环己酮单加氧酶的基因挖掘及高效催化合成奥美拉唑的研究

Gene Mining of Cyclohexanone Monooxygenase and Efficient Catalytic Synthesis of Omeprazole

摘要:

【目的】为了提高奥美拉唑的合成效率。【方法】以来源于Acinetobacter johnsonii （A. johnsonii）的CHMO_{NCIMB 9871}和来源于Priestia megaterium （P. megaterium）的P450 BM3为模板，筛选出转化效率最高的单加氧酶并确定其蛋白质诱导的最适条件，通过单因素试验和响应面优化确定该重组酶催化合成奥美拉唑的最优条件。【结果】来源于Pseudomonas sp.的环己酮单加氧酶CHMO_KT2440转化效率最高，其蛋白质诱导的最适条件为：菌体OD₆₀₀值0.6，诱导温度17 ℃，IPTG终浓度0.15 mmol/L。单因素试验结果为：在33 ℃、pH 9.0、FAD添加量0.05 mmol/L、底物质量浓度3.0 g/L、反应时间10 h的条件下，奥美拉唑的产率为78.9%。CHMO_KT2440催化合成奥美拉唑的回归方程为：y=60.78+9.56A-19.74B+9.55C+1.80AB+5.13AC+1.48BC-18.04A²-0.79B²-9.41C²。结合单因素试验结果，确定最优转化条件为：底物质量浓度3.0 g/L，反应温度34 ℃，反应pH 9.0，FAD添加量0.05 mmol/L，反应时间10 h， 在此条件下奥美拉唑的产率高达79.9%，较优化前提高了90.23%。 【结论】 筛选的环己酮单加氧酶CHMO_KT2440为奥美拉唑的合成提供了一个优秀候选酶。

Abstract:

[Objective] The aim of the study is to synthesize omeprazole efficiently. [Method] With CHMO_{NCIMB 9871} from Acinetobacter johnsonii （A. johnsonii）and P450 BM3 from Priestia megaterium （P. megaterium） as templates, the monooxygenase with the highest transformation rate was screened out and the optimal conditions for inducing protein expression were determined. Single factor experiments and response surface methodology were employed to optimize the conditions of this recombinant enzyme for producing omeprazole. [Result] The cyclohexanone monooxygenase CHMO_KT2440 from Pseudomonas sp. synthesized omeprazole with the highest transformation rate. The optimal inducing conditions of CHMO_KT2440 expression were as follows: OD₆₀₀ value of 0.6, induction temperature of 17 ℃, and final IPTG concentration of 0.15 mmol/L. The single factor experiment results showed that the reaction with 0.05 mmol/L FAD, 3.0 g/L substrate mass concentration at 33 ℃ and pH 9.0 for 10 h produced omeprazole with a yield of 78.9%. The CHMO_KT2440-catalyzed synthesis of omeprazole was fitted by the regression equation: y=60.78+9.56A-19.74B+9.55C+1.80AB+5.13AC+1.48BC-18.04A²-0.79B²-9.41C². Based on the single factor experimental results, the transformation conditions were finally optimized as follows: the substrate mass concentration of 3.0 g/L, 34 ℃, pH 9.0, 0.05 mmol/L FAD, and reaction time of 10 h. Under these conditions, the yield of omeprazole reached 79.9%, which increased by 90.23% compared with that before optimization. [Conclusion] This study provides an elite cyclohexanone monooxygenase candidate CHMO_KT2440 for omeprazole synthesis.