Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China.
Biotechnol Lett. 2011 Nov;33(11):2209-16. doi: 10.1007/s10529-011-0680-y. Epub 2011 Jul 1.
The endoglucanase, EGA, from Bacillus sp. AC-1 comprises a glycosyl hydrolase family-9 catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). Seven aromatic residues were subjected to site-directed mutagenesis in both CBM3 and EGA to investigate their roles in enzyme thermostability. The complexes generated by mixing CBMY527G, CBMW532A, or CBMF592G with CM9 each lost their activities after 15 min at 45°C, while the wild-type complex retained >70% activity after 2 h. The mutants EGAY527G, EGAW532A, and EGAF592G showed little activity after 15 min at 60°C, whereas EGA remained 70% active after 2 h. Thus the residues Tyr(527), Trp(532), and Phe(592) contribute not only to CBM3-mediated stability of CM9 but also to EGA thermostability suggesting that hydrophobic interaction between the two modules, independent of covalent linkages, is important for enzyme thermostability.
来自芽孢杆菌 AC-1 的内葡聚糖酶 EGA 由糖苷水解酶家族 9 催化模块 (CM9) 和家族 3 碳水化合物结合模块 (CBM3) 组成。在 CBM3 和 EGA 中对 7 个芳香族残基进行了定点突变，以研究它们在酶热稳定性中的作用。在 45°C 下孵育 15 分钟后，与 CM9 混合的 CBMY527G、CBMW532A 或 CBMF592G 生成的复合物均失去了活性，而野生型复合物在 2 小时后保留了超过 70%的活性。突变体 EGAY527G、EGAW532A 和 EGAF592G 在 60°C 下孵育 15 分钟后几乎没有活性，而 EGA 在 2 小时后仍保持 70%的活性。因此，残基 Tyr(527)、Trp(532)和 Phe(592)不仅有助于 CBM3 介导的 CM9 稳定性，也有助于 EGA 的热稳定性，这表明两个模块之间的疏水相互作用（不依赖于共价键）对于酶的热稳定性很重要。
