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FEMS Microbiology Letters 77 (1991) 295-298 @1991 Federation of European Microbiological Societies 0378-1097/91/$03.

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295

ADONIS 037810979100060I
FEMSLE 04235

Levels of activity of enzymes involved in anaerobic utilization of sugars by six yeast species: observations towards understanding the Kluyver effect
A.P. Sims a n d J.A. B a r n e t t
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Received 27 July 1990 Accepted 29 July 1990

Key words: Yeast; Pyruvate decarboxylase; Alcohol dehydrogenase; Glycosidase; Kluyver effect

1. SUMMARY The activities of pyruvate decarboxylase, alcohol dehydrogenase and certain glycosidases were measured for six species of yeast. Five of these yeasts could utilize one or more disaccharides aerobically, but not anaerobically, although all could use D-glucose anaerobically. That is, each of the five showed the Kluyver effect; but the sixth yeast, Saccharomyces cerevisiae, did not do so. When grown on a glycoside with which it gave the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grown on D-glucose or another glycoside. There was no consistent corresponding lowering of activity of either alcohol dehydrogenase, o r of the appropriate glycosidase. Hence, pyruvate decarboxylase may have a role in producing the Kluyver effect.

2. I N T R O D U C T I O N Many yeasts can utilize particular disaccharides aerobically, but not anaerobically, although these yeasts can use one or more of the component monosaccharides anaerobically. For any given yeast, this Kluyver effect [1] may be found with one disaccharide, but not another. The effect is widespread amongst the fermenting yeasts, and has been reported in 97 species [1]. According to one survey [2], all the strains examined of 40 yeast species show the effect with maltose and 41 with cellobiose. Factors that might underlie the Kluyver effect include the following. (i) Aerobic catabolism may be necessary for the supply of ATP for the expulsion of protons from the cells; this is necessary in order to maintain the proton symport of active transport of glycosides across the plasmalemma into the yeast cell [1,3,4]. (ii) Levels of activity of the appropriate glycosidases, of pyruvate decarboxylase or of alcohol dehydrogenase may fall

Correspondence to: J.A. Barnett, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.

296 Table 1 Aerobic growth and anaerobic fermentation by six yeasts supplied with different sugars as sole source of carbon Yeast Carbon source a D-Glucose G F + + + + + + Maltose G + + + + + F + + + + + Cellobiose G + + + + + . F" + . . . Lactose G + + + F -

Candida v&wanathiiCBS 4024 Debaryomyces castetlii CBS 2923 Debaryomycespolymorphus CBS 4349 Kluyveromyces dobzhanskii CBS 2104 Kluyveromyces wickerhamii CBS 2745 Saccharomyces cerevisiae CBS 1171

+ + + + + +

a G = aerobic growth, F = anaerobic fermentation; results were consistent with those of Barnett et al. [2]; fermentation was confirmed by measuring ethanol production. below those required to m a i n t a i n catabolic flux. This paper reports m e a s u r e m e n t s of these e n z y m i c activities i n five yeasts that show the K l u y v e r effect a n d also in one that does n o t do so. m e d i u m , p r o d u c e d b y a n a e r o b i c f e r m e n t a t i o n , was m e a s u r e d e n z y m i c a l l y [5].

3.4. Enzymic assays


E n z y m e s were assayed in cell extracts o b t a i n e d b y harvesting the yeasts d u r i n g their e x p o n e n t i a l phase of growth a n d d i s i n t e g r a t i n g the cells with fine glass beads [6]. Glycosidases: 4 - n i t r o p h e n y l a-D-glucopyranoside. 4 - n i t r o p h e n y l fl-D-glucopyranoside or 4n i t r o p h e n y l fl-D-galactopyranoside were used to m e a s u r e a-glucosidase (EC 3.2.1.20), B-ghicosidase (EC 3.2.1.21) or fl-galactosidase ( E C 3.2.1.23) activities, respectively [1,3]. Alcohol dehydrogenase (EC 1.1.1.1) activity was m e a s u r e d with N A D * a n d e t h a n o l [5]. Pyruvate decarboxylase (EC 4.1.1.1) was estim a t e d as described b y Schmitt a n d Z i m m e r m a n n [7]. Protein c o n c e n t r a t i o n was estimated b y the m e t h o d of Lowry et al. [8], using b o v i n e serum a l b u m i n as standard.

3. M A T E R I A L S A N D M E T H O D S

3.1. Yeasts
The following yeasts, from the C e n t r a a l b u r e a u voor Schimmelcultures (CBS), were examined: Candida viswanathii S a n d h u a n d R a n d h a w a (CBS 4024), Debaryomyces castellii Capriotti (CBS 2923), Debaryomyces polymorphus (K15cker) Price a n d Phaff (CBS 4349), Kluyveromyces dobzhanskii (Shehata et al.) v a n der Walt (CBS 2104), Kluyveromyces wickerhamii (Phaff et al.) v a n der W a l t (CBS 2745), a n d Saccharomyces cerevisiae M e y e n ex H a n s e n (CBS 1171).

3.2. Media and growth conditions


The yeasts were m a i n t a i n e d a n d cultivated aerobically in shaken, liquid, chemically-defined m e d i u m at 25 C, as described previously [3], except that p r e - i n c u b a t i o n was o n slopes of Difco (Bacto) Yeast N i t r o g e n Base, to which were added 1% ( w / v ) sugar (D-glucose, maltose, cellobiose or lactose, c o r r e s p o n d i n g with that of the s u b s e q u e n t i n c u b a t i o n ) a n d 1.5% ( w / v ) agar.

