Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus.
A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia 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 with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADeltasp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees 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 the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.
Hobel
Cédric F V
CF
Prokaria Ltd., Gylfaflöt 5, 112 Reykjavik, Iceland.
Hreggvidsson
Gudmundur O
GO
Marteinsson
Viggó T
VT
Bahrani-Mougeot
Farah
F
Einarsson
Jón M
JM
Kristjánsson
Jakob K
JK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D002614:Chelating Agents / Q000494:pharmacology; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics*; D048271:Chitosan / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003102:Colloids / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004187:Disaccharides / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006027:Glycosides / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D045245:Rhodothermus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014783:Viscosity
D002412:Cations; D002614:Chelating Agents; D003102:Colloids; D017931:DNA Primers; D004187:Disaccharides; D006027:Glycosides; D010455:Peptides; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C032438:chitobiose; D004247:DNA; D048271:Chitosan; D004492:Edetic Acid; D002688:Chitinases; C119825:chitinase C; C094590:chitinase ChiA