Arikawa Hisashi, Sato Shunsuke, Fujiki Tetsuya, Matsumoto Keiji
GP Group, Corporate R&D Planning and Administration Division, Kaneka Corporation, 1-8 Miyamae-Cho, Takasago-Cho, Takasago, Hyogo 676-8688, Japan.
GP Group, Corporate R&D Planning and Administration Division, Kaneka Corporation, 1-8 Miyamae-Cho, Takasago-Cho, Takasago, Hyogo 676-8688, Japan.
J Biosci Bioeng. 2017 Aug;124(2):250-254. doi: 10.1016/j.jbiosc.2017.03.003. Epub 2017 Apr 26.
We developed a new method for isolation and quantitation of polyhydroxyalkanoate (PHA) from culture broth. In this method, the cells were sonicated in sodium dodecyl sulfate (SDS) solution and centrifuged to recover PHA. The recovered PHA was rinsed with deionized water and ethanol, and then weighed after drying. Hazardous chemicals such as chloroform, methanol, and sulfuric acid were not used, and no expensive analytical instruments were needed. We applied this method to Cupriavidus necator culture broths that included various amounts of poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) from flasks and jar fermentors. The quantitation by this method was practical for use with a wide range of production amounts and PHA monomer compositions compared to the conventional whole-cell methanolysis method with gas chromatographic analysis, and besides, the recovered PHAs were adequately pure (≥96% purity). Therefore, this new method would be valuable not only for quantitation of PHA but also for preparation of samples to characterize their mechanical properties.
我们开发了一种从培养液中分离和定量聚羟基脂肪酸酯(PHA)的新方法。在该方法中,将细胞在十二烷基硫酸钠(SDS)溶液中超声处理并离心以回收PHA。回收的PHA用去离子水和乙醇冲洗,然后干燥后称重。该方法不使用氯仿、甲醇和硫酸等危险化学品,也不需要昂贵的分析仪器。我们将此方法应用于来自烧瓶和罐式发酵罐的包含不同量聚(3-羟基丁酸酯)(PHB)或聚(3-羟基丁酸酯-co-3-羟基己酸酯)(PHBHHx)的食酸戴尔福特菌培养液。与采用气相色谱分析的传统全细胞甲醇解方法相比,该方法的定量对于广泛的产量和PHA单体组成而言切实可行,此外,回收的PHA纯度足够高(纯度≥96%)。因此,这种新方法不仅对于PHA的定量有价值,而且对于制备用于表征其机械性能的样品也有价值。