title: A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli.
abstract: Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of β-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.
authors.affiliation: Institute of Molecular Enzyme Technology Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich Germany.
authors.forename: Filip
authors.initials: F
authors.lastname: Kovacic
authors.affiliation: Institute of Molecular Enzyme Technology Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Bleffert
authors.affiliation: Institute of Molecular Enzyme Technology Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich Germany.
authors.forename: Muttalip
authors.initials: M
authors.lastname: Caliskan
authors.affiliation: Institute of Molecular Enzyme Technology Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilhelm
authors.affiliation: Institute of Complex Systems, ICS-6: Structural Biochemistry Forschungszentrum Jülich Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Granzin
authors.affiliation: Institute of Complex Systems, ICS-6: Structural Biochemistry Forschungszentrum Jülich Germany.
authors.forename: Renu
authors.initials: R
authors.lastname: Batra-Safferling
authors.affiliation: Institute of Molecular Enzyme Technology Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich Germany; Institute of Bio- and Geosciences IBG-1: Biotechnology Forschungszentrum Jülich GmbH Germany.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
keywords: (D)‐β‐acetylthioisobutyric acid; Escherichia coli; Pseudomonas aeruginosa; esterase; inhibition; membrane protein