Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 95, Issue 2, Pages 419-430Publisher
SPRINGER
DOI: 10.1007/s00253-011-3781-6
Keywords
Polyester degradation; Thermobifida alba AHK119; Cutinase-type polyesterase; Ca-dependent cutinase; Thermostable polyesterase
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Funding
- Institute for Fermentation, Osaka (Japan)
- JSPS (Japan)
- NRCT (Thailand)
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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 A degrees 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 alpha/beta-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second beta-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 Ca2+ and was stable up to 60 A degrees C with 150 mM Ca2+. Another identical gene was located in tandem in the upstream of est119.
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