葡萄糖基转移酶在α-葡萄糖基化丁香酚生物合成中的应用

Application of glucosyltransferase in biosynthesis of α-EG

丁香酚因具有抑菌、抗肿瘤、廉价易得等特性,被广泛应用于医药、香料及调味剂等领域。但因其不稳定、易升华、水溶性差等缺点,限制了它的进一步应用。糖基化修饰可显著提高丁香酚的应用特性,本研究利用重组大肠杆菌E.coli BL21(DE3)p ET28a-agl A高效表达的源自野油菜黄单胞菌的葡萄糖基转移酶,生物催化丁香酚生成α-葡萄糖基化丁香酚(α-EG)。同时对其生物合成中酶催化反应性质进行了研究。结果表明其最适反应条件:pH=8.5,温度为35℃,丁香酚与麦芽糖最适浓度分别为60 mmol/L及1.2 mol/L。该酶在pH 6~8及4℃~30℃内能保持较好的稳定性。1 mmol/L金属离子Cu^(2+)能强烈抑制糖基转移酶活性,而1 mmol/L金属离子Fe^(2+)对酶的催化反应有显著的促进作用。采用最优催化条件,反应100 min,α-葡萄糖基化丁香酚(α-EG)的转化率为75%。

Eugenol has been widely used in medicine,spice and condiment because of its antibacterial,anti-tumor,cheap and easy to obtain and so on. However,due to its instability,easy sublimation and poor water solubility,its further application is limited. Glycosylation can improve the application of eugenol. In this study,eugenol was biocatalyzed to α-glucosyleugenol（ α-EG） by recombinant E. coli BL21（ DE3） p ET28 a-agl A,a glucosyltransferase from Xanthomonas campestris. At the same time,the properties of enzyme-catalyzed reaction in biosynthesis were studied. The results showed that the optimum reaction conditions： p H 8. 5,temperature 35 ℃,the optimum concentration of eugenol and maltose were60 mmol/L and 1. 2 mol/L,respectively. The enzyme was stable at p H 6 - 8 and 4 ℃ - 30 ℃. The activity of glycosyltransferase was strongly inhibited by 1 mmol/L Cu^2＋,while 1 mmol/L Fe^2＋ promoted the catalytic reaction of the enzyme. The conversion of α-glucosylated eugenol（ α-EG） was 75% under the optimum reaction conditions for 100 min.