蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析

Expression and characterization of surface-displayed sucrose:sucrose 1-fructosyltransferase on Yarrowia lipolytica cells

摘要:

【目的】蔗糖:蔗糖-1-果糖基转移酶催化1分子蔗糖上的果糖基转移到另一个蔗糖分子上，形成1-蔗果三糖和葡萄糖。在低聚果糖中，1-蔗果三糖益生素活性最高。本研究将该酶展示在酵母菌细胞表面上，并用于1-蔗果三糖的制备。【方法】将来自莴苣的蔗糖:蔗糖-1-果糖基转移酶基因克隆到用于酵母细胞表面展示的表达载体上，并在解脂亚罗酵母菌中进行异源表达，表达的酶展示在该细胞表面上，然后以蔗糖为底物，研究表面展示的蔗糖:蔗 糖-1-果糖基转移酶的性质。【结果】免疫荧光实验结果表明蔗糖:蔗糖-1-果糖基转移酶已展示在酵母菌的细胞表面上，高效液相色谱结果表明酵母表面展示的该酶具有转移酶的催化活性。该酶的最适作用温度、最适作用pH分别为45 °C和7.5；该酶的催化活性受Zn2+和Cu2+的抑制，受Ca2+激活；该酶重复使用7次后，酶活下降50%。表面展示的蔗糖:蔗糖-1-果糖基转移酶和3%蔗糖混合后在40 °C条件下孵育30 min后，所产1-蔗果三糖含量最高为20.8 mmol/L。【结论】蔗糖:蔗糖-1-果糖基转移酶在解脂亚罗酵母菌中得到成功表达，并展示在其细胞表面上，生化研究表明该重组蛋白具有果糖基转移酶活性，且催化蔗果三糖的生成。表面展示的蔗糖:蔗糖-1-果糖基转移酶作为一种全细胞催化剂能够用于1-蔗果三糖的制备。

Abstract:

[Objective] Sucrose:sucrose 1-fructosyltransferase (1-SST) catalyzes the transfer of a fructosyl residue from one sucrose to another sucrose, forming 1-kestose and glucose. 1-Kestose has the highest prebiotic activity among fructooligosaccharides. In this study, 1-SST displayed on the cell-surface of Yarrowia lipolytica was used to prepare 1-kestose. [Methods] 1-SST gene from Lactuca sativa was cloned into the surface-display vector and expressed in the cells of Y. lipolytica. Biochemical characteristics of the displayed 1-SST were investigated with sucrose as its substrate. [Results] Immunofluorescence microscopy assay and high performance liquid chromatography (HPLC) indicated that the expressed protein was displayed on the cell-surface of Y. lipolytica and possessed 1-SST activity. The displayed 1-SST showed the highest activity at 45 °C and pH 7.5. The activity of the displayed 1-SST was inhibited by Zn2+ and Cu2+, while stimulated by Ca2+. The enzyme activity reduced 50% of initial activity after the displayed 1-SST was repeatedly used for seven times. The highest content of 1-kestose reached 20.8 mmol/L after the reaction mixture containing the displayed 1-SST and 3% sucrose was incubated at 40 °C for 30 min. [Conclusion] 1-SST was successfully expressed and displayed on the cells of Y. lipolytica, and the fructosyltransferase activity was detected. The surface-displayed 1-SST as a whole-cell catalyst can be applied to the 1-kestose preparation.