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The interaction of zymohexoses and maltose in maltose fermentation by baker's yeast
voL. 20 (1956)
BIOCHIMICA ET BIOPHYSICA ACTA
319
THE INTERACTION OF ZYMOHEXOSES AND MALTOSE IN MALTOSE FERMENTATION
BY BAKER'S YEAST
by H E I K K I SUOMALAINEN, E R I K A X E L S O N A~D E R K K I O U R A
Research Laboratories o/the State Alcohol 31onopoly, Helsinhi (Finland)
INTRODUCTION
The studies of LEIBOWITZ AND HESTRINz as well as of SCHULTZ AND ATKIN5 have revealed that an addition of a small an~ount of glucose is able to abolish the known induction period of maltose fermentation by commercial baker's yeast. This phenomenon is sc distinct that SCHULTZAND ATKIN recommended it for determination of small amounts of glucose present as impurit.ies in maltose preparations. The cause of this particular action of glucose has so far remained unsolved despite all attempts. In addition to glucose, LE1BOWlTZ AND HESTRIN3 (C[. also SPIEGELMAN e~ al. 9) have, however, been able to show t h a t other zymohexoses also, viz. fructose and mannose - - b u t not galactose--exert a similar effect. On the other hand, they claimed that maltose did not act as an inhibitor in glucose fermentation b y baker's yeast. The question of the mechanism of maltose fermentation is still open (c]. MYRBACK AND RENVALL4). We have in this case started from the idea introduced once by SLATOR7 with regard to the fermentation of glucose and galactose, viz. : "If these two reactions were quite distinct, and no interference between them took place, the rate of evolution of carbon dioxide from the fermentation of a mixture of the two sugars would be the sum of the rates of the two singly". And although the fermentation of maltose and zymohexoses were, to a slight extent only, regarded as separate reaction chains, the small amounts of zymohexoses in question are in no case sufficient for "the saturation of zymase complex. Its insufficiency cannot therefore restrict the fermentation rate. On the contrary, in view of the above-mentioned accelerating effect of zymohexoses on maltose fermentation, the fermentation rate of maltose solution containing zymohexoses should still be considerably higher than the sum of the rates of the components singly. EXPERIMENTAL
Material The baker's yeast used was commercial material, produced from beet molasses by the RajamAki Factories of the State Alcohol Monopoly, Rajam~kki. The yeast was received from the factory washed and pressed to yeast cake containing about 25 % dry m a t t e r and was used as such. The dry yeast preparations were prepared by pressing the fresh yeast through a wire net (8 mashes per cm) in thin threads upon a layer of filter paper, and air-dried at room temperature. Of the sugar preparations used both glucose and maltose were Kerfoots' "pure bacteriological",/_ructose was "cryst. for scientific purposes" from Schering-Kahlbaum, and mannose C.P.
Re/erences p. 32z.
320
H. SUOMALAINEN, E. AXELSON', E. OURA
VOL. 2 0
(1956)
f r o m H e m a D r u g Co. I n later e x p e r i m e n t s w i t h d r y y e a s t a n d in c h r o m a t o g r a p h i c studies, p u r e m a l t o s e from T h o m a s Morson & Son, Ltd., London, was used. O t h e r chemicals were of p u r e or C.P. grade.
