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In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate

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Abstract

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with β-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by 1H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction.

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Acknowledgments

This work was supported by a Grant-In-Aid for Scientific Research (KAKENHI 19681008) to T. Tsuge. S. Tomizawa was supported by a Grant-in-Aid for JSPS Fellows (Tokyo Institute of Technology G-COE Program: Education and Research Centre for Material Innovation).

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Authors and Affiliations

  1. Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan

    Satoshi Tomizawa, Hideki Abe & Takeharu Tsuge

  2. Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan

    Shun Sato

  3. Department of Chemical Engineering and Materials Science, College of Engineering, Yuan Ze University, No. 135 Yuan-Tung Road, Chungli, Taoyuan, 32003, Taiwan

    John Chi-Wei Lan

  4. Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan

    Yoshiyuki Nakamura

  5. Bioplastic Research Team, RIKEN Biomass Engineering Program, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan

    Hideki Abe

  6. Enzyme Research Team, RIKEN Biomass Engineering Program, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan

    Satoshi Tomizawa

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  1. Satoshi Tomizawa
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  2. Shun Sato
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  3. John Chi-Wei Lan
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  4. Yoshiyuki Nakamura
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  5. Hideki Abe
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  6. Takeharu Tsuge
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Correspondence to Satoshi Tomizawa.

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Tomizawa, S., Sato, S., Lan, J.CW. et al. In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate. Appl Microbiol Biotechnol 97, 4821–4829 (2013). https://doi.org/10.1007/s00253-013-4798-9

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  • DOI: https://doi.org/10.1007/s00253-013-4798-9

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