College of Chemical and Biological Engineering, Zhejiang University, Zheda Road 38, Hangzhou 310027, China; State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Changwang Road 18, Hangzhou 310032, China.
College of Chemical and Biological Engineering, Zhejiang University, Zheda Road 38, Hangzhou 310027, China.
J Chromatogr A. 2018 Jun 15;1554:92-100. doi: 10.1016/j.chroma.2018.04.043. Epub 2018 Apr 21.
Phenyllactic acid (PLA) is an important organic acid with wide antimicrobial activities against gram-positive and gram-negative bacteria and some fungi. This interesting compound can be synthesized by the microbial fermentation or the bioconversion using phenylpyruvic acid (PPA) as the key substrate and microorganisms as the whole-cell biocatalysts. However, the isolation of high-purity PLA with a high recovery from the crude fermentation or conversion broth is a challenging task. In this work, the separation of PLA from the crude conversion broth prepared by employing Lactobacillus buchneri cells as the whole-cell catalysts was achieved by the chromatography using the poly(hydroxyethyl methacrylate) (pHEMA)-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups. The static adsorption behaviors of PLA under different salt concentrations and the adsorption capacities of PLA on the cryogel were measured experimentally. The chromatographic performance of PLA from the crude conversion broth was compared with that from the clarified broth. The results showed that the pHEMA-based cryogel has a high capacity of PLA, i.e., 14.64 mg mL cryogel, and the adsorption of PLA was influenced by the salt concentration. By using deionized water as running buffer, PLA with a high purity of 97.6% was obtained with one step elution using 0.3 M NaCl as the elution solution with the recovery at the range of 80.2-90.8% from crude feedstock without any pretreatment at various flow velocities. These values were close to those obtained for the clarified broth, i.e., the purity of 98.4% and the recovery of 92.3% under the same chromatography conditions at 1 cm min. The cryogel was then applied to separate PLA from clarified feedstock, high purity (>96.7%) and recovery (>91.4%) of PLA were found with 20 cycles, which verified the selectivity and robustness of prepared pHEMA-VBTAC cryogel. Therefore, the chromatography using pHEMA-based cryogel with the dual functional groups is an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation and production of high-purity PLA in industry.
苯乳酸（PLA）是一种重要的有机酸，对革兰氏阳性和革兰氏阴性细菌以及一些真菌具有广泛的抗菌活性。这种有趣的化合物可以通过微生物发酵或生物转化来合成，以苯丙酮酸（PPA）为关键底物，微生物为全细胞生物催化剂。然而，从粗发酵或转化液中分离具有高回收率的高纯度 PLA 是一项具有挑战性的任务。在这项工作中，采用 Lactobacillus buchneri 细胞作为全细胞催化剂制备的粗转化液中的 PLA 通过使用基于 poly(hydroxyethyl methacrylate)（pHEMA）的 cryogel 结合阴离子交换和疏水性苄基基团的色谱法进行分离。在不同盐浓度下测量了 PLA 的静态吸附行为和 PLA 在 cryogel 上的吸附容量。比较了粗转化液中 PLA 的色谱性能和澄清液中 PLA 的色谱性能。结果表明，基于 pHEMA 的 cryogel 对 PLA 具有高容量，即 14.64 mg mL cryogel，并且 PLA 的吸附受盐浓度的影响。使用去离子水作为运行缓冲液，使用 0.3 M NaCl 作为洗脱液，在 0.3 M NaCl 作为洗脱液，在 0.3 M NaCl 作为洗脱液，在各种流速下，无需任何预处理，一步洗脱即可获得纯度为 97.6%的 PLA，回收率为 80.2-90.8%。这些值与在相同的色谱条件下，从澄清原料中获得的 98.4%的纯度和 92.3%的回收率接近。然后将 cryogel 应用于从澄清原料中分离 PLA，发现 PLA 的纯度（>96.7%）和回收率（>91.4%）超过 20 个循环，这验证了制备的 pHEMA-VBTAC cryogel 的选择性和稳健性。因此，使用具有双重功能基团的 pHEMA 基 cryogel 的色谱法是一种从粗生物转化液中直接分离 PLA 的有效方法，因此在工业上分离和生产高纯度 PLA 可能很有趣。
