KR100913233B1 - Beta-1,3-glucanase from cryptopygus antarcticus, gene encoding the same, and use thereof - Google Patents

Abstract

The present invention Antarctic toktogi ( Cryptopygus gene encoding beta-1,3-glucanase enzyme having beta-1,3-glucan degrading activity isolated from antarcticus ), expression vector comprising the gene, expression vector The present invention relates to a transformant, a beta (beta) -1,3-glucanase enzyme produced by the transformant, and a method for preparing the same. The beta-1,3-glucanase according to the present invention converts beta-1,3-glucan laminarin to oligosaccharides into functional carbohydrates with high intake or selectively cleaves only specific sites of carbohydrates in the field of glycobiology. It can be used as an enzyme, and furthermore can be usefully used for starch decomposition process, food, beverage, feed and textile industries.

Cryptopygus antarcticus, Beta-1,3-glucan, Beta-1,3-glucanase, Laminarin

Description

Beta-1,3-glucanase derived from Antarctic tokto group, gene encoding the same, and use thereof {BETA-1,3-GLUCANASE FROM CRYPTOPYGUS ANTARCTICUS, GENE ENCODING THE SAME, AND USE THEREOF}

The present invention Antarctic toktogi ( Cryptopygus gene encoding beta-1,3-glucanase enzyme having beta-1,3-glucan degrading activity isolated from antarcticus ), expression vector comprising the gene, expression vector The present invention relates to a transformant, a beta (beta) -1,3-glucanase enzyme produced by the transformant, and a method for preparing the same.

Beta-1,3-glucanase is the first enzyme reported by Liz et al. In 1959 (Reese, BT and M. Mandels, Can. J. Microbiol., 5, 173- 185, 1959), found in many microorganisms such as fungi, yeast, bacteria (Clarke and Stone, Bio, Chem. J., 96, 793-801, 1965; Abd-E1-A1 and Phaff, Bio, Chem. J. , 109, 347-360, 1968; Notario, V. et al., Biochem.J., 159, 555-562, 1976), beta in cell walls such as algae, soybeans, grains during germination, mold and yeast It is known as an enzyme that selectively cleaves beta-1,3-glucoside bonds of -1,3-glucan (Whistler RL and CL Smart, Polysaccharide Chemistry, Academic Press, New York, 350, 1953; Manners DJ et al., Biochem. J., 135, 19-30, 1973).

To date, beta-1,3-glucanase is a virus (Chlorella Virus PBCV-1, Virology, 276, 27-36, 2000), Pyrococcus furiosus , J. Biol. Chem., 272, 31258-31264, 1997), Trichoderma harzianum , J. Bacteriol., 177, 6937-6945, 1995) and various types of bacteria ( Risoctonia solani ; Phytophtaora infestans ; Bacillus circulans , Biochim. Biophys. Acta., 73, 267-275, 1963; Oerskovia xantineolytica , J. Bacteriol., 178, 4751-4757, 1996; Thermotoga neapolitana, Biochem. Biophys. Res. Commun., 194, 1359-1364, 1993; Rothothermus marinus Eur. J. Biochem., 224, 923-930, 1994), and marine invertebrates ( Strongylocentrotus purpuratus Proc. Natl. Acad. Sci. USA, 93, 6808-6813, 1996; Mizuhopecten yessonensis Comp. Biochem. Physiol. Part B. 143, 473-485, 2006) has been isolated and purified for enzymatic properties, but there have been no reports on the purification or gene sequence of the enzyme in other multicellular animals.

Therefore, the technical problem to be achieved by the present invention is to use beta-1,3-glucan laminarin oligosaccharides to convert into high-intake functional carbohydrates or to use as an enzyme to selectively cut only a specific site of carbohydrates in the field of glycobiology research It is possible to provide a beta-1,3-glucanase (β-1,3-glucanase) protein having beta-1,3-glucan degrading activity isolated from Cryptopygus antarcticus .

Another technical problem of the present invention is to provide a polynucleotide encoding the beta-1,3-glucanase protein.

Another object of the present invention is to provide an expression vector comprising the polynucleotide.

Another technical problem of the present invention is to provide a transformant transformed by introducing the expression vector.

Another technical problem of the present invention is to provide a method for producing beta-1,3-glucanase having beta-1,3-glucan degrading activity using the transformant.

In order to achieve the above object, the present invention provides a beta-1,3-glucanase protein consisting of the amino acid sequence of SEQ ID NO: 3 and having beta-1,3-glucan degrading activity. The protein is Cryptopygus antarcticus ).

A polynucleotide encoding beta-1,3-glucanase protein having beta-1,3-glucan degrading activity is provided. The polynucleotide may be a polynucleotide consisting of the nucleotide sequences of the 29 th to 841 th sequences of SEQ ID NO: 1.

In another aspect, the present invention provides a vector, preferably pET-Trx-CaLam expression vector containing a nucleic acid sequence of the gene.

In addition, the present invention provides a transformant transformed by introducing the expression vector.

In addition, the present invention a) culturing cells having the coding sequence of beta-1,3-glucanase protein consisting of the transformant or the amino acid sequence of SEQ ID NO: 3 and having beta-1,3-glucan degrading activity Making; b) inducing the expression of beta-1,3-glucanase protein in said cultured cells; c) eluting the protein from the supernatant obtained by pulverizing and then precipitating the cells expressing the beta-1,3-glucanase protein; And d) provides a method for producing beta-1,3-glucanase having a beta-1,3-glucan degrading activity, comprising the step of purifying the eluted protein.

Hereinafter, the present invention will be described in detail.

Polysaccharides are classified into various types according to the constituent sugars and their binding forms, and the storage of bioenergy (ex. Starch, glycogen, inulin, etc.) and structural support (ex. Cellulose, chitin, glycosaminoglycan, etc.) Perform biological functions such as In addition, the polysaccharide component of the cell membrane or cell wall is directly involved in the process of cell division or mutual recognition of the cells and cells, cells and viruses, cells and antibodies, and biological defense mechanisms.

Among these polysaccharides, glucan is a generic term for polysaccharides comprising glucose as a constituent sugar, and is classified according to the binding mode between D-glucose. In particular, glucan can be divided into α-glucan and β-glucan according to the arrangement of the sub-carbon atoms. Representative examples of α-glucan include amylose (α-1,4 bond), amylopectin (α-1,4 and α-1,6 bond), glycogen (α-1,4 and α-1,6 bond) and bacteria Dextran (α-1,6 bond) and the like, β-glucan is cellulose (β-1,4 bond), laminarin (β-1,3 bond) of brown algae and likenane (β) of lichens -1,3 and β-1,4 bonds), and the like.

In particular, beta-1,3-glucan is laminarin is present in large quantities in the marine brown algae, La minariah digital tartar (Laminaria digitata ), Laminaria It is a binding form (β-1,3 glucose) of laminarin, which is a major structure and storage polysaccharide such as saccarina ), and is well known as a cell wall component of pathogenic fungi and yeast together with chitin.

