KR20020075142A - Novel cellulomonas sp. gm13 strain producing chitinase - Google Patents

Abstract

PURPOSE: Provided is novel Cellulomonas sp. GM13 strain producing chitinase which has excellent chitin decomposability and is thus effectively used in manufacturing disaccharide of N-acetyl-beta-D-glucosamine(NAG), as well as oligosaccharide. CONSTITUTION: The novel Cellulomonas sp. GM13 strain(KCTC 18073P) is isolated from soil. It cultured in a medium containing chitin to produce endochitinase and exochitinase which are used for the manufacture of chitin oligosaccharide having high bioavailability. Wherein the endochitinase has molecular weight of 63kDa, optimum pH of 7.0, optimum temperature of 60 deg.C and amino acid sequence at N-end of alanine-glycine-threonine-phenylalanine-threonine-alanine-threonine-phenylalanine.

Description

A novel strain of cellulomonas GM13 that produces chitinase {Novel Cellulomonas sp. GM13 strain producing chitinase}

The present invention relates to a novel cellulomonas GM13 strain that produces chitinase, and more specifically, chitinase, which is produced by the strain, and chitin oligosaccharide and N-acetyl-β-D- using the same. It relates to a method for producing glucosamine (NAG).

Chitin, the next most abundant biomass on the earth, is a polysaccharide linked by β-1 and 4 bonds based on N-acetyl-β-D-glucosamine (NAG), and has a molecular weight of about 1 × 10 ⁶ . to be. Chitin is a major component of the shells of sea crustaceans such as crabs, krill, lobsters, and insects such as spiders, ants, and grasshoppers.

Such chitin is an important natural resource, its application is gradually increasing. In particular, in recent years, chitin oligosaccharides, which are degradation products of chitin, have been found to have antimicrobial activity, anticancer activity, immunopotentiation effect, plant resistance to pathogenic fungi, and lactic acid bacteria growth promoting action. Not only is it effective, it has also been shown to be effective in promoting anti-inflammatory, heart protection, hepatoprotection and metabolism.

These chitin oligosaccharides and NAG are produced from chitin by acid hydrolysis. However, the production of chitin oligosaccharides by acid decomposition has a problem that not only the yield of oligosaccharides is low but also a lot of low-polymerization oligosaccharides including monosaccharides. In addition, it is difficult to use a solvent to remove residual salt after the reaction or to fractionate chitosan decomposition products to obtain a relatively high degree of polymerization of oligosaccharides.A large amount of solvent is used to cause environmental problems. It is difficult to produce high purity oligosaccharides that can be used. On the other hand, in the case of NAG production by the acid decomposition method, chitin is decomposed into monosaccharides during acid decomposition of chitin, and at the same time, deacetylation occurs, resulting in D-glucosamine (DGA) and NAG. When the temperature is lowered, there is a problem that the efficiency of NAG obtained from chitin is lowered. In addition, the conventional NAG manufacturing process uses a high concentration of acid, which requires a complicated purification process for corrosion of equipment, risk of work, and removal of impurities. For this reason, in recent years, enzymatic methods for replacing chitosan oligosaccharide and NAG have been sought.

Chitin oligosaccharide preparation by the enzymatic method uses mild reaction conditions, high yield of oligosaccharides of two or more sugars, and depending on the enzyme used, there is an advantage of making a relatively high polymerization oligosaccharide. In addition, in the case of chitin degradation by enzymatic method, since deacetylation of the final degradation product NAG does not occur, impurities are not generated, and thus, the purification process is very simple and the NAG can be produced with high yield.

In order to completely decompose chitin by enzymatic method, endochitinase which randomly cuts the inside of chitin chain and chitin oligosaccharides produced by this endokinase are sequentially cut at non-reducing ends of chitin monomer. There is a need for an exochitinase that completely hydrolyzes with phosphorus NAG. Chitinase is a generic term for this chitinase. However, to prepare chitin oligosaccharides from chitin by the enzymatic method, endocytase is required. In order to completely decompose chitin to produce NAG, endocytase and exochitinase are required.

However, enzymes capable of effectively decomposing crystalline chitin into oligosaccharides to produce relatively high degree of chitin oligosaccharides directly from chitin have not yet been developed and can efficiently decompose high-polymerization chitin oligosaccharides into chitin monomers, NAG. Exocytinase development is also sluggish.

In particular, the chitinases reported so far have a problem that does not effectively degrade chitin that is difficult to decompose. Therefore, a process for producing chitin oligosaccharides and NAG efficiently by enzymatic method has not been developed yet.

