A broad pH range and processive chitinase from a metagenome library

Abstract

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Potential of bacterial chitinolytic, Stenotrophomonas maltophilia, in biological control of termites

“…5). Thimoteo et al (2017) determined 5 to 7.5 pH range optimum for chitinase activity. Temperature also affects the enzyme activity.…”

Anticoagulant activity of partially purified chitinase produced by Citrobacter freundii str. nov. haritD11 by fermentation of wheat bran coupled with fish scales

“…The enzyme was stable at 7.5-10 pH range for 1 h at 4 °C in various buffers (100 mM each of sodium acetate (pH 4-7) and Tris-HCl (pH 7-9); Correspondingly, chitinase from Alternaria infectoria was stable at alkaline pH [8] but some bacterial chitinases were found stable at acidic pH [9,10]. The optimum temperature for chitinase was recorded at 50 °C; likewise 50 °C has been reported for Aspergillus terreus [9], Paenibacillus pasadenensis CS0611 and Escherichia coli [31]. Chitinase maintained 80% stability between 45 °C and 60 °C in 50 mM sodium acetate buffer, lower or higher temperatures lead to activity inhibition.…”

Cloning, Purification, and Characterization of a Novel Metagenome-Sourced Microbial Chitinase with Dual Catalytic Domains

“…Metagenome-based approaches are the complete access to the entire community's genetic pool without the need of microbial cultivation [11], which have been used to discover a few chitinases [12]. These metagenome-sourced chitinases could be used for the biocontrol of pathogenetic fungi [13] and insects [12,14], an additive in the food and feed industries [15], as well as industrial processing of chitin [16].…”