Rational Design Of Glucose Oxidase Stability Based On Structure

Glucose oxidase（Gox）,as a new alternative to antibiotics and growth promoters,uses oxygen molecule as electron acceptors to catalyzeβ-D-glucose to generate gluconic acid and hydrogen peroxide.Gox can sterilize and inhibit bacteria through non-drug mechanisms,maintaining the balance of intestinal flora and gut health.As a feed additive,outstanding thermostability and acid stability are the key to improve the application value of Gox.In this study,the thermostable glucose dehydrogenase At GDH derived from Aspergillus terreus and glucose oxidase GoxM8 derived from Aspergillus niger were used as research objects and based on the structure,the stability of Gox was studied by rational design.So as to comprehensively improve the performance of Gox to meet the application requirements of feed industry,which has important theoretical significance and application value.Firstly,the mutants with Gox activity that can accept oxygen molecule as electron acceptor were obtained through molecular modification of the thermostable At GDH.The thermostable glucose dehydrogenase At GDH was excavated through BLAST and searching Uni Prot database.The optimal reaction temperature of At GDH is 55℃and the half-life at 60℃is 80 min.Predicted amino acids related to electron transport chain,near FAD and interacting with oxygen molecules in At GDH were modified to introduce oxygen molecule binding pockets,which failed to get positive mutant.Which may be due to their different catalytic mechanisms despite of similar structures.Furthermore,high-throughput screening was used for the directed evolution of At GDH and 6mutants with weak Gox activity were obtained while no mutants with improved Gox activity after saturated mutation at the 6 amino acid sites.Subsequently,we changed our research ideas to study the stability of Gox.The crystal structure of Gox M8 with resolution of 2.08（?）was obtained by crystal structure analysis,based on which the mutation hotspots related to the thermostability were explored.Among the 11 mutants designed and verified by experiments,the thermostability of five mutants was improved.Especially,the catalytic efficiency of the mutant E361P was 56.37 m M^-1s^-1,and the half-life at 65°C reached495 min,which was 2 and 1.3 times that of Gox M8,respectively.The mutant E361P/A419I was obtained by further combination of mutations,and the half-life at 65°C was extended to 578 min.The results indicated that the strategy can effectively improve the thermostability of Gox without losing catalytic activity.Finally,based on the crystal structure of GoxM8,the acid stability of Gox M8 was rationally designed by three strategies,including optimization of protein surface charge,replacement of amino acids associated with p H properties,and design of salt bridge network.After experimental verification,mutants of A4D/N471E,Q241E/R499E and D193N/D497N was obtained by each of the strategies,which had significantly improved acid stability.The optimal p H of mutant A4D/N471E was 6.0,consistent with Gox M8,while the optimum p H of mutants Q241E/R499E and D193N/D497N was 5.0.After treatment at p H 2.75 for 1 h,the relative residual enzyme activities of A4D/N471E,Q241E/R499E and D193N/D497N was increased by 150%,57%and 104%,respectively,compared with Gox M8.The results of simulated gastric juice stability test showed that under the condition of p H 2.5,the residual enzyme activity of the three mutants were significantly higher than that of Gox M8.GoxM9（GoxM8-E361P/A419I/D193N/D497N）was obtained by iteratively mutation with improved thermostability and acid stability at the same time.Compared with Gox M8,the half-life at 65℃of Gox M9 was 1.4 times higher,and the relative residual enzyme activity of Gox M9 after treatment at p H 2.75 for 1 h was 88%higher,under the condition of 13%higher catalytic efficiency.With the activity not reduced,the stability of mutant Gox M9 was comprehensively improved which was conducive to its application in feed industry.In summary,this study firstly explored to obtain mutants with Gox activity that can accept oxygen molecules as electron acceptors through directed evolution of the thermostable glucose dehydrogenase.Subsequently,the crystal structure of Gox M8 was analyzed,and the stability of Gox M8 was rationally designed based on the crystal structure.Finally,a mutant with improved catalytic activity,thermostability and acid stability was obtained,which not only improved the application value of Gox in feed industry,but also provided theoretical support for protein stability molecular design based on structure.