BIOTECHNOLOGY LETTERS Volume 18 No. 8 (August) pp.869-874 Received as revised 28 May
ESTER SYNTHESIS IN AQI!EOUS MEDIA IN THE PRESENCE OF VARIOUS LIPASES Clotilde Lecointe, Eric Dubreucq* and Pierre Galzy UFR de Microbiologie Industrielle et de Gendtique des Microorganismes ENSA-INRA, Place Viala, F-34060 Montpellier Cddex, France
SITMMARY The ability of seven lipase preparations to catalyse methyl ester synthesis in aqueous media was compared and the synthesis reaction (esterification or alcoholysis) determined. Three behaviours were observed: three enz)qnes catalysed ester synthesis by esterification of free fatty acids and one enzyme catalysed alcoholysis but the other three lipases did not catalyse a net ester synthesis under the conditions tested. The three groups also differed by the influence of methanol on the hydrolysis reaction. The first group was not significantly inhibited up to the highest methanol concentration tested (5 M). Hydrolysis in the presence of the enzyme of the second group was increasinglyinhibitedwith increasing methanol concentrations. In the presence of the third group, hydrolysis was 40 to 50% inhibited for all the concentrationstested (0.2-5 M).
INTRODUCTION Lipases (or glycerol ester hydrolases, EC 3.1.1.3) are characterized by their ability to catalyse the hydrolysis of triacylglycerols and, under certain conditions, ester production by esterification, alcoholysis, acidolysis or inter-esterification. Although most of the published studies of lipase-catalysed ester synthesis have been peIformed in non-aqueous media, some lipases have been shown to catalyse ester synthesis in aqueous media (Okumara et al. 1979; Nishio et al, 1987; Briand et al., 1994 : Boutur et al., 1994 ; McNeill and Berger, 1995 ; Linko et al., 1995). In the presence of alcohol in the aqueous phase, esters can be produced by two mechanisms : a direct transfer of acyl groups fiom an acyl donor ester by alcoolysis, or the esterification of free fatty acids, eventually resulting from the hydrolysis of an acyl donor ester. The first mechanism does not involve water as a reactant. It has been shown that in the presence of various
869
donor esters, free fatty" acids and alcohols, the slightly
1(3)-regioselective
lipase/acyltransferase from ("amtJdct parapsJlosis only catalyses alcoholysis in aqueous media (Briand el al., 1995), whereas the formation o f esters catalysed in aqueous media by the l(3)-regiospecific lipase from ("andJda d e / o r m a m is the result o f an esterification o f free fatty acids (Boutur et al., 1995). In the work reported here, the ester synthesis activities o f seven lipase preparations have been compared and the reactions that are involved have been determined,
MATERIALS AND METHODS Materials: One enzyme unit (U) releases one ~tmol fatty acid in one mm. In these conditions, units are determined using triolein as substrate, at 45°C and pH 65, according to the protocol described below. The enzymes from Ah~cor mJehci (Mm; specific acti~lty : 24 U.mg1 measured by Fluka / 0.45 U.mg"1 in our conditions). RhJzoplts delemar (Rd ; 46 U.mgl/0.85 U.mg'l). t3~eudomonas cepac:a (Pc ; 600 U.mg~ / 3.3 U.mg'~). Pseudomonas" fluorescens (Pf; 3560 U.mg"~/ 330 U mg"1) were purchased from Fluka Biochemika. The lipase from RhJzolms arrhJzus (Ra: type XI, 6600U.mg'~/1230 U.mg~) was that commercialized by Sigma Chemical. The lipase from ('amhdtt d~Jormcms (Cd; 16U.mg"~ protein) and the lipase/acyltransferase from ('andJda parapsilosJs (Cp; 20U.mg~ protein) were prepared according to Boutur et al. (10o5) and Briand et al. (1005) respectively. All these preparations were free enzymes and used as such. A SDS-PAGE electrophoregram of the enzyme preparations, revealed by silver staining, showed one major band for each. Enzyme assays: An emulsion of lipid substrates (200 ~tl of 20 g.l "l polyvmyl alcohol containing 10 lamol lipids), was added to 1 ml 50 mM potassium phosphate, pH 6.5, possibly containing alcohol, and about 0.25 enzyme units (U) contained in one ml of the same buffer. The reaction was carried out at 45°C for 15 min and stopped by the addition of 4 ml of an ethanol / acetone / sulphuric acid (50:50:0.1, v/v/v) mixture. Lipids were extracted twice with 1 ml hexane. Analysis of the products: The reaction products were separated by thin-layer chromatography as described by Boutur et at. (1005). The silice containing lipids was scraped off the plate and placed in a capped microtube. After the addition of I ml diethyl ether the tube was vortexed, then water were added (100 tal for 1 mg silice) in order to hydrate the silica gel and ensure a full recovering of the lipids, especially free fatty acids. After agitation, the organic phase was recovered. Two other extractions were realized with 1 ml diethyl ether only. The ether phases were then pooled and the solvent evaporated. The fatty acid composition of the separated lipids was then determined by gas chromatography after saponification and methylation according to Boutur et al. (1005).The analytical procedure, including TLC separation, was standardized for each class of lipids using pure commercial samples.
