Study Of Cloning And Expression Of Sucrose Phosphorylase And Biotransformation Of ?-arbutin

?-Arbutin is a high-value glycoside and has broad application prospects in the cosmetics and pharmaceutical industries.The enzymatic method is the main method for producing?-arbutin,but due to the lack of enzyme with high catalytic efficiency,the production of?-arbutin is still limited.In this paper,an environmentally friendly biotransformation method for the synthesis of?-arbutin is proposed.Strain Bacillus subtilis WB600 introduced with sucrose phosphorylase?SmSP?is used to transfer the glucosyl from the donor substrate sucrose to the hydroxyl group of the receptor substrate hydroquinone?HQ?,thereby efficiently synthesizing?-arbutin.The main results are as follows:1.The SmSP derived from Streptococcus mutans UA159 was heterologous expressed in B.subtilis WB600,and the?-arbutin was synthesized by whole-cell transformation using the successfully constructed recombinant BS-HT-SmSP.At the same time,the expression level of SmSP is optimized at the level of transcription and translation.Firstly,different expression vectors are screened.Secondly,different promoters are screened.Thirdly,different coding sequences are fused and expressed at N-terminus of SmSP.It can be concluded that when the expression vector is pP43NMK and the promoter P₄₃,after whole-cell transformation,the production of?-arbutin reached 40.2 g·L^-1,and the molar conversion rate of substrate HQ was32.5%.2.The optimal mutants SmSP^I4K?Mutation of Ile4 to Lys4?and SmSP^I336L?mutation of Ile336 to Leu336?were obtained by performing site-directed saturation mutations on the N-terminal flexible region and the loop A region of SmSP.Their corresponding recombinant strains BS-NMK-I4K,BS-NMK-I336L showed better catalytic activity compared with the original strain BS-NMK-SmSP and the production of?-arbutin were 61.1 g·L^-1 and 71.7 g·L^-1,respectively,and the molar conversion of HQ were increased to 49.4%and 58%,respectively.Then,we conducted a combination mutation on the two single mutants and obtained a combination mutant(SmSP^I336L-I4K).The corresponding strain BS-NMK-I336L-I4K was compared with the BS-NMK-I336L for catalytic verification.Finally,we determined that the optimal mutant was the single mutant SmSP^I336L.Subsequently,we purified the wild-type enzyme?SmSP?and the optimal mutant(SmSP^I336L),and found that the optimal temperature for catalyzing the formation of?-arbutin was 30?and the optimal pH was 7.0.Moreover,we measured the enzymatic kinetic parameters and found that the K_m value of the mutant SmSP^I336L336L decreased from 26.3 mmol·L^-1 of the original enzyme?SmSP?to 21.08 mmol·L^-1,k_cat increased from 316.06 min^-1 to 389.74 min^-1,the specific enzyme activity increased from 16.3 U·mg^-1 to18.7 U·mg^-1.3.The obtained recombinant strain BS-NMK-I336L was optimized for whole-cell catalytic conditions,and the optimal transformation conditions were as follows:the final concentration of substrate HQ was 50 g·L^-1,the final concentration of sucrose was 310.9 g·L^-1,the fermentation time to collect the bacteria is 12 h,the whole cell transformation time is 20 h,the cell addition amount(OD₆₀₀)is 40,and 1%volume fraction of surfactant Triton X-100 is added.The final production of?-arbutin reached 110.3 g·L^-1,and the molar conversion rate of substrate HQ was 89.2%.