Specific affinity of glycerol dehydrogenase from Geotrichum candidium for 10-carboxydecyl-sepharose: its application to chromatography for purification of the enzyme.
Glycerol dehydrogenase [EC 1.1.1.6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20 mM acute 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 0.26 M NaCl. 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 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.
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D005848:Geotrichum / Q000201:enzymology*; D003904:Mitosporic Fungi / Q000201:enzymology*; D009243:NAD; D011134:Polysaccharides; D011433:Propranolol; D012685:Sepharose / Q000031:analogs & derivatives; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification*
C032781:10-carboxydecyl-sepharose; D011134:Polysaccharides; D009243:NAD; D012685:Sepharose; D011433:Propranolol; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase