• 한국양식학회지
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• 제19권1호
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• pp.40-46
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• 2006

Bacillus subtilis strain with highly active chitosanase was isolated from the intestine of Sebastiscus marmoratus (scorpion fish). It was named as Bacillus subtilis CH1 by morphological, biochemical and 165 rRNA gene analysis. The optimal conditions for chitosanase production were investigated. The optimum carbon and nitrogen sources for Bacillus stibtilis CH1 were 2% starch and 1% yeast extract respectively. Unlike other chitosanases, the expression of this chitosanase was not induced or slightly induced with chitosan. The chitosanase secreted into the medium were concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular weight of purified chitosanase was 30 kDa. The optimum pH and temperature of purified chitosanase were 5.5 and $60^{\circ}C$ respectively. The purified chitosanase was continuously thermostable at $40^{\circ}C$ and showed stable activity between pH 6.0 and 8.0. Chitosanase activity of Bacillus subtilis CH1 under optimum condition was 4.1 units/ml.

• Archives of Pharmacal Research
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• 제26권12호
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• pp.1036-1041
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• 2003

Two chitosanases produced by Aspergillus fumigatus KB-1 were purified by ion exchange and size exclusion chromatographies. Molecular weights of chitosanases were 111.23 kDa (chitosanase I) and 23.38 kDa (chitosanase II). The N-terminal amino acid sequence of chitosanase II was determined as follows: YNLPNNLKQIYDKHKGKXSXVLAKGFTN. The optimum pH of the chitosanase I and II was 6.5 and 5.5, respectively. The optimum temperatures were $60^{\circ}C$ for chitosanase land $70^{\circ}C$ for chitosanase II. Hydrolysis products of two chitosanases were analyzed by HPLC and GPC. Chitosanase I hydrolyzed substrate to glucosamine. Chitosanase II produced chitooligosaccharides.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.568-574
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• 1998

Two types of chitosanases produced from Aspergillus fumigatus KH-94 were purified by ion exchange and gel permeation chromatography. Molecular weights of the enzymes are 22.5 kDa (chitosanase I) and 108 kDa (chitosanase II). pI, optimum pH, and temperature of chitosanase I are 7.3, 5.5, and 70-$80^{\circ}C$, respectively, and those of chitosanase II are 4.8, 4.5~5.5, and 50~$60^{\circ}C$, respectively. Activities of both chitosanases were increased by $Mn^{2+}$ but inhibited by $Cu^{2+}$ and $Hg^{2+}$ . Chitosanase I has endo-splitting activity that hydrolyzes chitopentaose, chitohexaose, and chitosan to chitobiose, chitotriose, and chitotetraose, whereas chitosanase II has exo-splitting activity that hydrolyzes chitobiose and chitosan to glucosamine. Chitosanase II was found to have transglycosylation activity also in the reaction of 2% more chitooligosaccharides as a substrate and at the initial reaction. The higher degree of deacetylation, the stronger activities of chitosanase Iand II toward chitosans. Both chitosanases could hydrolyze chitosan and glycol chitosan but not chitin, cellulose, and carboxymethyl cellulose. To produce higher degree of polymerization of chitooligosaccharides, chitosanase I was used and yielded 80% of recovery.

• 한국미생물·생명공학회지
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• 제31권3호
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• pp.216-223
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• 2003

새롭게 분리된 Bacillus cereus H-1으로부터 크기가 45-kDa인 chitosanase를 정제하여 특성을 파악하였고 1.3-kb의 chitosanase 유전자(choA)를 대장균에 클로닝하여 발현시켰다. H-1의 chitosanase 단백질(ChoA)은 ammonium sulfate 침전과 CM-sephadex칼럼 크로마토그래피에 의해 정제하였다. 최적 pH는 약 7이었고 pH 안정성은 $50^{\circ}C$에서 4-11로 나타났다. 최적 온도는 약 5$0^{\circ}C$였으며 효소 활성은 $45^{\circ}C$ 아래에서 비교적 안정하였다. H-1 chitosanase는 soluble 또는 glycol chitosan뿐만아니라 carboxymethyl cellulose(CMC)에 대한 활성도 나타내었다. 정제된 ChoA의 MALDI-TOF MS분석에 기초하여 이미 알려진 다른 Bacillus chitosanases와의 데이터베이스 검색을 통해 전체 아미노산 서열을 밝혀내었다. Chitosanase gene에 해당하는 1.6 kb의 PCR 산물을 얻었으며 그의 DNA 서열을 결정하였다. choA의 추정 아미노산은 Bacillus sp. No 7-M과 Bacillus sp. KCTC0377BP의 아미노산과 98%의 유사성을 나타내었다. 재조합 ChoA단백질은 E. coli DH5$\alpha$에서 원 균주와 동일한 크기로 발현되었다. N말단의 추정아미노산서열을 다른 chitosanas리 서열과 비교해 볼때 ChoA는 chitosanase-cellulase 활성을 갖는 family 8에 속하는 미생물 endo-chitosanaseT. 추정되었다.

