摘要
根据制革原理，将金属离子Fe~(3+)、Zr~(4+)和Al~(3+)固定在胶原纤维上制备了新型吸附材料，并研究了这类吸附材料的物化性质。实验结果表明，胶原纤维对金属离子具有较高的固载容量，其中锆和铁的固载量分别可达563mg／g和319mg／g，且固化的金属离子耐水溶液萃取。胶原纤维与金属离子反应后，其热稳定性和等电点明显比胶原纤维高，此外，比表面积增加，吸附孔径减小。这说明，作为吸附材料使用，胶原纤维固化金属离子具有更好的性能。
胶原纤维及其固化金属离子材料对蛋白质和酶的吸附实验结果表明：胶原纤维对多种蛋白质和酶具有一定的吸附作用，但其吸附容量远低于胶原纤维固化金属离子的吸附容量，说明固载的金属离子在吸附过程中起着重要作用。胶原纤维固化金属离子材料对蛋白质和酶的最佳吸附pH值随着蛋白质和酶以及金属离子的种类不同而不同。
研究了胶原纤维固化铁对溶菌酶的吸附特性，包括温度、pH、初始浓度和离子强度对吸附容量的影响，以及吸附等温线、吸附动力学、固定床吸附动力学和吸附剂的解析性能。结果表明：当pH为8.0时，胶原纤维固化铁对溶菌酶的吸附容量最大，这是由于溶菌酶在固定化金属离子材料上的吸附作用主要是通过金属离子与溶菌酶分子上的组氨酸之间的螯合作用而进行的，而大多数蛋白质分子上组氨酸残基的pKa值在5.5～8.5范围内。当氯化钠浓度为0和0.25M时，胶原纤维固化铁对溶菌酶的吸附容量分别为最大和最小，这是由于金属离子亲和色谱对蛋白质的吸附在低盐浓度下主要通过蛋白质与金属离子间的螯合作用进行，在高盐浓度下主要通过疏水作用进行。吸附容量随温度和起始浓度的升高而增大，表明有化学吸附存在。当溶菌酶的起始浓度为2.5 mg／mL，吸附液体积为25mL，吸附剂用量为0.100 g时，30℃条件下胶原纤维固化铁对其的吸附量可达395 mg／g。Langmuir方程可以很好地拟合胶原纤维固化铁对溶菌酶的吸附等温线。当吸附时间为8h时，基本达到吸附平衡，吸附动力学可以用拟二级速度方程进行拟合，计算得到的平衡吸附量与实测值吻合得很好。0.25mol／L氯化钠、0.3mol／L咪唑和0.01mol／L磷酸缓冲液(pH6.0)的混合溶液对溶菌酶的解吸效果最好，其解吸率和酶活保存率分别可达96.7％和94.1％；胶原纤维固化铁吸附柱对溶菌酶具有良好的吸附柱动力学特性，能将溶菌酶大大地浓缩。吸附柱可以再生和重复使用。
研究了胶原纤维固化铁对溶菌酶和白蛋白的选择性吸附。结果表明：当pH4.0时选择性吸附白蛋白，而当pH8.0时选择性吸附溶菌酶。以胶原纤维固化铁或固化锆为固定床填料，研究其从蛋清粉中分离纯化溶菌酶的效果。结果表明：这两种材料都能够一步从蛋清粉中分离纯化得到溶菌酶，HPLC检测所得溶菌酶的纯度均为100％。用于溶菌酶分离后，胶原纤维固化金属离子吸附材料易于再生。胶原纤维固化铁固定床初次和再生后用于分离纯化溶菌酶时，得率分别为70.5％和70.0％；胶原纤维固化锆固定床初次和再生后用于分离纯化溶菌酶时，得率分别为68.7％和68.4％。
研究了胶原纤维固化铁对细菌的吸附特性，包括温度、pH、菌龄、初始浓度和离子强度对吸附容量的影响，以及吸附等温线和吸附动力学。结果表明：该吸附材料对细菌具有很强的吸附能力，且吸附迅速。当大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体浓度分别为1.02×10~7cfu／mL和9.8×10~6cfu／mL时，10分钟后胶原纤维固化铁对它们的吸附量分别为2.23×10~9cfu／g和2.72×10~9cfu／g，90分钟基本达到吸附平衡，吸附容量分别达到2.94×10~9cfu／g和3.15×10~9cfu／g。由于其吸附速度非常快，吸附有可能是在材料的表面进行。细菌的培养时间对平衡吸附量具有显著的影响，对处于生长期的细菌吸附能力最强。吸附容量随着离子强度的增加而增加。在pH4.0～10范围内均有较强的吸附作用；温度对平衡吸附量的影响不明显；其吸附平衡符合Freundlich方程。进一步研究表明，胶原纤维固化铁对细菌的吸附动力学可以用拟二级速度方程来描述，由该方程计算得到的平衡吸附量与实测值非常吻合，误差在2％以内。
