Biochimica ef Biophysics Elsevier
231
Acfa, 1042 (1990) 237-240
BBALIP 53308
Lipase from Brassica napus L. discriminates against cis-4 and cis-6 unsaturated fatty acids and secondary and tertiary alcohols Matthew
J. Hills, Irmgard
Kiewitt and Kumar
D. Mukherjee
Federal Center for Lipid Research, Institute for Biochemistry and Technology, H.P. Kaufmann-Institute,
Miinster (F.R.G.)
(Received 21 July 1989)
Key words: Plant lipase; Esterification; y-Linoleneic acid; Polyunsaturated fatty acid; (Brassica
napus)
Lipase (EC 3.1.1.3) from oilseed rape (Bmsicu nupus L., cv Ceres) hydrolyzes triacylglycerols containing a broad range of fatty acids at similar rates. In esterification reactions carried out in hexane, rape lipase also uses a wide range of fatty acids and alcohols as reaction partners. However, the rates of esterification of petroselinic, y-linolenic, stearidonic and docosahexaenoic acids are only between 2 and 7% that of oleic acid. The common feature of these fatty acids is that the first double bond is cis4 or k-6. Petroselaidic acid with a tmnsd double bond is esterified about lo-times faster than petroselinic acid. Arachidonic and eicosapentaenoic acids, both with the first double bond being cisd, are esterified about 20-times faster than docosahexaenoic acid. By analogy, tripetroselinin and tri-y-linolenin are hydrolyzed at 14% and 1.5%, respectively, of the rate of triolein hydrolysis. The rape lipase esterifies primary alcohols but cannot esterify secondary and tertiary alcohols.
Introduction Lipases are finding many technical and industrial uses which rely on their enantiomeric, positional or fatty acid selectivity [l-3]. At present, much effort is being directed at understanding the basis for these selectivities in order that they might be enhanced [4]. With regard to fatty acids, the majority of lipases have a broad specificity, with the notable exception of that from Geotrichum candidurn [5]. Commercial lipase preparations are generally derived from fungi, but another source of lipase for use as biocatalysts for technical processes which as yet has received little attention, is that from reserve tissues of young oilseed plants [6]. These tissues are rich in lipase activity and on the basis of experiments examining the hydrolysis of various triacylglycerols it has been suggested that these lipases might display specificities for fatty acids not shown by most fungal lipases [7]. Recently we described the preparation and immobilization of lipase from rape seedlings for use in synthetic reactions in the presence of organic solvents [B]. This lipase, in common with those from fungi catalyzes
Correspondence: K.D. Mukbejee, Federal Center for Lipid Research, Institute for Biochemistry and Technology, H.P. Kaufmann-Institute, Piusallee 68, D-4400 Mtinster, F.R.G. 0005-2760/90/$03.50
esterification and transesterification reactions [9]. In the present work we show that the lipase preparation from rape cotyledons discriminates strongly against those fatty acids which have a cis-4 or cis-6 double bond. The lipase does not accept secondary and tertiary alcohols as substrates. Materials and Methods Materials Lipase from cotyledons of 6-d-old rape (Brassicu naps L., cv Ceres) seedlings was prepared up to the stage of polyethyleneglycol (PEG) precipitation as previously described [8]. The PEG precipitated lipase was resuspended in buffer [8] and precipitated onto Celite with 9 vols of acetone ( - 20 o C) at a protein loading of 20 mg . gg’ Celite. The acetone was removed under vacuum in a desiccator at 4 o C. Esterification reactions Esterification reactions were carried out as described [9]. Hexane saturated with water was used as solvent throughout. The esterification of fatty acids and butanol was measured in two ways: (1) Reaction mixture consisting of fatty acid (25 mM) and butanol (50 mM); and (2) Reaction mixture consisting of fatty acid (12.5 mM), myristic or oleic acid (12.5 mM) as internal standard and butanol (50 mM).
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
238 The esterification of alcohols and oleic acid was measured using a mixture of alcohol (25 mM), butanol (25 mM) as an internal standard and oleic acid (25 mM). Control reaction mixtures contained Celite without lipase. Unesterified fatty acids present in aliquots taken from reaction mixtures were converted to methyl esters with di~omethane. The fatty acid esters were analyzed by GLC on glass columns (1.8 m x 4 mm) packed with 10% (w/w) Silar 5CP on Gas-Chrom Q, 80-100 mesh from Applied Science Laboratories (State College, PA, U.S.A.) in Perk&Elmer F-22 instrument equipped with flame ionization detectors from Perkin-EImer (Uberlingen, F.R.G.). Nitrogen (40 ml/mm) was used as carrier gas and the colunm temperature programmed from 150 to 200°C at 2 C”/min. Esters from alcohols longer than C, were analyzed by GLC on columns packed with 3% (w/w) OVl on Gas-Chrom Q, loo-120 mesh, from Applied Science Laboratories; nitrogen (60 ml/mm) was used as carrier gas and the column temperature programmed from 90 to 300 o C at 6 C “/min. Data were quantified using a Spectra Physics SP 4290 Integrator from Spectra Physics (San Jose, CA, U.S.A.). Experiments were carried out in duplicate and results agreed to within 15%. Initial rates of esterification, which was a first-order reaction, were calculated in each case and for comparison, all results are given relative to the rate of este~fication of oleic acid and butanol.
