Modification Of Thermotropic β-galactosidase Tn1577 And Its Application In The Synthesis Of Alkyl Glycosides

Alkyl glycosides,as a new generation of non-ionic,non-toxic,low irritant and biodegradable surfactants,have a wide range of applications.The synthesis methods include chemical method and enzymatic method.Among them,chemical synthesis must be carried out under extreme conditions and requires protection and deprotection processes and toxic catalysts.The products of the reaction are complex mixtures of alkyl polyglycosides.The alkylglycosides with definite structure can be obtained by enzymatic synthesis,which overcomes the disadvantages of chemical synthesis.Glycosidase is easy to produce,economical and has substrate specificity and high stereoselectivity.It is a candidate enzyme source for synthesis of alkyl glycosides.In this study,β-galactosidase Tn1577 from Thermotoga naphthophila RUK10 catalyses the synthesis of alkyl glycosides at high temperatures above 75 ℃ and has good stability in organic solvents.However,because glycosidase has both hydrolytic activity and transglycoside activity,the yield of transglycosylation reaction is reduced,which limits the application of glycosidase in transglycosylation reaction.Therefore,in this paper,by molecular modification of Tn1577,it is expected to increase the wild-type transglycoside/hydrolysis ratio of Tn1577,so as to improve the yield of alkyl galactoside.Through molecular docking and sequence alignment of enzyme and substrate,9site-directed mutants were designed to improve the yield of alkyl glycosides.Site-specific mutation was performed by whole plasmid PCR to construct mutant protein expression strain.The purified enzyme was obtained by heat treatment and Ni-NTA affinity chromatography,and its basic enzymatic properties were characterized and alkyl glycosides were synthesized.The results showed that the activity of H271 S,V594 and R353 Q to hydrolyze o-NPG was 1.9 and 1.3 times higher than that of wild enzyme 2,R102 V,H271S and R102 V to hydrolyze o-NPG.The optimum temperature and p H of most mutants are basically the same or similar to that of wild enzymes.The yield of H271 was 1.6 times and 1.8 times higher than that of wild enzyme in the synthesis of butyl and hexyl galactoside,and 2.7 times higher than that of wild enzyme in the synthesis of octylgalactoside.The content of octylgalactoside synthesized by R102 W and R353 Q was 1.7 and 1.4 times of that of wild enzyme,and the transglycoside/hydrolysis ratio was 2.3 and 3.1 times of that of wild enzyme.The yield of hexyl galactoside synthesized by W183 F and R353 Q was the same as that of wild enzyme,but its activity of lactose hydrolysis was only 12.8% and 45.7% of that of wild enzyme,which could reduce the hydrolysis of raw lactose,continuously synthesize alkyl glycosides and reduce the production cost.Eleven three-dimensional structures of GH42 family glycoside hydrolases have been analyzed,but none of them revealed the function of domain C.Through the characterization of truncated mutant V594,the function of domain C was preliminarily explored.The results showed that the optimum temperature and p H of V594 did not change with that of o-NPG and lactose,but the activity of V594 to hydrolyze o-NPG was nearly 1 times higher than that of wild enzyme,and the activity of lactose hydrolysis was lost by about 20%.DSC analysis and stability experiments showed that the thermal stability and p H stability of V594 were significantly reduced,indicating that domain C was closely related to enzyme stability.In conclusion,we successfully obtained site-specific mutants with increased transglycoside/hydrolysis ratio,and improved the synthetic yield of alkyl glycosides,which is of great value for the biosynthesis of alkyl glycosides.It was found that domain C might be related to the stability of the enzyme,which also provided a basis for the structure-function relationship of GH42 family galactosidase and a new idea for its future transformation.