研究了胶原纤维固化锆对酵母细胞(S.cerevisiae)的吸附特性和吸附规律，包括温度、pH、菌龄、初始浓度和离子强度对吸附容量的影响，以及吸附等温线和吸附动力学，并研究了固定化酵母细胞连续发酵产乙醇的效果。结果表明：当酵母的起始浓度为5.26×10~5cfu／mL时，吸附量可以达到8.2×10~7cfu／g。当pH6.0时，胶原纤维固化锆对酵母细胞的吸附量最大。酵母的培养时间对吸附量的影响很大，对处于生长期的酵母吸附能力最强。而温度对吸附量的影响不大，其吸附平衡符合Freundlich方程。当溶液中NaCl的浓度大于0.1mol／L时，酵母细胞存活率降低，吸附量减少，溶液中NaCl的浓度小于0.1mol／L时，则影响不明显。胶原纤维固化锆对酵母的吸附较为迅速，5小时基本达到吸附平衡，且吸附动力学可用二级速度方程来描述。胶原纤维固化锆吸附固定化酿酒酵母能进行连续发酵，其发酵产乙醇的能力是游离酵母发酵能力的两倍。
通过测定微生物细胞表面的接触角和Zeta-电位确定其表面的疏水性及电荷，探讨胶原纤维固化金属离子材料对微生物的吸附机理。由接触角计算微生物细胞表面自由能的结果可知，S.aureus、E.coli和S.cerevisiae三者的表面均具有亲水性，且都具有很强的给电子特性。其表面疏水性大小为：S.aureus＞E.coli＞S.cerevisiae，这个顺序与胶原纤维固化金属离子对三者的吸附容量结果一致，即微生物细胞表面的疏水性越强，则胶原纤维固化金属离子材料对其的吸附容量越大。这表明，微生物与胶原纤维固化金属离子之间存在疏水作用。Zeta—电位滴定结果表明：在pH4～12范围内这三种微生物细胞表面均带负电荷。在相同条件下，三者表面所带负电荷按从多到少的顺序为：S.aureus、E.coli、S.cerevisiae，这与胶原纤维固化金属离子材料对三者吸附容量的大小顺序一致，即微生物细胞表面所带负电荷越多，胶原纤维固化金属离子材料对其的吸附容量越大。这表明，微生物和胶原纤维固化金属材料之间极可能存在静电吸附。所以，微生物在胶原纤维固化金属离子材料上的吸附是疏水和静电共同作用的结果。
本研究不仅为这类吸附材料在生物分离工程、医药、环保、生物发酵工程及化学工程等领域获得广泛应用奠定了基础，同时对丰富螯合金属亲和层析(IMAC)理论也具有一定的科学意义。
A series of novel adsorbents were prepared by immobilizing Fe(III), Zr(IV) or Al(III) on collagen fiber matrix according to leather-making principles, and the physical and chemical properties of these adsorbents were studied. The experimental results indicated that collagen fiber exhibited high immobilization capacity to metal ions. The immobilization capacities to Zr(IV) and Fe(III) were 563 mg/g and 319mg/g respectively, and the immobilized metal ion can withstand extraction of water. Both the denaturation temperature and the isoelectric point of collagen fiber were heightened through immobilizing reaction. Furthermore, surface area of metal ions-immobilized collagen fiber was bigger than that of collagen fiber, and porosity of metal ions-immobilized collagen fiber was smallar than that of collagen fiber. These results indicated that, as an adsorbent, metal ions-immobilized collagen fiber might exhibit better performance than collagen fiber.