870
RESULTS AND DISCI SSION Ester synthesis activity The influence of methanol concentration on ester synthesis in the presence of the various lipase preparations and 10 p.mol triolein is shown on Figure l. The water:alcohol molar ratio in the aqueous phase was initially between 11.2 and 280 (for respectively 5 M and 0.2 M methanol). A significant ester synthesis, increasing with methanol concentration, was obtained in the presence of the enzymes from ('. parapsJlosJs, M. mJeheJ, R. delemar and (~. deJbrmans. The three other lipases did not catalyse or only slightly catalysed a net methyl ester synthesis. The two categories remained the same when 1-butanol (0.15 M) was used instead of methanol, with the difference that the ester synthesis activity of the enzyme from ('. deformans was much higher, being equivalent to that of
M. mJeheJ and R. delemar. Methanol concentration also had different effects on the hydrolysis activity of the enzymes. Three behaviours can be distinguished on figure 2 : • the enzymes from M. miehei, R. delemar and ('. deJbrmans were only slightly influenced by the methanol concentrations tested : • the activity of the lipases that did not catalyse a net ester synthesis was about 40-50% inhibited in the presence of the lowest methanol concentration tested (0.2 M), and this inhibition was the same for higher concentrations ; • the hydrolysis activity of the enzyme from (~.parapsJlosis decreased continuously with increasing methanol concentrations. In the presence of 5 M methanol, when ester synthesis activity was maximum, hydrolysis was 80 % inhibited.
Ester synthesis reaction involved Table 1 gives the fatty acid composition of the methyl esters and the free fatty acids in the reactant medium after 15 rain of reaction in the presence of 0.25 U of
871
Esters formed (pmollmin) 0 30s - ~ - X
0.25-
020-
o
MmI
•
Rd I
--A--
0.15-
Ra J
•
Pc I
•
Pf J
x Cp ~3 Cd
0107
005
0 0
1
2
3
4
5
Methanol (M) Figure 1 : Influence of methanol concentration on methyl ester synthesis in the presence oftriolein
Fatty acids released (pmollmin) 0
.
3
0
~
0.25' .:~-..e..---~e
[]
X
0.20 ,~.
-~ . . . . . . .
---e-- Mrr ---4--- Rd
_&,
0 1 0 - t"
•
~'- "- { . . Q
....
-
Z
".': _ ~ ~
--.--
...... ~ x..
0.10-
• -x-
+ • ,.,
Pc pf
Cp
Cd
, "'-X
0.05 l I
0-
I
0
1
-I
2
~
~
3
4
---4
5
Methanol (M)
Figure 2 ' Influence of methanol concentration on the hydrolysis activity. The lipid substrate was triolein
872
the various lipases, equimolar quantities (5~tmol) o f a triacylglycerol and a free fatty acid, and 2.2 M methanol. The use o f a mixture o f a triacylglycerol and a free fatty acid as substrates was intended to permit the determination o f the origin o f the esters. In order to avoid misinterpretations due to the substrate specificity o f the enzymes, two sets of experiments were performed in parallel, one with tripalmitolein and oleic acid and the other with triolein and palmitoleic acid.
