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Evaluation of Vernonia galamensis lipase (acetone powder) for use in biotechnology
INDUSTRIALCROPS ANDPRODUCTS AN INTERNATIONAL
ELSEVIER
JOURNAL
Industrial Crops and Products 3 (1995) 2X5-292
Evaluation of Emonia galamensis lipase (acetone powder) for use in biotechnology Ignatious Ncube, John S. Read * Department of Biochemistv, Universily of Zimbabwe, PO. Box MP 167, Harare, Zimbabwe
Received 14 September 1994; accepted 26 January 1995
Abstract An acetone powder was prepared from Vemonia ga[amensis seed. This acetone powder was used as a source of crude immobilised lipase to characterise the Vemonia lipase for its potential use in biotechnological processes. The lipase shows no fatty acid specificity in hydrolysis of coconut and soya bean oils. Short chain triglycerides were the preferred substrates in transesterification reactions. In both transesterification and hydrolysis of 1,3-dipalmytoyl-2oleylglycerol the lipase shows selectivity for the l,(3) position of triglycerides. The acetone powder catalysed the hydrolysis of triglycerides in 2,2,4- trimethylpentane (TMP) and was evaluated for use in the continuous production of poly-unsaturated fatty acids from soya bean oil in a packed bed reactor. Complete hydrolysis of 2.5% w/v soya bean oil dissolved in TMP was achieved within 3 h of introduction of the oil solution and the hydrolysis decreased with time to between 60% and 80% depending on temperature conditions. The lipase activity found in the ungerminated seed and the characteristics that the lipase shows make Vemonia galamensis an attractive oilseed crop not only as an industrial oil source but also as a source of cheap lipase. Keywords: Lipase; timonia galamensis; Acetone powder; Transesterification;
1. Introduction Lipases (E.C.3.1.1.3) are enzymes that hydrolyze triglycerides to fatty acids, glycerides and glycerol and are active at oil-water interfaces. Lipases can show different specificities for different types of substrates, the position of the fatty acid esterified to glycerol, different fatty acids and different stereo isomers (Jensen et al., 1983, 1990). Although in aqueous media the reaction catalysed by lipases is the hydrolysis of triglycerides, the recently developed field of biotechnology which involves the use of enzymes in organic media * Corresponding author. Fax: +263 4 333-407.
Hydrolysis; Ttiglycerides; Fatty acids
has opened up various applications for lipases as biocatalysts in synthetic reactions (Mukhejee, 1990). In biotechnology the areas of application that have been investigated for lipases include ester synthesis (Bloomer et al., 1992), triglyceride modification (Macrae, 1983; Bloomer et al., 1990), phospholipid modification (Svensson et al., 1992), monoglyceride production and fatty acid production through triglyceride hydrolysis (Bell et al., 1981; Millqvist et al., 1994). Free fatty acids are widely used in industry but are only found in small quantities in nature. Most of the fatty acids used in industry are obtained by the hydrolysis of plant oils or animal fat. In industry the hydrolysis of
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triglycerides, or fat splitting, is carried out using the high-temperature steam treatment (Sontag, 1989). Besides being energy consuming the process is not suitable for poly-unsaturated fatty acids and substituted fatty acids which undergo degradation. An example is ricinoleic acid, a hydroxylated mono-unsaturated 18-carbon fatty acid found in the triglycerides of castor oil; another is the epoxidised fatty acid, vernolic acid, found in the seeds under study here. Ricinoleic acid which has a variety of applications in industry, has been found to form polymeric acids when castor oil is subjected to this process of hydrolysis (Lakshminarayana et al., 1984). For such oils cleavage at mild temperatures is necessary and enzyme-catalysed cleavage has been shown to be a promising method (Bell et al., 1981; Khor et al., 1986). One of the methods that is being developed for enzymatic hydrolysis of triglycerides is that of the use of hollow fibre membrane reactor (Malcata et al., 1990; Derksen et al., 1993). The efficiency of the method is highly dependent on a balance of pressure that is applied on the oil phase, the surfactant activity of the released fatty acids (Vaidya et al., 1994), and the integrity of the permeable membrane. High pressures can damage the membrane. Some commercial lipase preparations have been found to contain cellulases which alter the permeabilities of the cellulose-membranes (Derksen et al., 1992). Therefore if alternative inexpensive enzymatic methods of fat splitting are devised they could be more attractive. Plant lipases, despite their high catalytic activities and specificities, have not been widely investigated for use in the oil industry. This may be as a result of the absence of lipase activity in most ungerminated oilseeds and thus presenting a problem of the need to germinate the seed (Ncube et al., 1993), thereby compromising the economic value of the seed in the vegetable oil industry. Emonia galamensis has potential as an industrial oilseed crop because of the unique nature of its seed oil. The seed contains about 40% of its weight as oil and about 75% of the fatty acids are vemolic acid (cis-12,13-epoxyoleic acid). This unique low-viscosity oil has potential in the plastic and paint industry (Elmore et al., 1993).
Unlike most other oilseeds, such as rape seed and mustard seed (Huang, 1984), Wmonia seeds have lipase activity in the resting state. This is shown by the autolysis of the triglycerides in ground seeds (Afolabi et al., 1991). Lipid-free Vemonia seed meal is a by-product of the oil extraction, and at present research is continuing in trying to find some use for the lipid-free cake left after extraction of the oil. One of the areas of this research has been focused on the use of the seed meal as livestock feed (Ologunde et al., 1990; Afolabi et al., 1991). The presence of a lipase in Emonia seed has created an interest in our laboratory to investigate the possible use of a crude preparation of the lipase as a catalyst in biotechnological processes. The main advantage of using enzymes as catalysts in synthetic reactions that can otherwise be achieved by using conventional methods, is that the enzyme-catalysed reactions occur at mild temperatures; therefore, less degradation of substrates and products takes place and enzymes are thus very specific compared to conventional methods of organic synthesis (Sonnet, 1988). The potential for different specificities of lipases makes it necessary to determine the specificity of a lipase before it can be of application in industrial processes. In the initial investigations of the K+monia acetone powder for fatty acid analysis it was not shown whether the lipase had any specificity (Afolabi et al., 1991). An investigation on the possible use of the lipid-free ground Kmonia seed as a catalyst in the release of poly-unsaturated fatty acids from triglycerides of vegetable oil and a study on the specificity of the lipase in this preparation is presented here. 2. Materials timonia galamensis seed was a gift from Chiredzi Agricultural Research Station, Chiredzi, Zimbabwe. The standard triglycerides and fatty acids, soya bean and coconut oil were obtained from Sigma Chemical Co., USA. Palm oil mid fraction was a gift from Prof. Bo Mattiasson who received it from Karlshamns AB, Karlshamn, Sweden.