The adsorption capacities of proteins and enzymes on collagen fiber or metal ions-immobilized collagen fiber were determined. It was found that collagen fiber can adsorb certain amount of protein and enzyme, but the adsorption capacities were remarkably lower than that of metal ions-immobilized collagen fiber. These results indicted that the immobilized metal ions play an important role in adsorption process. The optimum pH for adsorption depended on the type of protein, enzyme and metal ion used.
The adsorption behaviors of Fe(III)-immobilized collagen fiber (FICF) to lysozyme were investigated. The effects of temperature, pH, initial concentration and ionic strength on adsorption capacities were studied. The adsorption isotherms, adsorption kinetics, column adsorption kinetics and adsorption-desorption behaviors of lysozyme on FICF were also investigated. The adsorption capacity reached a maximum value around pH8.0. The adsorption of lysozyme on metal-immobilized materials is mainly through chelating bonding between metal ion and histidine residue of lysozyme, and the pK_a values of histidine residue in most of proteins are in the range of 5.5 to 8.5. When the concentrations of NaCl were 0 and 0.25mol/L, the adsorption capacities of lysozyme on FICF were of maximum and minimum respectively. The reason is that proteins were bound by the adsorbent via metal chelating at lower salt concentration, while the adsorption mechanism was hydrophobic interaction at relative higher salt concentration. Adsorption capacity increases with the rise of temperature and the increase of initial concentration of lysozyme. The adsorption capacity of lysozyme on 0.100 g adsorbent was 395 mg/g at 303 K in 25 mL of 2.5 mg/mL lysozyme solution. The adsorption isotherms could be described by the Langmuir equation. The adorption capacity reached equilibrium in 8h in adsorption kinetics experiment. A further analysis indicated that the adsorption kinetics data could be well fitted by the pseudo-second-order rate model, and adsorption capacities calculated by the model were consistent with the actual measurements. The mixture solution of 0.25mol/L NaCl and 0.3mol/L imidazole in 0.01mol/L phosphate buffer (pH6.0) was a good eluant for recovery of lysozyme, and the extents of recovery of lysozyme and enzymic activity were 96.7% and 94.1 % respectively. In addition, FICF had excellent column adsorption kinetic properties and high binding capacity. The adsorptivety of the column was stable in repeated adsorption-desorption cycles.
The adsorption selectivity of FICF to lysozyme and albumin in their binary mixture solutions was investigated. The results indicated that albumin could be selectively adsorbed at pH4.0, while lysozyme could be selectively adsorbed at pH8.0. FICF and ZICF were used to separate lysozyme from chicken egg white. The experiments showed that they both could separate and purify lysozyme by one step. The purity of separated lysozyme measured by HPLC was 100%. The metal ion-immobilized collagen fiber used for separating lysozyme. could be easily regenareted. The recoveries of lysozyme by using new FICF column and the regenareted column were 70.5% and 70.0% respectively, and were 68.7% and 68.4% by using new ZICF column and regenareted column respectively.