Hydrolytic activity of non-i~obil~ed lipase was assessed using a copper soaps method to measure released fatty acids [lo]. Hydrolytic reactions were carried out at 30’ C in 100 ~1 of a reaction medium containing 50 mM Bis-Tris-propane (pH 7.5~)~2 mM DTT, 2 mM CaCl,, 2.5 mM ~tylt~oglucoside and 5 mM triacylglycerol. The triacylglycerols were prepared as a 100 mM stock emulsion in 5% (w/v) Gum Arabic by sonication for 1 min at 20 W with a Branson B-12 sonicator. Measurements were made in triplicate. Hydrolysis of p-nitrophenyl esters of caprylic acid (pNPC) and myristic acid (pNPM) catalyzed by the non-i~obi~zed Iipase was measured spectrophotomet~ rically at pH 7.5 in 50 mM Bis-T&propane buffer using a Pye-Unican SP S-200 spectrophotometer [ll]. Substrates were dissolved in acetonit~le and added to the reaction mixture to give a final concentration of I.2 mM in 4% acetonitrile. Results Esterificution: unsaturated fatty acids The specificity of the rape lipase preparation in este~fication of saturated and unsaturated fatty acids to butanol (Table I) and of oleic acid to a wide variety of alcohols (Table II) was assessed by measuring lipase
TABLE I Esterification of unsaturated fatly acids to butanol, colaiyzed by immobilized rape lipase
Reaction mixtures contained fatty acid (25 mM) and butanol (SO mM) (Method 1) or fatty acid (12.5 mM), intemai standard (myristic acid, 12.5 mM) and butanol (50 mM) or for experiments marked *, fatty acid (12.5 mM), internal standard (oleic acid, 12.5 mM) and butanol (50 mM) {Method 2). Initial rates of esterification relative to oleic acid (1.0) have been calculated for comparison. Results are means from duplicate experiments. Rates agreed to within 15%. Fatty acid
Caproic Enantbic Capryfic Pelargonic Capric Laurie Myristic Palmitic Stearic Oleic Petroselinic Elaidic Petroselaidic Vaccenic Erucic Linoleic a-Linolenic y-linolenic Stearidonic Aracbidonic Bcosapentaenoic Docosahexaenoic
catalyzed are given acid and pmol - (g
Relative esterification methodl
method2
1.0 0.05 0.5 0.7 0.6 0.3 1.1 2.0 0.03 0.03 0.6 0.4 0.03
0.4 * 3.7 * 8.2 * 2.4 * 1.7 * 1.3 * 1.7 * 0.7 0.6 * 1.0 0.07 0.5 0.7 0.5 0.2 1.0 1.3 0.02 0.07 0.5 0.5 0.03
6:O
7:o 8:0 9:o lo:o 12:o 14:o 16:O 18:O cis-9-18 : 1 cis-6-18 : I truns-9-18 : 1 rruns-6-18 : 1 cis-11-18 : 1 cis-13-22 : 1 all cis-9.12-18 : 2 all cis-9,12,15-18: 3 all cis-6,9,12-18 : 3 all cis-6,9,12,15-18:4 ali cis-5.8.11.14-20:4 all cis-5,8,11,14,17-20: 5 all cis-4,7,10,13,16,19-22: 6
monoester fo~atio~. For comp~ison, all data relative to the rate of esterification of oleic butanol which was in the range of 0.3 and 1.0 Celite-lipase)-’ . mm-’ depending on the preparation. We have recently found, however, that esterification activity can be stimulated by up to 35-fold by the addition of water (1.2%, v/v) to the reaction mixture (in preparation). The results given in Table I show that the majority of the l&carbon unsaturated fatty acids were esterified at rates ranging from 0.5- to %.O-fold of that of oleic acid (cis-9-18 : 1). Elaidic acid (trans-9-18 : 1) was esterified at a rate similar to stearic acid (18: 0). However, a few 18-carbon unsaturated fatty acids, namely petroselinic (cis-6-18 : l), y-linolenic (all &s-6,9,12-18 : 3) and stearidonic (all cis-6,9,12,15-18 : 4) acids were very poor substrates, being esterified at 2-7s of the rate of oleic acid. y-Linolenic acid (all cis-6,9,12-18 : 3) was esterified to butanol at less than 0.02-times the rate of cy-linolenic acid (all cis-9,12,15-18 : 3). The common feature of the unsaturated fatty acids which are dis-