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Crops and Products 3 (1995) 285-292
3. Methods
281
n-hexane. The reactions were done in capped vials in a shaking water bath at a temperature of 40°C.
3.1. Preparation of the acetone powder
The acetone powder was prepared by blending 100 g of Wrnonia seed in cold acetone (-20°C) using a Waring blender. The fine-ground seed particles, which sedimented slowly, were collected by vacuum filtration. The resulting filter cake of these particles was washed several times with acetone until the washings were colourless. This fat-free resulting powder was used as a crude lipase preparation. 3.2. Determination of fatty acid specificity in the hydrolysis of vegetable oils (a) In oil emulsions. Soya bean and coconut oil emulsions were prepared by vortexing 100 mg of oil, 50 mg of gum arabic, 100 ~1 of 1% deoxycholate and 0.9 ml of 100 mM NaCl. The reaction was started by the addition of 200 mg of the acetone powder. The reaction was carried out in a water bath at 30°C. Samples of 200 ~1 were withdrawn, acidified with 50 ~1 of concentrated HCl and extracted with 400 ~1 of chloroform/methanol (3 : 1). The fatty acids were separated from the lipids extracted into the organic phase by spotting 200 ~1 of the organic phase onto silica gel-G TLC plates. The free fatty acid spots were scraped off and converted to methyl esters and were analysed by gas chromatography (see below). (b) In 2,2,4_trimethylpentane (TMP). Hydrolysis in TMP was done by dissolving the oils in TMP to a concentration of 10% w/v and the reaction started by the addition of 200 mg of the acetone powder to 1 ml of oil solution. The reaction mixture was incubated in a water bath at 30°C and 100 ,~l samples were withdrawn and spotted on silica gel-G TLC plates for TLC separation of the lipids. The free fatty acids were converted to methyl esters and analysed by gas chromatography.
3.3. Transesterification reactions In transesterification reactions the reaction mixture consisted of 30% w/v enzyme preparation, 0.5 M triglyceride and 1.8 M lauric acid in
3.4. Positional selectivity of the lipase The positional selectivity of the lipase was investigated for both triglyceride hydrolysis and triglyceride transesterification reactions. In hydrolysis reactions the substrate was 10% of either emulsified oil or oil dissolved in TMI? In the transesterification reaction 1,3-dipalmitoyl 2oleylglycerol (POP) in palm oil mid fraction was transesterified with lauric acid. The reaction was done at 40°C and the reaction mixture was 0.5 M in POP and 1.8 M lauric acid in n-hexane with 0.4 g/ml of the acetone powder. 3.5. Continuous hydrolysis of soya bean oil in a packed bed reactor A packed bed reactor system was set up. The reactor was a jacketed glass column packed with 4 g of the acetone powder. Soya bean oil was dissolved in water-saturated TMP, to a concentration of 2.5% w/v. This solution was introduced into the column from the top by use of a peristaltic pump connected to the bottom of the column and maintained at a flow rate of 4 ml/h. Samples were taken from the effluent at various time intervals and analysed by TLC. 3.6. Analytical methods 3.6.1. TLC analysis and quantification of lipids
Lipid samples to be analysed by thin-layer chromatography were spotted onto silica gel-G plates and the plates developed using a solvent system of petroleum ether(60-80”C)/diethyl ether/acetic acid (80 : 20 : 1). The lipid fractions were visualised either by spraying with 0.1% of 1,7 dichlorofluorescein in methanol and viewing under UV light when the lipid fractions were to be analysed by GC, or by spraying with phosphomolybdic acid followed by heating at 110°C when the fractions were to be quantified calorimetrically. In the colorimetric quantification the separated lipid fractions were scraped off the plate and dissolved in 50% ethanol. The absorbance of the dissolved
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lipid-molybdenum complex was measured at 730 nm (Ncube et al., 1994). 3.6.2. Gas chromatography Fatty acid methyl esters were prepared by adding 1 ml of 4% &So4 to the fatty acid sample and heating at 70°C for 30 min. The methyl esters formed were extracted by addition of 0.5 ml of nhexane and washing with 3 ml of 5% bicarbonate solution. The hexane layer was analysed by GC. Fatty acids esterified to glycerol were converted to methyl esters by reacting about 20 mg of oil or triglyceride with 3 ml of 0.5 M sodium methoxide in methanoVdiethy1 ether (1: 1) and heating at 45°C for 5 min. The methyl esters were extracted by addition of 4 ml saturated NaCl followed by 250 ~1 of n-hexane. The organic layer was extracted for GC analysis. Gas chromatography was performed using a Shimadzu 4MPF gas chromatograph equipped with a 15% DEGS column and a FID detector. The peak areas were obtained using a Shimadzu CR4-A integrator.
C16:O
C16:0
C16:1
C16:2
C16:3
Fatty acid
4. Results and discussion Extraction of the ground Vemonia galamensis seed with acetone and collection of the filter cake by filtration resulted in a fine grey powder. This acetone powder catalysed the breakdown of triglycerides into fatty acids and glycerides as shown by TIC and GC analysis and also catalysed transesterification reactions. The acetone powder is stable for several months if stored at 4°C. A similar crude preparation of the lipase has been used in the fatty acid analysis of seed oils (Afolabi et al., 1991). 4.1. Fattyacid specificity The fatty acid content of both coconut oil and soya bean oil was determined by converting the fatty acids in the triglycerides of the oils to methyl esters and analysing the methyl esters by gas chromatography. The soya bean oil used consists mainly of unsaturated l&carbon fatty acids whereas the coconut oil used consists mainly of saturated 8- to l&carbon fatty acids. After about
c6:o
c1o:o
c12:o
c14:o
C16:O
C16:O
C16:1
Fatty acid
Fig. 1. Fatty acid content of (a) soya bean oil and (b) coconut oil and the nature of the fatty acids that are produced during hydrolysis of the oil by the I/emonia lipase. ‘Nglyceride fatty acids (left-hand columns). Released fatty acids (right-hand columns).
5% hydrolysis in both oil emulsions and watersaturated TMP there was no significant difference in the fatty acid constitution of the oil and the fatty acid constitution of the free fatty acids released during hydrolysis of the triglyceride (Fig. 1). The release of a certain fatty acid or a group of closely related fatty acids from an oil by a lipase indicates fatty acid specificity of the lipase (Jensen et al., 1983) and our results show that this Kwzonia lipase preparation has no preference for or discrimination against any of the fatty acids that were in the two different vegetable oils.
