以巨型桿菌及大腸桿菌系統表現幾丁聚醣酵素及其化學修飾後的催化性質之研究

Abstract

以巨型桿菌及大腸桿菌系統表現幾丁聚醣酵素及其化學修飾後的催化性 質之研究 學生：姚月雲 指導教授：李耀坤 博士 國立交通大學應用化學研究所 摘要 本研究首先將Bacillus circulans MH-K1幾丁聚醣酵素的基因(cns)重建在枯草桿菌以及大腸菌表現系統。目前已利用載體pWH1520，於枯草桿菌株之巨型細胞 (Bacillus megaterium WH320)中表現此重組蛋白質。而大腸桿菌部份，則成功地利用載體pET22b(+)，於BL21 (DE3) 胞內進行大量表現，經純化後可得約90 %的純度。 在催化特性研究方面，由於活化中心之催化殘基 (general acid和general base)的距離在催化之向位決定扮演了重要的角色，因此，欲利用突變及化學修飾法，將此間距縮短，使general base residue更靠近反構形中心（anomeric center）。目前已完成E37C、D55C和E37C/D55C突變酵素，更進一步利用標定物質如：Iodoacetic acid、2,2-dithiodiacetic acid、3,3-dithiodipropionic acid及4,4-dithiobutanoic acid完成不同鏈長之化學修飾。透過DNS活性測試及黏度測試分析其反應速率初步得知，其反應性強弱分別為D55C-G > D55C-P > D55C-B > D55C，而在突變株E37C的部份，則是E37C-P > E37C-B > E37C-G > E37C。產物仍維持為幾丁二醣與幾丁三醣。Wild type之反應機制由NMR實驗證實為反轉型(inversion)機制，而突變株酵素與其化學修飾後之酵素的催化機制仍需進一步研究。

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.