the sulfated-fucose-containing polysaccharide mixture.

  In the present invention, the polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity is not limited to a naturally-occurring sulfated-fucose-containing polysaccharide-degrading enzyme. Polypeptides whose amino acid sequence has been altered by insertion, addition, etc. are also meant to be included in the present invention.

  The natural fucose sulfate-containing polysaccharide-degrading enzyme mentioned here includes, for example, those derived from the genus Alteromonas and the genus Flavobacterium, but the present invention is not limited thereto, and other bacteria Of course, those derived from microorganisms such as yeasts, filamentous fungi, ascomycetes and basidiomycetes, and those derived from organisms such as plants and animals are also included.

  In the present specification, the polypeptide having functionally equivalent activity refers to the following.

  In addition to polymorphisms and mutations of the gene that encodes it in naturally occurring proteins, deletion and addition of amino acid residues in the amino acid sequence by in vivo and modification during purification of the protein after production It is known that some mutations such as insertion and substitution can occur, but nonetheless exhibit substantially the same physiological and biological activities as proteins having no mutation. Even if there is a structural difference, a polypeptide that has no significant difference in function is called a polypeptide having functionally equivalent activity.

  This is the same even when the above mutations are artificially introduced into the amino acid sequence of the protein. In this case, it is possible to produce a wider variety of mutants. As long as they exhibit equivalent physiological activity, these variants are interpreted as polypeptides having functionally equivalent activity.

  For example, a methionine residue present at the N-terminus of a protein expressed in E. coli is often removed by the action of methionine aminopeptidase, but depending on the type of protein, those having a methionine residue, Both are generated. However, the presence or absence of this methionine residue often does not affect the activity of the protein. It is also known that a polypeptide in which a certain cysteine residue in the amino acid sequence of human interleukin 2 (IL-2) is substituted with serine retains interleukin 2 activity [Science, 224]. Volume 1431 (1984)].

  In addition, when a protein is produced by genetic engineering, it is often expressed as a fusion protein. For example, in order to increase the expression level of the target protein, an N-terminal peptide chain derived from another protein is added to the N-terminus of the target protein, or an appropriate peptide chain is added to the N-terminus or C-terminus of the target protein. For example, the purification of the target protein is facilitated by using a carrier having an affinity for the added peptide chain.

  In addition, a polypeptide in which at least one of one or more amino acid residues has been deleted, added, inserted, or substituted in the amino acid sequence of the target protein has a functionally equivalent activity to the target protein. In many cases, such a polypeptide and a gene encoding the polypeptide, whether isolated from nature or artificially produced, are also encompassed in the present invention.

  Generally, it is known that there are 1 to 6 codons (a combination of three bases) for designating amino acids on a gene for each amino acid type. Therefore, a large number of genes encoding amino acid sequences can exist depending on the amino acid sequence. Genes never exist stably in nature, and it is not uncommon for mutations to occur in their nucleic acids. In some cases, the amino acid sequence encoded by the mutation occurring on the gene does not change (called a silent mutation), and in this case, it can be said that different genes encoding the same amino acid sequence are generated. Therefore, even if a gene encoding a specific amino acid sequence is isolated, there is no denying the possibility that many kinds of genes encoding the same amino acid sequence will be created as the organism containing it is passaged. .

  In addition, it is not difficult to artificially prepare many kinds of genes encoding the same amino acid sequence using various genetic engineering techniques.

  For example, in genetic engineering protein production, if the codon used on the original gene encoding the protein of interest is not used frequently in the host being used, the expression level of the protein is May be low. In such a case, high expression of the target protein can be achieved by artificially changing the codon to that frequently used in the host without changing the encoded amino acid sequence. It has been broken. Needless to say, it is possible to artificially produce many kinds of genes encoding specific amino acid sequences. Therefore, even these artificially prepared different polynucleotides are included in the present invention as long as the amino acid sequence disclosed in the present invention is encoded.

  In addition, polypeptides having functionally equivalent activities often have homology in genes encoding them. Therefore, a gene encoding a polypeptide capable of hybridizing with a gene used in the present invention under strict conditions and having a fucose sulfate-containing polysaccharide degrading activity is also included in the present invention.

  Hereinafter, Alteromonas sp. SN-1009 and Flavobacterium sp. The present invention will be specifically described with SA-0082 as an example.

  This Alteromonas sp. SN-1009 is an Alteromonas sp. SN-1009 is displayed, and from February 13, 1996 (original deposit date), Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3 1-3 Higashi, Tsukuba City, Ibaraki, Japan (zip code 305 -8566)] is internationally deposited as FERM BP-5747. Flavobacterium sp. SA-0082 is Flavobacterium sp. It is displayed as SA-0082 and is internationally deposited as FERM BP-5402 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry.

  Alteromonas sp. SN-1009 or Flavobacterium sp. In order to obtain a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity produced by SA-0082, for example, a hybridization method, a PCR method, or a combination of these methods can be used. These methods require a probe capable of hybridizing to the gene, or a primer capable of amplifying the gene or a part thereof by PCR. Polysulfate having fucose sulfate-containing polysaccharide-degrading activity produced by these strains is required. Since the amino acid sequence and gene structure of the peptide are not known at all, synthetic oligonucleotides that can be used as probes or primers cannot be prepared. First, the partial amino acid sequence of the sulfated-fucose-containing polysaccharide-degrading enzyme produced by the microorganism is determined, and the production of synthetic oligonucleotides that can be used as probes or primers is examined.

  First, for example, Alteromonas sp. SN-1009 or Flavobacterium sp. SA-0082 is cultured, and then the produced fucose sulfate-containing polysaccharide-degrading enzyme is isolated and purified from the culture.

  Next, the information regarding the partial amino acid sequence is obtained about each refined sulfated-fucose-containing polysaccharide-degrading enzyme. In order to determine the partial amino acid sequence, for example, by subjecting the sulfated-fucose-containing polysaccharide-degrading enzyme to amino acid sequence analysis by Edman degradation method (for example, Protein Sequencer 476A, Applied Biosystems, Inc. can be used) directly according to a conventional method. The N-terminal amino acid sequence of the sulfated-fucose-containing polysaccharide-degrading enzyme is determined. Alternatively, a highly specific protein hydrolase such as Achromobacter protease I, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) -trypsin, etc. acts on the purified fucose sulfate-containing polysaccharide-degrading enzyme If the obtained peptide fragments are separated and purified using reverse phase HPLC, and then amino acid sequence analysis is performed on the purified peptide fragments, a large amount of amino acid sequence information can be obtained.

  Information on partial amino acid sequences specific to the fucose sulfate-containing polysaccharide-degrading enzyme thus obtained is selected, and oligonucleotides with degenerate base sequences are designed and synthesized based on the information. At this time, it is necessary to synthesize an oligonucleotide with a low degree of degeneracy and, in other words, an oligonucleotide highly specific for a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity. Is an important factor in the cloning of a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity.

  Next, it is necessary to examine conditions for specific hybridization between a synthetic oligonucleotide and a gene encoding a polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity by Southern hybridization.

For example, Alteromonas sp. SN-1009 or Flavobacterium sp. SA-0082 genomic DNA is completely digested with an appropriate restriction enzyme, separated by agarose gel electrophoresis, and then blotted onto a nylon membrane or the like according to a conventional method. First, for example, 6 × SSC (1 × SSC is 8.77 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water), 1% sodium lauryl sulfate (SDS), Blocking nylon membranes by incubating at 65 ° C. for several hours in a prehybridization solution containing 100 μg / ml salmon sperm DNA, 5 × denharz (bovine serum albumin, polyvinylpyrrolidone, and ficoll at 0.1% each) Then, for example, a synthetic oligonucleotide labeled with ³² P or the like is added, and the mixture is incubated at 42 ° C. overnight. The nylon membrane is washed with 1 × SSC containing 0.1% SDS at 42 ° C. for 30 minutes, and then autoradiography is performed to detect a DNA fragment that hybridizes with the synthetic oligonucleotide probe. At this time, it is effective to examine the incubation temperature, the salt concentration of the washing solution, etc., and select the optimum conditions according to the length of the synthetic oligonucleotide used and the complementarity with the gene encoding a polypeptide having a sulfated-fucose-containing polysaccharide degradation activity. Is.

  As a method for obtaining a DNA fragment containing a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity thus detected, a DNA fragment corresponding to the position of the directly detected band was extracted from the gel and purified. Then, a library incorporated in a commonly used host-vector vector is prepared, and colony hybridization or plaque hybridization is performed under the same conditions as in the Southern hybridization method to obtain a clone containing the target DNA fragment. What is necessary is just to screen and isolate. Alternatively, directly Alteromonas sp. SN-1009 or Flavobacterium sp. After digesting SA-0082 genomic DNA with an appropriate restriction enzyme, a library incorporated into a commonly used host-vector vector is prepared, and a clone containing the target DNA fragment is similarly screened by a hybridization method. It may be isolated.

  As the host-vector system to be used, known ones can be used, and examples thereof include plasmid vectors such as pUC18 and pUC19 using Escherichia coli as a host, phage vectors such as lambda phage, and the like. Absent.

  For the types and handling methods of these host-vector systems, commonly used types and methods may be used. For example, Molecular Cloning Laboratory Manual, Second Edition (Molecular Cloning, A Laboratory Manual, Second Edition) [J. By J. Sambrook et al., Cold Spring Harbor Laboratory, published in 1989].

  If a vector containing the target DNA fragment can be selected, the base sequence of the target DNA fragment inserted into this vector can be determined by a conventional method such as the dideoxy method [Proceedings of the National Academy of Sciences of the USA (Proc. Nat. Acad. Sci, U.S.A.), Vol. 74, p. 5463 (1977)]. By comparing the determined base sequence with the N-terminal analysis, partial amino acid sequence, molecular weight, etc. of the sulfated-fucose-containing polysaccharide degrading enzyme, the structure of the gene in the DNA fragment obtained and the amino acid sequence of the polypeptide encoded by the gene Can know.

  In addition, the PCR method is used as a method for 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 above-described sulfated-fucose-containing polysaccharide-degrading enzyme. Can do. Among them, the PCR method using cassette DNA is a method for obtaining a fragment of a target gene that can be used in a hybridization method from a small amount of amino acid sequence information in a short time.

  For example, Flavobacterium sp. After digesting genomic DNA extracted from cultured SA-0082 cells by a conventional method with an appropriate restriction enzyme, synthetic DNA (cassette DNA) having a known sequence is ligated. Using this mixture as a template, a PCR reaction was carried out using the gene-specific oligonucleotide primer designed based on the above partial amino acid sequence information and an oligonucleotide primer (cassette primer) complementary to the cassette DNA. It is possible to amplify DNA fragments. As the cassette DNA or cassette primer, for example, those manufactured by Takara Bio Inc. can be used. The cassette DNA preferably contains sequences corresponding to two types of cassette primers. First, a first PCR reaction is performed using a primer far from the linked restriction enzyme sites, and a part of the reaction solution is obtained. It is effective to perform a second PCR reaction using the inner primer as a template. Furthermore, two types of the gene-specific oligonucleotide primer are also designed and synthesized side by side, the upstream primer is used for the first PCR reaction, and the downstream primer is used for the second PCR reaction. And the possibility of specific amplification of the target DNA fragment is increased.

  However, since the base sequence of the target gene is unknown, the restriction enzyme site used for linking the cassette DNAs is not always at an appropriate position for the amplification reaction by PCR from the region encoding the partial amino acid sequence. Therefore, it is necessary to use many types of restriction enzyme site cassette DNAs. PCR can be performed under commonly used conditions as described in, for example, PCR technology (edited by PCR Technology, Erlich HA, published by Stockton Press, 1989). However, the annealing temperature, the number of cycles, the magnesium concentration, the thermostable polymerase concentration, etc., were examined based on the length of the synthetic oligonucleotide used and the complementarity with the gene encoding a polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity. It is necessary to select an optimum condition that minimizes a typical amplification band.

  The PCR reaction solution is subjected to electrophoresis such as agarose gel to confirm the amplified DNA fragment. These fragments can be extracted and purified according to a conventional method, inserted into a commonly used cloning vector such as pUC18 or pUC19, and the nucleotide sequence can be analyzed, for example, by the dideoxy method. Alternatively, the base sequence of the recovered amplified DNA fragment may be directly analyzed using the cassette primer used in the PCR reaction. As a result, in addition to the primer sequence, if a product encoding the partial amino acid sequence of the sulfated-fucose-containing polysaccharide degrading enzyme determined previously is obtained, a gene encoding the enzyme or a fragment of a gene showing homology thereto can be obtained. It will be.

  When the DNA fragment obtained by the Southern hybridization method or the PCR method is a part of the gene encoding the target enzyme in this way, a genomic library by hybridization using the DNA fragment as a probe is used. A DNA fragment containing the full length of the gene encoding the target enzyme can be obtained by screening or performing PCR using an oligonucleotide prepared based on the base sequence of the DNA fragment as a primer. .

  Furthermore, using the fucose sulfate-containing polysaccharide-degrading enzyme gene obtained as described above or a part thereof as a probe, Alteromonas sp. SN-1009 or Flavobacterium sp. When the genomic DNA of SA-0082 is analyzed by Southern hybridization, Alteromonas sp. Containing a gene encoding a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity is detected from the position of the detected band. SN-1009 or Flavobacterium sp. Information on the size of the genomic DNA restriction enzyme fragment of SA-0082 is obtained, and the number of genes encoding a polypeptide having sulfated fucose-containing polysaccharide-degrading activity and the number of genes complementary thereto are determined depending on the number of detected bands. It is anticipated that DNA fragments containing these genes can be isolated by the same method as described above.

  Whether the DNA fragment thus obtained contains a gene encoding the target enzyme is determined by preparing an expression vector containing the finally isolated DNA fragment and transforming the host using the vector. And then culturing the transformant and measuring the sulfated fucose-containing polysaccharide-degrading 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 in the sequence listing and having a base sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide degradation activity was isolated. Examples of base sequences encoding polypeptides having the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively in the sequence listing.

  Flavobacterium sp. From SA-0082, a gene having an amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing and having a base sequence encoding a polypeptide having a fucose sulfate-containing polysaccharide degradation activity was isolated. Examples of base sequences encoding polypeptides having the amino acid sequences 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 sulfated-fucose-containing polysaccharide-degrading activity or a functionally equivalent activity using the nucleotide sequence of the gene of the present invention, for example, the following methods can be applied.

