KR20210006833A - Chitinolytic enzyme derived from Clostridium cellulovorans - Google Patents

Abstract

The present invention relates to a novel chitin-decomposing enzyme comprising, as an active ingredient, exo-beta-N-acetylglucosaminidase composing a cellulosome derived from Clostridium cellulovorans. According to the present invention, chitin-related biomass which has not been easily used as a raw material in the past can be used, and N-acetyl-glucosamine can be eco-friendly produced. In addition, Clocel_3193 has cell wall adhesion and resolution, and thus the Clocel_3193 can be used as an antifungal composition.

Description

Chitinase derived from Clostridium cellulovorans {Chitinolytic enzyme derived from Clostridium cellulovorans}

The present invention relates to a chitin-degrading enzyme comprising exo-beta-en-acetylglucosaminedase as an active ingredient constituting a cellulosome derived from Clostridium cellulovorans , as a novel chitin-degrading enzyme. will be.

Anaerobic microorganisms that degrade biomass generally have enzyme complexes called cellulosomes on the outer wall of cells. Depending on the type of substrate that can be used outside, related enzymes are expressed in various ways, attached to the cell wall, and efficiently obtained and used by using synergy between the attached enzymes.

A number of enzymes attached to cellulosomes are related to the components of lignocellulosic biomass such as Cellulose, Xylan, Mannan, and Pectin, which can obtain carbon sources. There are only a few known cellulosome-forming enzymes for obtaining nitrogen sources other than carbon sources, and a representative one is chitinolytic enzyme.

There is no nitrogen source in lignocellulosic biomass, but a nitrogen source other than a carbon source is required for normal growth of Clostridium. Until now, it has been used as a nitrogen source to obtain a sugar with an amine called N-acetyl glucosamine by decomposing fungi, yeast, and dead insects that may be incidental in lignocellulosic biomass to receive a nitrogen source. It is an estimate of the scientific community. In addition, an enzyme related to nitrogen source as a cellulosome constituting enzyme of Clostridium cellulose has not been discovered.

Chitin-degrading enzyme (Chitinolytic enzyme) decomposes a polysaccharide called chitin, which is present in the shells of insects and crustaceans, and makes a sugar with an amine called N-acetyl glucosamine. Chitobiase (Chitobiase) is a type of chitin-degrading enzyme known to recognize and cleave two yen-acetyl-glucosamine. In addition, beta-hexosaminidase (beta-hexosaminidase) is a generic name for an enzyme that cleaves hexose, and chitin-degrading enzymes also belong to this enzyme. Therefore, one of the chitinolidic enzymes, Exo-β-N-acetylglucosaminidase, is also a type of hexosaminidase.

Exo-beta-en-acetylglucosaminidase (Exo-β-N-acetylglucosaminidase) is one of the chitin-degrading enzymes and is an enzyme that recognizes and cuts one N-acetyl-glucosamine from the non-reducing sugar end.

N-acetyl glucosamine is one of the amino sugars, which is a combination of amino acids and sugars, and is known as an essential component of cartilage. It is a functional sugar that is industrially useful as a dietary supplement that helps with inflammatory and ulcerative bowel disease and arthritis, bioethanol through sugar fermentation, and a moisturizing material for functional cosmetics.

On the other hand, the cell wall of the fungus is composed of 22-44% of chitin. Therefore, if an enzyme having chitin-degrading activity is used, it can degrade the fungal cell wall to produce industrially useful en-acetyl-glucosamine or be used as an anti-fungal agent.

Fu et al. Biotechnology for Biofuels 2014, 7:174

The technical problem to be achieved by the present invention is to provide a novel chitin-degrading enzyme derived from Clostridium cellulovorans .

In addition, an object of the present invention is to provide a method for preparing the chitin-degrading enzyme and an antifungal composition comprising the chitin-degrading enzyme as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a chitin-degrading enzyme derived from Clostridium cellulovorans comprising the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a chitin-decomposing enzyme derived from Clostridium cellulose encoded in the nucleotide sequence of SEQ ID NO: 2.

According to an embodiment of the present invention, the enzyme may have both an adhesion and resolution to chitin.

According to another embodiment of the present invention, the enzyme is capable of decomposing the ends of chitin.

