Semi-rational design of sucrose phosphorylase and optimization of conditionsfor α-arbutin production
SHEN Yang1, LYU Xueqin1, LIN Lu1,2, LI Jianghua2, DU Guocheng1, LIU Long1*
1(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China) 2(Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
Abstract: α-arbutin is a high-value glycoside and has a wide range of applications in the cosmetics and pharmaceutical industries. The enzymatic method is the main way for the production of α-arbutin, but the production of α-arbutin is limited due to the lack of enzymes with high catalytic efficiency. In order to improve the biotransformation efficiency of sucrose phosphorylase (SmSP) from Streptococcus mutans UA159 and further improve the production of α-arbutin, four vectors expressing SmSP in Bacillus subtilis WB600 were screened, among which the recombinant plasmid pP43NMK-P43-gtfA achieved the highest α-arbutin yield (40.2 g/L) after 20 h of whole cell transformation. Secondly, site-directed saturation mutagenesis was performed on the loop A around the catalytic active center of SmSP to improve the affinity between the enzyme and the receptor, and a mutant SmSPI336L was obtained. Its α-arbutin production and the molar conversion of substrate hydroquinone (hydroquinone,HQ) were 71.7 g/L and 72.4%, respectively. The production increased by 78.4% compared to the control. Finally, the reaction conditions for α-arbutin synthesis were optimized by the recombinant strain B. subtilis WB600/pP43NMK-P43-SmSPI336L.The final α-arbutin productionreached110.3 g/L,and the molar conversion rate of HQ was 88.7%. The production was increased by 2.74 times compared to the control. The obtained high-catalytic efficiency recombinant strains and the analysis of mutant kinetics were of important research significance and application value for the biotransformation of α-arbutin.
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2020, Vol. 46 