• 미생물학회지
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• 제42권2호
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• pp.142-148
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• 2006

본 연구는 다양한 생리활성을 가지고 있는 chitosan oligosaccharides를 효소적 방법으로 생산하기 위한 기초 연구로서, 전통발효식품인 메주에서 chitosan 분해능이 우수한 균주를 분리하였다. 분리한 균주를 형태학적, 생화학적 및 분자생물학적 방법을 이용하여 동정한 결과, Bacillus amyloliquefaciene MJ-1으로 명명하였다. B. amyloliquefaciene MJ-1으로 부터 chitosanase 유전자를 포함하는 1,049 bp DNA 단편을 클로닝하였으며, chitosanase 유전자는 825 염기로서 274 개의 아미노산으로 구성되어 있었고, 예상 분자랸은 30.9 kDa이었다. 클로링한 chitosanase의 homology search 결과, glucoside hydrolase family 46에 속하는 chitosanase로 추정되었다. B. amyloliquefaciene MJ-1 chitosanase 유전자를 E. coli BLR (DE3)에 도입하였으며, 1 mM의 IPTG로 chitosanase 과잉 발현을 유도하고 정제한 후, pH및 온도에 대한 특성을 조사하였다. 효소 활성의 최적 온도는 $60^{\circ}C$이었으며, $80^{\circ}C$에서도 75%의 활성을 나타내었으므로 내열성을 가진 효소로 추정되었다. 한편, 최적 pH는 5.0 이었으며, pH $5{\sim}7$사이에서 80% 이상의 높은 활성을 유지하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제4권1호
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• pp.26-31
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• 1999

The advantages of the organism Streptomycs griseus HUT 6037 is that the chitinase and chitosanase using chitinaceouse substrate are capable of hydrolyzing both amorphous and crystalline chitin and chitosan. We attempted to investigate the optimization of induction protocol for high-level production and secretion of chitosanase and the influence of chitin and partially deacetylated chitosan sources (75∼99% deacetylation). The maximum specific activity or chitinase has been found at 5 days cultivation with the 48 hours induction time using colloidal chitin as a carbon source. To investigate characteristic of chitosan activity according to substrate, we used chitosan with various degree of deacetylation as a carbon source and found that this strain accumulates chitosanase in the culture medium using chitosanaceous substrates rather than chitinaceous substrates. The highest chitosanase activity was also presented on 4 days with 99% deacetylated chitosan. The partially 53% deacetylated chitosan can secrete both chitinase and chitosanase which was defined as a soluble chitosan. The specific activities of chitinase and chitosanase were 0.89 at 3 days and 1.33 U/mg protein at 5 days, respectively. It indicate that chitosanase obtained from S. griseus HUT 6037 can hydrolyze GlcNAc-GlcN and GlcN-GlcN linkages by exo-splitting manner. This activity increased with increasing degree of deacetylation of chitosan. It is the first attempted to investigate the effects of chitosanase on various degrees of deacetylations of chitosan by S. griseus HUT 6037. The highest specific activity of chitosanase was obtained with 99% deacetylated chitosan.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.161.1-161.1
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• 2003

Two chitosanses produced by Aspergillus fumigatus KB-1 were purified by ion exchange and size exclusion chromatographies. Molecular weights of chitosanses were 111.23 KDa (chitosanase I) and 23.38 KDa (chitosanase II). The N-terminal amino acid sequence of chitosanase II was determined: YNLPNNLKQIYDKHKGKXSXVLAK(\ulcorner)GFTN. The optimum pH of the chitosanase I and II were 6.5 and 5.5 respectively. The optimum temperatures were $60^\times$C and $70^\times$. Two chitosanases were most stable at $10^\times$C. (omitted)

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.208-214
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• 2000

A bacterial strain producing extracellular chitosanase was isolated from crab shells and identified as a member of the genus Aureobacterium The production of chitosanase was proportionally related to the microbial growth, induced by the presence of chitosan, and repressed by glucose at 0.5% (w/v) concentration or higher. The optimal culture conditions for the production of chitosase were 3$0^{\circ}C$ and pH 7.0. Among the nitrogen sources tested, incubation with 0.25% (w/v) concentrations of tryptone and casitione showed the best production of chitosanase. The chitosanase of Aureobacterium sp. YL produced chitobiose as a major product and glucosamine, chitotriose, chitotetraose, and chitopentaose as minor products from chitosan.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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• pp.18-24
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• 2001

The advantage of using Streptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystalline chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75-99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 dyas of cultivation with 99% deacetylated chitosan. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)$_3$, (GlcN)$_4$and (GlcN)(sub)5 were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)$_3$was homogeneous and those of (GlcN)$_4$and (GlcN)(sub)5 were heterogeneous.

• Journal of Microbiology and Biotechnology
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• 제21권10호
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• pp.1021-1025
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• 2011

A Bacillus subtilis strain was isolated from the intestine of Sebastiscus marmoratus (scorpion fish) that was identified as Bacillus subtilis CH2 by morphological, biochemical, and genetic analyses. The chitosanase of Bacillus subtilis CH2 was best induced by fructose and not induced with chitosan, unlike other chitosanases. The strain was incubated in LB broth, and the chitosanase secreted into the medium was concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular mass of the purified chitosanase was detected as 29 kDa. The optimum pH and temperature of the purified chitosanase were 5.5 and $60^{\circ}C$, respectively. The purified chitosanase was continuously thermostable at $40^{\circ}C$. The specific acitivity of the purified chitosanase was 161 units/mg. The N-terminal amino acid sequence was analyzed for future study.