Studies were carried out to increase the productivity of the $\Delta^1$ -dehydrogenation process of hydrocortisone leading to prednisolone by optimization of the reaction conditions, increasing the substrate concentration with organic solvents and using immobilized living whole-cell enzyme of $\underline{Arthrobacter}$ $\underline{simplex}$ (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℃.
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. Km 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, dimethylsulfoxide and dimethylformamide were more effective than other solvents used in the test. The optimum concentration of methanol and ethanol for the immobilized living whole-cell enzyme was 10% (v/v).
The reaction rate of the enzyme was inhibited by methanol and ethanol 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 showed that the inhibition mechanism is not a simple but a second-order inhibition type.
The first-order inhibition constant was 14 M for methanol and 2.4 M for ethanol. The second-order inhibition constant was 83.3 $M^2$ for methanol and 27.0 $M^2$ for ethanol.
The simulation results obtained by using those inhibition constants showed that there is an optimum methanol and ethanol concentrations in the reaction mixture of saturated substrate concentration, the productivity could be increased about 2 times in the optimum solvent concentration.
$\underline{Arthrobacter simplex}$ (ATCC 6946) 의 고정화 균체효소를 이용하여 hydrocortisone 으로 부터 Prednisolone 을 생산하는데 있어서, 최적 반응 조건을 결정했으며, 유기용매를 이용하여 기질농도를 증가시킴 으로써 생산성을 증가시켰다.
먼저 고정화 균체효소를 peptone 과 hydrocortisone 혼합액에서 진탕배양하여 효소활성을 증대시켰다. 고정화 균체효소의 최적반응 온도와 pH 는 40℃와 7.6 이었다. Casitone, peptone, 그리고 tryptone 과 같은 질소원을 반응액중에 첨가했을 때는 외부로부터 조효소의 공급 없이도 연속적인 효소활성을 나타내었다. 또한 $K^+$, $Na^+$ 과 같은 금속이온의 첨가에 의하여 효소활성이 증가하였다.
이 효소는 Michaelis-Menten 반응 기전을 따랐으며, 그 상수는 각각 3.30 mM (Intact Whole-Cell Enzyme), 1.25 mM (Immobilized Resting Whole-Cell Enzyme), 2.40 mM (Immobilized Living Whole-Cell Enzyme) 이었다.
Methanol, ethanol, dimethylsulfoxide 그리고 dimethylformamide 와 같은 유기용매는 기질농도를 높임으로써 효소의 초기반응 속도를 증가시켰다. 그러나 이들 유기용매는 일정한 기질 농도하에서는 이 효소에 대하여 제해제로 작용하였다.
Methanol 과 ethanol 의 이 효소에 대한 제해반응 기전과 저해 상수를 구하였다. 이들은 이 효소에 대하여 비경쟁적 저해제 였으며, methanol 의 이 효소에 대한 저해상수는 14 M 과 $83.3 M^2$ 이었다. 또한 ethanol 의 이 효소에 대한 저해상수는 2.4 M 과 $27.0 M^2$ 이었다.
이들 저해상수와 구해진 실험식으로부터 computer 를 이용하여 최적 유기용매 농도와 각 반응 시간에서의 생산성을 결정할 수 있었으며, 유기 용매를 씀으로써 생산성을 2 배 가량 증가시킬 수 있다는 결론을 내릴 수 있었다.