First, a chromosomal DNA obtained from a target gene source or cDNA prepared from reverse mRNA using mRNA 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, and the grown colonies or plaques are transferred to a nitrocellulose or nylon membrane, and DNA is fixed to the membrane by denaturation treatment. A probe labeled with, for example, ³² P or the like (a probe to be used may be an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 in the sequence listing, or a base sequence encoding a part thereof, For example, a base sequence represented by any one of SEQ ID NOs: 5 to 8 in the sequence listing, or a part thereof can be used) and hybridized between the DNA on the membrane and the probe To form. For example, the DNA-immobilized membrane 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 a clone hybridized with the probe is identified by autoradiography or the like. This operation is repeated until the hybridized clone becomes single. In the thus obtained clone, a gene encoding the target polypeptide is inserted.

  For example, the base sequence of the obtained gene is determined as follows, and it is confirmed whether the obtained gene is a gene encoding a polypeptide having a target fucose sulfate-containing polysaccharide-degrading activity or a functionally equivalent activity. To do.

  For determination of the base sequence, if the transformant is E. coli transformed with a plasmid, culture is performed in a test tube or the like, and the plasmid is extracted according to a conventional method. This is cleaved with a restriction enzyme, the inserted fragment is taken out, subcloned into an M13 phage vector or the like, and the base sequence is determined by the dideoxy method. When a phage vector is used as the recombinant, the base sequence can be determined basically by the same steps. For the basic experimental method from culture to sequencing, for example, Molecular Cloning Laboratory Manual Second Edition [J. Sambrook et al., Cold Spring Harbor Laboratory, published in 1989].

  In order to confirm whether or not the obtained gene is a gene encoding a polypeptide having the desired sulfated-fucose-containing polysaccharide degrading activity or functionally equivalent activity, the determined base sequence or the amino acid encoded by the nucleotide sequence is determined. The sequence is compared with the nucleotide sequence represented by any of SEQ ID NOs: 5 to 8 in the sequence listing of the present invention or the amino acid sequence represented by any of SEQ ID NO: 1 to 4 of the sequence listing.

  In the case where the obtained gene does not contain all of the region encoding a polypeptide having sulfated fucose-containing polysaccharide-degrading activity or functionally equivalent activity, a synthetic DNA primer is prepared based on the obtained gene, The fucose sulfate-containing polysaccharide-degrading activity or function that hybridizes to the gene of the present invention by amplifying the missing region by PCR, or by screening a DNA library or cDNA library using the obtained gene fragment as a probe Thus, the base sequences of all coding regions of polypeptides having substantially the same activity can be determined.

  On the other hand, a primer for PCR reaction can be designed from the base sequence of the gene of the present invention. By performing a PCR reaction using this primer, a gene fragment highly homologous to the gene of the present invention can be detected, or the entire gene can be obtained.

  Next, the obtained gene is expressed, the sulfated-fucose-containing polysaccharide degradation activity is measured, and the function of the obtained gene is determined.

  The following method is convenient for producing a polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity using a gene encoding a polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity of the present invention.

  First, the host is transformed with a vector containing a gene encoding a polypeptide having the desired fucose sulfate-containing polysaccharide-degrading activity, and then this transformant is cultured under the usual conditions. A polypeptide having a polysaccharide-degrading activity can be produced. At this time, 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 measuring, for example, the sulfated-fucose-containing polysaccharide degradation activity. The activity can be measured, for example, using a cell extract of recombinant E. coli as an enzyme solution.

  If expression of the polypeptide having the desired fucose sulfate-containing polysaccharide-degrading activity is observed, for example, if the transformant is Escherichia coli, the composition of the medium, the pH of the medium, the culture temperature, the amount and timing of use of the inducer, A polypeptide having fucose sulfate-containing polysaccharide degrading activity can be efficiently produced by determining optimum conditions for the culture time and the like.

  A usual method is used to purify a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity from a culture of a transformant. When a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity accumulates in the cell as in the case of E. coli, the transformant was collected by centrifugation after culturing and disrupted by sonication or the like. Thereafter, a cell-free extract is obtained by centrifugation or the like. From this, the target polypeptide can be purified using general protein purification methods such as salting out, various types of chromatography such as ion exchange, gel filtration, hydrophobicity, and affinity. Depending on the host-vector system to be used, the expression product may be secreted outside the transformant. In this case, purification may be similarly performed from the culture supernatant.

  A polypeptide having a fucose sulfate-containing polysaccharide-degrading activity produced by a transformant coexists with various enzymes in the cell when it is produced in the cell, but these are polypeptides having a fucose sulfate-containing polysaccharide-degrading activity Since the amount is only a small amount compared to the amount of, the purification is very easy. Furthermore, if a cell to be used as a host is selected, the host-derived enzyme that acts on the sulfated-fucose-containing polysaccharide is greatly reduced. In addition, when a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity is secreted outside the cells, medium 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 culturing, the cells are collected by centrifugation, disrupted by sonication or the like, and then centrifuged to collect the insoluble fraction containing inclusion bodies. After the inclusion body is washed, it is solubilized with a commonly used protein solubilizer such as urea or guanidine hydrochloride, and this is subjected to various chromatography such as ion exchange, gel filtration, hydrophobicity, affinity, etc. as necessary. After purifying by the above, a polypeptide having the desired fucose sulfate-containing polysaccharide-degrading activity that retains the activity can be obtained by performing a refolding operation using a dialysis method or a dilution method. If necessary, this preparation can be further purified by various chromatographic methods to obtain a highly pure fucose sulfate-containing polysaccharide-degrading activity.

  In addition, the same production method and purification method may be used when producing a polypeptide having an activity equivalent to that of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity.

  Thus, the present invention provides the primary structure and gene structure of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity. In addition, it is possible to produce a polypeptide having a sulfated-fucose-containing polysaccharide-degrading activity or a polypeptide having an activity functionally equivalent to the activity thereof by genetic engineering.

  By using the genetic engineering production method of the present invention, it is possible to obtain a polypeptide having a high-pure fucose sulfate-containing polysaccharide-degrading activity at low cost or a polypeptide functionally equivalent to the activity thereof.

  As described above, the method of producing a fucose sulfate-containing polysaccharide-degrading enzyme by culturing an Alteromonas or Flavobacterium bacterium that produces a sulfated-fucose-containing polysaccharide-degrading enzyme simultaneously produces proteases and other polysaccharide-degrading enzymes. In order to isolate the desired sulfated-fucose-containing polysaccharide-degrading enzyme, it is necessary to separate and purify it from these extremely troublesome enzymes. However, according to the present invention, it is possible to provide a polypeptide having a high-purity fucose sulfate-containing polysaccharide-degrading activity at low cost.

  EXAMPLES 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 gagome kelp was pulverized by a free crusher M-2 type (manufactured by Nara Machinery Co., Ltd.), treated in 4.5 times 80% ethanol at 80 ° C. for 2 hours, and then filtered. The above steps of 80% ethanol extraction and filtration were further repeated 3 times for the residue to obtain 1870 g of ethanol washing residue. 36 liters of water was added to the residue, treated at 100 ° C. for 2 hours, and an extract was obtained by filtration. After the salt concentration of the extract was the same as that of a 400 mM sodium chloride solution, 5% cetylpyridinium chloride was added until no further precipitation occurred, followed by centrifugation. The precipitate was washed repeatedly with 80% ethanol to completely remove cetylpyridinium chloride, then dissolved in 3 liters of 2M sodium chloride, insolubles were removed by centrifugation, and equilibrated with 2M sodium chloride. 100 ml of DEAE-Cellulofine A-800 was suspended, stirred and filtered to remove the resin. This filtrate was applied to a 100 ml DEAE-Cellulofine A-800 column equilibrated with 2M sodium chloride, and the passing fraction was desalted and low-molecular-weight removed by an ultrafilter (excluded molecular weight of filtration membrane of 100,000). The precipitate formed at this time was removed by centrifugation. This supernatant was lyophilized to obtain 82.2 g of a purified gagome kelp-derived fucose sulfate-containing polysaccharide mixture.

  (2) After dissolving 7 g of the above-mentioned gagome kelp-derived fucose sulfate-containing polysaccharide mixture in 700 ml of 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride, 20 mM acetic acid containing 0.2 M calcium chloride in advance. Apply to a column of 4000 ml DEAE-Sepharose FF equilibrated with sodium (pH 6.0), wash the column thoroughly with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride, and then add 0-4 M sodium chloride. Elute with a gradient of. Fractions eluted at a sodium chloride concentration of 0.9 to 1.5M are collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane with an excluded molecular weight of 100,000, freeze-dried, and a sulfated fucose-containing polysaccharide 4.7 g of a freeze-dried preparation of -F was obtained.

  Fractions eluted at a sodium chloride concentration of 0.05 to 0.8M are collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane with an excluded molecular weight of 100,000, and lyophilized to contain fucose sulfate. 2.1 g of freeze-dried preparation of polysaccharide-U was obtained.

  (3) Alteromonas sp. SN-1009 (FERM BP-5747) was dispensed into 600 ml of a medium consisting of artificial seawater (jamarin laboratory) pH 8.2 containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract. And inoculated into a 2 liter Erlenmeyer flask (120 ° C., 20 minutes) and cultured at 25 ° C. for 25 hours to form a seed culture solution. 18 liters of a medium consisting of artificial seawater pH 8.0 containing 200 g of peptone, 4 g of yeast extract, and 4 ml of antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. . After cooling, 20 g of fucose sulfate-containing polysaccharide-F derived from gagome kelp prepared by using the method of Reference Example 1- (1) dissolved in 2 liters of artificial seawater sterilized at 120 ° C. for 15 minutes, and The seed culture solution (600 ml) was inoculated and cultured at 24 ° C. for 20 hours under the conditions of an aeration rate of 10 liters per minute and a stirring speed of 250 revolutions per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells and culture supernatant.

  When the activity of endo-fucose sulfate-containing polysaccharide-degrading enzyme in the culture supernatant was measured by the method described in Reference Example 2 using fucose sulfate-containing polysaccharide-F as a substrate, it was 10 mU / ml · culture solution.

  The obtained culture supernatant was concentrated with an ultrafilter having a molecular weight cut off of 10,000, and the resulting precipitate was removed by centrifugation, 85% saturated ammonium sulfate was salted out, and the resulting precipitate was collected by centrifugation. The mixture was sufficiently dialyzed against 20 mM Tris-HCl buffer (pH 8.2) containing artificial seawater (Jamarine S) at a concentration of 1 part to obtain 400 ml of crude enzyme.

  The obtained crude enzyme solution was previously equilibrated with 20 mM Tris-HCl buffer (pH 8.2) containing 5 mM sodium azide and one-tenth artificial seawater (Jamarine S). DEAE-Cellulofine A -Adsorbed on a column of 800 (manufactured by Seikagaku Corporation), the adsorbate was sufficiently washed with the same buffer, and then eluted with a solution containing 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM sodium chloride in the same buffer. The active fraction was collected.

  When the enzyme activity of the obtained active fraction was measured by the method described in Reference Example 2, it was 20400 mU (20.4 U).

  The obtained active fraction was concentrated with an ultrafilter having a molecular weight cut off of 10,000, and then ultrafiltered with addition of 20 mM Tris-HCl buffer pH 8.2 containing 10 mM calcium chloride and 50 mM sodium chloride. And completely replaced the buffer.

  The obtained enzyme solution was adsorbed on a DEAE-Sepharose FF column equilibrated in advance with the same buffer, and the adsorbate was sufficiently washed with the same buffer, and further washed with the same buffer having a sodium chloride concentration of 150 mM. Thereafter, gradient elution of sodium chloride was performed with the same buffer containing 150 mM to 400 mM 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 solution pH 8.0 containing 5 mM sodium azide and 1/10 concentration of Jamarin S was used. The molecular weight determined by the chromatography was about 100,000.

  The obtained active fractions were collected and dialyzed sufficiently against 20 mM Tris-HCl buffer (pH 8.2) containing 10 mM calcium chloride, 10 mM potassium chloride, and 4.2 M sodium chloride. The column was applied to a column of phenyl sepharose CL-4B equilibrated with the same buffer solution having a concentration of 4M and eluted with the same buffer solution containing 4M, 3M, 2M, 1M, 0.5M, and 0.15M sodium chloride.

  The obtained active fractions were collected, concentrated by ultrafiltration, and limited while adding 20 mM Tris-HCl buffer (pH 8.2) containing 10 mM calcium chloride, 10 mM potassium chloride, and 150 mM sodium chloride. The solution was filtered out to completely replace the buffer. This enzyme solution was applied in advance to DEAE-Cellulofine A-800 equilibrated with the same buffer solution, washed with the same buffer solution, and then subjected to gradient elution from 150 mM to 350 mM sodium chloride.

  The obtained active fractions were collected, dialyzed sufficiently with the same buffer containing 50 mM sodium chloride, adsorbed to DEAE-Cellulofine A-800 previously equilibrated with the same buffer containing 50 mM sodium chloride, and After washing with buffer, gradient elution with 50 mM to 150 mM sodium chloride was performed. The obtained active fractions were combined to obtain a purified enzyme.

  Further, when the molecular weight of the purified enzyme was determined by SDS (sodium dodecyl sulfate) -polyacrylamide electrophoresis, it was about 90,000.

Reference example 2
Using the fucose sulfate-containing polysaccharide-F obtained by the step of Reference Example 1- (2), the endo-fucose sulfate-containing polysaccharide decomposition activity was measured as follows.

  That is, 12 μl of 2.5% sulfated fucose-containing polysaccharide-F solution, 6 μl of 1 M calcium chloride solution, 9 μl of 4 M sodium chloride solution, 60 μl of a buffer solution containing 50 mM acetic acid, imidazole and tris-hydrochloric acid (pH 7. 5) and 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. Then, the reaction solution was treated at 100 ° C. for 10 minutes, centrifuged, and 100 μl was analyzed by HPLC. And the degree of molecular weight reduction was measured.

  As a control, instead of the test solution for measuring the degradation activity, the reaction was carried out under the same conditions using the buffer used in the test solution, and water was used instead of the sulfated-fucose-containing polysaccharide-F solution. Prepared were reacted and analyzed by HPLC in the same manner.

