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Purification and Biochemical Characterization of a Novel Acido-Halotolerant and Thermostable Endochitinase From Melghiribacillus Thermohalophilus Strain Nari2A

Purification and Biochemical Characterization of a Novel Acido-Halotolerant and Thermostable Endochitinase From Melghiribacillus Thermohalophilus Strain Nari2A

Sara Mohamed, Khelifa Bouacem, Sondes Mechri, Nariman Ammara Addou, Hassiba Laribi-Habchi, Marie-Laure Fardeau, Bassem Jaouadi, Amel Bouanane-Darenfed, Hocine Hacène

Carbohydr Res. 2019 Feb 1;473:46-56.

PMID: 30616169

Abstract:

An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI-TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s-1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-β-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
LS74818	Chitin azure			Price

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