An enzyme that hydrolyzes beta-1,3-glucan, that is, beta-1,3-glucanase, is present in beta-1,3-glucan. An enzyme that hydrolyzes glycoside linkages (β-1,3-glycodic linkage), depending on the pattern of hydrolysis, exo-beta-1,3-glucanase (EC3.2.1.58) and endo-beta-1 , 3-glucanase (EC3.2.1.6 or EC3.2.1.39). Exo-beta-1,3-glucanase cleaves from the non-reducing end of beta-1,3-glucan to produce glucose and also acts on betalin with a beta-1,6-binding form. Endo-beta-1,3-glucanase, on the other hand, randomly hydrolyzes beta-1,3-glucan to produce glucose and some laminarioligosaccaride (J. Biol. Chem., 272). , 31258-31264, 1997), various forms of oligosaccharides degraded by these endo-beta-1,3-glucanase enzymes enhance the proliferation of useful fungi in the intestine and promote various physiological activities. Do it.

To date, beta-1,3-glucanase has been studied in microorganisms such as viruses, archaea, fungi and various kinds of bacteria, and recently, marine invertebrates ( strongylocentrotus) purpuratus ) and beta-1,3-glucanase enzymes have been known to exist in the insect river Colembola, but the base sequence encoding the enzyme has not been disclosed, in particular beta-1,3 It is not known whether the glucanase enzyme is derived from a symbiotic bacterium or a gene present in the toctogi itself. In addition, since the beta-1,3-glucanase enzyme has been mostly found from bacteria and fungi, it is possible to isolate genes encoding beta-1,3-glucanase derived from the Antarctic toxin group of the present invention, which is another species. It is significant in that it provides another novel beta-1,3-glucanase gene source, thereby increasing industrial applicability.

Therefore, the inventors of the Antarctic toktogi ( Cryptopygus beta-1,3-glucanase gene was isolated and cloned from antarcticus for the first time, expressed in E. coli, purified and purified to dissolve beta-1,3-glucanase to substrate beta-1,3-glucan. The present invention was completed by confirming the activity.

In a preferred embodiment of the present invention described below provides a beta-1,3-glucanase protein consisting of the amino acid sequence of SEQ ID NO: 3 and having beta-1,3-glucan degrading activity. The protein may be artificially synthesized, or may be isolated from Cryptopygus antarcticus .

Beta-1,3-glucanase according to the present invention has the following characteristics:

1) Beta-1,3-glucanase of the present invention has the specificity of acting only on laminarin, a glucan, as a substrate. In this regard, in the practice of the present invention described below, the enzyme activity of beta-1,3-glucanase on various substrates was measured. As a result, as shown in Table 2, laminarin is effectively hydrolyzed, but cellulose, etc. It can be seen that other forms of polysaccharide substrates are not hydrolyzed (Table 2). In addition, as a result of confirming the degradation product of laminarin by beta-1,3-glucanase of the present invention using thin layer chromatography (TLC), as shown in FIG. 8, laminaribiose (L2) , Laminaritriose (L3), and glucose (G1) was confirmed to produce mainly (Fig. 8). These reaction products are described by P. furiosus (Gueguen et al., J. Biol. Chem., 272, 31258-31264) and B. xylophilus (Kikuchi et al., Biochem. J., 389, 117-125, 2005). The results, ie, endo-beta-1,3-glucanase, are consistent with the production of glucose and laminaoligosaccharides upon hydrolysis of beta-1,3-glucan, which is the beta-1,3-glu of the present invention. It means that the kinase is endo beta-1,3-glucanase. In particular, various forms of oligosaccharides degraded by endo-beta-1,3-glucanase enzymes function to enhance the proliferation of useful fungi living in the intestine and to promote various physiological activities.

2) Beta-1,3-glucanase of the present invention has an optimal pH range of 4.5 to 9.5, preferably pH 5 to 7.5, more preferably pH 5.5 to 6.5, even more preferably pH 5.5. . This pH range is a result of FIG. 6A of the present invention, except for enzymes from some basophils (Bacillus sp., Nogi and Horikoshi, Appl. Microbiol. Biotechnol. 32, 704-707, 1990). Most are similar to existing bacteria with optimum enzyme activity at pH 4-6.

3) The beta-1,3-glucanase of the present invention has an optimum temperature range of 0 to 60 ° C, preferably 20 to 60 ° C, more preferably 40 to 55 ° C, even more preferably 50 ° C. to be. This temperature range is the result of FIG. 6b of the present invention, which differs from the preferred optimum temperature of 37 ° C. reported by Watanabe et al. (Watanabe T. et al., Agric. Biol. Chem., 53, 1759 , 1989; Watanabe T. et al., J. Bacteriol., 174, 186, 1992). In particular, beta-1,3-glucanase of the present invention is characterized by having thermal stability at 50 ℃.

4) In addition, the beta-1,3-glucanase of the invention is Ca ^{2 +,} Na ⁺ and K ⁺ at least one metal ion selected from the group consisting, preferably keeping the enzyme activity when Ca ^{2 +} metal ion treatment And increasing thermal stability. In this regard, in the Examples of the present invention described below, Ca ^{2 +} , Na ⁺ and K ⁺ and Mg ^{2 +} , Mn ^{2 +} when measuring enzymatic activity of beta-1,3-glucanase on a substrate laminarin , the Cu ^{2 +,} Zn ^{2 +,} and the Fe ⁺ of each treatment the metal ions were measured by enzymatic activity change, so that the table 3 114.7% of enzyme activity for Ca ^{2 +} compared to the untreated group, as shown in It can be seen that the increase (Table 3). In addition, the beta-1,3-glucanase of the present invention is untreated group at 15 to 35 ℃, preferably 20 to 30 ℃, and 45 to 55 ℃, preferably 50 ℃ when Ca ^{2 +} metal ion treatment It can be seen that the thermal stability is slightly increased compared to (FIG. 7).

The present invention also provides a polynucleotide consisting of the amino acid sequence of SEQ ID NO: 3 and encoding a beta-1,3-glucanase protein having beta-1,3-glucan degrading activity. The polynucleotide may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

