文献类型：期刊文献

英文题名：Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp ECU0401 and its potential for (R)-(-)-mandelic acid production

作者：Zhang, Zhi-Jun[1];Xu, Jian-He[1];He, Yu-Cai[2];Ouyang, Li-Ming[1];Liu, You-Yan[3]

机构：[1]E China Univ Sci & Technol, Lab Biocatalysis & Bioproc, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Changzhou Univ, Coll Chem & Chem Engn, Changzhou 213164, Peoples R China;[3]Guangxi Univ, Coll Chem & Chem Engn, Nanning 530004, Guangxi, Peoples R China

年份：2011

卷号：34

期号：3

起止页码：315

外文期刊名：BIOPROCESS AND BIOSYSTEMS ENGINEERING

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000288049700007)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 20672037, 20773038 and 20902023), Ministry of Science and Technology, P.R. China (Nos. 2009ZX09501-016 and 2009CB724706), China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204) and Shanghai Municipal Fund for Natural Science (No. 07ZR14030).

语种：英文

外文关键词：Alcaligenes sp.; Nitrilase; Thermostable; Enantioselective; Biochemical properties; (R)-(-)-mandelic acid

摘要：A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His(6)-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V-max and K-m parameters were determined to be 27.9 mu mol min(-1) mg(-1) protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 degrees C and 94 h at 40 degrees C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(-)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(-)-mandelic acid.