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4.2. Substrate specificity in transesterification reactions The rate of incorporation of lauric acid into triglycerides with fatty acid chain lengths varying from 4 carbon atoms to 18 carbon atoms was investigated, Short chain fatty acid triglycerides were found to be the preferred substrates in this reaction system (Table 1). This substrate specificity can be explained by considering the micro-structure of a biocatalytic system in organic media. When enzymes are in non-polar solvents like hexane, the enzyme is surrounded by a layer of water and the lipase-catalysed reaction takes place at the watersolvent interface. Short fatty acid chain triglycerides are more polar than long chain triglycerides and thus partition into the aqueous phase more than the less polar long chain triglycerides. This could explain the faster conversion of short chain triglycerides. Such a type of specificity is controlled by the nature of the substrate and not the structure of the enzyme. Although it may be possible that the small triglycerides are the preferred substrates, this is unlikely considering the presence of only long chain fatty acids in the physiological substrate of the lipase, the Vernonia seed oil. A system like this can be used to increase the long chain fatty acid content of an oil, as the triglycerides rich in short chain fatty acids would be transesterified faster. This would however be a rare application due to the infrequent occurrence of fats or oils with short chain fatty acids. An ex-
Table 1 Vernonia lipase-catalysed transesterification of triglycerides ‘Ikiglyceride
Relative transesterification
C4:o c4:oc4:0 C6:OC6:OC6:0 c!8:0C8:0C8:0 c10:0c10:0c10:0 c11:0c11:0c11:0 c14:o c14:o C16:OC16:0 C18:OC18:0 C18:l C18:l
100 75 63 37 16 18 14 14 18
c14:o C16:O C18:O C18:l
The transesterification was carried out with lauric acid in n-hexane. The triglyceride with the highest rate of transesterification was given a relative activity of 100.
289
ample of an attractive application would be the production of short chain fatty acid esters from butter fat using alcoholysis reactions. Butter fat is rich in short chain fatty acids and could be used in synthesis of various flavour compounds through alcoholysis with various alcohols. The rates of interesterification of milk fat using lipases that have preference for long chain esters may not be as fast as the rates for long chain fatty acid containing oils of fats (Reyes and Hill, 1994). 4.3. Positional specificity When triolein was hydrolysed for 5 min in emulsions and in TMP and the lipid constitution determined by TIC on silica gel plates impregnated with boric acid and visualised using PMA, and then quantified calorimetrically, monoand diglycerides were found to accumulate in the reaction mixture. Of the diglycerides 80% was 1,Zdiolein and 20% 1,3-diolein. The presence of 1,Zdiolein in greater amounts in the initial stages of hydrolysis shows that the lipase has some selectivity for the l,(3) position of the triglyceride. To further investigate the positional selectivity of the lipase, hydrolysis of palm oil mid fraction was studied. Palm oil mid fraction is a fraction of palm oil which consists mainly of 1,3-dipalmitoyl 2-oleylglycerol (POP). The oil used in our experiment had up to 90% of triglycerides as POP The amount of palmitate and oleate produced by the hydrolysis of POP in TMP, and in emulsions, by Emonia lipase was monitored. In both systems palmitate and oleate were released although the palmitate consisted of above 75% of the fatty acids (Fig. 2). When the monoglycerides were converted to methyl ester and analysed by GC palmitate could not be detected. These results indicate that the Vemonia lipase possibly acts on both the primary and secondary positions of the triglyceride but has high selectivity for the primary positions. The other possibility is that the presence of oleate in the fatty acids and 1,3-diglycerides is due to acyl migration of the oleate from the 2-position to the l,(3)-position resulting in the formation of 1,3-diglycerides and lipolysis of the isomerised diglyceride yielding oleate. The l,(3)-position of the triglyceride is the preferred position for most
I. Ncube, J.S. Read I Industrial Crops and Products 3 (1995) 285-292
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tions of the triglyceride. The ratio of palmitic acid to oleic acid decreased (Fig. 3) and though not shown, the amount of palmitic acid decreased as the amount of lauric acid incorporated increased.
5.0
b) POP in emulsion 4.5
4.4. Continuous hydrolysis of soya bean oil in a packed bed reactor
0.0
5.0
10.0
15.0
20.0 time
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
(mins)
25.0
30.0
35.0
40.0
time (mins)
Fig. 2. Positional specificity of the Vemonia lipase: (a) POP dissolved in TMP; (b) emulsified POP. The lipids were separated by TLC and the fatty acids converted to methyl esters and quantified by gas chromatography. Heptadecanoic acid was used as an internal standard. Palmitic acid (o), oleic acid (0).
lipases as these are the less sterically hindered positions of the triglyceride. One of the major applications of positional selective or specific lipases is in the interesterification of triglycerides and phospholipids to produce tailor-made triglycerides or phospholipids (Macrae, 1983; Bloomer et al., 1990). Transesterification of palm oil mid fraction with lauric acid revealed that the lauric acid was being incorporated in the primary posi-
When 4 g of the acetone powder was tightly packed into a column of an internal diameter of 1 cm, the bed height came to about 20 cm. For about 3 h of passing 2.5% soya bean oil solution there was complete hydrolysis of the triglycerides of the oil. Oil concentrations higher than 2.5% caused accumulation of diglycerides in the product and flow rates higher than 4 ml/h reduced the conversions. After 3 h, a decrease in the percentage hydrolysis was observed to about 40% at 2O”C, 60% at 30°C and 80% at 40°C (Fig. 4). In organic solvents the catalytic activity of enzymes is dependent on the water content of the reaction system; an essential amount of water is needed for the flexibility of the enzyme molecule so that it can attain its catalytic conformation. In a hydrolysis system, such as the one that is presented here, water is also a substrate. Since water is available in limiting amounts, as hydrolysis proceeds the reaction system is likely to dry up due to the use of water (Macrae, 1983,1985) and thus the hydrolytic activity of the lipase is reduced. The solvent used here was saturated with water but the solubility of water in these non-polar solvents is very low and is likely to be near limiting amounts for hydrolysis. However, as the percentage hydrolysis increases with temperature, it appears that a limiting factor could be the capacity of the system used in these experiments. Once the stable hydrolysis rate had been achieved the percentage hydrolysis remained stable for about three weeks at 40°C. The main advantage of using this preparation is that the acetone powder acts as an immobilised lipase and can be readily used as a solid catalyst in a packed bed reactor. While a packed bed reactor system has the disadvantage of mass transfer limitations compared to, for example, a stirred tank reactor (CSTR), this acetone powder would provide a cheap alternative with minimised downstream processing under continual operation. Moreover,
I. Ncube, J.S. Read IIndustrial Crops and Products 3 (1995) 285-292
I 100
1.6 f 0
I 200 time
291
I 400
I 300
(mitts)
Fig. 3. Change in the ratio of palmitate to oleate during the Vemonia lipase catalysed transesterification with lauric acid.
