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, 1911 bp 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 69kDa. 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 α/β 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-Asn⁴⁰-, was identified, and Ser ³⁸ was predicted to constitute a catalytic triad with As ^p162 and His³⁰³. 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 8 M 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-C ₆ acyl chain. The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10°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°C) were lower than those at higher temperatures.