title: Site-directed mutagenesis of aromatic residues in the carbohydrate-binding module of Bacillus endoglucanase EGA decreases enzyme thermostability.
abstract: 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.
authors: 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.
authors: Qiuyu
authors: Q
authors: Yin
authors: Yigang
authors: Y
authors: Teng
authors: Ming
authors: M
authors: Ding
authors: Fukun
authors: F
authors: Zhao
mesh_terms: D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D024322:Amino Acids, Aromatic; D050505:Mutant Proteins; D002480:Cellulase