Assessing the chitinolytic activity of recombinant ChiC from Escherichia coli pM3CRYY BL21(DE3)

Abstract

Chitin is an abundant polysaccharide that plays a structural role in many organisms, including being present in the cell walls of fungi and exoskeletons of insects. As fungi and insects are common pests of plants that pose a threat in agricultural contexts, chitin is a promising biopesticide target. Chitinases, such as ChiC from Pseudomonas aeruginosa PAO1, degrade chitin through hydrolysis, making them a potential biocontrol agent that offers a safer alternative to chemical pesticides. Previously, chiC was cloned into the expression vector pET-28a to generate the expression plasmid pM3CRYY. Transforming Escherichia coli BL21(DE3) with pM3CRYY produces recombinant ChiC (rChiC), which has recently been shown to retain its chitin-binding activity. However, the chitinolytic activity of rChiC has yet to be demonstrated. Our study aimed to determine if recombinant ChiC from E. coli pM3CRYY BL21(DE3) has chitinolytic activity using fungal inhibition, 3,5-dinitrosalicylic acid (DNS), and colloidal chitin-plate based assays. We found that rChiC-expressing pM3CRYY E. coli had moderate antifungal effects but that purified rChiC could not degrade colloidal chitin into reducing sugar monomers. We further found that fungal inhibition assays with purified protein and colloidal chitin plates were inadequate for assessing the chitinase activity of rChiC. These results suggest that rChiC likely has chitinolytic activity but that either our extraction and purification methods could not produce sufficient amounts of functional protein for detectable in vitro activity or that colloidal chitin is not a suitable substrate for rChiC.