Bae Jungu, Kuroda Kouichi, Ueda Mitsuyoshi
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Appl Environ Microbiol. 2015 Jan;81(1):59-66. doi: 10.1128/AEM.02864-14. Epub 2014 Oct 10.
Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.
邻近效应是纤维素酶彼此在短距离内起作用时表现出的一种协同效应形式，这种效应可能是提高糖化效率的关键因素。在本研究中，我们评估了酿酒酵母细胞表面展示的3种纤维素降解酶（即内切葡聚糖酶、纤维二糖水解酶和β-葡萄糖苷酶）之间的邻近效应。我们通过基因组整合构建了2种武装酵母：ALL-酵母，其同时展示3种纤维素酶（因此，不同的纤维素酶彼此靠近）；MIX-酵母，由3种展示单一纤维素酶的酵母混合而成（纤维素酶彼此相距较远）。纤维素酶用荧光蛋白或多肽进行标记，以可视化并定量其展示情况。为了评估邻近效应，我们在调整纤维素酶量后，比较了ALL-酵母和MIX-酵母降解磷酸膨胀纤维素的活性。在酵母浓度等于1 ml酵母悬液中酵母细胞数，600 nm处光密度（OD）为10（OD10）或OD0.1时，ALL-酵母的活性比MIX-酵母高1.25倍或2.22倍。在OD0.1时，MIX-酵母中3种纤维素酶之间的距离大于OD10时的距离，但ALL-酵母中的距离保持不变；因此，ALL-酵母和MIX-酵母之间纤维素降解活性的差异在OD0.1时增大（至2.22倍），这有力地支持了展示的纤维素酶之间的邻近效应。对于结晶纤维素（微晶纤维素）也观察到了邻近效应。我们预计，当酶展示效率提高时，邻近效应会进一步增强，这将进一步提高纤维素降解活性。这种武装酵母技术也可应用于研究其他不同领域中的邻近效应。
