Screening,enzymatic Properties And Heterologous Expression Of The Encoding Gene In The Glucose Oxidase Producing Strain

Glucose oxidase（EC 1.1.3.4,GOD）is a dimeric enzyme containing flavin adenine dinucleotide（FAD）,which specifically catalyzesβ-D-glucose to form gluconic acid and hydrogen peroxide（H₂O₂）.GOD has a wide range of applications in the food,feed,pharmaceutical,and textile industries.It was derived from in animals,plants,and microorganisms,and microorganisms are the main source of GOD.The main strains used in industry to produce GOD are Aspergillus niger and Penicillium.However,these strains are difficult to meet the market demand due to the complicated product purification steps and high production costs.Therefore,it is especially important to further screen and obtain excellent strains with high GOD production,and to simplify and optimize the fermentation process and establish a set of process technology more suitable for industrial production.In this study,a GOD-producing strain was screened from soil samples and identified by morphology and molecular biology method.The fermentation conditions for enzyme production of the strain were optimized through single-factor experiments;Then the GOD gene was cloned,and other GOD genes were synthesized.They were heterologous expressed in Escherichia coli and Pichia pastoris to improve GOD production;Finally,the enzyme from this strain was isolated and purified and its enzymatic properties were studied.All results were summarized as follows:（1）A strain capable of producing GOD was obtained from the soil sample of the tobacco field using the starch-potassium iodide chromogenic plates method.After morphological observation and ITS identification,the strain was identified as Talaromyces flavus,and named Talaromyces flavus YD-1.（2）In order to improve the yield of GOD,we firstly optimized the fermentation medium composition of T.flavus YD-1 using the single-factor optimization method.The optimal culture conditions were determined as 8.00%glucose,0.70%peptone,0.45%Ca CO₃,0.06%Mg SO₄·7H₂O,0.20%KH₂PO₄,and 0.06%KCl.Then,we optimized the fermentation conditions of T.flavus YD-1.The results showed that the optimal fermentation conditions were24 h of incubation time,initial fermentation p H 6,5%inoculum,and 25℃of incubation temperature.Finally,the enzyme activity could reach 2.70 U/m L,which was 2.25 times higher than the initial one.（3）The t GOD gene was obtained by amplifying with the T.flavus YD-1 genome as a template.And the a GOD gene from Aspergillus flavus,p GOD gene from Penicillium subrubescens,and s GOD gene from Salmonella enterica were synthesized.The p ET-28a（+）-s GOD,p ET-28a（+）-p GOD,p ET-28a（+）-a GOD,and p Cold-TF-t GOD plasmids were transferred into E.coli BL21（DE3）,and further fermented to verify the enzymatic activity.Then the expression of GOD was detected by SDS-PAGE electrophoresis.The results showed that all GOD was expressed in E.coli BL21（DE3）without detectable activity.Recombinant s GOD and p GOD were expressed in E.coli BL21（DE3）as inclusion bodies,and a GOD was not expressed.Recombinant t GOD obtained soluble expression,but no enzyme activity was detected.The recombinant plasmids p PICZαA-t GOD,p PICZαA-p GOD,and p PICZαA-a GOD were expressed in P.pastoris GS115.P.pastoris GS115/p PICZαA-t GOD strain showed an enzyme activity of 0.39 U/m L in fermentation broth and 0.61 U/m L in cell.While P.pastoris GS115/p PICZαA-p GOD and P.pastoris GS115/p PICZαA-a GOD strains did not detect enzyme activity in both fermentation broth and cell.（4）The fermentation broth was sequentially treated by ammonium sulfate graded precipitation and Sephadex G-75 gel filtration chromatography.A single band appeared at around 80 k Da detected by SDS-PAGE,and t GOD with a molecular weight of 160 k Da was obtained by active electrophoresis.The enzymatic properties of t GOD were investigated,and the results showed that the optimum temperature was 35℃,and the stability of the enzyme was better in 30-40℃.The optimum p H was 6,and the enzyme had good stability in the p H of 2-7,indicating that the enzyme has good acid resistance.Ca²⁺and Zn²⁺increased enzyme activity by 110%and 118%,respectively.Cu²⁺and Fe²⁺have the most obvious inhibitory effect on the enzyme,with only 14%and 8%of enzyme activity remaining;The surfactant Tween-80 could improve the enzyme activity,and the promotion effect was improved with the increase of concentration,enzyme activity could be increased by 167%,while SDS had an inhibitory effect on the enzyme activity.In summary,in this study we obtained a GOD-producing strain T.flavus YD-1 by screening,and optimized the fermentation conditions of the strain using single-factor experiments,which increased the enzyme activity by 2.25 times;Then the GOD gene of the strain was cloned,and it was expressed in P.pastoris;The pure GOD was obtained by two-step isolation and purification,which has good stability under acidic conditions（p H 2-7）,indicating that it has good application in animal feed additives.This study enriches the resources of GOD-producing species and has reference value for reducing the production cost of GOD.