Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108.
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.
Yoon
Ho-Geun
HG
Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, Seoul.
Lee
Kyung-Han
KH
Kim
Hee-Yun
HY
Kim
Hye-Kyung
HK
Shin
Dong-Hoon
DH
Hong
Bum-Shik
BS
Cho
Hong-Yon
HY
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
D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase