• Journal of the Korean Chemical Society
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• v.40 no.1
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• pp.72-80
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• 1996

A chitinase-producing bacterium, Serratia marcescens JM, was isolated from a seashore muds. A chitinase was purified by ammonium sulfate precipitation, affinity adsorption, hydroxylapatite and sephadex G-200 column chromatography. The chitinase obtained from Serratia marcescens JM was purified 42.2 folds with the overall yield of 7.1%. The purified chitinase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 59,000 and the apparent kinetic parameters $K_m\;and\;V_{max}$ for the purified chitinase were 5.17 mg/mL and 39.8 unit/mL, respectively. The optimum pH and temperature of the purified chitinase were 7.0 and 50$^{\circ}C$, respectively and the purified enzyme was stable on pH 7.0 up to 50$^{\circ}C$. The enzyme were activated by $Cu^{2+},\;Ca^{2+}\;and\;Mg^{2+}$ and inhibited by $Hg^{2+}$ respectively. In addition, Cysteine increased the chitinase activity and EDTA, MIA, PCMB and SDS inhibited enzyme activities. Major cations, $MG^{2+},\;Ca^{2+},\;K^+\;and\; Na^+$ present in seawater slightly stimulated the chitinase activity.

• The Plant Pathology Journal
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• v.21 no.3
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• pp.275-282
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• 2005

A chitinolytic bacterium, Chromobacterium sp. strain C-61, was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of eggplant. In this study, the biocontrol activity and enzymatic characteristics of strain C-61 were compared with its four Tn5 insertion mutants (C61-A, -B, -C, and -D) that had lower chitinolytic ability. The chitinase activity of a 2-day old culture was about $76\%,\;49\%\;and\;6\%$ level in C61-A, C61-B and in C61-C, respectively, compared with that of strain C-61. The $\beta-N-acetylhexosaminidase$(Nahase) activity was little detected in strain C-61 but increased largely in C-61A, C61-B and C61-C. Activities of chitinase and Nahase appeared to be negatively correlated in these strains. Another mutant, C-61D, produced no detectable extracellular chitinase and Nahase. The in vitro and in vivo biocontrol activities of strain C-61 and its mutants were closely related to their ability to produce chitinase but not Nahase. No significant differences in population densities between strain C-61 and its mutants were observed in soil around eggplant roots. The results of SDS-PAGE and isoelectrofocusing showed that a major chitinase of strain C-61 is 54-kDa with pI of approximately 8.5. This study provides evidence that the biocontrol activity of Chromobacterium sp. strain C-61 against Rhizoctonia solani depends on the ability to produce chitinase with molecular weight of 54-kDa and pI of 8.5.

• Microbiology and Biotechnology Letters
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• v.9 no.4
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• pp.179-183
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• 1981

An extracellular chitinase was purified from the culture fluid of Streptomyces sp. 115-5, and its inhibition mechanism by end product was studied. The activity of chitinase was suppressed by the reducing sugar as the reaction proceeded, and the activity was inhibited by the addition of D-glucose. Besides D-glucose, the rate of chitin hydrolysis was inhibited by D-glucuronic acid, D-sorbitol and D-xylose in the reaction system of the enzyme. it was found that the hydroxyl groups at the C-2, C-3 and C-4 position of D-glucose molecule play an important role in the inhibition of the chitinase activity. D-glucose was found to inhibit the enzyme activity by mixed type of competitive and non-competitive mode.

• Journal of Industrial Technology
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• v.30 no.B
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• pp.61-68
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• 2010

Three chitinase genes (chiA, chiB, and chiC) were cloned into E. coli by PCR amplification from Serratia marcescens KFRI314. The sizes of cloned chitinase genes were 1692 bp, 1500 bp, and 1443 bp which correspond to 563, 499, and 480 amino acids, respectively. Recombinant chitinases were overexpressed using pHCEIA expression vector and purified to homogenity. The molecular weights of chitinases were about 60kDa, 50 kDa, 52 kDa, respectively. Optimum pHs were around pH 5~6 and optimum temperatures were $45{\sim}50^{\circ}C$ while 90% of enzyme activities were stable up to $50^{\circ}C$. The specific activities of ChiA, ChiB, and ChiC were 233.1, 278.8, $111.3{\mu}mol\;(min)^{-1}\;mg^{-1}$ against colloidal chitin. From experiments using TLC and fluorescent substrate analogues, it was demonstrated that ChiA was endo-chitinase while ChiB and ChiC were chitobiosidase.

• Korean journal of food and cookery science
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• v.15 no.2
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• pp.102-107
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• 1999

Several ingredients of kimchi including chinese cabbage, garlic, leek, big green onion, and small green onion were assayed for their chitinase activities. Kimchi with various leek contents (4, 8, 12%) were fermented at 15$^{\circ}C$ for 9 days and the chitinase (EC 3.2.1.14) activity and textural properties were determined. The chitinase activity of the ingredients was in the order of garlic>leek>small green onion>chinese cabbage>big green onion. During fermentation, the chitinase activity of kimchi juice appeared more prominent than that of kimchr tissue, however, it was decreased in all kimchi samples among which the control sample showed a remarkable drop. The activity of chitinase in kimchi tissue increased until 3rd or 5th day of fermentation and then decreased. The puncture force of all kimchi samples decreased and those of leek-added kimchi were higher than those of control. The above results suggested that the addition of leek for kimchi preparation could contribute to the improvement of textural qualities of kimchi due to chitinase activities of leek during fermentation.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.647-652
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• 2004