Specifically, in order to isolate the gene encoding the beta-1,3-glucanase protein, the entire RNA was isolated from the Antarctic tokto group inhabiting Antarctica, and a cDNA library was cloned to clone approximately 2000 clones. After the sequencing was performed. As a result, as shown in FIG. 1, the full-length cDNA of the beta-1,3-glucanase gene of the present invention contains the signal peptide (underlined portion of FIG. 1) corresponding to 1 to 18 th of SEQ ID NO: 1. 270 amino acid sequence (SEQ ID NO: 3), ie, 813 bp open reading frame (SEQ ID NO: 2) corresponding to the 29 to 841 th of SEQ ID NO: 1 encoding the amino acid sequence, and 1 of SEQ ID NO: 28 'to 5'-UTR corresponding to 28th, and 85' to 3'-UTR corresponding to 842 to 926th of SEQ ID NO: 1 containing polyadenylation A (aataaa) at 3'-end. It can be confirmed that the nucleotide sequence of 926 bp (SEQ ID NO: 1) (FIG. 1). Although the gene sequence is not particularly limited, preferably beta-1,3-glucanase derived from molluscs, echinoderm (sea urchins), and bacteria, and glucanase-like proteins derived from arthropods of insect arthropods (beta- 1,3-glucanase like protein) can be compared with the gene sequence coding to determine the homology of the nucleotide sequence, and the amino acid sequence is also a strain belonging to glycosyl hydrolase group 16, that is, O. xanthineolytica ( 41.2%, GenBank accession numb.AAC44371), B. circulans (39.9%, GenBank accession numb.BAC06195), R. marinus (39.6%, GenBank accession numb.AAC69707), P. puriosus (39.5%, GenBank accession numb.AAC25554 ), B. xylophilus (38.4%, GenBank accession numb.BAE02683), T. neapllitana (36.9%, GenBank accession numb.CAA88008), T. tridentatus (35.3%, GenBank accession numb.BAA04044), X. axonopodis (33.9% , GenBank accession numb.AAM36156), B. circulans (33.6%, GenBan k accession numb.AAC60453), beta from S. purpuratus (34%, GenBank accession numb.AAC47235), P. sachalinensis (32.1% GenBank accession numb.AAP74223) and M. yessoensis (31.9%, GenBank accession numb. AAW34372) -1,3-glucanase amino acid sequence; And A. aegypti (36.4%, GenBank accession numb. AAL76017), E. fetida (35.5%, GenBank accession numb. CAB70460), P. leniusculus (34.9%, GenBank accession numb. CAB65353), L. terrestris (34.3%, From GenBank accession numb.AAL09587), P. monodon (34%, GenBank accession numb.AAM21213), A. gambiae (34%, GenBank accession numb.CAA04496) and L. stylirostris (34%, GenBank accession numb.AAM73871) Comparative analysis with the glucanase-like protein amino acid sequence can determine the homology of the amino acid sequence (Fig. 2).

In addition, the present invention provides an expression vector, preferably pET-Trx-CaLam, an expression vector comprising the nucleic acid sequence of the gene.

The expression vector of the present invention may be selected from the group consisting of plasmids, cosmids and phages, and preferably, a pET-32a (+) plasmid vector having a T7 lac promoter may be used.

In addition, the present invention provides a transformant transformed by introducing the expression vector.

The host cell capable of transforming the expression vector according to the present invention may be selected from the group consisting of E. coli, prokaryotes, fungi, plants and animal cells, preferably Rosetta-gami (DE3) E. coli. .

In addition, the present invention

a) culturing a cell comprising the transformant or the amino acid sequence of SEQ ID NO: 3 and having a coding sequence of beta-1,3-glucanase protein having beta-1,3-glucan degrading activity;

b) inducing the expression of beta-1,3-glucanase protein in said cultured cells;

c) eluting the protein from the supernatant obtained by pulverizing and then precipitating the cells expressing the beta-1,3-glucanase protein; And

d) provides a method for producing beta-1,3-glucanase having a beta-1,3-glucan degrading activity, comprising the step of purifying the eluted protein.

In the above production method, the cell culture of step a) can be cultured in LB (Luria-Bertani) broth medium, preferably LB broth medium containing antibiotics such as empicillin, kanamycin and tetracycline, more preferably May be cultured in LB broth medium containing kanamycin capable of selectively propagating the transformant or cells.

Expression of the protein of step b) can be induced by treatment with an inducer such as allolactose and IPTG (Isopropyl-β-D-Thiogalactopyranoside). Preferably, the final concentration of IPTG is 0.01 to 1 mM. It may be induced by treating to preferably 0.05 to 0.5 mM, more preferably 0.1 mM.

Protein elution in step c) is a buffer containing salts of inorganic or organic acids that do not impair the biological function of the protein, for example citrate, acetate, succinate, lactate, tartarate, formate, propionate, It may be a buffer solution containing phosphate, or borate.

In addition, the protein purification of step d) can be purified using protein purification methods known in the art, preferably ion exchange chromatography, gel-permeation chromatography, reverse phase-HPLC, SDS-PAGE for preparation, and It can be purified by one or more methods selected from the group consisting of affinity column chromatography.

The present invention also provides an antimicrobial agent against pathogens comprising beta-1,3-glucanase as an active ingredient. The pathogens refer to pathogenic fungi or yeasts that cause disease in animals or plants whose cell wall components are composed of beta-1,3-glucan, for example, Saccharomyces cerevisiae ), Debaryomyces hansenii ), Hansenula ciferi ciferrii ), Candida albicans ), Pyricularia oryzae ), Aspergillus oryzae ), and Neurospora Krasa crassa ) and the like.

The content of beta-1,3-glucanase contained in the antimicrobial agent varies depending on the target pathogen, but can be used within the range effectively showing the antimicrobial activity against the pathogen, preferably in the case of using in liquid form, the whole antimicrobial agent 0.01 to 99.99 parts by weight, preferably 0.1 to 25 parts by weight, more preferably 0.1 to 5 parts by weight, based on 100 parts by weight of beta-1,3-glucanase; When used as a powder or granular product in a lyophilized or hot air dried state, it is 0.002 to 20 parts by weight, preferably 0.01 to 10 parts by weight, more preferably 0.05 to 2.5 parts by weight, based on 100 parts by weight of the total antimicrobial agent. , 3-glucanase.

The antimicrobial agent includes beta-1,3-glucanase as an active ingredient, and may further include an appropriate diluent or excipient as an auxiliary component capable of enhancing antimicrobial activity against pathogens in addition to the active ingredient. The excipient may use a conventional material according to the formulation, and when formulated it may be used fillers, extenders, wetting agents, disintegrants or surfactants. Representative diluents or excipients used may include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin and physiological saline. The content of the auxiliary ingredient is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the beta-1,3-glucanase.

Formulation of the antimicrobial agent is granules (GRANULES), lotions (LPTIONS), aromatics (AROMATIC WATERS), powders (POWDERS), syrups (SYRUPS), liquids (LIQUIDS AND SOLUTIONS), aerosol (AEROSOLS), EXTRACTS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, TABLETS, SPIRITS, CAPSULES, or CREAMS It may be prepared as, but is not limited thereto.

In addition, the present invention provides a carbohydrate decomposition method characterized by using beta-1,3-glucanase in the carbohydrate decomposition method. That is, the beta-1,3-glucanase according to the present invention is an enzymatic process for converting a polysaccharide into a monosaccharide or a low molecular weight polysaccharide, wherein the beta-1,3-glucanase is converted to a polysaccharide in a suitable enzymatic reaction condition. By contacting, monosaccharides or low molecular weight polysaccharides can be easily obtained.

In the enzymatic reaction conditions, the optimum pH range of beta-1,3-glucanase is 4.5 to 9.5, preferably pH 5 to 7.5, more preferably pH 5.5 to 6.5, and even more preferably pH. 5.5; The optimum temperature range is 0 to 60 ° C., preferably 20 to 60 ° C., more preferably 40 to 55 ° C., even more preferably 50 ° C .; Ca is the ^{2 +,} Na ⁺ and K ⁺ at least one metal ion selected from the group consisting of, preferably it can be processed by adding the Ca ^{2 +} metal ions.