Accordingly, the present inventors have isolated and identified a novel Cellulomonas genus strain in the soil to obtain a high activity chitinase that can be used in the enzymatic process for chitin degradation, and from the culture of the strain By purifying chitinase and examining the reaction characteristics, the present invention was completed by confirming that the enzyme is different from the previously reported chitinases such as excellent chitin resolution and high oligosaccharides.

Accordingly, it is an object of the present invention to provide a novel Cellulomonas genus GM13 strain that produces chitinase.

Another object of the present invention is to provide a chitinase produced by the strain GM13 genus.

Another object of the present invention relates to a method for preparing chitinase using the strain.

Another object of the present invention relates to a method for efficiently preparing chitin oligosaccharides and NAG from chitin by enzymatic digestion using chitinase prepared by the above method.

Another object of the present invention is to provide a reaction mixture prepared by the above method, that is, a chitin oligosaccharide and a NAG mixture.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a diagram showing the 16S rDNA sequence (SEQ ID NO: 1) of the GM13 strain of the genus Cellulomonas.

Figure 2 is a schematic diagram of the strains of the genus Cellulomonas according to the 16S rDNA sequencing analysis (sticks shown below represents a 1% base diversity).

Figure 3 is a graph of the production of chitinase, endokinase and protein of GM13 strain according to the culture time in MC medium.

4 is a diagram showing phenyl-Toyopearl column chromatography for purifying chitinase in GM13 strain culture.

FIG. 5 is a diagram showing the turbidity reduction ability of colloidal chitin of the fractions (Set I, Set II, Set III) separated by phenyl-Toyopearl column chromatography.

Figure 6 shows the coomassie blue staining (g) and glycol chitin active staining (b) by electrophoresis of the protein fractionated through the culture supernatant of the GM13 strain and phenyl-Toyopearl column chromatography.

7 is a diagram showing the optimum temperature (a) and the optimum pH (b) of the purified purified endokinase.

FIG. 8 is a thin layer chromatography (TLC) showing a reaction product generated from colloidal chitin using fractions (Set I, Set II, Set III chitinase) separated through phenyl-Toyopearl column chromatography of the present invention. ) Picture.

The present invention relates to microorganisms having chitin-degrading activity and chitinases isolated therefrom, and more particularly, to ectokinase and exochitinases produced by the novel cellulosic genus G13 strain isolated from soil. It relates to a method for producing high polymerization degree chitin oligosaccharides with high bioactivity.

Separation and purification of chitinases can be performed using protein separation methods by various chromatographic methods using the properties of chitinases that have been known, or by modifying them slightly to suit experimental purposes. Purity assay of the chitinase of the present invention purely separated by chromatography method was demonstrated using electrophoresis. The chitinase molecular weight was determined by moving with standard molecular weight material and comparing the travel distance.

Cellulose genus G13 used to isolate chitinase in the present invention is a novel strain isolated from the soil, the samples collected from the soil suspended in sterile distilled water and then overnight at 4 ℃ and appropriately diluted supernatant MCG plate media (5 g colloidal chitin per liter; 6.7 g yeast extract; 12.8 g Na ₂ HPO ₄ ; 5 g KH ₂ PO ₄ ; NaCl 0.5 g; (NH ₄ ) ₂ SO ₄ 1 g; glucose 10 g) After plating at 30 ° C., the strains were formed relatively rapidly on the MCG plate medium and the strains having a relatively large size of the degraded rings were selected first, and the first isolated strains were selected from the MCG liquid medium. After 5 days of shaking culture, the cells were removed by centrifugation, and the chitinase activity of the culture supernatant was measured. The strain showing the highest chitinase activity was named GM13 ( Cellulomonas sp. GM13). It was deposited on February 5, 2001 in the Gene Bank of the Institute of Biotechnology (Accession No. KCTC 18073P).

Chitinase of the present cellulomonas GM13 strain is produced by chitin as a substrate, but the production of glucose is suppressed, and among these, endokinase is compared with other exokinase. Large amounts of chitin have been shown to be produced early in the breakdown.

As a result of investigating the productivity of chitinase according to the temperature of the G13 strain of the present invention, it showed the highest productivity of chitinase at 30 ° C. culture, and the culture of chitinase at 30 ° C. when cultured at 25 ° C., 37 ° C. and 40 ° C., respectively. Compared with the productivity, about 91%, 43%, 0% productivity was shown.