Thermophilic microorganisms capable of producing chitinase enzymes were screened from samples collected from several crater and geothermal areas. The chitinolytic microorganisms were grown in a selective medium containing colloidal chitin. The Bacillus K29-14 isolate was found to exhibit the highest chitinase and chitin deacetylase activities. When grown in a chitin-containing medium, the isolate produced extracellular chitinase after 24 h of incubation. The optimum temperature and pH for the chitinase were $55^\circ{C}$ and pH 7, respectively, while those for the chitin deacetylase were $55^\circ{C}$ and pH 8, respectively. The thermostable chitinase and chitin deacetylase also retained 80- 90% of their activity after incubation for 5 h at $70^\circ{C}$. The divalent cations $CoCl_2\;and\;NiCl_2$, increased the chitinase activity, while $ZnCl_2$, inhibited the enzyme. The chitin deacetylase was also activated by the presence of $MgCl_2$ and inhibited by $MnCl_2,\;NiCl_2,\;and\;CaCl_2$. A zymogram analysis revealed several forms of chitinase, with a 67 kDa form being the major enzyme.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.187-196
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• 1995

In order to produce fuctional chitooligosaccharides, a strain excreting mainly endo-type chitinase suitable for chitooligosaccharides production was newly screened and identified as Aspergillus fumigatus JC-19. The chitinase excretion was repressed in nutrient rich medium but stimulated by colloidal chitin indicating that the chitinase is inducible type enzyme. Maximum secretion of the enzyme was observed at pH 7.0 and 37$\circ$C . The growth and chitinase production patterns of Aspergillus fumigatus JC-19 showed that the cell growth reached maximum after 4-5 days with final chitinase concentration of 0.46 unit per ml. Excreted chitinase was purified by ammonium sulfate precipitation, colloidal chitin adsorption, anion exchange chromatography, and gel filtration, respectively, and measured M.W of 50 KDa. The enzyme reaction carried out both by crude and purified chitinase showed that the purified chitinase accumulated more chitooligosaccharides of 1-6 degree of polymerization than that of crude chitinase.

• Journal of Life Science
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• v.13 no.1
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• pp.90-98
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• 2003

An allium rhizobacterium Serratia plymuthica AL-1 was previously selected as a biocontrol agent of allium white rot. The chitinase from S. plymuthica AL-1 produced in medium containing colloidal chitin was purified by ammonium sulfate precipitation (40~70%), affinity adsorption, column chromatography on DEAE-sephadex A-50 and sephadex C-200 gel filtration. The enzyme was purified 10.8-fold with a yield of 7.3% from the starting culture broth. The purified chtinase gave a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it's molecular weight was estimated to be 55 kDa. The optimum pH and temperature of the purified enzyme were pH 5.5 and $55^{\circ}C$, respectively and it is stable up to $50^{\circ}C$ and maintains around 90% of its activity for 60min. The enzyme were activated by $Ca^{2+}$, $Mn^{2+}$ and $Mg^{2+}$ and inhibited by $Cu^{2+}$, SDS, $\rho$-CMB, MIA, respectively. The purified chitinase showed broad spectrum of antifungal activities against plant pathogenic fungi Sclerotium cepivoruin, Alternana alternnta, Colletotrichum glceosporioidrs, Phoma sp., Sclerotinia sclerotiorum, Stemphylium solani, Fusarium oxysporium f. sp. niveum but rarely inhibited Phytophthora capsici and Pythium ultimum.. The purified chitinase from S. plymuthica AL-1 caused swelling, lysis, deceleration and degradation of the hyphal tips of S. sczerotiorum causing allium white rot. It suggest that S. prymuthica AL-1 chitinase play an important part in the bifunctional chitinase / lysozyme activity.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.599-606
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• 1990

A chitinase-producing bacterium, Aeromonas salmonicida YA7-625, was isolated from domestic seashore muds. The preferable medium composition for the production of chitinase was as follows: colloidal chitin 1.26% (w/v), tryptone 2.95% (w/v), $MgSO_4-7H_20$ 0.15% (w/v) and $K_2HP0_4$, 0.15% (w/v) (pH 8.5). The highest enzyme production was observed after cultivation of 48 hours at 27OC. The chitinase of Aeromonas salmonicida YA7-625 was purified successively by ammonium sulfate precipitation, affinity adsorption, hydroxylapatite column chromatography and gel filtration. The optimal temperature and pH for the activity of purified chitinase were $50^{\circ}C$ and 7.0, respectively. The molecular weight of purified chitinase was ca. 200,000 daltons and apparent Km value of it was 1.276 mglml on colloidal chitin.

• BMB Reports
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• v.36 no.2
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• pp.185-189
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• 2003

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Streptomyces sp. M-20 and purified by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration. No exochitinase activity was found in the culture filtrate. The molecular mass of the purified chitinase was 20 kDa, estimated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed by activity staining with Calcofluor White M2R. Chitinase was optimally active at pH of 5.0 and at $30^{\circ}C$. The enzyme was stable from pH 4 to 8, and up to $40^{\circ}C$. Among the metals and inhibitors that were tested, the $Hg^+$, $Hg^{2+}$, and p-chloromercuribenzoic acid completely inhibited the enzyme activity. The chitinase activity was high on colloidal chitin, chitotriose, and chitooligosaccharide. The purified chitinase showed antifungal activity against Botrytis cinerea, and lysozyme activity against the cell wall of Botrytis cinerea.