KR20110117556A - A chitinase and a use of the same - Google Patents

Abstract

The present invention has chitin resolution at high temperature and acidic pH conditions, E. The present invention relates to a chitinase that can be expressed in a coli expression system and a recombinant biocatalyst prepared using the chitinase gene. The present invention relates to a novel chitinase or a gene encoding the chitinase. By providing the drug can be used in a variety of industries, such as the application of chitinases, including pharmaceuticals and functional foods.

Description

Novel chitinase and its use {A CHITINASE AND A USE OF THE SAME}

The present invention provides chitinase having chitin resolution and chitosan resolution at high temperature and acidic pH; Nucleotide sequences encoding them; A vector comprising said nucleotide sequence; Transformants transformed with the vector; At least one selected from the group consisting of the transformant, the culture thereof, the cell-free culture medium thereof, the concentrate of the culture medium, the concentrate of the cell-free culture medium, and the recombinant chitinase prepared by expressing the transformant as an active ingredient. It relates to a biocatalyst having chitin resolution and chitosan resolution comprising.

Chitin (GlcNAc) is N - acetyl glucosamine (N -acetylglucosamine, NAG) polymer linked by a linear β-1,4 bond (linear polymer) a natural polymeric substance (biopolymer). The chitin is a major shell component of crustaceans such as crabs, shrimps, insects, and insects such as spiders, ants, and grasshoppers, and is a substance constituting cell walls of microorganisms such as fungi, bacteria, or green algae, followed by cellulose in nature. It is a polymer that exists a lot. Chitosan is also a polymer in which the acetyl group of chitin is eliminated. It is estimated that more than 10 billion tons of chitin are produced annually in the water biosphere alone.

The chitin has recently been increasingly applied as an important natural resource. In particular, N -acetylglucosamine, an acetylated form of D-glucosamine (DGA) and D-glucosamine, produced by hydrolysis of chitin, has excellent effects on degenerative arthritis, as well as anti-inflammatory, cardioprotective, and hepatic It is also known to be effective in protecting and promoting material assimilation. N- acetylglucosamine, a monomer of chitin, acts as a major component to make glycoproteins such as connective tissues, tendons, and cartilage tissues in vivo. Glycoproteins containing N- acetylglucosamine are binding materials that connect cells to cells. Essential for the complete maintenance of an organization or institution. In addition, N- acetylglucosamine is a major component of the mucous membranes of the digestive tract and helps treat ulcers in the digestive tract. N -acetylglucosamine is known to have a higher rate of migration in mucosal cells than D-glucosamine in the body. Therefore, N -acetylglucosamine is believed to have a higher utility value as a biological new material than D-glucosamine.

In addition, the chitosan has been reported to regulate the biorhythm with the function of activating the natural healing ability of the living body. In addition, it has a decholesterol effect, excessive blood glucose control effect, liver function improvement effect and anticancer effect of excess harmful cholesterol in the body, and it is known to have an effect of inhibiting blood pressure increase by adsorbing chloride ions which cause blood pressure increase and discharged to the body. have.

As mentioned above, chitin is present in abundance in nature and continues to be produced not only in tremendous amounts, but also in a variety of physiological activities, and their treatment and recycling are required.

In connection with the use of chitin and chitosan, the chitin or chitosan is a polymer material that is not only soluble in water but also degraded by digestive enzymes secreted from the stomach, and absorbed in the gastrointestinal tract of humans such as cellulose. There is a drawback to not being. Therefore, in order to efficiently utilize chitin or chitosan which not only exist in abundance in nature but also have various physiological activities as an active substance in vivo, it is necessary to produce their oligosaccharides, that is, chitin oligosaccharides.

Chitin oligosaccharides or N- acetylglucosamine, which are rich in nature and have various physiological activities such as chitin or chitosan, have attracted attention for their useful value as medical and functional food materials.

The chitin oligosaccharide or N- acetylglucosamine is currently produced by acid hydrolysis, which hydrolyzes chitin with acid. The acid hydrolysis is mainly performed by treating chitin at 60 to 80 ° C. using hydrochloric acid (HCl) at a concentration of 6N to 12N.

In the case of the acid hydrolysis, chitin is decomposed into monosaccharides during acid hydrolysis, and deacetylation occurs, thereby simultaneously producing N- acetylglucosamine and D-glucosamine. If the acid hydrolysis conditions are different, the relative ratio of N- acetylglucosamine and D-glucosamine may be changed. However, in order to reduce the amount of D-glucosamine generated, the concentration of acid is inhibited. When lowering or lowering the reaction temperature, there is a problem that the amount of N- acetylglucosamine obtained from chitin, that is, the production efficiency of N- acetylglucosamine is lowered.

In addition to the above problems, the acid hydrolysis method uses a high concentration of acid, so that the corrosion of the device or the risk of operation is a process problem, environmental problems such as generation of acidic wastes that are harmful impurities to the human body, and acid. There are economic problems such as high production prices due to the complex purification process for the removal of waste and the need for a process for the purification of N- acetylglucosamine.

In order to solve the above problems, a method of decomposing chitin using an enzyme has recently been proposed. Enzymatic hydrolysis is a method of decomposing chitin using chitinase, a chitin hydrolase, and enzymatic hydrolysis by chitinase does not proceed with deacetylation of N- acetylglucosamine during chitin degradation. Since the corresponding D-glucosamine does not occur, the purification process is not only very simple, but also can produce chitin oligosaccharides or N- acetylglucosamine, which are high yields of chitin, and uses enzymes that exist in nature instead of high concentrations of acid. There is an advantage that no environmental problems or process problems occur.

However, the technology for producing these enzymes efficiently and economically producing chitin oligosaccharides or N -acetylglucosamine using them has not been developed industrially.

In order to develop a technology for economically producing chitin oligosaccharides or N- acetylglucosamine using the chitinases, Escherichia has high reaction efficiency at easy hydrolysis conditions of chitin, that is, high temperature and acidic pH. coli expression system ( E. There is an urgent need for the development of chitinase that can be expressed in an expression system capable of producing enzymes such as coli expression systems in a high efficiency and enhancing the biological activity of enzymes in cellular processes.

In order to solve the problems of the prior art as described above, the present invention has chitin decomposition activity, chitin decomposition is easy to an example of the high temperature and acidic conditions, for example, chitin resolution even at a temperature of 40 ℃ or more and pH 4 to pH 6 Is maintained and expressed system, specifically Escherichia coli expression system ( E. It is an object of the present invention to provide a chitinase that can be efficiently expressed in a coli expression system.

