Preparation Of Metal Ion-immobilized Collagen Fiber And Their Adsorption Behaviours To Protein, Enzyme And Microorganism

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.