The P450 cytochrome monooxygenase CYP102A1 from Bacillus megaterium, better known as P450BM3, is a heme-thiolate enzyme that catalyzes the hydroxylation of numerous substrates. Many of the resulting products are of commercial interest to the pharmaceutical and fine chemical industries. Unlike most other P450 cytochromes, P450BM3 is both soluble and fused to its natural redox partner, which supplies the necessary electrons from NADPH to drive its reaction forward. However, the industrial use of this enzyme is limited by its poor stability and its expensive cofactor. In this work, we explore the effects of buffer formulation and temperature on the stability of wildtype and P450BM3 mutant R966D/W1046S as well as on the stability of nicotinamide cofactors NADPH, NADH, and the biomimetic cofactor N-benzyl-1,4-dihydronicotinamide. We demonstrate that cofactor stability is more important to increase product yield than that of the enzyme. We also demonstrate that low temperatures enhance oxido-reduction reactions coupling, thus resulting in an increase in the molar ratio of p-nitrophenolate produced from 10-pNCA per oxidized cofactor. Overall, the optimized reaction conditions lead to a 2 to 2.6-fold increase in total product output when wildtype P450BM3 or R966D/W1046S mutant is used with either of these three aforementioned cofactors.

中文翻译：

---

优化操作条件以提高细胞色素 P450BM3 酶促反应产率

巨大芽孢杆菌的 P450 细胞色素单加氧酶 CYP102A1，更好地称为 P450BM3，是一种血红素硫醇酶，可催化许多底物的羟基化。许多由此产生的产品对制药和精细化工行业具有商业价值。与大多数其他 P450 细胞色素不同，P450BM3 既可溶又融合到其天然氧化还原伴侣上，后者从 NADPH 提供必要的电子以推动其反应进行。然而，这种酶的工业应用受限于其较差的稳定性和昂贵的辅因子。在这项工作中，我们探讨了缓冲液配方和温度对野生型和 P450BM3 突变体 R966D/W1046S 稳定性以及烟酰胺辅助因子 NADPH、NADH 和仿生辅助因子 N-benzyl-1,4-dihydronicotinamide 稳定性的影响. 我们证明辅因子稳定性对于提高产品产量比酶更重要。我们还证明了低温增强了氧化还原反应的耦合，从而导致摩尔比的增加每个氧化辅因子由 10- p NCA产生的对硝基苯酚盐。总体而言，当野生型 P450BM3 或 R966D/W1046S 突变体与上述三种辅因子中的任何一种一起使用时，优化的反应条件导致总产物产量增加 2 至 2.6 倍。