Experimental procedure T h e f e r m e n t a t i o n e x p e r i m e n t s were p e r f o r m e d as m e n t i o n e d previously TM. T h e f e r m e n t a t i o n vessel c o n t a i n e d 20 m l buffer-salt solution used b y SCHtrLTZ et al.e; weighed a m o u n t s of s u g a r were a d d e d to it in crystalline form a n d dissolved. T h e y e a s t suspension, c o n t a i n i n g 250 m g fresh y e a s t in 5 ml, was a d d e d to t h e f e r m e n t a t i o n solution at t h e s t a r t of t h e e x p e r i m e n t b y t u r n i n g t h e side bulb. T h e final f e r m e n t a t i o n solution, with a v o l u m e of 25 ml, t h u s was m a d e i % in r e s p e c t to yeast, 3 % in respect to s u g a r - - t h e hexose a d d i t i o n s of m a l t o s e f e r m e n t a t i o n s exceeded this a m o u n t while t h e parallel e x p e r i m e n t s c o n t a i n e d hexose " a d d i t i o n s " alone in t h e s o l u t i o n - - a n d c o n t a i n e d per 1oo ml t h e following a m o u n t s of chemicals listed: N H 4 H 2 P O 4 900 mg, (NH4)2HPO 4 360 mg, M g S O 4 - 7 H 2 0 lO5 mg, K H z P O 4 33 rag, KCI 25.5 mg, C a C 1 . 2 H , O 7.5 mg, F e C l s - 6 H , O o.x 5 mg, M n S O 4 . 4 H 2 0 o.15 rag, a n d nicotinic acid x mg. In t h e e x p e r i m e n t s with d r y y e a s t t h e v o l u m e of t h e buffer-salt solution in t h e f e r m e n t a t i o n vessel was 25 ml, to which weighed a m o u n t s of s u g a r were a d d e d in crystalline f o r m a n d dissolved. T h e d r y y e a s t - - i n t h e e x p e r i m e n t s reported 125 m g - - w a s a d d e d at t h e s t a r t of t h e e x p e r i m e n t b y t u r n i n g t h e side bulb. T h e c h r o m a t o g r a m s of t h e f e r m e n t a t i o n e x p e r i m e n t s were r u n with n - b u t a n o l - p y r i d i n e b e n z e n e - w a t e r {5 : 3 : 3 : I) solution I a n d developed w i t h silver n i t r a t e solution.
RESULTS
As can be seen from the diagrams (Fig. I) the observation of LEIBOWlTZAND HESTRINa and SPIEGELMANet al. ~ could be confirmed, viz. that all the three zymohexoses shorten the induction period of maltose fermentation by commercial baker's yeast. Maltose, on the other hand, has a distinctly retarding effect on the fermentation of small amounts of hexoses added (Fig. 2). Thus, not even in a single case does the fermentation rate of hexoses in the maltose solution reach--let alone exceed--the fermentation rate of the corresponding hexose amounts separately in the solution. It could be demonstrated by paper chromatography using a 3% suspension of baker's yeast that an 0.3% addition of glucose, which disappeared almost entirely within I h when added separately to the solution, required about 2 h to disappear from a 3% maltose solution. With mannose, the difference was still more pronounced: o.3% addition of mannose separately to the solution disappeared almost completely in about 2 h, in 3% maltose solution in not less than about 4 hours. Maltose has a distinct retarding effect on the fermentation of all zymohexoses, glucose, fructose, and mannose alike. It is of interest to note, that the abolishing C0,7-
" !" ° ~ L ~ . ~ o
-
"
!2
CO n
,
,
7-:f/:i t " to
'
•
/
/ O f +o ) + l
/e
$0 0
t
l
/
I+ / 2
$
dh,1
/
./
. / ®i~°+z <<.~
Q
Fig. i. Effect of small a d d i t i o n s of z y m o h e x o s e s on t h e i n d u c t i o n period of m a l t o s e f e r m e n t a t i o n b y c o m m e r c i a l b a k e r ' s yeast. I n t e r v a r i a t i o n s in t h e f e r m e n t a t i o n r a t e s of z y m o h e x o s e s are p a r t l y d u e to e x p e r i m e n t series being r u n with different lots of yeast.
Re/erences p. 322.
INTERACTION OF ZYMOHEXOSES AND MALTOSE
VOL. 20 (I956)
321
co,,
" .......... /t
~/ucose ferrnentst~n
Is
• --
• $~
Fructon fermentation
,.
o - - o
0.80Xfr~elom
o--o
t~
-
......... /
"
Mannose fermentation
o --
Is
*0.ao~
o oJo~neee
•--o
s~
.
