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PMID:22183084
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| Citation |
Thumarat, U, Nakamura, R, Kawabata, T, Suzuki, H and Kawai, F (2012) Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119. Appl. Microbiol. Biotechnol. 95:419-30 |
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| Abstract |
Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 °C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119. |
| Links |
PubMed Online version:10.1007/s00253-011-3781-6 |
| Keywords |
Actinomycetales/enzymology; Amino Acid Substitution; Calcium/metabolism; Carboxylic Ester Hydrolases/chemistry; Carboxylic Ester Hydrolases/genetics; Carboxylic Ester Hydrolases/metabolism; Chromatography/methods; Electrophoresis, Polyacrylamide Gel; Enzyme Activators/metabolism; Enzyme Stability; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins/chemistry; Mutant Proteins/genetics; Mutant Proteins/metabolism; Protein Conformation; Substrate Specificity; Temperature |
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