It is also an object of the present invention to provide an amino acid sequence of the chitinase and a polynucleotide sequence encoding the same.

In addition, an object of the present invention is to provide a polynucleotide sequence including a signal sequence in a polynucleotide sequence encoding the chitinase.

Further, the vector comprising the respective polynucleotide sequence, the transformants transformed with the respective vectors, the respective transformants, the culture thereof, the cell-free culture medium, the concentrate of the culture, the cell-free culture solution It is an object of the present invention to provide a biocatalyst having chitin resolution and chitosan resolution as an active ingredient comprising at least one selected from the group consisting of a concentrated solution and a recombinant chitinase prepared by expression in the transformant.

In order to achieve the above object, the present invention has a chitin hydrolytic ability, not only excellent hydrolytic activity is maintained even at high temperature conditions of 40 ℃ to 60 ℃ and acidic pH conditions of pH 4 to pH 6, as well as a high efficiency expression system Escherichia It provides a chitinase that can be expressed with high efficiency in the coli expression system ( E. coli expression system).

The present invention also provides an amino acid sequence of the chitinase and a polynucleotide sequence encoding the same. The present invention also provides a polynucleotide sequence in which the signal sequence is included in the polynucleotide sequence encoding the chitinase.

In addition, the present invention provides a vector comprising the respective polynucleotide sequence, transformants transformed with each of the vectors, each transformant, its culture, its cell-free culture, the concentrate of the culture, Provided are biocatalysts having chitin resolution and chitosan resolution, each containing at least one selected from the group consisting of concentrated cell-free culture medium and recombinant chitinase prepared by expressing the transformant as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In addition to being abundant in nature, the inventors of the present invention not only have to produce a huge amount every year in nature and provide a treatment method, but also have various functionalities, and especially the hydrolyzate (chitin oligosaccharide or N- acetylglucosamine) Among the enzymes produced by the human body, the hydrolysis method of chitin, a non-insoluble, insoluble natural polymer, which is not hydrolyzed, in particular, is being studied. In addition to maintaining excellent hydrolytic activity even at high temperature conditions of ℃ to 60 ℃ and acidic pH conditions of pH 4 to pH 6, unlike conventional chitin hydrolase Escherichia coli expression system ( E. By establishing conditions that can be expressed at high efficiency in a coli expression system), a chitinase that can be expressed at high efficiency through an Escherichia coli expression system was found and its gene sequence was confirmed, thereby completing the present invention.

In the present invention, chitin is a kind of mucopolysaccharide present in the cell walls of fungi such as fungi and bacteria, such as cyclic animals, prototype animals, arthropods or mollusks, and fungi, and N -acetylglucosamine binds β-1,4. It is a natural polymer polymerized with biopolymer. The chitin is a polymer present in nature after cellulose, which is insoluble in water, weak in reactivity, and more stable than cellulose. As mentioned above, the amount of chitin present on the planet is surprisingly high, and it is reported that even a single group of crustaceans, each species of wood, produces hundreds of billion tons of chitin in one year.

In the present invention, chitinase is a hydrolase that hydrolyzes chitin, and is generally an enzyme that hydrolyzes β-1,4 bonds of N- acetylglucosamine polymers. Lysozyme, an enzyme that hydrolyzes chitin, is distinguished, and it has been reported to hydrolyze chitin, chondroitin and glycol chitin, which are water-soluble derivatives thereof, but not peptideglycans.

In the present invention, the culture refers to a culture in which a transformant transformed with a vector comprising a nucleotide sequence encoding a cell, preferably a microorganism, more preferably a Bacillus sp microorganism-derived chitinase. Means a medium, the culture medium may be one containing cultured microorganisms.

In the present invention, the cell-free culture means removing the cultured microorganisms in a culture medium in which cells or microorganisms are cultured. The culture medium means a solid composition or a liquid composition containing nutrients necessary for culturing microorganisms including animal cells, plant cells or bacteria, and preferably, may be a liquid composition. In the present invention, the concentrate means that the culture or the cell-free culture is concentrated.

The chitinase of the present invention is cloned from Bacillus sp SC081 strain, which is encoded by a total of 1,791 bp of polynucleotides, translated into 596 amino acids, and is about 65.5 kDa. Has the size of. The chitinase has a signal sequence of 99 bp at the front, a glycosyl hydrolase domain, a fibronectin type 3 (FN3), and a chitin / cellulose-binding site (chitin.cellulose-binding). domains)

The polynucleotide encoding the chitinase including the signal sequence is a total of 1,791 bp polynucleotide having the sequence of SEQ ID NO: 3, the chitinase of the present invention is a chitin hydrolase having the amino acid sequence of SEQ ID NO: 2, Preferably a chitin hydrolase encoded by a total of 1,692 bp of polynucleotides having the sequence of SEQ ID NO: 1.

The chitinase has an optimum reaction temperature of 50 ° C., excellent hydrolytic activity at 40 to 60 ° C., and at least about 50% of enzyme activity is maintained based on the pH 8 activity, which is the optimum reaction pH, even at pH 4 or higher. Esherichia It has chitin resolution and chitosan resolution that can be produced with high efficiency in coli expression system.

The chitinase is excellent in chitin resolution and chitosan resolution, which can solve the problems of the conventional acid degradation method, and also maintains the activity of the enzyme even at high temperature and acidic conditions, and has excellent chitin degradation activity. Unlike high efficiency in the Escherichia coli expression system, it is possible to solve the problem of low efficiency of the enzyme digestion method, which has been a problem in the past, and unlike the previously reported chitinase, it can decompose both chitin and chitosan, so it can be used for various purposes. It can be applied to various industrially.

The agarase of the present invention is a chitinase having the amino acid sequence set forth in SEQ ID NO: 2. The chitinase may be one having an amino acid sequence preferably encoded by the polynucleotide set forth in SEQ ID NO: 1.

The invention also relates to a polynucleotide encoding a chitinase having the amino acid sequence set forth in SEQ ID NO: 2. Preferably the polynucleotide is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1.

The present invention also relates to a nucleotide sequence in which a signal sequence is included in the nucleotide sequence encoding the chitinase. The nucleotide sequence may preferably be a polynucleotide set forth in SEQ ID NO: 3.

