Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
Appl Microbiol Biotechnol. 2013 Jun;97(11):4821-9. doi: 10.1007/s00253-013-4798-9. Epub 2013 Mar 10.
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 (1)H-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.
一种聚羟基烷酸酯（PHA）在体外被酶促合成，并且研究了与链转移（CT）反应相关的 PHA 的末端结构。在 CT 反应中，PHA 从 PHA 合酶（PhaC）转移到 CT 试剂，导致 CT 试剂与 PHA 链在其羧基末端共价键合。由于体外合成的聚[(R)-3-羟基丁酸酯)]（P(3HB)）的产量相对较低，体外 CT 反应从未得到证明，这使得通过核磁共振（NMR）难以表征聚合物的末端结构。为了克服这些困难，通过使用来自 Delftia acidovorans 的 PhaCDa 和两种对映选择性烯酰基辅酶 A（CoA）水合酶（来自 Aeromonas caviae 的 R-水合酶（PhaJAc）和来自 Pseudomonas putida KT2440 的 S-水合酶（FadB1x）），以β-丁内酯和 CoA 为起始原料，开发了一种生产相对大量 P(3HB)的新型体外合成方法。使用该方法，使用四乙二醇（TEG）作为可区分的 CT 试剂进行 P(3HB)合成，并用（1）H-NMR 对所得 P(3HB)进行表征。NMR 分析表明，P(3HB)的羧基末端与 TEG 共价连接，提供了体外 CT 反应的第一个直接证据。
