Department of Chemical and Biochemical Engineering, Faculty of Engineering, The University of Western Ontario, London Ontario, Canada N6A 5B9.
Biotechnol Prog. 2010 May-Jun;26(3):734-42. doi: 10.1002/btpr.370.
Microbially produced gamma-polyglutamic acid (gamma-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. Owing to its increasing demand in various industries, production of gamma-PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of gamma-PGA from cell-free fermentation broth of Bacillus licheniformis. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of gamma-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified gamma-PGA biopolymer. The efficiency and selectivity of gamma-PGA recovery was compared with ethanol precipitation method. We found that 85% of the original gamma-PGA content in the broth was recovered by copper sulfate-induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of gamma-PGA precipitate was analyzed by measuring proteins that co-precipitated with gamma-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with gamma-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified gamma-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified gamma-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified gamma-PGA was characterized using infrared spectroscopy.
微生物合成的γ-聚谷氨酸（γ-PGA）是一种具有广泛应用的商业重要生物聚合物，可应用于生物制药、食品、化妆品和废水处理等行业。由于其在各个行业的需求不断增加，γ-PGA 的生产在文献中有详细记载，但很少有报道其回收方法。本文报道了一种从地衣芽孢杆菌无细胞发酵液中选择性回收和纯化γ-PGA 的新方法。将无细胞发酵液用二价铜离子处理，导致γ-PGA 沉淀，通过离心收集沉淀得到γ-PGA 颗粒。将沉淀重新溶解并透析到去离子水中，得到纯化的γ-PGA 生物聚合物。与乙醇沉淀法相比，比较了γ-PGA 回收的效率和选择性。我们发现，与乙醇沉淀法相比，硫酸铜诱导沉淀可回收发酵液中 85%的原始γ-PGA 含量，而乙醇沉淀法可回收 82%。由于乙醇是蛋白质沉淀常用的溶剂，因此通过测量与γ-PGA 共沉淀的蛋白质来分析γ-PGA 沉淀的纯度。在发酵液中存在的总蛋白质中，发现 48%的蛋白质与乙醇沉淀的γ-PGA 共沉淀，而在硫酸铜诱导沉淀中，仅在最终纯化的γ-PGA 中检测到 3%的蛋白质，表明硫酸铜诱导沉淀比乙醇沉淀法具有更好的选择性。纯化的γ-PGA 的总金属含量分析表明，铜离子的含量无法检测到，而检测到的其他金属离子的浓度都处于低浓度范围。使用红外光谱对纯化的γ-PGA 进行了表征。
