Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E, coll. Operational conditions for maximum production were determined with twelve parameters under ...Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E, coll. Operational conditions for maximum production were determined with twelve parameters under consideration： the concentration of FeCl3, induction at OD578 （optical density measured at 578 nm）, induction time and inoculum concentration. Initially, Plackett-Burman （PB） design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90×10^-3 U/ml by the best combinations of the physicochemical parameters at optimum levels of 0,12 mg/L FeCl3, inoculum concentration of 2.10%, induction at OD578 equal to 1.07, and with 6.05 h of induction.展开更多
Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michae...Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths.Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate,pH 7.4,but sigmoidal kinetics(with an apparent intercept)in low ionic strength buffers and at physiological phosphate concentrations.In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites,with high cooperativity for the binding of the fourth palmitate molecule,and the full-length enzyme showing tighter palmitate binding than the heme domain.The first flavin-to-heme electron transfer is faster for laurate,myristate and palmitate in 100 mmol/L phosphate than in 50mmol/L Tris(pH 7.4),yet each substrate induces similar high-spin heme content.For palmitate in low phosphate buffer concentrations,the rate constant of the first electron transfer is much larger than kcat.The results suggest that phosphate has a specific effect in promoting the first electron transfer step,and that P450_(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.展开更多
文摘细胞色素P450s是用于生物合成的多功能催化剂.在P450催化循环中,需要两个电子来还原血红素铁,并通过电子转移途径(eTPs)激活随后的还原,该步骤是反应的限速步骤.本文重新设计了巨大芽孢杆菌P450 BM3的e TPs,大幅提高了其催化性能.通过在P450 BM3的e TP中引入芳香族氨基酸,“最佳”变体P2H02(A399Y/Q403F)的催化效率比P450 BM3野生型催化效率提高了12.9倍(kcat/KM从65.8 L mol^(-1)s^(-1)提高到913.5 L mol^(-1)s^(-1)).分子动力学模拟和电子传递分析表明,在辅因子FMN和血红素之间引入的芳香族氨基酸可以显著提高电子转移速率和酶催化性能.同时,在电子传递途径中引入酪氨酸可以保护P450的催化中心,使其避免被氧化性中间产物所破坏,从而提高其催化效率.此外,引入芳香族氨基酸的策略被证明对其他P450(如CYP116B3)同样有效,改造后的酶表现出显著提高的催化效率.综上,本文策略有望拓展到其他带有长程电子传递链的氧化还原酶上,成为改造的通用策略.
基金Project (No. 30570411) supported by the National Natural ScienceFoundation of China
文摘Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E, coll. Operational conditions for maximum production were determined with twelve parameters under consideration： the concentration of FeCl3, induction at OD578 （optical density measured at 578 nm）, induction time and inoculum concentration. Initially, Plackett-Burman （PB） design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90×10^-3 U/ml by the best combinations of the physicochemical parameters at optimum levels of 0,12 mg/L FeCl3, inoculum concentration of 2.10%, induction at OD578 equal to 1.07, and with 6.05 h of induction.
基金supported by grants from the Natural Sciences and Engineering Research Council(NSERC)Canada,and the Rhodes Trust(JY)the Higher Educational Funding Council for England.
文摘Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths.Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate,pH 7.4,but sigmoidal kinetics(with an apparent intercept)in low ionic strength buffers and at physiological phosphate concentrations.In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites,with high cooperativity for the binding of the fourth palmitate molecule,and the full-length enzyme showing tighter palmitate binding than the heme domain.The first flavin-to-heme electron transfer is faster for laurate,myristate and palmitate in 100 mmol/L phosphate than in 50mmol/L Tris(pH 7.4),yet each substrate induces similar high-spin heme content.For palmitate in low phosphate buffer concentrations,the rate constant of the first electron transfer is much larger than kcat.The results suggest that phosphate has a specific effect in promoting the first electron transfer step,and that P450_(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
