枯草芽孢杆菌全细胞催化高效合成α-熊果苷

Highly efficient synthesis ofα-arbutin by whole-cell catalysis of Bacillus subtilis

α-熊果苷是一种糖苷类化合物,有较好的抗炎、修复和美白作用。通过生物转化法合成α-熊果苷具有高效、绿色环保的特点。该文以枯草芽孢杆菌为宿主,以对苯二酚(hydroquinone,HQ)和蔗糖为底物,异源表达来源于变异链球菌(Streptococcus mutans)的蔗糖磷酸化酶(SmSP^(I336L))作为生物催化剂,全细胞催化高效合成α-熊果苷。首先,通过对蛋白表达能力的比较确定了枯草芽孢杆菌(Bacillus subtilis)WB800是合适的宿主菌株,并对SmSP^(I336L)进行启动子优化及多拷贝整合表达,获得全细胞催化合成α-熊果苷的重组B.subtilis,α-熊果苷的产量为54.05 g/L,底物HQ的摩尔转化率为43.7%;然后,通过优化SmSP^(I336L)的核糖体结合位点(ribosome binding site,RBS)序列显著提高了α-熊果苷的产量和底物HQ的摩尔转化率,分别达到99.14 g/L和80.15%;最后,对全细胞催化的条件进行优化,扩大催化体系至20 mL并将催化时间从22 h延长至34 h,进一步提高了α-熊果苷的产量且产量稳定,最终优化后的α-熊果苷产量最高达到了119.44 g/L,底物HQ的摩尔转化率为96.56%。该研究成果对α-熊果苷的工业化生产具有重要意义。

α-Arbutin is a glycoside,which has good effects on anti-inflammatory,wound repair and whitening.The synthesis ofα-arbutin by biotransformation has the characteristic of high efficiency and environmentally friendly.This study used Bacillus subtilis as the host to express the heterologous enzyme,sucrose phosphorylase from Streptococcus mutans(SmSP^(I336L)),as a biocatalyst to convert sucrose and hydroquinone(HQ)intoα-arbutin with high efficiency.Firstly,by investigating the protein expression ability,Bacillus subtilis WB800 was determined as a suitable host.The promoter optimization of SmSP^(I336L) and genome integration were performed in B.subtilis,obtaining the recombinant B.subtilis that synthesizedα-arbutin by whole-cell catalysis.The yield ofα-arbutin was 54.05 g/L,and the molar conversion rate of substrate HQ was 43.7%.Then,by optimizing the ribosome binding site(RBS)sequence of SmSP^(I336L),the yield ofα-arbutin and the molar conversion rate of substrate HQ were significantly improved,reaching to 99.14 g/L and 80.15%,respectively.Finally,the conditions of whole-cell catalysis were optimized.The catalytic volume was expanded to 20 mL and the catalytic time was extended from 22 h to 34 h,which further increased the production ofα-arbutin and stabilized the output.The optimized production ofα-arbutin reached to 119.44 g/L and the molar conversion rate of substrate HQ was 96.56%.The results of this study are of great significance to the industrial production ofα-arbutin.