Study On The Stability Improvement Of Glucose Oxidase From Aspergillus Niger

As a novel feed enzyme,glucose oxidase（Gox）can improve the intestinal environment,pr event intestinal infection and diarrhea in livestock and poultry production,thus to improve the utilization rate of diet and promote its growth.Gox has a broad application prospect in animal feed which should have the good thermostability and acid stability.In this study,we aimed to explore the mechanism of thermal and acid stability using the mutant Gox M4 from Aspergillus niger as material.Based on the rational design with computer aided,the mutants with improved thermal and acid stability were obtained.The thermostability improvement of GoxM4 was performed by computer aided rational desi gn.Combined the iterate mutation strategies of N-glycosylation modification,FAD binding-energ y optimization,hydrophobic forces introduction,and folding free energy optimization,the best mutant GoxM8（T31V/Q88R/S94A/T274F/Y278T）was obtained.The thermostability of GoxM8was significantly improved which remained the relative activity increased from 40%（GoxM4）t o 80%after heat treatment at 80^○C for 2 min.A simple and easy-to-perform method was use d to compare the thermostability parameters of GoxM4 and its mutants.Compared with GoxM4,the T_m value of GoxM8 was increased by 9.5^○C.What’s more,the catalytic efficiency(k_cat/K _m)of GoxM4 and its mutants were compared,Gox M8 was 6.8%higher than that of GoxM4,which was 28.3 mM^-1s^-11 and 26.5 mM^-1s^-1,respectively.The thermostablity improvement mecha nism of GoxM4 and its mutants was further analyzed by molecular dynamics simulations.The binding force between the FAD and enzyme was improved after the substitution of serine at po sition 94 with alanine which eliminated the steric hindrance of FAD.The stability of the intern al structure of the enzyme was improved after the substitution of hydrophilic threonine at positi on 31 with hydrophobic valine which enhanced the interaction of hydrophobic cores.The stabili ty of the surface structure of the enzyme was improved after the substitution of Gln88 with Ar g88 which increased the local electrostatic interaction.The results achieved the goal of improvi ng the thermostability of GoxM4 significantly with no expense of catalytic efficiency,which sol ved the problem of poor thermostability of Gox in the feed industrial.To promote the better application of Gox in the feed industry,the acid stability improveme nt of Gox M8 was studied.Three residues Ala14,Gln241 and Arg499 that related to acid stabili ty were identified based on eosinophilic mechanism of enzyme and the analysis of multiple seq uences.The three single mutant（A14E,Q241E,R499E）,and the two combined mutants A14E/R499E and A14E/Q241E/R499E were constructed for heterologous expression and property deter mination,which used compared with GoxM8.The optimal pH of all the five mutants was 5.0,which decreased 1 unit compared with GoxM8.After incubation at p H 3.0 for 1 h,GoxM8 ret ained 69%of the initial activity,while that of the mutants A14E,Q241E,R499E,A14E/R499E and A14E/Q241E/R499E were 70%,74%,100%,100%,100%,respectively,indicated the incre ase of acid stability.Furthermore,the specific activity of mutants A14E and R499E were 21%and 33%higher than that of GoxM8,and the mutant A14E/R499E and A14E/Q241E/R499E we re similar to that of Gox M8.The acid stability improvement mechanism for GoxM8 and its mu tants was further analyzed.Three additional hydrogen-bonds and a salt bond were formed with neighboring amino acids after the substitution of alanine at position 14 and glutamine at positio n 241 with acid glutamate.And,theα-helix（L498-Y504）was stabilized by the substitution of positive arginine at position 499 with negative glutamate,which removed unstabilizing factor of the dipole ofα-helix.The results achieved the goal of improving the acid stability of GoxM8without loss of enzyme activity.Based on the mutant Gox M4 from Aspergillus niger as material,the thermostability of Gox M4 was significantly improved with no expense of catalytic efficiency via the computer-aided r ational design strategy.The study solved the problem of the heat resistance of Gox in the proc ess of production and application.The acid stability improvement of Gox was carried out for t he first time.Based on the eosinophilic mechanism of enzyme and analysis of multiple sequenc es,the acid stability of GoxM8 was effectively enhanced without affecting the enzyme activity,which provided theoretical support for the acid stability improvement of similar enzymes.