Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7.
A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.
Bendt
A
A
Institute of Marine Biotechnology, Greifswald, Germany.
Hüller
H
H
Kammel
U
U
Helmke
E
E
Schweder
T
T
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001483:Base Sequence; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D014871:Water Microbiology
D004269:DNA, Bacterial; D002686:Chitin; D002688:Chitinases