Abstract
Glycerol dehydrogenase [EC 1. 1. 1. 6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20mM acetate buffer (pH 6.0) on either octyl-Sepharose or 10-carboxydecyl-Sepharose, among ten different kinds of n-alkyl-Sepharose derivatives tested, some of which have an amino or a carboxyl group as a terminal residue.
The enzyme adsorbed on 10-carboxydecyl-Sepharose is 95% eluted with 0.26M NaCl. n-Propanol (10% and 15%, but not 5%), and various nucleotides such as NAD-, NADH, NADP+, NADPH, AMP, ADP, and ATP (1mM) are also effective for elution. The elution with nucleotides is facilitated by 5%, n-propanol. On the other hand, the enzyme adsorbed on octyl-Sepharose is not eluted under the conditions described above.
These results suggest that the adsorption-elution of the enzyme on 10-carboxydecyl-Sepharose is due to a combination of hydrophobic interaction and electrostatic repulsion between a specific locus of the enzyme surface and the 10-carboxydecyl residue.
Experimental conditions are described under which the enzyme can be purified 266-fold with a yield of 79% by a single chromatography of the cell extract on a 10-carboxydecyl-Sepharose column.
