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

Abstract

Contamination of foods and feeds with Ochratoxin A (OTA) is a global problem, and its detoxification is challenging. In this study, Bacillus velezensis IS-6 culture isolate supernatant degraded 1.5 g/mL OTA by 89% after 24 h of incubation at 37 °C, whereas viable cells and intra-cell extracts were less effective. The OTA degradation by B. velezensis IS-6 was an enzymatic process mediated by the culture supernatant. The degradation activity was optimal at 37 °C and pH 7.0, and Fe 2+ and Cu 2+ ions enhanced the OTA degradation. The LC−MS/MS analysis confirmed that structure of OTA was modified, resulting in the production of OTα that was less toxic than OTA. The transcriptomic analysis of B. velezensis IS-6 showed that 38 differentially expressed genes (DEGs) were significantly up-regulated, and 24 DEGs were down-regulated after treatment with OTA. A novel OTA degradation enzyme Nudix hydrolase Nh-9 was successfully cloned and characterized from the up-regulated genes. The recombinant Nh-9 enzyme was overexpressed in Escherichia coli BL21 and purified by affinity chromatography, exhibiting 68% degradation activity against 1.0 μg/ mL OTA at 37 °C in 24 h. The degraded product by the Nh-9 enzyme was identified as the less toxic OTα by LC−MS/MS. According to the findings, it can be inferred that Nh-9 is the main 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 are yet to be discovered in this strain.

Mining Lactonase Gene from Aflatoxin B1-Degrading Strain Bacillus megaterium and Degrading Properties of the Recombinant Enzyme

“…The degradation of these mycotoxins by AttM was evaluated based on the degradation efficiency under the experimental conditions. The presence of OTA was assessed via HPLC with an excitation wavelength of 330 nm and an emission wavelength of 460 nm . The mobile phase was composed of A (acetonitrile) and B (a 1% acetic acid solution) at a ratio of 60:40 (v/v).…”

Bioenzymatic detoxification of mycotoxins

“…Lipases and Nudix family hydrolases can also detoxify OTA. The Nudix hydrolase Nh-9 gene from the Bacillus velezensis IS-6 strain was expressed in E. coli , the recombinant Nh-9 enzyme was purified to degrade 1.0 μg/mL OTA by 68% within 24 h at 37°C and pH 7.0, and OTA was detoxified to OTα; Fe 2+ and Cu 2+ enhanced the degradation of OTA ( Jahan et al, 2023 ). Amano lipase A from Aspergillus niger (ANL) and porcine pancreas lipase (PPL) hydrolyzed OTA, but the enzymes exhibited different degrees of hydrolysis; ANL completely degraded OTA and OTB after 3 and 10 h, respectively, whereas PPL failed to completely degrade OTA (only 43% of OTA was degraded after 25 h) but completely degraded OTB within 9 h ( Santos et al, 2023 ).…”

Mechanisms by which microbial enzymes degrade four mycotoxins and application in animal production: A review

“…(2021) ADH3 Stenotrophomonas acidaminiphila CW117 Amidohydrolase OTA The degradation efficiency reached 99% at pH 8.0 and 50 °C for 90 s. Luo et al. (2022) Nh-9 Bacillus velezensis IS-6 Nudix hydrolase OTA The degradation efficiency was 68% at pH 7.0 and 37 °C for 24 h. Jahan et al. (2023) Chr1_3858681_3267 Stenotrophomonas sp.…”