Abstract
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.
Highlights
Active alcohols are very useful chiral blocks in the special chemical and pharmaceutical industries [1,2,3]
This study demonstrates that it is possible to tailor WT SCRII, using a single mutation to yield a “synzyme”, which alters the enantiopreference of the enzyme and catalyzes acetophenone reduction to produce (R)-PE
The enzyme glucose dehydrogenase (GDH) was introduced into the pathway of (R)-PE with the mutant E228S SCRII for cofactor regeneration
Summary
Active alcohols are very useful chiral blocks in the special chemical and pharmaceutical industries [1,2,3]. Many catalysts including the microbial cell systems and carbonyl reductases have been reported to have improved functions after rational design, there are suffer several problems with regards to achieving effective enzymatic reduction. Cofactor regeneration has been successfully applied in vitro for the production of optically active alcohols by the introduction of glucose dehydrogenase (GDH) [24,25]. To satisfy the growing demand for novel reductive biocatalysts, we focus on the redesigning new function of SCRII by site mutation in or adjacent to the substrate-binding pocket located between αF and αFG [14,30]. The newly designed coexpression system altered the substrate specificity, and improved the enantioselective behavior of the desired enzyme. The system stimulated significant increases in acetophenone reductive activity and the yield of the product, (R)-PE, while simultaneously reducing the reaction time from 48 h to 1 h, when 0.06 mM NADP+ was added at the beginning of the reaction
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