Efficient Expression Of Sucrose Phosphorylase And Synthesis Of α-Arbutin

Sucrose phosphorylase（SPase,EC 2.4.1.7）,one of glycosyltransferase,is widely used in the food,cosmetics,and pharmaceutical industries due to its wide substrate specificity.α-arbutin is a safe and effective whitening additive and a glycosylated product of hydroquinone.Compared with chemical synthesis,enzymatic synthesis ofα-arbutin from sucrose by SPase is much more promising,due to its green properties and high efficiency.However,complex metabolic regulation mechanisms of wild-type strains usually lead to low yield of SPase,badly limiting its industrial production.Therefore,efficient expression and high catalytic activity of SPase appear to more important.In this study,SPase from Thermoanaerobacterium thermosaccharolyticum（TtSPase）was recombinantly expressed in E.coli BL21（DE3）and co-expressed with molecular chaperone.Then TtSPase was applied inα-arbutin synthesis,and its catalytic activitiy was improved via semi-rational design.The main results are as follows:（1）Recombinant strain E.coli BL21（DE3）/pET-20b（+）/spase was constructed and its optimal fermentation conditions were as follows:the final concentration of 0.05 mM IPTG was added in TB medium for induction when the OD600 reached 0.9,then 3.83 U/mg TtSPase was obtained after 20 h at 18°C.The enzymatic characterization of Tt SPase were further studied.The results showed TtSPase’s optimal temperature and pH were 60°C and 6.5,respectively.In particular,it had high thermostability of T50³⁰=67°C and half-life（t1/2 at 70°C）of 19 min.In addition,the melting temperature value is about 73°C.The kinetic parameters Km and kcat/Km of TtSPase were also determined,which were 55.6 mM and 1.8 s^-1·mM^-1,respectively.（2）Co-expressed strain of E.coli BL21（DE3）/pET-20b（+）/spase with four chaperone teams（pG-KJE8,pKJE7,pGro7,and pG-TF2）were constructed.Similarly,the induction conditions（inducer concentrations,addition order of different inducers,induction temperature）were also investigated.The results showed that the co-expression of pG-KJE8 and pG-TF2teams significantly improved the soluble expression of TtSPase and reduced inclusion body formation.The soluble TtSPase activities of pG-KJE8 and pG-TF2 co-expression strains were18.5 and 25.7 U·mg^-1 for PG-KJE8 and pG-TF2 co-expression respectively,which were 4.85-and 6.22-fold higher than that without chaperone co-expression.The soluble expression of TtSPase is only 10%,while the pG-KJE8-SPase and pG-TF2-SPase had about 40%and 85%soluble SPase.The positive chaperone teams（pG-KJE8 and pG-TF2）contain two similar chaperones（GroES and GroEL）,revealing that the GroES-GroEL chaperone team plays an important role on the soluble expression of TtSPase.Meanwhile,GroES-GroEL could accompanied by TF and DnaK-DnaJ-GrpE to help the new peptide chain to fold correctly,prevent protein aggregation,and reduce the formation of inclusion bodies.（3）In order to investigate the transglycosylation of TtSPase,the reaction conditions forα-arbutin synthesis were optimized.The main optimization conditions contained reaction temperature,pH,donor-acceptor ratio,reaction progress and the concentration of hydroquinone.The reaction system forα-arbutin synthesis by TtSPase was as follows:the reaction mixture（1.5 mL）including 400 mM sucrose,100 mM hydroquinone and TtSPase（50 units）in PBS buffer（50 mM,pH 6.6）was incubated in 25°C for 16 h,and the highest yield ofα-arbutin（22.1%）was obtained.（4）To improve the catalytic activity of TtSPase,the composite crystal structure of the enzyme and substrate was obtained by homology modeling and molecular docking,and the key amino acids for interaction between TtSPase and substrate were explored.Through directed evolution and random mutation,key amino acids were mutated into other different types of amino acids.It was found that the enzyme activities of V239A and Y201L were 1.3-and 1.2-folds that of WT,which were 494 U·mg^-1 and 454 U·mg^-1,respectively.