• The Plant Pathology Journal
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• 제33권3호
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• pp.318-328
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• 2017

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was $50^{\circ}C$. However, it was inactivated with time when it was incubated at $40^{\circ}C$ and $50^{\circ}C$. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.307-311
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• 2000

Abstract Extracellular chitinase was purified from the culture liquid of the marine bacterium, Bacillus sp. LJ-25 , and its enzymatic properties were examined. The purified chitinase exhibited a single band on SDS-PAGE and the molecular weight was estimated to be approximately 50 kDa. The optimum pH and temperature for the enzymatic activity were 7.0 and $35^{\circ}C$, respectively. The activity of the chitinase was strongly inhibited by $Zn^{2+}$ and slightly inhibited by $Ba^{2+},{\;}Co^{2+},{\;}Mn^{2+},{\;}and{\;}Cu^{2+}$. The purified chitinase did not hydrolyze $p-nitrophenolN-acetyl-{\bata}-D-glucosaminide{\;}(GlcNAc)_2$ and Micrococcus lysodeikticus cells, which are known to be the substrates for exo-type chitinase. Among the hydrolyzates of colloidal chitin, $(GlcNAc)_2$ was in the highest concentration with small amounts of GlcNAc and $(GlcNAc)_3$..

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.439-444
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• 1996

A bacterial strain, designated as WY22, producing extracellular chitinase was isolated from the soil around the Youngduck area, after enrichment culture in a medium containing $1{\%}$ (w/v) wet colloidal chitin as a sole carbon source. The isolate was identified as a strain of Bacillus sp. based on its morphological and physiological characteristics. It was observed that Bacillus sp. WY22 could inhibit the growth of Fusarium oxysporum with hyphal extention-inhibition assay on potato dextrose agar plate supplemented with $1{\%}$ collidal chitin. Optimum culture conditions of Bacillus sp. WY22 were examined for chitinase production in a chitin medium. High level production of chitinase was observed not only in the chitin medium but in a medium supplemented with $1{\%}$ N-glucosamine or lactose instead of chitin. The optimum concentrations of colloidal chitin and yeast extract were 3.0 and $0.5{\%}$, and the optimum culture conditions for initial pH of medium and temperature were 7.0 and $30^{\circ}C$, respectively, for the production of chitinase.

• 한국미생물·생명공학회지
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• 제9권4호
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• pp.179-183
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• 1981

진균류의 세포벽제거 및 이에 따른 protoplast생성 등에 많이 이용되어지고 있는 chitinase를 Streptomyces sp. 115-5 균주로부터 생산하여 순수 정제한 다음, 이 chitinase의 작용을 저해하는 포도당의 저해양상을 조사하였다. 포도당 외에 D-glucuronic acid, D-sorbitol및 D-xylose등도 chitinase의 활성을 저해하였다. 그런고로, 포도당 분자에 의한chitinase의 활성 저해 효과에는 위의 분자들의 공통부분인 2번, 3번 및 4번 탄소의 hydroxyl group들의 구조위치가 중요한 영향을 가진다는 것을 알 수 있다. 그리고 포도당에 의한 chitinase의 저해양상은 competitive inhibition 과 non-competitive inhibition 과의 혼합 저해형으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.134-140
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• 1994

An antagonistic bacterium Pseudomonas stutzeri YPL-1 liberated extracellular chitinase and $\beta$-1,3-glucanase which are key enzymes in the decomposition of fungal hyphal walls. The lytic enzymes caused abnormal swelling and retreating at the hyphal tips of plant pathogenic fungus Fusarium solani in a dual culture. Scanning electron microscopy revealed the hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. The production of chitinase and properties of a crude preparation of the enzyme from P. stutzeri YPL-1 were investigated. Peak of the chitinase activity was detected after 4 hr of cultivation. The enzyme had optimum temperature and pH of 50$^{\circ}C$ and pH 5.3, respectively. The enzyme was stable in the pH range of 3.5 to 6.0 up to 50$^{\circ}C$. The enzyme was significantly inhibited by metal compounds such as $HgCl_2$, but was stimulated by $CoCl_2$. P. stutzeri YPL-1 produced high levels of the enzyme after 84 hr of incubation. Among the tested carbon sources, chitin was the most effective for the enzyme production, at the concentration level of 3%. As a source of nitrogen, peptone was the best for the enzyme production, at the concentration level of 4%. The maximum amount of enzyme was produced by cultivating the bacterium at a medium of initial pH 6.8.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.96-104
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• 2019

Beauveria bassiana, one of the most common entomopathogenic fungi, has been isolated, pre defined and characterized in-house from soil of tea cultivation area. Experiments have been performed to verify the presence of chitinase as intracellular metabolite and its release as extracellular product rendering the spores with biopesticide activity. Although there are many responsible enzymes for the pest killer action of B. bassiana, binding property of chitinase depending on presence as well as absence of serine supplemented in the media has been studied with respect to the production and kinetics. A programmed investigation conclusively indicates that the isolated spore (hyphae) of B. bassiana has been metabolically enriched with the enzyme chitinase in presence of an externally added amino acid serine with its inhibitory kinetics.

• KSBB Journal
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• 제29권3호
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• pp.131-138
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• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.

• The Plant Pathology Journal
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• 제28권4호
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• pp.439-445
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• 2012

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.87-91
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• 1995

The chitinase gene was cloned from Acinetobacter sp. WC-17 for investigating the genetic control and enzymatic properties of bacterial chitinase. A genomic library of Acinetobacter sp. WC-17 was prepared in E.coli JM109 by using pUC18 as a vector. The chitinase-positive clone containing 3.2kb insert fragment was obtained from 5, 000 insert-bearing transformants. The optimum pH and temperature of cloned enzyme were 6.0 and $55^{\circ}C$, respectively. Almost all the chitinase activity of E.coli recombinant was localized in the periplasmic fraction, while most of the enzyme activity of Acinetobacter sp. WC-17 was found in the extracellular fraction.

• Journal of Microbiology
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• 제38권4호
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• pp.224-229
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• 2000

Chitin-degrading marine bacterial strain 98CJ11027 was isolated from bryozoa from the coastal area of Cheju Island, Korea, and identified as a member of the genus Vibrio. The molecular mass of the main extracellular chitinase (chitinase I), purified from strain 98CJ11027, was estimated to be 98 kDa. The optimal condition for chitinase I activity is pH 6.0 and 45$^{\circ}C$. The activity was inhibited by Fe$\^$+2/ and Cu$\^$+2/. Chitinase I displayed the hydrolysis type of chitobiosidase and catalyzed reversed hydrolysis leading to the synthesis of tetraacetylchitotetraose.