| 蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析 |
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| 引用本文： | 李慧娟,邵先祥,孙云鹏,高军,丁瑞,张碧凝,张治山,池振明. 蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析[J]. 微生物学通报, 2014, 41(11): 2190-2197 |
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| 作者姓名： | 李慧娟 邵先祥 孙云鹏 高军 丁瑞 张碧凝 张治山 池振明 |
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| 作者单位： | 1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;3. 汕头大学 香港中文大学联合汕头国际眼科中心 广东 汕头 515041;1. 山东科技大学 化学与环境工程学院 山东 青岛 266590;2. 中国海洋大学 联合国教科文组织中国海洋生物工程中心 山东 青岛 266100 |
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| 基金项目： | Higher School Science and Technology Plan Project of Shandong Province (No. J11LC08); Postdoctoral Innovation Project of Shandong Province (No. 201001008) |
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| 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p H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。
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| 关 键 词： | 蔗糖 蔗糖--果糖基转移酶 表面展示 解脂亚罗酵母菌 -蔗果三糖 酶学性质 |
Expression and characterization of surface-displayed sucrose:sucrose 1-fructosyltransferase on Yarrowia lipolytica cells |
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| LI Hui-Juan,SHAO Xian-Xiang,SUN Yun-Peng,GAO Jun,DING Rui,ZHANG Bi-Ning,ZHANG Zhi-Shan and CHI Zhen-Ming. Expression and characterization of surface-displayed sucrose:sucrose 1-fructosyltransferase on Yarrowia lipolytica cells[J]. Microbiology China, 2014, 41(11): 2190-2197 |
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| Authors: | LI Hui-Juan SHAO Xian-Xiang SUN Yun-Peng GAO Jun DING Rui ZHANG Bi-Ning ZHANG Zhi-Shan CHI Zhen-Ming |
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| Affiliation: | 1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;3. Joint Shantou International Eye Center, Shantou University & The Chinese University of Hong Kong, Guangdong, Shantou, 515041, China;1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, China;2. UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao, Shandong 266100, China |
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| 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. |
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| Keywords: | Sucrose:sucrose 1-fructosyltransferase Surface-display Yarrowia lipolytica 1-Kestose Enzyme characterization |
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