of palm oil mid fraction
60-
2oI 0
10
20
30
40
50
60
time (hrs)
Fig. 4. Effect of temperature acetone-extracted
on the Vemonia lipase catalysed hydrolysis of soya bean oil in a fixed bed reactor
of the
acetone powder. 20°C (O), 30°C (A), 40°C (0).
the granular nature of the powder offers very little restrictions to fluid flow. The material prepared here, if considered as a waste product of oil extraction, reduces the cost of the immobilised lipase to insignificant amounts. The use of lipases in organic solvents to synthesise organic compounds is a promising route of obtaining products of high purity in the chemi-
cal and pharmaceutical industry and products that are more “natural” and more acceptable to the consumer in the food industry. In these areas of research, plant lipases such as the Vemonia lipase should not be overlooked as they may possess some interesting properties and further investigations are proceeding on the purified lipase. Acetone powders like the one prepared here, have
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a good operational stability and may be a good source of cheap and immobilised lipase capable of carrying out a number of lipase-catalysed transesterification and hydrolysis reactions. The ability of the lipase to hydrolyse lipids with high conversions in organic media, a monophasic system, is particularly attractive and may be a possible alternative to harsh chemical methods and the expensive hollow fibre membrane reactors. Acknowledgements The authors thank the Research Board of the University of Zimbabwe and the Swedish Agency for Research Cooperation with Developing Countries (SAREC) for providing the financial support for the research project, and Bo Mattiasson and Patrick Adlercreutz for technical discussions. References Afolabi, O.A., Ologunde, M.O., Anderson, W.A., Read, J.S., Dacosta, M.D., Epps, EA. and Ayorinde, F.O., 1991. Use of lipase (Acetone powder) from Vemonia galamensis in fatty acid analysis of seed oils. J. Chem. Tech. Biotechnol., 51: 41-46. Bell, G., Todd, J., Blain, J., Patterson, J. and Shaw, C., 1981. Hydrolysis of triglycerides by solid phase lipolytic enzymes of Rhizopus arhizus in continuous reactor systems. Biotechnol. Bioeng., 23: 1703-1719. Bloomer, S., Adlercreutz, P. and Mattiasson, B., 1990. Cocoa butter equivalents from a fraction of palm oil. J. Am. Oil Chem. Sot., 67: 519-524. Bloomer, S., Adlercreutz, P and Mattiasson, B., 1992. Facile synthesis of fatty acid esters in high yields. Enzyme Micrab. Technol., 14: 546-552. Derksen, J.T.P., Boswinkel, G. and Cuperus, F.P., 1992. Cellulase activity in commercial lipase preparations. Biotechnol. Bioeng., 40: 858-860. Derksen, J.TP., Muuse, B.G., Cuperus, EP. and van Gelder, W.M.J., 1993. New seed oils for the oleochemical industry: evaluation and enzyme bioreactor mediated processing. Ind. Crops Prod., 1: 133-139. Elmore, J.D., DeGooyer, W.J., Tipton, M.B. and Kaiser, J.H., 1993. U.S. Patent No. 5227453. Huang, A.H.C., 1984. Plant lipases. In: B. Borgstrom and H.L. Brockman (Editors), Lipases. Elsevier Science Publishers, Amsterdam, pp. 420-441.
Jensen, R.G., Dejong, EA. and Clark, R.M., 1983. Determination of lipase specificity. Lipids, 18: 239-252. Jensen, R.G., Galluzo, R.D. and Bush, V.J., 1990. Selectivity is an important characteristic of lipases (acylglycerol hydrolases). Biocatalysis, 3: 307-316. Khor, H., ‘Ban, N. and Chua, C., 1986. Lipase catalysed hydrolysis of palm oil. J. Am. Oil Chem. Sot., 63: 538-540. Lakshminarayana, G., Subbarao, R., Sastry, Y.S.K., Rao, ‘PC., Kale, V and Vijayalakshmi, P., 1984. High pressure splitting of castor oil. J. Am. Oil Chem. Sot., 61: 1204-1206. Macrae, A.R., 1983. Lipase catalysed interesterification of fats and oils. J. Am. Oil Chem. Sot., 60: 243A-246A. Macrae, A.R., 1985. Interesterification of fats and oils. In: J. ‘Barnper, H.C. van der Plas and P. Linko (Editors), Biocatalysts in Organic Synthesis. Proc. Int. Symp., Elsevier Science., Amsterdam, pp. 195-208. Malcata, F.X., Reyes, H.R., Garcia, H.S., Hill, C.G. and Amundson, C.H., 1990. Immobilised lipase reactors for modification of fats and oils. J. Am. Oil Chem. Sot., 67: 890-910. Millqvist, A., Adlercreutz, P and Mattiasson, B., 1994. Lipasecatalysed alcoholysis of triglycerides for the preparation of 2-monoglycerides. Enzyme Microb. Bxhnol., 16: 10421047. Mukherjee, K.D., 1990. Lipase catalysed reactions for the modifications of fats and other lipids. Biocatalysis, 3: 277293. Ncube, I., Adlercreutz, I?, Read, J. and Mattiasson, B., 1993. Purification of rape (Bra&a napus) seedling lipase and its use in organic media. Biotechnol. Appl. Biochem., 17: 327-336. Ncube, I., Chikunguwo, S. and Read, J.S., 1994. An economic calorimetric method for the quantification of total lipid and assaying for lipase activity (unpubl. results). Ologunde, M.O., Ayorinde, F.O. and Shepard, R.L., 1990. Chemical evaluation of defatted Vemonia galamensismeal. J. Am. Oil Chem. Sot., 67: 92-95. Reyes, H.R. and Hill, C.G., 1994. Kinetic modeling of interesterification reactions catalysed by immobilised lipase. Biotech. Bioeng., 43: 171-182. Sonnet, PE., 1988. Lipase selectivities. J. Am. Oil Chem. Sot., 65: 900-904. Sontag, N.O.V., 1989. Fat splitting and glycerol recovery. In: R.W. Johnson and E. Fritz (Editors), Fatty Acids in Industry. Marcel Dekker, Inc., New York, pp. 23-72. Svensson, I., Adlercreutz, P. and Mattiasson B., 1992. Lipasecatalysed transesterification of phosphatidylcholine at controlled water activity. J. Am. Oil Chem. Sot., 69: 986991. Vaidya, A.M., Halling, P.J. and Bell, G., 1994. Surfactantinduced breakthrough effects during the operation of twophase biocatalytic membrane reactors. Biotechnol. Bioeng., 44: 765-771.
