• 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.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.191-195
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• 2003

The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diretyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N'-diacetyl chitobiose was about $60\%$. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylaton, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of tie sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.19-25
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• 1996

Chitosanase from Bacillus sp. HW-002 was purified with CM-cellulose column chromatography, and HPLC with DEAE- TSK gel and YMC-pack Diol 120. The purified enzyme appeared as a single band on SDS-polyacrylamide gel. The molecular weight of the enzyme was estimated to be about 46 kDa on SDS-polyacrylamide gel, and was estimated to be about 23 kDa by GFC. The optimal pH of chitosanolytic activity was about pH 5.5-6.0, and the purified enzyme was most stable at pH 5.0. The optimal temperature of chitosanolytic activity was $65^{\circ}C$ and the enzyme was stable at $45^{\circ}C$ for 1 h. Chitosan was the most favorable substrate among various $\beta$-glucan. UVmax of the purified enzyme was 195 nmand was not noted around 280 nm. The main product of enzyme reaction with chitosan was chitobiose.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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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.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.563-566
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• 2000

In order to obtain microbial endochitosanase for enzymatic production of chitooligosaccharides from chitosan, we screened four microbes from soil and selected. Bacillus sp. HSB-21 which showed highest activity. Chitosanase, produced from isolating microbe, was endo-type and molecular mass of the enzyme was estimated as 21,000 by active staining. Its optimum pH and temperature were 5.5 and $50^{\circ}C$, respectively. It was stable in the pH range of 3.0 to 8.0 and up to $40^{\circ}C$. It did not produce chitomonosaccharide and produced chitooligosaccharide ranging from chitobiose to chitooctaose as major end-products from chitosan. The chitosanase from Bacillus sp. HSB-21 can be applicable to enzymatic production of chitooligosaccharide which has high degree of polymerization .

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.38-42
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• 2002

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from the Serratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids. Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide and N-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a $98\%$ similarity to that of S. marcescens OMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N'-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount of N-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer $(analogue\;of\;NAG_2)$, thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were $50^{\circ}C$ and 5.0, respectively.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.358-361
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• 2009

The purified endochitosanase(Mw 41 kDa) from bacterium Bacillus cereus D-11 hydrolyzed chitooligomers $(GlcN)_{5-7}$ into chitobiose, chitotriose, and chitotetraose as the final products. The minimal size of the oligosaccharides for enzymatic hydrolysis was a pentamer. To further investigate the cleavage pattern of this enzyme, chitooligosaccharide alcohols were prepared as substrates and the end products of hydrolysis were analyzed by TLC and HPLC. The chitosanase split $(GlcN)_4GlcNOH$ into $(GlcN)_3+(GlcN)_1GlcNOH$, and $(GlcN)_5GIcNOH$ into $(GlcN)_4+(GlcN)_1GlcNOH$ and $(GlcN)_3+(GlcN)_2GlcNOH$. The heptamer $(GlcN)_6GlcNOH$ was split into $(GlcN)_5$ [thereafter hydrolyzed again into $(GlcN_3+(GlcN)2]+(GlcN)_1GlcNOH$, $(GlcN)_4+(GlcN)_2GlcNOH$, and $(GlcN)_3+(GlcN)_3GlcNOH$, whereas $(GlcN)_{1-3}GlcNOH$ was not hydrolyzed. The monomers GlcN and GIcNOH were never detected from the enzyme reaction. These results suggest that D-11 chitosanase recognizes three glucosamine residues in the minus position and simultaneously two residues in the plus position from the cleavage point.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.560-567
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• 1998

We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.

• Applied Biological Chemistry
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• v.17 no.2
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• pp.125-137
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• 1974

The chemical structure of glycolipid of Selenomonas ruminantium cell wall was to be elucidated. The bacterial cells were treated in hot TCA and the glycolipid fractions were extracted by the solvent $CHCl_3\;:\;CH_3OH$ (1 : 3). The extracted glycolipids fraction was further separated by acetone extraction. The acetone soluble fraction was named as the spot A-compound. The acetone insoluble but ether soluble fraction was named as the spot B-compound. These two compounds were examined for elucidation of their chemical structure. The results were as follows: 1. The IR spectral analysis showed that O-acyl and N-acyl fatty acids were linked to glucosamine moiety in the spot A-compound. However in the spot B-compound in addition to O and N-acyl acids phosphorus was shown to be attached to glucosamine. 2. It was recognized by gas liquid chromatography that spot A compound contained beta-OH $C_{13:0}$ fatty acid in predominance in addition to the fatty acid with beta-OH $C_{9:0}$, whereas the spot B compound was composed of the predominant fatty acid of beta-OH $C_{13:0}$ with small amount of beta-OH $C_{9:0}$. 3. According to the paper chromatographic analysis of hydrazinolysis products of the spot A compound, a compound of a similar Rf value as the chitobiose was recognized, which indicated a structure of two molecules glucosamine condensed. The low Rf value of the hydrazinolysis product of the spot B-compound confirmed the presence of phosphorus attached to glucosamine. 4. The appearance of arabinose resulting from. ninhydrin decomposition of the acid hydrolyzate of the spot A compound indicated that the amino group is attached to $C_2$ of glucosamine. 5. The amount of glucosamine in the N-acetylated spot A compound decreased in half of the original content by the treatment. with $NaBH_4$, indicating that there are two molecules of glucosamines in the spot A compound. The presence of 1, 6-linkage between two molecules of glucosamine was suggested by the Morgan-Elson reaction and confirmed by the periodate decomposition test. 6. By the action of ${\beta}-N-acetyl$ glucosaminidase the N-acetylated spot A compound was completely decomposed into N-acetyl glucosamine, whereas the spot B compound was not. This indicated the spot A compound has a beta-linkage. 7. When phosphodiesterase or phosphomonoesterase acted on $^{32}P-labeled$ spot B compound, $^{32}P$ was not released by phosphodiesterase, but completely released by phosphomonoesterase. This indicated that one phosphorus is linked to glucosamine moiety. 8. The spot A compound is assumed to have the following chemical structure: That is glucosaminyl, ${\beta}-1$, 6-glucosamine to which O-acyl and N-acyl fatty acids are linked, of which the predominant fatty acid is beta-OH $C_{13:0}$ fatty acid in addition to beta-OH $C_{9:0}$ fatty acid 9. The spot B compound is likely to have the linkage of $glucosaminyl-{\beta}-1$, 6-glucosamine to which phosphorus is linked in monoester linkage. Furthermore both O-acyl and N-acyl fatty acids contained beta-OH $C_{13:0}$ fatty acid predominantly in addition to beta-OH $C_{9:0}$ fatty acid.

• Journal of Life Science
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• v.12 no.1
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• pp.77-86
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• 2002

Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.