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- Title
Reduction of m-chlorophenacyl chloride coupled with regeneration of NADPH by recombinant Escherichia coli cells co-expressing both carbonyl reductase and glucose 1-dehydrogenase.
- Authors
Yu, Tao; Li, Jian-Fang; Zhu, Li-Juan; Hu, Die; Deng, Chao; Cai, Yu-Ting; Wu, Min-Chen
- Abstract
Both Sys1 and Sygdh, two codon-optimized genes encoding SyS1 and SyGDH, were synthesized based on the carbonyl reductase (S1) and glucose 1-dehydrogenase (GDH) gene sequences, respectively, from Candida magnoliae and Thermoplasma acidophilum, and co-expressed in Escherichia coli BL21(DE3) using two strategies. One strategy involved a recombinant E. coli strain ( E. coli/ Sygdh- Sys1) constructed by transforming a recombinant plasmid, pETDuet- Sygdh- Sys1, into E. coli BL21. The other strategy involved another recombinant E. coli strain ( E. coli/ Sys1/ Sygdh) obtained by co-transforming the recombinant plasmids pET-22b- Sys1 and pET-28a- Sygdh into E. coli BL21. The enzyme activity assays indicated that the activities of recombinant SyS1 and SyGDH (3.7 and 56.3 U/g wet cells) expressed in E. coli/ Sygdh- Sys1 were higher than those (2.8 and 44.1 U/g wet cells) in E. coli/ Sys1/ Sygdh. Accordingly, E. coli/ Sygdh- Sys1 was chosen, and its whole cells were used as catalysts for the asymmetric reduction of m-chlorophenacyl chloride ( m-CPC) to the corresponding ( R)-2-chloro-1-(3-chlorophenyl)ethanol [( R)-CCE] coupled with the regeneration of NADPH in situ. Under the optimized reaction conditions of 30 mM m-CPC, 50 mg/ml wet cells, 40 mM glucose and 0.2 mM NADP at pH 7.0 and 35 °C for 3 h, ( R)-CCE was obtained with a molar yield of 99.2 % and an enantiomeric excess (e.e.) value of more than 99 %.
- Publication
Annals of Microbiology, 2016, Vol 66, Issue 1, p343
- ISSN
1590-4261
- Publication type
Article
- DOI
10.1007/s13213-015-1114-1