• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.35-42
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• 2003

A 1,3 kb cellulase gene encoding novel bifunctional cellulase-chitosanase activity was cloned from biopolymer-producing alkali-tolerant B. lichenformis NBL420 in E. coli. A recombinant cellulase-chitosanase, named CelA, was expressed and purified to homogeneity. The activity staining and the enzymatic characterization of the purified CeIA revealed bifunctional activities on carboxymethyl cellulose (CMC) and glycol-chitosan. The similar characteristics of the enzymatic activities at the optimum pH, optimum temperature, and thermostability Indicated that CelA used a common catalytic domain with relaxed substrate specificity. A comparison of the deduced amino acids in the N-terminal region revealed that the mature CelA had a high homology with the previously identified bifunctional cellulase-chitosanase of Myxobacter sp. AL- 1.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.12-18
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• 1996

A chitosanase-producing bacteria was isolated on chitosan agar plate from soil samples. The strain was spore-forming gram positive bacteria, catalase positive, and rod shape. The strain was identified as Bacillus cereus. The strain did not need an inducer for the synthesis of chitosanase. Chitosanase from Bacillus sp. HW-002 was constitutive enzyme. The optimal medium for the production of the enzyme was composed of 0.5$\%$ sucrose and $1.5\%$ yeast extract-tryptone (1:1 w/w) mixture at pH 6.5. After Bacillus sp. HW-002 was cultivated at $32^{\circ}C$ for 32 h, maximal productivity was gained to be about 27, 200 U/l. Chitosanase from Bacillus sp. HW-002 was a mixed growth-linked metabolite.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.337-343
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• 2004

A bacterial strain concurrently producing extracellular chitosanase and chitinase was isolated from soil and identified as a member of the $\beta$-subgroup of Proteobacteria through its 16S rRNA analysis and some biochemical analyses. The newly discovered strain, named as KNU3, had 99% homology of its 16S rRNA sequence with chitinolytic $\beta$-Proteobacterium CTE108. Strain KNU3 produced 34 kDa of chitosanase in addition to two chitinases of 68 kDa and 30 kDa, respectively. The purified chitosanase protein (ChoK) showed activity toward soluble, colloidal, and glycol chitosan, but did not exhibit any activity toward colloidal chitin. The optimum pH and temperature of ChoK were 6.0 and $70^{\circ}C$, respectively. The chitosanase was stable in the pH 4.0 to 8.0 range at $70^{\circ}C$, while enzyme activity was relatively stable at below $45^{\circ}C$. MALDI-TOF MS and N-terminal amino acid sequence analyses indicated that ChoK protein is related to chitosanases from Matsuebacter sp. and Sphingobacterium multivorum. HPLC analysis of chitosan lysates revealed that glucosamine tetramers and hexamers were the major products of hydrolysis.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.16-21
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• 2004

Six bacterial strains which formed large halo on chitosan-containing agar plate were isolated from beach mud and crabs at South coast of Korean peninsula. They were designated as Bacillus cereus KNUC51, B. cereus KNUC52, B. cereus KNUC53, B. cereus KNUC54, B. cereus KNUC55, and Paenibacillus favisporus KNUC56 by analysing their morphologies and 16S rDNA sequences. Chitosanase activities of all isolates were similar to that of B. subtilis 168. To enhance the activity of chitosanase, a powerful mutagen, MNNG was treated for P favisporus KNUC56. Three mutants showed higher activity of chitosanase than that of the original strain. The DNA fragments containing chitosanase gene from B. cereus sources were cloned, sequenced, and their deduced amino acid sequence analysis showed over 93% homologies with that of the known B. cereus ATCC14579. Extracellular sample from the isolates was incubated in proper reaction mixture including chitosan for 5 minutes at $37^{\circ}C$ to produce 3-10 chitosan oligomers which has been known to be active for clinical agents and agronomical agents.

• Applied Biological Chemistry
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• v.48 no.1
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• pp.16-22
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• 2005

Many chitosanases, glycosyl hydrolases that catalyze the degradation of chitosan, have been found in microorganism. In this paper, classification of the enzyme has been described, which is based on the amino acid sequence (families) and splitting patterns (subclasses). Glycohydrolytic mechanisms such as inversion and retention of the substrate anomer are also discussed in context of the families. Interrelationship among the primary structure, clan, anomeric conversion and the splitting patterns has been suggested. In addition, advanced definition of chitosanase was introduced through the investigation of enzymatic products from partially N-acetylated chitosan as a substrate.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.631-636
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• 1999

The thermostable endo-chitosanase gene from the isolated strain Bacillus sp. KFB-C108 was identified on the basis of a phylogenetic analysis of the 16S rRNA gene sequence, and was cloned into plasmid pUCl8 using E. coli $DH5\alpha$ as the host strain. Positive clones carrying recombinant plasmids (pKCHO I and pKCHO II) containing chitosanase activity were selected using the direct activity staining method. Detailed physical maps showed the two plasmid inserts were identical except that the KCHO II insert (2.6 kb) was 1.8 kb smaller than that of the KCHO I. The recombinant plasmids were analyzed to determine the essential region for chitosanase activity, and a 1.3-kb fragment (KCHO-6) was subcloned into pTrc99A using the EcoRI and BamHI sites to construct pTrc99A/KCHO-6(pTrEB13). The resulting plasmid exerted high chitosanase activity upon transformation of E. coli $DH5{\alpha}cells$, overproducing about 20 times more in the cloned cells than in the wild-type cells. The cloned chitosanase protein exhibited the same molecular weight and catalytic activity similar to those of Bacillus sp. KFB-C108. The cloned enzyme was an endo-type that produced a chitosan tetramer as the major reaction product; however, it produced no monomers or dimers.

• 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.

• 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 .

• 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.