• 한국식품영양학회지
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• 제16권4호
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• pp.437-443
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• 2003

Chitosan을 Bacillus pumilus BN-262 유래의 chitosanase로 처리하여 trimer, tetramer, pentamer가 전체 올리고당 중 64.3%에 달하는 비교적 저분자의 chitooligosaccharides로 구성된 LMW-chitooligosaccharides를 얻었으며, chitosan을 Trichodema viride 유래의 cellulase로 처리하여 중합도 7 이상의 것이 전체 올리고당 중 49.3%에 달하는 상대적으로 분자량이 큰 chitooligosaccharides로 구성된 HMW-chitooligosaccharides 를 얻었다. 제조된 Chitooligosaccharides의 마우스에 대한 급성경구독성을 조사하였다. Chitooligosaccharides는 마우스에 대하여 어떠한 독성 효과도 보이지 않았으며 반수치사량은 5g/kg 이상인 것으로 추정되었다.

• 한국식품영양학회지
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• 제14권6호
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• pp.579-584
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• 2001

Chitosna을 Bacillus pumilus BN-262 유래의 chitosanase로 처리하여 trimer, tetramer, pentamer가 전체 올리고당 중 64.3%에 달하는 비교적 저분자의 chitooligosaccharides로 구성된 LMW-chitooligosaccha-rides를 얻었으며, chitosan을 Trichoderma viride 유래의 cellulase로 처리하여 중합도7이상의 것이 전체 올리고당 중 49.3%에 달하는 상대적으로 분자량이 큰 chitooligosaccharides로 구성된 HMW-chitooligosac-charides를 얻었다. 제조된 chitooligosaccharides의 항균성과 콜로니 형성 저해 활성을 측정하였다. LMW chitooligosaccharides의 Bacillus cereus, Bacillus subtilis, Candida albicans, Escherichia coli, Escherichia coli O157:H7, Lactobacillus plantarum, Listeria mo-nocytogenes, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Streptococcus mutans에 대한 MIC(최소저해농도)는 각각 1.5%, 1.5%, above 2.0%, 1.5%, 1.5%, below 0.5%, 2.0%, 1.5%, above 2.0%. 1.0%, 1.5%, 1.0%였으며, HMW-chitooligosaccharides의 Bacillus cereus, Bacillus subtilis, Candida albicans, Esche-richia coli, Escherichia coli O157 : H7, Lactobacillus plantarum, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enteritidis, Samlmonella typhimurium, Staphylococcus aureus, Streptococcus mutans에 대한 MIC는 각각 1.0%, 1.0%, 2.0% 이상, 1.0%, 1.0%, 0.25% 이하, 1.0%, 1.0%, 2.0%, 1.0%, 1.0%, 0.5%였다.

• 한국미생물·생명공학회지
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• 제23권2호
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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.

• 한국식품영양학회지
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• 제8권1호
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• pp.43-49
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• 1995

This studies were carried out to purification and seperation of chitooligosaccharides which containing excellent biological active substance. After deacetylation of chitosan (DAC%), DAC-45%, DAC-70%, DAC-95% and DAC-99% were used substrates and hydrolyzed by chitosanase (Bacillus pumilus BN-262) DAC-99% has excellent hydrolyzate which contained several chitooligosaccharides. Therefore, chitosan was hydrolyzed DAC-90 as substrate by chitosanase, and then purified and seperated of chitooligosaccharides Gel filteration and HPLC. This oligosaccharides composed with GlcN0, GlcN2, GlcN3, Glc5 and GlcN6.

• Applied Biological Chemistry
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• 제36권1호
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• pp.1-6
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• 1993

Chitooligosaccharides 혼합물을 처리한 벼 세포 현탁배양액과 처리하지 않은 배양액의 단백질 및 효소활성을 비교하여 이 elicitor에 의해 유도되는 chitinase를 확인하였다. Chitooligosaccharide 혼합물 처리로 chitinase 활성은 비처리구에 비하여 3.5배 증가하였으며 증가율이 단백질 증가보다 10배 이상 높아 본 효소가 선택적으로 유도되는 것으로 나타났다. Polyacrylamide gel 전기영동상 나타나는 총 11종의 chitinase중 4종이 유도효소로 판명되었으며 DEAE-cellulose chromatography 결과 3개는 $26{\sim}58\;KD$의 분자량을 가진 acidic chitinase 분획에, 나머지 1개는 basic chitinase 분획에 속한 것으로 나타나서 주로 acidic chitinase가 유도되는 것으로 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.256-261
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• 2004