The present invention also provides a food or food additive having beta-1,3-glucan degrading activity comprising beta-1,3-glucanase as an active ingredient.

When the beta-1,3-glucanase of the present invention is used as a food additive, the beta-1,3-glucanase may be added as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. Can be used. The content of the beta-1,3-glucanase may be appropriately adjusted according to the purpose of use, for example, 0.001 to 99.99 parts by weight of the beta-1,3-glucanase, preferably 0.01 to the whole food. To 50 parts by weight, more preferably from 0.1 to 5 parts by weight can be added.

Examples of foods to which the beta-1,3-glucanase may be added include dairy products, soups, meats, sausages, breads, snacks, confections, rice cakes, noodles, gums, candy, chocolates and ice creams There may be, but not limited to, the kind of food, alcoholic beverages, alcoholic beverages and vitamin complexes, and preferably used as an additive of diet foods using seaweed. This means that when the beta-1,3-glucanase is used as an additive of diet foods using seaweed, it effectively decomposes the polysaccharide component of seaweed to enhance digestion efficiency, and various forms of oligosaccharides produced by decomposition are intestinal. This is because it promotes intestinal activity by propagating useful bacteria. For this reason, the beta-1,3-glucanase of the present invention may also be used as an additive for animal feed using seaweed, for example, the content of the beta-1,3-glucanase may be used in animal feed using seaweed. It may be added in the range of 0.001 to 50 parts by weight, preferably 0.01 to 25 parts by weight, more preferably 0.1 to 5 parts by weight per 100 parts by weight.

In addition, the beta-1,3-glucanase of the present invention can be used to selectively cleave specific portions of carbohydrates in the field of glycobiology, thus replacing high value enzymes used in similar applications, and fiber It can also be used in industry.

As described above, beta-1,3-glucanase isolated from the Antarctic tokto group according to the present invention is converted to functional carbohydrates having high intake rate by glycosylating laminarin, beta-1,3-glucan, or glycobiology. It can be used as an enzyme to selectively cut only a specific portion of carbohydrates in the research field, and furthermore, it can be useful for starch decomposition process, food, beverage, feed and textile industries.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

[ Example ]

Example  One: Antarctic Toktogi ( Cryptopygus antarcticus Beta-1,3- derived from Glucana Aze Gene Isolation and Amino Acid Sequencing

1-1: Sample Preparation and Overall RNA  detach

Cryptopygus antarcticus was collected from moss around the Antarctic King Sejong Science Base in South Shetland Island and immediately added to 99.9% alcohol and stored at -70 ° C. In order to separate total RNA from the rapidly frozen Antarctic tokto group, alcohol was completely removed and total RNA was isolated using Trizol solution (Invitrogen, USA) according to the manufacturer's instructions. The total RNA isolated was confirmed in purity on a 1% formaldehyde agarose gel, and then its amount was measured using an ND-1000 spectrometer (Nanodrop, USA).

1-2: cDNA  Library Authoring and Beta-1,3- Glucanase  Clone gene analysis

CDNA libraries were synthesized from the RNA using the Creator SMART cDNA Library Kit (Clontech, USA). After then synthesized cDNA was connected to an adapter DNA at both ends, by cutting it to the Sfi I restriction enzyme (Promega, USA) connected to pDNR-LIB vector it was transformed in DH5α strain. From this, clones of about 2000 expression genes were cloned, and the sequences of the clones were analyzed using a Big dye terminator kit (PE Applied Biosystems) and an automatic sequencer (ABI3100). The analyzed sequences were compared with sequences of other beta-1,3-glucanase genes using NCBI database, Pfam database and ExPASy proteomics database, respectively.

As a result of analyzing the nucleotide sequence of the about 2000 clones, as shown in Figure 1, the full-length cDNA of the beta-1,3-glucanase gene of the present invention is a signal peptide corresponding to 1 to 18th of SEQ ID NO: 1 270 amino acid sequence (SEQ ID NO: 3) including the underlined portion of FIG. 1, that is, an open reading frame of 813 bp corresponding to 29 to 841 th of SEQ ID NO: 1 encoding the amino acid sequence (SEQ ID NO: 2) And a 5'-UTR of 28 bp corresponding to 1 to 28 th of SEQ ID NO: 1, and corresponding to 842 to 926 th of SEQ ID NO 1 including polyadenylation A (aataaa) at the 3'-end It was confirmed that the nucleotide sequence of 926 bp consisting of 85 bp 3'-UTR (SEQ ID NO: 1) (Fig. 1).

1-3: Homology  analysis

The amino acid sequence of Antarctic toxin beta-1,3-glucanase is O. xanthineolytica (41.2%, GenBank accession numb. AAC44371), B. circulans belonging to glycosyl hydrolase group 16 enzyme. (39.9%, GenBank accession numb.BAC06195), R. marinus (39.6%, GenBank accession numb.AAC69707), P. puriosus (39.5%, GenBank accession numb.AAC25554), B. xylophilus (38.4%, GenBank accession numb. BAE02683), T. neapllitana (36.9%, GenBank accession numb.CAA88008), T. tridentatus (35.3%, GenBank accession numb.BAA04044), X. axonopodis (33.9%, GenBank accession numb.AAM36156), B. circulans (33.6%, GenBank accession numb. AAC60453), beta-1,3 from S. purpuratus (34%, GenBank accession numb.AAC47235), P. sachalinensis (32.1% GenBank accession numb.AAP74223) and M. yessoensis (31.9%, GenBank accession numb. AAW34372) -Glucanase amino acid sequence; And A. aegypti (36.4%, GenBank accession numb. AAL76017), E. fetida (35.5%, GenBank accession numb. CAB70460), P. leniusculus (34.9%, GenBank accession numb. CAB65353), L. terrestris (34.3%, From GenBank accession numb.AAL09587), P. monodon (34%, GenBank accession numb.AAM21213), A. gambiae (34%, GenBank accession numb.CAA04496) and L. stylirostris (34%, GenBank accession numb.AAM73871) Homology was compared with the glucanase-like protein amino acid sequence. The results are shown in FIG.

As shown in Figure 2, the three residues of Glu137, Asp139 and Glu142, which are important for enzymatic activity, were all conserved as in the strain of glycosyl hydrolase group 16, in addition to 15 residues (F28, Gly45, N48, Glu50, P111, W117, W121, W132, Gly136, His156, F224, N233, A235, Gly238 and Val263) was also conserved (Fig. 2). This means that beta-1,3-glucanase of the invention is an enzyme belonging to glycosyl hydrolase group 16.

