Identification Of AFB₁ Degradation Products,Toxicity Evaluation,and Clonal Expression Of Degradation Enzymes Gene From Pseudomonas Aeruginosa

Aflatoxins（AFs）are secondary metabolites produced by Aspergillus flavus and A.parasiticus,among which aflatoxin B₁（AFB₁）has the strongest toxicity and widely distributed.AFB₁ not only seriously threatens the safety and economy of food and feed,but also causes great harm to humans and animals.Therefore,it is very important to study the detoxification mechanism of AFB₁ in moldy grain.The biological method has the characteristics of high efficiency,green,safety,and mild treatment conditions,it has become the research hotspot of AFB₁ detoxification.However,there are few reports on the degradation of AFB₁ in moldy maize and the application of recombinant enzymes expressed by the cloning of degradation enzyme genes.In this study,the fermentation supernatant of Pseudomonas aeruginosa that can degrade AFB₁ was selected as the strain of biodegradation of AFB₁ in moldy maize,and the degradation conditions of AFB₁ in moldy maize were optimized,the degradation mechanism and toxicity of degradation products were preliminarily explored,and the degradation enzymes were identified on the basis of the feasibility of the biomethod.The gene of degradation enzyme was cloned and expressed,and the obtained recombinant protein was applied to the degradation of AFB₁ in moldy maize.（1）Optimization of experimental conditions for degradation of AFB₁ in moldy maize by fermentation supernatant of P.aeruginosa.The degradation rate of AFB₁ was used as the evaluation index,and the optimal parameters of each influencing factor were determined by a single factor test and response surface test.The optimal conditions were as follows:solid-liquid ratio 25 g/100 m L,temperature of 63℃,p H 11.0,degradation time of 51.50 h.Under these conditions,and the AFB₁ degradation rate was 99.67%.（2）Study on the structure of AFB₁ degradation products.Based on UV-Vis,thin-layer chromatography,and fluorescence,the results showed that the UV absorption of the degradation products at 365 nm decreased,the fluorescent spots on thin-layer chromatography significantly decreased,and the fluorescence intensity also decreased,but the fluorescence characteristics were still retained.It was assumed that the lactone ring of some degradation products was broken.The secondary mass spectrum fragment information of degradation products was obtained and analyzed by UPLC-Q-TOF technology,and four main degradation products were identified as P1（C17H16O6）,P2(C₁₆H₁₄O₅),P3(C₁₇H₁₄O₅),and P4(C₁₆H₁₀O₆),and speculated the degradation products are mainly formed by hydroxylation,reduction and dimethoxy of groups in the parent structure of AFB₁.（3）Safety of AFB₁ degradation products.The toxicity of degradation products was predicted through T.E.S.T software,and the results showed that the toxicity of AFB₁degradation products was lower than AFB₁.Ames and CCK-8 tests were used to analyze the mutagenicity and cytotoxicity of AFB₁ degradation products,respectively.The results showed that untreated AFB₁ showed strong mutagenicity positive and strong cytotoxicity,which the degradation products were mutagenic negative after 72 h of degradation,and the cytotoxicity was significant reduced.When the initial concentration of AFB₁ was 2.5μg/m L,the cell survival rate could be increased by 39.55%.（4）The degradation enzymes were identified by mass spectrometry,and the genes of degradation enzymes were cloned and expressed.On the basis of the feasibility of the degradation of AFB₁ by P.aeruginosa fermentation supernatant,the active proteins in the fermentation supernatant were separated and purified by Native-PAGE,and the four obvious protein bands obtained were cut and recovered.The degradation ability of AFB₁ was more than 55%,and the active protein was identified by mass spectrometry.Catalase,a degradation enzyme with good degradation effect,was selected for gene cloning and prokaryotic expression.The catalase gene and expression vector p ET32a were digested with Eco RⅠand HindⅢ,then connected in vitro to construct the recombinant prokaryotic expression vector p ET32a-Kat A.Then,the recombinant vector was transformed into Escherichia coli BL21,and the recombinant catalase was successfully induced and expressed by adding IPTG.（5）Optimization of induced expression conditions and verification of degradation activity of recombinant catalase.The expression of recombinant catalase was optimized by changing the induction temperature,IPTG concentration,OD value,shaking speed,and loading volume.The optimal induction conditions were optimized as follows:when the OD₆₀₀value of bacterial solution reached about 0.8 at 37℃and 200 r/min,IPTG concentration of 0.2m M,the temperature of 21℃,rotation speed 60 r/min,volume 50 m L,and induction for 15 h.The electrophoresis grade recombinant protein was purified by Ni²⁺column,and the degradation rate of AFB₁ in moldy maize was 52.81%.