In addition, the present invention (1) preparing a recombinant expression vector comprising the nucleotide sequence of SEQ ID NO: 2; (2) preparing a transformant by injecting the recombinant expression vector into a host cell; (3) culturing the transformant; And (4) crushing the transformant and then centrifuging to obtain a supernatant thereof.

According to an embodiment of the present invention, the vector may be a pColdII plasmid vector.

According to another embodiment of the present invention, the host cell may be Escherichia coli .

According to another embodiment of the present invention, the transformant may be a pTf16 chaperon vector injected.

In addition, the present invention provides an antifungal composition comprising the chitin-degrading enzyme as an active ingredient.

As an embodiment of the present invention, the fungus is cloud mushroom ( Trametes versicolor ) , Buhugae rice cake mushroom ( Fomitopsis palustris ) , and It may be one or more species selected from the group consisting of small seashell mushrooms ( Gloeophyllum trabeum ).

In another embodiment of the present invention, the antifungal composition may be an antifungal composition.

The present invention reveals the chitin resolution and adhesion ability of exo-beta-ene-acetylglucosaminedase encoded by the Clocel_3193 gene of Clostridium cellulovorans , and this is a chitin-degrading enzyme for the production of lignocellulosic biomass. As provided, it is possible to use chitin-related biomass composed of en-acetyl-glucosamine, which cannot be easily used as a raw material by the provision of the present invention, and environmentally friendly production of en-acetyl-glucosamine is possible. In addition, Clocel_3193 can be used as an antifungal agent that inhibits the proliferation and growth of fungi having chitin as a major component of the cell wall from its chitin decomposition and adhesion ability.

1 is a diagram predicting the function of the exo-beta-ene-acetylglucosaminidase ( Clocel_3193 ) gene through the sequence.
Figure 2 is a schematic diagram of the recombinant vector pColdII:: Clocel_3193 and pTf16 chaperon vector into which the Clocel_3193 gene is inserted.
3 is a view confirming the expression of Clocel_3193 in BL21(DE3)_Clocel_3193, a transformant into which pColdII:: Clocel_3193 has been introduced.
4 is a diagram illustrating the ability of Clocel_3193 to adhere to insoluble substrates related to lignocellulosic biomass.
5 is a view confirming the resolution of Clocel_3193.
6 is a view confirming the ability of Clocel_3193 to adhere to mold.
7 is a view confirming the production of yen-acetyl-glucosamine through the decomposition of the fungus Clocel_3193.

The present inventors analyzed the sequence of the Clocel_3193 gene (Gene ID: 9610087) of Clostridium cellulovorans , whose function has not yet been identified, and the protein encoded by the gene is exo-beta-ene-acetylglucosa. The present invention was completed by confirming that it is Mini Days (Exo-β), and by confirming the selective adhesion and resolution of Clocel_3193 to chitin.

Thus, the present invention provides a chitin-degrading enzyme derived from Clostridium cellulovorans .

The chitin-degrading enzyme is exo-beta-ene-acetylglucosaminedase (Exo-β), which may include or consist of the amino acid sequence of SEQ ID NO: 1, and the enzyme is encoded in the nucleotide sequence of SEQ ID NO: 2. I can.

On the other hand, woody biomass (wood, herbaceous, agricultural by-products such as rice straw and rice husk) is pyrolysis and fermentation along with food-based sugar-based biomass (sugarcane, sugar beet, etc.) and starchy biomass (grains, potatoes, etc.). It is used as a biofuel that can produce energy such as methane, ethanol, and hydrogen through the process. The major components of lignocellulosic biomass differ in composition and content of chemical components that make up wood depending on conifers and hardwoods, tree species, and age, but generally cellulose (40-50%), hemicellulose (25-35%), and lignin (15 ~ 20%). Cellulose is a polymer compound in which glucose is regularly bonded by hydrogen bonds and van der Waals forces, and hemicellulose is an adhesive between cellulose and lignin by combining pentoses such as xylose and arabinose in β form. Play a role. Lignin is an insoluble, hardly decomposable polymer compound in which aromatic substances having phenylpropanoid units are irregularly linked, and has a structure that prevents the decomposition of polysaccharides.