ELSEVIER
JOURNAL
Industrial Crops and Products 3 (1995) 2X5-292
Evaluation of Emonia galamensis lipase (acetone powder) for use in biotechnology Ignatious Ncube, John S. Read * Department of Biochemistv, Universily of Zimbabwe, PO. Box MP 167, Harare, Zimbabwe
Received 14 September 1994; accepted 26 January 1995
Abstract An acetone powder was prepared from Vemonia ga[amensis seed. This acetone powder was used as a source of crude immobilised lipase to characterise the Vemonia lipase for its potential use in biotechnological processes. The lipase shows no fatty acid specificity in hydrolysis of coconut and soya bean oils. Short chain triglycerides were the preferred substrates in transesterification reactions. In both transesterification and hydrolysis of 1,3-dipalmytoyl-2oleylglycerol the lipase shows selectivity for the l,(3) position of triglycerides. The acetone powder catalysed the hydrolysis of triglycerides in 2,2,4- trimethylpentane (TMP) and was evaluated for use in the continuous production of poly-unsaturated fatty acids from soya bean oil in a packed bed reactor. Complete hydrolysis of 2.5% w/v soya bean oil dissolved in TMP was achieved within 3 h of introduction of the oil solution and the hydrolysis decreased with time to between 60% and 80% depending on temperature conditions. The lipase activity found in the ungerminated seed and the characteristics that the lipase shows make Vemonia galamensis an attractive oilseed crop not only as an industrial oil source but also as a source of cheap lipase. Keywords: Lipase; timonia galamensis; Acetone powder; Transesterification;
1. Introduction Lipases (E.C.3.1.1.3) are enzymes that hydrolyze triglycerides to fatty acids, glycerides and glycerol and are active at oil-water interfaces. Lipases can show different specificities for different types of substrates, the position of the fatty acid esterified to glycerol, different fatty acids and different stereo isomers (Jensen et al., 1983, 1990). Although in aqueous media the reaction catalysed by lipases is the hydrolysis of triglycerides, the recently developed field of biotechnology which involves the use of enzymes in organic media * Corresponding author. Fax: +263 4 333-407.
Hydrolysis; Ttiglycerides; Fatty acids
has opened up various applications for lipases as biocatalysts in synthetic reactions (Mukhejee, 1990). In biotechnology the areas of application that have been investigated for lipases include ester synthesis (Bloomer et al., 1992), triglyceride modification (Macrae, 1983; Bloomer et al., 1990), phospholipid modification (Svensson et al., 1992), monoglyceride production and fatty acid production through triglyceride hydrolysis (Bell et al., 1981; Millqvist et al., 1994). Free fatty acids are widely used in industry but are only found in small quantities in nature. Most of the fatty acids used in industry are obtained by the hydrolysis of plant oils or animal fat. In industry the hydrolysis of
0926-6690/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved. SSDI 0926-6690(95)00013-5
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triglycerides, or fat splitting, is carried out using the high-temperature steam treatment (Sontag, 1989). Besides being energy consuming the process is not suitable for poly-unsaturated fatty acids and substituted fatty acids which undergo degradation. An example is ricinoleic acid, a hydroxylated mono-unsaturated 18-carbon fatty acid found in the triglycerides of castor oil; another is the epoxidised fatty acid, vernolic acid, found in the seeds under study here. Ricinoleic acid which has a variety of applications in industry, has been found to form polymeric acids when castor oil is subjected to this process of hydrolysis (Lakshminarayana et al., 1984). For such oils cleavage at mild temperatures is necessary and enzyme-catalysed cleavage has been shown to be a promising method (Bell et al., 1981; Khor et al., 1986). One of the methods that is being developed for enzymatic hydrolysis of triglycerides is that of the use of hollow fibre membrane reactor (Malcata et al., 1990; Derksen et al., 1993). The efficiency of the method is highly dependent on a balance of pressure that is applied on the oil phase, the surfactant activity of the released fatty acids (Vaidya et al., 1994), and the integrity of the permeable membrane. High pressures can damage the membrane. Some commercial lipase preparations have been found to contain cellulases which alter the permeabilities of the cellulose-membranes (Derksen et al., 1992). Therefore if alternative inexpensive enzymatic methods of fat splitting are devised they could be more attractive. Plant lipases, despite their high catalytic activities and specificities, have not been widely investigated for use in the oil industry. This may be as a result of the absence of lipase activity in most ungerminated oilseeds and thus presenting a problem of the need to germinate the seed (Ncube et al., 1993), thereby compromising the economic value of the seed in the vegetable oil industry. Emonia galamensis has potential as an industrial oilseed crop because of the unique nature of its seed oil. The seed contains about 40% of its weight as oil and about 75% of the fatty acids are vemolic acid (cis-12,13-epoxyoleic acid). This unique low-viscosity oil has potential in the plastic and paint industry (Elmore et al., 1993).
Unlike most other oilseeds, such as rape seed and mustard seed (Huang, 1984), Wmonia seeds have lipase activity in the resting state. This is shown by the autolysis of the triglycerides in ground seeds (Afolabi et al., 1991). Lipid-free Vemonia seed meal is a by-product of the oil extraction, and at present research is continuing in trying to find some use for the lipid-free cake left after extraction of the oil. One of the areas of this research has been focused on the use of the seed meal as livestock feed (Ologunde et al., 1990; Afolabi et al., 1991). The presence of a lipase in Emonia seed has created an interest in our laboratory to investigate the possible use of a crude preparation of the lipase as a catalyst in biotechnological processes. The main advantage of using enzymes as catalysts in synthetic reactions that can otherwise be achieved by using conventional methods, is that the enzyme-catalysed reactions occur at mild temperatures; therefore, less degradation of substrates and products takes place and enzymes are thus very specific compared to conventional methods of organic synthesis (Sonnet, 1988). The potential for different specificities of lipases makes it necessary to determine the specificity of a lipase before it can be of application in industrial processes. In the initial investigations of the K+monia acetone powder for fatty acid analysis it was not shown whether the lipase had any specificity (Afolabi et al., 1991). An investigation on the possible use of the lipid-free ground Kmonia seed as a catalyst in the release of poly-unsaturated fatty acids from triglycerides of vegetable oil and a study on the specificity of the lipase in this preparation is presented here. 2. Materials timonia galamensis seed was a gift from Chiredzi Agricultural Research Station, Chiredzi, Zimbabwe. The standard triglycerides and fatty acids, soya bean and coconut oil were obtained from Sigma Chemical Co., USA. Palm oil mid fraction was a gift from Prof. Bo Mattiasson who received it from Karlshamns AB, Karlshamn, Sweden.
