Combined Approach to Engineer a Highly Active Mutant of Processive Chitinase Hydrolyzing Crystalline Chitin.

ABSTRACT: Serratia marcescens chitinase A (SmChiA) processively hydrolyzes recalcitrant biomass crystalline chitin under mild conditions. Here, we combined multiple sequence alignment, site-saturation mutagenesis, and automated protein purification and activity measurement with liquid-handling robot to reduce the number of mutation trials and shorten the screening time for hydrolytic activity improvement of SmChiA. The amino acid residues, which are not conserved in the alignment and are close to the aromatic residues along the substrate-binding sites in the crystal structure, were selected for site-saturation mutagenesis. Using the previously identified highly active F232W/F396W mutant as a template, we identified the F232W/F396W/S538V mutant, which shows further improved hydro

SUBMITTER: Visootsat A 

PROVIDER: S-EPMC7581260 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature