F. Ergan

Lipase-catalyzed production of wax esters

The lipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) catalyzed synthesis of wax esters has been investigated via two different approaches. All studies were performed using an immobilized 1,3-specific lipase [Lipozyme from Novo Industries (Montréal, Québec, Canada)]. The first approach involves reacting stoichiometric amounts of a fatty acid and stearyl alcohol in the presence of lipase. The medium is solvent-free, which allows for high substrate concentrations (1.55 M) and use of 5% (w/w) Lipozyme. In this reaction, maximum wax ester synthesis was found to be dependent upon the efficient removal of the water produced by the reaction. Under optimal conditions, yields of 100% were routinely reached after only 2 hr. The medium was then exclusively composed of the wax and the enzyme, no purification was required. The second method involves alcoholysis of a triglyceride, in this case triolein, with stearyl alcohol to produce 1,2-diolein, 2-monoolein and the wax ester of oleic acid. Again, no organic solvent was used. The wax ester yield was found to be directly dependent upon the alcohol concentration that was used to modulate the outcome of the reaction towards either the wax or the partial glycerides. The process was applied to the synthesis of waxes from high erucic acid rapeseed oil.

Solvent free triglyceride synthesis using lipozymeTM IM-20

SummaryThe synthesis of triglycerides using LipozymeTM IM-20 (Mucor miehei lipase immobilized on weak anion exchange resins by Novo) is described. Use of pure substrates in stoichiometric amounts in the absence of any organic solvent enables high conversion to be obtained with the addition of molecular sieves to remove water produced by the reaction.

Simple high performance liquid chromatography methods for monitoring lipase reactions

This paper describes three simple high performance liquid chromatography methods to separate mixtures of free fatty acids, mixtures of different triglycerides and mixtures of all fat classes (monoglycerides, diglycerides, triglycerides and free fatty acids). It is possible with our methods to identify and quantify each peak of the chromatogram. These methods have been designed to monitor lipase reactions. Using a first set of conditions, we have been able to separate five fatty acids: linolenic, linoleic, palmitic, oleic and stearic, without any specific preparation of the samples. With a second set of conditions, we showed that the same mobile phase and the same column could separate both triglyceride species and fat classes. However, in the latter case, a flow gradient was used.

Use of lipases in multiphasic systems solely composed of substrates

Three lipase-catalyzed reactions have been investigated in relation to specificity and water dependence. The reactions in question include: the synthetic reaction between oleic acid and glycerol; the enzymatic hydrolysis of triolein; and alcoholysis/glycerolysis transesterification reactions. All reactions were carried out under solventfree conditions. In each case, the medium composition and reaction conditions were optimized in order to work at elevated substrate concentrations and to minimize the production of by-products. Different lipase preparations have been tested in each reaction. In the synthetic reaction, the effective removal of produced water was found to be vital for the production of triolein. With water removal and glycerol amounts not higher than required by the stoichiometry of the reaction, 95% of the available oleic acid was converted to triolein in 48 hr. The production of triolein was also found to be dependent on the availability of the 1,2-diglyceride to react with oleic acid. In the hydrolysis reaction, best conversion yields of triolein towards monoolein, diolein and free fatty acid were obtained when water was considered simply as a substrate of the reaction. In glycerolysis reactions, the reaction of triolein to give monoolein and diolein followed much the same pattern as for hydrolysis, when water was replaced by glycerol. It was shown again that near stoichiometric amounts of substrates led to the best conversion to mono- and diglycerides. A small excess of glycerol was found to be very inhibitory to the reaction. All possible isomers were formed during the reaction. Conversely, in alcoholysis reactions between triolein and stearyl alcohol the specificity of the lipase was upheld. Excess alcohol in this instance was found to be beneficial.

