Study On The Synthetic Activity And Thermostability Of ?-glucosidase

There are both hydrolysis and synthetic activity in ?-glucosidase.It can hydrolyze oligosaccharides into the corresponding monosaccharides,and monosaccharides can also be synthesized into oligosaccharides by ?-glucosidases.Currently,people mainly focused on improving the hydrolysis activity of ?-glucosidase,but research on its synthesis activity is relatively rare.In this study,BglA,a kind of ?-glucosidase derived from Caldicellulosiruptor sp.F32,was chosen for improving the synthetic activity of disaccharides produced by rational design.The three-dimensional structure of BglA was built by homology modeling.The structure of BglA with cellobiose bind to its active site was predicted by flexible docking.It was shown that the outer glucose moiety of cellobiose was surrounded by a generally hydrophilic binding pocket.Whereas some hydrophobic residues close to cellobiose,such as L170?C166 and Y165,are in the surrounding area.A hypothesis was proposed:The transglycosylation activity of ?-glucosidase is related to the hydrophilicity of the amino acid at the disaccharide binding site,and the decrease in the hydrophobicity of the binding site may help to increase its transglycosylation activity.We therefore performed the rational engineering of BglA by site-directed mutagenesis and obtained proteins with increasing the hydrophilic property in the binding pocket.They were named by L170SBglA?L170S/C166SBglA and L170S/C166S/Y165DBglA.Additionally,The N219IBglA,BglA variant,was constructed,whose hydrophobic residue in the binding pocket was improved over the wildtype.Cel1A,a kind of ?-glucosidase derived from Trichoderma reesei,has been reported to have synthetic activity.But the thermostability of Cel1 A is poor.To improve the thermostability of Cell A,the structure and functions of the protein were analyzed by bioinformatics methods.C-terminal of Cel1 A was replaced with a segment of the C-terminal of the ?-glucosidase from Pyrococcus furiosus,a kind of thermophiles.The recombinant protein was named Cel1 A-461R473.Besides,the replaced fragment was truncated off and the recombinant protein was renamed Cel1A-464T466.Further more,dynamics and thermodynamic analysis revealed that the kinetic stability has been improved.The half-life of CellA-461R473 and CellA-464T466 is 50h and 31h,both are increased compared with the half-life of Cel1A,which is 12h.Besides,the specific?-glucosidase activity of Cel1 A-461R473 and Cel1A-464T466 increased by 56.2%and 82.7%,respectively,compared with that of Cel1 A.Therefore,the protein modification brings us a deeper understanding of the thermal stability mechanism of the enzyme,and also provides reference for the thermal stability of other enzymes.