9?-Hydroxylation of 4-androstene-3,17-dione by gel-entrapped Corynebacterium sp. cells

Abstract

Whole cells of Corynebacterium sp. having steroid 9α-hydroxylation system were immobilized by entrapment with photo-crosslinkable resin prepolymers, urethane prepolymers or several kinds of polysaccharides. Of various entrapment methods tested, cells entrapped in photo-crosslinked gels showed the highest activity to hydroxylate 4-androstene-3,17-dione at 9α-position. The properties of the photo-crosslinkable resin prepolymers, such as the hydrophobicity and the chain length of the prepolymers, affected markedly the activity of the entrapped cells. Addition of dimethyl sulfoxide to a buffer system at 15 vol. % was effective to solubilize the product, 9α-hydroxy-4-androstene-3,17-dione, and gave the highest yield. In an aqueous system, the activity of hydrophilic gel-entrapped cells was higher than that of hydrophobic gel-entrapped cells. In 15% of dimethyl sulfoxide, the hydrophobic gel-entrapped cells showed almost the same activity as the hydrophilic gel-entrapped cells probably due to extraction of the product from gels to the external solvent before its metabolic degradation. Entrapment significantly enhanced the conversion ratio at high substrate concentrations and the operational stability of the 9α-hydroxylation system in the cells. In the presence of nutrients in the reaction mixture, entrapped growing cells maintained fully the original activity at least during 10 times of repeated batch reactions for 5 days.