Improving The Thermal Stability Of The β-galactosidase From Aspergillus Oryzae By Site-directed Mutagenesis

β-D-galactosidase (β-D-galactoside galactohydrolase, EC3.2.1.23) hydrolyzes the disaccharide lactose to glucose and galactose, and it can transfer the galactoside. Lactase can be used for the treatment of'lactose intolerance', and the poduction of low-lactose milk, whey and other dairy Products. In recent years, the technology of lactose has matured iin the low-lactose milk and galacto-oligosaccharide production process. Marketing demand of lactose increases year by year. So lactase study has important theoretical and practical value.This study aimed at the thermostability improvement ofβ-galactosidase from Aspergillus oryzae. Three-dimensional structure of lactose had been prediction by homology modeling. Using Bioinformatics software, the amino acid site of the enzyme which may affect the thermostability had been analyzed. Methods of site-directed mutagenesis was used for the purpose. Different mutants were analyzed to test and verify the effects of mutations.In this study, we get eighteen mutants. Compare to the wild-type enzyme, the thermostability of eighteen mutants has changed in various degree.The thermostability of the mutant T211P has a notable raise. The mutant T211P remain more than 90% enzyme activity after incubate 40 minutes at 60℃. Tne half-life of T211P was 177.73 minutes at 60℃compare with wild type. Tne half-life of T211P was 15.07 minutes at 65℃. During mensurate Km/Kcat of the T211P, its Km/Kcat increase from the 1.19×103 to 2.23×103. subsequently, the site of 211 had been studied. According to side chain of amino acid, the twenty amino acid was divided into eight groups. The 211Thr was mutated into the eight groups. The results showed that the thermostability of t nutants of T211N, T211D, T211S and T211G have some improvement.The thermostability of the mutant L117P,S162P and G207P has not raise, but the optimal temperature of the mutant L117P,S162P and G207P has lower. the optimal temperature of the mutant L117P and G207P reduced from 60℃to 50℃, and the optimal temperature of the mutant S162P and G246P-248P reduced from 60℃to 40℃. In addition to, the thermostability of the mutant 211P-G246P, T211P-G248P and 211P-G246P have some improvement compare with wild type, but the effect of improvement was not significant.The thermostability of lactase LACH was improved by protein engineering methods in this study. The site of 211 has a great effert for the thermostability of LACH. The mutants obtained in this study can be good materials for further research into the relationship between structure and function of lactase.