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3. Methods
281
n-hexane. The reactions were done in capped vials in a shaking water bath at a temperature of 40°C.
3.1. Preparation of the acetone powder
The acetone powder was prepared by blending 100 g of Wrnonia seed in cold acetone (-20°C) using a Waring blender. The fine-ground seed particles, which sedimented slowly, were collected by vacuum filtration. The resulting filter cake of these particles was washed several times with acetone until the washings were colourless. This fat-free resulting powder was used as a crude lipase preparation. 3.2. Determination of fatty acid specificity in the hydrolysis of vegetable oils (a) In oil emulsions. Soya bean and coconut oil emulsions were prepared by vortexing 100 mg of oil, 50 mg of gum arabic, 100 ~1 of 1% deoxycholate and 0.9 ml of 100 mM NaCl. The reaction was started by the addition of 200 mg of the acetone powder. The reaction was carried out in a water bath at 30°C. Samples of 200 ~1 were withdrawn, acidified with 50 ~1 of concentrated HCl and extracted with 400 ~1 of chloroform/methanol (3 : 1). The fatty acids were separated from the lipids extracted into the organic phase by spotting 200 ~1 of the organic phase onto silica gel-G TLC plates. The free fatty acid spots were scraped off and converted to methyl esters and were analysed by gas chromatography (see below). (b) In 2,2,4_trimethylpentane (TMP). Hydrolysis in TMP was done by dissolving the oils in TMP to a concentration of 10% w/v and the reaction started by the addition of 200 mg of the acetone powder to 1 ml of oil solution. The reaction mixture was incubated in a water bath at 30°C and 100 ,~l samples were withdrawn and spotted on silica gel-G TLC plates for TLC separation of the lipids. The free fatty acids were converted to methyl esters and analysed by gas chromatography.
3.3. Transesterification reactions In transesterification reactions the reaction mixture consisted of 30% w/v enzyme preparation, 0.5 M triglyceride and 1.8 M lauric acid in
3.4. Positional selectivity of the lipase The positional selectivity of the lipase was investigated for both triglyceride hydrolysis and triglyceride transesterification reactions. In hydrolysis reactions the substrate was 10% of either emulsified oil or oil dissolved in TMI? In the transesterification reaction 1,3-dipalmitoyl 2oleylglycerol (POP) in palm oil mid fraction was transesterified with lauric acid. The reaction was done at 40°C and the reaction mixture was 0.5 M in POP and 1.8 M lauric acid in n-hexane with 0.4 g/ml of the acetone powder. 3.5. Continuous hydrolysis of soya bean oil in a packed bed reactor A packed bed reactor system was set up. The reactor was a jacketed glass column packed with 4 g of the acetone powder. Soya bean oil was dissolved in water-saturated TMP, to a concentration of 2.5% w/v. This solution was introduced into the column from the top by use of a peristaltic pump connected to the bottom of the column and maintained at a flow rate of 4 ml/h. Samples were taken from the effluent at various time intervals and analysed by TLC. 3.6. Analytical methods 3.6.1. TLC analysis and quantification of lipids
Lipid samples to be analysed by thin-layer chromatography were spotted onto silica gel-G plates and the plates developed using a solvent system of petroleum ether(60-80”C)/diethyl ether/acetic acid (80 : 20 : 1). The lipid fractions were visualised either by spraying with 0.1% of 1,7 dichlorofluorescein in methanol and viewing under UV light when the lipid fractions were to be analysed by GC, or by spraying with phosphomolybdic acid followed by heating at 110°C when the fractions were to be quantified calorimetrically. In the colorimetric quantification the separated lipid fractions were scraped off the plate and dissolved in 50% ethanol. The absorbance of the dissolved
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lipid-molybdenum complex was measured at 730 nm (Ncube et al., 1994). 3.6.2. Gas chromatography Fatty acid methyl esters were prepared by adding 1 ml of 4% &So4 to the fatty acid sample and heating at 70°C for 30 min. The methyl esters formed were extracted by addition of 0.5 ml of nhexane and washing with 3 ml of 5% bicarbonate solution. The hexane layer was analysed by GC. Fatty acids esterified to glycerol were converted to methyl esters by reacting about 20 mg of oil or triglyceride with 3 ml of 0.5 M sodium methoxide in methanoVdiethy1 ether (1: 1) and heating at 45°C for 5 min. The methyl esters were extracted by addition of 4 ml saturated NaCl followed by 250 ~1 of n-hexane. The organic layer was extracted for GC analysis. Gas chromatography was performed using a Shimadzu 4MPF gas chromatograph equipped with a 15% DEGS column and a FID detector. The peak areas were obtained using a Shimadzu CR4-A integrator.
C16:O
C16:0
C16:1
C16:2
C16:3
Fatty acid
4. Results and discussion Extraction of the ground Vemonia galamensis seed with acetone and collection of the filter cake by filtration resulted in a fine grey powder. This acetone powder catalysed the breakdown of triglycerides into fatty acids and glycerides as shown by TIC and GC analysis and also catalysed transesterification reactions. The acetone powder is stable for several months if stored at 4°C. A similar crude preparation of the lipase has been used in the fatty acid analysis of seed oils (Afolabi et al., 1991). 4.1. Fattyacid specificity The fatty acid content of both coconut oil and soya bean oil was determined by converting the fatty acids in the triglycerides of the oils to methyl esters and analysing the methyl esters by gas chromatography. The soya bean oil used consists mainly of unsaturated l&carbon fatty acids whereas the coconut oil used consists mainly of saturated 8- to l&carbon fatty acids. After about
c6:o
c1o:o
c12:o
c14:o
C16:O
C16:O
C16:1
Fatty acid
Fig. 1. Fatty acid content of (a) soya bean oil and (b) coconut oil and the nature of the fatty acids that are produced during hydrolysis of the oil by the I/emonia lipase. ‘Nglyceride fatty acids (left-hand columns). Released fatty acids (right-hand columns).
5% hydrolysis in both oil emulsions and watersaturated TMP there was no significant difference in the fatty acid constitution of the oil and the fatty acid constitution of the free fatty acids released during hydrolysis of the triglyceride (Fig. 1). The release of a certain fatty acid or a group of closely related fatty acids from an oil by a lipase indicates fatty acid specificity of the lipase (Jensen et al., 1983) and our results show that this Kwzonia lipase preparation has no preference for or discrimination against any of the fatty acids that were in the two different vegetable oils.