The adsorption behaviors of FICF to bacteria were investigated. The effects of temperature, pH, the age of bacteria, initial concentration and ionic strength on adsorption capacity were studied. The adsorption isotherms and adsorption kinetics of bacteria on FICF were also investigated. The results indicated that FICF exhibited a high adsorption capacity to bacteria, and the adsorption rate was fast. When initial concentrations of E. coli and S. aureus were 1.02×10~7 cfu/mL and 9.8×10~6cfu/mL, their adsorption capacities were 2.23×10~9cfu/g and 2.74×10~9 cfu/g respectively in 10 min. The adsoption reached equilibrium in 90min in the adsorption kinetics experiment, where the adsorption capacities of E. coli and S. aureus were 2.94×10~9cfu/g and 3.15×10~9 cfu/g respectively. The adsorption rate of bacteria on FICF was fast, probably duo to the fact that the adsorption process took place at the surface of adsorbent. The adsorption capacity was influenced by culture age of the bacteria, which reached a maximum when the bacteria was in exponential phase. The adsorption capacity increased with the rise of ionic strength, but no considerable change was observed as varying temperature and pH(4.0～10.0). The adsorption isotherms could be described by the Freundlich equation. A further analysis indicated that the adsorption kinetics data could be well fitted by the pseudo-second-order rate model, and adsorption capacities calculated by the model were consistent with the actual measurements with error≤2%.
The adsorption behaviors of ZICF to S.cerevisiae were investigated. The effects of temperature, pH, age of S. cerevisiae, initial concentration and ionic strength on adsorption capacity were studied. The adsorption isotherms and adsorption kinetics of S. cerevisiae on ZICF were also investigated. The adsorption capacity of S. cerevisiae on ZICF were 8.20×10~7 cfu/g when the initial concentration was 5.26×10~5 cfu/mL. The adsorption capacity was significantly influenced by pH, and the optimal pH for adsorption was 6.0. The adsorption capacity was also influenced by culture age of the S. cerevisiae, which reached a maximum when the S. cerevisiae was in stationary phase. But it was almost unchangeable as varying temperature. The adsorption isotherms could be described by the Freundlich model. It was found that the adsorption capacity and the content of living cell were almost unchanged when the concentration of NaCl was smaller than 0.1mol/L, while they were badly decreases as furhter increase of NaCl concentration. The adsoption reached equilibrium in 5h in the adsorption kinetics experiment, and the adsorption kinetics data could be well fitted by the pseudo-second-order rate model. The fermentation experiments exhibited that the yield of ethanol by using immobilized S. cerevisiae was two times higher than that of using dissociative S. cerevisiae.
In order to investigate the adsorption mechanism between microbial and metal ion-immobilized collagen fiber, the cell surface hydrophobicty and electroatatic charge were determined by measuring contact angle and Zeta potential respectively. The results indicated that the cell surfaces of S. aureus, E. coli and S. cerevisiae was all hydrophilic and electron- offering. The hydrophobicty sequence of microbial was S. aureus>E. coli>S. cerevisiae, which is consistent with the adsorption capacity of metal ion-immobilized collagen fiber to them. That is, the stronger is the hydrophobicty of microbial surface, the higher is the adsorption capacity on metal ion-immobilized collagen fiber. The three kinds of microbial cells possessed a net negative electrostatic surface charge when pH ranged from 4.0 to 12.0. In the same condition, the sequence of net negative electrostatic surface charge of microbial was S. aureus>E. coli>S. cerevisiae, which is also consistent with the adsorption capacity of metal ion-immobilized collagen fiber to them. Namely, the more was net negative electrostatic surface charge of microbial , the higher was the adsorption capacity of metal ion-immobilized collagen fiber to them. So adsorption of microbial on metal ion-immobilized collagen fiber was an interplay of hydrophobic and electrostatic properties of the interacting surfaces.
These studies would be significant in exploring application of metal ion-immobilized collagen fiber on bio-separation engineering, medicine, environmental protection, fermentation engineering and chemical industry. At the same time, the experimental results could be scientifically valuable to enrich the knowledge of metal-chelated affinity chromatography.
引文
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