Table 1. Products formed after 15 minutes reaction time in two reactant media Mm
Rd
Ra
Pc
Pf
Cd
(~
tripalmitolein + oleic acid Free fatty acids (~tmol) C18:1 (%) C16:1 (%)
4.4 40 60
4.5 41 50
4.5 40 60
4.7 49 51
4.5 60 31
5.2 44 56
4.0 03 7
Methyl esters (~mol) C18:1 (%) Ci6:1 (%)
1.3 37 63
1.3 35 65
0
0 -
0
0.3 45 55
2.3 0 100
triolein + palmitoleic acid Free fatty acids (lamol) Cl8:l (%) C16:1 (%)
4.2 73 27
4.5 73 27
4.4 66 34
5.0 53 47
5.0 12 88
4.8 44 56
3.0 21 70
Methyl esters (lamol) C18:1 (%) C16:l (%)
1.6 68 32
0.7 67 33
0
0 -
0
0.4 45 55
1.7 75 25
Experimental conditions are described in Materials and Methods. 31m. 3htcor miehei lipase: Rd. Rhizopus delemar lipase: Ra, Rhizopus arrhizus lipase: Pc'. Pseudomonas cepacia lipase: PJ. Pseudomonas JTuorescens lipase: Cd. Candida deformans lipase: (~o. Candida parapsih~sis lipase. C 18:1. oleic acid: C 16:1. palmitoleic acid. Initial amounts ~veretriac.vlglyceroi : 5 lxmol : free fatty:acid : 5 ~tmol : methanol 2.2 M. A m o n g the
7 enzyme preparations
tested,
only the
enzymes
from
( : parapsilosis, C deJbrmans, M. miehei and Rh. delemar catalysed significant
ester
synthesis.
Except
in
the
case
of
the
lipase/acyltransferase
from
C. parapsilosis, the relative fatty acid composition o f the esters produced was
very close to that o f the free fatty acids present in the reactant medium, suggesting that the esters originated from the free fatty acids initially present in the reaction m e d i u m or released by the hydrolysis o f the acylglycerols. On the
873
contrary, esters produced in the presence of the enzyme from (~. parapsilosis were mainly of the same fatty acid composition as the triacylglycerol used as substrate, indicating an alcoholysis reaction as already observed by Briand (1995). It is to be noted that in the presence of triolein and palmitoleic acid, 25 % of the esters formed in the presence of this enzyme were esters of palmitoleic acid, suggesting the occurrence of an esterification reaction. A supplementary experiment using only free fatty acids as substrates confirmed that the esterproducing lipases, except for the enzyme from C. parapsJlosJs, were able to catalyse the esterification reaction ; the enzyme from C. parapsJlosJs displayed a slight esterification activity in the presence of palmitoleic acid but not oleic acid. Three groups of lipases can thus be distinguished in these reaction conditions. The first, consisting of the lipases from M. mJeheJ, Rh. delemar and
C. defi~rmans, catalyse the esterification of free fatty acids and methanol. The enzyme from CandidaparapsilosJs, which catalyses mainly alcoholysis in the conditions tested, constitutes a second group ; this enzyme is much more of an acyltransferase (EC 2,3.1), reacting with long chain acyl groups, than a lipase (EC 3.1.1.3). The enzymes belonging to the last group, i.e. the lipases from
Rhizopus arrhizus, Pseudomonas cepacia and Pseudomonas fluorescens, did not catalyse a net ester synthesis in the aqueous media tested.
REFERENCES Boutur O., Dubreucq E. and Galzy P. (1994). Biotechnol. Lett. 16, 1179-1182. Boutur O., Dubreucq E. and Galzy P. (1995). J. Biotechnol.42, 23-33 Briand D., Dubreucq E. and GaIzy P. (1994). Biotechnol Lett. 16, 813-818. Briand D., Dubreucq E. and Galzy P. (1995). Eur. J Biochem. 228, 169-175. Linko Y.Y., Lamsa M., Huhtala A, and Rantanen O. (1995).. J. Am. Oil Chem. Soc. 72, 1293-1299 MeNeill C.P., Berger R. (1995). OCL 2, 359-363. Nishio T., Chikano T. and Kamimura M. (1987). Agric Biol. Chem, 52, 1203-1208. Okumura S., Iwai m. and Tsujisaka Y. (1979). Biochim Biophys. acta, 575, 156-165.
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