4. R E S U L T S A N D D I S C U S S I O N T h e characteristics (taken from B a r n e t t et al. [2]) of each of the six yeasts e x a m i n e d (Table 1), were c o n f i r m e d e x p e r i m e n t a l l y ; the occurrence or a b s e n c e of a n a e r o b i c f e r m e n t a t i o n was det e r m i n e d b y m e a s u r i n g the f o r m a t i o n of ethanol enzymically. Candida viswanathii, Debaryomyces

3.3. Anaerobic conditions and measurement of ethanol in the suspending medium


A n a e r o b i c c o n d i t i o n s were u n d e r argon, as described previously [3]; ethanol in the s u s p e n d i n g

polymorphus, Kluyveromyces dobzhanskii and K. wickerhamii all showed the K l u y v e r effect with

297 Table 2 Specific activities a of three enzymes in six yeasts grown on different sugars as sole sources of carbon Yeast name and strain number Carbon source b for growth D-Glucose Maltose Cellobiose K D-Glucose Maltose Lactose K D-Glucose Maltose Cellobiose K D-Glucose Maltose CeUobiose K D-Glucose Cellobiose K Lactose K D-Glucose Maltose Pyruvate decarboxylase 0.13 0.11 0.039 0.17 0.067 < 0.001 0.18 0.35 0.008 0.083 0.31 0.040 0.16 0.020 0.020 1.62 0.40 Alcohol dehydrogenase 1.04 0.32 0.23 0.19 0.088 < 0.006 0.24 0.37 0.038 0.48 0.070 1.07 1.17 0.88 0.21 0.57 0.64 Glycosidase c

Candida viswanathii
CBS 4024 0.33 0.33 0.48 0.03 0.15 0.28 0.19 0.31 0.14 0.34 1.01

Debaryomyces castellii
CBS 2923

Debaryomyces polymorphus
CBS 4349

Kluyveromyces dobzhanskii
CBS 2104

Kluyveromyces wickerhamii
CBS 2745

Saccharomyces cerevisiae
CBS 1171

a Means of at least duplicate experiments, expressed as nmol substrate catalysed min-1 (mg protein)-1. b Substrates that give the Kluyver effect are indicated by a bold letter K. Yeasts grown on maltose were tested for a-glucosidase activity, yeasts grown on cellobiose for fl-glucosidase activity, yeasts grown on lactose for fl-galactosidase activity.

cellobiose; Debaryomyces castellii and K. wickerhamii gave the effect with lactose. Pyruvate decarboxylase is involved in metabolic regulation in yeasts [9] and is necessary for glycolysis to proceed under anaerobic, but not aerobic, conditions. In every case, pyruvate decarboxylase activities (Table 2) were lower in each yeast grown on a glycoside with which that yeast gave the Kluyver effect, than when grown on D-glucose or on a glycoside that was used anaerobically. The range of differences were from 48% of the higher rate for cellobiose-grown Kluyveromyces dobzhanskii to 0.6% for lactose-grown Debaryomyces castellii. Such differences did not generally apply to the activities of either alcohol dehydrogenase or the glycosidases. So the levels of pyruvate decarboxylase activity were highly associated with those of anaerobic fermentation and, hence, were determined by what

sugar was supplied exogenously. In relation to the Kluyver effect, it was the activity of pyruvate decarboxylase, rather than glycosidase or alcohol dehydrogenase, that appeared to be rate-limiting. This observation is consistent with the findings of van Urk and his colleagues [10] for Saccharornyces cerevisiae and another strain of Candida utilis that pyruvate decarboxylase levels are associated with the rate of catabolic flux in the anaerobic utilization of D-glucose. Accordingly, the role of the regulation of pyruvate decarboxylase in the Kluyver effect and the factors affecting its activity are being investigated in detail.

REFERENCES
[1] Sims, A.P. and Barnett, J.A. (1978) J. Gen. Microbiol. 106, 277-288.

298 [2] Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: Characteristics and Identification, 2nd edn. Cambridge University Press, Cambridge. [3] Barnett, J.A. and Sims, A.P. (1982) J. Gen. Microbiol. 128, 2303-2312. [4] Schulz, B. and HSfer, M. (1986) Arch. Microbiol. 145, 367-371. [5] Bernt, E. and Gutmann, I. (1974) in Methods of Enzymatic Analysis, vol. 3 (Bergmeyer, H.U., ed.), pp. 14991502. Academic Press, New York. [6] Ciriacy, M. (1976) Mol. Gen. Genet. 145, 327-333. [7] Schmitt, H.D. and Zimmermann, F.K. (1982) J. BacterioL 151, 1146-1152. [8] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. (1951) J. Biol. Chem. 193, 265-275. [9] Schmitt, H.D, ciriacy, M. and Zimmerrnann, F.K. (1983) Mol. Gen. Genet. 192, 247-252. [10] van Urk, H., Schipper, D., Breedveld, G.V., Mak, P.R., Scheffers, W.A. and van Dijken, J.P. (1989) Biochim. Biophys. Acta 992, 78-86.