Sequencing and Expression of the Gene Encoding a Cold‐Active Citrate Synthase from an Antarctic Bacterium, Strain DS2‐3R

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Abstract
The gene encoding citrate synthase from a novel bacterial isolate (DS2‐3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2‐3R, are cold‐active, with a temperature optimum of 31°C. In addition the enzymes are rapidly inactivated at 45°C, and show significant activity at 10°C and below. Comparison of amino acid sequences indicates that DS2‐3R citrate synthase is most closely related to the enzyme from gram‐positive bacteria. The amino acid sequence of the DS2‐3R enzyme shows several features previously recognised in other cold‐active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.

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