Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7
Abstract. A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5°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 librar...
Published in: | Extremophiles |
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Main Authors: | , , , , |
Format: | Article in Journal/Newspaper |
Language: | unknown |
Published: |
2001
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Subjects: | |
Online Access: | https://epic.awi.de/id/eprint/4310/ https://doi.org/10.1007/s007920100179 https://hdl.handle.net/10013/epic.14885 |
Summary: | Abstract. A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5°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°C and pH 8. Activity of the chitinase at 5°C was 40% of that observed at 35°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+. |
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