~ojo~
.
t
04"7
$
/"/
,
,
Fig. 2. Fermentation rates of small amounts of zymohexoses by commercial baker's yeast as such and in maltose solution. effect of zymohexoses on the induction period of maltose fermentation and the retarding effect of maltose on the fermentation of zymohexoses are inversely proportional. Thus, the effect of maltose on the mannose fermentation is most retarding and the effect of mannose on the maltose fermentationleast activating. The retarding effect of maltose being detectable also with dry yeast (Fig. 3) it cannot be thought that the entrance of sugar molecules into the cells is prevented by the effect of unfermentable s u g a r - - i . e . maltose during its induction p e r i o d - - a s SOBOTKA, HOLZMANAND REINERs assumed with regard to the "pentose effect" they noted with brewer's yeast. There is presumably a competition for a certain factor connected with the fermentation enzyme complex. If maltose were fermented by baker's yeast after preceding hydrolysis to glucose, this ought to be detectable in the fermentation solution unless the activity of aglucosidase (maltase) were the limiting factor for fermentation. It has not been possible, however, to prove the appearance of glucose in the fermentation solution of maltose fermentation b y baker's yeast as has been done with brewer's yeast n. If again, the a-glucosidatic activity of baker's yeast were the limiting factor of maltose fermentation, glucose added to the fermentation solution ought to disappear rather quickly, its rate of fermentation being greater than the rate of formation. Yet, if maltose i t s e l f - - a s has been s h o w n - - a c t s as an inhibitor for glucose fermentation, maltose might also retard its own fermentation, especially with increasing coneen-
°-°
i$
-• --
....
• 9 ~ u ~ n o x *o35~.gluc • 3x ,..O,30X
dlfJnncwe fwment~'/on +
/
+ o~g
4D O--o
9x
.
*0.30~'.
/-°-//
//2" ..... o
!
~
j
d~
Fig. 3- Fermentation rates of small amounts of zymohexoses by dried commercial baker's yeast as such and in maltose solution. Re/erences p. 32z.
322
H. SUOMALAINEN, E. AXELSON, E. OURA
VOL. 2 0
(1956)
trations. It can at least be assumed that the more concentrated the maltose solution, the more effective the competitive effect in respect to glucose fermentation. LEIBOWITZ AND HESTRIN3 reported that high concentrations of maltose may inhibit the rate at which maltose is fermented by aged baker's yeast. We have noted that high concentrations of maltose retard its own fermentation by f r e s h - - a n d d r y - baker's yeast, whereas glucose fermentation in glucose solutions of even twice the same molar concentration is not notably retarded 1.. Yet, after glucose which followed maltose as an impurity had fermented away we were unable to show by paper chromatography the presence of glucose in the mMtose fermentation solution, to say nothing of its enrichment. The problem of the inhibitory effect of maltose on its own fermentation by baker's yeast will be returned to in another paper. Since the presence of maltose, as already stated, retards the fermentation of hexoses, the absence of glucose from the fermentation solution suggests that maltose fermentation is retarded for some other reason than the depressing action of maltose on the fermentation of glucose formed through hydrolysis or phosphorolysis. SUMMARY Zymohexoses activate maltose fermentation by commercial baker's yeast, glucose in the highest degree, m a n n o s e in the least. Maltose on the other hand, retards the f e r m e n t a t i o n of small a m o u n t s of zymohexoses to the extent t h a t the rate of carbon dioxide formation is distinctly higher when zymohexoses are present alone in the solution t h a n w h e n maltose is also present. The p h e n o m e n o n is m o s t pronounced with regard to mannose, least so with glucose. I t is also detectable with dried yeast. The retarding effect of maltose on the fermentation of zymohexoses has also been proved by paper c h r o m a t o g r a p h y . Higher concentrations of maltose retard its own fermentation b u t it has not been possible to show the occurrence of glucose in the fermentation solutions by p a p e r c h r o m a t o g r a p h y . This suggests t h a t the retardation would be ascribable to soine other reason t h a n the depressing effect of maltose on the fermentation of glucose formed either by hydrolysis or phosphorolysis.
