Enzymatic Synthesis Of ?-arbutin

?-Arbutin,a glycosylated derivative of hydroquinone(HQ),is a new non-irritating and natural whitening active substances,which is widely used in the cosmetics industry.The enzymatic conversion synthesis of ?-arbutin has the advantage of being safe and efficient.In this study,we optimized the synthesis of ?-arbutin by Cyclodextrin Glycosyltransferase(CGTase),and the molecular transformation of the enzyme with high conversion rate was studied.Sucrose phosphorylase(SPase)was used to catalyze the synthesis of ?-arbutin,and the purified ?-arbutin was obtained.The main research contents are as follows:(1)?-CGTase of Paenibacillus macerans,?-CGTase of Bacillus circulans 251,?/?-CGTase of B.stearothermophilus NO2 and Anaerobranca gottschalkii were selected to study the effects of CGTase on production of ?-arbutin.The optimum conditions for the synthesis of ?-arbutin were optimized,and the highest conversion rate of HQ was 25% by CGTase from A.gottschalkii.(2)To improve the conversion of HQ,we conducted site-directed mutagenesis of CGTase from A.gottschalkii to reduce the steric hindrance of amino acids near +2 receptor sites and improve its affinity to substrate,we obtain mutant Y299 A,the molar conversion of the mutant to HQ was up to 40% under the same optimized reaction.(3)The optimized sucrose phosphorylase gene from Leuconostoc mesenteroides ATCC 12291 was cloned and expressed in E.coli BL21(DE3).The recombinant strain was cultured in TB medium at 25°C and the enzyme activity was 40 U·m L-1 after cultivation of 24 h.SPase was further scaled up in 3-L batch fermentations for increasing its expression level.(4)SPase was effectively catalyzed the glucosyl residue transglycosylation from sucrose to hydroquinone.In addition,the enzymatic synthesis of ?-arbutin was performed under the optimal conditions:buffer pH 6.0,reaction at 40°C for 24 h and the conversion rate reached 91% and the yield of a-arbutin can reach 90 g·L-1.(4)The purification of ?-arbutin was carried out with large pore size resin AB-8.The optimum conditions were as follows: ?-arbutin concentration was 10 mg·ml-1 and loading rate was 1 BV?h-1.the resin is eluted with 30% ethanol solution and the purity of the production was 97%.