Spherical phenyl-regenerated cellulose stationary phases used for hydrophobic chromatography to extract bramelain from crude stem bramelain]
球状苯基纤维素填料在低压疏水制备色谱分离天然菠萝酶中的应用

The low-pressure preparative hydrophobic chromatography packing materials have been successfully synthesized by grafting hydrophobic phenyl group to the macro-pore Spherical Regenerated Cellulose Resin (diameter 0.7-1.3 mm, pore size 4.3-15.7 microns, specific surface area 88 m2/g). The optimum adsorption-desorption conditions have been got by using 10 mL of this hydrophobic resin to deal with 100 mL of the crude stem bramelain: the mobile phase pH is around 5.0, concentration of (NH4)2SO4 is 1.5 mol/L; and the pH of total eluate is 8.0. While the hydrophobic column is operated according to the following steps: 1. use 0.1 mol/L NaAc-HAc(pH 4.8) and 1.5 mol/L (NH4)2SO4 solution to soak resin for more than 30 min; 2. let 100 mL of the extracted liquor of pineapple skin, which contains the same concentration of salt as 1, pass through the hydrophobic column; 3. use 20 mL of the same solution as 1 to wash the impurities in proteins, which are not adsorbed by the hydrophobic column; 4. use 30 mL of eluant, which contains 67 mmol/L NaOH and 50 mmol/L Na2CO3-NaHCO3(pH 10.28), to elute the adsorbed bramelain, and the initial 5 mL of the eluate is discarded; 5. soak the hydrophobic column with 0.5 mol/L NaOH + 20% (volume fraction) C2H5OH for 30 min, then wash it with water, and soak it with 0.5 mol/L HCl for 30 min, at last use water to wash it to neutrality for next use. We can get enriched bramelain of 4.4 fold activity, moreover the total enzyme activity recovery is more than 120%. The Phenyl-Spherical Regenerated Cellulose Resin can be used at the pH from 1 to 13; As it has a high rigidity and big size, it can be operated under low-pressure condition; It has a larger specific surface area and a higher separation efficiency for natural bramelain; After it has been used repeatedly for more than 50 times, its efficiency does not fall. Moreover, it is cheap to be produced.