According to an embodiment of the present invention, Clocel_3193 is reacted with the substrates Avicel, Xylan, and Chitin, which are substrates through function prediction of Clocel_3193, which are the main foods of Clostridium cellulose. Through quantitative analysis of the amount of protein that did not bind to each substrate, it was confirmed that Clocel_3193 has excellent selective adhesion to chitin. The adhesion ability of Clocel_3193 is predicted to be due to the unknown part, and from the above results, it can be seen that the unknown part of Clocel_3193 exhibits a unique adhesion ability to chitin, which is not directly related to general lignocellulosic biomass.

Accordingly, the chitin-degrading enzyme of the present invention can rapidly decompose a chitin-related substrate by the above adhesion.

In addition, according to an embodiment of the present invention, focusing on the fact that the cell wall of the fungal cells belonging to Fungi is composed of chitin, as a result of confirming the adhesion and resolution of Clocel_3193 to the fungus, Clocel_3193 is Trametes versicolor, It was confirmed that it was attached to Fomitopsis palustris, Gloeophyllum trabeum , decomposed the cell wall, and produced N-acetyl-glucosamine as a product thereof.

Thus, the chitin-degrading enzyme of the present invention may be used as an antifungal composition, and in particular, may be used as an antifungal composition.

When the chitin-degrading enzyme of the present invention is used as an antifungal composition, the target fungus (Fungi) is not limited as long as the main component of the cell wall is chitin.

Further, according to one embodiment of the present invention, by inserting a gene Clocel_3193 removal of the gene coding for the signal peptide from the gene Clocel_3193 pColdII vector by transformation of E. coli, it was confirmed that the mass production Clocel_3193.

Thus, the present invention (1) preparing a recombinant expression vector comprising the nucleotide sequence of SEQ ID NO: 2;

(2) preparing a transformant by injecting the recombinant expression vector into a host cell;

(3) culturing the transformant; And

(4) After crushing the transformant, centrifugation and obtaining a supernatant thereof; provides a method for producing a chitin-degrading enzyme comprising.

In a specific embodiment of the present invention, the "vector" uses a pColdII plasmid, but any DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host is limited thereto. It is not. Thus, the vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since the plasmid is the most commonly used form of the current vector and is used in specific examples of the present invention, in the specification of the present invention, "plasmid" and "vector" are sometimes interchangeably Used. However, the present invention encompasses other forms of vectors that have functions equivalent to those known or become known in the art.

In addition, in the present specification, "recombinant expression vector" refers to a fragment of double-stranded DNA as a recombinant carrier into which a fragment of a heterogeneous DNA is inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA that is not naturally found in host cells. The expression vector, once in the host cell, can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be generated. In a specific embodiment of the present invention, E. coli is used as a host cell, but the recombinant expression vector is not limited as long as it is a cell in an environment capable of being replicated within a cell and expressing a target gene.

The vector may include a promoter operatively linked to the gene to be cloned. In the present specification, the term "promoter" promotes expression of the gene to be transfected, and the promoter includes a basic factor required for transcription (Basal element), as well as an enhancer that can be used to promote and regulate expression. In a specific embodiment of the present invention, a pTf16 chaperon vector was additionally injected into the transformant in order to increase the expression level of the target gene.

In addition, in the present specification, "transformation" or "transfection" means that DNA is introduced into a host so that the DNA becomes replicable as an extrachromosomal factor or by chromosomal integration completion.

In the present invention, various transformations can be applied and various embodiments can be applied, and specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

[Example]

Example 1.Clostridium cellulose-derived novel exo-beta-ene-acetylglucosaminedase sequence analysis through function prediction and gene security

From the entire genome of Clostridium cellulovorans, the location and sequence of the exo-beta-ene-acetylglucosaminedase gene ( Clocel_3193, Gene ID: 9610087) were confirmed. Sequence analysis was performed based on Signal P and NCBI Blast data base. As a result, an unknown sequence with 74.2% homology with a protein containing an F5/8 type C domain known to have a signal peptide (amino acids 1-21) involved in protein exocrine at the front and an adhesion ability to en-acetyl-glucosamine. (Amino acids 22-469), Chitobiase/beta-hexosaminidase C-terminal domain (amino acids 470-536), and repeated docurin (amino acids 578-598) required for cellulosome linkage as an enzyme complex were expected to be located ( 1). The final expression part, excluding the signal peptide that affects the enzyme expression, was amplified by PCR by preparing a forward primer in the 5` direction (restriction enzyme BamH Ⅰ) and a reverse primer in the 3` direction (restriction enzyme EcoR Ⅰ) from Genomic DNA . Specific information of each primer is shown in Table 1 below, and the underlined is the restriction enzyme recognition sequence used. Restriction enzymes are BamH Ⅰ and EcoR Ⅰ sequences in order.

