肠膜明串珠菌蔗糖磷酸化酶的酶学表征及在催化合成α-熊果苷中的应用

Characterization of a sucrose phosphorylase from Leuconostoc mesenterides for the synthesis of α-arbutin

摘要:

将来源于肠膜明串珠菌Leuconostoc mesenteroides ATCC 12291的蔗糖磷酸化酶 (Sucrose phosphorylase, SPase) 基因进行密码子优化后将其插入到pET-28a中构建表达载体pET-28a-spase，诱导大肠杆菌Escherichia coli BL21(DE3)/pET-28a-spase表达制得SPase粗酶液，将重组SPase纯化后进行酶学表征。结果表明，重组SPase的比酶活为213.98 U/mg，纯化倍数为1.47倍，酶活回收率达87.80%。该酶最适温度为45 ℃，最适pH为6.5，该酶对蔗糖的Km为128.8 mmol/L，Vmax为2.167 μmol/(mL·min)，kcat为39 237.86 min-1，利用重组SPase催化氢醌合成α-熊果苷，最优条件为：氢醌添加量为40 g/L，蔗糖/氢醌的摩尔比为5:1，重组SPase 250 U/mL。在25 mmol/L的吗啉乙磺酸 (MES) 缓冲液 (pH 7.0) 中，反应温度30 ℃，避光反应24 h后终止反应，再用500 U/mL的糖化酶40 ℃处理2.5 h。α-熊果苷产量达98 g/L，氢醌的转化率接近99%。综上所述，文中克隆并研究了重组SPase，并建立了其在α-熊果苷生产中的应用。

Abstract:

Sucrose phosphorylase (SPase) gene from Leuconostoc mesenteroides ATCC 12291 was synthesised after codon optimization, and inserted into pET-28a plasmid to generate pET-28a-spase. The recombinant strain Escherichia coli BL21 (DE3)/pET-28a-spase was induced for Spase expression. The recombinant protein Spase was purified and characterized. The specific enzyme activity of SPase was 213.98 U/mg, the purification ratio was 1.47-fold, and the enzyme activity recovery rate was 87.80%. The optimal temperature and the optimal pH of the SPase were identified to be 45 °C and 6.5 respectively, and Km, Vmax and kcat of the SPase for sucrose was 128.8 mmol/L, 2.167 μmol/(mL·min), and 39 237.86 min–1. The recombinant SPase was used for α-arbutin production from hydroquinone and the reaction process was evaluated. The optimal conditions for synthesis of α-arbutin by SPase were 40 g/L hydroquinone, 5:1 molar ratio of sucrose and hydroquinone, and 250 U/mL recombinant SPase at pH 7.0 and 30 °C for 24 h in the dark, and then 500 U/mL glucoamylase was added at 40°C for 2.5 h. Under the optimized process, the yield of α-arbutin reached 98 g/L, and the hydroquinone conversion rate was close to 99%. In summary, the recombinant SPase was cloned and characterized, and its application for α-arbutin production was feasible.