Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells

• Published in: Biotechnology and bioengineering 1980-01, Vol.22 (1), p.119-136
• Main Author: Constantinides, Alkis
• Format: Article
• Language: English
• Subjects: Biotransformation; Cells, Immobilized; Corynebacterium - metabolism; Hydrocortisone - metabolism; Membranes, Artificial; Prednisolone - antagonists & inhibitors; Prednisolone - metabolism; Solubility; Space life sciences; Time Factors
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.260220110

The steroid transformation of hydrocortisone to prednisolone, combining the two techniques of immobilized whole cells and high steroid concentrations, was investigated and found to be a feasible process. Freeze‐dried Corynebacterium simplex cells were immobilized in collagen, tanned with glutaraldehyde, and cast into a membrane. The reaction was studied at hydrocortisone concentrations ranging from 5 to 50 mg/ml. The following aspects of the system were examined: (1) the substrate concentration effect upon the reaction; (2) the effect of enzyme concentration; (3) the rate‐concentration relationship; and (4) the product inhibition characteristics of the system. The optimal substrate concentration was found to be 15 mg/ml of a membrane concentration of 80 mg/ml. This reaction attained an 80% conversion in 48 hr. A liner relation was found between the initial reaction rate and membrane concentration. One can thus increase the net production of steroid per unit volume and time by increasing the membrane levels. A physical limit to this increase occurred at membrane concentrations greater than 125 mg/ml. The rate‐concentration relationship was linear when graphed on a Line weaver‐Burk plot: giving a Km′ and Vm′ value of 5.39 mg/ml and 0.556 mg/ml/hr, respectively. When the data were tested for competitive product inhibition, the curves fitted the experimental points fairly well and produced Km′ and Vm′ values of 4.52 mg/ml and 0.566 mg/ml/hr, respectively. Product inhibition experiments showed that the inhibition was not purely competitive. At low substrate concentrations, product inhibited the enzyme; at high substrate concentrations, the enzyme was first stimulated and then depressed by increasing levels of products. This behavior has been analyzed and shown to be possibly a result of the information of a tertiary intermediate produced during the reaction.