According to the chitinase productivity according to the pH of the G13 strain of the present invention, the initial pH of the medium showed the highest chitinase productivity, and when cultured at pH 5.0, 6.0, 8.0, and 9.0, each cultured at pH 7.0. It showed about 0%, 88%, 82%, 65% productivity compared to chitinase productivity.

On the other hand, most known chitinase producing strains are known to be unable to use the crystalline chitin, the crab shell chitin and the powder chitin, which are not expanded to the colloidal state, but the GM13 strain of the present invention uses the above crystalline chitin as a carbon source. It showed usable properties and showed higher chitinase productivity when powder chitin was used as carbon source than when colloidal chitin was used as carbon source.

In addition, chitinase productivity was examined by varying the concentration of powder chitin. When the concentration of powder chitin was 1.5% (w / v), it showed the highest productivity of chitinase.

As a result of investigating the productivity of chitinase according to the nitrogen source of the G13 strain of the present invention, when the pharmamedia was added to the nitrogen source among the tested nitrogen sources, the chitinase productivity was the highest.

Electrophoresis of the kind of chitinase produced by the G13 strain of the present invention resulted in at least five kinase activity bands of 63 kDa, 50 kDa, 37 kDa, 33 kDa, and 20 kDa. It was found that Naase is produced. The first fraction (Set I) fractionated by Toyo-Pear Chromatography showed no endokinase activity but only chitinase activity, and the second fraction ( In the case of Set II), low endokinase activity was shown, but in the third fraction (Set III), the turbidity of colloidal chitin was markedly decreased as well as chitinase activity. The third fraction (Set III) chitinase of the purified molecular weight 63 kDa was classified as endokinase, and the optimum reaction temperature and pH were investigated, and the optimum reaction temperature was 60 ° C., and the optimum pH was 7.0. As a result of examining substrate specificity, it was found that high activity of colloidal chitin and N -acetyl-chito-oligosaccharides of 3 or more sugars.

When analyzing the reaction products of colloidal chitin by chitinases produced by the G13 strain of the present invention by TLC method, colloidal chitin was not only decomposed to produce the most NAG disaccharide, but also a small amount of NAG trisaccharide. It was also found that tetrasaccharide, pentasaccharide, and six sugars were also produced. Since the known chitinases rarely produce high-polymerization oligosaccharides of NAG trisaccharide or higher by degrading chitin, it was determined that the chitinase of the present invention would be useful for producing high-polymerization chitin oligosaccharides having high bioactivity.

On the other hand, after culturing GM13 strain in MC medium to which 0.5% (w / v) colloidal chitin was added, exocytinase activity of the culture supernatant and cell extract was measured. On the other hand, exochitinase activity was detected in cell extracts, especially in cell extracts cultured in chitin-free LB medium (per liter; Bakto tryptone, 10 g; yeast extract, 5 g; NaCl, 5 g). Exokininase productivity is shown to be different from inducible exokinases whose productivity is increased by the addition of chitin reported so far, so that the intracellular exokinase of the present invention has been reported so far. It was judged as chitinase.

Various industrial applications are possible using the chitinase activity of the chitinase proteins of the invention. The water-soluble low molecular chitosan, chitin and oligosaccharides thereof prepared using the kinase of the present invention can be widely used in the fields of food, cosmetics, medicine, agriculture and the like. For example, the chitinase of the present invention may be selected or used in combination to manufacture actual NAG monomers and / or chitin oligosaccharides, which may be used for health drinks and food development, and fungi causing disease in crops using chitin decomposition activity. It can also be used in biopesticides for the control of nematodes, development of chitin-resistant transformed plants transformed with chitinase genes, and medicines.

The present invention comprises the steps of isolation and identification of GM13 strain of Cellulomonas from soil; Chitinase productivity of GM13 strain according to culture medium and culture conditions; Characterizing the chitinase purified from the culture medium of the GM13 strain; This step consists in examining the productivity of exokinase produced by GM13 strain.

Hereinafter, the strain of the present invention, purification of chitinase enzymes having chitinase activity, and characteristics of the enzymes will be described in more detail with reference to Examples. However, the following examples are merely for the purpose of illustrating the present invention, the scope of the present invention is not limited to the following embodiments.

