Geng Yawei, Zhang Rongzhen, Wang Shanshan, Xu Yan
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Wei Sheng Wu Xue Bao. 2010 Jan;50(1):60-6.
A novel (S)-specific carbonyl reductase gene (scr II) was cloned from the genome of Candida parapsilosis CCTCC M203011, and its catalytic function for the biotransformation of chiral alcohol was verified.
The possible carbonyl reductase gene scr II was amplified by PCR method from the C. parapsilosis genome. Using the recombinant Escherichia coli BL21/pET28a-scr II as the catalyst and 2-hydroxyacetophenone as the substrate, the biotransformation was carried out. The optical purity and yield of the final product were investigated by HPLC analysis. The optimal pH and temperature of the reaction were also determined.
The gene scr II coded 279 amino acids with an open reading frame of 837 bp. It shared 85% identity with the reported gene scr. By analysis, SCR II contained 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. SDS-PAGE results showed that SCR II was overexpressed at 30 degrees C after the induction of 0.1 mmol/L IPTG. When the concentration of 2-hydroxyacetophenone was 6 g/L, 10% (w/v) wet recombinant E. coli cells showed excellent performance to give (S)-1-phenyl-1,2-ethanediol with high optical purity of 99.1% enantiomeric excess in a yield of 89.60% under the optimal conditions of pH 5.5 and 35 degrees C. SCR II catalyzed the transformation of (S)-1-phenyl-1, 2-ethanediol more efficiently than SCR. When compared with SCR, its substrate concentration was increased by two-fold, and the optical purity and yield of (S)-1-phenyl-1,2-ethanediol were improved by 10% and 28%, respectively.
The gene coding for novel carbonyl reductase SCR II was isolated using the molecular cloning technique and its discovery supplied a solid research foundation for chiral alcohol preparation efficiently.
从近平滑念珠菌CCTCC M203011基因组中克隆一个新的（S）-特异性羰基还原酶基因（scr II），并验证其对手性醇生物转化的催化功能。
采用PCR方法从近平滑念珠菌基因组中扩增可能的羰基还原酶基因scr II。以重组大肠杆菌BL21/pET28a-scr II为催化剂，以2-羟基苯乙酮为底物进行生物转化。通过HPLC分析研究最终产物的光学纯度和产率。还确定了反应的最佳pH值和温度。
基因scr II编码279个氨基酸，开放阅读框为837 bp。它与已报道的基因scr具有85%的同一性。经分析，SCR II包含短链羰基还原酶的两个典型基序，包括一个罗斯曼折叠Thr40-Gly41-（X）3-Gly45-X-Gly47和一个保守的催化三联体Ser172-（X）n-Tyr187-（X）3-Lys191。SDS-PAGE结果表明，在0.1 mmol/L IPTG诱导后，SCR II在30℃下过表达。当2-羟基苯乙酮浓度为6 g/L时，10%（w/v）湿重组大肠杆菌细胞表现出优异的性能，在pH 5.5和35℃的最佳条件下，以89.60%的产率得到对映体过量99.1%的高光学纯度（S）-1-苯基-1,2-乙二醇。SCR II催化（S）-1-苯基-1,2-乙二醇的转化比SCR更有效。与SCR相比，其底物浓度提高了两倍，（S）-1-苯基-1,2-乙二醇的光学纯度和产率分别提高了10%和28%。
利用分子克隆技术分离得到了编码新型羰基还原酶SCR II的基因，其发现为高效制备手性醇提供了坚实的研究基础。
