title: Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108.
abstract: The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90 degrees C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.
authors.affiliation: Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, Seoul.
authors.forename: Ho-Geun
authors.initials: HG
authors.lastname: Yoon
authors.forename: Kyung-Han
authors.initials: KH
authors.lastname: Lee
authors.forename: Hee-Yun
authors.initials: HY
authors.lastname: Kim
authors.forename: Hye-Kyung
authors.initials: HK
authors.lastname: Kim
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Shin
authors.forename: Bum-Shik
authors.initials: BS
authors.lastname: Hong
authors.forename: Hong-Yon
authors.initials: HY
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase