Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells

Abstract

SummaryThe 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 Cor.vnebactuiurn 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 mgiml. The following aspects of the system were examined: I) 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 mgiml of a membrane concentration of 80 mgiml. This reaction attained an 80% conversion in 48 hr. A linear 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 mgiml. The rate-concentration relationship was linear when graphed on a Lineweaver-Burk plot; giving a K,' and V,' value of 5.39 mgiml and 0.556 mgimllhr, respectively. When the data were tested for competitive product inhibition, the curves fitted the experimental points fairly well and produced K,' and V,' values of 4.52 mgiml and 0.566 mgimlihr, 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 product. This behavior has been analyzed and shown to be possibly a result of the formation of a tertiary intermediate produced during the reaction.

Transformation of microcrystalline hydrocortisone by free and immobilized cells of Arthrobacter simplex

“…Thus, the presence of menadione in the medium had no effect on the completeness of hydrocortisone conversion. These results are in agreement with those of Constantinides (1980).…”

“…Longer duration of the process did not lead to a more complete conversion of the substrate. Similar data have been reported by other authors (Borman et al 1975;Skryabin et al 1979;Constantinides 1980). …”

Optimization of cholesterol oxidase production and 16S rRNA partial sequence of Bacillus cereus strain KAVK4 isolated from butter

“…It can also be employed for degrading dietary cholesterol to improve human health associated with cardiovascular disease (Kaunitz et al, 1978). Many microorganisms have an ability to decompose cholesterol that has been due to the enzyme source in steroid biotransformations, this was achieved by cholesterol oxidase (Constantinides, 1980). Cholesterol oxidase can be produced by bacteria of the genera Arthrobacter, Brevibacterium, Pseudomonas, Nocardia, Rhodococcus, Streptomyces, Corynebacterium and Shizophyllum (Yazdi et al, 2008).…”

Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp

“…Cholesterol decomposition ability is widespread among microorganisms that have been explored as free and immobilized cells (15,29) or as enzyme source (12) in steroid biotransformations. Cholesterol may be completely oxidized by microbial cells to carbon dioxide and water by the action of a complex enzyme system in which cholesterol oxidase is the first enzyme involved.…”