In vitro screening of Pseudomonas aeruginosa PAO1 chitinase C

Abstract

Chitin is an abundant aminopolysaccharide constituent of many structural elements in crustaceans, insects, and fungi. They are critical for maintaining structural integrity. As such, chitin is an attractive target for agricultural pest control. Chitinases like chitinase C (chiC) from the Pseudomonas aeruginosa PAO1 genome are enzymes that exhibit chitinolytic activity. Many chitinase candidates have been screened in vitro and in vivo to identify key chitinases for downstream biotechnological applications. Previously, Rocha et al. cloned chiC into a pET-28a expression vector and subsequently confirmed expression in E. coli BL21(DE3). However, chiC was not purified and its chitinolytic properties remain unknown. Here we aimed to validate previous chiC experimental results as well as confirm chitinase activity. We identified a small number of mutations in the pM3CRYY backbone via plasmid sequencing. Following protein expression with IPTG, we purified ChiC and performed in vitro colloidal chitin agar assays. We were unable to detect any observable changes (zones of clearing for colloidal chitin plates and color change for colorimetric chitin plates) in our assays. Our findings indicate that currently established chitinolytic assays may need to be further optimized and chiC may undergo non-canonical secretion from P. aeruginosa which can enhance our understanding of secretion mechanisms.