1-4: Genome DNA Beta-1,3- amplified at Glucanase  Genetic analysis

In order to investigate whether the beta-1,3-glucanase gene of the present invention is derived from the symbiotic bacteria in the intestine of the Antarctic Toktogi group or from the Antarctic Tokto group itself, the genomic DNA is extracted from the Antarctic Tokto group and then beta The sequence was analyzed by amplifying the -1,3-glucanase gene. The gene uses a forward primer (5'-ATG AAC GCA TTT ACA TTT CCC CT-3 ', SEQ ID NO: 5) and a reverse primer (5'-TTA ATT CCA GCT CCA TTT CTT GA-3', SEQ ID NO: 6). By amplification, it was confirmed that the fragment of about 1.2kb amplification (Fig. 3a). The amplified gene fragment was cloned into pCR2.1-Topo (Iinvtrogen, USA) vector and subjected to sequencing. The results are shown in FIGS. 3B and 3C, respectively.

As shown in Figures 3b and 3c, it was confirmed that the beta-1,3-glucanase gene is a 1,117 bp nucleotide (SEQ ID NO: 4) consisting of five introns and six exons (Fig. 4). 3b and 3c). In particular, the gene structure composed of introns and exons is specific to the eukaryotic genome, and cannot be seen in the bacterial genome, considering the feeding habits of Antarctic toxins that mainly feed bacteria and algae (Broady, Br. Antarct. Surv). Bull. 48, 37-46, 1979), the beta-1,3-glucanase gene of the present invention can be seen that the gene present in the Antarctic toktogi itself.

Example  2: Antarctic Toktogi  Derived beta-1,3- Glucanase  gene Cloning , Protein expression and purification, and enzyme activity analysis

Antarctic springtail beta-1,3-glucanase gene in order to clone the gene encoding the mature protein, the signal sequence has been removed from the open reading frame of the forward primer (5'-NNNNNN GGATCC GCATGGGTGTTGGACTGGGAGGA-3 ', SEQ ID NO: 5 ) And reverse primers (5'-NNNNNN AAGCTT TTAATTCCACGTCCATTTCTTGA-3 ', SEQ ID NO: 6) to amplify the gene. The underlined portions of the forward and reverse primers indicate the recognition sites of Bam HI and Hind III restriction enzymes, respectively, and the pET-32a (+) vector (Novagen) was digested with Bam HI and Hind III and then digested with the same enzyme. , US) (Fig. 4). Figure 4 shows the cloning process and cleavage map of the expression vector expressing beta-1,3-glucanase of the present invention.

After cloning in the same manner as in FIG. 4, pET-Trx-CaLam, which is a final expression vector, was prepared, and transformed into host cell Rosetta-gami (DE3) Escherichia coli to confirm sequencing and expression of the vector. Transformants were prepared. Beta-1,3-glucanase protein expression in transformed cells was determined by addition of 0.1 mM Isopropyl-β-D-Thiogalatopyranoside (IPTG). Induction by incubation at 18 ℃ for 24 hours. The cells were precipitated by centrifugation at 4,000 g for 20 minutes at 4 ° C., and then resuspended in 20 mM Tris-HCl buffer (pH 7.9) to which 5 mM imidazole (Sigma, USA) was added and ground using ultrasonic waves. .

After grinding, the supernatant obtained by centrifugation at 17,000 g at 4 ° C. for 20 minutes was passed through a column having a metal affinity resin (Ni-NTA His-Bind Resin, Novagen, USA), followed by 100 mM addition of 60 mM imidazole. It was washed with 20 mM Tris-HCl buffer containing sodium chloride (pH 7.9) and thiolidoxin-beta-1,3-glucanase fusion protein (Trx-CaLam) with the buffer added with 300 mM imidazole. Eluted. The eluted protein solution was exchanged with 20 mM Tris-HCl buffer (pH7.9) with 10% glycerol added.

In addition, to obtain the beta-1,3-glucanase protein (CaLam) from which the thiolidoxin of the thiolidoxin-beta-1,3-glucanase fusion protein (Trx-CaLam) is removed, the eluted protein The solution was exchanged with 20 mM Tris-HCL buffer solution (pH7.9) added with 10% glycerol, followed by thrombin treatment to cleave thiolidoxin. The cleaved protein solution was concentrated using centricon (Centricon, USA), exchanged with 20 mM Tris-HCl buffer (pH7.9) with 10% glycerol, followed by histidine-tagged affinity chromatography. Purification was carried out.

Protein concentration was measured using the Bradford (Bradford, M. M., Ana. Biochem., 72: 248-254, 1976) method, and the purified protein was electrophoresed on SDS-PAGE to confirm its purity. The results are shown in FIG.

As shown in FIG. 5, the separated and purified thiolidoxin-beta-1,3-glucanase fusion protein (Trx-CaLam) and beta-1,3-glucanase (CaLam) were each 41 kDa in size. And 29 kDa (FIG. 5). In terms of protein recovery, Trx-CaLam was 12.06%, CaLam was 9.22% (Table 1), Trx-CaLam was 15.1 U / mg, and CaLam was 20.6 U / mg (Table 1). One).

Purification step Total protein (mg) Total activity (U) Specific activity (U / mg) Percentage yield (%) Crude extract 112.74 145.23 1.29 100 Trx-CaLam (1 ^st His-tagged affinity chromatography) 1.16 17.52 15.1 12.06 CaLam (Thrombin removal and 2 ^nd His-tagged affinity chromatography) 0.65 13.39 20.6 9.22

Example  3: Antarctic Toktogi  Origin Thiolidoxin -Beta-1,3- Glucanase  And / or beta-1,3- Glucanase  Biochemical Characterization

Investigation of the biochemical characteristics of thiolidoxin-beta-1,3-glucanase (Trx-CaLam) and / or beta-1,3-glucanase (CaLam) of the Antarctic Toto group separated and purified in Example 2 To 1) enzymatic activity against the substrate; 2) changes in enzyme activity depending on pH and temperature; And 3) changes in enzyme activity according to metal ions, respectively. Specifically, 1% (w / v) and 10 μl of the polysaccharides Laminarin, Lichenan, CM cellulose, Pustulan, and Locust bean gum substrates (0.04 U) of the enzyme was mixed in 50 mM sodium phosphate buffer (pH 5.0), and then incubated at 50 ° C. for 30 minutes to determine the reducing equivalent of glucose released by the enzymatic digestion. The reducing equivalents were measured using Dinitrosalicylic acid (DN) method (Stalbrand, H., et al., J. Biotechnol., 29: 229-242, 1993), beta-1,3 One unit of glucanase enzymatic activity was defined as the amount of enzyme that liberated glucose and equivalent 1 μmole of reducing sugar per minute.

Regarding the enzymatic activity to the substrate, as shown in Table 2, beta-1,3-glucanase (CaLam) of the present invention cellulose cellulose only effectively hydrolyzing only laminarin (enzyme activity: 20.6 U / mg) It was found that other forms of polysaccharide substrates did not hydrolyze (Table 2).

