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.

Abstract

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, ChiCWdeltaFC (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since ChiCWdeltaAC (lacking the chitin-binding domain) could not be expressed in Escherichia coli as ChiCWdeltaFC did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW (ChBD(ChiCW)) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with ChBD(ChiCW). When the chitin-binding domain of ChiA1 was replaced with ChBD(ChiCW), the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that ChBD(ChiCW) may play an important role in the antifungual activity of ChiCW.