One unit of enzyme is the amount of enzyme that cleaves the fucosyl bond of 1 μmol of fucose sulfate-containing polysaccharide-F per minute in the above reaction system. The quantification of the cleaved fucosyl bond was determined by the following formula.
{(12 × 2.5) / (100 × MF)} × {(MF / M) −1} × {1 / (180 × 0.01)} × 1000 = U / ml
(12 × 2.5) / 100: Fucose sulfate-containing polysaccharide-F (mg) added to the reaction system
MF: Average molecular weight of substrate-fucose sulfate-containing polysaccharide-F M: Average molecular weight of reaction product (MF / M) -1: Number of 1-molecule fucose sulfate-containing polysaccharide-F cleaved by enzyme 180: Reaction time (minutes) )
0.01: Amount of enzyme solution (ml)
The HPLC conditions were as follows.
Apparatus: L-6200 type (manufactured by Hitachi, Ltd.)
Column: OHpak SB-806 (8 mm × 300 mm) (Showa Denko)
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)
Flow rate: 1 ml / min Column temperature: 25 ° C

  In order to measure the average molecular weight of the reaction product, a commercially available pullulan (STANDARD P-82, manufactured by Showa Denko KK) with a known molecular weight was analyzed under the same conditions as in the above HPLC analysis, and the molecular weight of pullulan and OHpak SB-806 The relationship with the retention time is represented by a curve, which is a standard curve for measuring the molecular weight of the enzyme reaction product.

Reference example 3
Using the fucose sulfate-containing polysaccharide-U obtained in the step of Reference Example 1- (2), the sulfated fucose-containing polysaccharide-degrading activity was measured as follows.
50 μl of a 2.5% sulfated-fucose-containing polysaccharide-U solution, 10 μl of a test solution for measuring degradation activity, and 60 μl of 83 mM phosphate buffer pH 7.5 containing 667 mM sodium chloride were mixed at 37 ° C. for 3 hours. After the reaction, 105 μl of the reaction solution and 2 ml of water are mixed and stirred, and the absorbance (AT) at 230 nm is measured. As controls, instead of the test solution for measuring the degradation activity, the reaction was carried out under the same conditions using only the buffer solution used for the test solution, and only the water instead of the fucose sulfate-containing polysaccharide-U solution was used. Are prepared, and the absorbance is measured in the same manner (AB1 and AB2).

  One unit of enzyme is the amount of enzyme that releasably cleaves a glycosidic bond between 1 μmol of mannose and uronic acid per minute in the above reaction system. Quantification of the cleaved bond is performed by calculating the molar extinction coefficient of unsaturated uronic acid generated during the elimination reaction as 5.5. The enzyme activity was calculated according to the following formula.

(AT-AB1-AB2) × (2.105 × 120 / 5.5 × 105 ×)
0.01 × 180) = U / ml
Where
2.105 is the liquid volume (ml) of the sample for measuring absorbance,
120 is the volume of the enzyme reaction solution (μl),
5.5 is the molar extinction coefficient (/ mM) of unsaturated uronic acid at 230 nm,
105 is the volume of the reaction solution used for dilution (μl),
0.01 is the amount of enzyme solution (ml),
180 is the reaction time (minutes)
It is.

Reference example 4
(1) When the molecular weights of the sulfated-fucose-containing polysaccharide-F and the sulfated-fucose-containing polysaccharide-U were determined by gel filtration using Sephacryl S-500, molecular weight distributions centered on about 190,000 respectively were shown.
(2) Precipitate formation in the presence of an excess amount of cetylpyridinium chloride at each sodium chloride concentration of sulfated fucose-containing polysaccharide-U and sulfated-fucose-containing polysaccharide-F is shown in FIG.

  The vertical axis in FIG. 2 indicates the precipitation rate (%), and the horizontal axis indicates the sodium chloride concentration (M). In the figure, solid lines and white circles indicate the rate of precipitation formation at each sodium chloride concentration of sulfated-fucose-containing polysaccharide-U, and dotted lines and white triangles at each sodium chloride concentration (M) of sulfated-fucose-containing polysaccharide-F. The rate of precipitation formation is shown.

The precipitation rate was measured at a solution temperature of 37 ° C. as follows.
Fucose sulfate-containing polysaccharide-U and sulfated-fucose-containing polysaccharide-F are dissolved in water and 4M sodium chloride at a concentration of 2%, respectively, and mixed in various proportions to dissolve fucose in various concentrations of sodium chloride. 125 μl each of sulfuric acid-containing polysaccharide-U and fucose sulfate-containing polysaccharide-F solutions were prepared. Next, cetylpyridinium chloride is 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. did.

  It took 3.2 times by volume to completely precipitate 2% fucose sulfate-containing polysaccharide-U and fucose sulfate-containing polysaccharide-F dissolved in water with 1.25% cetylpyridinium chloride. Therefore, after adding 400 μl of cetylpyridinium chloride solution dissolved in each concentration of sodium chloride to 125 μl each of 2% sulfated fucose-containing polysaccharide-U and sulfated fucose-containing polysaccharide-F dissolved in each concentration of sodium chloride, Stir well, allow to stand for 30 minutes, centrifuge and measure the sugar content in the supernatant by the phenol-sulfuric acid method [Analytical Chemistry, 28, 350 (1956)]. The precipitation rate of each sulfated fucose-containing polysaccharide under the concentration was calculated.

(3) The component of the sulfated-fucose-containing polysaccharide-F was analyzed by the following method.
First, the amount of fucose was quantified according to the description of Journal of Biological Chemistry, Vol. 175, p. 595 (1948).

A dried preparation of sulfated-fucose-containing polysaccharide-F was dissolved in 1N hydrochloric acid at a concentration of 0.5%, treated at 110 ° C. for 2 hours, and hydrolyzed to a constituent monosaccharide. Glycotag (GlycoTAG ^™ ) and Glycotag ^™ Reagent Kit (both manufactured by Takara Bio Inc.) were used to hydrolyze the reducing end of the monosaccharide obtained by pyridyl- (2) -amination ( PA) and the ratio of the constituent sugars was examined by HPLC.

Next, the amount of uronic acid was quantified as described in Analytical Biochemistry, Vol. 4, page 330 (1962).
Further, the sulfuric acid content was quantified according to the description in Biochemical Journal, Vol. 84, page 106 (1962).

  The constituent sugars of this fucose sulfate-containing polysaccharide-F were fucose and galactose, and the molar ratio was about 10: 1. Uronic acid and other neutral sugars were not substantially contained. Further, the molar ratio of fucose and sulfate group was about 1: 2.

  The component of sulfated-fucose-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. Other neutral sugars were not substantially contained. The main components of fucose: mannose: galactose: uronic acid: sulfate group were about 10: 7: 4: 5: 20 in molar ratio.

(4) The specific rotation of the freeze-dried product of this fucose sulfate-containing polysaccharide-F was measured with a high-speed and high-sensitivity polarimeter SEPA-300 (manufactured by Horiba Ltd.) and found to be -135 degrees.
The specific rotation of the sulfated-fucose-containing polysaccharide-U was −53.6 °.

  (5) Endo-type fucoidan degradation according to Reference Example 5 described below in Reference Example 5 with 16 ml of 1% fucose sulfate-containing polysaccharide-F solution, 12 ml of 50 mM phosphate buffer (pH 8.0), 4 ml of 4M sodium chloride, and 32 mU / ml 8 ml of the enzyme solution was mixed and reacted at 25 ° C. for 48 hours. Formation of decomposition products due to the reaction was not observed.

  Under the above conditions, the fucose sulfate-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 sulfated-fucose-containing polysaccharide-U was decomposed by this enzyme.

Reference Example 5
The fucoidan degrading enzyme described in Reference Example 4 is prepared by the following method. The strain used for production of the fucoidan degrading enzyme may be any strain as long as it has the ability to produce the enzyme. Specific examples include, for example, Flavobacterium sp. SA-0082 strain (FERM BP-5402) can be mentioned.

  This strain was newly obtained by searching from seawater in Aomori Prefecture, and this strain is Flavobacterium sp. SA-0082 is displayed, and from March 29, 1995 (original deposit date), it has been deposited internationally with the accession number of FERM BP-5402 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry.

  The nutrient source added to the culture medium of this strain may be any strain that can be used by the strain to be used and produces a fucoidan degrading enzyme. Examples of the carbon source include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, Maltose, lactose, starch and the like can be used, and as the nitrogen source, yeast extract, peptone, casamino acid, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like are suitable. In addition, minerals such as sodium salts, phosphates, potassium salts, magnesium salts, zinc salts, and metal salts may be added.

  When cultivating the production strain of this fucoidan degrading enzyme, the production amount varies depending on the culture conditions. Generally, the culture temperature is 15 ° C. to 30 ° C., the pH of the medium is preferably 5 to 9, and aeration stirring is performed for 5 to 72 hours. The production of this fucoidan-degrading enzyme reaches its maximum in culture. Naturally, the culture conditions are set so as to maximize the production amount of the present fucoidan-degrading enzyme according to the strain used, the composition of the medium, and the like.

  The fucoidan-degrading enzyme is present in the cells and in the culture supernatant.

  Flavobacterium sp. The SA-0082 strain is cultured in an appropriate medium, the cells are collected, and the cells are disrupted by a commonly used cell disruption means such as ultrasonic treatment to obtain a cell-free extract.

  Next, a purified enzyme preparation can be obtained from this extract by a commonly used purification means. For example, the purified fucoidan-degrading enzyme can be obtained by purification by salting out, ion exchange column chromatography, hydrophobic binding column chromatography, gel filtration or the like.

  In addition, since a large amount of the present enzyme (extracellular enzyme) is also present in the culture supernatant obtained by removing the cells from the above culture solution, the enzyme can be purified by the same purification means as the enzyme in the cells.

An example of purification of fucoidan degrading enzyme is shown.
Flavobacterium sp. SA-0082 (FERM BP-5402) was dispensed with 600 ml of an artificial seawater (made by Jamarin Laboratory) pH 7.5 containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract. Sterilized (120 ° C., 20 minutes) was inoculated into a 2-liter Erlenmeyer flask and cultured at 24 ° C. for 24 hours to obtain a seed culture solution. Consists of artificial seawater (jamarin laboratory) 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). Twenty liters of medium was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture solution was inoculated and cultured at 24 ° C. for 24 hours under conditions of an aeration rate of 10 liters per minute and a stirring speed of 125 revolutions per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells.

  This microbial cell was suspended in 20 mM acetic acid-phosphate buffer (pH 7.5) containing 200 mM sodium chloride, subjected to ultrasonic disruption, and centrifuged to obtain a microbial cell extract. When the activity of the fucoidan degrading enzyme in this bacterial cell extract was measured by the method described in Reference Example 3, an activity of 5 mU was detected in 1 ml of the medium. The activity measurement will be described later.

  To this extract, ammonium sulfate is added so that the final concentration becomes 90% saturation, and after stirring and dissolving, the mixture is centrifuged. The precipitate is suspended in the same buffer as the above-mentioned cell extract and 20 mM containing 50 mM sodium chloride. Dialyzed thoroughly with an acetic acid-phosphate buffer solution (pH 7.5). After removing the precipitate generated by dialysis by centrifugation, the precipitate is adsorbed on a DEAE-Sepharose FF column equilibrated in advance with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride. After thoroughly washing with a buffer solution, the active fraction was collected by elution with a linear concentration gradient of 50 mM to 600 mM sodium chloride. Next, sodium chloride was added to this active fraction to a final concentration of 4M, and phenyl sepharose CL-4B (Pharmacia) previously equilibrated with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbate was sufficiently washed with the same buffer and eluted with a linear concentration gradient of 4M to 1M sodium chloride to collect the active fraction. Next, after concentrating this active fraction with an ultrafilter, the active fraction was collected by performing gel filtration with Sephacryl S-300 (Pharmacia) equilibrated in advance with 10 mM phosphate buffer containing 50 mM sodium chloride. It was. When the molecular weight of this enzyme was determined from the retention time of Sephacryl S-300, it was about 460,000. Next, the active fraction was dialyzed against 10 mM phosphate buffer (pH 7) containing 25 mM sodium chloride. This enzyme solution was adsorbed onto a mono (Mono) Q HR5 / 5 (Pharmacia) column previously equilibrated with 10 mM phosphate buffer (pH 7) containing 250 mM sodium chloride, and the adsorbate was adsorbed on the buffer solution. After sufficiently washing with, elution was performed with a linear concentration gradient of 250 mM to 450 mM sodium chloride, and the active fractions were collected to obtain a purified enzyme. The above purification steps are 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 a 1 mg / ml protein solution is calculated as 1.0.

Further, fucoidan degrading enzyme can also be purified by the following method.
Flavobacterium sp. SA-0082 (FERM BP-5402) was dispensed into 600 ml of a medium consisting of artificial seawater (made by Jamarin Laboratory) pH 7.5 containing 0.1% glucose, 1.0% peptone and 0.05% yeast extract. Sterilized (120 ° C., 20 minutes) into a 2-liter Erlenmeyer flask and cultured at 24 ° C. for 20 hours to form a seed culture solution. Artificial seawater (jamarin laboratory) pH7 containing fucoidan 0.3% derived from gagome kelp, 0.5% peptone, 0.01% yeast extract, and 0.01% antifoam (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) 20 liters of the medium consisting of 5 was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture solution was inoculated and cultured at 24 ° C. for 20 hours under conditions of an aeration rate of 10 liters per minute and a stirring speed of 125 revolutions per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells and a culture solution supernatant. The cells obtained in the main culture were suspended in a 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM sodium chloride, subjected to ultrasonic disruption, and centrifuged to obtain a cell extract. When the activity of the fucoidan-degrading enzyme in this bacterial cell extract was measured, 20 mU of activity was detected in 1 ml of the medium.

  On the other hand, when this culture supernatant was concentrated by ultrafiltration (exclusion molecular weight of filtration membrane 10,000) (Amicon) and fucoidan degrading enzyme was measured, an activity of 6 mU per 1 ml of the culture was detected.