Forward primer 5`-ATA GGATCC AAAATAAATTTCACTGTAA-3` Reverse primer 5`-CG GAATTC ACTAAGTAATTTTTTCTTTAAA-3`

Example 2. Cloning for expression of the exo-beta-ene-acetylglucosamidase gene in E. coli and introduction into E. coli

Exo-beta-ene-acetylglucosaminedase gene ( Clocel_3193 ) obtained in Example 1 was amplified through PCR except for the signal peptide, which is an enzyme expression inhibitor, and pColdII vector, one of the E. coli expression vectors, was amplified by BamH I and EcoR. After cutting into I, it was ligated using an ligation kit to transform Escherichia coli BL21 (DE3). In addition, in order to increase the expression level of Clocel_3193, a pTf16 chaperon vector was introduced together (see Fig. 2). The recombinant vector was named pColdII:: Clocel_3193 , and the E. coli transformant was named BL21(DE3)_Clocel_3193.

Example 3. Expression of recombinant Clocel_3193 in E. coli transformants

In order to confirm the expression of Clocel_3193 of the transformant obtained in Example 2, purification and SDS-PAGE were performed using His-Tag. More specifically, in order to induce protein expression of the recombinant strain, it was inoculated into an LB medium containing ampicillin and pre-cultured at 37°C for 24 hours. Afterwards, inoculate the pre-culture solution in 250 ml of LB medium containing ampicillin, and when the optical density (OD600) at 600 nm is about 1.0, IPTG, an inducer, is added at a final concentration of 1 mM to induce the expression of Clocel_3193. I did. After incubation for 18-20 hours, the cells were obtained by centrifugation. Thereafter, the cells were crushed using ultrasound and centrifuged to prepare a supernatant. It was purified with His-tag linked to the N-terminal of the protein using an affinity chromatography method based on the difference in the concentration of imidazol (~20 mM). Electrophoresis was performed with 10% SDS-PAGE gel, and it was confirmed that the result of staining with Cossie blue dye appeared at the same location as the expected protein size (65 kDa) (see FIG. 3).

Example 4. Confirmation of adhesion ability to selected insoluble substrates through functional prediction with representative lignocellulosic biomass of Clocel_3193

In order to confirm the selective attachment ability of Clocel_3193 to chitin obtained in Example 3, the Clocel_3193 was reacted with Avicel, Xylan, and Chitin, respectively. Prepare 0.06 g of each substrate, adjust the final concentration of protein (Albumin) as a control group and enzyme (Clocel_3193) as an experimental group to about 100 μg/ml, and select and use 100 mM sodium phosphate pH 7.0 as the adhesion reaction buffer. I did. The reaction temperature was 15° C., the stirring speed was carried out at 300 rpm for 45 minutes, centrifuged at 1,000 g for 1 minute, and the amount of protein remaining in the supernatant was measured through the Breadford method. 10 μl of protein or enzyme was treated with 400 μl of Breadford's reagent, mixed well, and absorbance was measured with a spectrophotometer at 595 nm. As a reference protein, albumin was used to set the baseline and concentration, and the concentration of the protein and the enzyme used was measured through quantitative analysis. As a result, it was confirmed that Clocel_3193 has excellent adhesion to chitin and selective adhesion to chitin (see FIG. 4).

Example 5. Confirmation of decomposition pattern of Clocel_3193

Clocel_3193 has C-terminals found in chitobiase and β-hexosaminidase, and is predicted to have catalytic activity similar to those of the enzymes specified above. Thus, in order to confirm whether Clocel_3193 of the present invention also has a similar decomposition pattern to Chitobiase and β-hexosaminidase, 1.5 mM 4-nitrophenyl N-acetyl beta-D-glucosaminide (ρNPβG ₁ ) and 4-nitrophenyl N , N -diacetyl- The reaction was carried out with beta-D-chitobioside (ρNPβG ₂ ), 4-nitrophenyl beta-D- N , N , N -triacetyl chitotriose (ρNPβG ₃ ). The reaction proceeded at 35° C. for 16-24 hours. In the reaction solution, an equal amount of 0.1 N NaOH reaction solution was added to terminate the reaction. The reaction liquid after the reaction was measured through a spectrophotometer at 405 nm. As a result of calculating the result value in units (nmole/h·L), it was confirmed that Clocel_3193 recognized one sugar and had a decomposition pattern of cutting from the end to produce en-acetyl-glucosamine (see FIG. 5).

