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F E M S M i c r o b i o l o g y L e t t e r s 7 7 ( 1 9 9 1 ) 2 9 5 - 2 9 8@ 1 9 9 1 F e d e r a t i o n o f E u r o p e a n M i c r o b i o l o g i c a l S o c i e t i e s 0 3 7 8 - 1 0 9 7 / 9 1 / $ 0 3 . 5 0A D O N I S 0 3 7 8 1 0 9 7 9 1 0 0 0 6 0 IF E M S L E 0 4 2 3 5

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Levels o f act iv i ty of enzymes involved in anaerobic ut i l i zat ionof sugars by s ix yea st species: ob serva t ions tow ards un derstan dingthe Kluyver e f fectA . P . S i m s a n d J . A . B a r n e t t

School of Biological Sciences, U niversity of East Anglia, Norwich, U.K.

Received 27 Ju ly 1990Ac c e p t e d 2 9 Ju l y 1 9 90

K e y w o r d s : Y e a s t ; P y r u v a t e d e c a r b o x y l a se ; A l c o h o l d e h y d r o g e n a s e ; G l y c o s i d a s e ; K l u y v e r e f f ec t

1 . S U M M A R YT h e a c t i v i t i e s o f p y r u v a t e d e c a r b o x y l a s e , a l -c o h o l d e h y d r o g e n a s e a n d c e r t a i n g l y c o si d a s es w e r e

m e a s u r e d f o r s i x s p e c i e s o f y e a s t . F i v e o f t h e s ey e a s t s c o u l d u t i l i z e o n e o r m o r e d i s a c c h a r i d e sa e r o b i c a l l y , b u t n o t a n a e r o b i c a l l y , a l t h o u g h a l lc o u l d u s e D - g l u c o s e a n a e r o b i c a l l y . T h a t i s , e a c h o ft h e f i v e s h o w e d t h e K l u y v e r e f f e c t ; b u t t h e s i x t hy e a s t , Saccharomyces cerevisiae, d i d n o t d o s o .W h e n g r o w n o n a g l y c o s i d e w i t h w h i c h i t g a ve t h eK l u y v e r e f fe c t , e a c h y e a s t h a d m u c h l es s p y r u v a t ed e c a r b o x y l a s e a c t iv i t y t h a n w h e n g r o w n o n D - gl u-c o s e o r a n o t h e r g l y c o s i d e . T h e r e w a s n o c o n s i s t e n tc o r r e s p o n d i n g l o w e r i n g o f a c t i v i t y o f e i t h e r a l -c o h o l d e h y d r o g e n a s e , o r o f t h e a p p r o p r i a t e g l y c o-s i da s e. H e n c e , p y r u v a t e d e c a r b o x y l a s e m a y h a v e ar o l e i n p r o d u c i n g t h e K l u y v e r e f f e c t .

Correspondence to: J .A. Barne t t , School of Bio logica l Sc iences ,U n i v e r s i t y o f E a s t A n g l ia , N o r w i c h N R 4 7 T J , U . K .

2. I N T R O D U C T I O N

M a n y y e a s t s c a n u t i l iz e p a r t ic u l a r d i s a c c h a r i d e sa e r o b i c a l l y , b u t n o t a n a e r o b i c a l l y , a l t h o u g h t h e s ey e a s t s c a n u s e o n e o r m o r e o f t h e c o m p o n e n tm o n o s a c c h a r i d e s a n a e r o b i c a l l y . F o r a n y g i v e ny e a s t , t h i s K l u y v e r e f f e c t [ 1 ] m a y b e f o u n d w i t ho n e d i s a c c h a r i d e , b u t n o t a n o t h e r . T h e e f f e c t i sw i d e s p r e a d a m o n g s t t h e f e r m e n t i n g y ea s ts , a n dh a s b e e n r e p o r t e d i n 9 7 s p e c i e s [1 ] . A c c o r d i n g t oo n e s u r v e y [ 2 ], a l l t h e s t r a i n s e x a m i n e d o f 4 0 y e a s ts p e c i es s h o w t h e e f f e c t w i t h m a l t o s e a n d 4 1 w i t hce l lo b io se .