Example 1 Isolation and Identification of GM13 Strains of Cellulomonas Species

About 1 g of the sample collected from the soil of the south coast of Gyeongnam was suspended in 30 ml sterile distilled water, and then allowed to stand overnight at 4 ° C., and the supernatant was diluted appropriately. 12.8 g Na ₂ HPO ₄ ; 5 g KH ₂ PO ₄ ; 0.5 g NaCl; (NH ₄ ) ₂ SO ₄ 1 g; glucose 10 g). During stationary culture at 30 ° C., strains were formed relatively rapidly on MCG plate media and strains with relatively large size of the degraded rings were selected first. The primary isolate was shaken and cultured in MCG liquid medium for 5 days, centrifuged to remove the cells, and the final selection of the GM13 strain with the highest activity was performed by measuring the chitinase activity of the culture supernatant.

At this time, the chitinase activity is 1% (w / v) colloidal chitin by adding an appropriate amount of enzyme and reacted for 30 minutes at 50 ℃ and then the reducing sugar of the reaction product of dinitrosalilic acid (dinitrosalicylic acid: DNS) method ( See GL Miller, Analytical Chemistry, 31: 426-428 (1959). The chitinase active unit (unit, U) by this method was defined as the amount of enzyme that produced 1 μmole NAG per hour. And the endokinase activity was determined by adding an appropriate amount of enzyme to 0.05% (w / v) colloidal chitin at 50 ° C. and measuring the absorbance at 510 nm to reduce the turbidity of the reaction solution. The active unit was defined as the amount of enzyme that decreases the turbidity of the reaction solution per minute by 0.1.

The morphological and biochemical characteristics of GM13 strains isolated from soils were shown in Tables 1 and 2 below, and the method of buggy (see Bergey's Manual of Determinative Bacteriology, 8th edition, Springer-Verlag Co. 1974) was identified as Cellulomonas sp. In addition, for more accurate identification, 16S rDNA of the isolate was cloned, and then the nucleotide sequence was analyzed and a phylogenetic tree was prepared (FIGS. 1 and 2). Based on these results, the chitinase producing strain of the present invention was found to be a novel strain having different microbiological characteristics from known chitinase producing strains and belonging to the genus Cellulomonas. Therefore, the novel soil isolation strain of the present invention was named Cellulomonas sp. GM13, and was deposited on February 5, 2001 in the Gene Bank of the Institute of Biotechnology (Accession No. KCTC 18073P).

Example 2: Investigation of the productivity of chitinase of GM13 strain of Cellulomonas according to medium and culture conditions

Example 2-1. Investigation of Changes in Chitinase Productivity by Colloidal Chitin Addition

All the chitinases reported to date are known as inducible enzymes in which mass production of chitin used as a substrate is required in the medium. Therefore, GM13 strains in the GM13 strain of the present invention to determine whether chitinase production is induced by chitin, MCG medium, MC medium (0.05% medium instead of glucose in MCG medium), MG medium ( Samples were inoculated in each MCG medium) and inoculated in a chitin-excluded medium) and shaken at 30 ° C for 12 hours. After that, the cells were removed, and the chitinase activity and the endokinase activity of the culture supernatant were measured.

Experimental results showed that the chitinase activity and endokinase activity in MC medium gradually increased over the incubation time, and showed 13 U / mL and 2.2 U / mL, respectively.

On the other hand, MG medium showed 4.4 U / mL and 0.0 U / mL after 36 hours of incubation, and MC U medium showed 12 U / mL and 1.28 U / mL, respectively. These results indicate that chitinase production of the GM13 strain is induced by the substrate chitin, and inhibited the production of chitinase by glucose.

On the other hand, when the GM13 strain was cultured in MC medium, as shown in FIG. 3, the chitinase activity showed a maximum at 54 hours and the endotokinase activity showed a maximum at 48 hours. In the case of extracellular protein, there was a sharp increase between 36 and 42 hours and there was no significant change since then. Examining the ratio of endokinase activity to chitinase activity showed the highest value at 42 hours, indicating that endokinase was produced in the early stages of chitin digestion compared to other exokinase.

Example 2-2. Investigation of productivity of chitinase according to culture temperature and pH

GM13 strains were inoculated in MC medium and shaken for 5 days at four culture temperatures of 25 ° C., 30 ° C., 37 ° C. and 40 ° C., followed by chitinase productivity. As a result, incubation at 30 ° C. showed the highest chitinase productivity, and when incubated at 25 ° C., 37 ° C. and 40 ° C., respectively, about 91%, 43 %, 0% productivity.

