Research On Separation And Purification Of Human-like Collagen Ⅰ And Its Functional Properties

Separation and purification of Human-like collagen I (HCB I) and its functional properties were researched in this paper. Harvested recombinant Escherichia coli BL 21 cells were collected from high density fermentation and treated with high pressure homogenization, precipitation, ultra-filtration, and ion exchange chromatography. After a series of processes the highly purified target protein could be received.For cell disruption process, a systematic study was carried out on the influence of physical and chemical factors including homogenization pressure, homogenization passes, cell concentration and suspension system during homogenization processes. The techniques could be confirmed as 15% cell concentration, distilled water suspending system, high pressure homogenization twice continuously under a pressure of 80Mpa and an icewater bath was used to cool the suspensions through the homogenizer.For precipitation process, L₁₆(4⁵) orthogonal design was used and the optimal parameters were confirmed then. pH 2.8, 3% NaCl concentration, 18℃, and 5g·L^-1 protein concentration were choosed.For ultra-filtration process, the membrane package with a 30kD cut-off molecular weight was used to filtrate the supernatant from precipitation. The optimal parameters then were established: 0.2% protein concentration, 0.3 Mpa operating pressure, 3 L·min^-1 flow velocity, 25℃and pH 3.For ion exchange chromatography process, cation exchange resin, CM-1, was adopted to adsorb impurities for 1h firstly on the condition of 4mg·mL^-1 feeding concentration, which was made by 20 mmol·L^-1 phosphate buffer with 0.03 mol·L^-1 NaCl at pH 6.5; and then the target protein with a 4mg·mL^-1 concentration was adsorbed by Fracogel EMD using 50 mmol·L^-1 Tris-HCl buffer of pH 8.0 in a flow rate of 3.0 mL·min^-1 and eluated by stepwise elution. The purity and recovery of the target protein could reach to 96.8% and 80.47% respectively. A single band could be gained via SDS- PAGE dying with Coomassie brilliant G-250.According to methods from references, water solubility, water absorption, water-holding ability, wettability, fat absorption, emulsifying property, foam formation and foam keeping ability of Human-like collagen I were determined and compared with gelatin, which had a close molecular weight. The results showed that HCB I had high water solubility, water absorption, wettability, fat absorption, emulsifying property, foam formation and foam keeping ability, and some water-holding ability.