Development of a recombinant Chitinase C chitinolytic activity assay using the DNS method for detection of reducing sugars

Abstract

Chitin is a polymer consisting of N-acetyl-D-glucosamine (GlcNAc) monomers. Chitinolytic organisms such as various Pseudomonas spp. are promising in pest biocontrol research due to their ability to break down chitin-containing exoskeletons of insects. Previous studies have designed a chiC expression plasmid, pM3CRYY, which encodes chiC from Pseudomonas aeruginosa PAO1. Purified recombinant chitinase C (rChiC) expressed in E. coli BL21 (DE3) pM3CRYY A3 has been shown to possess a functional chitin binding domain (CBD). However, the chitinolytic activity of rChiC in this model has not been explored. Our study aims to develop a functional assay to measure chitinolytic activity of rChiC expressed by E. coli BL21 (DE3) pM3CRYY A3. We used dinitrosalicylic acid (DNS) reagent to assay for GlcNAc with spectrophotometry, as DNS reagent detects reducing sugars such as GlcNAc at 540 nm. Our substrate was chitosan, a popular chitin derivative in chitin-requiring experiments due to its solubility in acetic acid. Streptomyces griseus positive control chitinase and rChiC exhibited chitinolytic activity against chitosan, which was inhibited by heat denaturation of the chitinases. A small amount of chitinolytic activity was also detected in E. coli BL21 (DE3) pM3CRYY A3 culture supernatant, which suggests rChiC may be secreted by E. coli. Confirmation that rChiC is functional highlights the potential to develop an eco-friendly insecticide that can be upscaled through an E. coli expression system.