文献类型：期刊文献

英文题名：Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production

作者：Fan, Li-Qiang[1];Li, Ming-Wei[1];Qiu, Yong-jun[1,2];Chen, Qi-ming[1,2];Jiang, Si-Jing[1];Shang, Yu-Jie[1];Zhao, Li-Ming[1,2]

机构：[1]East China Univ Sci & Technol, R&D Ctr Separat & Extract Technol Fermentat Ind, State Key Lab Bioreactor Engn, Sch Biotechnol, Shanghai 200237, Peoples R China;[2]SCICBT, Shanghai 200237, Peoples R China

年份：2018

卷号：278

起止页码：1

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20181805132115);WOS:【SCI-EXPANDED(收录号:WOS:000434095300001)】；

基金：This work was partially supported by the National High Technology Research & Development Program of China (863 Program) (No. SS2014AA021202), "Shuguang Program" project supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (NO. 15SG28), and Fundamental Research Funds for the Central Universities (222201717026).

语种：英文

外文关键词：Glutamate decarboxylase; gamma-Aminobutyrate; Site-directed saturation mutagenesis; Thermostability; Hydrogen bonds

摘要：Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert L-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 degrees C, 8.7 degrees C rise in melting temperature (Tm) and a 14.3 degrees C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T-50(15)), compared to wild-type enzyme. Protein 3D structure analysis showed that the induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in E. coli GadB homo-hexamer may be responsible for the improved thermostability. Increased thermostability contributed to increased GABA conversion ability. After 12 h conversion of 3 mol/L L-glutamate, GABA produced and mole conversion rate catalyzed by M6 whole cells was 297 g/L and 95%, respectively, while those by wild-type GAD was 273.5 g/L and 86.2%, respectively.