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

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)1119-26
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume93
Issue number3
DOIs
Publication statusPublished - 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

Keywords

  • Alcohols
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Biocatalysis
  • Cloning, Molecular
  • Escherichia coli
  • Esterases
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Pseudomonas putida
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Substrate Specificity
  • Journal Article
  • Research Support, Non-U.S. Gov't

Fields of Expertise

  • Human- & Biotechnology

Cite this

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, Vol. 93, No. 3, 02.2012, p. 1119-26.

Research output: Contribution to journalArticleResearchpeer-review

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 ; Vol. 93, No. 3. pp. 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 - Kourist, Robert

AU - Bornscheuer, Uwe T

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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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DO - 10.1007/s00253-011-3464-3

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EP - 1126

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

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