In addition, after adjusting the initial pH of the MC medium to five kinds of 5.0, 6.0, 7.0, 8.0, 9.0 and inoculated GM13 strain and incubated at 30 ℃ for 3 days to investigate the chitinase productivity, the initial pH of the medium was 7.0 Cases showed the highest productivity of chitinase, and when cultured at pH 5.0, 6.0, 8.0, and 9.0, they were approximately 0%, 88%, 82%, and 65% more productive than the chitinase productivity at pH 7.0 culture, respectively. Indicated.

Example 2-3. Investigation of productivity of chitinase according to carbon source

Crab shell chitin extracted from crab shell and finely powdered powder instead of 0.5% (w / v) colloidal chitin added into MC medium as chitinase production inducer Chitinase productivity of GM13 strain was investigated after adding chitin (powder chitin) to 0.5% (w / v), respectively.

As a result, most known chitinase producing strains are known to not use the crystalline chitin, crab shell chitin and powder chitin, which have not been expanded into a colloidal state, but the GM13 strain of the present invention has the same crystalline chitin as described above. It showed the characteristics that can be used as a carbon source, the use of powder chitin as a carbon source showed higher chitinase productivity than when using the colloidal chitin as a carbon source. In particular, the chitinase productivity was examined by varying the concentration of powder chitin. As shown in Table 3, the highest chitinase productivity was obtained when the concentration of powder chitin was 1.5% (w / v).

Example 2-4. Investigation of the productivity of chitinase by nitrogen source

0.5% (w / v) powder Chitinase was added to the MC medium using chitin as a carbon source by adding various nitrogen sources to 0.5% (w / v) instead of 0.5% (w / v) yeast extract. The productivity of the was investigated.

As shown in Table 4, the highest chitinase productivity was obtained when pharmamedia was added as a nitrogen source.

Example 3 Isolation and Purification of Chitinase from Cellulose

Chitinase was purified from the GM13 strain culture by the procedure shown in Table 4 below. In particular, a sample of chitinase dissolved in 10 mM sodium phosphate (pH 6.8) with 1 M ammonium sulfate was adsorbed onto a phenyl-Toyopearl column equilibrated with the same buffer, followed by 0 M at 1 M. When the enzyme adsorbed by the ammonium sulfate linear concentration tool was eluted, three kinds of fractions having chitinase activity in the concentration range of 0.2 M and 0 M ammonium sulfate were obtained (FIG. 4).

However, as shown in Table 5 and FIG. 5, in the first fraction (Set I), there was no endokinase activity (ie, turbidity reducing ability of chitin), but only chitinase activity, and the second fraction ( Set II) showed low endokinase activity. However, in the third fraction (Set III), the turbidity of colloidal chitin was markedly decreased by showing not only chitinase activity but also very high endokinase activity.

When cultured supernatant of GM13 strain was electrophoresed and then Coomassie blue staining and glycol chitin activation staining, 5 kinds of chitinase activity bands of 63 kDa, 50 kDa, 37 kDa, 33 kDa, 20 kDa Therefore, it can be seen that the GM13 strain produces at least five chitinases (FIG. 6).

By the way, when Coomassie blue staining and active staining were performed for Set III chitinase, which had high endokinase activity, only a 63 kDa chitinase band appeared, indicating that the chitinase was purely separated. On the other hand, the Set I chitinase, which had no endokinase activity, contained four kinds of chitinases except 50 kDa, 37 kDa, 33 kDa, and 20 kDa, except for 63 kDa chitinase. These results suggest that Set III chitinase is an endokinase. Also, when comparing the degradability of 4-methylumbelliferyl (MU)-(NAG) ₂ and 4-MU- (NAG) _{3 using} the Set III enzyme, the Set III enzyme was not reactive with 4-MU- (GlcNAc) ₃ . It was found that about 3.5 times higher than the reactivity for 4-MU- (GlcNAc) ₂ . Considering these results and the ability to reduce chitin turbidity, Set III chitinases were classified as endokinase.

Example 4 Characterization of Endotokinase Purified from GM13 Strain Cultures

Example 4-1. N-terminal amino acid sequence of endokinase

Examination of the N-terminal amino acid of Set III chitinase with 63 kDa molecular weight showed alanine-glycine-threonine-phenylalanine-threonine-alanine-threonine-phenylalanine in order.

Example 4-2. Optimum Temperature and Optimum pH Thermal Stability of Endokinase

In order to determine the optimum reaction temperature and the optimum pH of the purified purified endokinase, the enzyme activity was measured at various temperatures and pH conditions. As shown in (a) of FIG. 7, the optimum reaction temperature of the purified enzyme was It was 60 ℃, the optimum pH was 7.0 (Fig. 7 (b)).

