(A) study on the immobilized whole-cell enzyme of arthrobacter simplex in steroid transformation

Abstract

Studies were carried out to increase the productivity of Predinisolone from hydrocortisone by optimization of the $\Delta$`` -dereaction conditions and by increasing the substrate concentration using organic solvents and immobilized living whole-cell enzyme of $\mbox{\underline{Arthrobacter}}$ $\mbox{\underline{simplex}}$ (ATCC 6946) The immobilized resting whole-cell enzyme was activated by incubation in media containing 0.5\% peptone and 0.7 mM hydrocortisone, and the activity was increased 12 fold. The optimum pH and temperature of the immobilized living whole-cell enzyme was 7.6 and 40$^\circ$C. Nitrogen sources such as casitone, peptone, and tryptone were good for the enzyme activity and there was no need to add the cofactor of the enzyme in presence of these media. Metal ions such as potassium and sodium showed favorable effect on the enzyme activity. The reaction kinetics of the enzyme followed the Michaelis-Menten type. $K_m$ values for the intact whole-cell enzyme, the immobilized resting whole-cell enzyme, and the immobilized living whole-cell enzyme were 3.30, 1.25 and 2.40 mM respectively. Some organic solvents were tested to increase the productivity by increasing the substrate concentration. Ethanol, methanol, dimethlsulfoxide and dimethlformamide were more effective than other solvents tested. The optimum concentration of methanol and ethanol for the immobilized living whole-cell enzyme was 10\% (v/v). The enzyme was inhibited by methanol and ethanol beyond the optimal concentration in the reaction mixture of constant substrate concentration, but the organic solvents could increase the substrate concentration remarkably. The enzyme kinetics of solvent inhibition was studied and it was found that methanol and ethanol was non-competitive inhibitors of the intact whole-cell enzyme system. An empirical equation of the enzyme reaction kinetics was derived from the experimental data and the inhibition constants were determined. The empirical equation s...