The present invention also relates to a biocatalyst having chitin resolution and chitosan resolution. The biocatalyst is a vector comprising each polynucleotide sequence, specifically, a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: 3, a transformant transformed with the respective vector, each transformant, Chitin comprising at least one selected from the group consisting of a culture thereof, a cell-free culture medium, a concentrate of the culture, a concentrate of the cell-free culture medium and a recombinant chitinase prepared by expressing each transformant as an active ingredient Biocatalysts having resolution and chitosan resolution.

The chitinase may be preferably composed of the amino acid sequence of SEQ ID NO: 2, more preferably may have an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 1, to maintain the activity of the enzyme As long as it is a method known to those skilled in the art to which the present invention belongs may be modified by deletion, substitution or insertion of amino acids.

The polynucleotide encoding the chitinase according to the present invention may have a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, preferably may have a polynucleotide sequence described in SEQ ID NO: 1, As long as the activity of the enzyme is maintained, the nucleotide may be modified by deletion, substitution or insertion by a method known to those skilled in the art.

The polynucleotide having a signal sequence in the polynucleotide sequence encoding the chitinase according to the present invention may have a nucleotide sequence as set forth in SEQ ID NO: 3, by the activity of the enzyme and the signal sequence site to the polynucleotide sequence As long as the kinase having the amino acid sequence encoded by the protein is retained to be released into the cell, the nucleotide may be modified by deletion, substitution or insertion by a method known to those skilled in the art. have.

In addition, the present invention, in order to solve the problem of increased cost and activity loss by the separation and purification process for the development of enzyme biocatalyst, the polynucleotide encoding the chitinase or polynucleotide sequence encoding the chitinase An expression vector comprising a polynucleotide containing a signal sequence, a transformant transformed with the expression vector, and a recombinant chitinase using the transformant and a method for producing the same. Preparation of the vector and preparation of the transformant are prepared according to conventional genetic engineering methods.

The expression vector may be appropriately selected according to the host to be used, and all of the usual expression vectors capable of expressing the protein in the transformant may be applicable. For example, the protein expressed by the transformant may be purified. It may be an expression vector comprising a polynucleotide encoding a fusion partner (fusion-tag). Specifically, in the case of Escherichia coli, pBADNH, pET 11a, pET 16b, pET 21a (Novagen Co., Germany), pGEX 4T-1 (GE) capable of expressing the introduced foreign genes in a highly soluble form in a transformant. Healthcare, USA) or pHCE 19 (BioLeaders Co., South Korea), preferably pBADNH, pET 11a or pET 16b.

The transformant was prepared using the expression vector, and the host used for the production of the transformant includes prokaryotic cells such as E. coli or Bacillus sp., Or eukaryotic cells such as yeast and fungus. One is not limited thereto.

Preferably, the expression vector is a polynucleotide having a gene sequence encoding a chitinase of SEQ ID NO: 2 in a pBADNH vector, more preferably a polynucleotide having a sequence of SEQ ID NO: 1 or SEQ ID NO in a pBADNH vector A polynucleotide having a sequence of 3 is inserted, and the host is preferably E. coli , for example, E. coli MC1061 or E. coli DE5α.

In the method for producing chitinase using the transformant, the transformant is cultured using a culture medium, and the chitinase contained in the culture medium or the chitinase present in the transformant is obtained. It may be to include the step.

The culture medium, culture conditions and culture methods may be appropriately selected depending on the type of transformant, for example, a polynucleotide having a gene sequence encoding the chitinase of SEQ ID NO: 2 or the sequence of SEQ ID NO: 3 Inserting the polynucleotide having pBADNH into a recombinant vector; The prepared recombinant vector may be introduced into E. coli MC1061 to prepare a transformant and culturing the transformant in a culture medium.

Specifically, the present invention comprises the steps of preparing a recombinant vector by inserting a polynucleotide having a gene sequence encoding the chitinase of SEQ ID NO: 2 or a polynucleotide having a sequence of SEQ ID NO: 3 into pBADNH; The recombinant vector may be introduced into E. coli MC1061 to produce a transformant, and the method may further comprise a method for producing a recombinant chitinase comprising culturing the transformant in a culture medium.

The recombinant chitinase has motility, chitin degrading activity, and it has been confirmed that the soluble chitinase can be produced in high yield by the synthetic chitinase method.

In relation to the step of culturing the transformant in the culture medium, the culture medium contains arabinose in an amount of 0.175% (w / v) to 0.215% (w / v) (arabinose weight / culture volume) Medium may be, the culture conditions may be 26 ℃ to 30 ℃, the incubation time may be 3 hours 30 minutes to 4 hours 30 minutes.

Recombinant chitinase prepared by the method expressed by the transformant may be purified by a conventionally known method, but may be used in the form of whole cells or cultures thereof or cell-free cultures thereof without purification.

In addition, the present invention relates to a recombinant chitinase prepared by expressing in a transformant transformed with the expression vector.

The expression vector may be a recombinant vector in which a polynucleotide having a sequence of SEQ ID NO: 3 is inserted into a pBADNH vector, a pET 11a vector, or a pET 16b vector. The transformant is a recombinant vector in which the expression vector is transformed into a host cell, preferably E. coli transformed with the expression vector, and more preferably a polynucleotide having a sequence of SEQ ID NO: 3 is inserted into a pBADNH vector. May be E. coli MC1061 transformed.

In addition, the present invention is chitin degradation activity or chitosan degradation comprising at least one selected from the group consisting of the transformant, the culture of the transformant, its cell-free culture, the concentrate of the culture and the concentrate of the cell-free culture. It relates to a biocatalyst having activity.

The present invention also provides a composition for decomposing chitin and / or a composition for decomposing chitosan, containing at least one selected from the group consisting of the chitinase, the recombinant chitinase and the biocatalyst. Since the decomposition composition can decompose both chitin and chitosan, it can be used to decompose chitin or chitosan independently, or to decompose both chitin and chitosan.

As mentioned above, the novel chitinase of the present invention has an optimum activity at 50 ° C. at high temperatures, in particular, the hydrolytic reactivity of chitin is improved, and excellent hydrolytic activity is maintained at 40 ° C. to 60 ° C., and acidic pH In particular, in addition to maintaining excellent hydrolytic activity even at pH 4 to pH 6, Escherichia is a high efficiency expression system coli expression system ( E. In the coli expression system), the transformant and the biocatalyst using the vector containing the novel chitinase of the present invention and the polynucleotide encoding the chitinase can be expressed as chitin oligosaccharide or N- acetylglucosamine. Since it can be used in various fields such as pharmaceuticals, functional foods or cosmetics used, its industrial value will be very large.

