Analysis of the Involvement of Chitin-Binding Domain of ChiCW in Antifungal Activity, and Engineering a Novel Chimeric Chitinase with High Enzyme and Antifungal Activities

摘要

An antifungal chitinase, ChiCW, produced by Bacillus cereus28-9 is effective against conidial germination of Botrytiselliptica, the causal agent of lily leaf blight. ChiCW as amodular enzyme consists of a signal peptide, a catalyticdomain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCWwere truncated, ChiCWΔFC (lacking the chitin-bindingdomain and fibronectin type III-like domain) lost itsantifungal activity. Since ChiCWΔC (lacking the chitin-binding domain) could not be expressed in Escherichiacoli as ChiCWAFC did, a different strategy based onprotein engineering technology was designed to investigatethe involvement of the chitin-binding domain of ChiCW(ChBD_(chicw)) in antifungal activity this study. BecauseChiAl of Bacillus circulans WL-12 is a modular enzymewith a higher hydrolytic activity than ChiCW but notinhibitory to conidial germination of Bo. elliptica and thesimilar domain composition of ChiAl and ChiCW, the C-terminal truncated derivatives of ChiAl were generatedand used to construct chimeric chitinases with ChBD_(chicw).When the chitin-binding domain of ChiAl was replacedwith ChBD_(chicw),the chimeric chitinase named ChiAAAWexhibited both high enzyme activity and antifungal activity.The results indicate that ChBD-(chicw)may play an importantrole in the antifungal activity of ChiCW.