• Applied Biological Chemistry
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• v.46 no.3
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• pp.171-175
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• 2003

The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{\circ}C$.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.246-249
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• 2000

We investigated the production of chitin oligosaccharides using food grade papain. A solution of commercial food grade papain (FGP) was dialyzed for 12 h before measuring its chitinolytic activity. The effects of enzyme concentration, reaction temperature, and pH on the endochitinase and $\beta$-N-acetylglucosaminidase activities and the thermostability of these enzymes were investigated. In adddition, the reaction products were analyzed with gel filtration on a Bio-Gel P2. The endochitinase activity was twentyfold higher than that of $\beta$-N-acetylglucosaminidase. The optimal endochitinase activity was at pH 3.0, while the maximal $\beta$-N-acetylglucosaminidase activity was at pH 6.0. The reaction product consisted mainly of the dimer of N -acetylglucosamine, with a small amount of its trimer. Under the experimental conditions, $120{\mu}g$ of chitin oligomers were obtained with 1 mg of FGP protein after an incubation of 2 h.

• 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.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.466-471
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• 1996

The survey on the chitinolytic activity of some plants was performed for the purpose of obtaining some reliable and inexpensive sources of chitinase. Rice, soybean for sprouting, kiwi fruit, almond and crude papain were investigated. Rice bran, seed coat of the soybean and the pericarp of kiwi fruit showed a considerable activity, while the bean after the removal of the seed coat, the mixture of rice integument and endosperm, polished rice, and defatted soybean powder didn't have any detectable activity. These crude enzymes have shown to contain both endo- and exochitinase activity. The effects of pH and temperature on the enzyme activity were variable. Furthermore we have observed the chitosanolytic activity from these enzyme Preparations. The rice bran had the highest activity in the enzymatic degradation of chitosan, and seed coat of soybean and the pericarp of kiwi fruit followed. On the basis of the fact that crude papain was not only commercially available but also the most potent in the endochitinase activity and the lowest in the exochitinase activity, we could conclude that crude papain was considered as the most suitable source of the chitinase among plants studied in this paper. In addition, rice bran was worth further investigation from the point of utilizing agricultural by-product.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.107-113
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• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.

• Applied Biological Chemistry
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• v.40 no.2
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• pp.95-100
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• 1997

This study was aimed to survey inexpensive and reliable sources of chitinase from the animal origin. The stomach and its content of the broiler, the cod, the yellowtail and ${\beta}-glucuronidase$ from snail gut showed a considerable chitinolytic activity, while those of the bas didn't have any detectable activity. These crude enzymes was found to have both endo- and exochitinase activity. The effects of pH and temperature on the enzyme activity were variable. The hydrolytic products of colloidal chitin by the enzyme preparation from the broiler and the cod were chitooligomers having the degree of polymerization between 3 and 5. Furthermore we observed the chitosanolytic activity from these enzymes. In the degradation of chitosan the chyme of the broiler had the highest activity and ${\beta}-glucuronidase$ from snail gut followed. On the basis of the fact that the by-product of the broiler was not only commercially available but also the most potent in the endochitinase activity and the lowest in the exochitinase activity, we conclude that the gizzard and its chyme are considered as the most suitable source of the industrial chitinase among animals studied in this paper.

• Applied Biological Chemistry
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• v.37 no.4
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• pp.255-258
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• 1994

Using the enzyme activity staining, we studied the induction and distribution of chitinase isozymes, pathogenesis-related proteins, in intercellular fluids of bean and soybean leaves under stress conditions. The chitinase in intercellular fluids was barely detected in healthy plant leaves. By treatment of ethylene, pathogen (Fusarium oxysporum), or wounding, only 34 kD intercellular endochitinase was induced in bean leaves, while 30 kD and 36 kD intercellular endochitinases were induced in soybean leaves.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.38-42
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• 2002

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from the Serratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids. Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide and N-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a $98\%$ similarity to that of S. marcescens OMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N'-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount of N-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer $(analogue\;of\;NAG_2)$, thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were $50^{\circ}C$ and 5.0, respectively.

• Applied Biological Chemistry
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• v.37 no.1
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• pp.49-55
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• 1994

An acidic protein, RCG-2, containing chitinase and ${\beta}-1,3-glucanase$ activity conccurrently was purified from rice leaves by chromatofocusing and gel slicing. The purified enzyme gave a single band on polyacrylamide gel electrophoresis and its molecular weight was appeared to be 29.7 kd using SDS-PAGE. This enzyme also had lysozyme activity. The optimal temperature for both enzyme activities was $40^{\circ}C$, optimal pH were 4.0 for chitinase activity and 7.0 for ${\beta}-1,3-glucanase$ activity. $K_M$ and $V_{max}$ values for chitinase were 7.86 mM and $0.025\;{\mu}M/min.$, and those for ${\beta}-1,3-glucanase$ were 5.95 mM and $0.16\;{\mu}M/min.$ respectively. TLC analysis of the enzyme hydrolysates of chitooligosaccharides indicated that this enzyme acts as endochitinase.

• Applied Biological Chemistry
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• v.35 no.3
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• pp.191-195
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• 1992

In order to elucidate the plant-microorganism relationship, we purified an ethylene-inducible, basic 30 KD endochitinase from bean leaves and studied its antifungal activity by a hyphal extension-inhibition assay. The purified chitinase was effective in the inhibition of hyphal growth of Aspergillus fumigatus, Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, while microbial chitinases of Serratia marcescens and Streptomyces griceus, egg white lysozyme and papya protease didn't affect hyphal growth of the fungi. The chitinase degraded the cell walls of Micrococcus lysodeikticus, suggesting the lysozyme activity of the chitinase. We discussed the implication of the bifunctional chitinase/lysozyme activities of the protein with hydrolysis of chitin in the rapidly extending hyphae of the fungi.