239 criminated against by the rape lipase is that the first double bond is cis-6. Petroselaidic acid (truns-6-18 : 1) was esterified lo-times faster than cis-6-18 : 1. Thus, it seems that the cis configuration of the double bond in position A6 causes the low rate of esterification. It was also found that arachidonic and eicosapentaenoic acids, in which the first double bond is c&5, were esterified 20-times faster than docosahexaenoic acid in which the first double bond is cis-4 (Table I). It was possible that the low rate of esterification of the unsaturated fatty acids containing a cis-4 or cis-6 double bond (Table I, method 1) was due either to inhibition of the enzyme in the presence of such fatty acids, or that the lipase was not activated for some reason. Therefore, the rates of esterification were also measured in the presence of an internal standard, i.e., my&tic acid (method 2). It is clear from the data given in Table I that the relative activity of the lipase in the esterification to the different fatty acids is very similar in both cases. Thus, the discrimination against fatty acids with cis-4 or cis-6 double bonds was not due to lack of activation of the enzyme. Hydrolysis: triacylglycerols containing unsaturated fatty acids The data on esterification reactions presented in Table I show that the lipase has a broad specificity towards the common saturated and unsaturated fatty acids. These data compare well with those for hydrolytic activity of lipase from B. napus reported previously, which was found to hydrolyze triacylglycerols containing a wide range of fatty acids [12,13]. In the present work, the hydrolytic activity of rape lipase was determined from triacylglycerols containing petroselinic or y-linolenic acids which were found to be very poor substrates in the esterification reactions (Table I). Fig. 1 shows the kinetics of hydrolysis of t~petroselinin and tri-y-linolenin as compared to triolein. Triolein was hydrolyzed rapidly but tripetroselinin and tri-y-linolenin were hydrolyzed at only 14% and 1.5%, respectively, of the rate of triolein. Thus, the specificity of the rape lipase found in the esterification reaction under non-aqueous conditions was also reflected in its hydrolytic activity in an aqueous medium. Esteri~cation /hydrolysis: saturated fatty acids Esterification of most even numbered (C&s) saturated fatty acids to butanol was catalyzed by the rape lipase at rates similar to the esterification of oleic acid (Table I). However, the lipase esterified caprylic acid more than 8-times faster than oleic acid; heptanoic and nonanoic acids were esterified at faster rates than most acids but not as fast as the caprylic acid (Table I). The rates of hydrolysis of p-nitrophenyl esters of caprylic acid (pNPC) and myristic acid ( pNPM) catalyzed by non-immobilized rape lipase were measured
lime
lmin)
Fig. 1. Kinetics of hydrotysis of triolein {A), t~petroseIi~n (a) and tri-y-linoienin (m) catalyzed by rape lipase in an aqueous system. Each point is a mean of three determinations with standard deviation.
and were found to be quite similar (10.8 pmol- (mg protein)-’ . min- ’ for pNPC and 12.3 pmol - (mg protein)-’ . mm-’ for pNPM). Thus, the comparatively rapid rate of ~te~fi~tion of caprylic acid to butanol (Table I) was not reflected in the rate of hydrolysis of a caprylate ester. Esterification: alcohol specificity The esterification of different alcohols to oleic acid (Table II) revealed that butan-l-01 and propan-l-01
TABLE
II
Esterificakn lipase
of alcohols to oleic acid catalyzed
by immobiiized
rape
Reaction mixtures contained 25 mM alcohol, 25 mM butanol and 25 mM oleic acid. Initial rates of esterification expressed relative to formation of butyl oleate (1.0). Alcohol
Relative esterification
Ethanol Propanol Butanol Octanol Dodecanol Hexadecanol Geraniol Propan-2-01 Butan-Z-01 Benzyl alcohol Propan-2-methyl-2-01 Terpineol
0.2 0.8 1.0 0.5 0.4 0.1 0.6 0.0 0.0 0.0 0.0 0.0