  Ammonium sulfate is added to the concentrated solution of the culture broth so that the final concentration becomes 90% saturation, and after stirring and dissolving, the solution is centrifuged. The precipitate is suspended in the same buffer as the above-mentioned cell extract, and 50 mM Dialyzed thoroughly with 20 mM acetate-phosphate buffer (pH 7.5) containing sodium chloride. After removing the precipitate generated by dialysis by centrifugation, the precipitate is adsorbed onto a DEAE-Sepharose FF column equilibrated in advance with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride, and the adsorbate is adsorbed to the same buffer. After sufficiently washing with the solution, the active fraction was collected by eluting with a gradient of 50 mM to 600 mM sodium chloride. Next, sodium chloride is added to the active fraction to a final concentration of 4M, and the active fraction is adsorbed to a column of phenyl sepharose CL-4B equilibrated in advance with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbate was thoroughly washed with the same buffer and eluted with a 4M to 1M sodium chloride gradient to collect the active fraction. Next, this active fraction was concentrated with an ultrafilter (Amicon), and then gel filtered with Sephacryl S-200 equilibrated in advance with a 10 mM phosphate buffer containing 50 mM sodium chloride to collect the active fraction. Next, sodium chloride is added to this active fraction to a final concentration of 3.5M and adsorbed on a column of phenyl sepharose HP equilibrated with 10 mM phosphate buffer (pH 8.0) containing 3.5M sodium chloride in advance. The adsorbate was washed with the same buffer and eluted with a 3.5M to 1.5M sodium chloride gradient, and the active fraction was collected to obtain a purified enzyme. When the molecular weight of this enzyme was determined from the retention time of Sephacryl S-200, it was about 70,000.

Example 1
(1) Arteromonas sp., Which is a production strain of endo-fucose sulfate-containing polysaccharide-degrading enzyme SN-1009 (FERM BP-5747) was dispensed in 500 ml of a medium consisting of artificial seawater (jamalin laboratory) pH 8.0 containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract. And inoculated into a 2 liter Erlenmeyer flask (120 ° C., 20 minutes) and cultured at 25 ° C. for 23 hours. After completion of the culture, the culture solution is centrifuged to collect the cells, and half of the cells are suspended in 10 ml of extraction buffer [50 mM Tris-HCl buffer (pH 8.0), 100 mM ethylenediaminetetraacetic acid (EDTA)]. After turbidity, a 20 mg / ml lysozyme solution dissolved in 1 ml of extraction buffer was added, and the mixture was kept on an ice bath for 30 minutes. Subsequently, 10 ml of proteinase K solution [1 mg / ml proteinase K, 50 mM Tris-HCl buffer (pH 8.0), 100 mM EDTA, 1% SDS] was added, and the mixture was kept at 50 ° C. for 2 hours. After returning to room temperature, an equal volume of TE buffer [10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA] saturated phenol was added, gently stirred for 1 hour, centrifuged at 10000 rpm for 20 minutes, and the upper layer was collected. (Hereafter, this operation is abbreviated as phenol extraction).

  To this upper layer, an equal volume of TE buffer saturated phenol / chloroform 1: 1 was added, and the mixture was gently stirred and centrifuged at 10,000 rpm for 20 minutes, and then the upper layer was recovered (hereinafter this operation is abbreviated as phenol / chloroform extraction). After extraction with phenol / chloroform again, sodium chloride was added to the aqueous layer to a concentration of 0.1 M, and further doubled ethanol was added to precipitate the DNA, which was wound up with a glass rod and rinsed with 80% ethanol. And lightly air dried. This genomic DNA was dissolved in 20 ml of TE buffer in which 20 μg / ml of 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 described above, and DNA was collected and suspended in 5 ml of TE buffer. About 20 mg of genomic DNA was obtained by the above operation.

(2) 100 μg of the genomic DNA prepared in Example 1- (1) was digested with 10 units of the restriction enzyme Sau3AI at 37 ° C. for 1 minute and 40 seconds, partially decomposed, extracted with phenol / chloroform, and the upper layer was recovered. Add 1/10 volume of 3M sodium acetate aqueous solution (pH 5.0) and 2.5 volumes of ethanol to the upper layer to precipitate DNA, collect the precipitate by centrifugation, rinse with 80% ethanol and air dry. (This operation is hereinafter abbreviated as ethanol precipitation). The obtained partially decomposed product was subjected to size fractionation by 1.25-5M sodium chloride density gradient ultracentrifugation, and DNA was collected by ethanol precipitation from a fraction containing a size of 10 to 20 kbp. After mixing 0.18 μg of the resulting genomic DNA partial degradation product and 0.6 μg of Novagene's Lamb Double Star BamHI arm (λ Blue STAR BamHI arm) and ligating them using a DNA ligation kit manufactured by Takara Bio. The lambda phage was packaged using a Gigapack II Gold kit manufactured by Stratagene, and Alteromonas sp. A genomic DNA library of SN-1009 was prepared.

  (3) Alteromonas sp. Obtained in Reference Example 1- (3) After applying 200 pmol of SN-1009 purified endo-fucose sulfate-containing polysaccharide-degrading enzyme protein to a desalting column equilibrated with 20 mM ammonium hydrogen carbonate (first desalting column PC3.2 / 10, manufactured by Pharmacia), the same Elute with buffer to replace the buffer. The eluate is collected in a glass vial and concentrated to dryness. The sample is then placed in a large glass test tube containing 10 μl of pyridine, 2 μl of 4-vinylpyridine, 2 μl of tri-N-butylphosphine, and 10 μl of water. After sealing the glass test tube, it was reacted at 95 ° C. for 10 minutes for pyridylethylation. After completion of the reaction, the glass vial was taken out and azeotroped with water several times to remove volatile components.

  The obtained pyridylethylated fucose-containing polysaccharide-degrading enzyme protein was mixed with 40 μl of 8 mM urea-containing 10 mM Tris-HCl buffer (pH 9.0), 90 μl of 10 mM Tris-HCl buffer (pH 9.0), and 0.5 pmol of Achromobacter protease I (manufactured by Takara Bio Inc.) was added and digested overnight at 30 ° C., and the peptide fragment was purified from the resulting digest by HPLC system (Smart System, Pharmacia). As the column, μRPC C2 / C18 SC2.1 / 10 (Pharmacia) was used at a flow rate of 100 μl / min. For elution, 0.12% trifluoroacetic acid aqueous solution (eluent A) and acetonitrile containing 0.1% trifluoroacetic acid (eluent B) were used as eluent, and the sample was applied at a ratio of eluent B of 0%. Then, elution was performed by a linear concentration gradient method in which the ratio of the eluate B was increased to 55% in 90 minutes, followed by separation and purification. Amino acid sequence analysis was performed on each peptide fraction, and 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) were determined.

  (4) After digesting 20 μg of the genomic DNA prepared in Example 1- (1) with 100 units of restriction enzymes BamHI, EcoRI, HindIII, PstI, SacI, SalI, SphI, and XbaI for 4 hours each at 37 ° C., phenol / chloroform Extracted. The digest was recovered by ethanol precipitation, and 10 μg of each was further digested with 50 units of the same restriction enzyme at 37 ° C. for 16 hours, followed by phenol / chloroform extraction, and the digest was recovered by ethanol precipitation. Each 5 μg of this digest was subjected to 0.8% agarose gel electrophoresis, and a nylon membrane [trade name: High Bond N +, manufactured by Amersham Co., Ltd.] by Southern blotting (Reference: Genetic Research Method II, pages 218-221, Tokyo Chemical Dojin). The DNA was transferred to (Hybond-N +)].

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). 20 pmol of the synthetic oligonucleotide was labeled with ³² P using a megalabel kit (MEGALABEL KIT, manufactured by Takara Bio Inc.).

  The prepared filter was prehybridized at 65 ° C. for 3 hours in a solution containing 6 × SSC, 1% SDS, 100 μg / ml salmon sperm DNA, 5 × Denharz, and then labeled probe at 0.5 pmol / ml. Then, hybridization was performed overnight at 42 ° C. After completion of hybridization, first wash in 6 × SSC at room temperature for 10 minutes, in 1 × SSC, 0.1% SDS for 10 minutes at room temperature, in 1 × SSC, 0.1% SDS for 30 minutes at 42 ° C. After removing excessive moisture, the film was exposed to an imaging plate manufactured by Fuji Film Co., Ltd. for 30 minutes, and then detected with a BAS2000 imaging analyzer manufactured by Fuji Film Co., Ltd.

  As a result, the BamHI, EcoRI and SalI digests have positions of 23 kbp or more, the HindIII digests have positions of about 4.8, 1.4, and 0.3 kbp, the PstI digests have positions of 23 kbp or more and 3.6 kbp, Digested at positions 23 kbp and 9.8 kbp, SphI digested at positions 23 kbp and above, 4.9 kbp and 3.0 kbp, XbaI digested at positions 12, 5.2 and 3.5 kbp, respectively. A band hybridizing with the probe was observed.

  Arteromonas sp. Prepared in Example 1- (2) Clones containing a sulfated-fucose-containing polysaccharide-degrading enzyme gene were screened from the SN-1009 genomic DNA library according to Novagen's Lamb double star instruction manual by plaque hybridization. First, the phage library was infected with E. coli ER1647, and about 500 plaques were formed on four L medium plates with a diameter of 8.5 cm. This plate was brought into contact with Amersham's nylon membrane [trade name: High Bond N +] for about 30 seconds for the first sheet and for about 2 minutes for the second sheet, and the phages were copied on two plates. This nylon membrane was denatured for 5 minutes on a filter paper soaked in a solution of 0.5 M sodium hydroxide and 1.5 M sodium chloride, and then 0.5 M Tris-HCl buffer (pH 7.0), 3 M sodium chloride solution The sample was neutralized on the filter paper soaked in 5 minutes and rinsed with 2 × SSC. When this nylon membrane and synthetic oligonucleotide pFDA27 (SEQ ID NO: 13) were hybridized, washed and detected under the same conditions as in the Southern hybridization described above, 18 positive signals were obtained. From the original plate, plaques in the vicinity of the positive signal were scraped, suspended in SM buffer, again formed into plaques on a new plate, and the same operation was repeated to isolate phages that gave 14 positive signals.

According to Novagen's Lamb Double Star instruction manual, each phage obtained was infected with E. coli BM25.8, and then an ampicillin resistant colony was selected, and the phage was converted into a plasmid form. The colonies of the obtained clones were inoculated into L medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) containing 100 μg / ml ampicillin and cultured overnight at 37 ° C. Plasmid DNA was prepared by the lysis method. Using this plasmid DNA, Escherichia coli JM109 (manufactured by Takara Bio Inc.) was transformed, and colonies of each clone obtained were inoculated into L medium containing 100 μg / ml ampicillin and cultured at 37 ° C. overnight. Plasmid DNA was prepared again by the alkaline lysis method. The plasmids of the obtained clones were named pSFDA1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16, and pSFDA17, respectively.

  Each plasmid was digested with the restriction enzyme NotI at both ends of the cloning site of the lambda double star vector and analyzed by 0.3% agarose gel electrophoresis. As a result, a fragment of 8.4 to 17.4 kbp was inserted. It became clear. Further, each plasmid was digested with the restriction enzyme HindIII, and after Southern blotting as described above, hybridization was performed with the synthetic oligonucleotide pFDA27 (SEQ ID NO: 13). As a result, a band of about 4.8 kbp was obtained. Give (pSFDA1, and pSFDA17), give 4.8 and 0.3 kbp bands (pSFDA5, 6, 7, 11, and pSFDA13), give about 1.4 kbp bands (pSFDA10), about 0 A band giving a band of .3 kbp and other sizes (pSFDA2 and pSFDA14), a band giving a band of about 4.8 kbp and other sizes (pSFDA16), a band of a size of 5.0 kbp or more What to give (pSFDA4, , And they were divided into six groups of pSFDA12). However, except for pSFDA10 which gives a band of about 1.4 kbp, a plurality of bands having similar sizes are detected by ethidium bromide staining of agarose gel electrophoresis. It was estimated that approximately 4.8 and 0.3 kbp of the HindIII fragment or either one or a part thereof was inserted. In addition, it was speculated that the approximately 4.8 and 0.3 kbp HindIII fragments that hybridize with this pFDA27 are in very close portions on the genome. Therefore, the 14 plasmids obtained were roughly grouped into pSFDA10 and other plasmids that give a band of about 1.4 kbp, and were detected by Southern hybridization of genomic DNA HindIII digestion at about 4.8, 1.. It was speculated to have either 4, and 0.3 kbp, or both about 4.8 and 0.3 kbp. Among the resulting plasmids, pSFDA7 having an insert of about 10.2 kbp, including both HindIII fragments of about 4.8 and 0.3 kbp, and an insert of about 8.4 kbp, about 1.4 kbp PSFDA10 containing the HindIII fragment of the above was digested with several types of restriction enzymes and then analyzed by agarose gel electrophoresis to produce a restriction enzyme map, and the region hybridized with synthetic oligonucleotide pFDA27 was subcloned into plasmid pUC119 and the like, followed by the dideoxy method. The base sequence was analyzed by Among the approximately 1.4 kbp HindIII fragment of pSFDA10, a sequence in which 14 bases out of 17 bases of pFDA27 matched was found, but this fragment does not match the sequence encoding the amino acid sequence of F27. However, it was considered to be a fragment unrelated to the endo-fucose sulfate-containing polysaccharide degrading enzyme gene. On the other hand, the partial amino acid sequence F27 determined in Example 1- (3), which is completely identical to one sequence of pFDA27 from both the approximately 4.8 and 0.3 kbp HindIII fragments of pSFDA7, including the surrounding sequences. A sequence encoding an amino acid matching (SEQ ID NO: 9) was found.

  Further, the HindIII fragments of about 4.8 kbp and 0.3 kbp are separated by about 3 kbp or more on the restriction enzyme map, and the Alteromonas sp. Since it is larger than the size of the enzyme gene expected from the molecular weight of about 100,000 measured by gel filtration of the endo-fucose sulfate-containing polysaccharide-degrading enzyme purified from SN-1009, at least two similar genes are present. It was expected to be.

  The Escherichia coli JM109 strain into which pSFDA7 has been introduced is designated as Escherichia coli JM109 / pSFDA7. In addition, Escherichia coli JM109 strain into which pSFDA7 has been introduced is designated as Escherichia coli JM109 / pSFDA7 and has been deposited as FERM P-16362 on August 1, 1997 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. FERM BP-6340 (date of transfer to international deposit: May 6, 1998) has been deposited internationally at the Biotechnology Institute of Technology.

