?-glucosidase: A Site-directed Mutagenesis And Study Of Galacto-oligosaccharides Synthesis

?-glucosidases,which exist in animals,plants and microorganisms,have widespread use in food industry,pharmacy industry,agriculture and chemical engineering.They show functions in removing the non-reductive glucosyl from the end of carbohydrate and glycoside,assisting glycolipid and exogenous glycoside metabolism,activating plant hormone as well as releasing aromatic compounds and biomass conversion.The galactooligosaccharides(GOS),which is a kind of undigested oligosaccharide from lactose,has a prominent prebiotic characteristics,and has been widely applied in infant formula as a substitute of human milk oligosaccharides.Till now,the production of GOS mainly relies on hydrolysis of lactose using glycoside hydrolase,forming various products with different degrees of polymerization.Further research about GOS focus more on higher catalyzing efficiency and purity of products.In the present study,?-glucosidase TN0602 from Thermotoga naphthophila RUK10 was used as research object.All the recombinant plasmids with site mutation were obtained by whole plasmid PCR using recombinant plasmid p ET 28b-tn0602 as template.After T4 polynucleotide kinase phosphorylation and ligation,those PCR products were transformed into E.coli DH5?.The DNA sequencing results showed that the 11 mutants were successfully constructed.Those plasmids were eventually expressed in E.coli BL21(DE3).After expression in large scale,thermal treatment purification and lyophilization,those mutants could be used for catalyzing the GOS reactions.Besides,the hydrolysis and transglycosylation activity of these mutants were measured.The effect of the mutants on enzyme activity and substrate specificity were determined,finding that the exchange of one phenylalanine to serine(F414S)increased the GOS production(207.63 m M)by inhibiting hydrolysis activity.Additionally,the initial concentration of lactose increased to 1169 m M,and yield promoted to 4.79 times compared with former type.The optimal temperature for F414 S was 75 °C,and the reaction reached equilibrium after 40 h,making a yield promotion from 65.77 % to 82.29 % and synthesized GOS4 with the foundation of GOS3.If using cellobiose as substrates,the reaction would reach equilibrium after 30 h(GOS3:127.43 m M,GOS4:1.2 m M).The F414 S with His-tag was further purified by Ni2+-IDA and got electrophoresis pure protein,accompanied by assessment of the enzymology characterization.Both optimized temperatures for F414 S hydrolyzing p-nitrophenyl-?-D-glucopyranoside(p NPGlu)and o-Nitrophenyl-?-D-Galactopyranoside(o NPGal)were higher than 95 °C,and the optimized p H were 7.0 and 7.5.The thermal stability research indicated that the half-life of F414S(0.2 mg/m L)at 75,80 and 85 °C were 55,12.5 and 6 h,respectively.p H stability measurement showed that F414 S had high stability at p H 4.5-7.5,while F414 S had decreasing catalyzing efficiency.Taken together,our innovative study not only provided new thinking about GOS industrial production,but also obtained ?-glucosidase with better performance,which laid the foundation for further research.