College of Life Science, Hubei University, Wuhan 430062, People's Republic of China.
Biotechnol Appl Biochem. 2009 Oct 13;54(3):171-6. doi: 10.1042/BA20090222.
A novel surface-display system was constructed using the cell-wall anchor protein Flo1p from Saccharomyces cerevisiae, the mannanase (man1) from Bacillus subtilis fused with the C-terminus of Flo1p and the 6xHis tag was inserted between Flo1p and man1. The fusion protein was displayed on the cell surface of Yarrowia lipolytica successfully, and it was confirmed by immunofluorescence. In succession, the surface-displayed mannanase was characterized. The optimum catalytic conditions for the recombinant mannanase were 55 degrees C at pH 6.0, and it exhibited high stability against pH variation. The highest activity of the recombinant mannanase reached 62.3 IU/g (dry cell weight) after the recombinant was cultivated for 96 h in YPD medium [1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose]. To our knowledge, the present paper is the first to report that high-activity mannanase is displayed on the cell surface of Y. lipolytica with Flo1p.
构建了一种新型的表面展示系统，该系统使用来自酿酒酵母的细胞壁锚定蛋白 Flo1p、与 Flo1p 的 C 末端融合的来自枯草芽孢杆菌的甘露聚糖酶（man1）和 6xHis 标签。融合蛋白成功地在解脂耶氏酵母的细胞表面展示，并通过免疫荧光进行了确认。随后，对表面展示的甘露聚糖酶进行了表征。重组甘露聚糖酶的最佳催化条件为 55°C、pH6.0，并且对 pH 变化具有很高的稳定性。在 YPD 培养基[1%（w/v）酵母提取物/2%（w/v）蛋白胨/2%（w/v）葡萄糖]中培养 96 小时后，重组甘露聚糖酶的最高活性达到 62.3IU/g（干细胞重量）。据我们所知，本文首次报道了高活性的甘露聚糖酶通过 Flo1p 在解脂耶氏酵母的细胞表面展示。
