• Fisheries and Aquatic Sciences
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• 제2권1호
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• pp.105-111
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• 1999

In order to produce functional chitin oligosaccharides, a chitinolytic bacterium was newly screened from the viscera of Korean bony fishes, and identified as Bacillus sp. LJ-25. For the production of chitinolytic enzymes, $1.0\%$ nutrient broth and $0.3\%$ colloidal chitin were used as nitrogen and carbon source, respectively. The optimal temperature, initial pH and concentration of NaCl for the enzyme production by Bacillus sp. LJ-25 were $30^{\circ}C$ 6.5-7.0 and $1.0\%$, respectively. The enzyme activity of Bacillus sp. LJ-25 increased until the incubation time of 168 hr, followed by a decrease in activity.

• 한국식물병리학회지
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• 제11권1호
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• pp.47-52
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• 1995

One hundred and thirty bacterial isolates with high chitinolytic activity on chitin agar media were isolated and identified. Most of the isolates were Aeromonas hydrophila (110 isolates), and the others were Serratia marcescens (11 isolates), Aeromonas caviae (3 isolates), Chromobacterium violaceum strain C-61 (2 isolates), Chromobacterium violaceum strain C-72 (1 isolate) and unknown species (3 isolates). Among them, C. violaceum strain C-61 had highest chitinolytic activity and fungal growth inhibition on PDA. This bacterium also inhibited the growth of Rhizoctonia solani, Sclerotinia scelrotiorum, Phytophthora capsici and Pythium ultimum, but it didn't inhibit the growth of Fusarium oxysprum and Fusarium solani. C. violaceum strain C-61 suppressed damping-off of eggplant caused by R. solani. Populations of the chitinolytic bacteria such as Aeromonas hydrophila, Serratia marcescens, Aeromonas caviae, Chromobacterium violaceum strain C-61 and Chromobacterium violaceum strain C-72 introduced into R. solani-infested soil were continuously decreased until 20 days after treatment, but their populations except A. caviae were not changed significantly and maintained over 5$\times$104 CFU per g of soil thereafter.

• Fisheries and Aquatic Sciences
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• 제6권1호
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• pp.1-6
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• 2003

A chitinolytic enzyme-producing bacterium was isolated from sea water on the coast of Busan. The bacterium was identified as Aeromonas sp. based on its morphological, cultural and biochemical characteristics and designated Aeromonas sp. J-5003. The strain produced two chitinoloytic enzymes: chitinase and chitobiase. The optimum culture conditions of the strain for production of chitinoloytic enzymes were investigated. For the production of chitinase, the major components of medium were colloidal chitin $0.5\%$, glucose $0.2\%$, yeast extract $0.25\%$ and peptone $0.25\%$ while for the production of chitobiase, they were colloidal chitin $0.5\%$, galactose and tryptone $0.2\%$. The optimum cultural temperature and initial pH for the production of chitinase and chitobiase were $30^{\circ}C$ and pH 7.0, respectively.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.865-871
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• 2002

A bacterium having strong chitinolytic activity on $0.2\%$ colloidal chitin-containing agar medium was isolated from coastal soil in Korea. Based on the nucleotide sequence of conserved segment of a 165 rRNA gene, the bacterium was identified as Paenibacillus illinoisensis KJA-424. The population of P. illinoisensis KJA-424 and chitinase activity significantly increased for the first 2 days of incubation. On SDS-PACE analysis with $0.01\%$ glycol chitin, three protein bands (63, 54, and 38 kDa) with chitinolytic activity were detected tooted. The effect of P illinoisensis KJA-424 on the egg hatch of root-knot nematode (Meloidogyne incognita) was investigated. After 7 days of incubation with the chitinase-producing P. illinoisensis KJA-424, none of the eggs hatched, whereas a $39.8\%$ egg hatching rate was observed in the water control. Inverted and scanning electron microscopic observations demonstrated that P. illinoisensis KJA-424 deformed and destroyed the eggshell of M. incognita. In conclusion, chitinase-produced by p. illinoisensis KJA-424 caused the lysis of M. incognita eggshell and resulted in the inhibition of egg hatching in vitro.

