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气助超顺磁性萃取技术用于蛋白质规模化连续分离的应用基础研究
Alternative TitleApplying Basic Research on Gas-assisted Superparamagnetic Extraction for Scalable and Continuous Sep
李文松
Subtype博士
Thesis Advisor刘会洲
2014-04
Degree Grantor中国科学院研究生院
Degree Discipline化学工程
Keyword气助超顺磁性萃取 气助磁分离器 浮选 磁分离技术 生物分离
Abstract进入新世纪以来,生物技术引领着化学工业的绿色革命。从复杂生物体系中获得生物产品通常需要一个步骤繁多的分离纯化过程,分离过程的成本构成了生物产品的主要生产成本。因此,发展高效分离提纯技术是生物技术实现产业化的关键。 基于超顺磁性载体的分离技术具有分离快速简单、高选择性和可直接适用于复杂体系的分离等突出优点,已在蛋白质和酶的分离纯化、细胞分离、核酸纯化、固定化酶和生物检测等生物分离领域得到了广泛的研究并展现出广阔而诱人的前景。然而超顺磁性载体分离技术在生物分离中的应用仍主要局限在小规模的实验室分析和分离,规模化应用该技术的主要瓶颈是目前通用的高梯度磁分离器(HGMS)无法实现生物分离过程中超顺磁性载体的规模化连续回收。针对这一问题,本论文将浮选技术引入到超顺磁性载体分离技术中,提出可实现生物产品规模化连续分离的气助超顺磁性萃取新技术,以蛋白质作为模型生物分离体系,系统地开展了以下几方面的创新性研究: 基于浮选对溶液中颗粒的高度富集作用,提出了一种低成本规模化分离新工艺:气助超顺磁性萃取。将其用于单一蛋白质的分离和混合蛋白质的选择性分离,分别以10 nm的柠檬酸钠修饰Fe3O4(CMNs)分离牛血清蛋白(BSA)和偶联Zn2+磁性聚合物微球(MPMs)选择性分离牛血红蛋白(BHb)作为模型,考察了蛋白质负载磁性载体的可浮性,浮选过程与超顺磁性载体分离过程耦合的可行性,并优化了浮选工艺条件,结果表明:无需添加任何起泡剂和浮选剂,只需简单地调节溶液pH值和离子强度,浮选即可快速高度富集稀溶液中蛋白质负载的磁颗粒,在优化条件下,对于BSA负载CMNs,可在1.5min内实现富集比为31的富集且回收率达99%;对于混合蛋白溶液中BHb负载的MPMs,可在1.3 min内实现富集比为39的富集且回收率达98%,而且还能通过多次浮选富集来更进一步提高其富集比。浮选能以泡沫浮选和非泡沫浮选两种方式进行,浮选过程条件和磁颗粒对蛋白质的选择性吸附条件相一致,而且浮选过程不会导致蛋白质从磁颗粒表面的脱落。上述结果充分证明了气助超顺磁性萃取用于规模化分离蛋白质的可行性。 将气泡对颗粒的作用力用于辅助磁力共同进行磁分离,提出了可实现规模化连续磁分离的气助磁分离新方法并开发出了低成本的气助磁分离器新设备。以分离稀溶液中BSA负载的10 nm的弱磁性CMNs为模型,在间歇条件下详细考察了各分离参数对气助磁分离性能的影响,并同单纯磁分离性能进行了对比,结果表明:在间歇条件下,气体能大大加速磁分离过程且能实现磁颗粒的远距离捕获。与单纯磁分离比,气助磁分离在分离速率上可以达到其100倍(气速为80 ml/min条件下),对磁场的依赖性也大大下降,并且分离过程易于放大。在此基础上进一步开发设计了一套小型中试规模的连续气助磁分离器,考察了各工艺参数对分离效果的影响及该磁分离器的分离性能,将其用于0.5 mg/ml浓度的BSA负载弱磁性CMNs的连续分离,处理规模能达18 L/h,首次实现了生物分离过程中弱磁性纳米颗粒的规模化连续回收。该磁分离器具有以低场强的永磁铁作为场源、无需填充聚磁介质、能连续化操作且易于放大、分离规模不受磁场限制和成本低廉等一系列优点,有望能成为一种可替代高梯度磁分离器实现生物分离应用中规模化连续磁分离的很有前景的磁分离器。 将所开发的气助低场磁分离器用于实现蛋白质萃取、洗涤和反萃单元过程的规模化连续分离,并进一步设计开发了可实现蛋白质全流程规模化连续分离的气助超顺磁性萃取集成装置。以2.5 μm磁性微球MPMs用于BHb的选择性分离作为模拟体系,间歇条件下考察并优化了萃取、洗涤和反萃各单元过程主要工艺参数,以此为基础获得优化的连续操作条件,详细研究了连续气助磁分离器用于蛋白质萃取、洗涤和反萃过程料液规模化连续分离的可行性,结果表明:连续气助磁分离器用于蛋白质的萃取、洗涤和反萃过程不仅能实现从各单元过程料液中规模化连续回收磁颗粒而且能实现磁颗粒与料液中蛋白质的良好分离,对于含1 mg/ml MPMs浓度的料液,在250 ml/min气速下,连续处理萃取过程和洗涤过程的料液规模为12 L/h,反萃过程的规模达18 L/h, 95%以上的磁颗粒能从上部磁辊处回收,98% 以上的蛋白质能从底部清液中流出。在此基础上进一步设计开发出了一套最大处理规模为300 L/h且能实现蛋白质萃取、洗涤和反萃过程全流程规模化连续分离的气助超顺磁性萃取集成装置,并已获连续化试水成功。 从气泡和蛋白质负载磁颗粒间表面相互作用力入手,结合前期实验结果的印证,对气助超顺磁性萃取过程的机理进行了探索和解析。以BSA负载CMNs和BHb负载MPMs为模型,通过Zeta电位和接触角测量等表征手段,主要考察了pH值、蛋白质和离子强度三个重要因素对气泡和蛋白质负载磁颗粒间静电力和疏水作用力的影响规律,进而推断出各因素对总表面力的影响规律,并结合实验结果进行了相互印证,综合两个体系共同规律,结果表明:从静电力角度,在气泡和蛋白质负载磁颗粒二者等电点之外,越酸或越碱会越显著地降低气泡和蛋白质负载磁颗粒间总表面吸引力,因而通常在太酸或太碱条件下气助效果会显著变差;蛋白质对磁颗粒表面具有强的改性作用,随蛋白质吸附量的增加磁颗粒表面表现出越来越趋近于直至等同于蛋白质的表面性质,蛋白质能显著增加气泡和亲水磁颗粒间总表面吸引力,蛋白质的表面活性为气助超顺磁性萃取能在无任何表面活性剂添加的情况下顺利进行提供了保障;当气泡和蛋白质负载颗粒表现为静电排斥力时,增加离子强度能较显著增加他们间总表面吸引力,表现为静电吸引力时,这种增加效果会下降。离子强度对气助超顺磁性萃取的强化效果在远离气泡和蛋白质负载磁颗粒二者等电点的条件下较好。这些规律与相关实验结果很吻合。
Other AbstractBiotechnology in the new century has brought chemical industry to a green revolution. A purification procedure including many separation steps is generally required to obtain bio-products from complex biological matrix. Due to this interminable purification process, cost for separation process constitutes major production cost for bio-products. Hence, the key to turning industrialization of biotechnology into reality is to develop separation and purification technology with high efficiency. Superparamagnetic carrier based separation technology has extensive and prospective applications in the field of bio-separation, such as protein and enzyme purification, cell isolation, nucleic acid purification, enzyme immobilization and bioassay, due