Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity.
An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8, and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent. The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).
Wood
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Department of Biochemistry and Molecular Biology, University College London, UK.
Fernandez-Lafuente
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Cowan
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D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010666:Phenylpropionates / Q000378:metabolism; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010666:Phenylpropionates; C017568:4-nitrophenyl propionate; D012710:Serum Albumin, Bovine; D002265:Carboxylic Ester Hydrolases; C096434:Tok19A1 carboxylesterase