Screening Of Cyciohexanone Monooxyaes For Catalytic Synthesis Of Omeprazole

Omeprazole,as a proton pump inhibitor,is the main drug in the treatment of acute gastritis.When combined with the proton pump in the stomach,this drug can effectively block gastric acid secretion by reducing gastric pH,gastric mucosal inflammation to promote gastric mucosal repair.In this study,the cyclohexanone monooxygenase was screenced to catalyze the asymmetric synthesis of omeprazole to sulfur omeprazole amidine sulfoxides.The optimal reaction conditions were obtained by single factor experiment and response surface optimization method.The main contents as follows:1)With amino acid sequence of CHMO_{NCIMB 9871}from Acinetobacter sp.NCIMB 9871as a template,seven candidate enzymes were screened.With amino acid sequence of P450BM3 from Priestia megaterium as a template,five candidates were screened.Twelve recombinant plasmids were constructed and transformed into Escherichia coli BL21（DE3）.SDS-PAGE results showed that 12 recombinant enzymes could be expressed in E.coli,and their molecular weights were consistent with their theoretical values.The protein bands of ScCHMO,BsCHMO,CHMO_KT2440,P450 and 4O4P and PmP450 BM3 were more obvious with the higher expression level.2)Twelve candidate enzymes catalyzed the substrate omeprazolthiamidine and the product yield was detected with a high performance liquid chromatography（HPLC）.The results showed that 10 of the 12 candidates had the ability to catalyze omeprazole thiamidin to synthesize omeprazole.The conversion efficiency of omeprazole by CHMO_KT2440 from Pseudomonas sp.reached the highest value of 42.4%.4O4P as a P450 candidate have no ability to synthesize the target product omeprazole,but it could convert the substrate to the by-products at a high yield rate.4O4P was confirmed that the substrate was directely converted to the other by-product without the target product omeprazole through checking the transformation dynamics.3)The protein expression conditions of CHMO_KT2440including the final IPTG concentrations and induction temperature were optimized,and the conversion conditions of omeprazole catalyzed by CHMO_KT2440 were also optimized.The optimal expression conditions were as follows:0.15 mmol×L^-1IPTG,induction temperature 17℃and incubation time 16 h.The optimal conversion conditions of omeprazole by CHMO_KT2440 were obtained as follows:substrate concentration 3 g×L^-1,reaction temperature 33℃,reaction pH 9.0,FAD concentration 0.05 mmol×L^-1,reaction time 10 h.Under the optimal conditions,the final conversion rate reached 78.9%.4)Response surface optimization was carried out for the transformation conditions of omeprazole synthesis catalyzed by CHMO_KT2440.According to the single factor experimental results,the main factors affecting the reaction were determined including reaction temperature,pH and coenzyme addition,and the variation range of these variables was also confirmed.The reaction temperature（A）,pH（B）and coenzyme additionwere were set as independent variables,and the omeprazole yield was taken as dependent variables.The above factors were optimized by response surface methodology（RSM）.The response surface optimization model was significant,and the regression equation of the final fit was solved:Y=60.78+9.56A-19.74B+9.55C+1.8AB+5.13AC+1.48BC-18.04A²-0.79B²-9.41C².The optimal reaction conditions were predicted and combined with the single factor experimental conditions to obtain the optimal transformation conditions as follows:substrate 3 g×L^-1,reaction temperature 34℃,reaction pH 9.0,FAD addition amount 0.05 mmol×L^-1,reaction time 10 h.The final yield reached 79.9%,which was in good agreement with the theoretical predicted value（80.82%）.The protein structure of CHMO_KT2440 was simulated and the substrate and coenzyme were docked into CHMO_KT2440.Its structure was compared with CHMO 5M10,which could only catalyze small molecule substrate cyclohexanone analogues.The results showed that CHMO_KT2440 had a larger substrate binding pocket for accepting macromolecular substrate omeprazole thioamidine.