Expression of chitosanase by Bacillus megaterium and Escherichia coli and study on the catalytic activity by chemical modification

• Main Authors: Yueh -Yun Yao, 姚月雲
• Other Authors: Yaw-Kuen Li
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/63792054874749786492

碩士 === 國立交通大學 === 應用化學系 === 91 === Expression of chitosanase by Bacillus megaterium and Escherichia coli and study on the catalytic activity by chemical modification Student：Yueh-Yun Yao Advisor：Dr. Yaw-Kuen Li Department of Applied Chemistry National Chiao Tung University ABSTRACT This study presented the construction of a chitosanase from Bacillus circulans MH-K1 in Bacillus megaterium and E. coli expression systems. The pWH1520 vector and WH320 strain were used as the expression vector and host of the Bacillus system, respectively. Whereas, pET22b(+) vector and BL21(DE3) strain were employed in E. coli system. Both systems have been shown to successfully express the target gene. The recombinant chitosanase can be easily purified by a single Q column chromatography. The potentially essential amino acid residues, E37 and D55, were replaced with cysteine by site-directed mutagenesis. Two mutated enzymes, E37C and D55C, were successfully labeled with 2,2-dithiodiacetic acid, 3,3-dithiodipropionic acid, and 4,4-dithiodibutanoic acid through the formation of a disulfide bond between protein and the dithioalkyl acids. ESI-MS analyses confirmed that the mutant enzymes can only be labeled stoichiometrically. Although all of the mutants (E37C, D55C) and the chemical-modified enzymes retained only less than 1% of the wild type enzyme, their enzymatic products, DP2 and DP3, are virtually identical to that of wild type chitosanase. Since the wild type chitosanase is an inverting enzyme. Chemical labeling at the essential groups may shorten the distance of the two catalytic residues, with which the mechanism may be converted into a retaining type. Unfortunately, no transferase activity can be observed for all chemically labeled enzymes. Low enzymatic activity limited the possibility to observe the anomeric feature by time-course NMR study. The expected retaining mechanism of the modified enzymes cannot be confirmed.