Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44

• Published in: International journal of biological macromolecules Vol. 106; pp. 338 - 350
• Main Authors: Bouacem, Khelifa, Laribi-Habchi, Hassiba, Mechri, Sondes, Hacene, Hocine, Jaouadi, Bassem, Bouanane-Darenfed, Amel
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2018
• Subjects: Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Biocatalysis; Chitin; Chitin - chemistry; Chitinase; Chitinases - antagonists & inhibitors; Chitinases - chemistry; Chitinases - isolation & purification; Enzyme Inhibitors - chemistry; Enzyme Stability; Ethylmaleimide - chemistry; Gene Expression; Hot Temperature; Hydrogen-Ion Concentration; Hydrogenophilaceae - chemistry; Hydrogenophilaceae - classification; Hydrogenophilaceae - enzymology; Hydrogenophilus hirschii; Hydrolysis; Kinetics; Molecular Weight; p-Chloromercuribenzoic Acid - chemistry; Phylogeny; Substrate Specificity; Chitinase; Hydrogenophilus hirschii; Chitin
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2017.08.026

•Purification of endo-chitinase (ChiA-Hh59) from a H. hirschii KB-DZ44 was carried out.•The molecular weight and the NH2-terminal sequence of the chitinase were determined.•Optimum pH and temperature values for activity were pH 5.0 and 85°C, respectively.•The kinetic parameters and the TLC of hydrolysis products of the enzyme were studied.•ChiA-Hh59 may be used as potential candidate for the enzymatic degradation of chitin.
An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s−1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.