FEMS Microbiology Letters 111 (1993) 207-212
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
207
FEMSLE 05540
Mechanism of ethyl acetate synthesis
by Kluyveromyces fragilis
H61a Kallel-Mhiri and Andr6 Miclo
Laboratoire de Biologie industrieUe et alimentaire, ENSAIA, Vandoeuvre-les-Nancy, France
(Received 2 April 1993; revision received 30 April 1993; accepted 1 May 1993)
Abstract: The enzymes implicated in ethyl acetate synthesis and the catabolism of ethanol by Kluyveromyces fragilis were
investigated under varying growth conditions. The culture was grown continuously to D = 0.25 h - i on diluted whey permeate. The
results showed that ethyl acetate synthesis by Kluyveromyces fragilis is catalysed by both an esterase and an alcohol acetyltransferase. The esterase is a constitutive enzyme, while alcohol acetyltransferase is inducible. The catabolism of ethanol by
Kluyveromyces fragilis resulted in production of ethyl acetate, acetate and acetaldehyde. The glyoxylic shunt is totally inactive in
these conditions. The production of acetaldehyde is only governed by an alcohol dehydrogenase.
Key words: Kluyveromyces fragilis; Whey permeate; Ethyl acetate; Esterase; Alcohol acetyltransferase
Introduction
In previous work [1] we have studied the synthesis of ethyl acetate by Kluyveromyces fragilis
on diluted whey permeate. We have shown that
the synthesis of ethyl acetate by this yeast requires in the broth both ethanol and lactose. To
further optimize ethyl acetate synthesis by this
yeast, the elucidation of the possible pathways for
ester production is necessary. The mechanism of
ethyl acetate synthesis by yeasts is not yet elucidated. Most of the studies were carried out with
the brewer's yeast. According to Howard and
Correspondence to: A. Miclo, Laboratoire de Biologic industrielle et alimentaire, ENSAIA, avenue de la for~t de Haye 2,
54500 Vandoeuvre-les-Nancy, France.
Anderson [2], Yoshioka and Hashimoto [3], Malcorps and Dufour [4] and Ramos-Jeunehomme et
al. [5], ethyl acetate synthesis is only due to an
alcohol acetyltransferase (condensation of acetylCoA with ethanol). However, Schermers et al. [6]
and Suomalainen [7] demonstrated the existence
of an esterase activity in Saccharomyces cerevisiae, which allowed the synthesis of ethyl acetate from ethanol and acetate. Thomas and
Dawson [8] showed that ethyl acetate synthesis by
Candida utilis is governed by an alcohol acetyltransferase. In Hansenula anomala ethyl acetate
production is due to an esterase [9].
The aim of this paper was to elucidate the
mechanism of ethyl acetate synthesis by Kluyveromyces fragilis grown on diluted whey permeate
supplemented with ethanol. The activities of several enzymes implicated in the synthesis of ethyl
208
acetate and the catabolism of ethanol (alcohol
acetyltransferase, esterase, alcohol dehydrogenase, alcohol oxidase and isocitrate lyase) were
investigated.
Materials and Methods
Microorganism
Kluyveromyces fragilis isolated from a dairy
product was maintained on yeast malt agar slopes
at 4°C.
Growth conditions
The yeast was grown continuously at a dilution
rate of 0.25 h - 1 in a 2-I Biolafitte fermentor (LSL
Biolafitte, Saint-Germain-en-Laye, France). The
working volume was 1 1. The medium contained
whey permeate diluted to a lactose concentration
of 15 g 1-1 supplemented with 1 g 1-1 of yeast
extract and ammonium sulfate at a concentration
of 1 g 1-1 . Temperature was kept at 35°C, a
condenser was placed at the gas outlet to minimize the stripping of the volatiles, pH was regulated at 4.5 by N a O H 2 N. Dissolved oxygen was
recorded by a polarographic probe.
Residual lactose
Culture samples were filtered through a 0.22/~m Millipore filter. Lactose was determined using an enzymatic kit (Boehringer, Meylan, France,
ref. 176-303).
