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Kluyveromyces Fragilis: Mechanism of Ethyl Acetate Synthesis by
Kluyveromyces Fragilis: Mechanism of Ethyl Acetate Synthesis by
Kluyveromyces Fragilis: Mechanism of Ethyl Acetate Synthesis by
FEMSLE 05540
(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 acetyltrans-
ferase. 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
acetone were added as an internal standard. Then The stripping coefficients of acetaldehyde,
2 g of NaC1 were added. The vials were then ethyl acetate and ethanol were determined exper-
heated at 45°C for 30 min. The isoamyl acetate imentally. They are respectively 0.035, 0.09 and
content was determined by head space gas liquid 0.016 h - 1.
chromatography under the following conditions:
column 4 m × 0.0032 m stainless steel packed
with 10% Carbowax 20 M on chromosorb W Results
80/100 mesh AW-DMCS, oven temperature
80°C; injector and detector temperatures 220°C. We have shown in previous studies [1] that
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 Specific activity (U mg protein - 1)
Alcohol Alcohol Alcohol Esterase Isocitrate
dehydrogenase oxidase acetyltransferase lyase
Ethanol production 3.5 nd a 4.5 4.3 nd
Ethyl acetate synthesis 3.6 nd 5.2 3.6 nd
Acetaldehyde accumulation 36 nd 2.9 3.7 nd
Times of cells harvesting are indicated by arrows in Fig. 2.
a nd, not detected.
21i
Ethyl acetate and acetaldehyde production a carbon balance. The carbon balance indicates
To,favour acetaldehyd6 accumula'tiOn, the level that in the case of ' e t ~ l acetate production77%
of dissolved oxygen was naaintaine~i a t 100%. of the carbon supplied as ethanol was converted
Both ethyl acetate and acetaldehyde were synthe- to ethyl acetate and acetate. When growth condi-
sized by Kluyveromyces fragilis. At the .steady tions allowed an accumulation of acetaldehyde,
state, their levels were 1.5 and 0.4 g l -l, respec- 99% of the carbon provided from ethanol was
tively. The activities of the five enzymes have converted to ethyl acetate, acetaldehyde and iac-
been characterized. etate. ~
Table 1 shows that alcohol acetyltransferase The analysis of acetaldehyde synthesis by
mala the synthesis is related to an esterase activ- (1989) The relationship of subcellular localization/ester
ity. In Kluyverornyces fragilis the two enzymes are synthesizing activity of alcohol acetyltransferase in brewery
fermentations. In: Proceedings of the European Brewery
implicated in the process of ethyl acetate synthe- Convention Congress, Ziirich, pp. 513-519. IRL Press,
sis. London.
6 Schermers, F.H., Duffour, J.H. at/d MacLeod, A.M. (1976)
Studies on yeast esterase. J. Inst. Brew. 82, 170-174.
7 Suomalainen, H. (1981) Yeast esterases and aroma esters
Acknowledgements in alcoholic beverages. J. Inst. Brew. 87, 296-300.
8 Thomas, K.C. and Dawson, P.S.S. (1978) Relationship
between iron-limited growth and energy limitation during