F a c t o r s t h a t m i g h t u n d e r l i e t h e K l u y v e r e ff e c ti n c l u d e t h e f o ll o w i n g . ( i) A e r o b i c c a t a b o l i s m m a yb e n e c e s s a r y f o r th e s u p p l y o f A T P f o r t h e ex p u l -s i o n o f p r o t o n s f r o m t h e c e l l s ; t h i s i s n e c e s s a r y i no r d e r t o m a i n t a i n t h e p r o t o n s y m p o r t o f a c t i v et r a n s p o r t o f g ly c o s i d e s a c r o s s t h e p l a s m a l e m m ain to the y e as t c e l l [ 1,3,4] . ( i i) Lev e l s o f a c t iv i ty o ft h e a p p r o p r i a t e g l y c o s i d a s e s , o f p y r u v a t e d e c a r -b o x y l a s e o r o f a l c o h o l d e h y d r o g e n a s e m a y f a l l

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296Table 1Aerobic growth and anaerobic fermentation by six yeasts supplied with different sugars as sole source of carbonYeast Carbon source a

D-Glucose Maltose Cellobiose LactoseG F G F G F" G FCandida v&wanathiiCBS 4024 + + + + + - - -Debaryomyces castetliiCBS 2923 + + + + + + + -Debaryomyces polymorphus CBS 4349 + + + + + - + -Kluyveromyces dobzhanskiiCBS 2104 + + + + + - - -Kluyveromyces wickerhamiiCBS 2745 + + - - + - + -Saccharomyces cerevisiaeCBS 1171 + + + + . . . .

a G = aerobic growth, F= anaerobic fermentation; results were consistent with those of Barnett et al. [2]; fermentation wasconfirmed by measuring ethanol production.below those required to m aint ain catabolic f lux.This paper reports measu rement s of these enzymicactivities in five yeasts that show the K luy vereffect and also in one that does not do so.

3. MATER IALS AND METHO DS3.1. Yeasts

The fol lowing yeasts, from the Centraa lbure auvoor Sch immelc ulture s (CBS), were exami ned:Candida viswanathii Sandhu and Randhawa (CBS4024), Debaryomyces castellii Capriot t i (CBS2923), Debaryomyces polymorphus (K15cker) Priceand Phaff (CBS 4349), Kluyveromyces dobzhanskii(Shehata et al.) van der Walt (CBS 2104), Kluy-veromyces wickerhamii (Phaff et al.) van der Walt(CBS 2745), and Saccharomyces cerevisiae Meyenex Hansen (CBS 1171).3.2. Media and growth conditions

The yeasts were maintained and cult ivatedaerobical ly in shaken, l iquid, chemically-definedme di um at 25 C, as descri bed previousl y [3], ex-cept that p re-i ncub atio n was on slopes of Difco(Bacto) Yeast Nitrogen Base, to which were added1% (w /v ) sugar (D-glucose, maltose, cellobiose orlactose, correspondi ng with that of the subse quentincubati on) and 1.5% (w/v) agar.3.3. Anaerobic conditions and measurement ofethanol in the suspending medium

Anaerobic condit ions were under argon, as de-scribed previously [3]; ethanol in the suspending

medium, produced by anaerobic fe rmenta t ion , wasmeasu red enzy mica lly [5].3.4. Enzymic assays

Enzymes were assayed in cell extracts obt ainedby harvest ing the yeasts during their exponentialphase of growth and disinteg rat ing the cells withfine glass beads [6].

Glycosidases: 4-ni t rophenyl a -D-glucopyrano-side. 4-ni t rophen yl f l -D-glucopyranoside or 4-ni tro pheny l f l -D-galactopyranoside were used tomeasu re a-glu cosida se (EC 3.2.1.20), B-ghico-sidase (EC 3.2.1.21) or fl-galactosida se (EC3.2.1.23) activities, respectively [1,3].Alcohol dehydrogenase (EC 1.1.1.1) activity wasmeasured with NA D* and ethanol [5].

Pyruvate decarboxylase (EC 4.1.1.1) was esti-mated as descr ibed by Schmit t and Zimmermann[7].

Protein concent ra t io n was es t imated by themet hod of Lowry et al. [8], using bovi ne serumalbumin as standard.

4 . RESULTS AND DISCUSSIONThe characterist ics ( taken from Barnett et al .

[2]) of each of the six yeasts examined (Table 1),were confirmed experimental ly; the occurrence orabsence of anaerobic fe rmenta t ion was de-termined by measuring the formation of ethanolenzymically. Candida viswanathii, Debaryomycespolymorphus, Kluyveromyces dobzhanskii and K.wickerhamii all showed the K luyv er effect with

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Table 2Specific activities a of three enzymes in six yeasts grown on different sugars as sole sources of carbon

297

Yeast name and Carbon source b Pyruvate Alcohol Glycosidase cstrain number for growth decarboxylase dehydrogenaseCandida viswanathiiCBS 4024