Dry weight
10 ml of cell suspension were centrifuged at
7000 × g for 10 min, washed with distilled water,
centrifuged again then dried at 105°C for 24 h.
Analysis of volatiles
Ethanol, acetaldehyde and ethyl acetate were
detected by a gas membrane sensor as described
in previous work [1]. Acetate was assayed by a gas
chromatograph equipped with a flame ionization
detector and 2 m x 2 mm nickel column packed
with Porapak Q 80. The carrier gas used was
nitrogen. The temperature of the oven was set at
170°C, the injector and the detector at 220°C.
Preparation of cell extract
Cells were harvested from the continuous culture, washed twice with distilled water then suspended in 0.02 M phosphate buffer (pH = 7.4)
containing 0.1 M 2-mercaptoethanol and 20 mg
Pronase (Merck, Darmstadt, F R G ) per g wet
weight cells. This suspension was incubated at
30°C for 30 min with slight shaking. The pretreated cells were washed with an osmotic solution containing 1.4 M KCI and suspended in the
same solution. Then 22 mg Cytohelicase (I.B.F.,
Villeneuve-la-Garenne, France) per g wet weight
cells was added to the suspension and the mixture was incubated at 30°C under slight stirring
for 2.5 h. Lysis of sphaeroplasts was obtained by
suspension in 0.1 M phosphate buffer containing
0.01 M KCI adjusted to p H 7. The homogenate
obtained was treated with a sonic desintegrator
for 2 min then centrifuged at 20 000 x g for 20
min. The supernatant obtained was used for the
enzyme activity assays.
Enzyme assays
Alcohol dehydrogenase. The assay mixture contained glycine-KOH buffer (pH 10, 0.1 M), NAD +
(1 mg m1-1) and the extract. The reaction was
started with 200 mM ethanol. Enzyme activity is
expressed in /zmol N A D H produced min -1 (mg
protein)- 1.
Isocitrate lyase. This activity was measured following the method of Dixon and Kornberg [10].
The assay mixture contained phosphate buffer 0.1
M (pH 7.0), phenylhydrazine 0.1 M, MgC1 z 0.1
M, cysteine 0.06 M and the extract. The final
volume was 3 ml. The reaction was started by
addition of isocitrate 0.1 M. Enzyme activity is
expressed as /xmol of glyoxylate formed min -~
(mg protein)- 1.
Alcohol acetyltransferase. Alcohol acetyltransferase activity was determined following the
method of Yoshioka and Hashimoto [3]. The reaction mixture contained 0.8 mM acetyl-CoA, 15
mM isoamyl alcohol and the cell extract suspended in phosphate buffer M / 1 5 (pH 7.5), in a
10-ml reaction flask with a silicone rubber stopper. The final volume was 2 ml. This mixture was
incubated for 30 min at 30°C. The reaction was
stopped by adding 10/zl of H2SO 4 6 N. 20/xl of
209
acetone were added as an internal standard. Then
2 g of NaC1 were added. The vials were then
heated at 45°C for 30 min. The isoamyl acetate
content was determined by head space gas liquid
chromatography under the following conditions:
column 4 m × 0.0032 m stainless steel packed
with 10% Carbowax 20 M on chromosorb W
80/100 mesh AW-DMCS, oven temperature
80°C; injector and detector temperatures 220°C.
The carrier gas was nitrogen, head space sample
size was 0.5 ml. Alcohol acetyltransferase activity
is expressed in /xmol of isoamyl acetate formed
m i n - 1 (mg protein)- ~.
Esterase. The esterase activity was assayed in 2
ml of 0.1 M acetate buffer containing 15 mM of
isoamyl alcohol, 7 mM MgCI 2 and cell extract.
The reaction mixture was incubated for 30 min at
30°C. The isoamyl acetate content was determined in the same manner as for the ~dcohol
acetyltransferase assay.
Alcohol oxidase. Alcohol oxidase was assayed
following the method used by Glenwyn et al. [11].
The assay contained in 1.5 ml: 50 mM Na 2
H P O 4 / N a H 2 P O 4 buffer, 0.8 mM 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS),
60 tzg peroxidase and cell extract. The reaction
was started with 50 mM ethanol.
