Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
复制标题
DOI：
10.1371/journal.pone.0083586
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Xiao R
中科院分区：
文献类型：
--
作者：
Zhang R;Zhang B;Xu Y;Li Y;Li M;Liang H;Xiao R
The NADPH-dependent (S)-carbonyl reductaseII from Candida parapsilosis catalyzes acetophenone to chiral phenylethanol in a very low yield of 3.2%. Site-directed mutagenesis was used to design two mutants Ala220Asp and Glu228Ser, inside or adjacent to the substrate-binding pocket. Both mutations caused a significant enantioselectivity shift toward (R)-phenylethanol in the reduction of acetophenone. The variant E228S produced (R)-phenylethanol with an optical purity above 99%, in 80.2% yield. The E228S mutation resulted in a 4.6-fold decrease in the K M value, but nearly 5-fold and 21-fold increases in the k cat and k cat/K M values with respect to the wild type. For NADPH regeneration, Bacillus sp. YX-1 glucose dehydrogenase was introduced into the (R)-phenylethanol pathway. A coexpression system containing E228S and glucose dehydrogenase was constructed. The system was optimized by altering the coding gene order on the plasmid and using the Shine–Dalgarno sequence and the aligned spacing sequence as a linker between them. The presence of glucose dehydrogenase increased the NADPH concentration slightly and decreased NADP+ pool 2- to 4-fold; the NADPH/NADP+ ratio was improved 2- to 5-fold. The recombinant Escherichia coli/pET-MS-SD-AS-G, with E228S located upstream and glucose dehydrogenase downstream, showed excellent performance, giving (R)-phenylethanol of an optical purity of 99.5 % in 92.2% yield in 12 h in the absence of an external cofactor. When 0.06 mM NADP+ was added at the beginning of the reaction, the reaction duration was reduced to 1 h. Optimization of the coexpression system stimulated an over 30-fold increase in the yield of (R)-phenylethanol, and simultaneously reduced the reaction time 48-fold compared with the wild-type enzyme. This report describes possible mechanisms for alteration of the enantiopreferences of carbonyl reductases by site mutation, and cofactor rebalancing pathways for efficient chiral alcohols production.
登录
查看更多内容
影响因子：
5
作者：
Kataoka, M;Yamamoto, K;Shimizu, S
通讯作者：
Shimizu, S
影响因子：
4.8
作者：
Blazyk, JL;Lippard, SJ
通讯作者：
Lippard, SJ
影响因子：
2.9
作者：
JORNVALL, H;PERSSON, B;GHOSH, D
通讯作者：
GHOSH, D
影响因子：
2.1
作者：
Gröger, H;Hummel, W;Buchholz, S
通讯作者：
Buchholz, S
大肠杆菌中芽孢杆菌 G3 葡萄糖脱氢酶的克隆、表达和表征
影响因子：
--
作者：
Chen, Xuejiao;Ding, Haitao;Zhao, Yuhua
通讯作者：
Zhao, Yuhua