Fujita Yasuya, Ito Junji, Ueda Mitsuyoshi, Fukuda Hideki, Kondo Akihiko
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan.
Appl Environ Microbiol. 2004 Feb;70(2):1207-12. doi: 10.1128/AEM.70.2.1207-1212.2004.
A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
通过在酿酒酵母细胞表面共展示三种纤维素分解酶，构建了一种具有诱导协同和顺序纤维素降解反应能力的全细胞生物催化剂。当使用基于α-凝集素的细胞表面展示系统时，里氏木霉内切葡聚糖酶II、纤维二糖水解酶II和棘孢曲霉β-葡萄糖苷酶1作为与α-凝集素C端半区的单个融合蛋白同时共展示。通过荧光显微镜观察免疫荧光标记的细胞，证实了这三种酶在细胞表面的共展示。与仅展示内切葡聚糖酶II的酵母菌株相比，共展示内切葡聚糖酶II和纤维二糖水解酶II的酵母菌株对无定形纤维素（磷酸膨胀纤维素）具有显著更高的水解活性，其主要产物是纤维二糖；共展示β-葡萄糖苷酶1、内切葡聚糖酶II和纤维二糖水解酶II使酵母菌株能够直接从无定形纤维素生产乙醇（共展示β-葡萄糖苷酶1和内切葡聚糖酶II的酵母菌株则不能），在40小时内从10克/升的无定形纤维素中产生约3克/升的乙醇。产率（每消耗1克碳水化合物产生的乙醇克数）为0.45克/克，相当于理论产率的88.5%。这表明使用共展示三种纤维素分解酶的酵母菌株可以有效地实现无定形纤维素同时协同糖化和发酵生产乙醇。