240 were esterified most efficiently. Alcohols having longer chains were esterified more slowly. Secondary and tertiary alcohols were not esterified by the rape lipase. Benzyl alcohol, a secondary alcohol, was not esterified and its presence caused the almost total inhibition of the esterification of n-butanol. Discussion The rape lipase has a very unusual specificity in accepting most natural saturated and unsaturated fatty acids but discriminating against those with the initial double bond being cis-4 or cis-6. Such a fatty acid specificity has not previously been reported for any lipase. The characteristics of the rape lipase can be compared with those of Geotrichum candidum [?I]. In the case of G. candidum, C,, acyl moieties containing cis-9 double bonds such as oleoyl and linoleoyl moieties were preferentially hydrolyzed from triacylglycerols, whereas, elaidoyl moieites (trans-9-18 : 1) and acyl groups having cis double bonds in other positions were much more slowly hydrolyzed. In the case of pancreatic lipase it was found that a cis double bond at any carbon up to C, caused inhibition of lipase activity [14]. In view of these findings, the lipase from oilseed rape might well prove useful for the isolation of unusual fatty acids, such as y-linolenic or petroselinic acids from seed oils of evening primrose (Oenothera biennis L.) or coriander (Car~and~m sativ~ IL.), respectively. For example, enrichment of fatty acids with cisd double bonds from fatty acid mixtures made from these oils could be achieved by esterifying all fatty acids except the cis-6 unsaturated fatty acid to butanol or another alcohol. The enrichment of docosahexaenoic acid from fish oils could also be envisaged. At present it is not possible to give a definite explanation for the selectivity shown by the rape lipase. It is interesting to note, however, that only those fatty acids with the first double bond being cis-4 or cis-6 are poor substrates for the lipase, whereas those in which the first double bond is cis-9 or cis-5 are utilized quite rapidly. If one considers that fatty acids with cis double bonds at odd or even number carbons have syn- or antiorientations, respectively, with respect to the earboxyl group ]lS], then from the data reported here it appears that rape lipase discriminates against anti- oriented cis-unsaturated fatty acids. It is possible that the direc-
tion of twist of the carbon chain after the first double bond of these fatty acids might hinder binding of the reactive group to the lipase. In a similar manner, but opposite way, an acyltransferase was shown to select anti, rather than syn conformations [15]. Further study with a range of synthetic isomers of monoenoic fatty acids, as reported for pancreatic lipase [ 141, would clarify this point. An unsaturated fatty acid with a trans double bond has a configuration similar to a saturated fatty acid and we found that the rates of esterification of elaidic and petroselaidic acids were similar to that of stearic acid.
This work was supported in part by a research grant from the Bundesrninisterium fir Ernlhrung, Landwirtschaft und Forsten, Bonn, Federal Republic of Germany. References 1 Kloosterman, M., Elferrink, V.H.M., Van Iersal, J., Roskam, J., Meijer, E.M., Hulshof, L.A. and Sheldon, R.A. (1988) Trends Biotechnol. 5, 251-254. 2 Macrae, A.R. and Hammond, R.C. (1985) Biotechnol. Genet. Eng. Rev. 3, 193-217. 3 Haraldsson, G.G., Hiiskuldsson, P.A., Sigurdsson, S.Th., Thorsteinsson, F. and Gudbjarnason, S. (1989) Tetrahedron Lett. 30, 1671-1674. 4 Sonnet, P.E. and Welch Baiilargeon, M. (1989) Lipids 24,434-437. 5 Brockerhof, H. and Jensen, R.G. (1974) in Lipolytic Enzymes, pp. 140-146, Academic Press, New York. 6 Hassanien, F.R. and Mukhejee, K.D. (1986) J. Am. Oil Chem. Sot. 63, 893-897. 7 Huang, A.H.C., Lin, Y. and Wang, S. (1988) J. Am. Oil Chem. Sot. 65, 897-899. 8 Hills, M.J. and O’Sullivan, J.N. (1989) B&hem. Sot. Trans. 17, 480. 9 Hills, M.J., Kiewitt, I. and Mukherjee, K.D. (1989) B&hem. Sot. Trans. 17, 478. 10 Hills, M.J. and Beevers, H. (1987) Plant Physiol. 85, 1084-1088. 11 Chapus, C., Semeriva, M., Bovier-Lapierre, C. and DesnueIle, P. (1976) Biochemistry 15, 4980-4987. 12 Lin, Y.H., Yu, C. and Huang, A.H.C. (1986) Arch. Biochem. Biophys. 244, 346-356. 13 Hills, M.J. and Murphy, D.J. (1988) Biochem. J. 249, 687-693. 14 Heimermann, W.H., Holman, R.T., Gordon, D.T., Kowalyshyn, D.E. and Jensen, R.G. (1973) Lipids 8, 45-46. 15 Lands, W.E.M. (1979) in Geometrical and Positional Fatty Acid Isomers (Emken, E.A. and Dutton, H.J., eds.), pp. 181-212, American Oil Chemists’ Society, Champaign, IL.