Example 4-3. Substrate Specificity of Endokinase

As shown in Table 6, purified endokinase showed high activity against colloidal chitin and three or more N -acetyl-chitooligosaccharides. Although it showed low activity for glycol chitin, it did not show activity for crab shell chitin, chitosan, and N -acetyl-chitobiose.

Example 4-4. Degradation Product Analysis of Colloidal Chitin

When the reaction product of the colloidal chitin by the chitinases of Example 3 was analyzed by thin layer chromatography (TLC) method (FIG. 8), the endocytase (Set III chitinase) and the Set II chitinaa were analyzed. Zease decomposed the colloidal chitin to produce the most NAG disaccharide. In addition, a large amount of NAG was produced as a reaction product, and a small amount of NAG trisaccharide, tetrasaccharide, pentasaccharide, and hexasaccharide were also observed. However, since the known chitinases rarely produce high-polymerization oligosaccharides of NAG trisaccharide or higher by decomposing chitin, it was determined that the chitinase of the present invention would be useful for producing high-polymerization chitin oligosaccharides having high bioactivity.

On the other hand, Set I chitinase, which did not show any endocytase activity, produced only NAG and NAG disaccharides as a reaction product by degrading colloidal chitin, and did not produce oligosaccharides of three or more sugars.

Example 5. Investigation of productivity of exokinase produced by GM13 strain

After culturing the GM13 strain in MC medium to which 0.5% (w / v) colloidal chitin was added, the exokinase activity of the culture supernatant and the cell extract was measured. At this time, the exokininase activity was added to 50 mM sodium phosphate buffer (pH 6.8) to add 2 mM of p -nitrophenyl-β-DN-acetylglucosamine, an artificial substrate for measuring exokinase activity. After 0.2 ml of enzyme solution was added to 0.2 ml of this substrate solution, the mixture was reacted at 50 ° C for 15 minutes. Thereafter, 1 ml of 0.1 M glycine-NaOH buffer (pH 10.5) was added to stop the enzymatic reaction, and the amount of p- nitrophenol released upon hydrolysis at 420 nm was measured. Enzyme units were defined as the amount of enzyme that produced 1 μ mole of p- nitrophenol per minute.

As a result of measuring the exokinase activity by the above method, the exokinase activity was not detected in the culture supernatant, while the exokinase activity was detected in the cell extract. In particular, 1.37 unit / ml for the cell extract of GM13 strain obtained by culturing in LB medium (per liter; Bakto tryptone, 10 g; yeast extract, 5 g; NaCl, 5 g) without chitin added High exochitinase productivity was shown. By the way, most of the reported exokinases were inducible enzymes whose productivity was increased by the addition of chitin added in the medium, and no exokinase present in cells in Gram-positive bacteria has been reported. Therefore, the intracellular exokinase of the present invention was determined to be a novel chitinase that has not been reported so far. Passing the cell extract was Superose 12HR column when the active fraction purified by electrophoretic separation of pure exo kitty the coma better stain and 4-MU-GlcNAc (4- methylumbelliferyl- N -acetyl-β-D-glucosamine) activity staining The molecular weight was found to be about 54,000 Daltons.

As described in detail above, the chitinase produced by the novel cellulomonas genus GM13 strain isolated from the soil has been shown to be excellent in chitin resolution and to produce oligosaccharides of high polymerization as well as NAG disaccharides. The chitinase produced will be useful for the production of chitin oligosaccharides and NAG.

Claims (7)

Cellulose genus G13 strain (KCTC18073P) having chitinase producing ability with chitin degrading activity. The method according to claim 1, wherein the strain comprises the step of culturing in a medium containing chitin. A chitinase selected from the group consisting of chitinases or mixtures thereof having molecular weights of 63 kDa, 50 kDa, 37 kDa, 33 kDa and 20 kDa as a result of the electrophoresis measurement prepared by the method of claim 1. According to the electrophoresis measurement, the molecular weight is approximately 63kDa, optimum pH 7.0, optimum temperature 60 ℃, N-terminal amino acid sequence is characterized in that the alanine-glycine-threonine-phenylalanine-threonine-alanine-threonine-phenylalanine Endokinase. 4. The exokinase of claim 3, wherein the molecular weight (electrophoresis) produced in the cells of the GM13 strain is about 54 kDa. A method of producing chitin oligosaccharides or NAG from chitin using the chitinase of claim 3. A strain culture or composition comprising a chitinase prepared by the method of claim 2.