We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B´-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11±2 µM (mean±S.D., n = 3) and 6610±880 s-1 (mean±S.D., n = 3) respectively at 70 °C and pH 7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic MoraxellaTA144lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (±)-3-bromo-5-(hydroxymethyl)-Δ2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity–stability–temperature relationship is discussed in relation to those of the homologous members of the HSL group.
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May 1998
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Research Article|
May 15 1998
Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily
Giuseppe MANCO;
Giuseppe MANCO
*Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
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Elena ADINOLFI;
Elena ADINOLFI
*Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
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Francesca M. PISANI;
Francesca M. PISANI
*Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
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Gianluca OTTOLINA;
Gianluca OTTOLINA
†Istituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9, 20131, Milano, Italy
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Giacomo CARREA;
Giacomo CARREA
†Istituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9, 20131, Milano, Italy
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Mosè ROSSI
Mosè ROSSI
1
*Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
‡Università degli Studi di Napoli Federico II, Via Mezzocannone 16, 80100, Naples, Italy
1To whom correspondence should be addressed at the Istituto di Biochimica delle Proteine ed Enzimologia (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
December 17 1997
Revision Received:
February 09 1998
Accepted:
February 13 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 332 (1): 203–212.
Article history
Received:
December 17 1997
Revision Received:
February 09 1998
Accepted:
February 13 1998
Citation
Giuseppe MANCO, Elena ADINOLFI, Francesca M. PISANI, Gianluca OTTOLINA, Giacomo CARREA, Mosè ROSSI; Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem J 15 May 1998; 332 (1): 203–212. doi: https://doi.org/10.1042/bj3320203
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