Example 6. Confirmation of adhesion ability of Clocel_3193 to mold

The cell walls of fungi are made of chitin. Thus, in order to confirm the fungal adhesion ability of Clocel_3193 obtained in Example 3, Trametes versicolor, Fomitopsis palustris, and Gloeophyllum trabeum were cultured in PDB medium for 2 weeks, and the mycelium of the fungus was collected through gauze. The hyphae was washed 3 times with sterilized distilled water, and the remaining moisture was evaporated through a dry oven and crushed into powder. Prepare 0.06 g of mold powder prepared in this way, and set the final concentration of protein (Albumin) as a control group and enzyme (Clocel_3193) as an experimental group to about 100 μg/ml, and use 100 mM sodium phosphate pH 8.0 as a buffer. I did. The reaction temperature was 15° C., the stirring speed was performed at 300 rpm for 45 minutes, and centrifuged at 1,000 g for 1 minute. The amount of protein remaining in the supernatant was measured by the Breadford method. 10 μl of protein or enzyme was treated with 400 μl of bradford reagent, mixed well, and absorbance was measured with a spectrophotometer at 595 nm. As a reference protein, albumin was used to set the baseline and concentration, and the concentration of the protein and the enzyme used was measured through quantitative analysis. As a result, it was possible to confirm the adhesion ability to the mold in Clocel_3193 (see Fig. 6).

Example 7. Confirmation of the fungal resolution of Clocel_3193

We tried to check whether Clocel_3193 attached to the mold can decompose it, that is, whether Clocel_3193 has the ability to resolve mold. Specifically, Trametes versicolor, Fomitopsis palustris, Gloeophyllum trabeum were cultured in PDB medium for 2 weeks, and the mycelium was collected using gauze, and the hyphae was washed 3 times with sterile distilled water, and the residual moisture was evaporated through a dry oven. It was ground and made into powder. 0.41 g of this powder was prepared as a substrate. 100 mM sodium citrate pH 6.0 was used as a reaction buffer to 0.41 g of mold powder, and the reaction temperature was performed at 35° C., 200 rpm, for 4 days. The amount of enzyme used at this time is about 100 μg/ml. After the reaction, the reaction solution was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was filtered and analyzed by HPLC. As the analysis equipment, refractive index detector (RID) was used, and an Aminex HPX-87H ion exchange column (300 mm 7.8 mm) was used as a column. Sugar separated at a rate of 0.5 ml/min was measured using 5 mM sulfuric acid as the mobile phase. 10 μl of the sample was injected. As a result, it was found that Clocel_3193 decomposes fungi to produce N-acetyl-glucosamine (see Fig. 7).