REFERENCES 1 N. ALBON AND D. GROSS, Analyst, 75 (195 o) 454. 2 j LEIBOWITZ AND S. HESTRIN, Enzymologia, 6 (1939) I5. 3 j. LEIBOWITZ AND S. HESTRIN, Biochem. J., 36 (1942) 772. 4 K. MYRB~,CK AND S. RENVALL, Arkiv Kemi, Mineral. Geol., 23A, No. 6 (1946). 5 A. S. SCHULTZ AND L. ATKIN, J. Am. Chem. Soc., 61 (1939) 291. e A. S. SCHULTZ, L. ATKIN AND C. N. FREY, Ind. Eng. Chem., Anal. Ed., 14 (I942) 35. A. SLATOR, J. Chem. Soc., 93 (19o8) 2i 7. 8 H. SOBOTKA, M. HOLZMAN AND M. REINER, Biochem. J., 3° (1936) 933. 9 S. SPI/~GELMAN, J. M. REINER AND R. COHNBERG, J. Gen. Physiol., 31 (i948) 27. 10 H. SUOMALAINEN ANn E. AXELSON, Biochim. Biophys. Acta, 2o (I956) 315. ll H. SUOMALAINEN,The Role of a-Glucosidase in the F e r m e n t a t i o n of Oligosaccharides b y Living Yeast. Dissertation, Helsinki, 1948. li H. SUOMALAINEN AND E. OURA, unpublished.
Received August 3rd, 1955
BIOCHIMICA ET BIOPHYSICA ACTA
319
THE INTERACTION OF ZYMOHEXOSES AND MALTOSE IN MALTOSE FERMENTATION
BY BAKER'S YEAST
by H E I K K I SUOMALAINEN, E R I K A X E L S O N A~D E R K K I O U R A
Research Laboratories o/the State Alcohol 31onopoly, Helsinhi (Finland)
INTRODUCTION
The studies of LEIBOWITZ AND HESTRINz as well as of SCHULTZ AND ATKIN5 have revealed that an addition of a small an~ount of glucose is able to abolish the known induction period of maltose fermentation by commercial baker's yeast. This phenomenon is sc distinct that SCHULTZAND ATKIN recommended it for determination of small amounts of glucose present as impurit.ies in maltose preparations. The cause of this particular action of glucose has so far remained unsolved despite all attempts. In addition to glucose, LE1BOWlTZ AND HESTRIN3 (C[. also SPIEGELMAN e~ al. 9) have, however, been able to show t h a t other zymohexoses also, viz. fructose and mannose - - b u t not galactose--exert a similar effect. On the other hand, they claimed that maltose did not act as an inhibitor in glucose fermentation b y baker's yeast. The question of the mechanism of maltose fermentation is still open (c]. MYRBACK AND RENVALL4). We have in this case started from the idea introduced once by SLATOR7 with regard to the fermentation of glucose and galactose, viz. : "If these two reactions were quite distinct, and no interference between them took place, the rate of evolution of carbon dioxide from the fermentation of a mixture of the two sugars would be the sum of the rates of the two singly". And although the fermentation of maltose and zymohexoses were, to a slight extent only, regarded as separate reaction chains, the small amounts of zymohexoses in question are in no case sufficient for "the saturation of zymase complex. Its insufficiency cannot therefore restrict the fermentation rate. On the contrary, in view of the above-mentioned accelerating effect of zymohexoses on maltose fermentation, the fermentation rate of maltose solution containing zymohexoses should still be considerably higher than the sum of the rates of the components singly. EXPERIMENTAL
Material The baker's yeast used was commercial material, produced from beet molasses by the RajamAki Factories of the State Alcohol Monopoly, Rajam~kki. The yeast was received from the factory washed and pressed to yeast cake containing about 25 % dry m a t t e r and was used as such. The dry yeast preparations were prepared by pressing the fresh yeast through a wire net (8 mashes per cm) in thin threads upon a layer of filter paper, and air-dried at room temperature. Of the sugar preparations used both glucose and maltose were Kerfoots' "pure bacteriological",/_ructose was "cryst. for scientific purposes" from Schering-Kahlbaum, and mannose C.P.