Debaryomyces castelliiCBS 2923

Debaryomyces polymorphusCBS 4349

Kluyveromyces dobzhanskiiCBS 2104

Kluyveromyces wickerhamiiCBS 2745

Saccharomyces cerevisiaeCBS 1171

D-Glucose 0.13 1.04 -Mal tos e 0.11 0.32 0.33Cellobiose K 0.039 0.23 0.33D-Glucose 0.17 0.19 -Mal tos e 0.067 0.088 0.48Lac tose K < 0.001 < 0.006 0.03D-Glucose 0.18 0.24 -Mal tose 0.35 0.37 0.15Cellobiose K 0.008 0.038 0.28D-Glucose 0.083 0.48 -Mal tose 0.31 0.070 0.19CeUobiose K 0.040 1.07 0.31D-Glucose 0.16 1.17 -Cellob iose K 0.020 0.88 0.14Lac tos e K 0.020 0.21 0.34D-Glucose 1.62 0.57 -Maltose 0.40 0.64 1.01

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.

c e l l o b i o s e ; Debaryomyces castellii a n d K. wicker-hamii g a v e t h e e f f e c t w i t h l a c t o s e .

P y r u v a t e d e c a r b o x y l a s e i s i n v o l v e d i n m e t a -b o l i c r e g u l a t i o n i n y e a s t s [ 9 ] a n d i s n e c e s s a r y f o rg l y c ol y s is t o p r o c e e d u n d e r a n a e r o b i c , b u t n o ta e r o b i c , c o n d i t io n s . I n e v e r y ca s e , p y r u v a t e d e -c a r b o x y l a s e a c t iv i ti e s ( T a b l e 2 ) w e r e l o w e r i n e a c hy e a s t g r o w n o n a g l y c o s i d e w i t h w h i c h t h a t y e a s tg a v e t h e K l u y v e r e f f e c t, t h a n w h e n g r o w n o nD - g l u c o s e o r o n a g l y c o s i d e t h a t w a s u s e da n a e r o b i c a l l y . T h e r a n g e o f d i f fe r e n c e s w e r e f r o m4 8 % o f t h e h i g h e r r a t e f o r c e l l o b i o s e - g r o w n Kluy-veromyces dobzhanskii t o 0.6 % f o r l a c t o s e - g r o w nDebaryomyces castellii. S u c h d i f f e r e n c e s d i d n o tg e n e r a l l y a p p l y t o t h e a c t i v i t i e s o f e i t h e r a l c o h o ld e h y d r o g e n a s e o r t h e g l y c o s i d a s e s .

S o t h e l e v e ls o f p y r u v a t e d e c a r b o x y l a s e a c t i v it yw e r e h i g h l y a s s o c i a t e d w i t h t h o s e o f a n a e r o b i cf e r m e n t a t i o n a n d , h e n c e , w e r e d e t e rm i n e d b y w h a t

s u g a r w a s s u p p l i e d e x o g e n o u s l y . I n r e l a t i o n t o t h eK l u y v e r e f f e c t , i t w a s t h e a c t i v i t y o f p y r u v a t ed e c a r b o x y l a s e , r a t h e r t h a n g l y c o s i d a s e o r a l c o h o ld e h y d r o g e n a s e , t h a t a p p e a r e d t o b e r a t e - l i m i t i n g .T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e f i n d i n g s o fv a n U r k a n d h i s c o l l e a g u e s [ 1 0 ] f o r Saccharornycescerevisiae a n d a n o t h e r s t r a in o f Candida utilis t h a tp y r u v a t e d e c a r b o x y l a s e l e v e l s a r e a s s o c i a t e d w i t ht h e r a t e o f c a t a b o l i c f l u x i n t h e a n a e r o b i c u t i l i z a -t i o n o f D - g l u c o s e . A c c o r d i n g l y , t h e r o l e o f t h er e g u l a ti o n o f p y r u v a t e d e c a r b o x y l a s e in t h eK l u y v e r e f f e c t a n d t h e f a c t o r s a f f e c t i n g i ts a c t i v i tya r e b e i n g i n v e s t i g a t e d i n d e t a i l .

RE F E RENCE S

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

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29 8[2] Barnett, J .A., Payne, R.W. and Yarrow, D. (1990) Yeasts:Charac te r is t ics and Ident i f ica t ion , 2nd edn . Cambr idgeUniv ers i ty Press , Cam br idge .[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] Bern t , E . and Gutmann, I . (1974) in Methods of En-zym atic Analy sis , vol. 3 (Bergmeyer, H.U ., ed.) , pp. 14 99-1502. Academic Press , New York.

[6] Cir iacy , M. (1976) Mol. Ge n. Gen et. 145, 327-33 3.[7] Schmit t , H .D. and Z imm erma nn, F .K. (1982) J . Bac ter ioL151, 1146-1152.[8] Lowry, O.H. , Rosebrough, N.J . , Far r , A .L. and Randa l l ,R J . (1951) J . B io l . Chem. 193, 265-275.[9] Schmit t , H .D, c i r iacy , M. and Zimmerrnann, F .K. (1983)Mol. Gen. G ene t . 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 , 78-8 6 .