Immobilized Rhizopus Nigricans For C11α Hydroxylation

Steroidal drug was widely used in the medical field，C11α hydroxylation is acritical step in the production of steroidal drugs. Rhizopus nigricans has a very strongcapacity in the steroidal C11α hydroxylation. C11α hydroxylation byRhizopusnigricans can eliminate the14-step reaction needed for chemical synthesis.The polyurethane foam has the advantages of a low-cost, low density, the stabilityof the physical and chemical properties, porosity. It can be a good carrier for the cellsimmobilization.In this paper, Rhizopus nigricans spores were fixed onto the polyurethane foamby adsorption. Among the five kinds of the polyurethane foam, the best immobilizedone can absorb14.25×10~6spores per cubic centimeter. The adsorption capacityreached the maximum in about60minutes. Treating the surface of the polyurethanefoams by polyethylenimine (PEI) and polyacrylamide (PAM) can significantlyimprove the adsorption density of the R. nigricans spores on the foams.The conversion of the immobilized cells was higher than that of the free cells inthe shake flasks. The substrate, Wolfowitz oxides (16,17-α-Epoxyprogesterone), hada significant inhibitory effect on the cell growth. The optimal substrate conversionconditions were: the feeding time of the substrate was20h; the conversion time was48h; the rotating speed was200r/min; pH4.5; the optimal amount of the immobilizedmaterial was3.5g/L. The conversion was42%under these conditions.The immobilized cells could retain46%of the initial activity after it was usedonce. Its activity would reduce down to13%after it was used twice. The immobilizedcells could retain34.8%activity after it was stored at4℃for three weeks.In the substrate transformation by the resting immobilized cells, Addition of thecorn syrup powder into the substrate solution can significantly improve the conversion rate because the cells needed the energy and the cofactor for the substrateconversion. Addition of0.05mol/L acetone as a cosolvent could improve the abilityof the cells for the steroidal C11α hydroxylation. Addition of0.07g/L magnesiumsulfate and magnesium chloride can improve the conversion rate of the substrate;Excessiveaddition of these metal ions had the negative impacts.In a2L fermentor, the best aeration rate for C11α hydroxylation by theimmboilized Rhizopus nigricans was2.5v/v min. Under this condition the conversionreached45%.Within a certain range, increasing the stirring speed can increase theconversion rate per mass of the cells, but excessive stirring speed may inhibit the cellgrowth.