231
Acfa, 1042 (1990) 237-240
BBALIP 53308
Lipase from Brassica napus L. discriminates against cis-4 and cis-6 unsaturated fatty acids and secondary and tertiary alcohols Matthew
J. Hills, Irmgard
Kiewitt and Kumar
D. Mukherjee
Federal Center for Lipid Research, Institute for Biochemistry and Technology, H.P. Kaufmann-Institute,
Miinster (F.R.G.)
(Received 21 July 1989)
Key words: Plant lipase; Esterification; y-Linoleneic acid; Polyunsaturated fatty acid; (Brassica
napus)
Lipase (EC 3.1.1.3) from oilseed rape (Bmsicu nupus L., cv Ceres) hydrolyzes triacylglycerols containing a broad range of fatty acids at similar rates. In esterification reactions carried out in hexane, rape lipase also uses a wide range of fatty acids and alcohols as reaction partners. However, the rates of esterification of petroselinic, y-linolenic, stearidonic and docosahexaenoic acids are only between 2 and 7% that of oleic acid. The common feature of these fatty acids is that the first double bond is cis4 or k-6. Petroselaidic acid with a tmnsd double bond is esterified about lo-times faster than petroselinic acid. Arachidonic and eicosapentaenoic acids, both with the first double bond being cisd, are esterified about 20-times faster than docosahexaenoic acid. By analogy, tripetroselinin and tri-y-linolenin are hydrolyzed at 14% and 1.5%, respectively, of the rate of triolein hydrolysis. The rape lipase esterifies primary alcohols but cannot esterify secondary and tertiary alcohols.
Introduction Lipases are finding many technical and industrial uses which rely on their enantiomeric, positional or fatty acid selectivity [l-3]. At present, much effort is being directed at understanding the basis for these selectivities in order that they might be enhanced [4]. With regard to fatty acids, the majority of lipases have a broad specificity, with the notable exception of that from Geotrichum candidurn [5]. Commercial lipase preparations are generally derived from fungi, but another source of lipase for use as biocatalysts for technical processes which as yet has received little attention, is that from reserve tissues of young oilseed plants [6]. These tissues are rich in lipase activity and on the basis of experiments examining the hydrolysis of various triacylglycerols it has been suggested that these lipases might display specificities for fatty acids not shown by most fungal lipases [7]. Recently we described the preparation and immobilization of lipase from rape seedlings for use in synthetic reactions in the presence of organic solvents [B]. This lipase, in common with those from fungi catalyzes
Correspondence: K.D. Mukbejee, Federal Center for Lipid Research, Institute for Biochemistry and Technology, H.P. Kaufmann-Institute, Piusallee 68, D-4400 Mtinster, F.R.G. 0005-2760/90/$03.50
esterification and transesterification reactions [9]. In the present work we show that the lipase preparation from rape cotyledons discriminates strongly against those fatty acids which have a cis-4 or cis-6 double bond. The lipase does not accept secondary and tertiary alcohols as substrates. Materials and Methods Materials Lipase from cotyledons of 6-d-old rape (Brassicu naps L., cv Ceres) seedlings was prepared up to the stage of polyethyleneglycol (PEG) precipitation as previously described [8]. The PEG precipitated lipase was resuspended in buffer [8] and precipitated onto Celite with 9 vols of acetone ( - 20 o C) at a protein loading of 20 mg . gg’ Celite. The acetone was removed under vacuum in a desiccator at 4 o C. Esterification reactions Esterification reactions were carried out as described [9]. Hexane saturated with water was used as solvent throughout. The esterification of fatty acids and butanol was measured in two ways: (1) Reaction mixture consisting of fatty acid (25 mM) and butanol (50 mM); and (2) Reaction mixture consisting of fatty acid (12.5 mM), myristic or oleic acid (12.5 mM) as internal standard and butanol (50 mM).
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
238 The esterification of alcohols and oleic acid was measured using a mixture of alcohol (25 mM), butanol (25 mM) as an internal standard and oleic acid (25 mM). Control reaction mixtures contained Celite without lipase. Unesterified fatty acids present in aliquots taken from reaction mixtures were converted to methyl esters with di~omethane. The fatty acid esters were analyzed by GLC on glass columns (1.8 m x 4 mm) packed with 10% (w/w) Silar 5CP on Gas-Chrom Q, 80-100 mesh from Applied Science Laboratories (State College, PA, U.S.A.) in Perk&Elmer F-22 instrument equipped with flame ionization detectors from Perkin-EImer (Uberlingen, F.R.G.). Nitrogen (40 ml/mm) was used as carrier gas and the colunm temperature programmed from 150 to 200°C at 2 C”/min. Esters from alcohols longer than C, were analyzed by GLC on columns packed with 3% (w/w) OVl on Gas-Chrom Q, loo-120 mesh, from Applied Science Laboratories; nitrogen (60 ml/mm) was used as carrier gas and the column temperature programmed from 90 to 300 o C at 6 C “/min. Data were quantified using a Spectra Physics SP 4290 Integrator from Spectra Physics (San Jose, CA, U.S.A.). Experiments were carried out in duplicate and results agreed to within 15%. Initial rates of esterification, which was a first-order reaction, were calculated in each case and for comparison, all results are given relative to the rate of este~fication of oleic acid and butanol.