Figure 1 is a diagram showing the nucleotide sequence of the chitinase (scchi-1) according to an embodiment of the present invention, in the base sequence underline the amino acid sequence encoded by the base sequence (codon), point ( Dotted areas indicate the signal sequence, stop codons are indicated by an asterisk, and squared areas indicate the glycosyl hydrolase domain of the chitinase. The indicated portion refers to fibronectin type 3 (FN3), and the portion indicated in bold denotes chitin / cellulose-binding domains.
Figure 2 is transformed cells according to an embodiment of the present invention ( E. coli MC1061) was used to measure the production of recombinant protein (H ₆ SCChi-1) according to the content of arabinose, the number on the left means the protein size (kDa), the number on the top means the content of arabinose. .
Figure 3 is transformed cells according to an embodiment of the present invention ( E. coli MC1061) using a temperature and time to measure the production of recombinant protein (H ₆ SCChi-1), the number on the left means the protein size (kDa), the M on the top (marker) , The underlined number indicates the reaction temperature, and the underlined number indicates the reaction time.
Figure 4 is transformed cells according to an embodiment of the present invention ( E. coli MC1061) using a recombinant protein (H ₆ SCChi-1) produced on the SDS-PAGE showing the results of purification, the lane M is a protein marker (size marker), the Lane 1 and Lane 2 is After SDS-PAGE, stained with Coomassie Blue dye, and Lane 3 and Lane 4 is the result of SDS-PAGE using SDS-PAGE gel containing glycol chitin, Lane 5 is Ni ^{2 +} affinity After the SDS-PAGE of the purified recombinant protein using chromatography, the result of staining with Coomassie blue dye, Lane 1 and Lane 3 is a cell lysate that does not induce expression with arabinose, Lane 2 and Lane 4 refer to cell lysates of transformants incubated for 4 hours at 28 ° C. after inducing expression using arabinose.
Figure 5 is a graph showing the result of measuring the optimum reaction temperature of the purified recombinant chitinase (H ₆ SCChi-1) according to an embodiment of the present invention as a comparison value based on the optimum reaction temperature (50 ℃).
Figure 6 is a graph showing the result of measuring the optimum reaction pH of the purified recombinant chitinase (H ₆ SCChi-1) according to an embodiment of the present invention as a comparison value based on the optimum reaction pH (pH 8).
Figure 7 is a photograph showing the results of measuring the hydrolysis activity of each substrate of the chitinase of the present invention according to an embodiment of the present invention, 1 is a cell lysate of the transformant induced enzyme production with arabinose 2 represents a cell lysate of a transformant that did not induce enzyme production with arabinose, 3 means an extracellular protein (cell culture medium) of B. atrophaeus SC081, and A represents a substrate of 0.1% of the colloidal chitin. , B means 0.1% glycol chichi (glycol-chitin) is the substrate, C means 0.1% of the soluble chitosan (soluble chitosan) is the substrate, D is 0.1% of Glycol-chitosan means that the substrate.
8 is a graph showing the results of measuring the hydrolysis activity of each substrate of the chitinase of the present invention according to an embodiment of the present invention, SCChi-1 is a cell lysate of the transformant induced enzyme production with arabinose SC081 refers to extracellular protein (cell culture medium) of B. atrophaeus SC081, the value on the left means the relative enzyme activity measured on the basis of optimal enzyme activity, and the value below is the reaction time (min) Means.

Hereinafter, the preparation examples and examples are presented in order to explain the present invention in more detail. However, the following Preparation Examples and Examples are merely illustrated to aid the understanding of the present invention, and may be modified in various other forms, and the scope of the present invention is not limited to the following Examples.

Example  1: Isolation of Microorganisms with Chitin Resolution

1-1. sample

Reagents and chemicals used in the experiment were all purchased from Sigma (Sigma Chemical Co., USA) and Difco laboratories (USA). In addition, glycol chitosan for the production of L-Arabinose (L (+)-arabinose) and glycol chitin (Glycol chitin) that induced enzyme expression in the transformants was purchased from Sigma, and water-soluble chitosan (soluble chitosan, pH 5.0) was prepared from chitosan powder.

The plasmid containing the expression vector used in the experiment is as follows. The pGEM-T vector used as a cloning vector was purchased from Promega Co., USA, and the vector used as an expression vector of fusion protein was inserted into a gene. The pBADNH vector (pBADNH vector) having 6 X His (hexahistidine tag) as a fusion partner at the amino-terminus of was used. Ni-NTA matrices (Qiagen, Canada) and imidazole (Sigma, USA) were used for the purification of 6 X His-tagged hydrolase produced by the pBADNH vector with the fusion partner.

The host used for the production and expression of the recombinant vector is E. coli DH5α and E. coli MC1061 were used. Transformant transformed host with the recombinant vector was isolated by incubating in LB agar plate (AMP agar plate) containing ampicillin (50 ㎍ / ml).

1-2. Isolation of Microorganisms with Chitin Resolution

In order to clone the new chitinase of the present invention, samples were collected around Korean traditional soy sauce, and 10 g of trypton, 10 g of NaCl, and yeast extract in 1 L of distilled water. Strains with chitinolytic activity were isolated from the samples using a medium in which 1% of colloidal chitin was added to LB agar medium containing 5 g and 15 g of agar. By separating the strains which form the greatest degradation ring (clear zone) to the medium in the strain was identified, the strain has been identified as a search result, the strain B. atrophaeus using 16S rRNA as gram positive (gram-positive) B It was named atrophaeus SC081.

< Example  2> Chitinase  Gene sequencing

In order to analyze the nucleotide sequence of the B. atrophaeus SC081 strain-derived chitinase, the primers of Table 1 were prepared, and PCR (polymer chain reaction) was performed using the primers to amplify the chitinase nucleotide sequence. It was.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: F ATGAAAAAAGTGTTTTCAA 4 R TTATTTGCAATCACCAAT 5

In more detail, the gene sequence encoding the amino acid of the previously reported chitinase was examined to prepare a primer capable of amplifying the sequence as shown in Table 1 above.

