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

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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 Ca2+ and was stable up to 60 °C with 150 mM Ca2+. Another identical gene was located in tandem in the upstream of est119.

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Acknowledgements

The study was supported by funds from the Institute for Fermentation, Osaka (Japan) to F. K. We appreciate Prof. Y. Yamamoto, Institute for Plant Resources, Okayama University, for her kind help in atomic absorption spectrometry measurements. We are grateful to Dr. T. Nakajima-Kambe, University of Tsukuba, for his kind supply of PBSA (Bionolle™EM-301). This work is linked with the Asia Core Program supported by JSPS (Japan) and NRCT (Thailand).

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Authors and Affiliations

  1. Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan

    Uschara Thumarat, Ryota Nakamura & Hideyuki Suzuki

  2. Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Takeshi Kawabata

  3. Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan

    Fusako Kawai

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  1. Uschara Thumarat
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  2. Ryota Nakamura
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  3. Takeshi Kawabata
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  4. Hideyuki Suzuki
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  5. Fusako Kawai
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Corresponding author

Correspondence to Fusako Kawai.

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Fig. 1

SDS-PAGE of Est119 purified by one-step and two-step chromatographies. 1, protein standards; 2, crude cell lysate from E. coli Rosetta-gami B (DE3) harboring pQE80L-est119 induced with 0.1 mM IPTG; 3, Purified Est119 by one-step chromatography on a Ni-Sepharose 6 Fast Flow column; 4, Purified Est119 by the second step chromatography on a Mono Q anion exchange column. (DOC 300 kb)

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Thumarat, U., Nakamura, R., Kawabata, T. et al. Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119. Appl Microbiol Biotechnol 95, 419–430 (2012). https://doi.org/10.1007/s00253-011-3781-6

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