Substrate Main linkage type (monomer) Specific activity (Units / mg) Laminarin β-1,3 (Glucose) 20.6 Lichenan β-1,3-1,4 (Glucose) 0 CM cellulose β-1,4 (Glucose) 0 Pustulan β-1,6 (Glucose) 0 Locust bean gum β-1,4 (Manose) 0

In addition, with regard to changes in enzymatic activity according to pH (FIG. 6A) and temperature (FIG. 6B), thioridoxin-beta-1,3-glucanase (■) and beta-1,3-glucana of the present invention The enzyme (□) all showed the optimum activity at pH 5.5, the enzyme activity was relatively stable under weakly alkaline conditions from pH 4.5-9.5 or less (Fig. 6a), and showed optimum activity at 50 ℃ (Fig. 6b), 0 It was observed that the enzyme activity was maintained in the temperature range of -60 ℃ (Fig. 6b).

In addition, as shown in Table 3, in the group treated with Ca ^{2 +} , Na ⁺ and K ⁺ , the enzyme activity was slightly increased compared to the untreated group, but the other metal ions ( ^{Mg 2 +, Mn 2 +,} Cu 2 +, Zn 2 ⁺ in, and the group Fe ⁺⁾ handles could be rather suppressed the enzyme activity observed compared to the untreated group (Table 3).

Metal ion Residual activity (%) None 100 Ca ^{2 +} 114.7 Mg ^{2 +} 93.2 Mn ^{2 +} 64.2 Cu ^{2 +} 53.3 Zn ^{2 +} 74.3 Fe ⁺ 62.5 Na ⁺ 105.4 K ⁺ 103.3 EDTA 98.9

In particular, in the group treated with Ca ^{2 +} increased by 114.4% compared to the untreated group, the results were examined from the change in thermal stability of beta-1,3-glucanase of the invention in accordance with the presence or absence of Ca ^{2 +} and, as a result, as shown in Figure 7, it could be observed that the slight increase in the thermal stability of the beta-1,3-glucanase by Ca ^{2 +} (Fig. 7).

Figure 7 is a graph showing the change in thermal stability of beta-1,3-glucanase according to the presence or absence of Ca ^{2 +} according to an embodiment of the present invention, (●) is added to 2.5 mM Ca ^{2 +} is a measure of the thermal stability changes in beta-1,3-glucanase graph, (○) is Ca ^{2 +} a non-doped (EDTA addition of 1 mM) thermal stability changes in beta-1,3-glucanase Is a graph measured.

In addition, as shown in Table 4, the beta-1,3-glucanase derived from the Antarctic tokto group of the present invention showed similar biochemical characteristics of beta-1,3-glucanase derived from the known bacteria (Table 4) This means that the beta-1,3-glucanase of the present invention can replace the beta-1,3-glucanase previously used.

Phylum Species Optimum pH Optimum temperature (℃) Specific activity (U / mg) References Arthropoda Cryptopygus antarcticus 5.5 50 20.6 This study Nematoda Bursaphelenchus xylophilus 4.9 65 337 T. Kikuchi et al., 2005 Mollusca Mizuhopecten yessoensis 4.5 45 6.0 for laminaran S. Ootsuka et al., 2006 Spisula sacchalinensis 5.8 45 350 B. Valeri et al., 2004 Fungi Sclerotium rofsii 2.9 (41 kDa) 72 380 G.M. Gubitz et al., 1996 Penicillum wortmanni 6 60 n.a E.T. Reese and Y. Shibata, 1965 Bacteria Oerskovia xanthineolytica 8.0 37 8 Bacillus circulans 5.5 4.1 Y. Nogi and K. Horikoshi, 1990 Bacillus sp . 9.0 60 396.6 Thermotoga neapolitana 85 V. Zverlov et al., 1997 Aeromonas hydrophila 5.5 40 201 T. Araki, 1983 Vibrio sp . 6.5 40 51.9 Y. Tamaru et al., 1995 Rhodothermus marinus 7.0 85 542 R. Spilliaert et al., 1994 Archaea Pyrococcus furiosus 6.0 100 922 Y. Gueguen et al., 1997

Example  4: Thin layer chromatography  Beta-1,3- On glucanase  by Laminarin Decomposition products  Confirm

In one embodiment of the present invention, beta-1,3-produced by the transformant prepared in Example 2 to identify the degradation product of laminarin hydrolyzed by beta-1,3-glucanase Laminar hydrolyzate by glucanase was analyzed using commonly used thin layer chromatography (TCL) (AI Vogel et al., Vogel's Textbook of Practical Organic Chemistry, 5 ^th Edition). The results are shown in FIG.

8 is a result of analyzing the decomposition product of laminarin hydrolyzed by beta-1,3-glucanase according to an embodiment of the present invention by thin layer chromatography, MK is a marker, G1 Silver glucose (glucose), L2 refers to Laminaribiose, L3 refers to Laminaritriose, L4 refers to Laminaritetroose, L5 refers to Laminalipentose, L6 refers to Laminarihexose, and Lam. Refers to Laminarin. .

As shown in Fig. 8, When L2 to L6 oligosaccharides were decomposed using beta-1,3-glucanase, L2 oligosaccharides were not cleaved, but L3 to L6 oligosaccharides were cleaved to reduce glucose (G), laminaribiose (L2) and laminatritri. It was confirmed that the decomposition product of laminarin of os (L3) was produced (FIG. 8). This means that the beta-1,3-glucanase enzyme of the present invention is endo-beta-1,3-glucanase, and in order for the substrate to be degraded, it must be an oligosaccharide having at least 3 glucose, In the case of narin, the degradation products are mostly glucose, laminaribis and laminaritriose.

1 is an Antarctic toktogi ( Cryptopygus) according to an embodiment of the present invention antarcticus ) shows the full-length cDNA sequence of beta-1,3-glucanase, where the single line represents the predicted signal sequence, the * marks the stop codon (TAA), and the double line represents the predicted polyanimation sequence. Point to each.

Figure 2 is an amino acid sequence of beta-1,3-glucanase isolated from the Antarctic toxin group (denoted as C. antarcticus ) according to an embodiment of the present invention and other species of beta-1,3 (4) -glu Figure showing the homology analysis of the amino acid sequence of the kinase and glucanase-like protein, the * symbol indicates a conserved residue between each enzyme, the # symbol indicates a catalytically active residue important for enzymatic activity, respectively.

Figure 3 is an analysis of the beta-1,3-glucanase gene isolated from the Antarctic toktogi according to an embodiment of the present invention, Figure 3a amplifies genomic DNA and cDNA of beta-1,3-glucanase 3 shows a nucleotide sequence of exons (in capital letters) and introns (in lowercase letters) of the beta-1,3-glucanase gene amplified from genomic DNA. Figure 3c shows the structure of the beta-1,3-glucanase gene present on the genome.

Figure 4 shows the cloning process and cleavage map of the expression vector expressing beta-1,3-glucanase according to an embodiment of the present invention.

5 shows thiolidoxin-beta-1,3-glucanase fusion protein (Trx-CaLam) and thioridoxin of the protein in a transformant transformed with an expression vector according to an embodiment of the present invention. It is a gel photograph obtained by SDS-PAGE analysis of the removed beta-1,3-glucanase (CaLam) expression.

Figure 6 is thioridoxin-beta-1,3-glucanase (■) and beta-1,3-glucana of the present invention according to pH (a) and temperature (b) according to an embodiment of the present invention It is a graph showing the change of enzyme activity of the enzyme (□).

