氨氧化亚硝化螺菌蔗糖合酶的性质研究及应用

Properties and application of sucrose synthase from Nitrosospira multiformis

尿苷二磷酸(uridine diphosphate,UDP)-葡萄糖是核苷酸依赖性糖基转移酶(“Leloir”glycosyltransferases,GTs,EC 2.4)进行糖基化反应的重要糖供体。蔗糖合酶(sucrose synthase,SuSy)可以利用蔗糖和UDP为底物,通过一步反应可逆生成UDP-葡萄糖和果糖。该文对来源于氨氧化亚硝化螺菌蔗糖合酶(Nitrosospira multiformis,NmSuSy)的基因在大肠杆菌中进行了异源表达、纯化、酶学性质分析以及功能研究。通过克服可逆反应的热力学和动力学限制实现了UDP-葡萄糖的大量合成。结果表明,该酶的分子质量为89 kDa,最适反应pH为6.5,在pH 6~7.5内有较好的稳定性。最适反应温度为55℃,温度低于40℃时具有较好的稳定性。在最佳反应条件pH 6、温度40℃、1 mol/L蔗糖、100 mmol/L UDP下反应,100 mg/L的NmSuSy催化反应1 h可生成35.8 mmol/L(20.2 g/L)的UDP-葡萄糖,反应6 h即可达到平衡状态,生成41.5 mmol/L(23.5 g/L)的UDP-葡萄糖。该研究为后续进一步实现UDP-葡萄糖的高效合成奠定了理论基础,为糖生物学的研究提供了更为经济的供体底物原料。

UDP-glucose is an important sugar donor for glycosylation of“Leloir”glycosyltransferases(GTs).Sucrose synthase(SuSy)can utilize both sucrose and UDP as substrates to reversibly generate UDP-glucose in a one-step reaction.The SuSy gene from Nitrosospira multiformis(NmSuSy)was heterologously expressed in Escherichia coli.The purified NmSuSy was analyzed for its enzymatic properties and functions.The efficient synthesis of UDP-glucose was achieved by overcoming the thermodynamic and kinetic constraints of the reversible reaction.Results showed that the molecular weight of SuSy was 89 kDa,and the optimum reaction pH for SuSy was 6.5 and it was stable ranging from pH 6.0 to 7.5;while the optimal temperature was 55℃and it behaved better with temperature lower than 40℃.Under the optimized conditions(pH 6,temperature 40℃,1 mol/L sucrose,100 mmol/L UDP),reactions with NmSuSy(100 mg/L)could produce 35.8 mmol/L(20.2 g/L)of UDP-glucose in 1 h.When the reversible reaction reached equilibrium within 6 hours,41.5 mmol/L(23.5 g/L)of UDP-glucose could be produced.This study lays a theoretical foundation for the efficient synthesis of UDP-glucose,and provides a more economical donor substrate material for the research of glycobiology.