• Journal of Life Science
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• v.9 no.4
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• pp.341-347
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• 1999

Insects use chitinolytic enzyme to digest chitin in the exoskelton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the silkworm, Bombyx mandarina, compared its sequenced with genes encoding chitinolytic enzymes from other sources. The insert DNA in the clone is 2,675 nucleotides long with an open reading frame of 1,695 uncletides that encodes a protein of 565 amino acids with a molecuar weight of 63.4 kDa. The 3' -untranslated region of 889 nucleotides is AT-rich and contains two putative polyadenylation signals. The N-terminal sequence of the encoded protein contains numerous hydrophobic residues characteristic of a leader peptide. The amino acid alignment revealed that the endo-$\beta$-N-acetylglucosaminidase had 83% and 97% homology with M. sexta and B. mori, respectively. The deduced amino acid had two highly conserved region at the amino acid residues 97-111 and 139-148 that were related to the existing chitinase.

• 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.

• The Korean Journal of Mycology
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• v.31 no.3
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• pp.168-174
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• 2003

In this study, Cordyceps militaris was grown in a liquid medium containing colloidal chitin. A chitinase was purified from the supernatant or cultured medium by ammonium sulfate fractionation, DEAE-Sephadex A-25 and Sephadex G-50 column chromatography. Optimum temperature and pH of this enzyme were $35^{\circ}C$ and 5.5, respectively. The molecular weight of the chitinase was estimated to be 48.5 kDa by SDS-PAGE and its Km value was 0.57 mM. The activity of this enzyme was inhibited by $Cu^{2+},\;Mn^{2+},\;Hg^{2+},\;Zn^{2+},\;CO_{3}^{2-},\;SO_4^{2-},\;CN^-,\;ion,\;and\;OCN^-$ maleic anhydride, acetic anhydride or N-bromo succinimide, especially strongly inhibited by sodium cyanate for 84.0 percentage. But its activity wag slightly stimulated by $Mg^{2+}\;and\;K^+$ ion, respectively. The products formed during hydrolysis of the hexa-N-acetylchitohexaose with this enzyme were N,N'-diacetylchitobiose and N,N',N'-triacetylchitotriose. These results imply that this purified enzyme may be an endo-chitinase.

• Korean Journal Plant Pathology
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• v.10 no.4
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• pp.277-283
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• 1994

An antimicrobial chitinase was purified from grapefruit extract and its properties were characterized. The chitinase was purified with a single step chromatography on regenerated chitin affinity gel column. The molecular weight of the purified chitinase was 29 kDa. The grapefruit extract contained the chitinase protein more than 50% of its total soluble proteins measured by coomassie stained protein bands. When the purified chitinase was incubated with polymers of N-acetylglucosamine (NAG), such as mycelia of Fusarium oxysproum and swollen chitin, they were degraded to oligosaccharides, and the oligosaccharides were then further hydrolyzed by the same enzyme to monomer and dimer of NAG. This result suggests that the chitinase contained both endo- and exo- chitinase activities. The chitinase was stable to heat and pH treatment; its activity was not diminished by the heat treatment upto 7$0^{\circ}C$ for 1 hr, and it showed a pH stability in the range of pH 4.0 to 12.0.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.560-567
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• 1998

We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.

• 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.

• Archives of Pharmacal Research
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• v.14 no.3
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• pp.255-260
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• 1991

Chitinase was partially purified from Allium fistulosum L (green onion_. Protein fraction precipitated from ammonium sulfate was passed through CM-Sepharose and Sephacryl HR-200. The specific activity of the chitinase was 6.4 units/mg and total recovery was 6.3%. The analysis of the products from the digestion of N-acetychitohexaose indicated that chitinase was endo in action, with oligerms from N-acetylchitobiose to chitotetraose. N-Acetylglucosaminidase from the same species hydrolyzed oligomers obtained from chitinase reaction to lower oligosaccharides. These data demonstrated that chitinolytic system exists in green onion.

• Journal of Industrial Technology
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• v.30 no.B
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• pp.69-74
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• 2010

Chitinolytic activity was enhanced by coexpression of endo-chitinase gene (chiA) and chitobiosidase gene (chiB) from Serratia marcescens KFRI314 using constitutive expression vector, pHCEIA, in E. coli. Coexpression vector was constructed by inserting ribosome binding site (RBS) into junction between two chitinase genes. SDS-PAGE analyses showed that two chitinase were constitutively expressed while E. coli clones expressing two chitinases simultaneously increased halo size on colloidal chitin plate. Furthermore, the chitinolytic activities were much enhanced in coexpressed clones when degradation patterns of substrate analogues such as 4-MU-(NAG), $4-MU-(NAG)_2$,$4-MU-(NAG)_3$ were used. Consequently, the combined use of endochitinase and chitobiosidase greatly increased overall chitinolytic activities on recombinant E. coli clones.

• Korean Journal of Food Science and Technology
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• v.35 no.6
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• pp.1188-1192
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• 2003

Chitinase producing bacteria, Arthrobacter nicotianae CH4 and A. nicotianae CH13, were isolated from small crabs by an enrichment culture using chitin as the sole carbon source. Crude chitinases from the two isolated strains, A. nicotianae CH4 and A. nicotianae CH13, were stable in the pH range of $3.0{\sim}9.0$ and in the temperature range of $20{\sim}60^{\circ}C$. The reducing sugar $(GlcNAc)_1$, or $(GlcNAc)_4$, corresponding to over 98% of the enzyme reaction products, was obtained. The production of functional $(GlcNAc)_1$ and $(GlcNAc)_4$ from A. nicotianae CH13 and A. nicotianae CH4, respectively, from the chitinases was useful. The chitinase system of A. nicotianae CH13 was supposed to be endo- and exo-chitinase, and N-acetylglucosaminidase.

• Journal of Life Science
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• v.22 no.6
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• pp.837-843
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• 2012

This study was carried out to investigate the morphological and physiological characteristics of six new cultivars of Hypsizygus marmoreus (Hm) and measure endo-, exo-cellular enzyme-specific activity. The domestic wild stain (Hm3-10) and commercial strain in Japan (Hm1-1) were mated by crossing monokaryon mycelia. We gained 58 strains from one of 400 crosses through the $1^{st}$ cultivation experiment, and selected six strains from one of 58 strains through the $2^{nd}$ cultivation experiment. When six of the selected new strains were grown during several spawn culture periods (60, 70, 80, 90, and 100 days), a spawn culture period of more 80 days was considered to be excellent as being shorter than 19~20 days. Therefore, we determined the period of spawn culture as 80 days. Three strains such as Hm15-3, Hm15-4, and Hm17-5 showed an excellent result. When endo-cellular enzyme activity measured eight strains, we obtained a result of that specific activity of ${\alpha}$-amylase at the highest as 73.9~102.2 unit/mg protein, and chitinase is lower than ${\alpha}$-amylase at 8.1~13.1 unit/mg protein. When exo-cellular enzyme activity measured eight strains, we determined the result of that specific activity of ${\alpha}$-amylase is the highest at 5,292~1,184 unit/mg protein, and CMCase and xylanase were 1,140~245 unit/mg protein, 94~575 unit/mg protein, compared to each other. However, the enzyme activity of ${\beta}$-glucosidase and chitinase is low.