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- Title
Cloning, expression, and characterization of a highly thermostable family 18 chitinase fromRhodothermus marinus.
- Authors
Hobel, Cédric; Hreggvidsson, Gudmundur Ó.; Marteinsson, Viggó T.; Bahrani-Mougeot, Farah; Einarsson, Jón M.; Kristjánsson, Jakob K.
- Abstract
A family 18 chitinase genechiAfrom the thermophileRhodothermus marinuswas cloned and expressed inEscherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity withBacillus circulanschitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant,chiA?sp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiA?sp were 70°C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that theR. marinuschitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.
- Subjects
THERMOPHILIC microorganisms; BODY temperature regulation; CHITINASE; ENZYMES; CLONING; ESCHERICHIA coli; GENETIC engineering
- Publication
Extremophiles, 2005, Vol 9, Issue 1, p53
- ISSN
1431-0651
- Publication type
Article
- DOI
10.1007/s00792-004-0422-3