Fidaleo M, Charaniya S, Solheid C, Diel U, Laudon M, Ge H, Scriven L E, Flickinger M C
BioTechnology Institute, University of Minnesota, 140 Gortner Laboratories, 1479 Gortner Avenue, St. Paul, 55108, USA.
Biotechnol Bioeng. 2006 Oct 20;95(3):446-58. doi: 10.1002/bit.21051.
We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.
我们开发了一种新型的厚度小于50微米的纳米多孔双层乳胶涂层，用于保存氧化葡萄糖杆菌（一种严格需氧菌）作为全细胞生物催化剂。氧化葡萄糖杆菌被包裹在丙烯酸酯/醋酸乙烯酯共聚物基质（玻璃化转变温度约为10℃）中，并浇铸成直径为12.7毫米的贴片涂层（细胞涂层），其中含有约10⁹CFU，表面覆盖有纳米多孔面漆。利用D - 山梨醇氧化为L - 山梨糖来研究涂层的催化性能。在微生物反应器中，于30℃使用pH 6.0的D - 山梨醇、磷酸盐、丙酮酸（SPP）非生长培养基研究本征动力学，并测定米氏常数。通过使用扩散池，测定了通过添加甘油和/或蔗糖阻止聚合物颗粒聚结而优化的细胞涂层和面漆的扩散率。低温场发射扫描电子显微镜图像显示出两层结构，氧化葡萄糖杆菌被小于40纳米的孔隙包围。研究了SPP培养基中>150小时的活细胞密度、细胞泄漏和氧化动力学。尽管对涂层的渗透性进行了优化，但在细胞涂层干燥过程中约50%的氧化葡萄糖杆菌活力丧失，添加面漆后活力进一步降低。观察到每单位涂层体积的高反应速率（80 - 100 g/L·h），这与使用独立实验估计的参数的扩散 - 反应模型的预测结果一致。观察到细胞涂层的有效因子为0.22 - 0.49，比此前报道的该氧化葡萄糖杆菌氧化反应的任何有效因子大20倍。这些纳米结构涂层以及提高其保存氧化葡萄糖杆菌活力能力的可能性，可能有助于设计用于多相微通道和膜生物反应器的高活性粘合剂涂层，以显著提高全细胞氧化的强度。
