Biochemical and thermostability features of acetyl esterase Aes from Escherichia coli.
Previously we characterized an acetyl-esterase from Escherichia coli, formally Aes, from a thermodynamic point of view in comparative studies with thermophilic homologs. Since the enzyme appeared unusually resistant to the thermal denaturation we analysed the kinetic behaviour with respect to the temperature. The enzyme displays a surprising optimal temperature at 65 degrees C, showing a specific activity of 250 U/mg using pNP-butanoate as substrate, but a low kinetic stability at the same temperature (t(1/2) of inactivation=5 min). By a random mutagenesis approach we searched for mutated versions of Aes with increased thermostability. We found the mutant T74A, which shows the same specific activity of wild type but a t(1/2) of inactivation of 30 min at 65 degrees C.
Farias
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Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche (CNR), Via Pietro Castellino 111, 80131 Naples, Italy.
Mandrich
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Rossi
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Manco
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D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D029968:Escherichia coli Proteins; C115513:AES protein, E coli; D000115:Acetylesterase