Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose.
The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase.
Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose. The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase.