Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon.
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 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees 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.
Hotta
Yuji
Y
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Ezaki
Satoshi
S
Atomi
Haruyuki
H
Imanaka
Tadayuki
T
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D019624:Thermoproteaceae / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases