【作者】 王良华；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2008， 博士

【摘要】 超大孔连续床晶胶层析分离技术是一种近几年才出现的新型生物分离技术,其晶胶介质内具有尺寸达数微米至数百微米的超大孔隙,允许发酵液中的微生物细胞、细胞碎片等固体颗粒顺利通过,柱压低、传质阻力小,目标物的吸附传质主要为对流传质,吸附分离迅速,可在高流速下直接从复杂料液中分离纯化生物分子或生物颗粒。研究晶胶基质的成孔规律和机理、功能基团固载和修饰方法、晶胶介质对蛋白质的吸附分离特性以及晶胶层析的应用等问题,具有十分重要的意义。本论文以聚丙烯酰胺基体系为反应液,用结晶致孔法制备了晶胶基质,研究了基质反应液的液—固相变特性、晶胶基质的成孔规律以及晶胶介质的基本性能和层析性能,考察了控制晶胶基质成孔和性能的影响因素,并探讨了晶胶基质内超大孔隙的形成机理;进而,通过固载金属离子,制备了金属螯合亲和晶胶介质;通过孔内直接接枝法,固载磺酸基和氨基功能基团,制备得到了离子交换晶胶介质;最后,以酵母发酵液中三磷酸胞苷（CTP）和三磷酸腺苷（ATP）的分离为例,探讨了晶胶介质的应用问题。对基质反应液液固相变特性的研究结果表明,基质反应液的起始结晶温度（T_c）和冰点温度(T_mc)随反应液浓度、冷冻速率和冷冻终温等的变化而呈现不同的规律:冷冻速率对T_c与T_mc的影响较小,T_c与T_mc随冷冻终温的下降略有降低,但T_mc随反应液浓度增大而减小。对基质成孔规律的研究结果表明,晶胶基质的成孔是溶剂结晶和单体聚合反应两个动态过程同时发生的复杂过程,晶胶基质的孔隙结构和性能受床柱尺寸、冷冻终温、冷冻速率、反应液组成、催化剂用量等因素的影响。通过条件优化,在一定内径的床柱内,于较合适的冷冻速率下,控制冷冻终温在-15℃以下,可得到孔隙大小在10～100μm、分布较均匀、连通性好、孔隙率82～85%、理论等板高度为0.5～1.1 mm的晶胶基质。对Cu²⁺-IDA（iminodiacetic acid）、Ni²⁺-IDA、Zn²⁺-IDA等不同金属螯合亲和晶胶介质的实验研究结果表明,螯合金属离子后,晶胶孔隙结构形态、理论等板高度、渗透率等基本不变;金属螯合亲和晶胶介质对牛血清白蛋白（BSA）的吸附和解吸特性受缓冲液pH值、离子强度、流速、洗脱液组成等因素的影响,其吸附机理主要为配位作用和静电作用。当缓冲液pH值在蛋白质等电点附近时,蛋白质吸附容量可达最大值,盐离子的存在会降低吸附容量,但上样流速对吸附容量的影响很小。用咪唑溶液可对蛋白质进行有效洗脱,较小的流速对洗脱有利。对基质孔内接枝方法和相应离子交换晶胶介质的研究结果表明,通过孔内直接接枝法,以K₅[Cu（HIO₆）₂]溶液为催化剂,可以顺利地将带磺酸基的2-羟基-3-烯丙氧基丙磺酸钠（AHPSA）和带氨基的甲基丙烯酸二甲氨基乙酯（DMAEMA）接枝到聚丙烯酰胺基晶胶基质上,得到离子交换晶胶介质。接枝AHPSA的阳离子交换晶胶介质的理论等板高度基本不受接枝反应时间和单体浓度的影响,其渗透率随接枝反应时间增大略有减小,其对溶菌酶的吸附容量与接枝单体的浓度成正比,受接枝反应时间的影响较小。接枝DMAEMA的阴离子交换晶胶介质对BSA的吸附容量随离子强度的增大而下降;料液中存在NaCl或CH₃COONa时,吸附容量随离子强度增大呈线性下降;存在C₆H₅Na₃O₇或Na₂SO₄时,吸附容量随离子强度增大呈指数下降。对酵母发酵液中CTP和ATP的晶胶层析分离研究结果表明,用带有氨基的阴离子交换晶胶介质,可以在较高的流速（2～10 cm/min）下,直接从含有酵母细胞的发酵料液中分离得到较高纯度的CTP和ATP产品。经过一次层析分离操作,分步洗脱,CTP纯度可达93.4%,回收率为35%;ATP纯度可达98.3%,回收率为58%。在高达10 cm/min的层析流速下,经过一次层析分离操作,ATP纯度仍可高达97.4%,回收率达49%。更多还原

【Abstract】 The continuous bed chromatography using supermacroporous cryogel is suggested as a novel bioseparation technique in downstream processes in recent years. The cryogel has interconnected supermacropores with diameter of several to hundreds micrometers,which allow large bioparticles such as cell debris and even the whole cells passing through without being blocked.Thus,the back pressure and the mass transfer resistance are low and convective mass transfer of biomolecules always dominates within the cryogel beds.In addition,fast adsorption equilibrium can also be achieved in cryogels,which makes it possible to directly separate and purify target biomolecules or bioparticles from unclarified particulate-contained feedstock or crude fermentation broth without being pretreated at high flow rate.It is of great importance to reveal the mechanism of the formation of supermacropores in the preparation of cryogel matrix,the immobilization of functional binding groups on the matrix,the protein adsorption characteristics in cryogel columns and the related applications of cryogels.In this paper,the cryogel matrix was produced by radical co-ploymerization of acrylamide（AAm）-based reactive system under frozen conditions.The liquid-solid phase transition characteristics of the aqueous solution system containing AAm,AGE and MBAAm for the production of polyacrylamide-based cryogels were studied and the pore formation mechanisms were discussed.The basic properties and the chromatographic performances of the cryogels prepared under various conditions were measured and the effects of preparation condition on the microstructures and properties of the cryogels were also investigated.Then,metal ions were immobilized on polyacrylamide-based cryogel matrix to produce the metal-chelate affinity cryogels and the functional binding groups of sulfoacid and amine groups,were grafted onto the cryogel matrixes in an in-situ manner to prepare ion-exchange cryogels.The anion-exchange chromatography using cryogel was also applied to directly isolate cytidine triphosphate（CTP）and adenosine triphosphate（ATP）from the yeast fermentation broths and the corresponding behaviors were obtained and discussed.From the results of the liquid-solid phase transition characteristics of the reactive solution,the actual initial solvent crystallization temperature T_c and the freezing point temperature T_mcchanged with the monomer concentration,the freezing rate and the freezing terminated temperature.Freezing rate had a limited effect on T_c and T_mc,while the values of T_c and T_mcdecreased with the decrease of freezing terminated temperature at a constant freezing rate and a given monomer concentration.T_m decreased with the increase of monomer concentration at a constant freezing rate.From the present work,the process of supermacropores formation was a complex process combining both solvent freezing crystallization and momomer copolymerization.The cryogel structure,the pore morphology and the cryogel bed properties were influenced by several factors,i.e.,the column diameter,the terminated freezing temperature,the freezing rate,the monomer concentration as well as the catalyst and its amount used.A cryogel bed with well interconnected supermacropores of diameters of 10 to 100 micrometers,porosity of 82-85%and height equivalent to theoretic plate（HETP）of 0.5-1.1 mm was obtained under suitable preparations.The terminated freezing temperature of less than -15℃was found to be effective for a suitable freezing rate in a column with given inner diameter.The measuremental results of the properties of the Cu²⁺-IDA（iminodiacetic acid）, Ni²⁺-IDA and Zn²⁺-IDA metal-chelate affinity cryogels showed that there are no obvious variations for the pore morphology,HETP and permeabilities between the metal-chelate affinity cryogels and the corresponding basic cryogel matrixes without immobilized metal ions.The adsorption and elution behaviors of bovine serum albumin（BSA）in these metal-chelate affinity cryogels were influenced by the buffer pH,the ionic strength,the flow rate and the eluent composition.The binding of BSA on the metal-chelate affinity cryogels was depended upon the cooperative effects of coordination and electrostatic interaction.The liquid flow velocity had a weak influence,while the buffer pH had an obvious effect on the binding capacity of protein in these cryogels.The protein binding capacity reached the maximum when the buffer pH was near the isoelectric point of BSA and decreased with the adding of salt to the loading liquid.The bound protein molecules were eluted effectively using imidazole solution and a low elution liquid flow velocity was found to be benefited to the elution process.The graft polymerization experiments showed that 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt（AHPSA）with sulfoacid group and 2-（dimethylamino）ethyl methacrylate（DMAEMA）with amine group can be successfully grafted onto the pore wall surface of the polyacrylamide-based cryogels to get the cation- and anion-exchange cryogels,respectively.The graft polymerization can be initiated by potassium diperiodatocuprate（K₅[Cu（HIO₆）₂]）in an in-situ manner.For the cation-exchange cryogels grafted with AHPSA,the HETP was not obviously influenced by the graft reaction time and monomer concentration,while the permeability slightly decreased with the increase of graft reaction time.The protein binding capacity of lysozyme increased linearly with the increase of the concentration of AHPSA and had no obvious change under different graft reaction times.For the anion-exchange cryogels grafted with DMAEMA,the binding capacity of BSA decreased with the increase of ionic strength.This parameter was observed to decrease linearly in the existing of NaCl or CH₃COONa and exponentially in the existing of C₆H₅Na₃O₇ or Na₂SO₄,with the increase of ionic strength respectively.The isolations of CTP and ATP from unclarified yeast fermentation broths were achieved successfully by one-step chromatography using the anion-exchange cryogel with amine group as chromatographic matrix at high flow rate（2 to 10 cm/min）.The results showed that high purify of CTP and ATP can be obtained by this method.The purity of CTP reached 93.4%and the recovery rate of CTP was 35%,while the purity of ATP reached 98.3%and the recovery rate of ATP was 58%.At high chromatographic flow rate of 10 cm/min,the purity of ATP still reached 97.4%and the recovery rate of ATP reached 49%after one-step separating operation in the present work.更多还原