Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP + cycling at high efficiency

Abstract

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP + was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD + oxidoreductase, EC 1.1.1.176), and the regeneration of NADP + was obtained through the glutamate dehydrogenase ( l-glutamate:NADP + oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained ∼70% of the initial activity after 2 months of continuous use.