The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltoge...The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412(LrMA)toward soluble starch was exactly enhanced through hotspot-based research.Based on multiple sequence alignment,three-dimensional structure and existed literature,thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis.After the screening of the mutants,LrMA^(D172A),LrMA^(G260A),LrMA^(K334A)and LrMA^(M477A)were selected with the activity accounted for 144-209% of that in wild-type.Among all the mutants,LrMA^(G260A) possessed the highest activity toward soluble starch,reached 133 U/mg,about twice as high as that in the wild-type.Its temperature for optimum activity still maintained at 60℃,while had no significant loss of thermal stability occurred.In addition,compared with the wild-type in pH stability,the mutant retained over 80% residual activity at a wider pH range of 4.5-8.5.Furthermore,the k_(cat)/K_(m)of LrMA^(G260A) was two times higher than that of the wild-type,indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.展开更多
基金supported by the Postdoctoral Science Foundation of China(2021M691278)the National Key Special Project for the 13th National 5-Year Plan Program of China(2016YFD0400500).
文摘The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412(LrMA)toward soluble starch was exactly enhanced through hotspot-based research.Based on multiple sequence alignment,three-dimensional structure and existed literature,thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis.After the screening of the mutants,LrMA^(D172A),LrMA^(G260A),LrMA^(K334A)and LrMA^(M477A)were selected with the activity accounted for 144-209% of that in wild-type.Among all the mutants,LrMA^(G260A) possessed the highest activity toward soluble starch,reached 133 U/mg,about twice as high as that in the wild-type.Its temperature for optimum activity still maintained at 60℃,while had no significant loss of thermal stability occurred.In addition,compared with the wild-type in pH stability,the mutant retained over 80% residual activity at a wider pH range of 4.5-8.5.Furthermore,the k_(cat)/K_(m)of LrMA^(G260A) was two times higher than that of the wild-type,indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.