Effect of lipase specificity on triglyceride synthesis

SummaryEnzymatic triglyceride synthesis from free fatty acid and glycerol is shown to be catalysed by both 1,3-specific lipase and non-specific lipase. This paper elucidates the mechanism of the reaction, showing that, with a 1,3-specific lipase, the 1,3-diolein enzymatically formed has to isomerise spontaneously to 1,2-diolein in presence of oleic acid, before being transformed to triolein by the enzyme.

Lipase Specificity against Some Fatty Acids

Lipases hydrolyze triglycerides according to their specificity, which has been defined by Jensen et al. as a comparative difference in rates of catalysis of certain reactions.’ For example, if the substrate contains three different fatty acids, A, B, and C, they can be hydrolyzed at three different rates. If one is able to follow the rate of appearance of A, B, and C, it will be possible to take advantage of this fact to discriminate between the fatty acids. If A is hydrolyzed faster than B, and B is hydrolyzed faster than C, then the product of the hydrolytic reaction will be a mixture of a free fatty acid fraction, mainly A and B with very little C, and a glyceride fraction, enriched in C. One can then separate the two fractions in order to produce the fatty acid C which is of interest. A number of oil crops contain in their triglycerides a fatty acid of interest: crarnbe and high erucic acid rapeseed (HEAR) both contain erucic acid (C22:1), whereas evening primrose oil (EPO) and borage oil each contain y-linolenic acid, or GLA (C18:3). The use of lipases to hydrolyze with discrimination the triglycerides of these oils allows for the separation of the fatty acid of interest from all the other fatty acids contained in the oil.

Enzymatic synthesis of trierucin from high-erucic acid rapeseed oil

The lipase fromCandida rugosa has been shown to discriminate against erucic acid. Advantage of this property has been taken to produce trierucin from high-erucic acid rapeseed (HEAR) oil. A method has been developed for extracting erucic acid from the oil as dierucin and subsequently enzymatically converting it to trierucin. Unrefined HEAR oil was hydrolyzed with lipase fromC. rugosa to produce a mixture of free fatty acids and dierucin. Precipitation and filtration from cold ethanol gave 73% pure dierucin, free of fatty acids. This dierucin was treated in two ways to produce trierucin. First, in the presence of an immobilized lipase and a known amount of water, some trierucin is produced by interesterification. Second, a more efficient route to trierucin utilizedRhizopus arrhizus lipase to completely hydrolyze dierucin to erucic acid, which was then combined with an appropriate amount of dierucin in the presence of an immobilized lipase to produce trierucin in a quantitative yield.

Enzymatic Triglyceride Synthesis with Total Consumption of Concentrated Substrates

The goal of this work was to find the right conditions to completely reverse hydrolysis of triglycerides using the lipase from Mucor miehei (Lipozyme from Novo). A model system, the synthesis of triolein from oleic acid and glycerol (TABLE l ) , was used. The role of water in this reaction was examined. The overall goal of this research was to gain expertise and know-how in the synthesis and modification of fats using enzymes, as well as developing appropriate technologies for the synthesis and transformations of lipids, glycerides, and other esters. A possible application of this work would be the processing of an oil with a lipase in low-water media. This allows for the reesterification of partial glycerides with free fatty acids, thus reducing the acidity of the oil. The characteristics that we wanted to achieve are as follows: no organic solvent should be used; the work should be performed at high substrate concentrations; and complete conversions have to be achieved. The medium will then be solely composed of the necessary substrates and the reaction will be performed with a lipase, that is to say, the medium will be triphasic. There is no example in the literature’-* of the synthesis of triglycerides in these conditions and giving the results presented here.

Lipase Catalyzed Triglyceride Synthesis. The Role of Isomerization

The synthesis of triolein from oleic acid and glycerol catalyzed by an immobilized Mucor miehei lipase has been studied. The equilibrium constants for the synthesis of mono-, di- and triglycerides were determined. The rate of isomerization of 1,3-diolein and its dependence on oleic acid concentration has been measured. A kinetic study of the synthesis of triolein from 1,3-diolein and oleic acid showed that the isomerization to 1,2-diolein is the rate limiting step.