【作者】 缪志俊；

【导师】 姚善泾； 林东强；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 硕士

【摘要】 扩张床吸附(expanded bed adsorption,EBA)技术是一种新型的生物分离技术,集固液分离、浓缩和初步纯化于一个单元操作之中,能直接从含有细胞和细胞碎片的发酵液或匀浆液中提取目标产物,而不必事先除去悬浮的固体颗粒。吸附介质是决定扩张床吸附技术是否成功应用的关键因素。本文以不锈钢粉为增重剂,将其包埋于纤维素黏胶中,采用“反相悬浮热再生”法制备了一种新型扩张床复合基质。考察了该复合基质的制备工艺、理化性质和扩张床特性,随后将其制备成染料亲和介质,研究了亲和介质的吸附性能和扩张床纯化应用实例。全文分为五部分。第一部分综述了扩张床吸附技术的历史沿革、基本原理和操作方法,重点介绍了扩张床吸附基质的特性要求、常见种类和制备方法。在叙述纤维素介质理化性质和制备应用情况的基础上,提出了本文的研究思路,即以不锈钢粉为增重剂,通过“反相悬浮热再生”技术包埋于纤维素骨架之中,制备一种新型的亲水性扩张床吸附基质。第二部分综述了一套扩张床吸附介质的表征方法。该方法包括了对空白基质的基本物理性质(湿真密度、含水率、收缩率、机械强度)、孔结构特征(孔度、孔容、孔径、孔比表面积)、仪器分析(外观形态、粒径分布、Zeta电位)、扩张床特性(扩张性能、流体混合性能)及染料亲和介质的吸附性能(染料配基含量、酶活测定、总蛋白测定、凝胶电泳、批吸附性能、柱层析性能)等方面性能的表征。通过这些表征方法,可以评价出所制备吸附介质的应用潜力。第三部分考察了纤维素/不锈钢粉复合基质的制备工艺,得到较优的工艺条件:老成温度为15℃,时间为24 hr,黄化温度为30℃,时间为3 hr;分散相与聚合相质量比为6:1;搅拌速度为500-600 rpm。在此工艺条件下,详细考察了不锈钢粉加量对两种不同粒径分布复合基质的基本性质和孔结构特征的影响,结果表明:不锈钢粉颗粒起到很好的增重作用,且对孔结构特征影响较小;复合基质具有规则的球形外观、良好的基本性质、孔结构特征和粒径分布,与商品化扩张床基质相近。第四部分研究了上述两种不同粒径分布复合基质的扩张床特性。首先,通过Richardson-Zaki方程式对基质在扩张床内的扩张行为进行了描述,发现此方程式可以较好地描述扩张率与流动相流速的关系。然后用RTD测试对扩张床内的流体混合性能进行了研究,表明流动相的流速是影响扩张床操作稳定性的重要参数,其值的增大会引起轴向分散系数的增大。在所研究的实验范围内,采用本文所制备基质的扩张床内的流体流动可以近似为平推流。第五部分讨论了染料亲和介质的制备,提出较优的工艺条件:黏胶中纤维素含量为8 wt%,空白基质的不锈钢粉与黏胶质量比为0. 25,粒径分布为60-150 μm,初始染料溶液浓度为20 mg/mL。考察了所制备的染料亲和介质的酶活和蛋白吸附性能,摘要包括吸附等温线和吸附动力学曲线,并以Streamline OEAE为对照。结果表明,亲和介质对乳酸脱氢酶的吸附符合Langmuir等温式,且具有较高的吸附容量;酶活吸附动力学曲线较好,与动力学速率参数模型吻合。进行了固定床层析和扩张床层析的实验,实现了直接从兔肌匀浆液中分离兔肌乳酸脱氢酶,纯化倍数和回收率分别为3.65和85.2%。505一PAGE和等电聚焦凝胶电泳结果证实了扩张床吸附的分离效果。关键词:扩张床吸附,复合基质,纤维素,不锈钢粉,染料亲和介质,乳酸脱氢酶工更多还原

【Abstract】 Expanded bed adsorption (EBA) is a novel bioseparation technique, which integrates the clarification, concentration and initial purification into a single step operation unit. It enables the target products to be recovered directly from unclarified feedstock (such as the cultivations of microorganisms or cells and homogenates of disrupted cells) without the prior removal of suspended cells or cell debris.The specially-designed matrix is the principal "hardware" pillar supporting the successful application of expanded bed adsorption. In this work, a new type of composite matrix was prepared with cellulose as the framework and stainless steel powder as the inert weighted through the water-in-oil suspension thermal regeneration method. Then, the physical properties and expansion characteristics of cellulose/stainless steel powder composite matrix were investigated systematically. Finally, a dye-affinity expanded bed adsorbent based on the cellulose/stainless steel powder composite matrix was developed, and its adsorption properties and application in expanded bed were studied subsequently.The thesis is divided into five sections. Section 1 is the review of expanded bed adsorption, including the history, principles and operation modes. The advances in the matrices of expanded bed adsorption are introduced with the emphasis on the physicochemical properties required, familiar types as well as their preparation methods. Furthermore, on the base of physical properties, preparation methods and applications of cellulose matrix, the idea is put forward, which adopted the composite of regenerated cellulose and stainless steel powder to prepare a novel hydrophilic porous matrix for expanded bed adsorption through the water-in-oil suspension thermal regeneration method.Section 2 summarizes a series of methods for the analysis and characterization of the composite matrix and dye-affinity adsorbent, including basic physical properties, pore structures, instrument analysis, expansion characteristics and adsorption abilities. The application potential of the prepared matrix can be estimated through these methods mentioned above.Section 3 studies the preparation of the composite matrix. The optimal reaction conditions were found to be as follows: the ageing temperature and time at 15 ℃ and 24 hours, the xanthation temperature and time at 30 ℃ and 3 hours, the mass ratio of disperse phase to water phase at 6:1 and the mixing speed at 500-600 rpm. Under these conditions, the effects of stainless steel powder contents on the physicalproperties and pore structures of two matrices containing different size distribution were studied. The results showed that both matrices have regular sphericity, good properties, pore structures and size distributions, which are similar to the commercial Streamline matrix.Section 4 investigates the expansion characteristics of the matrices prepared. The results indicated that the expansion behavior of both matrices agreed well with the Richardson-Zaki law and the relationship of expansion factor E and flow velocity U could be described. The liquid mixing performances in the bed were studied through retention time distribution (RTD) test, which showed that the flow velocity was an important parameter effecting on the stability of expanded bed. The increase of flow velocity would increase the degree of the axial mixing in the bed. Under these experiment conditions, the flow in expanded bed was proved to be plug flow for the composite matrices prepared.Section 5 involves the preparation and adsorbability of dye-affinity adsorbent based on the cellulose/stainless steel powder composite matrix. The optimized reaction conditions were found to be as follows: the cellulose content in viscose at 8 wt%, the mass ratio of stainless steel powder and viscose at 1:4, the size distribution of composite matrix at 60~150 (im and the initial concentration of dye solution at 20 mg/mL The lactate dehydrogenase (LDH) adsorption isotherms of the prepared dye-affinity adsorbent followed the Langmuir adsorption equation and the a更多还原