Heterologous Expression,Characterization And Application Of Zearalenone Lactonase

Zearalenone,also known as F-2 toxin(Zearalenone,ZEN),is a non-steroidal estrogen mycotoxin,mainly produced by Fusarium during its secondary metabolism process.ZEN has a phenolic dihydroxylactone structure similar to estradiol and can competitively bind to estrogen receptors in the body,thereby causing a series of reproductive toxicity to humans and animals.ZEN is widely found in moldy corn,wheat,and other grains and their by-products.It is one of the most contaminated mycotoxin worldwide,causing huge safety hazards and economic losses.Presently,the main enzymes that can degrade ZEN are:lactonase,laccase and peroxidase.Among them,lactonase has received wide attention because of its clear degradation mechanism,thorough detoxification,and clarified crystal structure.In this study,four assumed lactonase genes were discovered through the NCBI database.The p ET-22b(+)was used as vector to construct the recombinant plasmid,which was then transformed into E.coli BL21.Ni⁺column was used to obtain pure enzyme.As a result,only the recombinant ZENG from Gliocladium roseum was successfully expressed and purified.SDS-PAGE showed that the molecular weight of ZENG was about 28.5 k Da,and it was dimer.The identification results of ZENG showed that the optimal p H was 7.0.After incubation for 3hours in the buffer of p H 7.0 and p H 8.5,the residual activity was still above 90%,meaning it with a high p H stability;the optimal temperature was 38�C,the T_m value was 50.82�C,and 20%residual activity were remained after incubation at 48�C for 7 min,which all meant it had a poor thermostability;The ZENG was metal-independent enzyme,metal ions had no activating or promoting effects on its catalytic activity;Enzyme kinetics studies showed that its K_m value was 129.03�14.90?mol L^-1,and the K_cat value was 0.501 s^-1;The enzymatic activity of the ZENG to ZEN was 315�1.03 U/mg,and the relative activity of ZENG against?-ZOL and?-ZAL was about 53%and 37%of that it against ZEN.Then,the p UB110,of which antibiotic resistance gene was replaced by D-alanine racemase gene(dal),was used as vector to construct the p UB-p43-Glro-dal.The food-grade expression of ZENG in B.subtilis 1A751(dal^-)was realized.The SR medium was selected for shake flask fermentation and no need inducer.The OD₆₀₀ value and fermentation enzyme activity both showed an overall trend of increasing first,then gradually stabilizing,and finally decreasing during fermentation.The highest OD₆₀₀ value reached about 5.8 after 14 h,and the highest fermentation enzyme activity reached about 5.2 U/m L after 16 h.It improved the application safety of enzyme.Finally,we improved the activity against?-ZOL and the thermostability of ZENG by molecular biology methods combined with computational simulation and rational design.The degradation activity to?-ZOL of the mutant V153H enzyme was increased to 1.46 times than that of WT which may be attributed to the formation of hydrogen bonds between the mutation point and?-ZOL;the residual activity of the mutant H134L/S136L enzyme was about 1.39times that of the wild enzyme after incubating at 53�C for 2 min,The improvement of thermostability may be due to the hydrophobic residues changed the hydrophobic microenvironment at the corner between the?helix and the Loop;the residual activity of the mutant S162P enzyme was about 1.52 times that of the wild enzyme after incubating at 50�C for 2 min,the half-life at 48�C was 3.6 times that of the wild enzyme.The T_m value increased by 1.74�C.Molecular dynamics simulation and structural analysis showed that:the degree of freedom of amino acids residues near the mutation point was decreased,the overall rigidity was improved,and the structure was more compacted.