• The Korean Journal of Food And Nutrition
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• v.16 no.4
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• pp.437-443
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• 2003

Chitooligosaccharides were prepared by enzymatic hydrolyzing of crab shell chitosan. Low molecular weight chitooligosaccharides(LMW-chitooligosaccharides), 64.3% of which was composed of trimer, tetramer, and pentamer, was obtained by hydrolyzing chitosan with the chitosanase originated Bacillus pumilus BN-262. High molecular weight chitooligosaccharides(HMW-chitooligosaccharides), 49.3% of which was composed of chitooligosaccharides over heptamer, was obtained by hydrolyzing chitosan with the cellulase originated Trichoderma viride. Acute oral toxicity of chitooligosaccharides were tested in mice. Chitooligosaccharides did not have any toxic effect in mice and oral LD$\_$50/ value of chitooligosaccharides was over 5.0g/kg in mice.

• The Korean Journal of Food And Nutrition
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• v.14 no.6
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• pp.579-584
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• 2001

Chitooligosaccharides were prepared by enzymatic hydrolyzing of crab shell chitosan. Low Molecular Meight chitooligosaccharides(LMW-chitooligosaccharides) , 64.3% of which was composed of trimer, tetramer, and pentamer, was obtained by hydrolyzing chitosan with the chitosanase originated Bacillus pumilus BN -262. High Molecular Meight chitooligosaccharides ( HMW-chitooligosaccharides ) , 49.3% of which was composed of chitooligosaccharides over heptamer, was obtained by hydrolyzing chitosan with the cellulase originated Trichoderma viride. Antimicrobial activity and colony forming inhibitory activity of chitooligosaccharides were tested. MIC of LMW-chitooligosaccharides against Bacillus cereus, Bacillus subtilis, Candida albicans, Escherichia coli, Escherichia coli O157 : H7, Lactobacillus plantarum, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus and Streptococcus mutans was 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% and 1.0% respectively. .

• Microbiology and Biotechnology Letters
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• v.23 no.2
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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.

• The Korean Journal of Food And Nutrition
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• v.8 no.1
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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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• v.36 no.1
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• pp.1-6
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• 1993

Chitinase was induced in rice cell suspension culture with treatment of chitooligosaccharides mixture. Among eleven isozymes found in 10% polysacrylamide gel electropherogram, four isozymes were identified as induced enzymes. Acidic chitinase fraction separated in DEAE-cellulose column chromatography, includes three induced chitinase, while basic fraction contains only one induced isozyme. Treatment of chitooligosaccharides mixture enhanced the contents in both protein and chitinase activity in cell suspension culture media, but increase in chitinase activity was much higher than in protein.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.345-351
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• 1998

In an effort to develop a potent system for the production of various dp (degree of polymerization) chitooligosaccharides, 32 enzymes or microbial systems were screened for chitosanolytic acitivity using chitosan as a substrate. The efficiency of each enzyme system was evaluated by the changes of turbidity and viscosity of chitosan solution, the amount of precipitate and the reducing sugar-producing activity in the enzymatic reaction mixture. Based on these assay methods for the chitosanase activity, Bacillus sp. P2l out of 32 screened systems showed highly potent endochitosanase, which was comparable with a commercially available enzyme (E7). Chitooligosaccharides of dp 3-7 were separated by TLC as major enzymatic reaction products, suggesting that the chitosanase from Bacillus sp. P2l be endo-splitting type.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.6
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• pp.696-701
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• 2002

Optimal conditions for preparing of chitooligosaccharides from chitosan with cellulase was researched by response surface methodo-logy, Penicillium funiculosum derived cellulase was most effective for chitooligosaccharides production as the point of hydrolyzing activity and commercial utility. The result which measures the change of degrading ratio at time course, 10 hr reaction showed a exponential increase and after that time degrading ratio was not changed. The optimal conditions determined by response surface methodology with central composite design of total 26 species were $0.5\%$ of chitosan, 143 U enzyme, 49$^{\circ}C$ of reaction temperature, 13.2 hr of reaction time and pH 3.8. Major chitooligosaccharides produced from chitiosan on optimal conditions were dimer and trimer.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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.

• Microbiology and Biotechnology Letters
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• v.46 no.1
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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.