Hydrolytic activity of non-i~obil~ed lipase was assessed using a copper soaps method to measure released fatty acids [lo]. Hydrolytic reactions were carried out at 30’ C in 100 ~1 of a reaction medium containing 50 mM Bis-Tris-propane (pH 7.5~)~2 mM DTT, 2 mM CaCl,, 2.5 mM ~tylt~oglucoside and 5 mM triacylglycerol. The triacylglycerols were prepared as a 100 mM stock emulsion in 5% (w/v) Gum Arabic by sonication for 1 min at 20 W with a Branson B-12 sonicator. Measurements were made in triplicate. Hydrolysis of p-nitrophenyl esters of caprylic acid (pNPC) and myristic acid (pNPM) catalyzed by the non-i~obi~zed Iipase was measured spectrophotomet~ rically at pH 7.5 in 50 mM Bis-T&propane buffer using a Pye-Unican SP S-200 spectrophotometer [ll]. Substrates were dissolved in acetonit~le and added to the reaction mixture to give a final concentration of I.2 mM in 4% acetonitrile. Results Esterificution: unsaturated fatty acids The specificity of the rape lipase preparation in este~fication of saturated and unsaturated fatty acids to butanol (Table I) and of oleic acid to a wide variety of alcohols (Table II) was assessed by measuring lipase
TABLE I Esterification of unsaturated fatly acids to butanol, colaiyzed by immobilized rape lipase
Reaction mixtures contained fatty acid (25 mM) and butanol (SO mM) (Method 1) or fatty acid (12.5 mM), intemai standard (myristic acid, 12.5 mM) and butanol (50 mM) or for experiments marked *, fatty acid (12.5 mM), internal standard (oleic acid, 12.5 mM) and butanol (50 mM) {Method 2). Initial rates of esterification relative to oleic acid (1.0) have been calculated for comparison. Results are means from duplicate experiments. Rates agreed to within 15%. Fatty acid
Caproic Enantbic Capryfic Pelargonic Capric Laurie Myristic Palmitic Stearic Oleic Petroselinic Elaidic Petroselaidic Vaccenic Erucic Linoleic a-Linolenic y-linolenic Stearidonic Aracbidonic Bcosapentaenoic Docosahexaenoic
catalyzed are given acid and pmol - (g
Relative esterification methodl
method2
1.0 0.05 0.5 0.7 0.6 0.3 1.1 2.0 0.03 0.03 0.6 0.4 0.03
0.4 * 3.7 * 8.2 * 2.4 * 1.7 * 1.3 * 1.7 * 0.7 0.6 * 1.0 0.07 0.5 0.7 0.5 0.2 1.0 1.3 0.02 0.07 0.5 0.5 0.03
6:O
7:o 8:0 9:o lo:o 12:o 14:o 16:O 18:O cis-9-18 : 1 cis-6-18 : I truns-9-18 : 1 rruns-6-18 : 1 cis-11-18 : 1 cis-13-22 : 1 all cis-9.12-18 : 2 all cis-9,12,15-18: 3 all cis-6,9,12-18 : 3 all cis-6,9,12,15-18:4 ali cis-5.8.11.14-20:4 all cis-5,8,11,14,17-20: 5 all cis-4,7,10,13,16,19-22: 6
monoester fo~atio~. For comp~ison, all data relative to the rate of esterification of oleic butanol which was in the range of 0.3 and 1.0 Celite-lipase)-’ . mm-’ depending on the preparation. We have recently found, however, that esterification activity can be stimulated by up to 35-fold by the addition of water (1.2%, v/v) to the reaction mixture (in preparation). The results given in Table I show that the majority of the l&carbon unsaturated fatty acids were esterified at rates ranging from 0.5- to %.O-fold of that of oleic acid (cis-9-18 : 1). Elaidic acid (trans-9-18 : 1) was esterified at a rate similar to stearic acid (18: 0). However, a few 18-carbon unsaturated fatty acids, namely petroselinic (cis-6-18 : l), y-linolenic (all &s-6,9,12-18 : 3) and stearidonic (all cis-6,9,12,15-18 : 4) acids were very poor substrates, being esterified at 2-7s of the rate of oleic acid. y-Linolenic acid (all cis-6,9,12-18 : 3) was esterified to butanol at less than 0.02-times the rate of cy-linolenic acid (all cis-9,12,15-18 : 3). The common feature of the unsaturated fatty acids which are dis-
