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J. Biochem, 1982, Vol. 91, No. 1 211-217
© 1982 Japanese Biochemical Society

research-article

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

Ikuharu SASAKI*,1, Nobuya ITOH**, Hirosi GOTO*,1, Ryohei YAMAMOTO*,1, Hideyuki TANAKA*,1, Kanzo YAMASHITA**, Jinpei YAMASHITA* and Takekazu HORIO*

*Division of Enzymology, Institute for Protein Research, Osaka University Suita, Osaka 565
**Amano Pharmaceutical Co., Ltd. Nagoya, Aichi 460

Glycerol dehydrogenase EC 1.1.1.6 [EC] ] (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.

1 On leave of absence from Amano Pharmaceutical Co., Ltd., Nagoya, Aichi 460.


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