Gene Cloning And Expression Of A Novel (s)- Carbonyl Reductase From Candida Parapsilosis

Carbonyl reductases are considered as a group of promising biocatalysts with high chemo-, regio-, and stereo-selectivity. Researches on gene cloning and expression of microbial carbonyl reductases are of great significance or value for both the development of carbonyl reductases to apply in industry and the explanation of molecular mechanism of asymmetric reduction for carbonyl reductases. In the present study, a novel (S)-specific carbonyl reductase gene (scrⅡ) was cloned from Candida parapsilosis CCTCC M203011 and overexpressed in Escherichia coli BL21 and Pichia pastoirs GS115. Using 2-hydroxyacetophenone as substrate, enzymatic properties of recombinant enzyme were investigated. When using whole recombinant cells as catalysts, reaction conditions for the reduction of 2-hydroxyacetophenone were optimized. In order to gain efficient whole-cell catalyst, (S)-specific carbonyl reductase (SCRⅡ) and glucose 6-phosphate dehydrogenase (G6PDH) were coexpressed in P. pastoirs to strengthen cofactor regeneration. The research in details are as follows:(1) Sequence alignment results showed that a possible gene was found in C. parapsilosis genome, it shared 85% identity with the reported gene scr (GenBank ID: DQ675534). The gene coded 278 amino acids with an open reading frame of 840 bp. By analysis, SCRⅡcontained two typical motifs of the short-chain carbonyl reductase, including a Rossmann-fold Thr40-Gly41-(X)₃-Gly45-X-Gly47 and a conserved catalytic triad Ser172-(X)_n-Tyr187-(X)₃-Lys191. This indicated SCRⅡbelonged to the short-chain carbonyl reductase family.(2) SCRⅡwas overexpressed in E.coli BL21 (DE3). When using 2-hydroxyacetophenone as substrate, 10% (w/v) of wet recombinant E. coli cells showed excellent performance to give (S)-1-phenyl-1,2-ethanediol (PED) with high optical purity of 99.1% enantiomeric excess in a yield of 89.6% under the optimal conditions.(3) Recombinant carbonyl reductase (EcSCRⅡ) was purified and its specific activity was 6.12 U/mg. The The optimal pH and temperature of EcSCRⅡwere 6.0 and 35°C, respectively. It showed good pH stability from 5.0 to 7.0 and good thermal stability when the temperature was below 40°C. However, Co²⁺, Cu²⁺, Ni²⁺ and Zn²⁺ had negative effect on the enzyme activity.(4) Analysis of quaternary structure suggested that Ala220 and Glu228 were adjacent to the substrate-binding loop. The A220 and E228 mutations almost lost activity for the reduction of 2-hydroxyacetophenone. The catalytic efficiencies (Kcat/Km) for the A220 or E228 variants were 7% that of the unmutated enzyme.(5) In order to gain more efficient whole-cell catalyst, a recombinant P. pastoris/pPIC3.5K-scrⅡwas also constructed to express SCRⅡ. When using 6 g/L of 2-hydroxyacetophenone as substrate and 10% (w/v) of wet P. pastoris cells as catalyst, it took only 24 hours to obtain (S)-PED with 100% e.e in a yield of 93.1%. Compared to the recombinant E.coli, not only was the catalytic efficency improved, but also the reaction time decreased by half.(6) To improve catalytic stability, the gene ZWF1 from Saccharomyces cerevisiae was integrated into the genome of P. pastoris/pPIC3.5K-scrⅡand another recombinant cells P. pastoris/SCRⅡG was constructed to coexpresse SCRⅡand glucose 6-phosphate dehydrogenase (G6PDH). When 5% (w/v) of glucose was added to reaction system, the catalytic stability of P. pastoris/SCRⅡG was improved a lot. For the reduction of 2-hydroxyacetophenone, this recombinant P. pastoris cells could be reused for ten times, the optical purity and yield of (S)-PED kept at 100% e.e. and above 85%, respectively.