Diclofenac exerts the highest level of acute toxicity among tested non-steroidal anti-inflammatory drugs. Although there are many reports on the microbial degradation of diclofenac, gaps remain in the knowledge related to the degradation process, especially that by Bacillus subtilis. In this study, we used B. subtilis 168trpc2 and its cytochrome P450 genetic defect strains B. subtilis $\Delta$yrhJ and B. subtilis $\Delta$yjiB to identify the key limiting protein and factor in diclofenac degradation. The results showed that in 72 h, B. subtilis 168trpc2 degraded 88.3% of diclofenac, whereas B. subtilis $\Delta$yrhJ and B. subtilis $\Delta$yjiB degraded 10% and 21%, respectively. These results illustrate that cytochrome P450 is essential for diclofenac degradation. The hydroxylation product of B. subtilis 168trpc2 is 5-OH-diclofenac, whereas the genetic defect strains generate the hydroxylation products 5-OH-diclofenac and 4ʹ-OH-diclofenac. Hydroxydiclofenac can subsequently be dechlorinated; through CO₂ loss and C-N bond cleavage wherein dechlorination is the central step. This study provides direct evidence that cytochrome P450 determines the position of diclofenac hydroxylation and that hydroxydiclofenac can be subsequently degraded.

在测试的非甾体抗炎药中，双氯芬酸的急性毒性水平最高。尽管有许多关于双氯芬酸微生物降解的报道，但是关于降解过程的知识仍然存在空白，尤其是枯草芽孢杆菌。在这项研究中，我们使用枯草芽孢杆菌168trpc2及其细胞色素P450遗传缺陷菌株枯草芽孢杆菌 $\Delta$yrhJ和枯草芽孢杆菌 $\Delta$yjiB可确定双氯芬酸降解的关键限制蛋白和因子。结果表明，在72 h内，枯草芽孢杆菌168trpc2降解了双氯芬酸88.3％，而枯草芽孢杆菌 $\Delta$yrhJ和枯草芽孢杆菌 $\Delta$yjiB分别降低了10％和21％。这些结果说明细胞色素P450对双氯芬酸降解至关重要。枯草芽孢杆菌168trpc2的羟化产物为5-OH-双氯芬酸，而遗传缺陷菌株产生5-OH-二氯芬那和4 + -OH-双氯芬酸的羟化产物。羟基双氟芬酸可随后脱氯；通过CO ₂损失和CN键裂解，其中脱氯是主要步骤。这项研究提供了直接的证据，表明细胞色素P450决定了双氯芬酸的羟基化位置，并且羟基双氯芬酸随后可以被降解。