Bacillus Velezensis IS-6 Broad-spectrum Mechanism Analysis Of Degrading Mycotoxins And Functional Verification Of Degrading Enzymes

Mycotoxins are produced by a variety of widespread microscopic toxigenic strains of Aspergillus,Penicillium and Fusarium.Mycotoxins are toxic metabolites that cause serious problems for human health.Mycotoxins such as aflatoxins B₁（AFB₁）,zearalenone（ZEN）and ochratoxin A（OTA）are toxic compounds mainly produced by Aspergillus,Fusarium and Penicillium species.Contamination of foods and feeds with mycotoxins is a global problem,and its detoxification is a challenging task.Ochratoxin A,among the most prevalent toxicants,is present in a variety of foods.An isolated Bacillus velezensis IS-6 displaying high OTA degradation activity was obtained.Its culture supernatant could degrade1.5μg/m L OTA by 89%when incubated at 37℃for 24 h,while viable cells and intra cell extracts were less effective.The degradation activity was dramatically reduced by proteinase K,proteinase K plus SDS,and heat treatment,suggesting the degradation of OTA by B.velezensis IS-6 is an enzymatic process.The culture supernatant exhibited the highest degradation activity at 37℃and p H 7.0 with ions Fe²⁺and Cu²⁺as enhancers.The degradation of OTA by strain IS-6 was studied using HPLC analysis,which indicated a new chromatographic peak.The LC-MS/MS mass spectrometry analysis confirmed the molecular identity of the less toxic product as OTα,with[M⁺H⁺]m/z 386.075,m/z 358.0844,m/z 341.0581,and m/z 256 as the precursor ions in the MS spectrum,were consistent with OTαmass spectrometry.These findings suggest that OTA has been degraded into lower toxic metabolites.The comparative transcriptome 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.The cellular component category revealed that cell binding and cell separation were the most significantly enriched Gene Ontology（GO）terms.The results of the GO and KEGG pathway analyses indicated that AAV34＿RS03650（encoding carboxylesterase Ce-3）,AAV34＿RS08565（encoding signal peptidase Sp-2）,and AAV34＿RS17190（Nudix hydrolase）were highly expressed under stress conditions.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 enzyme could degrade 1.0μg/m L OTA by 68%at 37℃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 Ce-3 signal peptidase Sp-2 and other degrading agents that has yet to be discovered in this strain.The bacterium B.velezensis IS-6 has demonstrated an ability to degrade AFB₁ and ZEN toxins by 58%and68%respectively,when exposed to 1μg/ml of these toxins for 24 h at 37°C.The purified enzymes Nudix hydrolase Nh-9 and Carboxylesterase Ce-3 derived from this bacterium were found to be effective in degrading AFB₁（Nh-9 by 46%and Ce-3 by 35%）and ZEN（Nh-9 by 54%and Ce-3 by 45%）.Optimal enzyme activity was observed at 37℃and p H 7.0,with different metal ions enhancing their activity.Mg²⁺enhanced the activity of Nh-9,while Ca²⁺and Fe²⁺enhanced the activity of Ce-3 for ZEN degradation.For AFB₁ degradation,Ni²⁺enhanced the activity of Nh-9,while Fe³⁺and Fe²⁺enhanced the activity of Ce-3.