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Title: Chitinase from Vibrio carchariae : purification, gene isolation, and sequence determination
Author: Suginta, Wipa
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Fourteen species of Vibrio were screened for chitin-induced chitinase activity in the culture medium. V. carchariae and V. alginolyticus 283 showed high levels of activity, and screening on agar plates containing swollen chitin showed high expression of chitinase by the same two species, and also by V. fishceri and V. alginolyticus 284. An affinity purification procedure was developed for the major chitinase from V. carchariae. The purified chitinase was active as a monomer with Mr 63000-66000, and displayed activity toward both chitin and 4-methyl-umbelliferyl-chitooligosaccharides. N-terminal sequence analysis confirmed that the enzyme belongs to the ChiA family of chitinases. The gene encoding chitinase was isolated from a V. carchariae genomic library and cloned in pBluescript KS II- vector and Escherichia coli XL1 Blue. The gene had an open reading frame (2,550 bp) that encodes 850 amino acids. E. coli harbouring the vector with a 4.0-kb DNA insert expressed a 95-kDa precursor chitinase which was accumulated in inclusion bodies. The polypeptide sequence of V. carchariae chitinase showed 54 %, 53 %, and 52 % identity with ChiA from Alteronomas sp. strain O-7, ChiA from Enterobacter agglomerans, and ChiA from Serratia marcescens, respectively. Homology modelling of the three-dimensional structure of V. carchariae chitinase revealed extensive similarity with the known structure of ChiA from S. marcescens. The structure comprises three major domains, N-terminal domain, catalytic α/β-barrel domain, and α+β-small domain. Glu315 and Asp391 located at the active site are suggested to play important roles in the enzyme catalysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available