239 criminated against by the rape lipase is that the first double bond is cis-6. Petroselaidic acid (truns-6-18 : 1) was esterified lo-times faster than cis-6-18 : 1. Thus, it seems that the cis configuration of the double bond in position A6 causes the low rate of esterification. It was also found that arachidonic and eicosapentaenoic acids, in which the first double bond is c&5, were esterified 20-times faster than docosahexaenoic acid in which the first double bond is cis-4 (Table I). It was possible that the low rate of esterification of the unsaturated fatty acids containing a cis-4 or cis-6 double bond (Table I, method 1) was due either to inhibition of the enzyme in the presence of such fatty acids, or that the lipase was not activated for some reason. Therefore, the rates of esterification were also measured in the presence of an internal standard, i.e., my&tic acid (method 2). It is clear from the data given in Table I that the relative activity of the lipase in the esterification to the different fatty acids is very similar in both cases. Thus, the discrimination against fatty acids with cis-4 or cis-6 double bonds was not due to lack of activation of the enzyme. Hydrolysis: triacylglycerols containing unsaturated fatty acids The data on esterification reactions presented in Table I show that the lipase has a broad specificity towards the common saturated and unsaturated fatty acids. These data compare well with those for hydrolytic activity of lipase from B. napus reported previously, which was found to hydrolyze triacylglycerols containing a wide range of fatty acids [12,13]. In the present work, the hydrolytic activity of rape lipase was determined from triacylglycerols containing petroselinic or y-linolenic acids which were found to be very poor substrates in the esterification reactions (Table I). Fig. 1 shows the kinetics of hydrolysis of t~petroselinin and tri-y-linolenin as compared to triolein. Triolein was hydrolyzed rapidly but tripetroselinin and tri-y-linolenin were hydrolyzed at only 14% and 1.5%, respectively, of the rate of triolein. Thus, the specificity of the rape lipase found in the esterification reaction under non-aqueous conditions was also reflected in its hydrolytic activity in an aqueous medium. Esteri~cation /hydrolysis: saturated fatty acids Esterification of most even numbered (C&s) saturated fatty acids to butanol was catalyzed by the rape lipase at rates similar to the esterification of oleic acid (Table I). However, the lipase esterified caprylic acid more than 8-times faster than oleic acid; heptanoic and nonanoic acids were esterified at faster rates than most acids but not as fast as the caprylic acid (Table I). The rates of hydrolysis of p-nitrophenyl esters of caprylic acid (pNPC) and myristic acid ( pNPM) catalyzed by non-immobilized rape lipase were measured
lime
lmin)
Fig. 1. Kinetics of hydrotysis of triolein {A), t~petroseIi~n (a) and tri-y-linoienin (m) catalyzed by rape lipase in an aqueous system. Each point is a mean of three determinations with standard deviation.
and were found to be quite similar (10.8 pmol- (mg protein)-’ . min- ’ for pNPC and 12.3 pmol - (mg protein)-’ . mm-’ for pNPM). Thus, the comparatively rapid rate of ~te~fi~tion of caprylic acid to butanol (Table I) was not reflected in the rate of hydrolysis of a caprylate ester. Esterification: alcohol specificity The esterification of different alcohols to oleic acid (Table II) revealed that butan-l-01 and propan-l-01
TABLE
II
Esterificakn lipase
of alcohols to oleic acid catalyzed
by immobiiized
rape
Reaction mixtures contained 25 mM alcohol, 25 mM butanol and 25 mM oleic acid. Initial rates of esterification expressed relative to formation of butyl oleate (1.0). Alcohol
Relative esterification
Ethanol Propanol Butanol Octanol Dodecanol Hexadecanol Geraniol Propan-2-01 Butan-Z-01 Benzyl alcohol Propan-2-methyl-2-01 Terpineol
0.2 0.8 1.0 0.5 0.4 0.1 0.6 0.0 0.0 0.0 0.0 0.0
