Expression And Application Of Deoxynivalenol Degrading Enzyme Gene In Bacillus Subtilis

Deoxynivalenol（DON）is a toxic secondary metabolite produced by fungi such as Fusarium graminearum and Fusarium culmorum.DON mainly contaminates wheat,corn,and other food crops,seriously threatening human and livestock health.Currently,the method of biological detoxification using microorganisms and enzymes shows good application prospects.The biotransformation of DON by microbes or biological enzymes has received wide attention because it has high catalytic efficiency,strong specificity,less nutrient loss,and no pollution of the environment.However,there is still a lack of efficient detoxifying microorganisms and enzymes that can be applied to complex substrates.In this study,based on the screening of the DON-degrading bacterium D-8 in the laboratory at an early stage,its degradation enzyme gene was cloned and heterologously expressed in Bacillus subtilis.The degradation products were analyzed,and recombinant strains were used for preliminary detoxification applications on moldy wheat to provide a reference for the biological detoxification of DON in food and feed.In this study,the previously screened bio-degrading bacterium D-8 was used for cloning and heterologous expression of its degrading enzyme genes in Bacillus subtilis,and its degradation products were analyzed.The recombinant strain was used for the preliminary application of detoxification of moldy wheat,and it is hoped that the research results will provide a reference for the biological detoxification of DON in food and feed.Details are as follows:（1）The cellular localization and gene cloning were performed for the degradation of active substances by Devosia sp.D-8.The active substance with DON degradation activity in this strain is located intracellularly.The D-8 intracellular fluid can degrade 5μg of DON in 2h,and also almost completely degrade 18719.78μg/kg DON in highly contaminated wheat in4 h.Through literature research,database gene sequence retrieval,and bioinformatics software analysis,dehydrogenase and aldo-keto reductase genes are predicted.Using D-8 strain DNA as a template,three candidate genes D15（Gen Bank:OQ603484）,D16（Gen Bank:OQ603485）,and D17（Gen Bank:OQ603486）were cloned,encode dehydrogenase D15,aldo-keto reductase D16 and D17,respectively.（2）The recombinant E.coli strain was constructed and the expression and degradation activity of the degradation enzyme were analysed.The E.coli strain expressing the degradation enzyme D15 could degrade 3.6μg of DON to 3-keto-DON in 2 h with pyrroloquinoline quinone（PQQ）and Ca²⁺,and the degradation rate was 72.48%.The degradation enzymes D16 and D17 could degrade 1.7μg and 1.3μg of 3-keto-DON to DON and 3-epi-DON in 8 h,respectively.with reduced nicotinamide adenine dinucleotide phosphate（NADPH）,and the degradation rate was 33.96%and 27.63%,respectively.Meanwhile,degrading enzyme enzymatic property results showed that the specific activities of D15,D16,and D17 were 313.4±22.76 U/g,49.31±0.83 U/g,and 30.02±1.53 U/g,respectively.The optimum reaction conditions are p H values of 6-7 and temperatures of 35-40℃.（3）Using the high safety and secretion expression characteristics of Bacillus subtilis to study the heterologous expression characteristics of degradation enzymes with various recombinant B.subtilis strains.The recombinant B.subtilis strains successfully expressed the degradation enzymes D16 and D17.The recombinant strain ASAG17D could degrade 3-keto-DON to 3-epi-DON,a low-toxicity product,without the addition of coenzyme NADPH.The maximum activity of degrading enzyme D17 in recombinant strain ASAG17D was 12.64±0.06U/L,when fermented for 18 hours,the OD₆₀₀ value was 8.62±0.08 at the same time.However,degradation enzyme D15 could not be expressed in B.subtilis.We will try to solve this problem in future studies with other expression systems.（4）Using D-8 as the degrading microorganism to conduct research on the preparation of DON degradation agents.The preparation process was optimized through single-factor and orthogonal experiments.The best preparation process for high live bacteria involved concentrating the fermentation of D-8 for 21 hours by ten times and using 40-80 mesh corn flour as the carrier material,with drying temperature was 40℃and the moisture content was10%.The D-8 agent in a live bacterial count of（12.38±0.11）lg CFU/g could reduce DON in contaminated wheat from 18719.78μg/kg to 585.93μg/kg in 24 hours,and the degradation rate was 96%.（5）Conducting preliminary application research on DON detoxification in complex substrates.A three-system enzyme synergy detoxification method was established with using recombinant strain ASAG17D,degradation enzymes D15 and intracellular fluid of D-8 in contaminated wheat.The DON content in contaminated wheat was reduced to 8556.81μg/kg and 9515.26μg/kg in 24 hours,with degradation rate was 49.17±3.60%and 54.29±3.62%,respectively,without the addition of coenzymes NADPH and PQQ respectively.This breakthrough addresses the innovative addition of coenzymes to DON-degrading enzymes in complex substrates.The results also provide technical reference for the development of an effective and safe enzyme-based DON bio-degradation agent that will be applied in feed and food.