Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols
Autor: | Uwe T. Bornscheuer, Giang-Son Nguyen, Geoffrey A. Behrens, Robert Kourist, Jessica Rehdorf |
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Rok vydání: | 2011 |
Předmět: |
Models
Molecular Amino Acid Motifs Molecular Sequence Data Applied Microbiology and Biotechnology Esterase Substrate Specificity Kinetic resolution Hydrolysis Affinity chromatography Catalytic triad Escherichia coli Organic chemistry Amino Acid Sequence Cloning Molecular biology Pseudomonas putida Chemistry Esterases Sequence Analysis DNA General Medicine biology.organism_classification Kinetics Biocatalysis Alcohols Specific activity Sequence Alignment Biotechnology |
Zdroj: | Applied Microbiology and Biotechnology. 93:1119-1126 |
ISSN: | 1432-0614 0175-7598 |
Popis: | 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 e-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. |
Databáze: | OpenAIRE |
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