Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7

• Published in: Extremophiles : life under extreme conditions 2001-04, Vol.5 (2), p.119-126
• Main Authors: BENDT, Anne, HÜLLER, Heike, KAMMEL, Ulrike, HELMKE, Elisabeth, SCHWEDER, Thomas
• Format: Article
• Language: English
• Subjects: Adaptation, Physiological; Amino Acid Sequence; Antarctic Regions; Bacteriology; Base Sequence; Biological and medical sciences; Chitin - metabolism; Chitinases - genetics; Chitinases - isolation & purification; Chitinases - metabolism; Cloning, Molecular; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Genetics; Hydrolysis; Microbiology; Molecular Sequence Data; Sequence Homology, Amino Acid; Space life sciences; Vibrio - enzymology; Vibrio - genetics; Vibrio - physiology; Water Microbiology
• ISSN: 1431-0651; 1433-4909
• DOI: 10.1007/s007920100179

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+.