【作者】 薛文娇；

【导师】 范代娣； 尚龙安；

【作者基本信息】 西北大学 ， 生物化工， 2006， 硕士

【摘要】 本文利用成本相对低廉的离子交换方法纯化重组类人胶原蛋白Ⅱ（Human-like Collagen Bioprotein Ⅱ,HCB Ⅱ）,将菌体经高压匀浆、沉淀、超滤浓缩,得到HCBⅡ的粗品,然后通过离子交换法进一步纯化,获得的HCB Ⅱ达到电泳纯。 通过对6种树脂进行静态筛选,得出阳离子交换树脂CM52对HCB Ⅱ的分离效果最佳。该树脂对HCB Ⅱ有较高的吸附容量和选择性:当pH为4.0缓冲液不加NaCl时,其对HCB Ⅱ的吸附量为54.8mg/g湿树脂,分离因数2.64。 CM52树脂对HCB Ⅱ的吸附平衡研究表明:HCB Ⅱ在CM52上的吸附基本符合Langmuir方程;pH、NaCl浓度对Langmuir方程参数影响较大;pH低于HCB Ⅱ等电点（pI=5.0）时,pH对吸附平衡的影响显著:即在4<pH<5范围内,随着pH增大,吸附容量减小,解离系数K₄增大;在3≤pH<4范围内,随着pH增大,吸附容量增大,解离系数K₄减小,而pH大于等于5.0时Langmuir方程参数变化不明显;随着NaCl浓度增大,吸附容量减小,解离系数K₄增大;温度对Langmuir方程参数的影响较小,温度每升高10℃,吸附容量增加3%左右,解离系数K_d的变化不显著。 以CM52对HCB Ⅱ的吸附平衡数据为基础,优化了CM52对HCB Ⅱ的纯化工艺。通过实验,分别确定了静态吸附和柱层析吸附的操作条件,即静态吸附:在pH4.0、NaCl0.15mol/L、进料浓度为7g/L,处理量为17.26mL/g的条件下吸附60min:柱层析:在pH4.0、NaCl 0.15mol/L、进料浓度5g/L、流速5mL/min的条件下进行吸附。然后上柱,在pH4.0、NaCl 0.30mol/L下进行脱附。实验表明:采用静态吸附的方式吸附HCB Ⅱ,经上柱洗脱后,HCB Ⅱ的吸附量可达到48.6mg/（g树脂）,回收率83.8%,操作时间短,分辨率高,最终纯化的HCB Ⅱ达电泳纯,相对分子量为97kD。 对CM52吸附HCB Ⅱ的动力学研究表明:搅拌速度和料液浓度决定了其动力学控制机理。以简单线性推动力模型拟合膜扩散控制（film diffusion control,FDC）时的动力学数据,其线性关系良好,模型准确度较高;对于颗粒扩散控制（particle diffusion control,PDC）的吸附,则以粒内表面扩散控制模型拟合,求出表面扩散系数,模型较好的描述了CM52对HCB Ⅱ的PDC吸附。同时,考察了搅拌速度、溶液浓度和温度对FDC模型参数的影响以及溶液浓度和温度对PDC模型参数的影响。更多还原

【Abstract】 Ion exchange chromatography with low cost, was used to purify Human-like Collagen Bioprotein Ⅱ (HCB Ⅱ) , harvested recombinant E.coli cell was lysed by high pressure homogenizer, supernatant was collected by centrifuging. After salt precipitation, the crude product solution of HCB II was purified with ion exchange chromatography, the purity of HCB II could reach electrophoresis grade purity.Cation exchange resin CM52 was suitable for the purification of HCB II than the other five kind resins used in the experiment. The CM52 cation exchange resin was of better capacity of adsorption and selectivity for HCB Ⅱ. Under the conditon of pH 4.0, buffer free of NaCl, the adsorption capacity of CM52 for HCBⅡ was 54.8mg/g.wet resin and seperation factor was 2.64.The experiments of adsorption equilibrium showed that the adsorption of HCB II onto CM52 obeyed Langmuir model. Ionic strength and pH value were two important factors affecting the equilibrium characteristics. The model parameters hardly vary with pH value unless pH<pI=5.0;within 4.0<pH<pI=5.0,the higher pH value, the lower binding strength (higher Kd) and binding capacity, within 3.0≤pH<4.0, the higher pH value, the higher binding strength and binding capacity. The higher ionic strengths, the higher binding strength and binding capacity. Meanwhile, the increased temperature leads to slightly higher binding strength (lower Kd) and higher binding capacity (increased by 3% per 10°C).On the basis of adsorption isotherms data, purification process of HCBⅡ was optimized. The batch operation and normal chromatography column method were used in experiments, i.e., Batch operation of chromatography: HCB II was adsorbed under pH 4.0, NaCl (ionic strength) 0.15mol/L, 17.26mL(protein volume)/g (resin )and concentration of crude protein liquid 7g/L;Column chromatography: HCB II was adsorbed under pH 4.0, NaCl (ionic strength) 0.15mol/L, flow rate 5mL/min and concentration of crude protein liquid 5g/L, the elution of HCB II was pH 4.0, the concentration of NaCl (ionic strength) 0.30mol/L. As a result, the CM52 cation exchange chromatography was of better capacityof adsorption for HCB II and the elution was simple;the total purity process time would be shorten and resolution was high, The recovery of aim protein HCB II with a molecular weight around 97kD was above 83.8% and purity of HCB II could reach electrophoresis grade purity.The ion exchange kinetics for HCB II onto CM52 were developed. The results showed that seperation mechanism mainly depended on stirring speeds and feeding concentration. The kinetics obeys linear driving force model in case of FDC (film diffusion control) and intraparticle surface diffusion model in case of PDC (particle diffusion control). The film diffusion constant becomes higher with the temperature, concentration and stirring speeds increased. The higher temperature,the higher intraparticle surface diffusion coefficient ,but little influence on intraparticle surface diffusion coefficient when concentration increased.更多还原