Purification andbiochemicalcharacterizationofaneworganicsolvent- tolerant chitinasefrom Paenibacillus timonensis strain LK-DZ15isolatedfrom the DjurdjuraMountainsinKabylia,Algeria
Loading...
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Université de M'sila
Abstract
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.
Description
Keywords
Chitinase Paenibacillus timonensis Endo-splitting enzyme