Improving Transglycosylation Activity Of Thermotolerant ?-glucosidase By Site-Directed Mutagenesis

Galactooligosaccharides is a kind of functional oligosaccharides with a variety of physiological functions,Alkyl Polyglycoside is a new generation of environmental surfactant,which used in foods and detergents industry widely.Traditionally,the compound method of these two types of glycoside were chemical synthetic methods,but the complicated pretreatment limits the industrial production greatly,so the use of mild conditions and safety enzymatic method has become the first choice,generally,glycosyltransferases and glycosidases are two major types.Due to the rare types and other restrictions,the glycosyltransferase were not suitable for mass production,so glycosidases became the new choice of industrial production of glycosides,which due to its abundant sources and the activity of transglycosylation.Since the high temperature help to improve the solubility of the substrate,reduce the viscosity,the industry costs and prevent microbial contamination,therefore,this paper choose ?-glucosidase from Thermoanaerobacter ethanolicus JW200 and Thermotoga maritima MSB8 as objects.1?Study the transglycosylation activity of Recombinant Te-BglA and Tm-BglA.Recombinant plasmids were overexpressed in E.coli and purified by Ni affinity chromatography to purify recombinant Te-BglA and Tm-BglA.Analyse the effect of monosaccharides on ?-glucosidase,found that compare with Te-BglA,Tm-BglA was activated by L-glucose.Study the substrate specificity of recombinational ?-glucosidase,finding that Te-BglA and Tm-BglA have better specifiity for pNPGlc and cellobiose than other substrates.By thin-layer chromatography and high performance liquid chromatography,analyse the transglycosylation activity of Te-BglA and Tm-BglA with lactose,pNPG,glucose and cellobiose,finding that Te-BglA has better activity of transglycosylation than Tm-BglA,especially,while 34 mM cellobiose,10 ?g/ml enzyme,at pH 6.2 and 60 C,react 8 h,the concentration of HG was 18.2 mM.2?Site-directed mutagenesis and research the characterization of mutants.Tm-BglA as a template with a better thermostability,by multiple sequences alignment,mutated sites Glu166,Asn222,Tyr295,Glu351,Phe414 were determined,obtaining mutants E166A,N222F,N223C,N223Q,G224A,Y295F,E351A,F414S and W168S-N246S by inverse PCR techniques,and characterizing enzymatic properties.The optimal pH of N223Q was 6.6,while others had not obvious change with Tm-BglA which pH 6.2.The pH stability of Y295F was better than others,keeping over 60%residue activity range in pH from 4.2 to 8.2.The optimal temperature of G224A and Y295F were 95?,while others had not obvious change with Tm-BglA which 90?.The thermal stability of Y295F was higher than Tm-BglA,keeping over 50%residue activity at 95? for 1 h,while others could not keep 40%residue activity.The ethanol tolerance of Y295F was better than others.The activity of each mutants were activated by L-glucose as additive.Study the substrate specificity of mutants,finding that N222F?N223C?N223Q?G224A and W168Y-N246S have better specifiity for pNPGlc than Tm-BglA,and the Km value of these mutants below Tm-BglA,and kcat/Km value is higher than Tm-BglA.3?Study the transglycosylation activity of Tm-BglA and mutants.By thin-layer chromatography to screen qualitatively and high performance liquid chromatography to analyze transglycosylation reaction mechanism with pNPG,glucose and cellobiose quantitatively.Finding that N222F has better activity of transglycosylation than Tm-BglA,especially,while 34 mM pNPG,10 ?g/ml enzyme,at pH 6.2 and 60?,react 8 h,the concentration of HG was 28.0 mM,equivalent to twice Tm-BglA.And research factor of transglycosylation,find the optimal pH is 6.2,the optimal moisture content is 20%,the optimal temperature is 80?,the optimal concentration of pNPG is 45 mM,and the optimal N222F content is 15 ?g/mL,that provides a theoretical basis for industrial production.4?The molecular docking of subtrate with Tm-BglA.Using Autodock to simulate the binding status within the enzyme and the substrate,in case to study the difference of binding within the enzyme and the substrate before mutation and after mutation.In this study,we found that compared to Tm-BglA,the binding free energy of the substrate pNPG and N222F decreased significantly,the distance of space to the pNPG glycoside bond,indicating strong hydrophobic phenyl group projecting more favorable substrate pNPG glycoside bond cleavage,promote transglycosylation reaction by combined with hexanol,it's also have important application value to improve the surface-active agent-biological conversion of hexyl glycosides.