Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia.
Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia.
Enzyme Microb Technol. 2019 Apr;123:8-14. doi: 10.1016/j.enzmictec.2019.01.005. Epub 2019 Jan 6.
In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to β-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1, or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the K values for NADPH of both immobilized enzymes were somewhat higher than the K of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization.
为了在酿酒酵母细胞表面展示木糖还原酶，我们构建了两种不同的基因构建体。在第一种构建体中，木糖还原酶基因 GRE3 与 CCW12 基因的两个部分融合，N 端编码分泌信号序列，C 端编码糖基磷脂酰肌醇锚定信号。转化细胞合成木糖还原酶并通过糖基磷脂酰肌醇锚的残基将其整合到细胞壁中。另一种构建体是通过将 GRE3 与 PIR4 基因融合构建的，PIR4 基因编码 Pir 家族的一种蛋白，该蛋白包含特征性的 N 端重复序列，将 Pir 蛋白锚定到β-1,3-葡聚糖上。通过这种方式，木糖还原酶通过其 N 端共价连接到葡聚糖上。对于这些构建体的表达，我们使用了 GAL1 或 PHO5 启动子。两种菌株都显示出活性木糖还原酶，并且比较了它们的酶性质与仅带有分泌信号序列而没有锚定信号的对照酶，该对照酶分泌到培养基中。通过与 PIR4 的 N 端融合表达的酶对木糖的亲和力比另一种构建体更高，但它们的亲和力都比对照酶稍低。类似地，两种固定化酶的 NADPH 的 K 值都比对照 XR 的 K 值稍高。与对照酶相比，两种固定化酶，特别是与 Pir4 融合的酶，具有更高的热稳定性和 pH 稳定性，而其他酶学性质则不受表面固定化的显著影响。