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4.2. Substrate specificity in transesterification reactions The rate of incorporation of lauric acid into triglycerides with fatty acid chain lengths varying from 4 carbon atoms to 18 carbon atoms was investigated, Short chain fatty acid triglycerides were found to be the preferred substrates in this reaction system (Table 1). This substrate specificity can be explained by considering the micro-structure of a biocatalytic system in organic media. When enzymes are in non-polar solvents like hexane, the enzyme is surrounded by a layer of water and the lipase-catalysed reaction takes place at the watersolvent interface. Short fatty acid chain triglycerides are more polar than long chain triglycerides and thus partition into the aqueous phase more than the less polar long chain triglycerides. This could explain the faster conversion of short chain triglycerides. Such a type of specificity is controlled by the nature of the substrate and not the structure of the enzyme. Although it may be possible that the small triglycerides are the preferred substrates, this is unlikely considering the presence of only long chain fatty acids in the physiological substrate of the lipase, the Vernonia seed oil. A system like this can be used to increase the long chain fatty acid content of an oil, as the triglycerides rich in short chain fatty acids would be transesterified faster. This would however be a rare application due to the infrequent occurrence of fats or oils with short chain fatty acids. An ex-
Table 1 Vernonia lipase-catalysed transesterification of triglycerides ‘Ikiglyceride
Relative transesterification
C4:o c4:oc4:0 C6:OC6:OC6:0 c!8:0C8:0C8:0 c10:0c10:0c10:0 c11:0c11:0c11:0 c14:o c14:o C16:OC16:0 C18:OC18:0 C18:l C18:l
100 75 63 37 16 18 14 14 18
c14:o C16:O C18:O C18:l
The transesterification was carried out with lauric acid in n-hexane. The triglyceride with the highest rate of transesterification was given a relative activity of 100.
289
ample of an attractive application would be the production of short chain fatty acid esters from butter fat using alcoholysis reactions. Butter fat is rich in short chain fatty acids and could be used in synthesis of various flavour compounds through alcoholysis with various alcohols. The rates of interesterification of milk fat using lipases that have preference for long chain esters may not be as fast as the rates for long chain fatty acid containing oils of fats (Reyes and Hill, 1994). 4.3. Positional specificity When triolein was hydrolysed for 5 min in emulsions and in TMP and the lipid constitution determined by TIC on silica gel plates impregnated with boric acid and visualised using PMA, and then quantified calorimetrically, monoand diglycerides were found to accumulate in the reaction mixture. Of the diglycerides 80% was 1,Zdiolein and 20% 1,3-diolein. The presence of 1,Zdiolein in greater amounts in the initial stages of hydrolysis shows that the lipase has some selectivity for the l,(3) position of the triglyceride. To further investigate the positional selectivity of the lipase, hydrolysis of palm oil mid fraction was studied. Palm oil mid fraction is a fraction of palm oil which consists mainly of 1,3-dipalmitoyl 2-oleylglycerol (POP). The oil used in our experiment had up to 90% of triglycerides as POP The amount of palmitate and oleate produced by the hydrolysis of POP in TMP, and in emulsions, by Emonia lipase was monitored. In both systems palmitate and oleate were released although the palmitate consisted of above 75% of the fatty acids (Fig. 2). When the monoglycerides were converted to methyl ester and analysed by GC palmitate could not be detected. These results indicate that the Vemonia lipase possibly acts on both the primary and secondary positions of the triglyceride but has high selectivity for the primary positions. The other possibility is that the presence of oleate in the fatty acids and 1,3-diglycerides is due to acyl migration of the oleate from the 2-position to the l,(3)-position resulting in the formation of 1,3-diglycerides and lipolysis of the isomerised diglyceride yielding oleate. The l,(3)-position of the triglyceride is the preferred position for most
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tions of the triglyceride. The ratio of palmitic acid to oleic acid decreased (Fig. 3) and though not shown, the amount of palmitic acid decreased as the amount of lauric acid incorporated increased.
5.0
b) POP in emulsion 4.5
4.4. Continuous hydrolysis of soya bean oil in a packed bed reactor
0.0
5.0
10.0
15.0
20.0 time
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
(mins)
25.0
30.0
35.0
40.0
time (mins)
Fig. 2. Positional specificity of the Vemonia lipase: (a) POP dissolved in TMP; (b) emulsified POP. The lipids were separated by TLC and the fatty acids converted to methyl esters and quantified by gas chromatography. Heptadecanoic acid was used as an internal standard. Palmitic acid (o), oleic acid (0).
lipases as these are the less sterically hindered positions of the triglyceride. One of the major applications of positional selective or specific lipases is in the interesterification of triglycerides and phospholipids to produce tailor-made triglycerides or phospholipids (Macrae, 1983; Bloomer et al., 1990). Transesterification of palm oil mid fraction with lauric acid revealed that the lauric acid was being incorporated in the primary posi-
When 4 g of the acetone powder was tightly packed into a column of an internal diameter of 1 cm, the bed height came to about 20 cm. For about 3 h of passing 2.5% soya bean oil solution there was complete hydrolysis of the triglycerides of the oil. Oil concentrations higher than 2.5% caused accumulation of diglycerides in the product and flow rates higher than 4 ml/h reduced the conversions. After 3 h, a decrease in the percentage hydrolysis was observed to about 40% at 2O”C, 60% at 30°C and 80% at 40°C (Fig. 4). In organic solvents the catalytic activity of enzymes is dependent on the water content of the reaction system; an essential amount of water is needed for the flexibility of the enzyme molecule so that it can attain its catalytic conformation. In a hydrolysis system, such as the one that is presented here, water is also a substrate. Since water is available in limiting amounts, as hydrolysis proceeds the reaction system is likely to dry up due to the use of water (Macrae, 1983,1985) and thus the hydrolytic activity of the lipase is reduced. The solvent used here was saturated with water but the solubility of water in these non-polar solvents is very low and is likely to be near limiting amounts for hydrolysis. However, as the percentage hydrolysis increases with temperature, it appears that a limiting factor could be the capacity of the system used in these experiments. Once the stable hydrolysis rate had been achieved the percentage hydrolysis remained stable for about three weeks at 40°C. The main advantage of using this preparation is that the acetone powder acts as an immobilised lipase and can be readily used as a solid catalyst in a packed bed reactor. While a packed bed reactor system has the disadvantage of mass transfer limitations compared to, for example, a stirred tank reactor (CSTR), this acetone powder would provide a cheap alternative with minimised downstream processing under continual operation. Moreover,
I. Ncube, J.S. Read IIndustrial Crops and Products 3 (1995) 285-292
I 100
1.6 f 0
I 200 time
291
I 400
I 300
(mitts)
Fig. 3. Change in the ratio of palmitate to oleate during the Vemonia lipase catalysed transesterification with lauric acid.
