Thermal Stability And Hydrolysis Activity Improvement Of Medium-temperature Amylase

?-amylase is a class of enzymes that hydrolyze starch 1,4 glycosidic bonds to produce dextrins,oligosaccharides,and monosaccharides.With the increase of application demand and the maturity of biotechnology,it is of great significance to improve its stability and hydrolysis activity.This paper which used industrial production from Cytophaga sp.and Bacillus?Bacillus amyloliquefaciens?of amylase gene CamyA and BamyA as material,successfully realized the heterologous expression in Bacillus subtilis.Through the rational design,effectively improved the thermal stability and hydrolytic activity and explored the amylase CamyA thermal stability and hydrolytic mechanism by dynamics simulation.The optimal temperature for amylase CAMYA is 55°C,the optimum pH of 7.0.Treatment at 70°C for 5 min,residual enzyme activity was 26%,at pH 1.0 to 6.0 is not unstable.When soluble starch was used as substrate,the specific activity was 5,550 U·mg^-1.The kinetic constants were determined,K_m was 3.19 mg·mL^-11 and V_max was 6,534 mol·min^-1·mg^-1.Through the different properties of amylase sequence and structure analysis,design of mutant CAMYA-?R178/?G179 and CAMYA-S33A/S34E/V35H.Mutant?R178/?G179 under 70°C for 5 min,the residual enzyme activity is 70%;80°C treatment for 5 min,?R178/?G179 and wild type residual enzyme activity were 28%and 8%,respectively.The mutant S33A/S34E/V35H showed higher catalytic activity,which was 1 times higher than that of the wild type(10,725 U·mg^-11 vs.5,550 U·mg^-1),and the catalytic efficiency was 2.3 times higher.The combination of S33A/S34E/V35H/?R178/?G179,treatment at 70°C and 80°C for 5 min,the residual enzyme activity were 90%and 40%respectively,specific activity was 15,594 U·mg^-1,2.8times that of wild type.The simulated trajectory showed that after removing the two amino acids R178and G179,the Loop region became shorter and the ability of amylase to bind Ca²⁺was enhanced,thus improving the thermal stability.After mutation,the amylase changed from Ca²⁺-dependent to Ca²⁺-independent,possibly because the binding Ca²⁺was not easy to be lost after the enhancement of Ca²⁺binding force,so additional addition was needed in the middle of the later reaction.The mutation site S33A/S34E/V35H was located in the N-terminal domain of amylase,and the K_m value was significantly reduced after mutation.It was speculated that this partial mutation enhanced the affinity between enzyme and substrate,thus improving the catalytic activity.After 90 h fermentation in 15 L fermenter,the enzyme activity of the combined mutant was 355,800 U·mL^-1,and the protein content was 23mg·mL^-1.To verify the universal effect of S33A/S34E/V35H site mutation,amylase BAMYA from Bacillus amylophilus,which is widely used in industry,was selected for mutation at the same site.The designed mutant S61A/D62E/I63H was expressed in Bacillus and the specific activity of recombinase was 10,744U·mg^-1,which was 57%higher than that of the wild type(6,835 U·mg^-1).The K_m value,V_max and catalytic efficiency of the wild-type BAMYA and mutant BAMYA-S61A/D62E/I63H were 3.58mg·mL^-11 and 2.97 mg·mL^-1,6,534 mol·min^-1·mg^-1,16,628 mol·min^-1·mg^-1,and 1,577 mL·s^-1·mg^-1 and3,042 mL·s^-1·mg^-1,respectively.In addition,the sequence and structure analysis with high specific activity showed that the L,K and N sites at 473-475 of BAMYA C-terminal site may be related to the catalysis of the enzyme.The designed mutation site L473K/K474H/N475K showed a specific activity of 10,148 U·mg^-1,which was 48%higher than that of the wild type.K_m value and catalytic efficiency were 2.21 mg·mL^-1 and 4,760 mL·s^-1·mg^-1,respectively.However,the mutation effect of the above two combinations was not obvious.In this study,site-directed mutagenesis was used to effectively improve the thermal stability and catalytic activity of amylase in industrial application.The obtained mutants could effectively improve the hydrolysis efficiency in industrial production,and provide reference for amylase mutation research.