Template DNA (template DNA) used in the PCR was extracted from the B. atrophaeus SC081 strain genomic DNA using G-spin ^TM for Bacteria kit (iNtRON biotechnology, South Korea). The PCR reaction was denatured at 95 ° C. for 5 minutes, then repeated 30 times for 40 seconds at 40 ° C. and 56 seconds at 56 ° C. and finally at 72 ° C. for 2 minutes.

The PCR product was purified and cloned into pGEM easy T vector, and the cloned vector was transformed into E. coli DH5α. After culturing the transformed E. coli DH5α, the cloned vector was collected using a QIAprep spin miniprep kit (Qiagen, USA).

The collected cloning vector was commissioned by Macrogen (Korea) and analyzed for sequencing using an ABI 377 DNA sequencer. In addition, the ORF (open reading frame) of the B. atrophaeus SC081 strain-derived chitinase was analyzed using the BLAST (N) program of the (ORF) NCBI, and the amino acid sequence analysis was performed using Swiss-Prot databases of GenBank. The signal sequence was analyzed using Signal P3.0 Server (http://www.cbs.dtu.dk/services/SignalP/), and the specific domain was BLAST program (http: //blast.ncbi). .nlm.nih.gov / Blast.cgi).

The analysis results including the sequence analysis results are shown in FIG. 1.

As shown in FIG. 1, the analysis of the chitinase derived from the B. atrophaeus SC081 strain showed that 1,692 bp (SEQ ID NO: 1), which theoretically encodes 563 amino acids (SEQ ID NO: 2) having a size of 65.5 kDa Naase partial nucleotide sequences have been identified. In addition, the polynucleotide encoding the chitinase was identified as a polynucleotide (SEQ ID NO: 3) to which a signal sequence having 99 base sequences at the N-terminus was added, which is shown together in FIG. 1. The chitinase from which the sequence was identified was named scchi-1.

A 99 bp signal sequence is present in the N-terminal of the scchi-1 nucleotide sequence, between the 33rd alanine (Ala-33) and the 34th serine (Ser-34). It was expected to be cut. Since the sequence includes an Ala-X-Ala sequence in which one amino acid is present between alanine and alanine, it is assumed that the sequence is a signal sequence related to secretion of proteins. Therefore, it was expected that the above-described chitinase of the present invention, that is, scchi-1, could be released extracellularly.

In addition, as shown in FIG. 1, the scchi-1 is a glycosyl hydrolase domain located at the 448 th proline (Pro-448) residue from the 34 th serine (Ser-34) residue, and the 458 th serine. From fibronectin type 3 (FN3) and 549 lysine (Lys-549) residues to the 582 leucine (Leu-582) residues located at the 539 th threonine (Thr-539) residue from the (Ser-458) residue. It was found to have located chitin / cellulose-binding sites (chitin.cellulose-binding domains).

< Example  3> Chitinase  gene Cloning  And In E. coli  Protein expression analysis

Expression of a PCR amplification product having a nucleotide sequence (SEQ ID NO: 3) of the polynucleotide comprising a coding region and a signal sequence of the chichiase scchi-1 derived from the B. atrophaeus SC081 strain amplified in Example 2 PBADNH was cloned using a DNA binding enzyme (T4 DNA ligase, Takara, South Korea), and then transformed into E. coli MC1061, an expression host.

More specifically, the signal of SEQ ID NO: 3 amplified by using PCR in Example 2 Sac I and Sph I restriction enzymes that can cut the restriction enzyme sites contained in the primers of SEQ ID NO: 4 and SEQ ID NO: 5 After treatment with scchi-1 and pBADNH containing sequences, the restriction enzyme-treated scchi-1 PCR product and pBADNH vector were ligation with T4 DNA ligase (Takara, South Korea). The prepared recombinant vector is E. coli MC1061 was transformed. The transformed E. coli MC1061 was incubated for 16 hours at 37 ℃ using LB medium (LB medium) containing ampicillin, the transformant is 37 ℃ to OD ₆₀₀ = 0.5 (mid-log phase) Shaking culture was performed.

After the incubation, arabinose was added at concentrations of 0.1% (w / v), 0.15% (w / v) and 0.2% (w / v), respectively, for 2 hours at 28 ° C and 37 ° C. Incubated for 4 and 6 hours. After the incubation, the cells were obtained by centrifugation for 5 minutes under conditions of 15,000 x g. The cells were added with lysis buffer (pH 7.5) containing 25 mM Tris-Cl and 1 mM EDTA and sonicated, followed by centrifugation for 25 minutes at 15,000 xg to obtain precipitated insoluble material. Removed, only the supernatant was separated to obtain a cell lysate to prepare a crude enzyme solution. The protein content of the obtained cell lysate was stained after electrophoresis, and measured using bovine serum albumin as a standard material using a Bio-Rad protein assay kit (Bio-Rad, USA). The measurement results are shown in FIGS. 2 and 3.

As shown in FIG. 2 and FIG. 3, the optimum production condition for the enzyme was found to add arabinose at a concentration of 0.2% (w / v). In addition, the optimum reaction conditions related to the reaction conditions, it was confirmed that incubation for 4 hours at 28 ℃.

SDS-PAGE and zymogram analysis were performed to analyze H ₆ SCChi-1, a 6 His-tag-coupled recombinant enzyme, using the pBADNH vector.

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 10% SDS-polyacrylamide gel and a Multiphor II system (Amersham Pharmacia Biotech, USA). After the SDS-PAGE, the gel was stained using 0.05% Coomassie brilliant blue R-250. The zymogram analysis was performed by adding the coenzyme solution to SDS-PAGE sample buffer, heating at 80 ° C. for 5 minutes, separating using a 10% polyacrylamide gel containing 0.1% glycol chitin, and then 50mM Tris. The reaction was overnight at 37 ° C. by immersion in refolding buffer containing -HCl (pH 7.5) and 2.5% Triton X-100. After the reaction, the gel was washed with distilled water and stained using Coomassie brilliant blue R-250. After 2 hours, the dye was removed by washing with distilled water containing methanol and acetic acid, and then the lytic zone was confirmed. The confirmed result is shown in FIG.

