Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp. strain B11-1.

A gene coding for an esterase (PsEst1, 1911bp in length) of the psychrotrophic bacterium Pseudomonas sp. B11-1 isolated from Alaskan soil was cloned and sequenced. The deduced amino acid sequence revealed a protein of 637 amino acid residues with a molecular mass of 69 kDa. Although the expression product, PsEst1, showed no appreciable sequence similarity (less than 15% identity) to any known proteins with the established biochemical functions, it is expected to be related to the alpha/beta hydrolase superfamily because it shared sequence motifs that have been identified with this superfamily. For example, a unique 'nucleophilic elbow' motif, -Gly(36)-Asp-Ser-Leu-Asn(40)-, was identified, and Ser(38) was predicted to constitute a catalytic triad with Asp(162) and His(303). PsEst1 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3) cells as an inclusion body. A soluble denatured form of the enzyme was purified to homogeneity in the presence of 8M urea, and the catalytically active form of the enzyme could be obtained by subsequent removal of urea by dialysis, where the addition of 0.1% Triton X-100 was essential for the efficient renaturation of the enzyme. To our knowledge, this was the first example of the successful renaturation of the recombinant cold-adapted enzyme. The enzyme efficiently hydrolyzed vinyl and aryl esters with the C4-C6 acyl chain. The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10 degrees C) was significantly lower than the value (79.9 kcal/mol) of the mesophilic lipase. It was observed that the K(m) values for p-nitrophenyl butyrate in the growth temperature range of strain B11-1 (5-15 degrees C) were lower than those at higher temperatures.