240 were esterified most efficiently. Alcohols having longer chains were esterified more slowly. Secondary and tertiary alcohols were not esterified by the rape lipase. Benzyl alcohol, a secondary alcohol, was not esterified and its presence caused the almost total inhibition of the esterification of n-butanol. Discussion The rape lipase has a very unusual specificity in accepting most natural saturated and unsaturated fatty acids but discriminating against those with the initial double bond being cis-4 or cis-6. Such a fatty acid specificity has not previously been reported for any lipase. The characteristics of the rape lipase can be compared with those of Geotrichum candidum [?I]. In the case of G. candidum, C,, acyl moieties containing cis-9 double bonds such as oleoyl and linoleoyl moieties were preferentially hydrolyzed from triacylglycerols, whereas, elaidoyl moieites (trans-9-18 : 1) and acyl groups having cis double bonds in other positions were much more slowly hydrolyzed. In the case of pancreatic lipase it was found that a cis double bond at any carbon up to C, caused inhibition of lipase activity [14]. In view of these findings, the lipase from oilseed rape might well prove useful for the isolation of unusual fatty acids, such as y-linolenic or petroselinic acids from seed oils of evening primrose (Oenothera biennis L.) or coriander (Car~and~m sativ~ IL.), respectively. For example, enrichment of fatty acids with cisd double bonds from fatty acid mixtures made from these oils could be achieved by esterifying all fatty acids except the cis-6 unsaturated fatty acid to butanol or another alcohol. The enrichment of docosahexaenoic acid from fish oils could also be envisaged. At present it is not possible to give a definite explanation for the selectivity shown by the rape lipase. It is interesting to note, however, that only those fatty acids with the first double bond being cis-4 or cis-6 are poor substrates for the lipase, whereas those in which the first double bond is cis-9 or cis-5 are utilized quite rapidly. If one considers that fatty acids with cis double bonds at odd or even number carbons have syn- or antiorientations, respectively, with respect to the earboxyl group ]lS], then from the data reported here it appears that rape lipase discriminates against anti- oriented cis-unsaturated fatty acids. It is possible that the direc-
tion of twist of the carbon chain after the first double bond of these fatty acids might hinder binding of the reactive group to the lipase. In a similar manner, but opposite way, an acyltransferase was shown to select anti, rather than syn conformations [15]. Further study with a range of synthetic isomers of monoenoic fatty acids, as reported for pancreatic lipase [ 141, would clarify this point. An unsaturated fatty acid with a trans double bond has a configuration similar to a saturated fatty acid and we found that the rates of esterification of elaidic and petroselaidic acids were similar to that of stearic acid.
This work was supported in part by a research grant from the Bundesrninisterium fir Ernlhrung, Landwirtschaft und Forsten, Bonn, Federal Republic of Germany. References 1 Kloosterman, M., Elferrink, V.H.M., Van Iersal, J., Roskam, J., Meijer, E.M., Hulshof, L.A. and Sheldon, R.A. (1988) Trends Biotechnol. 5, 251-254. 2 Macrae, A.R. and Hammond, R.C. (1985) Biotechnol. Genet. Eng. Rev. 3, 193-217. 3 Haraldsson, G.G., Hiiskuldsson, P.A., Sigurdsson, S.Th., Thorsteinsson, F. and Gudbjarnason, S. (1989) Tetrahedron Lett. 30, 1671-1674. 4 Sonnet, P.E. and Welch Baiilargeon, M. (1989) Lipids 24,434-437. 5 Brockerhof, H. and Jensen, R.G. (1974) in Lipolytic Enzymes, pp. 140-146, Academic Press, New York. 6 Hassanien, F.R. and Mukhejee, K.D. (1986) J. Am. Oil Chem. Sot. 63, 893-897. 7 Huang, A.H.C., Lin, Y. and Wang, S. (1988) J. Am. Oil Chem. Sot. 65, 897-899. 8 Hills, M.J. and O’Sullivan, J.N. (1989) B&hem. Sot. Trans. 17, 480. 9 Hills, M.J., Kiewitt, I. and Mukherjee, K.D. (1989) B&hem. Sot. Trans. 17, 478. 10 Hills, M.J. and Beevers, H. (1987) Plant Physiol. 85, 1084-1088. 11 Chapus, C., Semeriva, M., Bovier-Lapierre, C. and DesnueIle, P. (1976) Biochemistry 15, 4980-4987. 12 Lin, Y.H., Yu, C. and Huang, A.H.C. (1986) Arch. Biochem. Biophys. 244, 346-356. 13 Hills, M.J. and Murphy, D.J. (1988) Biochem. J. 249, 687-693. 14 Heimermann, W.H., Holman, R.T., Gordon, D.T., Kowalyshyn, D.E. and Jensen, R.G. (1973) Lipids 8, 45-46. 15 Lands, W.E.M. (1979) in Geometrical and Positional Fatty Acid Isomers (Emken, E.A. and Dutton, H.J., eds.), pp. 181-212, American Oil Chemists’ Society, Champaign, IL.
