Specific Affinity of Glycerol Dehydrogenase from Geotrichum candidium for 10-Carboxydecyl-Sepharose: Its Application to Chromatography for Purification of the Enzyme

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Abstract
Glycerol dehydrogenase EC 1.1.1.6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20 m 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 lO-carboxydecyl-Sepharose is 95% eluted with 0.26 M NaCI. n-Propanol (10% and 15%, but not 5%), and various nucleotides such as NAD+ NADH, NADP+ NADPH, AMP, ADP, and ATP (1 mM) 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 condi tions described above. These results suggest that the adsorption-elution of the enzyme on 10-carboxy decyl-Sepharose is due to a combination of hydrophobic interaction and electro static repulsion between a specific locus of the enzyme surface and the 10-carboxy decyl 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.