重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of α-arbutin

摘要:

利用重组大肠杆菌Escherichia coli Rosetta(DE3)/pET-SPase发酵生产蔗糖磷酸化酶 (EC 2.4.1.7，Sucrose phosphorylase，SPase)。收集的菌体经高压破碎后离心得到粗酶液，通过镍NTA亲和层析、超滤除盐后得到电泳纯的SPase，纯化后的SPase的比酶活是原来的2.1倍，酶活回收率达到82.7％。经SDS-PAGE电泳测定，重组SPase的分子量约为59 kDa。该酶在不高于37 ℃，pH 6.0~6.7的条件下比较稳定，最适催化温度与最适催化pH分别为37 ℃，pH 6.7，该酶对蔗糖的米氏常数 (Km) 为7.3 mmol/L，最大反应速率 (Vmax) 为0.2 μmol/(min·mg)。此外文中还以蔗糖和氢醌为底物，利用重组SPase催化合成α-熊果苷。其最佳反应条件为：20％蔗糖，200 U/mL的酶液，1.6％氢醌，pH 6.0~6.5，25 ℃，反应21 h。α-熊果苷的摩尔产率为78.3％，α-熊果苷的产量为31 g/L。

Abstract:

Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA af?nity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 °C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 °C and 6.7, respectively. The Km of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 μmol/(min·mg). Besides, α-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of α-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6?6.5 and 25 °C for 21 h. Under these conditions, α-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of α-arbutin was about 31 g/L.