| α-arbutin is a glycosylated derivative of hydroquinone.α-arbutin is commonly used in the cosmetic industry and clinical drug therapy because of its broad mechanism.Enzymatic method and whole-cell biotransformation are the main production methods ofα-arbutin,while enzymes with low catalytic efficiency limit the production ofα-arbutin.In this study,the sucrose phosphorylase（SPase）from Leuconostoc mesenteroides was used for the efficient production ofα-arbutin in 5 L fermenter by combining semi-rational enzyme modification and synthesis process optimization.The main findings were as follows:（1）The gene gtfA was cloned in Escherichia coli,Bacillus subtilis and Corynebacterium glutamicum,respectively.By optimizing the whole-cell transformation cell biomass of each host strain,theα-arbutin yield of B.subtilis 168/p MA5-gtfA was as high as 80.9 g·L-1 after 24h of transformation,with a molar conversion rate of 81.6%,which was 2.9 times higher than that of E.coli BL21/p ET28a-gtfA(27.9 g·L-1).（2）The enzymatic properties of wild-type sucrose phosphorylase were determined,and its specific enzyme activity was 48.6 U·mg-1.To address the problem of low enzymatic activity of wild-type sucrose phosphorylase,computer simulation was used to design mutation sites semi-rationally to screen for mutants with higher enzymatic activity.Potential beneficial mutation sites R137,F160 and L337 were first found by molecular docking.The saturable mutant libraries were established for these sites,and the mutant R137F with 161%enzyme activity of the wild type was obtained.（3）Molecular dynamics simulations and kinetic parameters were performed for the mutant R137F and the wild-type.The Km of R137F for hydroquinone was almost 2.4-fold less,and the kcat/Km was 4.6-fold higher by contrast to the wild-type.Meanwhile,the RMSF of R137F was higher than that of the wild-type,indicating an increased flexibility of the protein binding pocket of R137F mutant,which explains the increased catalytic efficiency of mutant R137F.（4）It was verified that theα-arbutin production of recombinant strain B.subtilis168/p MA5-gtfAR137F was found to be 1.2 times as high as the original strain B.subtilis168/p MA5-gtfA.The reaction conditions for the whole-cell transformation of recombinant strain B.subtilis 168/p MA5-gtfAR137F were optimized as follows:p H 7.0,temperature 30℃,biomass OD600 of 50;initial hydroquinone concentration of 45 g·L-1,and hydroquinone was added by batch feeding.The yield ofα-arbutin after the optimization of reaction conditions was107.9 g·L-1 with a molar conversion rate of 87.3%.（5）Under optimal whole-cell transformation conditions,the recombinant strain B.subtilis168/p MA5-gtfAR137F was amplified for production in a 5 L bioreactor for 24 h.The final titer ofα-arbutin was 119.3 g·L-1 with a molar conversion of 96.5%,which was a 47.5%increase in yield compared to the original strain B.subtilis 168/p MA5-gtfA. |
