This research is concerned with production of medically important steroid compound that is one of the anti-inflammatory agents. It is of some industrial importance to develop a process of enzymatic transformation of steroid. In this research 3-ketosteroid-delta-1-dehydrogenase was first produced by using $\underline{Arthrobacter}$ $\underline{sim}$ $\underline{plex}$ strain. In the production of the enzyme, several important process variables have been optimized in order to maximize the enzyme productivity. These variables include fermentation conditions made of enzyme induction, and preparation of whole-cell-enzyme. The whole-cell-enzyme was prepared by washing the cells with buffer and acetone followed by heat treatment under mild conditions. The whole-cell-enzyme so obtained retained high enzymatic activity and could be used in both batch and continuous reaction system. The whole-cell-enzyme offers significant advantages over the other forms of enzyme preparation. For instance, continuous operation, reusability, easy storage, free from contamination, etc. With such advantages, the use of whole-cell-enzyme proves to be more economical than the purified enzyme, purified-immobilized enzyme, and the growing cells in fermentation system.
In order to develop the steroid process by the enzymatic transformation, it was important to determine kinetic constants of the whole-cell-enzyme produced. Km constant and Vm(Maximum velocity)determined were $1.57\times10^{-3}M$ and $2.07\times10^{-4}M/min$ respectively.
These values of the kinetic constants determined were found to be significantly different from those constants obtained for the partially purified enzyme. This difference was believed to be due to the mass transfer limitation through the cell membrane barrier and steroid solubility under the conditions of experiments used.
We found that the dehydrogenase enzyme prepared from $\underline{A}$. ${\underline{simplex}$ cells did not show any inhibition by either product or substrate. Other workers previously reported that the dehydrogenase enzyme obtained from other microbial strain showed inhibition by substrate and product.
Another important result that is worth noting is the development of new assay method for the steroid in the presence of whole-cell-enzyme in the reaction system. This assay method is based on U.V. spectrophotometric analysis. We found that fractional conversion of steroid showed linear correlation with the U.V. absorption ratios (the absorption at 265nm the absorption at 242nm). This new assay method will be very useful in determining the steroid conversion in the presence of extractable cellular material from whole-cell-enzyme in the reaction system. In view of the fact that the whole-cell-enzyme is used very widely in industry, this kind of $\underline{in}$ $\underline{situ}$ assay technique will be of practical importance.
In addition, based on the experimental results the process of enzymatic transformation of steroids was simulated with the aid of a computer and the method of predicting optional conditions and the performance of batch and continuous enzyme reacter systems.
All of the results obtained for the enzymatic transformation of steroid with the use of whole-cell-enzyme will be of practical importance to the industrial application of these research results.
본 연구는 steroid 의 이중결합을 도입하여 efficient 한 antiinflamnatonry drug (항염제) 을 제조하는 공정에 관한 것이다. 대개 이 반응은 미생물을 이용하고 있으며 이 반응은 산업적으로 많이쓰이는 steroid 생산에 관계되는데 그 중요성이 있다.
이 연구에서는 미생물 Arthrobacter simplex 를 이용하여 3-keto steroid - delta - dehydrogenase 를 생산하였다. 활성이 높은 효소를 제조하기위하여 효소유도물질과 효소와의 접촉시간에 대해 조사하였더니 적어도 20시간 접촉하는 것이 효율적임을 알았다.
이 효소를 완충용액으로 세척 acetone으로 세척한후 열처리 하여서 wholecell-enzyme 을 제조하였는데 이whole-cell-enzyme 을 이용하여 효율적인 steroid dehydrogenation 을 할 수 있었다. 이 whole-cell-enzyme을 이용하는 방법은 대사하고 있는 세포를 이용한 발효 방법에 의한 제조 방법에 비해 효율적이었고 이 whole-cell-enzyme 의 kinetic constant를 조사하여 본 결과 Km 은 $1,57\times10^{-3}M$, Vm 은 $2,07\times10^{-4} mole/min$ 으로서 다른 사람들이partcially purified enzyme 을 이용하여서 구한 kinetic constant와 비교할때 값은 약10배, maximum velocity 는 약 $\frac{1}{2}$에 해당하였다. 또한Mosbach 등의 acrylamide gel 에 immobilize 시킨 enzyme 의 activity 비교할때, 이 whole-cell-immobilized enzyme은 immobilized enzyme 의 성질을 가지고 있어 recovery 및production cost 를 싸게 할수 있고 다른 immobilized enzyme 에 비해 activity 가 높아 효율적임을 알수 있었다. 이 연구에서 $\underline{A}$. $\underline{simplex}$ 에서 제조한 3-ketosteroid-deltagl-dehydrogenase 는 substrate 나 product inhibition 이 없음을 알수 있었다.
이 연구에서는 특히 enzyme assay 방법에 대해서 조사하였는데 이것은 U.V absorption 을 이용하여 product 와 substrate 의 U.V maxima absorption wave length 인 242 nm 의 absorbancy 로 2개의 compound 의 U.V absorption difference maximum 인 265nm의 U.V absorption을 나눈값이fractional conversion 에 정비례하는 것을 이용하였다.
이 방법에서 cellular material 에 의한 disturbance 를 calibration curve 에 adjust 하는 equation 을 만들어 사용하였다 이렇게 얻은결과는 steroid 의 산업적 생산에 whole-cell-enzyme 을 이용하여 효과적으로 사용될수 있는 근본 실험치를 얻었다는데 그 중요성이 있다고하겠다.
