Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop.

摘要: Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, role played Arg60 Tyr61 in unique surface loop was investigated. The results are consistent previously observed hydrogen bonding stacking interactions between these two residues substrate. Interestingly, mutant Y61G had highest activity when compared wild-type enzyme other mutants. It also shows wider range working temperatures than does wild type, along retention hyperthermostability. kcat Km values both higher those type. In conjunction structure Y61G–substrate complex, kinetic data suggest that endoglucanase probably due to facile dissociation cleaved sugar moiety at reducing end. Additional crystallographic analyses indicate insertion deletion mutations site did not affect overall protein structure, but local perturbations might diminish substrate-binding strength. likely catalytic efficiency subtle balance substrate binding product release. enhancement single mutation provides good example engineered for industrial application.