Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols

Jessica Rehdorf, Geoffrey A Behrens, Giang-Son Nguyen, Robert Kourist, Uwe T Bornscheuer

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

Abstract

An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.

Originalspracheenglisch
Seiten (von - bis)1119-26
Seitenumfang8
FachzeitschriftApplied Microbiology and Biotechnology
Jahrgang93
Ausgabenummer3
DOIs
PublikationsstatusVeröffentlicht - Feb 2012

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Pseudomonas putida
Esterases
Acetates
Carboxylic Ester Hydrolases
Alcohols
Mixed Function Oxygenases
Affinity Chromatography
Esters
Hydrolysis
Escherichia coli
Enzymes
Proteins
4-hydroxyacetophenone
caprolactone
4-nitrophenyl acetate
4-nitrophenyl butyrate

Schlagwörter

    Fields of Expertise

    • Human- & Biotechnology

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    Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols. / Rehdorf, Jessica; Behrens, Geoffrey A; Nguyen, Giang-Son; Kourist, Robert; Bornscheuer, Uwe T.

    in: Applied Microbiology and Biotechnology, Jahrgang 93, Nr. 3, 02.2012, S. 1119-26.

    Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

    Rehdorf, Jessica ; Behrens, Geoffrey A ; Nguyen, Giang-Son ; Kourist, Robert ; Bornscheuer, Uwe T. / Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols. in: Applied Microbiology and Biotechnology. 2012 ; Jahrgang 93, Nr. 3. S. 1119-26.
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    abstract = "An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.",
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    AU - Rehdorf, Jessica

    AU - Behrens, Geoffrey A

    AU - Nguyen, Giang-Son

    AU - Kourist, Robert

    AU - Bornscheuer, Uwe T

    PY - 2012/2

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    N2 - An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.

    AB - An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.

    KW - Alcohols

    KW - Amino Acid Motifs

    KW - Amino Acid Sequence

    KW - Biocatalysis

    KW - Cloning, Molecular

    KW - Escherichia coli

    KW - Esterases

    KW - Kinetics

    KW - Models, Molecular

    KW - Molecular Sequence Data

    KW - Pseudomonas putida

    KW - Sequence Alignment

    KW - Sequence Analysis, DNA

    KW - Substrate Specificity

    KW - Journal Article

    KW - Research Support, Non-U.S. Gov't

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    JO - Applied Microbiology and Biotechnology

    JF - Applied Microbiology and Biotechnology

    SN - 0175-7598

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    ER -