Spacer-mediated display of active lipase on the yeast cell surface
Spacer-mediated display of active lipase on the yeast cell surface
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DOI：
10.1007/s002530100718
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发表时间：
2001-09-01
影响因子：
5
通讯作者：
Tanaka, A
中科院分区：
文献类型：
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作者：
Washida, M;Takahashi, S;Tanaka, A
我们利用细胞表面工程技术，构建了一株在细胞表面展示活性脂肪酶的酿酒酵母。将编码米根霉脂肪酶（ROL）的基因与编码前α因子前导序列和α-凝集素C末端一半（包括糖基磷脂酰肌醇锚定附着信号）的基因融合。构建的基因在甘油醛-3-磷酸脱氢酶启动子的控制下过表达。将由Gly/Ser重复序列组成的接头肽（间隔子）插入ROL的C-末端部分，以通过保持C-末端部分附近的活性位点的构象来增强脂肪酶活性。通过免疫荧光显微镜证实表达的ROL在细胞表面上的定位。展示在酵母细胞壁上的ROL表现出对可溶性2，3-二巯基丙-1-醇三丁酯（BALB）和不溶性三油酸甘油酯的活性。接头肽的插入影响对BALB的活性，从而证明存在接头肽的最佳长度。对三油酸甘油酯的活性在具有较长接头肽的脂肪酶中更高。展示在细胞壁上的ROL对三油酸甘油酯表现出比分泌形式的酶相当和/或更高的活性。这是第一个报告的活性脂肪酶展示在细胞表面。此外，插入适当长度的接头肽作为间隔物可以是在细胞表面上有效展示酶，特别是在C-末端部分具有活性区的酶的改进方法。
We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha -factor leader sequence and the C-terminal half of alpha -agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.