Proteins
Protein concentrations in cell-free extracts
were determined by Lowry method [12] with
bovine serum albumin as a standard.
Carbon balance
Carbon balance for ethanol was calculated according to:
The stripping coefficients of acetaldehyde,
ethyl acetate and ethanol were determined experimentally. They are respectively 0.035, 0.09 and
0.016 h - 1.
Results
We have shown in previous studies [1] that
ethyl acetate synthesis by Kluyveromyces fragilis
required the presence of ethanol in the medium.
For the degradation of ethanol by this yeast,
three pathways are possible, all summarized in
Fig. 1. In order to quantify the contribution of
each pathway in the catabolism of ethanol by
Kluyveromyces fragilis, the activities of the enzymatic markers of each pathway have been followed.
The first pathway leads to ethyl acetate synthesis by condensation of ethanol with acetyl-CoA
(alcohol acetyltransferase) or with acetate (esterase). The second consists in the transformation of
ethanol to acetaldehyde. For this pathway, the
activity of alcohol dehydrogenase and alcohol oxidase are investigated. As a last possibility, ethanol
can be used via the glyoxylic shunt, therefore the
activity of isocitrate lyase is followed.
The activities of these enzymes were studied in
a continuous culture at D = 0.25 h -~ (Fig. 2).
The environment of the yeast was varied to favour,
successively, ethanol production, ethyl acetate
Lactose
C metabolites + Cstripping of metabolites
Cethanol -- Cstripping of ethanol
where Cmetabolites, Cstripping of metabolites a r e the
ethanol carbon transformed in the different
metabolites (ethyl acetate, acetate and acetaldehyde) and the carbon of metabolites lost by the
stripping, respectively. Cethano I represents the
ethanol carbon which disappears from the culture
medium and Cstripping of ethanol is the ethanol carbon lost by the stripping.
Fig. 1. Possible pathways for ethanol metabolism in Kluyveromyces fragilis grown on whey permeate.
210
~120
a
tion were varied to have in each case only one of
the three pathways mentioned above.
I
~ go0
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Ethanol production
The production of ethanol by Kluyveromyces
fragilis on diluted whey permeate is enhanced by
• 1,2
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Ethyl acetate production
-0
20
40
60
80
100
limiting the level of dissolved oxygen. For D =
0.25 h-1, at the steady state, the activities of the
five enzymes mentioned above were determined.
The results are presented in Table 1.
These results indicate that despite low levels
of ethyl acetate (0.1 g 1-1) and acetaldehyde (0.1
g 1-1), alcohol acetyltransferase and esterase activities (ethyl acetate synthesis) are at a high
level. The same observation is reported for alcohol dehydrogenase activity (acetaldehyde synthesis). Neither activity for alcohol oxidase (acetaldehyde synthesis) nor for isocitrate lyase (glyoxylic shunt) was detected in these conditions•
120
T i m e (hours)
Fig. 2. Continuous culture o f Kluyverornyces fragilis at D =
0.25 h - i on diluted whey permeate. (a) Evolution of dissolved
oxygen; (b) evolution of volatiles products; and (c) evolution
of biomass and residual lactose. The arrows indicate the time
of cells harvesting for the enzymatic assays.
synthesis, acetaldehyde accumulation or yeast
growth without any accumulation of metabolites.
The level of dissolved oxygen and broth composi-
To favour ethyl acetate production, ethanol at
4 g 1-1 was added to the feed, oxygen level was
maintained at 40%. At the steady state, ethyl
acetate level reached 3 g 1-1. The activities of the
five enzymes were measured. Table 1 shows that
both alcohol acetyltransferase and esterase were
active. Nevertheless, compared to the results obtained in the case of ethanol production, the
activity of alcohol acetyltransferase was higher,
whereas the activities of esterase and alcohol
dehydrogenase had the same levels.
The activities of alcohol oxidase and of isocitrate lyase were not detected.
