The Mechanism Study Of Protein Separation Using Ultrafiltration And Its Application

Ultrafiltration is a novel bioseparation technology, with advantages of simple operation, easy scale-up and so on. It has a huge potential for large-scale production of biomacromolecules. Our efforts are therefore to study the mechanism of protein separation using ultrafiltration and offer suggestions to the further experimental design and industrial application; such work will be certainly of great significance.In this work, the process of protein approaching and getting through a membrane was firstly divided into three stages (Ⅰ, elastic impact stage;Ⅱ,concentration polarization stage;Ⅲ,distortion in pore stage ). The physical and mathematical models for each of the stages were developed. In stageⅡ, the resistance for protein approaching the membrane surface came from the―concentration polarization effect‖, while the resistance for protein getting through the membrane in stageⅢwas mainly from the deformation of protein moleculars, and the electrostatic interactions among the proteins and between the membrane and individual proteins as well. When the pore resistance was greater than that from the concentration polarization effect, the control step would switch from stageⅡto stageⅢ.The effects of solution pH, ionic strength, stirring speed and permeate flux on the transmission of protein were examined with single protein (bovine serum albumin, BSA) solutions. The reults showed that the highest transimission of BSA was obtained at its isoelectric point (pH 4.9). In addition, the BSA apparent sieving coeffeicient decreased at lower ionic strength(<100mM) and maintained around a certain value at higher ionic strength(>100mM). The self-electrostatic exclusion and the electrostatic shielding were the reasons for the trsanmission change of BSA at different ionic strength levels. The BSA apparent sieving coeffeicient decreased with stirring speed or permeate flux increase. To study the interactions among the four parameters, response surface methodology was employed using a central composite design. The experimental results demonstrated that srirring speed had independent effect on BSA transmission. The interactions between solution pH and ionic strength (permeate flux) were moderate and the interaction between ionic strength and permeate flux was not significant. All these results were consistent with the rules induced from the developed physcial models.The effects of solution pH, ionic strength, stirring speed and permeate flux on the selectivity of ultrafiltration process were also examined with binary protein solutions (BSA and lipase). Based on the experimental results, the following deductions were obtained: the interactions between the membrane and individual proteins have significant effects on the selectivity of ultrafiltration at lower salt concentrations(<100mM). The protein with opposite charges to membrane has electrostatic adsorption on the membrane surface and gets more opportunity to approach and get access into the pores.With the guidance of the outcomes obtained above, the 100 kDa-regenerated cellulose membrane and 50 kDa-polyethersulfone membrane were successfully employed to isolate superoxide dismutase ( SOD ) from garlic by removing the bigger macromolecules and smaller molecules. Under optimized conditions, the purity of SOD obtained was greater than 98.0%, the recovery of SOD from the feedstock was close to 90%, and the specific activity was 396.59 U mg^-1.