As shown in FIG. 4, the recombinant protein was about 62.5 kDa, and was measured using a standard calilbration protein to have motility and chitin resolution. As a result of the experiment, it was confirmed that the expression level is significantly increased when the expression is induced as compared with the case where the expression of the recombinant protein is not induced. (Lane 1 and Lane 2)

In addition, chitinase, which was expected to be secreted extracellularly from Bacillus SC081 by the method for producing a recombinant protein of the present invention, is generally produced in an insoluble colony when producing a recombinant protein using E. coli . As a result, it was confirmed that it is secreted in the host E. coli , and this result was evaluated to be able to produce recombinant chitinase, a recombinant protein with high yield.

< Example  4> Recombination Chitinase  Check for optimum conditions and decomposition patterns

In order to confirm the optimum reaction conditions of the chitinase of the present invention, the chitinase activity prepared by the method of Example 3 was measured while varying the conditions of temperature, pH, and metal ion addition.

4-1 Purification of Recombinant Enzymes

E. coli transformed with a recombinant pBADNH vector containing scchi-1 comprising the signal sequence of SEQ ID NO: 3 as in Example 3 After MC1061 was shaken at 37 ° C. until OD ₆₀₀ = 0.5 (mid-log phase), arabinose was added at a concentration of 0.2% (w / v) and incubated at 28 ° C. for 4 hours. After the incubation, the cells were obtained by centrifugation for 5 minutes at 10,000 xg. Protein extraction lysis buffer (pH 7.5) containing 50 mM NaH ₂ PO ₄ , 300 mM NaCl and 20 mM imidazole was added to the cells, sonicated, and centrifuged for 25 minutes at 15,000 xg. SDS-PAGE was performed by removing the insoluble material and separating only the supernatant. Purification of the recombinant chitinase was performed by affinity chromatography using a nickel column (Ni-NTA) and separated using 250 mM imidazole, followed by 25 mM Tris-Cl (pH 7.5) and 1 Dialysis was performed using a protein buffer containing mM EDTA. The result of purifying the recombinant protein to which the 6his-tag is linked is shown in FIG. 4 (Lane 5). As a result of SDS-PAGE of the purified protein shown in FIG. 4, the purity of the protein was about 99%.

4-2 Optimal Temperature Condition Measurement

Chitin degrading activity was measured using colloidal chitin as a substrate.

More specifically, the purified recombinant enzyme was added together to 25 mM Tris-Cl (pH 7.5) containing 1.0% of the colloidal chitin, followed by 20 ° C, 30 ° C, 37 ° C, 40 ° C, 50 ° C and 60 ° C. After the reaction was carried out for 30 minutes under the temperature condition of the temperature fluctuated by the furnace and pH 7.5 of pH 7.5, the reaction solution was boiled for 5 minutes to terminate the reaction. After cooling, the reaction was centrifuged and the reducing sugars contained in the supernatant were measured by the modified dinitrosalicylic acid method. The activity of the enzyme was determined using the result of performing the same method using D-glucose as a reference curve. The activity of the enzyme measured was expressed as a value compared to 100% of the optimum activity, and the results are shown in FIG. 5.

As shown in FIG. 5, the optimum temperature condition was found to be 50 ° C. In addition, in relation to the reaction activity after maintaining for 30 minutes at the temperature condition, it was confirmed that at the temperature of 30 ° C to 60 ° C, the activity at 50 ° C which is the optimum temperature condition, that is, about 60% or more of the initial activity was maintained. .

4-3 optimal pH  Condition measurement

Chitin degrading activity was measured using colloidal chitin as a substrate. The measurement was carried out by reacting the reaction conditions at 37 ℃ temperature conditions and pH conditions in which the pH was changed in the range of pH 2.0 to pH 10.0, and then measuring the enzyme activity by the method of Example 4-2, The results are shown in FIG.

As shown in FIG. 6, the optimum pH condition was found to be pH 8, and the activity at pH 8, that is, the optimal pH condition, that is, about 60% or more of the initial activity was maintained at the conditions of pH 5.5 to pH 10. It became. In particular, at pH 4 or higher, the reaction activity of about 50% or more was maintained even after 30 minutes of treatment, and it was confirmed that the reaction was effective under acidic conditions.

According to the experimental results, it was confirmed that the chitinase of the present invention stably reacts at various temperature conditions and pH conditions and maintains reactivity. In addition, the optimum reaction conditions can be effectively reacted at a temperature of 50 ℃ and pH 8, a temperature of 40 ℃ to 60 ℃ and a pH of pH 4 to pH 10, can be used under various reaction conditions, decomposition of chitin It can be used in high temperature and acidic conditions with high activity.

4-4 Substrate Characterization 1

Degradation activity of the chitinase of the present invention was measured using the agar diffusion method using a variety of substances as a substrate.

More specifically, 5 cm thick with 0.1% colloidal chitin, 0.1% glycol-chitin, 0.1% soluble chitosan (pH 5.0) and 0.1% glycol chitosan (glycol-chitosan) Enzyme production was carried out with 20 μg of the cell lysate enzyme of the transformant which induced enzyme production with arabinose before purification in Example 4-1 on 1% agar plate of 20 μg of the cell lysate of the uninduced transformant and the extracellular protein of B. atrophaeus SC081 of Example 1, ie, the culture solution, were spotted. The medium was incubated at 37 ° C. for 1 or 2 hours and then stained with 0.01% Calcofluor white M2R dissolved in 25 mM Tris-Cl (pH 7.5). After the dyeing, the dyeing solution was removed and the plate was washed with distilled water. The clearing zone was confirmed using a UV-transilluminator, and the results are shown in FIG. 7. The measured activity is obtained by measuring the result of the same method using D-glucose as a relative value by measuring the reducing sugar by the modified dinitrosalicylic acid method (modified dinitrosalicylic acid method). Indicated.

As shown in FIG. 7, the cell lysates of the transformants inducing enzyme production with arabinose showed the highest degradation activity against the colloidal chitin, and the superior degradation activity against the colloidal chitin and water-soluble chitosan. (FIG. 7A and FIG. 7C). As confirmed in FIG. 7C, it was confirmed that the hydrolyzability of glycol chitinic acid in an acidic condition was excellent. On the other hand, the cell lysates of transformants that did not induce enzyme production with arabinose and the extracellular protein of B. atrophaeus SC081, that is, the hydrolysis capacity were not confirmed. From the above results, it was confirmed that when inducing enzyme production with arabinose, in terms of the amount of enzyme production, that is, the amount of enzyme per unit volume and the activity of the enzyme.