  The base sequence of pSFDA7 was further analyzed by the dideoxy method using the primer extension method, and the base sequence of 8.3 kbp was determined from the 10.2 kbp insert of pSFDA7. As a result, 2646 bases (including the stop codon) were obtained. Reading frame 1 (hereinafter abbreviated as ORF-1) and reading frame 2 (hereinafter abbreviated as ORF-2) of 2445 bases (including the stop codon) were found. The approximately 0.3 and 4.8 kbp HindIII fragments detected by Southern hybridization of the genomic DNA HindIII digest are exactly 140 and 4549 base pairs in length, respectively, and are part of ORF-1 and ORF-2, respectively. Or it included the full length. In addition to the sequence corresponding to the endo-fucose sulfate-containing polysaccharide degrading enzyme partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3) in the amino acid sequence encoded by ORF-2, the partial amino acid sequence F34 ( Sequences highly homologous to SEQ ID NO: 10), F47 (SEQ ID NO: 11), and F52 (SEQ ID NO: 12) were found. Therefore, ORF-2 is related to Alteromonas sp. It is considered that SN-1009 substantially encodes an endo-fucose sulfate-containing polysaccharide-degrading enzyme. On the other hand, in the amino acid sequence encoded by ORF-1, a partial amino acid other than the sequence corresponding to the endo-fucose sulfate-containing polysaccharide degrading enzyme partial amino acid sequence F27 (SEQ ID NO: 9) determined in Example 1- (3) A sequence having high homology with sequences F34 (SEQ ID NO: 10) and F52 (SEQ ID NO: 12) was found, but a sequence showing high homology with partial amino acid sequence F47 (SEQ ID NO: 11) was not found. . When comparing the amino acid sequences encoded by ORF-1 and ORF-2, there is an insertion sequence of 67 amino acid residues not present in ORF-2 near the N-terminal of ORF-1, and the amino acid sequence after the insertion sequence is 70% or more. Therefore, it was considered that ORF-1 also encodes a polypeptide having a fucose sulfate-containing polysaccharide degrading activity. As described above, it is considered that a gene (ORF-2) that is thought to encode a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity and a fucose sulfate-containing polysaccharide-degrading activity that exhibits very high homology to the gene. The entire base sequence of the gene (ORF-1) encoding the novel polypeptide was determined.

  The result is shown in FIG. That is, FIG. 1 is a diagram showing the positions of ORF-1 and ORF-2. The black arrows in the figure indicate the ORF-1 code area and orientation, and the hatched arrows in the figure indicate the ORF-2 code area and orientation, respectively. The base 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. Further, 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 described above, according to the present invention, a gene (ORF-2) that is considered to substantially encode an endo-type fucose sulfate-containing polysaccharide-degrading enzyme and a novel polypeptide that exhibits homology to the gene and has fucose sulfate-containing polysaccharide-degrading activity A gene (ORF-1) that is thought to encode is isolated and purified.

Example 2
In order to construct a direct expression vector of the gene (ORF-2) obtained in Example 1, which is considered to encode a substantially endo-fucose sulfate-containing polysaccharide-degrading enzyme, the start codon of ORF-2 was placed on the expression vector. The plasmid pEFDA-N was constructed by inserting a part of the 5 ′ region of ORF-2 in accordance with the optimized start codon.

  First, synthetic DNAs, FDA-N1 (SEQ ID NO: 14) and FDA-N2 (SEQ ID NO: 15) were synthesized. FDA-N1 is a 15-mer synthetic DNA containing the sequence of SEQ ID NO: 5 of the sequence listing and the sequence of SEQ ID NO: 1-13, and FDA-N2 is complementary to the sequence of SEQ ID NO: 5 of the sequence listing 5-4 This is a 15-mer synthetic DNA containing a unique sequence.

  These synthetic DNAs were kept in a solution containing 0.2 M Tris-HCl buffer (pH 7.5) and 0.3 M sodium chloride at 70 ° C. for 10 minutes, and then slowly cooled to room temperature to form double strands. The linker FDA-N was prepared. The obtained FDA-N contains a SnaBI site at nucleotide sequence numbers 8-13 of SEQ ID NO: 5 in the sequence listing, and is a synthetic DNA that can be linked to the NcoI site at the start codon and the BamHI site immediately downstream of the SnaBI site. It is a linker.

  On the other hand, pET21d (manufactured by Novagen), which is an expression vector using the T7 promoter, is divided into an NcoI site containing an initiation codon optimized for expression downstream of the T7 promoter and a BamHI site in the multicloning site. Disconnected. This digested product and the previously prepared synthetic DNA linker FDA-N were ligated using a DNA ligation kit (manufactured by Takara Bio Inc.), and then transformed into E. coli JM109, on an L medium plate containing 100 μg / ml ampicillin. Growing colonies were selected. Each transformant was inoculated into L medium containing 100 μg / ml ampicillin, cultured at 37 ° C. overnight, and plasmid DNA was prepared from the cultured cells by the alkaline lysis method. The plasmid DNA was digested with SnaBI and subjected to 1% agarose gel electrophoresis, a plasmid that can be cleaved with SnaBI was selected, the nucleotide sequence of the inserted fragment was confirmed by the dideoxy method, and the ORF-2 between the NcoI-BamHI sites of pET21d. Plasmid pEFDA-N in which the region from the start codon to the SnaBI site was inserted was obtained.

  Next, about 5 μg of the plasmid pSFDA7 obtained in Example 1 was digested with 30 units of SnaBI at 37 ° C. for 2 hours, separated by 1% agarose gel electrophoresis, and about 2 containing the almost full length region of ORF-2. A .5 kbp SnaBI fragment was excised and extracted and purified. This SnaBI fragment was mixed with the previously constructed SnaBI digest of plasmid pEFDA-N, ligated using a DNA ligation kit, transformed into E. coli JM109, and grown on an L medium plate containing 100 μg / ml ampicillin. Selected. Plasmid DNA was prepared in the same manner as above, digested with SnaBI, and subjected to 1% agarose gel electrophoresis to select a plasmid that released a 2.5 kbp SnaBI fragment. Further, the orientation of the inserted fragment was confirmed by the dideoxy method, and a plasmid in which ORF-2 was inserted in the same direction as the T7 promoter was selected. The thus obtained expression plasmid into which the full length of ORF-2 was inserted from the start codon at the NcoI site of pET21d was named pEFDAII103.

  The pEFDAII103 thus obtained was used to transform E. coli BL21 (DE3) strain (Novagen). The obtained Escherichia coli BL21 (DE3) / pEFDAII103 was inoculated into 5 ml of L medium containing 100 μg / ml ampicillin and 5 mM calcium chloride and cultured with shaking at 37 ° C. D. At the stage of 600 = 0.8, IPTG was added so that the final concentration was 1 mM, and then the culture temperature was 15 ° C., followed by further overnight shaking culture. After completion of the culture, the culture solution is centrifuged to collect the cells and suspended in 1 ml of a cell disruption buffer (20 mM Tris-HCl buffer (pH 7.5), 10 mM calcium chloride, 10 mM potassium chloride, 0.3 M sodium chloride). It became cloudy and the cells were crushed by ultrasonication. This was centrifuged and the supernatant was collected to obtain an 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, as a result of analysis of the E. coli extract by SDS polyacrylamide gel electrophoresis, a band with a molecular weight of about 90,000 that was not found in the extract of E. coli BL21 (DE3) / pEFDAII103 was found in the extract of E. coli BL21 (DE3) / pET21d. Was observed. This molecular weight is in good agreement with the molecular weight 88210 of the polypeptide calculated from the amino acid sequence that can be encoded by ORF-2 shown in SEQ ID NO: 1 in the Sequence Listing. This coincides with the molecular weight of about 90,000 analyzed by SDS polyacrylamide gel electrophoresis of endo-fucose sulfate-containing polysaccharide-degrading enzyme purified from SN-1009. As described above, it was confirmed that Escherichia coli BL21 (DE3) / pEFDAII103 expresses the polypeptide encoded by ORF-2.

  Next, the endo-fucose sulfate-containing polysaccharide degrading activity of the E. coli extract was measured by the method described in Reference Example 2.

  As a result, an endo-fucose sulfate-containing polysaccharide-degrading activity of 520 mU / ml was detected in the extract of Escherichia coli BL21 (DE3) / pEFDAII103. That is, the polypeptide encoded by ORF-2 has a fucose sulfate-containing polysaccharide-degrading activity, and about 104 mU of fucose sulfate-containing polysaccharide-decomposing activity in 1 ml of a culture solution of E. coli BL21 (DE3) / pEFDAII103 having the gene of the present invention. It can be seen that the polypeptide encoded by ORF-2 having the above has been produced.

  On the other hand, no endo-fucose sulfate-containing polysaccharide degradation activity was detected from the extract of E. coli BL21 (DE3) / pET21d.

Example 3
The sequences from the start codon of ORF-1 and ORF-2 to the 15 bases are completely identical, and the restriction enzyme SnaBI site is in that region. That is, since the insertion sequence in the plasmid pEFDA-N constructed in Example 2 is exactly the same for ORF-1 and ORF-2, this pEFDA-N can be used for the construction of an ORF-1 expression vector.

  First, about 5 μg of the plasmid pSFDA7 obtained in Example 1 was digested with 30 units of SnaBI at 37 ° C. for 2 hours, separated by 1% agarose gel electrophoresis, and about 3 including the almost full length region of ORF-1. A 2 kbp SnaBI fragment was excised and extracted and purified. This SnaBI fragment was mixed with a SnaBI digest of plasmid pEFDA-N, ligated using a DNA ligation kit, E. coli JM109 was transformed, and colonies that grew on L medium plates containing 100 μg / ml ampicillin were selected.

  Plasmid DNA was prepared in the same manner as in Example 2, digested with SnaBI, and subjected to 1% agarose gel electrophoresis to select a plasmid that releases a 3.2 kbp SnaBI fragment. Further, the orientation of the inserted fragment was confirmed by the dideoxy method, and a plasmid in which ORF-1 was inserted in the same direction as the T7 promoter was selected. The expression plasmid obtained by inserting the full length of ORF-1 from the start codon at the NcoI site of pET21d thus obtained was designated as pEFDAI103.

  Using the thus obtained pEFDAI103, expression of the polypeptide encoded by ORF-1 and confirmation of the fucose sulfate-containing polysaccharide degradation activity were confirmed in the same manner as in Example 2.

  That is, first, Escherichia coli BL21 (DE3) strain was transformed with pEFDAI103. The obtained Escherichia coli BL21 (DE3) / pEFDAI103 was inoculated into 5 ml of L medium containing 100 μg / ml ampicillin and 5 mM calcium chloride and cultured with shaking at 37 ° C. D. At the stage of 600 = 0.8, IPTG was added so that the final concentration was 1 mM, and then the culture temperature was 15 ° C., followed by further overnight shaking culture. After completion of the culture, the culture solution was centrifuged to collect the cells, suspended in 1 ml of a cell disruption buffer, and disrupted by sonication. This was centrifuged and the supernatant was collected to obtain an E. coli extract.

  As a result of analysis of the E. coli extract by SDS polyacrylamide gel electrophoresis, a band with a molecular weight of about 100,000 that was not found in the extract of E. coli BL21 (DE3) / pETDAd103 was observed in the extract of E. coli BL21 (DE3) / pEFDAI103. It was done. This molecular weight is in good agreement with the molecular weight 94910 of the polypeptide calculated from the amino acid sequence that can be encoded by ORF-1 shown in SEQ ID NO: 2 in the Sequence Listing, and ORF-1 is encoded by E. coli BL21 (DE3) / pEFDAI103. It was confirmed that the polypeptide was expressed.

  Next, the endo-fucose sulfate-containing polysaccharide degrading activity of the E. coli extract was measured by the method described in Reference Example 2.

  As a result, 35.8 mU / ml of endo-fucose sulfate-containing polysaccharide-degrading 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-degrading activity, and about 7.2 mU of fucose sulfate-containing polysaccharide is contained 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 degradation activity was produced.

  On the other hand, no endo-fucose sulfate-containing polysaccharide degradation activity was detected from the extract of E. coli BL21 (DE3) / pET21d.

Example 4
(1) Flavobacterium sp., A strain producing fucoidan degrading enzyme SA-0082 (FERM BP-5402) was dispensed with 500 ml of a medium consisting of artificial seawater (made by Jamarin Laboratory) pH 8.0 containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract. And inoculated into a 2 liter Erlenmeyer flask (120 ° C., 20 minutes) and cultured at 25 ° C. for 23 hours. After completion of the culture, the culture solution is centrifuged to collect the cells, and half of the cells are suspended in 10 ml of extraction buffer [50 mM Tris-HCl buffer (pH 8.0), 100 mM ethylenediaminetetraacetic acid (EDTA)]. After turbidity, a 20 mg / ml lysozyme solution dissolved in 1 ml of extraction buffer was added, and the mixture was kept on an ice bath for 30 minutes. Subsequently, 10 ml of proteinase K solution [1 mg / ml proteinase K, 50 mM Tris-HCl buffer (pH 8.0), 100 mM EDTA, 1% SDS] was added, and the mixture was kept at 50 ° C. for 2 hours. After returning to room temperature, an equal volume of TE buffer [10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA] saturated phenol was added, gently stirred for 1 hour, centrifuged at 10000 rpm for 20 minutes, and the upper layer was collected. . To this upper layer, an equal volume of TE buffer saturated phenol / chloroform 1: 1 was added, gently stirred, and centrifuged at 10,000 rpm for 20 minutes, and then the upper layer was recovered. After extraction with phenol / chloroform again, sodium chloride was added to the aqueous layer to a concentration of 0.1 M, and further doubled ethanol was added to precipitate the DNA, which was wound up with a glass rod and rinsed with 80% ethanol. And lightly air dried. This genomic DNA was dissolved in 20 ml of TE buffer in which 20 μg / ml of 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 described above, and DNA was collected and suspended in 5 ml of TE buffer. About 20 mg of genomic DNA was obtained by the above operation.

  (2) 100 μg of the genomic DNA prepared in Example 4- (1) was digested with 10 units of restriction enzyme Sau3AI at 37 ° C. for 1 minute and 40 seconds for partial degradation, followed by phenol / chloroform extraction, and the upper layer was recovered. Add 1/10 volume of 3M sodium acetate aqueous solution (pH 5.0) and 2.5 volumes of ethanol to the upper layer to precipitate DNA, collect the precipitate by centrifugation, rinse with 80% ethanol and air dry. did. The obtained partially decomposed product was subjected to size fractionation by 1.25-5M sodium chloride density gradient ultracentrifugation, and DNA was collected by ethanol precipitation from a fraction containing a size of 10-20 kbp. After mixing with 0.2 μg of the obtained genomic DNA partial degradation product and 0.6 μg of Lamb Double Star BamHI arm manufactured by Novagen and ligating them using a DNA ligation kit manufactured by Takara Bio, Gigapack II Gold kit manufactured by Stratagene was used. And packaging into lambda phage, Flavobacterium sp. A genomic DNA library of SA-0082 was prepared.

