G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues

Citation
Fp. Lin et al., G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues, ARCH MICROB, 175(3), 2001, pp. 220-225
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ARCHIVES OF MICROBIOLOGY
ISSN journal
0302-8933 → ACNP
Volume
175
Issue
3
Year of publication
2001
Pages
220 - 225
Database
ISI
SICI code
0302-8933(200103)175:3<220:GSDMCF>2.0.ZU;2-R
Abstract
A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-d irected deletion mutagenesis in order to study the role of the 304 C-termin al amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The r ecombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expre ssion system. The His-Tag-affinity-purified recombinant ChiAG561 had a calc ulated 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 optimu m pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degr eesC instead of 42.5 degreesC). Biochemical properties of the recombinant C hiAG561 suggested that deletion of the 304 C-terminal amino acid residues o f ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relati ve activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(G lcNAc)(3)] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest t hat the active region of A. caviae ChiA is located in the region before G56 1 of the protein molecule.