4-5 Substrate Characterization 2

In order to further confirm the results of Examples 4-4, Example 4 in 25 mM Tris-Cl (pH 7.5) containing 0.1% colloidal chitin and 0.1% soluble chitosan (pH 5.0) 20 μg of the cell lysate enzyme of the transformant which induced enzyme production with arabinose prior to purification at −1 and the extracellular protein of B. atrophaeus SC081 of Example 1, respectively, were added and the reducing sugar was measured by dinitrosalicylic acid ( The hydrolysis activity was measured by the method of Example 4-2 measured by the modified dinitrosalicylic acid method), and the result of performing the same method using D-glucose was obtained by using a calibration curve. The results are shown in FIG. 8.

As shown in FIG. 8, as the time was increased, the degradability was also increased, and in the case of the cell lysate of the transformant which induced enzyme production with arabinose, it was confirmed that the hydrolysis activity was remarkably excellent.

More specifically, the cell lysates of the transformants induced enzyme production with the arabinose, all of the wild type chitinase of B. atrophaeus SC081 derived from the recombinant enzyme of the present invention, that is, all strain cultures It was found to have remarkably good activity against the type of substrate. In particular, it was confirmed that hydrolysis activity was superior to colloidal chitin than chitosan, that is, water-soluble chitosan.

In general, it is reported that the chitinase has a hydrolytic activity against chitin, but the recombinant enzyme of the present invention has a hydrolytic activity not only for chitin but also for chitosan, and thus, the range of the substrate is common. It is excellent in being wider than the first.

