Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae
Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae
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DOI：
10.1007/s002530051086
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发表时间：
1997-10-01
影响因子：
5
通讯作者：
Tanaka, A
中科院分区：
文献类型：
--
作者：
Murai, T;Ueda, M;Tanaka, A
我们尝试通过基因方法将纤维素酶蛋白以其活性形式固定在酿酒酵母的细胞表面上，即编码真菌棘孢曲霉的 FI-羧甲基纤维素酶 (CMCase) 的 cDNA。与其分泌信号肽一起，与编码酵母α-凝集素C端一半（C端的320个氨基酸残基）的基因融合，酵母α-凝集素是一种参与交配并共价锚定在细胞壁上的蛋白质。将构建的含有该融合基因的质粒导入酿酒酵母并在来自酿酒酵母的甘油醛-3-磷酸脱氢酶启动子的控制下表达。在细胞沉淀部分中检测到 CMCase 活性。通过葡聚糖酶处理而非十二烷基硫酸钠处理，CMCase 蛋白从细胞壁部分中溶解，表明融合蛋白与细胞壁共价结合。通过免疫荧光显微镜和免疫电镜进一步证实了融合蛋白在细胞表面的出现。这些结果证明CMCase以其活性形式锚定在细胞壁上。
We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form, A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus. with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.