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea University Research and Buisness Foundation <120> Chitinolytic enzyme derived from Clostridium cellulovorans <130> DHP19-300-P <150> KR 1020190082666 <151> 2019-07-09 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 568 <212> PRT <213> Clostridium cellulovorans_Clocel_3193 <400> 1 Lys Ile Asn Phe Thr Val Ser Tyr Thr Ser Ala Val Gly Lys Thr Glu 1 5 10 15 Asn Tyr Thr Cys Ala Thr Arg Asp Ser Phe Tyr Asp Leu Asn Asp Asn 20 25 30 Ser Val Asn Arg Ala Thr Ser Glu His Phe Gln Ile Ile Trp Gly Asn 35 40 45 Gly Asp Thr Thr Gly Thr Val Asn Gln Glu Leu Val Lys Gly Asn Leu 50 55 60 Gln Asn Leu Glu Ala Ile Arg Asp Phe Tyr Val Asn Val Met Gly Phe 65 70 75 80 Val Asp Thr Ser Val Ser Val Asn Ser Pro Leu Thr Ser Ser Asn His 85 90 95 Tyr Lys Thr Asn Val Tyr Ile Ser Asn Thr Gly Leu Ser Lys Ile Thr 100 105 110 Asp Asp Trp Ala Tyr Met Ser Ser Asp Gly Glu Gly Phe Ala Phe Leu 115 120 125 Val Leu His Pro Gly Ala Met Arg Val Asp Pro Pro Ser Trp Val Val 130 135 140 Pro His Glu Tyr Ala His Ala Ile Thr Met His Gln Arg Gly Val Ile 145 150 155 160 Asp Ala Pro Trp Tyr Glu Val Thr Ala Asn Trp Phe Arg Asp Gln Tyr 165 170 175 Leu Gly Ser Ser Phe Tyr Lys Tyr Gly Asn Asn Val Tyr Gly Pro Asp 180 185 190 Ser Asp Phe Phe Arg Pro Ile Val Leu Asn Ser Asp Tyr Tyr Phe Pro 195 200 205 His Leu Lys Asn Tyr Tyr Asp Ala Trp Pro Phe Leu Leu Tyr Val Thr 210 215 220 Glu Asn Pro Asp Gln Met Lys Gly Leu Gly Leu Glu Val Met Lys Ala 225 230 235 240 Met Phe Lys Asp Thr Asn Asn Glu Val Met Phe Lys Lys Leu Glu Arg 245 250 255 Leu Ser Gly Thr Ser Ala Lys Asp Met Leu Gly Gly Tyr Ala Arg Arg 260 265 270 Met Val Thr Phe Asp Phe Lys Arg Gln Ala Asn Tyr Lys Lys Tyr Tyr 275 280 285 Asp Glu Leu Ile Ala Glu Asp Ser Ala Asn Tyr Asn Lys Ile Tyr Thr 290 295 300 Thr Leu Glu Asn Asp Ser Asn Gly Trp Phe Lys Val Pro Ser Ser Arg 305 310 315 320 Ala Pro Gln Gln Gly Gly Tyr Asn Ile Ile Pro Leu Asn Ile Asp Leu 325 330 335 Lys Ser Lys Gln Val Val Val Asn Phe Gln Gly Asn Ser Ser Glu Val 340 345 350 Gly Ala Asp Trp Arg Ala Ser Ile Val Ala Lys Thr Lys Thr Gly Gln 355 360 365 Thr Arg Tyr Ser Thr Met Trp Asn Ser Gly Thr Asn Thr Leu Asn Leu 370 375 380 Gln Gly Asp Glu Glu Lys Val Tyr Leu Val Val Cys Ala Thr Pro Lys 385 390 395 400 Glu Met Leu Asn Leu Thr Ser Phe Asp Leu Asp Val Val Gly Thr Arg 405 410 415 Tyr Pro Tyr Lys Val Gln Ile Ser Thr Asn Ser Ala Val Ser Lys Val 420 425 430 Glu Met Pro Thr Val Ser Val Ala Ala Gly Ser Tyr Asn Ala Ala Gln 435 440 445 Thr Ile Ser Leu Ser Ser Lys Thr Ser Gly Thr Thr Ile Tyr Tyr Thr 450 455 460 Leu Asp Gly Ser Thr Pro Thr Ala Ser Ser Thr Ala Tyr Ser Ser Pro 465 470 475 480 Ile Val Val Ser Lys Asn Thr Thr Ile Lys Ala Val Ala Ile Lys Ser 485 490 495 Gly Met Thr Asn Ser Asp Ile Thr Ser Ala Thr Tyr Thr Ile Ser Ile 500 505 510 Ile Gly Asp Val Asn Glu Asp Gly Arg Val Asn Ala Ile Asp Tyr Ala 515 520 525 Asn Ile Lys Ser Tyr Leu Leu Ala Asn Ser Thr Lys Ile Asn Leu Lys 530 535 540 Asn Ala Asp Met Asn Asn Asp Ser Lys Val Asn Ala Ile Asp Leu Ala 545 550 555 560 Leu Leu Lys Lys Lys Leu Leu Ser 565 <210> 2 <211> 1707 <212> DNA <213> Clostridium cellulovorans_Clocel_3193 <400> 2 aaaataaatt tcactgtaag ttatacttca gcagtgggaa agactgaaaa ttatacttgt 60 gcaacaagag attcctttta tgatttgaat gataattcag ttaacagagc tacttcagaa 120 catttccaaa taatttgggg aaatggtgat actacaggaa ctgttaatca agaattagta 180 aaaggtaatt tacaaaacct agaggcaata agagatttct atgttaatgt aatgggcttt 240 gtagatacta gtgtatctgt taatagtcca ctaactagca gcaatcacta caaaacaaat 300 gtgtacatat ccaatactgg actttcaaag attacagatg attgggcata tatgtcttct 360 gacggagaag gctttgcttt cttagtttta catccaggag ctatgcgtgt tgacccacca 420 agttgggtag ttcctcacga atatgctcat gccattacta tgcatcaacg tggtgttatc 480 gatgctcctt ggtatgaagt aacagcaaat tggttcagag atcaatattt aggaagtagt 540 ttttataagt atggaaataa tgtttatgga cctgattcag acttctttag gccaatagtc 600 ttgaactcag actactactt tccacatttg aagaactatt atgatgcttg gccattctta 660 ctatatgtaa ctgaaaatcc tgaccaaatg aaaggattag gtcttgaagt aatgaaagcg 720 atgtttaagg acactaataa tgaagttatg tttaagaaat tagagagatt atctggaaca 780 tctgctaaag atatgttagg tggctatgct agaagaatgg taacctttga ttttaagaga 840 caagctaatt ataaaaaata ttatgatgaa ttaatagcag aggacagtgc aaattataat 900 aaaatttata caaccttaga aaatgatagt aatggatggt ttaaggttcc tagctcaagg 960 gctccacaac aaggtggata taatattatt ccacttaaca tagatctaaa gagtaagcag 1020 gtagtcgtta attttcaagg aaatagttca gaagtagggg cagattggcg tgcaagcatt 1080 gttgctaaaa caaaaactgg acaaactaga tattctacaa tgtggaatag tggaactaac 1140 actttgaatc tacaaggaga tgaagaaaaa gtttatcttg tagtatgtgc aactccgaag 1200 gaaatgctta atttaacttc atttgattta gatgtagtag gaacaagata tccatataaa 1260 gtgcaaatat caacgaatag cgctgtttca aaggtagaaa tgccaactgt tagtgtagca 1320 gcaggtagtt acaatgctgc acaaacaata tcacttagca gtaaaacttc aggtacaact 1380 atatattata cacttgacgg aagtactcca acagcttcat ctactgccta tagtagtccg 1440 atagtagtat caaaaaatac aactattaag gctgttgcaa taaagagtgg aatgacaaac 1500 tctgatataa cttctgctac atatacaatt tctataattg gtgatgttaa tgaagatggt 1560 cgtgtaaatg caattgatta tgccaatatt aagagctatt tactggctaa ttcaacaaag 1620 ataaatttaa agaacgcaga tatgaataat gatagtaaag taaatgccat cgatttagca 1680 cttttaaaga aaaaattact tagttaa 1707 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 ataggatcca aaataaattt cactgtaa 28 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 cggaattcac taagtaattt tttctttaaa 30

