Mutation,Expression And Enzymatic Properties Of α-Amylase Gene In Bacillus Velezensis

α-amylase is a kind of industrial enzyme with wide application value.Its acid and alkali resistance and heat stability determine the application prospect of α-amylase.In order to obtain high efficiency used in feed industry of amylase,40 ℃ at low p H condition and environment with high energy and high stability of alpha amylase,this study were applied to site-directed mutation,the structural biology analysis,and computer simulation.E.coli BL21 was used to express α-amylase and its enzymatic properties were studied.The α-amylase ORF of B.velezensis was amplified and sequenced,with a total length of 1980 bp,659 coding amino acids and a molecular weight of 66 k Da.Four mutants,P546 E,H572D,A614 E and K622 E,were designed in the C domain,and three mutants,Mut1(E),Mut2(ED)and Mut3(EDEE),were obtained by site-directed mutation in sequence.The results were as follows.(1)The optimum temperature of Mut3 reached 65℃,5℃ higher than Ori.(2)There was no significant change in the optimal p H,which ranged from p H 6.0 to 7.0,but Mut2 and Mut3 showed small peaks at p H 5.0.(3)The specific activity of Ori,Mut1 and Mut2 at 60℃ and p H 7.0 was about 500 U/mg,while the specific activity of Mut3 was about 600 U/mg.(4)The thermal stability of Mut3 was higher than that of Ori at 40 to 55℃ for 4 h,and the specific activity of Mut3 was 50 U/mg higher than Ori at 40℃ for 4 h.(5)The acid tolerance of Mut1,Mut2 and Mut3 was higher than that of the original amylase at p H 2.0 to 5.0 for 4 h at 40℃,and Mut3 was the highest.This study also compared the effects of Mut3 and Ori on the enzymatic hydrolysis of laying hens’ feed in vitro.By designing the orthogonal experiment of enzymolysis time,solid-liquid ratio and amylase addition level,it was found that Mut3 had a higher enzymatic hydrolysis effect on laying hens’ feed than Ori group.In both groups,the time and feed to liquid ratio had significant effects on the enzymolysis efficiency(P <0.01),the amount of Mut3 had a significant effect(P <0.05),but the amount of Ori had no significant effect.The four sites P546 E,H572D,A614 E and K622 E mutated effectively improved the acid resistance and thermal stability of amylase.This study provides a basis for the application of α-amylase in feed industry,and has important reference significance for improving the acid resistance and thermal stability of other industrial enzymes.