to its outstanding merits including rapid and simple separation, high selectivity, and direct separation of complex matrix. However, until now, superparamagnetic separation technology is still limited to laboratory-scale analysis and separation. A main bottleneck for this is that universal high gradient magnetic separator (HGMS) is not suitable for large-scale and continuous recovery of superparamagnetic particles from bio-suspensions. In this paper, a novel gas-assisted superparamagnetic extraction technology, which combines flotation technology and superparamagnetic separation technology, is developed to provide a solution to this scalable and continuous separation problem. Protein is selected as a model bio-molecule, and systematical study on scalable separation of this bio-molecule has been carried out, as follows: Firstly, a novel scalable separation technology named gas-assisted superparamagnetic extraction with low cost was proposed. This technology combines flotation for high concentration effect of particles solution and HGMS to obtain scalable magnetic separation of magnetic carriers. The feasibility of this technology for separation of a single protein and selective separation of mixing proteins was investigated, and 10-nm citrate-modified superparamagnetic nanoparticles (CMNs) for separation of bovine serum albumin (BSA) and magnetic PGMA-IDA-Zn2+ microspheres (MPMs) for selective separation of bovine hemoglobin (BHb) were selected as models, respectively. Main research included the flotability of protein loaded magnetic carriers, the feasibility of coupling flotation process and magnetic separation process, and the optimization of flotation technological conditions. The results indicated that flotation could be used to well concentrate protein loaded magnetic particles from dilute solution by simple adjustment of pH values and ionic strength, without additive foaming agent and flotation agent. Under optimal conditions, for BSA loaded CMNs, the enrichment ratio of 31 and recovery of 99% was obtained within 1.5 min, and for BHb loaded MPMs in mixing proteins solution, the enrichment ratio of 39 and recovery of 98% was obtained within 1.3 min. Furthermore, this enrichment ratio could be further improved by added flotation steps. More importantly, flotation could be carried out in either foaming flotation or non-foaming flotation, flotation conditions were consistent with the selective adsorption ones, and no proteins were desorbed from magnetic particles after flotation process. These results well demonstrated the feasibility of gas-assisted superparamagnetic extraction for scalable separation of proteins. Secondly, to achieve scalable and continuous magnetic separation of magnetic particles, a novel gas-assisted magnetic separation method with low field was proposed and a novel gas-assisted magnetic separator with low field was developed by combining the acting force of bubble on the particles and magnetic force for magnetic separation. Selecting 10-nm BSA loaded CMNs with weak magnetic response as a separation model, the effects of process parameters on separation performance of interval gas-assisted magnetic separation were investigated, and separation performance between gas-
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15535
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
李文松. 气助超顺磁性萃取技术用于蛋白质规模化连续分离的应用基础研究[D]. 中国科学院研究生院,2014.
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