Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
Biotechnol J. 2011 Apr;6(4):410-9. doi: 10.1002/biot.201000266. Epub 2011 Mar 7.
Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ÄKTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.
最近，人们评估了在分批补料实验中生产荚膜多糖 K4（一种果糖化的软骨素）的可能性。在本研究中，开发了一种从大肠杆菌 K4 发酵液中获得软骨素的新型下游工艺。该工艺简单、可扩展且经济。特别是，下游工艺经过了优化，目的是纯化出适合进一步化学修饰的产品，试图开发一种用于硫酸软骨素生产的生物技术平台。在工艺开发过程中，利用了膜设备（超滤/渗滤），为不同的纯化阶段选择了合适的膜组件截留分子量。该工艺的操作条件（错流速率和跨膜压力）是在 AKTA 切向流仪器（GE Healthcare，美国）上确定的，这是一种实验室规模的自动切向流过滤系统。此外，还根据 K4 和去果糖化 K4 以及主要污染物的分析定量计算了选择性和通量等参数。完整的下游工艺可得到约 75%纯度高于 90%的软骨素。
