The Purification, Enzymatic Characteristics, And Degradation Products Of Recombinant ZEN-degrading Enzyme A4-Prx

Zearalenone, a mycotoxin with two hydroxy benzoic acid lactone structure, mainly found in moldy corn, wheat and other cereal crops, is a harm to the poultry and livestock and a big threat to human health, because of its reproductive toxicity, endocrine and liver toxicity. ZEN detoxification methods include physical, chemical and biological methods. Among those methods, biological degradation methods are popular in ZEN detoxification researchers, advantage of reaction specificity, less loss of nutrients to foods and nontoxic reaction product. Our laboratory workers isolated a strain named Acinetobacter sp. SM04 from soil which can degrade ZEN into nontoxic products with very low estrogenic activity. Then they isolated a kind of peroxidase from culture broth of SM04, cloned and expressed the gene of the peroxidase into Pichia pastoris, resulting into GS115/p PIC9k-A4-Prx, which can secrete recombinant A4-Prx after optimizing its culture conditions. This paper is commol/Litted to purify A4-Prx from GS115/p PIC9k-A4-Prx culture broth, study its enzymatic properties and preliminary study its degradation products and mechanism through the reaction of ZEN.In this paper, peroxidase A4-Prx is got from using a series of protein separation and purification methods to the induced secrete culture broth of recombinant Pichia pastoris strain GS115 / p PIC9K-A4-Prx. Through experimental study, we got A4-Prx with high purity and biological activity by the purified methods as follow:firstly 60% ammol/Lonium sulfate precipitation, secondly Then Sephadex G-100 column elution by 0.02 M Tris-HCl(p H8.0), finally Sephadex DEAE-52 column elution by gradient concentration buffer. SDS-PAGE experiment said that molecular weight of A4-Prx is 41 k Da. Detecting peroxidase activity in the purification process, arrive at a we know final purification factor is 12 and purification effect is obvious.In this paper, we study the enzymology properties of peroxidase A4-Prx, resulting that p H and temperature have a significant impact on peroxidase activities of A4-Prx, in p H 9.0 and 60℃ the enzyme activity is the highest.Under a certain concentration, the peroxidase activity of A4-Prx increase with the concentration of H2O2, which comform to mie equation of first order reaction kinetics, but when the concentration of H2O2 is higher than 50 mmol/L, peroxidase activities begin to decline. According to double-reciprocal plot, Vmax of peroxidase activity is 204.1U, while Km is 4.85 mmol/L. Increasing the concentration of phenol and 4- aminoantipyrine, Km decrease but Vmax basically remain unchanged. EDTA inhibits the peroxidase activity of A4-Prx significantly, when EDTA concentration is 10 mmol/L, enzyme activity of A4-Prx quickly fell by 80%. Unlike EDTA, the inhibition action of sodium azide is not significant, when the concentration of sodium azide is 60 mmol/L, inhibition rate is less than 40%. A4-Prx not include heme, which means A4-Prx is not belong to the hemoglobin dependent peroxidase, it belongs to thioredoxin dependent peroxidase.This paper includes ZEN degradation experiments of peroxidase A4-Prx, analyzing ZEN content by HPLC, indicating that peroxidase A4-Prx can degrade ZEN, and the degradation rate is 63%. The reaction solution of peroxidase A4-Prx degrading ZEN is extracted three times with ethyl acetate, then the extract is separated by thin layer chromatography, resulting that the reaction may give two products. Using infrared spectrometer to get the infrared absorption spectrum of the products, we know that the ester bond of ZEN is cleavage in the peroxidase A4-Prx catalytic reation of ZEN, and generating a carboxyl group COOH, and the phenolic hydroxyl group O-H of ZEN is reduced to –O-, exposing active site which bond other free ions in the reaction system.