Matsuoka Hiroyuki, Hashimoto Kazuya, Saijo Aki, Takada Yuki, Kondo Akihiko, Ueda Mitsuyoshi, Ooshima Hiroshi, Tachibana Taro, Azuma Masayuki
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Yeast. 2014 Feb;31(2):67-76. doi: 10.1002/yea.2995. Epub 2014 Jan 13.
A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes.
已开发出一种用于在微生物细胞表面添加新蛋白质功能的展示系统，并提出了该系统在各个领域的应用。为了构建适合在酿酒酵母中进行蛋白质展示的细胞表面环境，对细胞壁突变体的细胞表面结构进行了研究。通过使用基于糖基磷脂酰肌醇（GPI）锚定的绿色荧光蛋白（GFP）展示系统进行分析，选择了四种细胞壁突变菌株。在这四种突变体中，β-葡萄糖苷酶和内切葡聚糖酶II展示在细胞表面，并对它们的活性进行了评估。mnn2缺失菌株对这两种酶均表现出最高活性。特别是，在突变菌株中以羧甲基纤维素为底物时，内切葡聚糖酶II的活性比野生型菌株高1.9倍。此外，用溶壁酶20T处理从mnn2缺失菌株释放的内切葡聚糖酶II的活性高于野生型菌株。绿色荧光蛋白（GFP）和内切葡聚糖酶展示的结果表明，mnn2缺失增加了细胞表面展示的酶量。将mnn2缺失菌株的酶活性与野生型菌株进行了比较。以羧甲基纤维素为底物时内切葡聚糖酶II活性的相对值（mnn2缺失突变体/野生型菌株）高于以对硝基苯基-β-D-吡喃葡萄糖苷为底物时β-葡萄糖苷酶活性的相对值，这表明mnn2缺失菌株的细胞表面环境有利于高分子量底物与展示酶的活性位点结合。
