Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Uschara Thumarat, Ryota Nakamura, Takeshi Kawabata, Hideyuki Suzuki, Fusako Kawai

研究成果: ジャーナルへの寄稿学術論文査読

85 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)419-430
ページ数12
ジャーナルApplied Microbiology and Biotechnology
95
2
DOI
出版ステータス出版済み - 2012 7月

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