In order to detect chitooligosaccharides (COS) in soybean paste, tandem immunoaffinity chromatography and enzyme-linked immunosorbent assay (ELISA) were developed. Polyclonal anti-chitooligosaccharides mixture (CaSM) antibody specific to COSM was attached to Sepharose gel for initial sample cleanup and concentration of COS in soybean paste. COS was eluted and quantified by competitive direct ELISA (cdELISA). Average ELISA recoveries from the column using binding buffer spiked with COSM at levels of 0.5, 2.0, 5.0, and $10.0\mu$g/ml were 79.8, 72.0, 77.7, and 60.6%, respectively, with a mean recovery of 72.5%. Mean inter-well and inter-assay coefficients of variation (CV) were 7.7% and 10.3%, respectively. Average recoveries from soybean paste spiked with COSM at levels of 2, 6, 20, and $60\mu$g/g were 115, 91.7, 91, and 73.3%, respectively, with a mean recovery of 92.8%. Mean inter-well and inter-assay CV were 12.9% and 16%, respectively. The COS was detected from 24 out of 25 homemade Korean soybean paste samples at an average of $14.0\mu$g/g (n, 25; range, $0-51.2 \mu$g/g) and from 13 out of 14 commercially made soybean paste samples at an average of $4.1\mu$g/g(n, 14; range, $0-18.4\mu$g/g). The tandem immunoaffinity chromatography-cdELISA that was developed in this study showed that the level of COS eluted from homemade soybean paste was higher than that of the commercially made ones. In addition, the level of COS eluted from commercially available soybean paste in Korea was higher than that of the ones in Japan.

• 한국미생물·생명공학회지
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• 제26권4호
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• pp.345-351
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• 1998

다양한 중합도의 키토산올리고당의 생산에 적합한 효소원을 개발하기 위해서 23종의 시판효소와 토양에서 분리한 8종의 Bacillus sp.와 1종의 Aspergillus sp.가 생산하는 조효소에서 키토산 분해효소의 활성을 검색하였다. 각 효소의 키토산에 대한 가수분해활성은 탁도의 변화, 반응 후에 생성되는 침전물의 양, 총환원당 생성능력, 점도의 감소 속도 등을 기준하여 평가하였다. 시험한 효소원 가운데서 키토산에 대한 강한 분해활성을 가지는 효소는 P2l이 생산하는 것이었다. 이 균주가 생성하는 chitosanase는, HPLC와 TLC에 의한 분해산물의 분석과, 점도 변화의 측정과 활성염색 등에 근거하여, 주로 내부 가수분해활성을 갖는 것으로 판단되었다. 이 효소는 키토산 올리고당의 생산에 적합한 효소 소재로 가능성이 큰 것으로 평가되었다.

• 한국수산과학회지
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• 제35권6호
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• pp.696-701
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• 2002

Cellulase를 이용하여 키토산 올리고당을 제조하기 위한 최적조건을 반웅표면 분석법을 이용하여 설정하였다 키토산 분해능과 산업적 활용의 측면에서 유리한 Pen. funiculosum 유래의 cello-lase를 예비 실험을 통해 결정하였다. 이 효소의 적정 반응 조건에서 경시적 키토산 분해율을 측정한 결과, 반응 10시간까지는 대수적 증가를 보였으나, 10시간 이후로는 완만한 분해율을 나타내었다. 중심합성계획에 의해 총 26개의 실험을 행한뒤 반응표면분석법으로 설정한 키토산 분해 최적 조건은 기질 농도 $0.5\%$에 대해 효소 농도 143(U), 반응 온도 $49^{\circ}$, 반응 시간 13.2hr및 pH는 3.8이었다. 본 연구에서 설정된 최적의 조건에서 얻어진 키토산 분해물은 dimer와 trimer가 주된 올리고당이었고, 소량의 tetramer와 hexamer 그리고 monomer 등의 총 8종 정도의 올리고당이 검출되었다.

• 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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• 제46권1호
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• pp.45-50
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• 2018

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.