A membrane‐bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli. Issue 5 (19th April 2016)

• Record Type: Journal Article
• Authors: Kovacic, Filip; Bleffert, Florian; Caliskan, Muttalip; Wilhelm, Susanne; Granzin, Joachim; Batra‐Safferling, Renu; Jaeger, Karl‐Erich
• Is Part Of: FEBS open bio. Volume 6:Issue 5(2016)
• Page Start: 484
• Page End: 493
• Publication Date: 2016-04-19
• Subjects: (D)‐β‐acetylthioisobutyric acid -- Escherichia coli -- esterase -- inhibition -- membrane protein -- Pseudomonas aeruginosa; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1002/2211-5463.12061
• Languages: English
• ISSNs: 2211-5463

Abstract

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. Abstract : Pseudomonas aeruginosa PA01 produces a membrane‐bound esterase (PA2949) that can efficiently be expressed in E. coli . Triton X‐100‐aided extraction and purification of PA2949 led to catalytically active and highly stable enzyme, which can be used for the enantioselective hydrolysis of the racemic methyl ester of β‐acetylthioisobutyrate to produce a precursor of captopril, a drug for treatment of hypertension.