title: G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues.
abstract: A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.
authors.affiliation: Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, Taiwan. fp15505@mail.ntou.edu.tw
authors.forename: F P
authors.initials: FP
authors.lastname: Lin
authors.forename: W Y
authors.initials: WY
authors.lastname: Juang
authors.forename: K H
authors.initials: KH
authors.lastname: Chang
authors.forename: H C
authors.initials: HC
authors.lastname: Chen
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
chemical_list: D002686:Chitin; D002688:Chitinases