  (3) Flavobacterium sp. Obtained as in Reference Example 5 200 pmol of purified fucoidan-degrading enzyme protein of SA-0082 was applied to a desalting column equilibrated with 20 mM ammonium bicarbonate (first desalting column PC3.2 / 10, manufactured by Pharmacia) and then eluted with the same buffer. The buffer was replaced. The eluate is collected in a glass vial and concentrated to dryness. The sample is then placed in a large glass test tube containing 10 μl of pyridine, 2 μl of 4-vinylpyridine, 2 μl of tri-N-butylphosphine, and 10 μl of water. After sealing the glass test tube, it was reacted at 95 ° C. for 10 minutes for pyridylethylation. After completion of the reaction, the glass vial was taken out and azeotroped with water several times to remove volatile components.

  The obtained pyridylethylated fucoidan-degrading enzyme protein was mixed with 40 μl of 8 mM urea-containing 10 mM Tris-HCl buffer (pH 9.0), 90 μl of 10 mM Tris-HCl buffer (pH 9.0) and 0.5 pmol of achromo. Bacter protease I (manufactured by Takara Bio Inc.) was added and digested overnight at 30 ° C., and the peptide fragment was purified from the digested product with an HPLC system (Smart System, Pharmacia). As the column, μRPC C2 / C18 SC2.1 / 10 (Pharmacia) was used at a flow rate of 100 μl / min. For elution, 0.12% trifluoroacetic acid aqueous solution (eluent A) and acetonitrile containing 0.1% trifluoroacetic acid (eluent B) were used as eluents, and the sample was applied at a ratio of eluent B of 0%. The elution fractions L27, L31, L36 and the like are eluted as a peak eluted at a linear concentration gradient method in which the ratio of the eluate B is increased to 55% in 80 minutes and the ratio of the eluate B is 27% or more. Got. Furthermore, fractions with a poorly separated eluent B ratio of 17-27% were collected, concentrated and added to the same HPLC system, and then the elution pattern was changed from 15% to 40% for 87 minutes in 87 minutes. The elution fractions LR8, LR9, LR14, LR16 and the like were obtained by elution with a linear concentration gradient up to%. Amino acid sequence analysis was performed on each of the obtained peptide fractions, and 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: 19) 20) was determined.

  (4) 2.4 μg of genomic DNA prepared in Example 4- (1) was digested with 30 units of restriction enzymes EcoRI, HindIII, MunI, SpeI, XbaI and Sau3AI for 3 hours each at 37 ° C., followed by phenol / chloroform extraction. . The digest was collected by ethanol precipitation. About 0.5 μg of each digest, EcoRI cassette for EcoRI and MunI digests, HindIII cassette for HindIII digests, XbaI cassette for SpeI and XbaI digests, and Sau3AI cassette for Sau3AI digests (each cassette DNA Were mixed with 20 ng each (200 ng for Sau3AI cassette) after being mixed with each other using Takara Bio's DNA ligation kit. The reaction product was recovered by ethanol precipitation, dissolved in 10 μl of water, and used as a template DNA for the PCR method using cassette DNA.

  On the other hand, from the amino acid sequence of 1 to 6 of the partial amino acid sequence LR14 (SEQ ID NO: 19) determined in Example 4- (3), pL14F17 (SEQ ID NO: 21) and from the amino acid sequence of 3 to 11 of pL14F26 (SEQ ID NO: 22). Each mixed oligonucleotide was synthesized in the same orientation as the amino acid sequence.

  1 μl of the template DNA prepared previously, 20 pmol of the cassette primer C1 (Takara Bio Inc.), 100 pmol of the mixed oligonucleotide pL14F17 (SEQ ID NO: 21), and sterilized water were added to make 22 μl, followed by heating at 94 ° C. for 10 minutes and then rapidly cooling. 10 μl of 10-fold concentration Ex-tack amplification buffer (Takara Bio Inc.), 16 μl of 1.25 mM dNTP mixed solution, 2.5 units of Takara Ex Tac (Takara Ex Taq, Takara Bio Inc.), and sterilized water Was added to make 100 μl, and further mineral oil was overlaid. This mixture was subjected to an amplification reaction using an automatic gene amplification apparatus thermal cycler (DNA Thermal Cycler 480, manufactured by Takara Bio Inc.). In the PCR reaction, 25 cycles of denaturation at 94 ° C. for 0.5 minute, annealing at 45 ° C. for 2 minutes, and synthesis reaction at 72 ° C. for 3 minutes were performed for 25 cycles. Finally, incubation was continued at 72 ° C. for 7 minutes to complete the synthesis. It was.

  Next, a second PCR reaction was performed using a first PCR reaction solution diluted 10-fold with sterilized water as a template DNA after heating at 94 ° C. for 10 minutes and then rapidly cooling. That is, 10 μl of the heat-treated first PCR reaction solution, cassette primer C2 (Takara Bio) 20 pmol, mixed oligonucleotide pL14F26 (SEQ ID NO: 22) 100 pmol, 10 μl of 10-fold concentration Ex-tack amplification buffer, 16 μl of 1 A 25 mM dNTP mixed solution, 2.5 units of Takara Ex tack, and sterilized water were added to make 100 μl, and mineral oil was further layered thereon. This mixture 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. Finally, the mixture was incubated at 72 ° C for 7 minutes to complete the synthesis. It was. At the same time, a PCR reaction was carried out on the reaction solution of only the cassette primer C2 or only the mixed oligonucleotide pL14F26 as a control for the non-specific amplification product of each primer.

  When the reaction solution was analyzed by agarose gel electrophoresis and compared with the control of the nonspecific amplification product, multiple amplification bands were detected in many reaction solutions. Among these, a band of about 0.7 kbp was extracted and purified from the second PCR reaction solution of MunI digest-EcoRI cassette, which had a relatively low background and one band was strongly amplified. This DNA fragment and pT7 blue T-vector (pT7blue T-Vector, manufactured by Novagen) were mixed and ligated using a DNA ligation kit, and then transformed into Escherichia coli JM109. White colonies that grow on L media plates containing X-Gal and 1 mM IPTG were selected. Each transformant is inoculated into L medium containing 100 μg / ml ampicillin, cultured at 37 ° C. overnight, plasmid DNA is prepared from the cultured cells by the alkaline lysis method, and a plasmid into which a band of about 0.7 kbp is inserted is prepared. Selected and named pT7-Mun. When this pT7-Mun insertion sequence was subjected to base sequence analysis by the dideoxy method, the 12th and subsequent amino acid sequences of the partial amino acid sequence LR14 (SEQ ID NO: 19) following the pL14F26 sequence used in the second PCR reaction were obtained. A coding region was found. Further downstream, a region encoding an amino acid sequence showing very high homology with partial amino acid sequence L27 (SEQ ID NO: 16) was found. These facts revealed that this approximately 0.7 kbp DNA fragment was part of the fucoidan degrading enzyme gene. The sequence from the primer pL14F26 of this approximately 0.7 kbp DNA fragment to the junction of the EcoRI cassette is shown in SEQ ID NO: 23.

  (5) After digesting 20 μg of the genomic DNA prepared in Example 4- (1) with 100 units of restriction enzymes BamHI, EcoRI, HindIII, PstI, SacI, SalI, SphI, and XbaI for 4 hours each at 37 ° C., phenol / Extracted with chloroform. The digest was recovered by ethanol precipitation, and 10 μg of each was further digested with 50 units of the same restriction enzyme at 37 ° C. for 16 hours, followed by phenol / chloroform extraction, and the digest was recovered by ethanol precipitation. Each 5 μg of the digest was subjected to 0.8% agarose gel electrophoresis, and the DNA was transferred to a nylon membrane high bond N + manufactured by Amersham 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 the vector, and a fragment containing a part of about 0.7 kbp fucoidan-degrading enzyme gene was extracted and purified. . This DNA fragment was labeled with ³² P using a Bucabest labeling kit (BcaBEST Labeling Kit, manufactured by Takara Bio Inc.) to prepare a probe DNA for hybridization.

  The prepared filter was prehybridized at 65 ° C. for 1 hour in a solution containing 6 × SSC, 1% SDS, 100 μg / ml salmon sperm DNA, 5 × denharz, and then the labeled probe was about 1 million cpm / ml. The mixture was added to a concentration of ml, and hybridization was performed overnight at 60 ° C. After completion of the hybridization, first at room temperature in 6 × SSC for 10 minutes, in 2 × SSC, 0.1% SDS at room temperature for 10 minutes, in 0.2 × SSC, 0.1% SDS at room temperature for 5 minutes, further. After washing for 30 minutes at 45 ° C. in 2 × SSC, 0.1% SDS to remove excess water, the film was exposed to an imaging plate made by Fuji Film for 30 minutes, and then detected with a BAS2000 imaging analyzer made by Fuji Film. .

  As a result, BamHI, SalI and SphI, one digest at a position of 23 kbp or more in the SalI digest, two at the positions of about 8 and 3 kbp with the EcoRI digest, two at the positions of about 11 and 4 kbp with the HindIII digest, PstI The probe hybridizes strongly with two probes at positions about 12 and 2.5 kbp, two at positions about 10 and 9.5 kbp with the SacI digest, and one at about 6 kbp with the XbaI digest. Admitted the band. From this, Flavobacterium sp. It was strongly suggested that there are two types 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 SA-0082 genomic DNA library were screened by plaque hybridization according to the instruction manual of Novagene Lamb Double Star.

  First, the phage library was infected with E. coli ER1647, and about 300 plaques were formed on 5 L medium plates with a diameter of 8.5 cm. The plate was contacted with Amersham nylon membrane high bond N + for about 30 seconds to copy the phages. This 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 in a solution of 0.5 M Tris-HCl buffer (pH 7.0) and 3 M sodium chloride for 5 minutes. Treated, rinsed with 2 × SSC and air dried. After this nylon membrane was subjected to ultraviolet irradiation treatment to immobilize DNA, hybridization, washing and detection were carried out under the same conditions as Southern hybridization in Example 4- (5). As a result, 36 positive signals were obtained. . Plaques near the positive signal were scraped from the original plate and suspended in SM buffer to recover phages. For some of the obtained phage solutions, plaques were again formed on a new plate, and the same procedure was repeated to isolate phages that gave 9 positive signals.

  According to Novagen's Lamb Double Star instruction manual, each phage obtained was infected with E. coli BM25.8, spread on L medium plate containing 100 μg / ml ampicillin, and phages were selected by selecting ampicillin resistant colonies. Converted to plasmid form. The obtained clone colonies were inoculated into L medium containing 100 μg / ml ampicillin, and plasmid DNA was prepared from the culture solution cultured overnight at 37 ° C. by the alkaline lysis method. Using this plasmid DNA, Escherichia coli JM109 (manufactured by Takara Bio Inc.) was transformed, and colonies of each clone obtained were inoculated into L medium containing 100 μg / ml ampicillin and cultured at 37 ° C. overnight. Plasmid DNA was prepared again by the alkaline lysis method. The obtained plasmids of the respective clones were named pSFLA1, 5, 10, 11, 12, 13, 15, 17, and pSFLA18, respectively.

  Each plasmid was digested with an appropriate combination of the restriction enzymes KpnI, SacI, and XbaI, and then subjected to agarose gel electrophoresis. Southern hybridization was performed as described above, and a restriction enzyme map of each insert was prepared and analyzed. did. As a result, a plurality of bands having similar sizes were detected by ethidium bromide staining of agarose gel electrophoresis, and two bands hybridized with the about 0.7 kbp insert fragment of pT7-Mun by SacI digestion were obtained. 2 of the set of pSFLA5, 11, 13, and pSFLA17, in which two bands hybridize with the approximately 0.7 kbp insert of pT7-Mun by KpnI digestion as well as the set of 10, 12, 15, and pSFLA18. It became clear that they were grouped into groups. Furthermore, it was revealed that the three plasmids pSFLA5, pSFLA10, and pSFLA17 release an approximately 6 kbp fragment by XbaI digestion, and an approximately 6 kbp XbaI digest detected by Southern hybridization in Example 4- (5). It is suggested that the two bands overlap each other, and Flavobacterium sp. It was revealed 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 insert fragment was further analyzed. That is, about 3 μg of these plasmids were digested with XbaI, and a free fragment of about 6 kbp was extracted and purified. These XbaI fragments were mixed with an XbaI digest of pHSG399 (Takara Bio Inc.), which is a chloramphenicol resistant vector, and ligated using a DNA ligation kit (Takara Bio Inc.), followed by transformation of E. coli JM109, 30 μg White colonies were selected that grew on L media plates containing / ml chloramphenicol, 0.004% X-Gal, and 1 mM IPTG. Each transformant is inoculated into L medium containing 30 μg / ml chloramphenicol, cultured at 37 ° C. overnight, plasmid DNA is prepared from the cultured cells by alkaline lysis, digested with restriction enzymes, and subjected to agarose gel electrophoresis. Went and analyzed. As a result, pH10X6-1 and pH10X6-2 were obtained in which about 6 kbp DNA derived from pSFLA10 was inserted in the reverse direction with respect to the vector DNA. Similarly, pH17X6-7 and pH17X6-11 into which about 6 kbp DNA derived from pSFLA17 was inserted were obtained. Escherichia coli JM109 strain into which pH10X6-1 has been introduced is designated as Escherichia coli JM109 / pH10X6-1 and deposited as FERM P-16659 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry. FERM BP-6341 (Request date for transfer of international deposit: May 6, 1998) is deposited at the Institute of Biotechnology, Institute of Biotechnology. The Escherichia coli JM109 strain into which pH17X6-7 has been introduced is designated as Escherichia coli JM109 / pH17X6-7, deposited as FERM P-16660 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry. FERM BP-6342 (Date of request for transfer of international deposit: May 6, 1998) has been deposited internationally at the Institute of Biotechnology. When these nucleotide sequences were analyzed in detail by the direct primer extension method or by sub-cloning after digestion with restriction enzymes and the dideoxy method, a reading frame of 2094 bases (including a stop codon) was obtained from the DNA fragment of about 6 kbp derived from pSFLA10. However, a reading frame of 2115 bases (including a stop codon) was found from the DNA fragment of about 6 kbp derived from pSFLA17, and the partial amino acid sequence determined in Example 4- (3) in the amino acid sequence encoded by these reading frames And a region with very high homology was found.

  The reading frame derived from pSFLA10 was named fdlA, and the reading frame derived from pSFLA17 was named fdlB.