Table 1
Specific activities (/zmol (mg protein)-1 m i n - I) of alcohol dehydrogenase, alcohol oxidase, alcohol acetyltransferase, esterase and
isocitrate lyase measured in Kluyveromyces fragilis grown continuously at D = 0.25 h - 1 on diluted whey permeate under different
growth conditions
Growth stage
Ethanol production
Ethyl acetate synthesis
Acetaldehyde accumulation
Specific activity (U mg protein - 1)
Alcohol
dehydrogenase
Alcohol
oxidase
Alcohol
acetyltransferase
Esterase
Isocitrate
lyase
3.5
3.6
36
nd a
nd
nd
4.5
5.2
2.9
4.3
3.6
3.7
nd
nd
nd
Times of cells harvesting are indicated by arrows in Fig. 2.
a nd, not detected.
21i
Ethyl acetate and acetaldehyde production
To,favour acetaldehyd6 accumula'tiOn, the level
of dissolved oxygen was naaintaine~i a t 100%.
Both ethyl acetate and acetaldehyde were synthesized by Kluyveromyces fragilis. At the .steady
state, their levels were 1.5 and 0.4 g l -l, respectively. The activities of the five enzymes have
been characterized.
Table 1 shows that alcohol acetyltransferase
and esterase are still active. However, the activity
of alcohol acetyltransferase was reduced by 44%
and the esterase activity remained unchanged.
In these growth conditions, a high increase in
alcohol dehydrogenase activity (10-fold) was
noted, no activity of alcohol oxidase and isocitrate lyase was detected.
Biomass production
In previous work, we showed that iron
markedly decreases ethyl acetate synthesis and
favours an oxidative catabolism of lactose [1]. The
activities of the enzymes responsible for ethyl
acetate synthesis in Kluyveromyces fragilis w e r e
followed. This yeast was cultivated in batch culture on the medium mentioned in Materials and
Methods supplemented with 4 mg 1'-1 of FeCI 3.
At the end of the exponential growth, cells were
harvested to measure the activities of the two
enzymes. We obtained 2.6 U mg-1 of protein for
alcohol acetyltransferase activity and 3.8 U mg-1
of protein for the activity of esterase.
These results show that in spite of presence of
iron, which strongly reduces ester synthesis, the
activity of esterase was at the same level as in the
conditions of ethanol production and ethanol
catabolism. Nevertheless, the alcohol acetyltransferase activity was reduced by 50%.
Discussion
Our results show that the activity of isocitrate
lyase was not detected, so growth of Kluyveromyces fragilis did not occur on ethanol via the
glyoxylic shunt. Ethanol degradation serves mainly
to ethyl acetate, acetaldehyde or acetate synthesis
and slightly for energy production via the tricarboxylic acid cycle. This hypothesis was verified by
a carbon balance. The carbon balance indicates
that in the case of ' e t ~ l acetate production77%
of the carbon supplied as ethanol was converted
to ethyl acetate and acetate. When growth conditions allowed an accumulation of acetaldehyde,
99% of the carbon provided from ethanol was
converted to ethyl acetate, acetaldehyde and iacetate.
~
The analysis of acetaldehyde synthesis by
Kluyveromyces fragilis revealed that this pathway
is governed by alcohol dehydrogenase. No activitY
of alcohol oxidas~ was observed. The ~ame result
was reported in Candida utilis and ~Saccharomyces cerevisiae [3,14]. In contrast, in methylotrophic yeasts such as Pichia pastoris acetaldehyde synthesis results from an alcohol oxidase
activity [13].
The evolution of alcohol acetyltransferase and
esterase activities indicated that the first enzyme
is inducible whilst the second enzyme is constitutive. Esterase activity remained coilstant for all
the cases studied whereas alcohol acetyltransferase changed with growth Conditions. Compared to the results obtained by Ramos-Jeunehomme et al. [5] for the brewer's yeast, the activity of alcohol acetyltransferase in Kluyveromyces
fragilis is 100-fold,higller.
The synl;hesis o~,ethyl acetate occurred on
both lactose and ethanol. Lactose supplies acetylCoA to produce ethyl~ acetate by condensation
with ethanol. This result was confirmed by the
evolution of the alcohol acetyltransferase activity
and residual lactose (Fig. 2). We observed that
when the ethyl acetate concentration is high, the
level of residual lactose was low and alcohol
acetyltransferase activity was high. In the case of
a high concentration of residual lactose in the
broth, both the synthesis of ethyl acetate and the
activity of alcohol acetyltransferase were low.
