Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Organization of African Unity St., Abbassia, 11566, Cairo, Egypt.
Mol Biol Rep. 2019 Jun;46(3):3357-3370. doi: 10.1007/s11033-019-04798-4. Epub 2019 Apr 17.
About 24 h incubation of Azomonas (A.) macrocytogenes isolate KC685000 in 14L fermenter produced 22% poly (3-hydroxybutyrate) (PHB) per cell dry weight (CDW) biopolymer using 1 vvm aeration, 10% inoculum size, and initial pH of 7.2. To control the fermentation process, Logistic and Leudeking-Piret models were used to describe the cell growth and PHB production, respectively. These two models were in good agreement with the experimental data confirming the growth associated nature of PHB production. The best method for recovery of PHB was chemical digestion using sodium hypochlorite alone. The characterization of the produced polymer was carried out using FT-IR, HNMR spectroscopy, gel permeation chromatography and transmission electron microscope. The analysis of the nucleotide sequences of PHA synthase enzyme revealed class III identity. The putative tertiary structure of PHA synthase enzyme was analyzed using Modular Approach to Structural class prediction software, Tied Mixture Hidden Markov Model server, and Swiss model software. It was deduced that PHA synthases' structural class was multidomain protein (α/β) containing a conserved cysteine residue and lipase box as characteristic features of α/β hydrolase super family. Taken together, all the results of molecular characterization and transmission electron microscope images supported that the PHB formation was attained by the micelle model. To the best of our knowledge, this is the first report on production of growth associated PHB polymer using A. macrocytogenes isolate KC685000, and its class III PHA synthase.
在 14L 发酵罐中，24 小时培养 Azomonas (A.) macrocytogenes KC685000 分离株，使用 1 vvm 通气、10%接种量和初始 pH 值为 7.2，每细胞干重 (CDW) 生物聚合物产生 22%的聚 (3-羟基丁酸酯) (PHB)。为了控制发酵过程，使用 Logistic 和 Leudeking-Piret 模型分别描述细胞生长和 PHB 生产。这两个模型与实验数据非常吻合，证实了 PHB 生产与生长相关。回收 PHB 的最佳方法是单独使用次氯酸钠进行化学消化。使用傅里叶变换红外光谱、HNMR 光谱、凝胶渗透色谱和透射电子显微镜对所生产聚合物的特性进行了表征。对 PHB 合酶酶的核苷酸序列分析表明其具有 III 类同一性。使用 Modular Approach to Structural class prediction 软件、Tied Mixture Hidden Markov Model 服务器和 Swiss model 软件对 PHB 合酶酶的三级结构进行了分析。推断 PHB 合酶的结构类为多结构域蛋白 (α/β)，含有保守的半胱氨酸残基和脂肪酶盒，这是 α/β 水解酶超家族的特征。总的来说，所有分子特征分析和透射电子显微镜图像的结果都支持 PHB 的形成是通过胶束模型实现的。据我们所知，这是首次报道使用 A. macrocytogenes KC685000 及其 III 类 PHB 合酶生产与生长相关的 PHB 聚合物。