Figure 7 is a graph showing the change in thermal stability of beta-1,3-glucanase according to the presence or absence of Ca ^{2 +} according to an embodiment of the present invention, (●) is added to 2.5 mM Ca ^{2 +} the thermal stability changes in beta-1,3-glucanase, (○) is a measure of the thermal stability changes in Ca ^{+ 2} is added to the non-beta-1,3-glucanase (EDTA addition of 1 mM), respectively It is a graph.

8 is a result of analyzing the decomposition product of laminarin hydrolyzed by beta-1,3-glucanase according to an embodiment of the present invention by thin layer chromatography, MK is a marker, G1 Silver glucose (glucose), L2 refers to Laminaribiose, L3 refers to Laminaritriose, L4 refers to Laminaritetroose, L5 refers to Laminalipentose, L6 refers to Laminarihexose, and Lam. Refers to Laminarin. .

<110> Korea Ocean Research & Development Institute <120> BETA-1,3-GLUCANASE FROM CRYPTOPYGUS ANTARCTICUS, GENE ENCODING          THE SAME, AND USE THEREOF <130> DPP-2007-2598-KR <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 926 <212> DNA <213> Full length sequence for beta-1,3-glucanase gene <400> 1 acagtcagtc ggaattaatc tggaaaacat gaacgcattt acatttcccc tacttttggc 60 gttctgcgcc tttgcccacg gtgcatgggt gttggactgg gaggatgaat tcaacggagg 120 aaatttagca gatagatgga atttcgagtt ggggtgcaat ggttggggaa acaatgagct 180 tcaatgctac accgacaaca gaggtgccaa tgccagacaa gaggacggaa aattggtcat 240 ctcggccgtg agggagtggt ggggcgatgg agttaatcca gacaaagaat tcacctctgc 300 ccgtatgacc acaaaggcta attggcttca tggaaagttt gagatgagag cacgattgcc 360 caagggtaaa catctctggc ctgcattctg gatgatgccc caaaattcag aatacggcgg 420 ttggccccgg agtggagaaa ttgatattac tgaatacagg gggcaaaagc cacaacagat 480 tttgggaacc cttcactttg gagctgcatg ggacaacaag ggtgatgccg gaactggcgc 540 aagagacttt ccaatcgact tttctgccga tttccacact tttggattgg actggtcccc 600 tgattccatt caatggcttt tggatgacca agtttaccac acagagtctc ttcaaagaaa 660 cttctgggat ggcgtctaca accaaaatgg gtctcccttt gataaaaact ttttcatcat 720 tttgaacttg gccgttggtg gaaacttctt tgggggtgaa cctttcgatc ccagtgagtc 780 tgatggctgg gcaaagaata cttttgaagt tgaatatgtc aagaaatgga cgtggaatta 840 aatcttttca tctagaaatt tttatcttaa tattcccgtt gttaaattcg agctattaat 900 aaattttgcc aaattgttat tttgag 926 <210> 2 <211> 813 <212> DNA <213> Coding sequence for beta-1,3-glucanase gene <220> <221> CDS (222) (1) .. (810) <400> 2 atg aac gca ttt aca ttt ccc cta ctt ttg gcg ttc tgc gcc ttt gcc 48 Met Asn Ala Phe Thr Phe Pro Leu Leu Leu Ala Phe Cys Ala Phe Ala   1 5 10 15 cac ggt gca tgg gtg ttg gac tgg gag gat gaa ttc aac gga gga aat 96 His Gly Ala Trp Val Leu Asp Trp Glu Asp Glu Phe Asn Gly Gly Asn              20 25 30 tta gca gat aga tgg aat ttc gag ttg ggg tgc aat ggt tgg gga aac 144 Leu Ala Asp Arg Trp Asn Phe Glu Leu Gly Cys Asn Gly Trp Gly Asn          35 40 45 aat gag ctt caa tgc tac acc gac aac aga ggt gcc aat gcc aga caa 192 Asn Glu Leu Gln Cys Tyr Thr Asp Asn Arg Gly Ala Asn Ala Arg Gln      50 55 60 gag gac gga aaa ttg gtc atc tcg gcc gtg agg gag tgg tgg ggc gat 240 Glu Asp Gly Lys Leu Val Ile Ser Ala Val Arg Glu Trp Trp Gly Asp  65 70 75 80 gga gtt aat cca gac aaa gaa ttc acc tct gcc cgt atg acc aca aag 288 Gly Val Asn Pro Asp Lys Glu Phe Thr Ser Ala Arg Met Thr Thr Lys                  85 90 95 gct aat tgg ctt cat gga aag ttt gag atg aga gca cga ttg ccc aag 336 Ala Asn Trp Leu His Gly Lys Phe Glu Met Arg Ala Arg Leu Pro Lys             100 105 110 ggt aaa cat ctc tgg cct gca ttc tgg atg atg ccc caa aat tca gaa 384 Gly Lys His Leu Trp Pro Ala Phe Trp Met Met Pro Gln Asn Ser Glu         115 120 125 tac ggc ggt tgg ccc cgg agt gga gaa att gat att act gaa tac agg 432 Tyr Gly Gly Trp Pro Arg Ser Gly Glu Ile Asp Ile Thr Glu Tyr Arg     130 135 140 ggg caa aag cca caa cag att ttg gga acc ctt cac ttt gga gct gca 480 Gly Gln Lys Pro Gln Gln Ile Leu Gly Thr Leu His Phe Gly Ala Ala 145 150 155 160 tgg gac aac aag ggt gat gcc gga act ggc gca aga gac ttt cca atc 528 Trp Asp Asn Lys Gly Asp Ala Gly Thr Gly Ala Arg Asp Phe Pro Ile                 165 170 175 gac ttt tct gcc gat ttc cac act ttt gga ttg gac tgg tcc cct gat 576 Asp Phe Ser Ala Asp Phe His Thr Phe Gly Leu Asp Trp Ser Pro Asp             180 185 190 tcc att caa tgg ctt ttg gat gac caa gtt tac cac aca gag tct ctt 624 Ser Ile Gln Trp Leu Leu Asp Asp Gln Val Tyr His Thr Glu Ser Leu         195 200 205 caa aga aac ttc tgg gat ggc gtc tac aac caa aat ggg tct ccc ttt 672 Gln Arg Asn Phe Trp Asp Gly Val Tyr Asn Gln Asn Gly Ser Pro Phe     210 215 220 gat aaa aac ttt ttc atc att ttg aac ttg gcc gtt ggt gga aac ttc 720 Asp Lys Asn Phe Phe Ile Ile Leu Asn Leu Ala Val Gly Gly Asn Phe 225 230 235 240 ttt ggg ggt gaa cct ttc gat ccc agt gag tct gat ggc tgg gca aag 768 Phe Gly Gly Glu Pro Phe Asp Pro Ser Glu Ser Asp Gly Trp Ala Lys                 245 250 255 aat act ttt gaa gtt gaa tat gtc aag aaa tgg acg tgg aat 810 Asn Thr Phe Glu Val Glu Tyr Val Lys Lys Trp Thr Trp Asn             260 265 270 taa 813 <210> 3 <211> 270 <212> PRT <213> Coding sequence for beta-1,3-glucanase gene <400> 3 Met Asn Ala Phe Thr Phe Pro Leu Leu Leu Ala Phe Cys Ala Phe Ala   1 5 10 15 His Gly Ala Trp Val Leu Asp Trp Glu Asp Glu Phe Asn Gly Gly Asn              20 25 30 Leu Ala Asp Arg Trp Asn Phe Glu Leu Gly Cys Asn Gly Trp Gly Asn          35 40 45 Asn Glu Leu Gln Cys Tyr Thr Asp Asn Arg Gly Ala Asn Ala Arg Gln      50 55 60 Glu Asp Gly Lys Leu Val Ile Ser Ala Val Arg Glu Trp Trp Gly Asp  65 70 75 80 Gly Val Asn Pro Asp Lys Glu Phe Thr Ser Ala Arg Met Thr Thr Lys                  85 90 95 Ala Asn Trp Leu His Gly Lys Phe Glu Met Arg Ala Arg Leu Pro Lys             100 105 110 Gly Lys His Leu Trp Pro Ala Phe Trp Met Met Pro Gln Asn Ser Glu         115 120 125 Tyr Gly Gly Trp Pro Arg Ser Gly Glu Ile Asp Ile Thr Glu Tyr Arg     130 135 140 Gly Gln Lys Pro Gln Gln Ile Leu Gly Thr Leu His Phe Gly Ala Ala 145 150 155 160 Trp Asp Asn Lys Gly Asp Ala Gly Thr Gly Ala Arg Asp Phe Pro Ile                 165 170 175 Asp Phe Ser Ala Asp Phe His Thr Phe Gly Leu Asp Trp Ser Pro Asp             180 185 190 Ser Ile Gln Trp Leu Leu Asp Asp Gln Val Tyr His Thr Glu Ser Leu         195 200 205 Gln Arg Asn Phe Trp Asp Gly Val Tyr Asn Gln Asn Gly Ser Pro Phe     210 215 220 Asp Lys Asn Phe Phe Ile Ile Leu Asn Leu Ala Val Gly Gly Asn Phe 225 230 235 240 Phe Gly Gly Glu Pro Phe Asp Pro Ser Glu Ser Asp Gly Trp Ala Lys                 245 250 255 Asn Thr Phe Glu Val Glu Tyr Val Lys Lys Trp Thr Trp Asn             260 265 270 <210> 4 <211> 1230 <212> DNA <213> Genome sequence for beta-1,3-glucanase gene <400> 4 acagtcagtc ggaattaatc tggaaaacat gaacgcattt acatttcccc tacttttggc 60 gttctgcgcc tttgcccacg gtgcatgggt gttggactgg gaggatgaat tcaacggagg 120 aaatttagca gatagatgga atttcgagtt ggggtgcaat ggtaagttat ttaaaggtat 180 ttttttttaa acatataaac aagattgtat tcccaaaggt tggggaaaca atgagcttca 240 atgctacacc gacaacagag gtgccaatgc cagacaagag gacggaaaat tggtcatctc 300 ggccgtgagg gagtggtggg gcgatggagt taatccagac aaagaattca cctctgcccg 360 tatgaccaca aaggctaatt ggcttcatgg aaagtttgag atgagagcac gattgcccaa 420 gggtaaaatt tgaaacttat caaaaaatgc aatgatacta atcttaagat tccttttata 480 aattataggc aaacatctct ggtaacattt taaagcctat ttaaacatac tgtgttttta 540 tctttctata tgatctaggc ctgcattctg gatgatgccc caaaattcag aatacggcgg 600 ttggccccgg agtggagaaa ttgatattac tgaagtatgt atagtttatt ttaaatatta 660 atgattttta tttttatttt taactcttta tttagtacag ggggcaaaag ccacaacaga 720 ttttgggaac ccttcacttt ggagctgcat gggacaacaa gggtgatgcc ggaactggcg 780 caagagactt tccaatcgac ttttctgccg atttccacac tgtaatttca aatttttatt 840 ttattagatt acattttctt aatttatttg aattatttga atagtttgga ttggactggt 900 cccctgattc cattcaatgg cttttggatg accaagttta ccacacagag tctcttcaaa 960 gaaacttctg ggatggcgtc tacaaccaaa atgggtctcc ctttgataaa aactttttca 1020 tcattttgaa cttggccgtt ggtggaaact tctttggggg tgaacctttc gatcccagtg 1080 agtctgatgg ctgggcaaag aatacttttg aagttgaata tgtcaagaaa tggacgtgga 1140 attaaatctt ttcatctaga aatttttatc ttaatattcc cgttgttaaa ttcgagctat 1200 taataaattt tgccaaattg ttattttgag 1230 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for beta-1,3-glucanase gene <400> 5 atgaacgcat ttacatttcc cct 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-1,3-glucanase gene <400> 6 ttaattccag ctccatttct tga 23 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for signal sequence deleted beta-1,3-glucanase          gene <400> 7 nnnnnnggat ccgcatgggt gttggactgg gagga 35 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for signal sequence deleted beta-1,3-glucanase          gene <400> 8 nnnnnnaagc ttttaattcc acgtccattt cttga 35  

