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(SmSPI336L)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SmSPI336L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SmSPI336L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 of α-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 (SmSPI336L), as a biocatalyst to convert sucrose and hydroquinone (HQ) into α-arbutin with high efficiency. Firstly, by investigating the protein expression ability, Bacillus subtilis WB800 was determined as a suitable host. The promoter optimization of SmSPI336L and genome integration were performed in B. subtilis, obtaining the recombinant B. subtilis that synthesized α-arbutin by whole-cell catalysis. The yield of α-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 SmSPI336L, the yield of α-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 of α-arbutin and stabilized the output. The optimized production of α-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 of α-arbutin.
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