For the efficient production of phospholipase D from Streptomyces sp., the suitable composition of the culture medium was determined by a fractional factorial experimental design. The composition of the medium was as follows; 15 g/L of Bacto-peptone, 5 g/L of yeast extract, 20 g/L of glucose, 6 g/L of $CaCO_3$. The activity of phospholipase D from Streptomyces sp.in a culture supernatant was 5.62(unit/mL). The synthesis of phosphatidylserine with phospholipase D from Streptomyces sp in the two-phase system was carried out by using phosphatidylcholine as the donor and serine as the acceptor. The yield of phosphatidylserine in a two-phase system was found to be better than that obtained in an emulsion system. In the case of serine, the initial rate of transphosphatidylation was higher with D-serine than with L-serine when the PLD from Streptomyces sp was used. Among the various solvents tested, PLD showed the most efficient catalytic activity and stability in butylacetate. In order to find the optimum conditions for the synthesis of PS several different conditions were tried. Among these the highest yield of PS was obtained at 40℃, pH of 5.5 - 7.5, 400 rpm, above 0.1 mmol of $Ca^{2+}$ and 1.5 mmol of L-serine. This experiment showed the ratios of the solvent/water to be very important e.g. the higher the ratios of the solvent/water, the better the catalytic activity and selectivity. When the ratios of the solvent/water increased ranging from 1:1 to 5:1, the conversion and selectivity of PS were notably improved and in these conditions a smaller amount of serine could produce the same yield of product. The reusability of PLD was also examined; this was found not to be efficient because of the instability of PLD in butylacetate.
본 실험에서는 PLD 생산의 양상과 ps 합성에 관한 여러 특성을 고찰하여 다음과 같은 결과를 얻었다.
1. Fractional factorial design에 의해 플라스크 배양 조건에서 배지성분의 최적농도를 결정하였다.
: 20 g/L glucose, 15 g/L bacto-peptone, 5 g/L Yeast extract, 6 g/L $CaCO_3$
2. 30℃, pH 7, 400 rpm 조건으로 3 L 발효를 통해 접종 30시간 후에 최대의 효소 활성을 보였다.(분해활성 5.62 U/mL)
3. 에멀전 반응계 보다 2상 용매 반응계에서 전이활성이 월등이 우세하다.
4. 비교 실험한 여러 용매 중에서 butylacetate가 PS합성이 가장 우세하고 효소에 대한 안정성이 좋았다.
5. 각각 40℃, pH 5.5-7.5, 400 rpm, 0.1 mmol $Ca^{2+}$, 1.5 mmol L-serine에서 전이활성이 가장 좋은 것을 보였다.
6. 유기용매층과 수용액층의 비가 1:1에서 5:1로 높였을 때 conversion과 selectivity가 현저하게 증진될 뿐만 아니라 serin의 양을 줄인 조건에서도 높은 전이 활성을 보였다.
7. 반복 회분식 반응을 통한 PLD의 재이용성은 효소의 butylacetate에 의한 불활성화로 비효율적인 것으로 고찰되었다.
