Degradation of diclofenac by B. subtilis through a cytochrome P450-dependent pathway

Abstract

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 Δ yrhJ and B. subtilis Δ 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 Δ yrhJ and B. subtilis Δ 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 CO 2 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.