  The results are shown in FIGS. 3 is a diagram showing the position of fdlA in pSFLA10, and FIG. 4 is a diagram showing the position of fdlB in pSFLA10. Black arrows in FIG. 3 indicate the code region and orientation of fdlA, respectively, and black arrows in FIG. 4 indicate the code region and orientation of fdlB, respectively.

  In the amino acid sequence encoded by fdlA, a sequence corresponding to the fucoidan degrading enzyme partial amino acid sequences L36 (SEQ ID NO: 17) and LR14 (SEQ ID NO: 19) determined in Example 4- (3) is also included. A highly homologous sequence was found with L27 (SEQ ID NO: 16), LR9 (SEQ ID NO: 18) and LR16 (SEQ ID NO: 20). Furthermore, in the peptide fraction named LR8 in Example 4- (3), two amino acid derivatives in each cycle were detected by amino acid sequence analysis, and the amino acid sequence was not uniquely determined. Since the amino acid derivatives detected in each cycle can be explained by applying the two sequences in the amino acid sequence to be identified, it was revealed that the peptide containing these two sequences was contained in this fraction. The respective sequences were named LR8-1 (SEQ ID NO: 24) and LR8-2 (SEQ ID NO: 25). Thus, since the amino acid sequence encoded by fdlA is identical to or highly homologous to all amino acid sequences revealed by the fucoidan-degrading enzyme partial amino acid sequence analysis of Example 4- (3), Flavobacterium sp . It is thought that it substantially encodes the fucoidan degrading enzyme of SA-0082.

  On the other hand, in the amino acid sequence encoded by fdlB, a sequence corresponding to the fucoidan degrading enzyme partial amino acid sequence LR14 (SEQ ID NO: 19) and LR8-1 (SEQ ID NO: 24) is also included, and the partial amino acid sequence L27 (SEQ ID NO: 16) , LR16 (SEQ ID NO: 20), and LR8-2 (SEQ ID NO: 25) were found to be highly homologous to the partial amino acid sequences L36 (SEQ ID NO: 17) and LR9 (SEQ ID NO: 18). The sequence shown was not found. However, when fdlA and fdlB were compared, the base sequence and amino acid sequence homology were as high as about 67% and about 56%, respectively. Therefore, it was considered that fdlB also encodes a polypeptide having fucoidan degradation activity. As described above, the entire base sequence of the gene (fdlA) considered to encode a polypeptide having fucoidan degrading activity and the gene (fdlB) showing very high homology to the gene was determined. The base sequence of fdlA is shown in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence encoded by fdlA is shown in SEQ ID NO: 3 in the sequence listing. Further, the base sequence of fdlB is shown in SEQ ID NO: 8 in the sequence listing, and the amino acid sequence encoded by fdlB is shown in SEQ ID NO: 4 in the sequence listing.

  As described above, the gene (fdlA) considered to substantially encode fucoidan-degrading enzyme according to the present invention and a gene that shows homology to the gene and encodes a novel polypeptide considered to have a fucose sulfate-containing polysaccharide-degrading activity ( fdlB) was isolated and purified.

Example 5
First, a direct expression vector of a gene (fdlA) obtained in Example 4 and considered to substantially encode a fucoidan-degrading enzyme was constructed.

  In the plasmid pH10X6-1 obtained in Example 4, an approximately 6 kbp XbaI fragment derived from pSFLA10 was inserted in a direction facing the fdlA gene and the lac promoter on the vector. This pH10X6-1 was digested with SacI inside the fdlA gene, further digested with BspHI located at the start codon position of the fdlA gene, separated by 1% agarose gel electrophoresis, and about 400 bp encoding the N-terminal region of fdlA. The DNA fragment was excised and extracted and purified.

  On the other hand, pET21d (manufactured by Novagen), which is an expression vector using the T7 promoter, is converted into an NcoI site containing an initiation codon optimized for expression downstream of the T7 promoter and a SacI site in the multicloning site. After cleavage, an about 400 bp BspHI-SacI fragment encoding the N-terminal region of fdlA prepared above was inserted to construct plasmid pEFLA10-N.

  Next, the plasmid pH10X6-1 was digested with HindIII to cleave the inside of the fdlA gene and at the vector-derived HindIII site, and an about 2 kbp DNA fragment encoding the C-terminal region containing the stop codon of the fdlA gene was excised and purified. . This fragment was ligated with the HindIII digest of the previously constructed plasmid pEFLA10-N, and a plasmid having the 2 kbp HindIII fragment inserted in the direction in which the full length of the fdlA gene was regenerated was selected. The expression plasmid obtained by inserting the full length of fdlA from the start codon at the NcoI site of pET21d thus obtained was named pEFLA10.

  Escherichia coli BL21 (DE3) strain (manufactured by Novagen) was transformed with pEFLA10 thus obtained. The obtained Escherichia coli BL21 (DE3) / pEFLA10 was inoculated into 5 ml of L medium containing 100 μg / ml ampicillin and cultured at 37 ° C. with shaking. D. At the stage of 600 = 0.8, IPTG was added so as to give a final concentration of 1 mM, and then further cultured overnight with shaking at a culture temperature of 15 ° C. After completion of the culture, the culture solution is centrifuged to collect the cells, suspended in 0.5 ml of a cell disruption buffer (20 mM sodium phosphate buffer (pH 7.0), 0.3 M sodium chloride), and sonicated. The cells were crushed by. A part of this suspension was taken as an E. coli disruption solution. Further, this was centrifuged to remove insoluble matters to obtain an E. coli extract.

  As a control, Escherichia coli BL21 (DE3) / pET21d transformed with pET21d was cultured under the same conditions at the same time to prepare an Escherichia coli disrupted solution and an extract and used for the following analysis.

  First, as a result of analyzing the E. coli disrupted solution by SDS polyacrylamide gel electrophoresis, a molecular weight of about 7 which is not found in the E. coli BL21 (DE3) / pEFLA10 disrupted solution and in the E. coli BL21 (DE3) / pET21d disrupted solution. 60,000 bands were observed. This molecular weight is in good agreement with the molecular weight 75,740 of the polypeptide calculated from the amino acid sequence that can be encoded by fdlA shown in SEQ ID NO: 3 of the Sequence Listing, and 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 described above, it was confirmed that Escherichia coli BL21 (DE3) / pEFLA10 expresses the polypeptide encoded by fdlA.

  Next, the fucose sulfate-containing polysaccharide degrading 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 degradation activity in the extract of Escherichia coli BL21 (DE3) / pEFLA10 was 2,300 mU / ml. That is, the polypeptide encoded by fdlA has fucoidan-degrading activity, and is encoded by fdlA having about 230 mU of fucose sulfate-containing polysaccharide-degrading activity in 1 ml of a culture solution of E. coli BL21 (DE3) / pEFLA10 having the gene of the present invention. It can be seen that the polypeptide was produced.

  On the other hand, no fucoidan degradation 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 thought to have a fucose sulfate-containing polysaccharide-degrading activity obtained in Example 4, was constructed. A recognition sequence for BspHI exists at the position of the start codon of this fdlB gene. However, since it is methylated due to the upstream sequence, the plasmid recovered from Escherichia coli JM109 used as the host could not be directly digested with BspHI. . Therefore, construction was performed using a DNA fragment amplified by the PCR method.

  In the plasmid pH17X6-7 obtained in Example 4, an about 6 kbp XbaI fragment derived from pSFLA17 was inserted in the same orientation as the fdlB gene and the lac promoter on the vector. First, pH17X6-7 was cleaved at the inside of the fdlB gene and at a vector-derived KpnI site, and self-ligated to construct plasmid pH17X6-7K. A set of synthetic DNA primer 17X6F4 (SEQ ID NO: 26) in the same direction as fdlB and M13 primer M4 (manufactured by Takara Bio Inc.) that can be annealed to the vector at a position upstream from the coding region of fdlB using this pH17X6-7K as a template A PCR reaction was performed. The PCR reaction was performed in the same reaction solution composition as in Example 4- (4), with denaturation at 94 ° C for 0.5 minutes, primer annealing at 55 ° C for 0.5 minutes, and synthesis reaction cycle at 72 ° C for 1 minute. 25 cycles were performed, and finally, the temperature was kept at 72 ° C. for 7 minutes to complete the synthesis. 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 BspHI located at the start codon position of the fdlB gene and MflI site located within the coding region, and then separated by 3% agarose gel electrophoresis. The fragment was excised and extracted and purified.

  On the other hand, pET21d, which is an expression vector using the T7 promoter, was cleaved at the NcoI site and the BamHI site in the multicloning site in the same manner as in Example 5, and then about 450 bp encoding the N-terminal region of fdlB prepared earlier. The BspHI-MflI fragment was inserted to construct plasmid pEFLA17-N.

  Next, the plasmid pH17X6-7 is cleaved at the NspV site inside the fdlB gene and the EcoRI site downstream of the fdlB gene, and an approximately 2 kbp NspV-EcoRI fragment encoding the C-terminal region containing the stop codon of the fdlB gene is excised. Extracted and purified. This fragment was inserted between NspV-EcoRI of the previously constructed plasmid pEFLA17-N. The expression plasmid obtained by inserting the full length of fdlB from the start codon at the NcoI site of pET21d thus obtained was named pEFLA17.

  Using the thus obtained pEFLA17, expression of the polypeptide encoded by fdlB and confirmation of fucoidan degradation activity were carried out in the same manner as in Example 3.

  That is, first, Escherichia coli BL21 (DE3) strain was transformed with pEFLA17. The obtained Escherichia coli BL21 (DE3) / pEFLA17 was inoculated into 5 ml of L medium containing 100 μg / ml ampicillin and cultured with shaking at 37 ° C. D. At the stage of 600 = 0.8, IPTG was added so as to give a final concentration of 1 mM, and then further cultured overnight with shaking at a culture temperature of 15 ° C. After completion of the culture, the culture was centrifuged to collect the cells, suspended in 0.5 ml of a cell disruption buffer, and disrupted by sonication. A part of this suspension was taken as an E. coli disruption solution. Further, this was centrifuged to remove insoluble matters to obtain an E. coli extract.

  As a result of analyzing the E. coli disrupted solution by SDS polyacrylamide gel electrophoresis, the molecular weight of about 77,000 which is not found in the E. coli BL21 (DE3) / pEFLA17 disrupted solution is not found in the E. coli BL21 (DE3) / pET21d disrupted solution. A band was observed. This molecular weight is in good agreement with the molecular weight 76,929 of the polypeptide calculated from the amino acid sequence that can be encoded by fdlB shown in SEQ ID NO: 4 in the sequence listing, and E. coli BL21 (DE3) / pEFLA17 is a polypeptide encoding fdlB. Was confirmed to be expressed.

  Next, the fucoidan degradation activity of the E. coli extract was measured by the method described in Example 3.

  As a result, 480 mU / ml fucoidan degradation activity was detected from the extract of Escherichia coli BL21 (DE3) / pEFLA17. That is, the polypeptide encoded by fdlB has a fucose sulfate-containing polysaccharide-degrading activity, and has about 48 mU of fucose sulfate-containing polysaccharide-degrading activity in 1 ml of a culture solution of E. coli BL21 (DE3) / pEFLA17 having the gene of the present invention. It can be seen that the polypeptide encoded by was produced.

  On the other hand, no fucoidan degradation activity was detected from the extract of E. coli BL21 (DE3) / pET21d.

Example 7
The action of the polypeptide having the activity of degrading a fucose sulfate-containing polysaccharide obtained in Example 5 and Example 6 on the fucose sulfate-containing polysaccharide-U was examined.

That is, 10 μl of Example 5 or Example in a mixture of 50 μl of 100 mM phosphate buffer (pH 7.5), 50 μl of 2.5% fucose sulfate-containing polysaccharide-U, and 10 μl of 4M sodium chloride. The polypeptide solution having the fucose sulfate-containing polysaccharide-degrading activity obtained in 6 was added and kept at 25 ° C. At 16, 40, and 65 hours after the start of the reaction, the reaction solution was collected and the reaction product was analyzed by HPLC. The HPLC conditions were as follows.
Column: Shodex SB802.5 (Showa Denko)
Column temperature: 25 ° C
Eluent: 50 mM sodium chloride aqueous solution containing 5 mM sodium azide Flow rate: 1 ml / min Detector: Differential refractive index detector (manufactured by Shodex RI-71, Showa Denko)

  In addition, since the thing of the following structure is known until now as a reaction product, the following substance was used as a standard substance. In addition, what was manufactured with the following method was used for the substance of the following formula [I]-[IV].

  The dried gagome kelp was pulverized with a free crusher M-2 type (manufactured by Nara Machinery Co., Ltd.), treated in 10 times the amount of 85% methanol at 70 ° C. for 2 hours, and then filtered. A 20-fold amount of water was added to the residue, treated at 100 ° C. for 3 hours, and an extract was obtained by filtration. After the salt concentration of the extract was the same as that of a 400 mM sodium chloride solution, 5% cetylpyridinium chloride was added until no further precipitation occurred, followed by centrifugation. The precipitate was thoroughly washed with ethanol to completely remove cetylpyridinium chloride, and then desalted and low-molecular-weight removed with an ultrafilter (excluded molecular weight of filter membrane: 100,000) (Amicon). The resulting precipitate was removed by centrifugation. This supernatant was freeze-dried to obtain purified gagome kelp fucoidan. The yield was about 4% based on the dry gagome kelp powder weight.

  Next, 600 ml of 5% purified Gagome kelp fucoidan solution, 750 ml of 100 mM phosphate buffer (pH 8.0), 150 ml of 4M sodium chloride, and 3.43 ml of fucoidan degrading enzyme solution described in Reference Example 5 of 1750 mU / ml were added. The mixture was mixed and reacted at 25 ° C. for 144 hours. The reaction solution was dialyzed using a dialysis membrane having a pore size of 3500, and fractions having a molecular weight of 3500 or less were collected. This fraction was desalted with a microacylator G3 (manufactured by Asahi Kasei Corporation) and 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 with a concentration gradient with ammonium acetate, and the eluate was fractionated by 50 ml. FIG. 5 shows the elution of the chromatography. Nine fractions (a) to (i) were obtained by the chromatography, and the fractions (a), (b), (c) and (f) among them were used for structural analysis.

Next, structural analysis was performed on the (a), (b), (c), and (f) fractions according to a conventional method, and (a) the fraction was represented by the following formula [I], ] (C) After confirming that the fractions are compounds of the following formula [IV] and (f) fractions of the following formula [II], each fraction was used as a standard for the analysis of the above reaction products. used.
The fraction numbers in the ion exchange chromatography using the DEAE-Sepharose FF column are as follows: (a): 42-43 (b): 84-91 (c): 51-52 (f): 62 -63.

