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dc.contributor.authorWipa Sugintaen_US
dc.contributor.authorArchara Vongsuwanen_US
dc.contributor.authorChomphunuch Songsiriritthigulen_US
dc.contributor.authorJisnuson Svastien_US
dc.contributor.authorHeino Prinzen_US
dc.contributor.otherSuranaree University of Technologyen_US
dc.contributor.otherNational Synchrotron Research Center, Thailanden_US
dc.contributor.otherMahidol Universityen_US
dc.contributor.otherMax Planck Institut fur molekulare Physiologieen_US
dc.date.accessioned2018-06-21T08:08:52Z-
dc.date.available2018-06-21T08:08:52Z-
dc.date.issued2005-07-01en_US
dc.identifier.citationFEBS Journal. Vol.272, No.13 (2005), 3376-3386en_US
dc.identifier.issn1742464Xen_US
dc.identifier.other2-s2.0-22144480668en_US
dc.identifier.urihttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=22144480668&origin=inwarden_US
dc.identifier.urihttp://repository.li.mahidol.ac.th/dspace/handle/123456789/16323-
dc.description.abstractThe enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of α and β anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated β-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed ≈20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme. © 2005 FEBS.en_US
dc.rightsMahidol Universityen_US
dc.source.urihttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=22144480668&origin=inwarden_US
dc.subjectBiochemistry, Genetics and Molecular Biologyen_US
dc.titleEnzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MSen_US
dc.typeArticleen_US
dc.rights.holderSCOPUSen_US
dc.identifier.doi10.1111/j.1742-4658.2005.04753.xen_US
Appears in Collections:Scopus 2001-2005

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