Claims (11)

A beta-1,3-glucanase protein consisting of the amino acid sequence of SEQ ID NO: 3 and having beta-1,3-glucan degrading activity. The method of claim 1, wherein the protein is Antarctic Toktogi ( Cryptopygus) protein, isolated from antarcticus ). A polynucleotide consisting of an amino acid sequence according to claim 1 and encoding a beta-1,3-glucanase protein having beta-1,3-glucan degrading activity and consisting of the 29th to 841th nucleotide sequences of SEQ ID NO: 1 Nucleotides. delete An expression vector comprising the polynucleotide of claim 3. The expression vector according to claim 5, wherein the expression vector is pET-Trx-CaLam represented by a cleavage map shown in FIG. A transformant transformed by introducing the expression vector of claim 5 or 6. a) culturing a cell comprising a transformant according to claim 7 or an amino acid sequence of SEQ ID NO: 3 and having a coding sequence of beta-1,3-glucanase protein having beta-1,3-glucan degrading activity step; b) inducing the expression of beta-1,3-glucanase protein in said cultured cells; c) eluting the protein from the supernatant obtained by pulverizing and then precipitating the cells expressing the beta-1,3-glucanase protein; And d) purifying the eluted protein Method for producing a beta-1,3-glucanase having a beta-1,3-glucan degrading activity, including. An antimicrobial agent against pathogenic fungi or yeast comprising the protein of claim 1 or 2 as an active ingredient. Food or food additives having beta-1,3-glucan degrading activity comprising the protein of claim 1 or 2 as an active ingredient. A feed having beta-1,3-glucan degrading activity comprising the protein of claim 1 or 2 as an active ingredient.

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