Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86)

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Abstract
The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58°C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8–15.4 μM and for acetyl CoA in the range 13.6–15.5 μM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50°C and appears to pass through a transition state between 48 and 50°C.

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