• BMB Reports
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• 제38권1호
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• pp.82-88
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• 2005

Bacillus cereus 28-9 is a chitinolytic bacterium isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. At least two chitinases (ChiCW and ChiCH) were excreted by B. cereus 28-9. The ChiCW-encoding gene was cloned and moderately expressed in Escherichia coli DH5$\alpha$. Near homogenous ChiCW was obtained from the periplasmic fraction of E. coli cells harboring chiCW by a purification procedure. An in vitro assay showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis elliptica, a major fungal pathogen of lily leaf blight.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.470-478
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• 2016

지렁이의 장내로부터 분리한 미생물 중에서 키틴 가수분해 활성이 우수한 미생물을 선발하였으며, 이를 동정하여 Bacillus licheniformis GA9으로 명명하였다. B. licheniformis GA9이 생산하는 키틴 분해효소의 정제는 배양상등액 40-60% 황산암모늄 침전, 음이온교환 크로마토그래피, 겔 크로마토그래피를 사용하여 정제하였다. 최종적으로 정제한 키틴 분해효소는 45.2배로 정제되었고 효소단백질 회수율은 20.0%를 나타내었다. 정제한 키틴 분해효소의 분자량은 약 52.1 kDa으로 나타났으며, N-terminal amino acid sequencing 분석결과, 아미노산 서열은 D-S-G-K-N-G-K-I-I-R-Y-Y-P-IR로 확인되었다. 키틴 분해효소의 최적반응 pH와 pH 안정성을 측정한 결과, pH 5.0에서 최대 활성을 나타내었으며 pH 5.0-6.0에서 안정성을 나타내었다. 키틴 분해효소의 최적반응 온도와 온도안정성의 경우, $40^{\circ}C$에서 최대 활성을 나타내었으며, $60^{\circ}C$까지 60%의 잔존 활성을 나타내었다. 정제한 키틴 분해효소는 10 mM $Co^{2+}$ 금속이온에 의해 효소활성이 증가하였으며, $Fe^{2+}$과 $Cu^{2+}$ 금속이온에 의해 효소활성이 감소하였으나, EDTA 첨가시 감소한 효소활성이 일부 회복되었다. 정제한 효소의 $K_m$ 및 $V_{max}$는 각각 4.02 mg/ml와 0.52 mg/min이었다. 또한 키틴 분해효소는 생명공학, 생물의약, 농업, 식품영양 등 다양한 산업분야에서 응용이 가능하다.

• KSBB Journal
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• 제11권6호
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• pp.696-703
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• 1996

S. marcescens QM 81466 균주는 chitin 분해 효소(1mg/Lmedium)를 선택적으로 높게 생성시킬 수 있는 균주로서, chitin을 N-acetyl-$\beta$-D-glucosa­m mine(NAG)으로 효소적 가수분해를 할 때 chitinase와 chitobiase의 두 가지 가수분해 효소계를 구 성시킨다. 본 연구에서는 이 균주의 chitinase/chitobiase 생성을 위한 chitin 입자크기에 대한 최적화와, 회분 발효계에서 이 균주의 세포 밀도 배양에 따른 두 효소 생성의 변화를 조사하여 NAG 생산성의 증대를 시도하였다. 아울러. chitin과 CM­ chitin이 chitinase/chito biase 생성비 와 NAG 생성 에 미치는 영향을 검토하였는데, CM-chitin을 colloidal 및 결정성 chitin 대신에 사용했을 때, chitinase 활성을 약 7~10U/mL 증가시켰다. 이 경우에 있어서, chitinase/chitobiase의 비는 9:1로 서 NAG의 생성량이 3.0g/L로서 높게 나타났다.

• 식물병연구
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• 제21권3호
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• pp.222-226
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• 2015

The chitinase-producing bacterial strain C-1 is one of the key chitinase-producing biocontrol agents used for effective bioformulations for biological control. These bioformulations are mixed cultures of various chitinolytic bacteria. However, the precise identification, biocontrol activity, and the underlying mechanisms of the strain C-1 have not been investigated so far. Therefore, we evaluated in planta biocontrol efficacies of C-1 and determined the draft genome sequence of the strain in this study. The bacterial C-1 strain was identified as a novel Serratia sp. by a phylogenic analysis of its 16S rRNA sequence. The Serratia sp. C-1 bacterial cultures showed strong in planta biocontrol efficacies against some major phytopathogenic fungal diseases. The draft genome sequence of Serratia sp. C-1 indicated that the C-1 strain is a novel strain harboring a subset of genes that may be involved in its biocontrol activities.

• The Plant Pathology Journal
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• 제21권3호
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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.

• The Plant Pathology Journal
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• 제25권4호
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.