Browsing by Author "Merzouk, Yahiaouia"

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    Purification andbiochemicalcharacterizationofaneworganicsolvent- tolerant chitinasefrom Paenibacillus timonensis strain LK-DZ15isolatedfrom the DjurdjuraMountainsinKabylia,Algeria
    (Université de M'sila, 2019) Merzouk, Yahiaouia
    A newextracellularchitinase(calledChiA-Pt70)wasproducedandpurifiedfromanewlyisolated Paenibacillus timonensis strain LK-DZ15.Themaximumchitinaseactivityrecordedafter44-hofincubationat30°Cwas11,500 U/mL. Pureenzymewasobtainedafterammoniumsulphateprecipitation(40–70%)followedbysequential column chromatographiesonfastperformanceliquidchromatography(FPLC)andhighperformanceliquid chromatography (HPLC).Basedonmatrixassistedlaserdesorptionionization-timeofflightmassspectrometry (MALDI-TOF/MS) analysis,thepurifiedenzymeisamonomerwithamolecularmassof70,166.11kDa.The sequence ofthe25NH2-terminal residuesofthematureChiA-70showedhighhomologywith Paenibacillus GH- 18 chitinasesfamily.OptimalactivitywasachievedatpH4.5and80°C.Thepureenzymewascompletely inhibited by p-chloromercuribenzoic acid(p-CMB), 5,5′-dithio-bis-2-nitrobenzoicacid(DTNB),and N-ethylmaleimide (NEM).Chitinaseactivitywashighoncolloidalchitin,chitinazure,glycolchitin,glycolchitosane, chitotriose, andchito-oligosaccharidewhileitdidnothydrolysechitibioseandamylose.Furthermore,thin-layer chromatography (TLC)analysisfromenzymaticcatalyzedhydrolysisofcolloidalchitinshowedthatChiA-Pt70 acted asan endo-splitting enzyme.Its Km and kcat values were0.611mgcolloidalchitin/mLand87,800s−1, respectively. Interestingly,itscatalyticefficiencywashigherthanthoseofchitinasesChiA-Mt45from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-β-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibitedremarkablebiochemicalpropertiessuggestingthatitissuitablefortheenzymaticde- gradation ofchitin.