Identification of a Novel Bacillus Velezensis Is-6 Nudix Hydrolase Nh-9 Involved in Ochratoxin a Detoxification by Transcriptomic Profiling and Functional Verification

An isolate Bacillus velezensis IS-6 displaying high OTA degradation activity was obtained. Its culture supernatant could degrade 1.5 µg/mL OTA by 89% when incubated at 37 °C for 24 h. The degradation activity was dramatically reduced by proteinase K, proteinase K plus SDS, and heat treatment, suggesting the degradation of OTA by IS-6 was an enzymatic process. The culture supernatant exhibited the highest degradation activity at 37 °C and pH 7.0 with ions Fe2+ and Cu2+ as enhancers. A degradation product OTα was observed using LC/TOF-MS analysis, suggesting OTA was degraded to lower toxic metabolites. The comparative transcriptome analysis of IS-6 showed 38 differentially expressed genes were significantly up-regulated after treatment with OTA. From the up-regulated genes, a novel OTA degradation enzyme Nudix hydrolase Nh-9 was successfully cloned and characterized. The recombinant protein was overexpressed in E. coli BL21, then was purified by affinity chromatography. The purified Nh-9 could degrade 1.0 µg/mL OTA by 68% at 37 °C in 24 h. According to the findings, the Nh-9 is the major OTA degrading enzyme in B. velezensis IS-6. Furthermore, OTA may be co-degraded by Nh-9, carboxylesterase, signal peptidase and other degrading agents that has yet to be discovered in this strain.