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重均分子量在5.0-8.0 kDa范围内的寡聚右旋糖酐可用于制备右旋糖酐铁注射液，在临床上用于治疗缺铁性贫血。目前主要采用微生物发酵生产大分子右旋糖酐，再通过酸解和多级乙醇沉淀来制备寡聚右旋糖酐。但这种生产工艺得到的寡聚右旋糖酐产品分子量不均一，还引入多种杂质，会造成临床上的免疫风险。同时生产过程中乙醇消耗量大，产生大量难处理的高盐废水，给环境治理带来极大的负担。本文构建了用于寡聚右旋糖酐生产的酶膜反应器（Enzymatic membrane reactor, EMR）原型，使用酶解法替代传统的酸解法，并采用超滤技术对寡聚右旋糖酐进行在线分离，避免目标产物的进一步水解，并通过调控EMR的过滤行为控制产品的分子量，使其均一性提高。实验结果表明，膜材料对渗透通量和寡聚右旋糖酐的跨膜传质影响显著。切割分子量为20 kDa的聚醚砜（PES）超滤膜对目标寡聚右旋糖酐分离选择性高，可将产品分子量控制在目标范围内且分布较窄，其渗透通量也有突出优势。在120分钟的酶解反应-分离后，较高跨膜压力（3 bar）和较低搅拌转速（160 rpm）下获得寡聚右旋糖酐产率超过50%。较高的渗透通量能将目标产物及时移出反应器，防止其被过度水解，但过高的渗透通量也会带来目标产物的高截留率，降低产率。膜分离过程中严重的浓差极化现象会导致大分子寡聚右旋糖酐在膜表面积累并扩散过膜，造成透过液中寡聚右旋糖酐分子量分布变宽，产品质量变差，而在膜面施加高剪切力可缓解这一现象。在酶解过程中，右旋糖酐酶和右旋糖酐均会对超滤膜造成不可逆污染。基于膜污染的机理，利用超滤膜对右旋糖酐酶溶液进行预过滤，不仅可以实现部分酶在膜表面的固定化，还可调节膜孔径分布，并透过杂蛋白从而提高酶比活力，从而构建出一种半固定化新型酶膜反应器。这种酶膜反应器生产的寡聚右旋糖酐分子量均一性更好，产品质量更高。本文通过选择合适的超滤膜构建酶膜反应器，并通过调控超滤膜的过滤行为来控制寡聚右旋糖酐的分子量及其分布，得到高质量的目标寡聚右旋糖酐产品，在工业上具有应用潜力。;With molecular weight (Mw) in range of 5.0-8.0 kDa, oligodextrans can be used as commercial precursors of iron-dextran for anemia treatment. Traditionally, fermentation is carried out to produce large dextran molecules which will be further hydrolyzed by acid. Several ethanol precipitations are coupled for final purification of oligodextran products. But the present process results in uneven Mw product and also safety hazards. Moreover, over consumption of ethanol and wastewater discharge would bring severe burden on environment. A novel enzymatic membrane reactor (EMR) prototype has been established in this work, conducting enzyme hydrolysis in place of acid hydrolysis to produce oligodextran, where membrane separation was coupled to in-situ remove the target products. The EMR can be manipulated to control the Mw distribution of the target products and improve their uniformity. Results showed that the permeate flux and oligodextran transmission were greatly affected by the membrane materials. A polyethersulfone (PES) membrane with a molecular weight cut-off (MWCO) of 20 kDa was found to be optimal for permeating the target oligodextran as it successfully controlled the oligodextran Mw within the desired range with a narrow distribution and high permeate flux. After an integrated hydrolysis and filtration for 120 min, a high transmembrane pressure (3 bar) and a low stirring rate (160 rpm) promoted yields over 50%. Efficient removal of target products by high permeate flux is benefit for promoting the product quality by preventing over enzyme hydrolysis, while too high permeate flux may increase the rejection of target products, thus resulting in a low yield. Moreover, the resulting concentration polarization (CP) on membrane surface should be reduced to prevent large oligodextran molecule accumulation, which might diffuse through the membrane leading to a wide Mw distribution of the products. High shear rate on the membrane could mitigate CP and improve the Mw distribution, and dextranase kept stable in the range of agitation speed tested.It was found that both dextran and dextranase caused membrane irreversible fouling during the enzyme hydrolysis process, and the latter favored the enzyme immobilization and contributed to membrane pore size narrowing. By simple pre-filtration of enzyme solution, part of enzyme could be immobilized on the membrane and the impurity proteins passed through the membrane, leading to an increase in specific activity in EMR. In this novel EMR with both immobilized and free enzymes, oligodextran products with higher uniformity were obtained. It was concluded that an EMR was established by selecting a suitable ultrafiltration membrane, and its filtration behavior could be manipulated to control the oligodextran Mw and distribution, producing target oligodextran products with high quality.
|苏子然. 酶膜反应器生产均一分子量的寡聚右旋糖酐[D]. 中国科学院大学,2018.|
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