Enzymatic synthesis of triglycerides has rarely been achieved with an immobilized lipase. Starting with glycerol and pure fatty acids without any organic solvent, and using Mucor meihei lipase immobilized onto exchange resins, production of commercially viable triglycerides was attempted.
Triolein production from a solvent free environment is greatly enhanced by the elimination of water through molecular sieves. In order to improve the yields of triglyceride synthesis the effect of temparature of the reaction as well as the effect of ratio of glycerol over oleic acid was studied.
Given quantities of glycerol and oleic acid were mixed together, followed by the addition of immobilized lipase. The three phase system was shaken at various temparatures in an eppendorf tube on a vortex shaker for several days, and the progress of the reaction was followed by the HPLC techique.
In a typical batch experiment 0.5g of oleic acid and 0.0543g of glycerol were mixed with 0.05g of Lipozyme in an eppendorf tube kept at 60 degree C. The amounts of substrates (177m moles of oleic acid and 590 m moles of glycerol) correspond to the exact stoichometric amounts necessary to synthesize 100% of triolein.
Oleic acid consumption starts immediately after the addition of enzyme, being initially incorporated into monolein and diolein. First 80 minutes there is no formation of triolein. Monolein is accumulated for 3 hours upto a max of 4.4% and is then consumed towards a higher level of synthesis to reach 0% level after 8hrs of reaction. During this time, diolein synthesis continues reaching a maximum level of 50% at around 5 hrs. After this the diolein is also consumed towards the higher degree of synthesis, triolein. Diolein decreases to 9% after 48hrs of reaction and remains constant thereafter. Triolein goes up to 86% within 48hrs.
When monolein reaches its maximum, 50% of initial oleic acid is still available, when diolein reaches its maximum 32% of oleic acid is still available. At the end of the reaction when all glyceride levels have stabilized 57% of oleic acid remains unused. This mixture is kept under the same conditions for 600 hrs without significant change in composition. The glyceride synthesis yields obtained here at 60 degree C with an open vial were found to be comparable to results obtained at 34 degree C using molecular sieves to eliminate excess water.
The batch experiment conducted without the removal of water had different results. The reactor in this case was covered at all times to prevent evaporation of water. Accumulation of water slows down the reaction. After 600 hrs of mixing at 60 degree C, equilibrium has not been reached as far as oleic acid and triolein are concerned. In the first 80 minutes, there is no triolein synthesis, while monolein and diolein are being synthesized aupto a maximum of 12% and 25% in 3 hrs. The monolein and dioleins formed are not consumed but remain at these levels, preventing the further synthesis of triolein which reaches 60% after 600 hrs.
Free evaporation of water is effecient in keeping the water content low, which is necessary for the reaction to proceed. The experiments conducted above also show that 0.06% of water in the reaction medium is low enough to allow excellent progress of synthesis reactions, however a reaction medium consisting of 1% of water will seriously inhibit the synthesis of the reaction.
Ratios of 0.23, 0.30, 0.46,0.70, 1, 1.5, 2, 3, 4 of glycerol to oleic acid (ratio of 1 corresponding to the exact stoichometry of the reaction) have been used as reaction media and experiments were carried out in open and closed eppendorf tubes. The effect of the ratio has been studied on the initial rate of the reaction as well as on the endpoint of the same reaction. Equilibrium of the reaction was affected by the initial ratio of glycerol to oleic acid. The main difference is between the amounts of triolein formed as well as a shift in monoloin and diolein synthesis towards a higher glycerol amounts in the case of open tubes.
For open tubes, for ratios in the range of 0.23 to 0.70, the only product of the reaction is triolein in amounts equivalent to the total available glycerol.
Whatever the ratio glycerol/oleic acid is, the reaction is pushed further when water as a product can leave the medium. When working with open tubes, the composition of the medium at equilibrium depends more on the initial ratio of glycerol/oleic acid.By choosing the appropriate amounts of glycerol and oleic acid available at the begining of the reaction
The synthesis of glycerides has been studied at different temparatures like 34, 50, 60, 70 and 80 degree C in open tubes and glycerol and oleic acid were present in stoichiometric amounts.As the temparature is raised, the removal of water is facilitated and the reactions are pushed towards a higher synthesis of triglycerides. The time required to attain the maximal yield of triolein is also shortened as the temparature is increased,until the minimum time required to produce triolein in greater than 86% yield is reduced to 48 hrs or less (60, 70 and 80 degree C). Temparature of 34 degree C is not sufficient to allow for an effecient removal of water.