Directed Evolution Of α-glucosidase From Baker’s Yeast

Maltose is the main fermentable sugar in unsweetened dough,so the utilization rate of maltose is the influencing factor of baker’s yeast’s fermentation ability.Alpha-glucosidase plays a leading role in maltose metabolism and the presence of glucose can inhibit the activity of alpha-glucosidase to some extent.Therefore,improving the activity of alpha-glucosidase and weakening the inhibition of glucose are effective ways to improve the speed of maltose metabolism.In this paper,three mutants of baker’s yeast,which have enhanced the activity of alpha-glucosidase and weakened the inhibitory effect of glucose,were obtained by high-throughput screening through the combination of error-prone PCR,point mutation and DNA rearrangement,and their enzymatic properties and structure-activity relationship were studied.The main results are as follows:(1)The mutation library encoding a-glucosidase was constructed by error-prone PCR,and the mutant library was screened by high-throughput screening.The 37 mutants obtained by the primary screening were screened to obtain the ratio of the relative enzyme activity of the two strains.The higher mutants had a relative specific activity that was 1.4 and 1.5-folds greater than the alpha-glucosidase wild type,respectively.The enzymatic properties of a-glucosidase wild-type and mutants were studied.It was found that the optimum reaction temperature of a-glucosidase in mutants MALep1 and MALep2 was 30℃,which was 5℃lower than that of wild type.At less than 35℃,the thermal stability of α-glucosidase in MALep1 is superior to that of wild type,and the thermal stability of the enzyme rapidly decreases when it exceeds 35℃.The thermal stability of MALep2 is lower than that of wild type.The optimum reaction pH of the mutant did not change,but the pH stability of the mutant enzyme deteriorated.The inhibitory effect of glucose on mutant enzyme activity is diminished.By sequencing,it was found that both mutants had sense mutations.The homology modeling analysis of the relationship between the mutation sites and the spatial structure revealed that the amino acid mutation caused the internal space of the protein to increase,which may make the substrate easier to enter the active pocket.It can better catalyze the hydrolysis of the substrate.(2)A single point site-directed mutagenesis study was performed on each mutant site of mutant MALep2 to analyze the relationship between the mutation site and α-glucosidase activity.The results showed that the D440V mutation resulted in an increase in α-glucosidase activity,and the mutations in the other three sites did not have a positive effect on α-glucosidase activity.Through homology modeling,it was found that the mutation resulted in a decrease in the hydrogen bond formed between the amino acid at position 440 and the surrounding amino acid,which increased the flexibility of the α-helix and increased the spacing between amino acids,possibly making the substrate easier to enter the protein.Internal,but the stability of the enzyme deteriorates due to a decrease in hydrogen bonding.(3)The DNA rearrangement was carried out by using the mutant of α-glucosidase obtained by error-prone PCR as a template,and 72 mutants obtained by primary screening were subjected to rescreening to obtain a mutant MALDs3,and the α-glucosidase ratio of the mutant was compared.The enzyme activity reached 1.7-folds that of the α-glucosidase wild type.By analyzing the effect of glucose on the activity of α-glucosidase wild type and mutant,it was found that the inhibition of a-glucosidase mutant by glucose was weakened.By sequencing,it was found that the mutation site not only came from the template but also introduced a new mutation site K425E compared to the amino acid sequence of the α-glucosidase wild type.Through homology modeling and protein activity pocket prediction,it was found that the mutation site is located near the active pocket.Therefore,it is speculated that the increase of the mutant enzyme activity may be due to the mutation leading to the change of the active pocket,thus affecting the ability of the catalytic site to hydrolyze the substrate.