  As a result of the analysis of the above reaction product, when the polypeptide having the fucose sulfate-containing polysaccharide-degrading activity obtained in Example 5 was used, the reaction product was only [I], [II], and [III]. However, in addition to the above [I], [II], and [III], the reaction product obtained using the polypeptide having the fucose sulfate-containing polysaccharide-degrading activity obtained in Example 6 was [IV]. [IV] was not decomposed into [I] even if the reaction was continued thereafter.

  That is, the polypeptide encoded by fdlA obtained as in Example 5 acts on the fucose sulfate-containing polysaccharide-U to produce trisaccharides such as [I], [II], and [III] above. Although released as a final cleavage unit, the polypeptide encoded by fdlB obtained in the same manner as in Example 6 acts on the fucose sulfate-containing polysaccharide-U, and the hexasaccharide such as [IV] above is also converted into the final cleavage unit. It was found to be liberated as

Example 8
In Example 5 and Example 6, polypeptides encoded by the respective genes are produced by the respective recombinant E. coli containing fdlA and fdlB genes, and the fucose sulfate-containing polysaccharide-degrading activity is detected from these E. coli extracts. And the polypeptide encoded by fdlB was confirmed to have a fucose sulfate-containing polysaccharide-degrading activity. However, as a result of analyzing the E. coli extract by SDS polyacrylamide gel electrophoresis, only a small amount of the recombinant polypeptide was detected as compared with the E. coli disrupted solution, and only a part of the produced polypeptide was active. It was suggested that it is expressed as a soluble polypeptide.

  Flavobacterium sp. The fucoidan-degrading enzyme of SA-0082 is an extracellular secretory enzyme and is expected to be expressed in the form of a precursor with a secretion signal for membrane permeation added to the N-terminus. Thus, when the N-terminal amino acid sequences encoded by fdlA and fdlB were examined, regions from the initiation methionine to the 25th and 24th alanine residues were predicted to be secretion signals. Since the presence of this hydrophobic secretion signal was considered to be related to the solubility of the respective recombinant polypeptides in Example 5 and Example 6, expression plasmids excluding the secretion signals of fdlA and fdlB were used. It was constructed.

  (1) First, primer FDL-Q-Bam (SEQ ID NO: 27) and primer 10X6R4 (SEQ ID NO: 28) are designed to introduce a restriction enzyme BamHI site immediately upstream of the 26th glutamine residue from the start methionine of fdlA. And synthesized. FDL-Q-Bam is a 28-mer synthetic DNA in which a BamHI site is arranged upstream of the sequence of SEQ ID NO: 76-95 in the sequence listing. 10X6R4 is a 21-mer synthetic DNA complementary to the sequence of nucleotide sequence numbers 1471 to 1491 of SEQ ID NO: 7 in the sequence listing.

  Using pEFLA10 constructed in Example 5 as a template, a PCR reaction was performed with a set of primers FDL-Q-Bam and 10 × 6R4. The PCR reaction was performed in the same reaction solution composition as in Example 4- (4), with 25 cycles of denaturation at 94 ° C. for 0.5 minute, primer annealing at 50 ° C. for 1 minute, and synthesis reaction at 72 ° C. for 2 minutes. The synthesis was completed by cycling for a final 7 minutes at 72 ° C. 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), a plasmid DNA was prepared by inserting this fragment into a pT7 blue T-vector (manufactured by Novagen), and the nucleotide sequence was confirmed. The obtained plasmid was cleaved at the BamHI site located in the primer FDL-Q-Bam and the SnaBI site in the fdlA coding region, and then separated by 5% polyacrylamide gel electrophoresis, and the 26th glutamine residue from the starting methionine of fdlA was separated. An about 480 bp BamHI-SnaBI fragment encoding the N-terminal region after the base was excised, extracted and purified.

  On the other hand, pET21a (manufactured by Novagen), an expression vector using the same T7 promoter as in Example 5, was cleaved at the HindIII site in the multicloning site, and the C-terminal containing the stop codon of the fdlA gene prepared in Example 5 was used. An approximately 2 kbp HindIII DNA fragment encoding the region was inserted so that the T7 promoter and the fdlA gene were in the same orientation, and a plasmid pEFDLA-C was constructed. This pEFDLA-C was cleaved at the BamHI site derived from the multicloning site and the SnaBI site in the fdlA coding region, and the about 480 bp encoding the N-terminal region after the 26th glutamine residue from the starting methionine of fdlA prepared earlier. The BamHI-SnaBI fragment was inserted to obtain plasmid pEFDLLA101.

  This plasmid pEFDLA101 encodes a polypeptide in which downstream of the T7 promoter is a 14-residue N-terminal leader sequence (SEQ ID NO: 29) derived from pET21a followed by the 26th and subsequent sequences of SEQ ID NO: 3 in the sequence listing. Yes.

  (2) The sequence of 20 bases after the 26th glutamine residue of fdlA used in the design of the primer FDL-Q-Bam is the same as the sequence after the 25th glutamine residue of fdlB (SEQ ID NO: 8 in the Sequence Listing). The nucleotide sequence number 73-92). Therefore, the primer FDL-Q-Bam used in Example 8- (1) can be used as it is for the construction of the fdlB expression vector, and basically the fdlB expression using the same method as in Example 8- (1). Vector pEFDLB101 was constructed.

  First, in order to perform a PCR reaction using FDL-Q-Bam, a 20-mer synthetic DNA, 17X6R1 (SEQ ID NO: 30) complementary to the sequence of base sequence numbers 1489-1508 of SEQ ID NO: 8 in the sequence listing was prepared. . Using pEFLB17 constructed in Example 6 as a template, a PCR reaction was performed with a set of primers FDL-Q-Bam and 17X6R1, and the amplified DNA fragment of about 1.4 kbp was extracted and purified. This fragment was inserted into a pT7 blue T-vector, plasmid DNA was prepared, and the nucleotide sequence was confirmed.

  Next, the obtained plasmid was cleaved at the BamHI site arranged in the primer FDL-Q-Bam and the NspV site in the fdlB coding region, and then separated by 5% polyacrylamide gel electrophoresis, and 25% from the starting methionine of fdlB. An approximately 210 bp BamHI-NspV fragment encoding the N-terminal region after the first glutamine residue was excised and extracted and purified.

  On the other hand, the plasmid pH17X6-7 obtained in Example 4 was cleaved at the EcoRI site, and an about 2.6 kbp EcoRI fragment containing the entire length of the fdlB gene was excised and extracted and purified. This fragment was inserted into the EcoRI site in the multi-cloning site of pET21a so that the T7 promoter and the fdlB gene were in the same direction to construct plasmid pEFDLB-W. This pEFDLB-W was cleaved at the BamHI site derived from the multicloning site and the NspV site in the fdlB coding region, and about 210 bp encoding the N-terminal region after the 25th glutamine residue from the starting methionine of fdlB prepared above. Of BamHI-NspV was inserted to obtain plasmid pEFDLB101.

  This plasmid pEFDLB101 is located at the 25th position of SEQ ID NO: 4 in the sequence listing following the 14-residue N-terminal leader sequence (SEQ ID NO: 29) identical to the plasmid pEFDLLA101 obtained in Example 8- (1) downstream of the T7 promoter. It encodes a polypeptide in which subsequent sequences are linked.

  (3) Using the pEFDLA101 and pEFDLB101 obtained in Example 8- (1) and (2), the recombinant was cultured and the bacterial cell extract was prepared in the same manner as in Example 5. The expression level of each recombinant polypeptide and the fucoidan degradation activity by pEFLA10 and pEFLA17 constructed in 5 and Example 6 were compared.

  That is, Escherichia coli BL21 (DE3) strain was transformed with pEFLA10, pEFLA17, pEFDLA101, or pEFDLB101. The obtained transformant was inoculated into 5 ml of L medium containing 100 μg / ml ampicillin and cultured at 37 ° C. with shaking. The turbidity of the culture is 0. D. At the stage of 600 = 0.8, IPTG was added so as to give a final concentration of 1 mM, and then further cultured overnight with shaking at a culture temperature of 15 ° C. After completion of the culture, the culture was centrifuged to collect the cells, suspended in 0.5 ml of a cell disruption buffer, and disrupted by sonication. A part of this suspension was taken as an E. coli disruption solution. Further, this was centrifuged to remove insoluble matters to obtain an E. coli extract.

  Each E. coli disrupted solution and extract were separated by SDS polyacrylamide gel electrophoresis and analyzed by Coomassie brilliant blue staining according to a conventional method. As a result, a large amount of polypeptide having the expected molecular weight was observed in all E. coli disrupted solutions. It was done. The expression level was estimated by comparison with the amount of bovine serum albumin migrated simultaneously, and the fucoidan degradation activity in each extract was measured by the method described in Reference Example 3. These results are summarized in Table 2.

  As shown in Table 2, the expression level of the target polypeptide contained in the disrupted solution is expressed by pEFDLA101 (fdlA), pEFDLB101 constructed such that an N-terminal leader sequence is added instead of the sequence expected to be a secretion signal. Compared with the direct expression plasmids pEFLA10 (fdlA) and pEFLA17 (fdlB) having the expected secretion signal, (fdlB) showed a production amount about twice as high for both genes.

  In addition, when there is a secretion signal, the amount of the target polypeptide present in the extract is extremely small compared to the disruption solution, and most of the expressed polypeptide is present as insoluble, whereas the secretion signal When was removed, the target polypeptide in an amount equivalent to that of the disrupted solution was detected in the extract, revealing that the expressed polypeptide was almost soluble. Furthermore, the amount of activity in the extract also greatly increased corresponding to the amount of the target polypeptide.

Example 9
The action of the polypeptides having a fucose sulfate-containing polysaccharide-degrading activity obtained in Example 2 and Example 3 on the fucose sulfate-containing polysaccharide-F was examined.

That is, 500 μl of 50 mM imidazole-HCl buffer (pH 7.5), 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 4M sodium chloride 100 μl of the 9.7 mU / ml polypeptide solution having polysaccharide-degrading activity containing fucose sulfate obtained in Example 2 or Example 3 was added, and distilled water was added to make the total volume to 1 ml. After reacting at 25 ° C. for 18 hours, the reaction product was analyzed by HPLC. The HPLC conditions were as follows.
Column: Shodex SB804 (made by Showa Denko)
Column temperature: 25 ° C
Eluent: 50 mM sodium chloride aqueous 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 analysis of the reaction product produced by the action of the polypeptide encoded by the gene of the present invention, the polypeptide having the fucose sulfate-containing polysaccharide-degrading activity obtained in Example 2 and Example 3 was used. In each reaction product, two peaks of 8.62 minutes and 9.30 minutes were detected by HPLC retention time.

  In addition, when the case where the polypeptide having the fucose sulfate-containing polysaccharide-degrading activity obtained in Example 2 is used is compared with the case where the polypeptide having the fucose-sulfate-containing polysaccharide-degrading activity obtained in Example 3 is used, Example 2 is compared. Produced more material at 9.30 minutes.

EFFECT OF THE INVENTION According to the present invention, the amino acid sequence and base sequence of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity have been revealed for the first time, thereby making it possible to provide a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity. An industrially advantageous genetic engineering method for producing a polypeptide having a sulfate-containing polysaccharide-degrading activity is provided.

  According to the present invention, there is no need to add a sulfated-fucose-containing polysaccharide to a medium for inductive production of a sulfated-fucose-containing polysaccharide-degrading enzyme, and productivity is high. In addition, enzymes such as protease and other polysaccharide-degrading enzymes are not produced at the same time, and purification is easy. By providing an amino acid sequence and a base sequence of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity, a polypeptide antibody having an anti-fucose sulfate-containing polysaccharide-degrading activity can be produced based on the amino acid sequence. In addition, it has become possible to prepare probes and primers specific to the base sequence of a polypeptide having a fucose sulfate-containing polysaccharide-degrading activity.

It is a figure which shows the position of ORF-1 and ORF-2. It is a figure which shows the precipitation rate of a fucose sulfate containing polysaccharide. It is a figure which shows the position of fdlA. It is a figure which shows the position of fdlB. It is a chromatogram using DEAE-Sepharose FF.

Claims (9)

A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 3 to 4 in the sequence listing, or an amino acid having at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the sequence An activity of desulfurizing a fucose sulfate-containing polysaccharide having a sequence and having the following physicochemical properties to release at least one compound selected from the following formulas [I], [II], [III] and [IV] An isolated gene comprising a DNA sequence encoding a polypeptide having
(A) Constituent sugar: contains uronic acid.
(B) Flavobacterium sp. It is degraded by fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402).

  The gene according to claim 1, wherein the polypeptide is derived from a genus Flavobacterium.   A DNA sequence represented by any one of SEQ ID NOs: 7 to 8 in the sequence listing, or a DNA sequence in which at least one of deletion, addition, insertion or substitution of one or more bases is added to the sequence The gene according to claim 1. It acts on a fucose sulfate-containing polysaccharide having the following physicochemical properties, which can hybridize with the gene of claim 1 to 3 under strict conditions, thereby under-differentiating the fucose sulfate-containing polysaccharide, and the following formula [I ], A gene encoding a polypeptide having an activity of releasing at least one compound selected from [II], [III] and [IV].
(A) Constituent sugar: contains uronic acid.
(B) Flavobacterium sp. It is degraded by fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402).

  A recombinant DNA comprising the gene according to any one of claims 1 to 4.   An expression vector having a microorganism, animal cell or plant cell as a host cell, into which the recombinant DNA according to claim 5 is inserted.   A transformant obtained by transformation with the expression vector according to claim 6.   A method for producing a polypeptide having a fucose sulfate-containing polysaccharide degrading activity, comprising culturing the transformant according to claim 7 and collecting a polypeptide having a fucose sulfate-containing polysaccharide degrading activity from the culture. An amino acid sequence represented by any one of SEQ ID NOs: 3 to 4 in the sequence listing, or an amino acid sequence in which at least one of deletion, addition, insertion or substitution of one or more amino acid residues is added to the sequence; And acting on a sulfated-fucose-containing polysaccharide having the following physicochemical properties to reduce the molecular weight of the sulfated-fucose-containing polysaccharide, and at least one selected from the following formulas [I], [II], [III] and [IV] A polypeptide having an activity of liberating the compound.
(A) Constituent sugar: contains uronic acid.
(B) Flavobacterium sp. It is degraded by fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402).

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