Cloning, Expression, Purification And Characterization Of ?-Glucosidases Tcur-1739 And AmyS

Background and purpose : ?-Glucosidase?EC.3.2.1.20?is a kind of starch hydrolases specifically hydrolyzes alpha-1,4 glycosidic bond of oligosaccharides such as maltose.It also has transglycosidation activity.It is a key enzyme in the production of isomaltooligosaccharide such as isomaltose isomaltotriose isomaltotetrose.With the catalysis of ?-glucosidase,the production such as isomaltose panose and isomaltotriose can improve the taste of beer.So?-glucosidase palys an important role in industrial applications.Compared with the normal temperature ?-glucosidase,thermophilic enzyme has some advantages such as high thermal stability,wide substrate adaptability and low cost of purification,make it more suitable for industrial application.There for this thesis intends to clone,express,purify and characterize ?-glucosidase Tcur-1739 and Amy S from thermophilic bacteria Thermomonospora curvata Henssen 1957 and Bacillus licheniformis Chester 1901.The results will provide new choice for the application of ?-glucosidase in industry.Experimental method:Got the gene of ?-glucosidase Tcur-1739 and Amy S by PCR.Constructed the expression vector p ET-21a-tcur-1739 and p ET-28a-amy S by using molecular biology technology.Expressed the protein using E.coli.BL21?DE3?as host.Purified protein by anion exchange chromatography column Q and Ni²⁺column.And then characterized ?-glucosidase Tcur-1739 and Amy S.Finally,the kinetic constant Km and kcat value were detected.Rusult:The sequencing result showed the recombinant protein and the sequence from NCBI was the same.The purity of recombinant proteins is over 93%.The optimum p H for Tcur-1739 and Amy S are both 7.0.The optimum temperature of Tcur-1739 is 50 ? and Amy S is 80 ?.The Km and kcat for Tcur-1739 against p NP-?-Glu are 0.23 m M and 2.94×106 s-1.The Km and kcat for Amy S against p NP-?-Glu are 0.21 m M and 1.08×105 s-1.Conclusion: Two ?-glucosidases were successfully expressed with high activity.After purification,the recombinant proteins were then characterized.Tcur-1739 showed high activity for p NP-?-Glu.Amy S showed good thermal stability and catalytic properties.