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Purification and biochemical characterization of a new organic solvent-tolerant chitinase from Paenibacillus timonensis strain LK-DZ15 isolated from the Djurdjura Mountains in Kabylia, Algeria
- Yahiaoui, Merzouk, Laribi-Habchi, Hassiba, Bouacem, Khelifa, Asmani, Katia-Louiza, Mechri, Sondes, Harir, Mohamed, Bendif, Hamdi, Aïssani-El Fertas, Radia, Jaouadi, Bassem
- Carbohydrate research 2019 v.483 pp. 107747
- Hydrogenophilus, Paenibacillus, Streptomyces griseus, Trichoderma viride, ammonium sulfate, amylose, benzoic acid, catalytic activity, chitin, chitinase, desorption, enzyme activity, high performance liquid chromatography, hydrolysis, mass spectrometry, molecular weight, mountains, pH, thin layer chromatography, Algeria
- A new extracellular chitinase (called ChiA-Pt70) was produced and purified from a newly isolated Paenibacillus timonensis strain LK-DZ15. The maximum chitinase activity recorded after 44-h of incubation at 30 °C was 11,500 U/mL. Pure enzyme was obtained after ammonium sulphate precipitation (40–70%) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). 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 70,166.11 kDa. The sequence of the 25 NH2-terminal residues of the mature ChiA-70 showed high homology with Paenibacillus GH-18 chitinases family. Optimal activity was achieved at pH 4.5 and 80 °C. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB), 5,5′-dithio-bis-2-nitro benzoic acid (DTNB), and N-ethylmaleimide (NEM). Chitinase activity was high on colloidal chitin, chitin azure, glycol chitin, glycol chitosane, chitotriose, and chito-oligosaccharide while it did not hydrolyse chitibiose and amylose. Furthermore, thin-layer chromatography (TLC) analysis from enzymatic catalyzed hydrolysis of colloidal chitin showed that ChiA-Pt70 acted as an endo-splitting enzyme. Its Km and kcat values were 0.611 mg colloidal chitin/mL and 87,800 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Mt45 from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59 from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-β-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibited remarkable biochemical properties suggesting that it is suitable for the enzymatic degradation of chitin.