Biotechnological approach to the lipid-hydrolyzing systems has advantages over chemical process. Recently the conversion of lipid by lipase has been studied intensively. Most of the work on this conversion has been performed in the conventional emulsion system, which has so many drawbacks to be improved. Moreover, little attention has been paid to bioprocess engineering for this reaction system. Thus an attempt has been made to develop the appropriate bioreactor system to overcome the drawbacks. A reverse phase system was developed for the hydrolysis of lipid at high substrate concentration, where lipases were immobilized on the gel particles containing water and dispersed in the continuous solvent phase containing olive oil.
Lipase from Candida rugosa was used because it lacks the stereospecificity of triglycerides and catalyzes the complete hydrolysis of several oils to free fatty acids and glycerol.
Three supports such as Sephadex LH-20, Sephadex LH-60, and Amberlite XAD-7 are selected, which are capable of containing much water and being dispersed in organic solvent. Lipase was immobilized simply by adsorption on these supports.
The binding of lipase to the supports reached equilibrium after 2 hr. Adsorption was consistentwith a Langmuir isotherm. It was found that the increase of temperature and ionic strength of buffer during the immobilization of lipase was disadvategeous because they resulted in reduction of available water for the hydrolysis of lipids.
Lipase immobilized on swollen Sephadex LH-20 and Sephadex LH-60 could almost completely hydrolyze 60 % (v/v) olive oil in isooctane. Sephadex LH-20 was selected as the most appropriate support, since immobilized lipase on this gel showed the highest activity and long term storage stability even though it has a low immobilization efficiency. Kinetic analysis of the lipase catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional up to the enzyme concentration of 720 ug per g wet gel. And specific activity of immobilized lipase was 217 units/mg protein at 60 %(v/v) olive oil concentration. The reaction rate of hydrolysis by lipase in the reverse phase system was about 4 or 5 times faster than in the emulsion system. When the initial rate is plotted against olive oil concentration, this system did not follow Michaelis-Menten kinetics. The maximum activity was obtained at pH 7, but the optimum temperature shifted toward higher one with the increase of olive oil concentration. Among the various chemical compounds tested, $Hg^{2+}$ and $Fe^{2+}$ inhibited the lipase seriously. In the batch hydrolysis of olive oil, as the concentration of olive oil increased, the rate of hydrolysis also increased, but degree of the hydrolysis observed to decrease.
To select the most suitable solvent in reverse phase system, the effect of various organic solvents on the stability and catalytic activity of lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to the other solvents for enzymatic fat splitting in reverse phase system. The effect of the solvent polarity on the hydrolysis of olive oil had been also examined in more detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times consecutively. Attainment of the chemical equilibrium took longer time when water was supplied to the gel through the reaction medium and the degree of hydrolysis decreased in the consecutive trials. These facts can be ascribed to the loss of enzyme activity and/or the accumultion of glycerol within the gel. After the second trial of hydrolysis the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05 M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one. Initial water content was found to be the most important factor that determined both the hydrolysis rate and the degree of hydrolysis; during the hydrolysis reaction in a reverse phase system the water content in the gel should be regarded another external parameter just like pH or temperature.
유지의 가수 분해를 위한 생물공정에 의한 접근은 화학공정보다 더 유리한 점이 있으며 최근들어 리파제에 의한 유지의 전환이 다각적으로 연구되어 오고 있다. 유지의 가수 분해를 위한 효과적인 반응계인 역상계에서 고농도 올리브유의 가수분해에 대하여 실험하였다. 즉, Candida rugosa 로 부터 얻은 리파제를 다량의 수분을 함유한 담체에 고정화시킨 후 올리브유를 고농도로 함유한 유기용매의 연속상에서 회분식 가수분해에 이용하였다. 고정화 방법으로는 흡착법을 이용하였다. Sephadex LH-20 에 고정화된 리파제는 이소옥탄 유기 용매내에서 60% (v/v) 올리브유를 완전하게 가수분해 시켰다. 가장 효율적인 리파제의 고정화 농도는 720 ug/g wet gel 이었으며, 효소역가는 217 units/mg protein 였다. 고정화는 두시간 만에 이루어졌으며 온도와 완충용액내의 이온강도를 높여줌에 따라 흡착정도는 상당히 증가하였다. 그러나 담체내 수분함량은 감소하였다. 담체내 수분함량의 감소는 지방산 생성에 불리하므로 고정화는 낮은 이온 강도 및 온도에서 수행하였다. 수소 이온의 변화에 따른 흡착 정도는 변화가 없었다. 기질의 농도가 초기속도에 미치는 영향은 Michaelis Menten 식을 따르지 않았다. 최적 수소이온 농도는 7 이었고, 최적 온도는 기질 농도의 증가에 따라 높은 온도쪽으로 이동하였다. 여러 금속이온 중 $Hg^{+2}$ 이온과 $Fe^{+2}$ 은 리파제의 활성을 매우 억제하였다. 회분식 가수분해의 결과, 기질의 농도가 증가함에 따라 지방산으로의 가수분해 속도 및 생산량은 증가하였지만 지방산으로의 가수분해율은 감소하였다. 또한, 유기용매가 효소활성 및 안정도에 미치는 영향을 기준으로 이소옥탄이 가장 적합한 유기용매로 선택되었다. 기질의 농도가 감소함에 따라 유기용매 효소활성에 미치는 정도는 현저하여졌다. 또한, 이 반응계는 유기용매의 극성에 효소의 활성도가 현저하게 영향을 받았고 비극성의 유기용매에서 일반적으로 리파제가 높은 활성을 나타내었다.
또한, 고정화된 담체를 반복하여 사용해 보았다. 반복사용함에 따라 올리브유의 가수분해율은 감소하였다. 연속된 사용에서 반응계에 수분을 첨가시킨 결과 더 높은 가수 분해율을 보여 주었고 평형에 도달하는데 더 많은 시간을 요구하였다. 이것은 담체내 글리세롤이 축적된 결과 및 반복사용에 의한 효소 역가의 감소 때문으로 생각된다. 글리세롤을 많이 함유한, 수분이 고갈된 담체는 이것을 칼럼에 넣고 에탄올과 완충용액 (pH 7) 을 차례로 흘려줌으로써 재생되었다. 수분이 고갈된 담체를 재생시킨 결과 재생 전의 고정화 효소와 거의 같은 활성을 보여 주었다.
또한, 초기의 담체내 수분함량이 가수분해 속도와 가수분해율을 결정하는 주요 인자임이 확인되었다. 담체 수분 함량의 약 54% 가 쉽게 가수분해에 이용이 되었다.