<110> Cheju National University Industry-Academic Cooperation Foundation <120> A CHITINASE AND A USE OF THE SAME <130> KP20100043 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1692 <212> DNA <213> sschi-1 <220> <221> CDS (222) (1) .. (1689) <400> 1 agt tct gac aaa agt tat aaa atc atc ggc tac tat ccg tcc tgg ggc 48 Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly   1 5 10 15 gct tat gga agg gat ttt caa gta tgg gat atg gat gct tct aaa gtc 96 Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val              20 25 30 agc cat att aat tat gca ttt gct gat att tgc tgg gag ggg agg cat 144 Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Glu Gly Arg His          35 40 45 gga aac cct gat ccg aca ggc ccg aat ccc cag aca tgg tct tgc cag 192 Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Thr Trp Ser Cys Gln      50 55 60 gat gaa aac gga gtg att gat gtt cca aat gga tca atc gtt atg ggg 240 Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly  65 70 75 80 gac cca tgg atc gat gtg caa aag tca aat gcc gga gat act tgg gat 288 Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp                  85 90 95 gag ccg att cgg ggc aac ttt aaa caa cta ttg aag ctg aaa aag aac 336 Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn             100 105 110 cat cct cat ttg aaa aca ttc ata tca gtt ggc gga tgg agt tgg tca 384 His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser         115 120 125 aat cgc ttt tca gat gtt gcg gca gat cct gcg gca aga gag aat ttt 432 Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe     130 135 140 gct gct tca gca gta gat ttt tta aga aaa tat ggg ttt gat ggg gtc 480 Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val 145 150 155 160 gac ctt gac tgg gaa tac ccg gtt agt gga ggg ctg ccg gga aac agc 528 Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser                 165 170 175 acc cgt ccg gag gat aag aga aac tac acg cta ctc ttg cag gat gtg 576 Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val             180 185 190 cga gaa aag ctt gac gcc gca gaa gcg aag gat ggc aag aaa tac ttg 624 Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu         195 200 205 ctg acg atc gcc tcc ggc gcc agt cct gaa tat gta agc aac act gaa 672 Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu     210 215 220 tta gat aag att gct gaa acc gtt gat tgg att aac att atg acc tat 720 Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr 225 230 235 240 gac ttt aat ggc gga tgg caa agt ata agc gca cat aat gcc cca tta 768 Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu                 245 250 255 ttc tat gat cca aaa gcg aaa gaa gcc ggc gtt cca aac gct gag aca 816 Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr             260 265 270 ttt aac att gaa agc acc gtg aag cgc tac aag gaa gcc ggt gtc aaa 864 Phe Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys         275 280 285 gcg gac aaa tta gtg ctt ggc aca ccg ttc tat ggc aga ggc tgg agc 912 Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser     290 295 300 aat tgt gag cct gca gac aac gga gaa tat caa aaa tgc cga ccg gct 960 Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Arg Pro Ala 305 310 315 320 aaa gaa ggg acg tgg gaa aag ggg gta ttt gat ttt tca gat ctt gaa 1008 Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu                 325 330 335 aga act aca tca ata aaa acg gat ata aaa gat att gga atg atc gag 1056 Arg Thr Thr Ser Ile Lys Thr Asp Ile Lys Asp Ile Gly Met Ile Glu             340 345 350 caa aag tgc cgt ttt tgt ata atg cgg aga atg gaa act tca tta cct 1104 Gln Lys Cys Arg Phe Cys Ile Met Arg Arg Met Glu Thr Ser Leu Pro         355 360 365 acg atg atg aag aat cat atg gat aca aaa ccg att tta att aaa tca 1152 Thr Met Met Lys Asn His Met Asp Thr Lys Pro Ile Leu Ile Lys Ser     370 375 380 aac gga tta agc ggg gct atg ttt tgg gat ttc agc ggt gat tca aat 1200 Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn 385 390 395 400 cag act ttg ctc aac aaa tta gca gcc gat tta ggg ttc gct ccg ggt 1248 Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly                 405 410 415 ggg ggt aat cca gag ccg cct tca tct gca ccg ggt aat ttg cat gtg 1296 Gly Gly Asn Pro Glu Pro Pro Ser Ser Ala Pro Gly Asn Leu His Val             420 425 430 acc gaa aaa acc gca acc agt atc agt ctg gca tgg gat gca ccg agc 1344 Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Ala Trp Asp Ala Pro Ser         435 440 445 gac gga gca aac atc gca gag tat gtg ctg tca tat gaa ggc ggg gcc 1392 Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala     450 455 460 gta tcg gtc aag gat aca tcg gcg aca atc ggg caa tta aag ccg aat 1440 Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn 465 470 475 480 acg aca tac tca ttt aca gta tcg gca aag gat gcg gac gga aag ctc 1488 Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu                 485 490 495 cat acc ggg cca acg ata gaa gca gca aca aac tct gat caa aca tgt 1536 His Thr Gly Pro Thr Ile Glu Ala Ala Thr Asn Ser Asp Gln Thr Cys             500 505 510 ggg tat aac gaa tgg aaa gat aca gcc gtc tac aca ggc gga gac cga 1584 Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg         515 520 525 gtc gtc ttt aac ggc aaa gtg tat gaa gcg aaa tgg tgg acg aaa gga 1632 Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly     530 535 540 gag cag ccg gat cag gct ggt gag tcg ggt gta tgg aaa tta att ggt 1680 Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly 545 550 555 560 gat tgc aaa t aa 1692 Asp Cys Lys <210> 2 <211> 563 <212> PRT <213> sschi-1 <400> 2 Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly   1 5 10 15 Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val              20 25 30 Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Glu Gly Arg His          35 40 45 Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Thr Trp Ser Cys Gln      50 55 60 Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly  65 70 75 80 Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp                  85 90 95 Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn             100 105 110 His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser         115 120 125 Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe     130 135 140 Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val 145 150 155 160 Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser                 165 170 175 Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val             180 185 190 Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu         195 200 205 Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu     210 215 220 Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr 225 230 235 240 Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu                 245 250 255 Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr             260 265 270 Phe Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys         275 280 285 Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser     290 295 300 Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Arg Pro Ala 305 310 315 320 Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu                 325 330 335 Arg Thr Thr Ser Ile Lys Thr Asp Ile Lys Asp Ile Gly Met Ile Glu             340 345 350 Gln Lys Cys Arg Phe Cys Ile Met Arg Arg Met Glu Thr Ser Leu Pro         355 360 365 Thr Met Met Lys Asn His Met Asp Thr Lys Pro Ile Leu Ile Lys Ser     370 375 380 Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn 385 390 395 400 Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly                 405 410 415 Gly Gly Asn Pro Glu Pro Pro Ser Ser Ala Pro Gly Asn Leu His Val             420 425 430 Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Ala Trp Asp Ala Pro Ser         435 440 445 Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala     450 455 460 Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn 465 470 475 480 Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu                 485 490 495 His Thr Gly Pro Thr Ile Glu Ala Ala Thr Asn Ser Asp Gln Thr Cys             500 505 510 Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg         515 520 525 Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly     530 535 540 Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly 545 550 555 560 Asp Cys Lys             <210> 3 <211> 1791 <212> DNA <213> scchi-1 + signalpeptide <400> 3 atgaaaaaag tgttttcaaa caaaaagttt ctcgtttttt ctttcatttt tgcgatgatt 60 ttaagtctgt ctttttttaa tggggaaagc gcaaaagcca gttctgacaa aagttataaa 120 atcatcggct actatccgtc ctggggcgct tatggaaggg attttcaagt atgggatatg 180 gatgcttcta aagtcagcca tattaattat gcatttgctg atatttgctg ggaggggagg 240 catggaaacc ctgatccgac aggcccgaat ccccagacat ggtcttgcca ggatgaaaac 300 ggagtgattg atgttccaaa tggatcaatc gttatggggg acccatggat cgatgtgcaa 360 aagtcaaatg ccggagatac ttgggatgag ccgattcggg gcaactttaa acaactattg 420 aagctgaaaa agaaccatcc tcatttgaaa acattcatat cagttggcgg atggagttgg 480 tcaaatcgct tttcagatgt tgcggcagat cctgcggcaa gagagaattt tgctgcttca 540 gcagtagatt ttttaagaaa atatgggttt gatggggtcg accttgactg ggaatacccg 600 gttagtggag ggctgccggg aaacagcacc cgtccggagg ataagagaaa ctacacgcta 660 ctcttgcagg atgtgcgaga aaagcttgac gccgcagaag cgaaggatgg caagaaatac 720 ttgctgacga tcgcctccgg cgccagtcct gaatatgtaa gcaacactga attagataag 780 attgctgaaa ccgttgattg gattaacatt atgacctatg actttaatgg cggatggcaa 840 agtataagcg cacataatgc cccattattc tatgatccaa aagcgaaaga agccggcgtt 900 ccaaacgctg agacatttaa cattgaaagc accgtgaagc gctacaagga agccggtgtc 960 aaagcggaca aattagtgct tggcacaccg ttctatggca gaggctggag caattgtgag 1020 cctgcagaca acggagaata tcaaaaatgc cgaccggcta aagaagggac gtgggaaaag 1080 ggggtatttg atttttcaga tcttgaaaga actacatcaa taaaaacgga tataaaagat 1140 attggaatga tcgagcaaaa gtgccgtttt tgtataatgc ggagaatgga aacttcatta 1200 cctacgatga tgaagaatca tatggataca aaaccgattt taattaaatc aaacggatta 1260 agcggggcta tgttttggga tttcagcggt gattcaaatc agactttgct caacaaatta 1320 gcagccgatt tagggttcgc tccgggtggg ggtaatccag agccgccttc atctgcaccg 1380 ggtaatttgc atgtgaccga aaaaaccgca accagtatca gtctggcatg ggatgcaccg 1440 agcgacggag caaacatcgc agagtatgtg ctgtcatatg aaggcggggc cgtatcggtc 1500 aaggatacat cggcgacaat cgggcaatta aagccgaata cgacatactc atttacagta 1560 tcggcaaagg atgcggacgg aaagctccat accgggccaa cgatagaagc agcaacaaac 1620 tctgatcaaa catgtgggta taacgaatgg aaagatacag ccgtctacac aggcggagac 1680 cgagtcgtct ttaacggcaa agtgtatgaa gcgaaatggt ggacgaaagg agagcagccg 1740 gatcaggctg gtgagtcggg tgtatggaaa ttaattggtg attgcaaata a 1791 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F-primer <400> 4 atgaaaaaag tgttttcaa 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R-primer <400> 5 ttatttgcaa tcaccaat 18

Claims (8)

A chitinase having the amino acid sequence set forth in SEQ ID NO: 2. The method of claim 1,
Wherein said chitinase has an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 1. A polynucleotide encoding a chitinase having the amino acid sequence set forth in SEQ ID NO: 2 The method of claim 3,
The polynucleotide has a nucleotide sequence of SEQ ID NO: 1. A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 3. A vector comprising the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 3. A transformant transformed with the vector of claim 6. At least one selected from the group consisting of the transformant of claim 7, the culture thereof, the cell-free culture medium thereof, the concentrate of the culture medium, the concentrate of the cell-free culture medium, and the recombinant chitinase produced by expression of the transformant of claim 7. Biocatalyst having chitin resolution and chitosan resolution comprising as an active ingredient.

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