The comparison of the mechanism of ethyl
acetate synthesis by Kluyueromyces fragilis to that
already known for other strains, Candida utilis,
Saccharomyces cerevisiae and Hansenula anomala,
indicated that each strain has a specific mechanism.
In Candida utilis [8,14] and Saccharomyces
cerevisiae [3] ethyl acetate is governed by an alcohol acetyltransferase, whilst in Hansenula ano-
212
mala the synthesis is related to an esterase activity. In Kluyverornyces fragilis the two enzymes are
implicated in the process of ethyl acetate synthesis.
6
7
Acknowledgements
8
The authors thank Dr. M. Ghoul for critically
reading the manuscript. They are also grateful to
the Institut de Biotechnologie de Nancy (Prof.
J.-M. Engasser) for financial support.
9
10
References
1 Kallel-Mhiri, H., Engasser, J.M. and Miclo, A. Continuous
ethyl acetate production by Kluyveromyces fragilis on whey
permeate. Appl. Microbiol. Biotechnol., submitted.
2 Howard, D. and Anderson, R.G. (1976) Ceil-free synthesis
of ethyl acetate extracts from Saccharomyces cerevisiae. J.
Inst. Brew. 82, 70-71.
3 Yoshioka, K. and Hashimoto, N. (1981) Ester formation by
alcohol acetyltransferase from brewers' yeast. Agric. Biol.
' Chem. 45, 2183-2190.
4 Malcorps, Ph. and Dufour, J.P. (1987) Ester synthesis by
Saccharomyces cerevisiae: localisation of the acetylCoA: isoamyl alcohol transfcrase ("AT")I In: Proceedings
of the European Brewery Convention Congress, Madrid,
pp. 377-384. IRL Press, London.
5 Ramos-Jeunehomme, C., Laub, R. and Masschelein, C.A.
11
12
13
14
(1989) The relationship of subcellular localization/ester
synthesizing activity of alcohol acetyltransferase in brewery
fermentations. In: Proceedings of the European Brewery
Convention Congress, Ziirich, pp. 513-519. IRL Press,
London.
Schermers, F.H., Duffour, J.H. at/d MacLeod, A.M. (1976)
Studies on yeast esterase. J. Inst. Brew. 82, 170-174.
Suomalainen, H. (1981) Yeast esterases and aroma esters
in alcoholic beverages. J. Inst. Brew. 87, 296-300.
Thomas, K.C. and Dawson, P.S.S. (1978) Relationship
between iron-limited growth and energy limitation during
phased cultivation of Candida utilis. Can. J. Microbiol. 24,
441-447.
Ranadive, K. and Tamhane, D.V. (1985) Flavour production by Hansenula anomala by directed fermentation. In:
Newer approaches of biological applications, Bombay, pp.
215-219.
Dixon, G.H. and Kornberg, H.L. (1959) Assay methOds for
key enzymes of the glyoxylate cycle. Biochem.~J. 72, 3p.
Glenwyn, D.K., Dickinson, M.F. and Ratledge, C. (1988)
Inducible long chain alcohol oxidase from alkane-grown
Candida tropicalis. Appl. Microbiol. Biotechnol. 29, 370374.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall,
R.J. (1951) Protein measurement with folin phenol reagent.
J. Biol. Chem. 193, 265-275.
Murray, W.D., Duff, S.J.B., Lanthier, P.H., Armstrong,
D.W., Welsh, F.W. and Williams, R.E. (1987) Development of biotechnological processes for the production of
natural flavours and fragrances. In: Proceedings of the 5th
International Flavour Conference, Porto Karras, Halkidiki,
Greece (Charalambous, Ed.), pp. 1-18. Elsevier, Amsterdam.
Armstrong, D.W., Martin, S.M. and Yamazaki, H. (1984)
Production of ethyl acetate from dilute ethanol solutions
by Candida utilis. Bioteehnol. Bioeng. 26, 1038-1041.