Claims (9)

Chitin degrading enzyme derived from Clostridium cellulovorans comprising the amino acid sequence of SEQ ID NO: 1.
The method of claim 1,
The enzyme is chitin-degrading enzyme, characterized in that it has both adhesion and resolution to chitin.
The method of claim 1,
The enzyme is characterized in that to decompose the terminal of chitin, chitin degrading enzyme.
Chitin-degrading enzyme derived from Clostridium cellulovorans encoded in the nucleotide sequence of SEQ ID NO: 2.
Chitin-degrading enzyme preparation method comprising the following steps:
(1) preparing a recombinant expression vector comprising the nucleotide sequence of SEQ ID NO: 2;
(2) preparing a transformant by injecting the recombinant expression vector into a host cell;
(3) culturing the transformant; And
(4) After crushing the transformant, centrifuging to obtain a supernatant.
The method of claim 5,
The vector is a pCold II plasmid vector, characterized in that, chitin degrading enzyme production method.
The method of claim 5,
The host cell is E. coli ( Escherichia coli ), characterized in that, chitin-degrading enzyme production method.
The method of claim 5,
The transformant is characterized in that the pTf16 chaperon (chaperon) vector is additionally injected, chitin-degrading enzyme production method.
An antifungal composition comprising the chitin-decomposing enzyme of claim 1 or 4 as an active ingredient.

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