of palm oil mid fraction
60-
2oI 0
10
20
30
40
50
60
time (hrs)
Fig. 4. Effect of temperature acetone-extracted
on the Vemonia lipase catalysed hydrolysis of soya bean oil in a fixed bed reactor
of the
acetone powder. 20°C (O), 30°C (A), 40°C (0).
the granular nature of the powder offers very little restrictions to fluid flow. The material prepared here, if considered as a waste product of oil extraction, reduces the cost of the immobilised lipase to insignificant amounts. The use of lipases in organic solvents to synthesise organic compounds is a promising route of obtaining products of high purity in the chemi-
cal and pharmaceutical industry and products that are more “natural” and more acceptable to the consumer in the food industry. In these areas of research, plant lipases such as the Vemonia lipase should not be overlooked as they may possess some interesting properties and further investigations are proceeding on the purified lipase. Acetone powders like the one prepared here, have
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a good operational stability and may be a good source of cheap and immobilised lipase capable of carrying out a number of lipase-catalysed transesterification and hydrolysis reactions. The ability of the lipase to hydrolyse lipids with high conversions in organic media, a monophasic system, is particularly attractive and may be a possible alternative to harsh chemical methods and the expensive hollow fibre membrane reactors. Acknowledgements The authors thank the Research Board of the University of Zimbabwe and the Swedish Agency for Research Cooperation with Developing Countries (SAREC) for providing the financial support for the research project, and Bo Mattiasson and Patrick Adlercreutz for technical discussions. References Afolabi, O.A., Ologunde, M.O., Anderson, W.A., Read, J.S., Dacosta, M.D., Epps, EA. and Ayorinde, F.O., 1991. Use of lipase (Acetone powder) from Vemonia galamensis in fatty acid analysis of seed oils. J. Chem. Tech. Biotechnol., 51: 41-46. Bell, G., Todd, J., Blain, J., Patterson, J. and Shaw, C., 1981. Hydrolysis of triglycerides by solid phase lipolytic enzymes of Rhizopus arhizus in continuous reactor systems. Biotechnol. Bioeng., 23: 1703-1719. Bloomer, S., Adlercreutz, P. and Mattiasson, B., 1990. Cocoa butter equivalents from a fraction of palm oil. J. Am. Oil Chem. Sot., 67: 519-524. Bloomer, S., Adlercreutz, P and Mattiasson, B., 1992. Facile synthesis of fatty acid esters in high yields. Enzyme Micrab. Technol., 14: 546-552. Derksen, J.T.P., Boswinkel, G. and Cuperus, F.P., 1992. Cellulase activity in commercial lipase preparations. Biotechnol. Bioeng., 40: 858-860. Derksen, J.TP., Muuse, B.G., Cuperus, EP. and van Gelder, W.M.J., 1993. New seed oils for the oleochemical industry: evaluation and enzyme bioreactor mediated processing. Ind. Crops Prod., 1: 133-139. Elmore, J.D., DeGooyer, W.J., Tipton, M.B. and Kaiser, J.H., 1993. U.S. Patent No. 5227453. Huang, A.H.C., 1984. Plant lipases. In: B. Borgstrom and H.L. Brockman (Editors), Lipases. Elsevier Science Publishers, Amsterdam, pp. 420-441.
Jensen, R.G., Dejong, EA. and Clark, R.M., 1983. Determination of lipase specificity. Lipids, 18: 239-252. Jensen, R.G., Galluzo, R.D. and Bush, V.J., 1990. Selectivity is an important characteristic of lipases (acylglycerol hydrolases). Biocatalysis, 3: 307-316. Khor, H., ‘Ban, N. and Chua, C., 1986. Lipase catalysed hydrolysis of palm oil. J. Am. Oil Chem. Sot., 63: 538-540. Lakshminarayana, G., Subbarao, R., Sastry, Y.S.K., Rao, ‘PC., Kale, V and Vijayalakshmi, P., 1984. High pressure splitting of castor oil. J. Am. Oil Chem. Sot., 61: 1204-1206. Macrae, A.R., 1983. Lipase catalysed interesterification of fats and oils. J. Am. Oil Chem. Sot., 60: 243A-246A. Macrae, A.R., 1985. Interesterification of fats and oils. In: J. ‘Barnper, H.C. van der Plas and P. Linko (Editors), Biocatalysts in Organic Synthesis. Proc. Int. Symp., Elsevier Science., Amsterdam, pp. 195-208. Malcata, F.X., Reyes, H.R., Garcia, H.S., Hill, C.G. and Amundson, C.H., 1990. Immobilised lipase reactors for modification of fats and oils. J. Am. Oil Chem. Sot., 67: 890-910. Millqvist, A., Adlercreutz, P and Mattiasson, B., 1994. Lipasecatalysed alcoholysis of triglycerides for the preparation of 2-monoglycerides. Enzyme Microb. Bxhnol., 16: 10421047. Mukherjee, K.D., 1990. Lipase catalysed reactions for the modifications of fats and other lipids. Biocatalysis, 3: 277293. Ncube, I., Adlercreutz, I?, Read, J. and Mattiasson, B., 1993. Purification of rape (Bra&a napus) seedling lipase and its use in organic media. Biotechnol. Appl. Biochem., 17: 327-336. Ncube, I., Chikunguwo, S. and Read, J.S., 1994. An economic calorimetric method for the quantification of total lipid and assaying for lipase activity (unpubl. results). Ologunde, M.O., Ayorinde, F.O. and Shepard, R.L., 1990. Chemical evaluation of defatted Vemonia galamensismeal. J. Am. Oil Chem. Sot., 67: 92-95. Reyes, H.R. and Hill, C.G., 1994. Kinetic modeling of interesterification reactions catalysed by immobilised lipase. Biotech. Bioeng., 43: 171-182. Sonnet, PE., 1988. Lipase selectivities. J. Am. Oil Chem. Sot., 65: 900-904. Sontag, N.O.V., 1989. Fat splitting and glycerol recovery. In: R.W. Johnson and E. Fritz (Editors), Fatty Acids in Industry. Marcel Dekker, Inc., New York, pp. 23-72. Svensson, I., Adlercreutz, P. and Mattiasson B., 1992. Lipasecatalysed transesterification of phosphatidylcholine at controlled water activity. J. Am. Oil Chem. Sot., 69: 986991. Vaidya, A.M., Halling, P.J. and Bell, G., 1994. Surfactantinduced breakthrough effects during the operation of twophase biocatalytic membrane reactors. Biotechnol. Bioeng., 44: 765-771.