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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

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

Merzouk Yahiaoui, Hassiba Laribi-Habchi, Khelifa Bouacem, Katia-Louiza Asmani, Sondes Mechri, Mohamed Harir, Hamdi Bendif, Radia Aïssani-El Fertas, Bassem Jaouadi

Carbohydr Res. 2019 Sep 1;483:107747.

PMID: 31349143

Abstract:

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.

Chemicals Related in the Paper:

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

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