Recombinant Chitinase C from Pseudomonas aeruginosa is expressed and potentially secreted in Escherichia coli BL21 (DE3)

Abstract

There have been growing efforts to develop natural alternatives to chemical pesticides to mitigate the repercussions of microbiological post-harvest loss on the agricultural industry. Chitin is an attractive target because it is abundant in organisms that are leading causes of post-harvest loss. Many organisms have evolved chitinases which hydrolyze chitin and may serve in the development of a natural biocontrol agent. In this study, we investigated recombinant chitinase C (ChiC) expressed from the previously established pM3CRYY expression vector. We hypothesized that pM3CRYY(+) E. coli BL21 (DE3) expresses and secretes recombinant ChiC. Ultimately, we confirmed the retention of chiC in the pM3CRYY expression vector through Sanger Sequencing and evaluated the expression and secretion of ChiC via western blotting. We determined that ChiC is strongly expressed in pM3CRYY(+) E. coli and potentially secreted in the extracellular medium. To determine if recombinant ChiC retains its chitinolytic activity, we piloted a functional assay that utilized media clearance on chitin-containing plates as a readout for enzymatic activity. Our findings were inconclusive; however, they serve as an important stepping stone for the improvement of future chitinolytic assays. Furthermore, our study supports the potential of recombinant ChiC expressed in non-pathogenic E. coli for the development of a natural biocontrol alternative to synthetic pesticides.