Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon

Abstract

ABSTRACT We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene ( est Pc ) from strain VA1. est Pc consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est Pc showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p -nitrophenyl caproate (C 6 ) the best substrate among the p -nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert -butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.