Genetic Engineering，Expression and Characterization of Chitinases from Vibrio parahaemolyticus

• Main Authors: Hung-Wen Wang, 王泓文
• Other Authors: Fu-Pang Lin
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/33273685804960845069

碩士 === 國立海洋大學 === 生物科技研究所 === 91 === The chitinase gene from Vibrio parahaemolyticus was cloned by PCR techniques.The full-length chitinase gene of V. parahaemolyticus was 1863 base pairs and encoded 621 amino acid residues. The gene was expressed in the pET prokaryotic expression system and the recombinant chitinase was enzymatically active. The site-directed deletion mutants of V. parahaemolyticus chitinases VpChiA del-1 and VpChiA del-2 were then performed by PCR based on the amino acid sequence homology comparison of the chitinases among Serratia marcescens, Aeromonas caviae D1 ChiAG561 and V. parahaemolyticus .The recombinant chitinase genes were cloned and were expressed in E. coli RosettaTM(DE3)pLysS.They were efficiently purified from the host cell lysate by His-tag affinity chromatography and were found enzymatically active. The recombinant VpChiA，VpChiA del-1 and VpChiA del-2 have silimar optimal temperature and pH . Kinetic parameters Km and kcat of VpChiA，VpChiA del-1 and VpChiA del-2 were characterized using the 4-MU-(NAG)2 and 4-MU-(NAG)3 as substrate，respectively . By HPLC analysis，the (NAG)，(NAG)2 and (NAG)3 were found in the (NAG)7 or (NAG)8 hydrolysis by VpChiA，VpChiA del-1 and VpChiA del-2 at 37℃ for 24 hr.The major product from these hydrolysis was (NAG)2 .