Re/erences p. 32z.
320
H. SUOMALAINEN, E. AXELSON', E. OURA
VOL. 2 0
(1956)
f r o m H e m a D r u g Co. I n later e x p e r i m e n t s w i t h d r y y e a s t a n d in c h r o m a t o g r a p h i c studies, p u r e m a l t o s e from T h o m a s Morson & Son, Ltd., London, was used. O t h e r chemicals were of p u r e or C.P. grade.
Experimental procedure T h e f e r m e n t a t i o n e x p e r i m e n t s were p e r f o r m e d as m e n t i o n e d previously TM. T h e f e r m e n t a t i o n vessel c o n t a i n e d 20 m l buffer-salt solution used b y SCHtrLTZ et al.e; weighed a m o u n t s of s u g a r were a d d e d to it in crystalline form a n d dissolved. T h e y e a s t suspension, c o n t a i n i n g 250 m g fresh y e a s t in 5 ml, was a d d e d to t h e f e r m e n t a t i o n solution at t h e s t a r t of t h e e x p e r i m e n t b y t u r n i n g t h e side bulb. T h e final f e r m e n t a t i o n solution, with a v o l u m e of 25 ml, t h u s was m a d e i % in r e s p e c t to yeast, 3 % in respect to s u g a r - - t h e hexose a d d i t i o n s of m a l t o s e f e r m e n t a t i o n s exceeded this a m o u n t while t h e parallel e x p e r i m e n t s c o n t a i n e d hexose " a d d i t i o n s " alone in t h e s o l u t i o n - - a n d c o n t a i n e d per 1oo ml t h e following a m o u n t s of chemicals listed: N H 4 H 2 P O 4 900 mg, (NH4)2HPO 4 360 mg, M g S O 4 - 7 H 2 0 lO5 mg, K H z P O 4 33 rag, KCI 25.5 mg, C a C 1 . 2 H , O 7.5 mg, F e C l s - 6 H , O o.x 5 mg, M n S O 4 . 4 H 2 0 o.15 rag, a n d nicotinic acid x mg. In t h e e x p e r i m e n t s with d r y y e a s t t h e v o l u m e of t h e buffer-salt solution in t h e f e r m e n t a t i o n vessel was 25 ml, to which weighed a m o u n t s of s u g a r were a d d e d in crystalline f o r m a n d dissolved. T h e d r y y e a s t - - i n t h e e x p e r i m e n t s reported 125 m g - - w a s a d d e d at t h e s t a r t of t h e e x p e r i m e n t b y t u r n i n g t h e side bulb. T h e c h r o m a t o g r a m s of t h e f e r m e n t a t i o n e x p e r i m e n t s were r u n with n - b u t a n o l - p y r i d i n e b e n z e n e - w a t e r {5 : 3 : 3 : I) solution I a n d developed w i t h silver n i t r a t e solution.
RESULTS
As can be seen from the diagrams (Fig. I) the observation of LEIBOWlTZAND HESTRINa and SPIEGELMANet al. ~ could be confirmed, viz. that all the three zymohexoses shorten the induction period of maltose fermentation by commercial baker's yeast. Maltose, on the other hand, has a distinctly retarding effect on the fermentation of small amounts of hexoses added (Fig. 2). Thus, not even in a single case does the fermentation rate of hexoses in the maltose solution reach--let alone exceed--the fermentation rate of the corresponding hexose amounts separately in the solution. It could be demonstrated by paper chromatography using a 3% suspension of baker's yeast that an 0.3% addition of glucose, which disappeared almost entirely within I h when added separately to the solution, required about 2 h to disappear from a 3% maltose solution. With mannose, the difference was still more pronounced: o.3% addition of mannose separately to the solution disappeared almost completely in about 2 h, in 3% maltose solution in not less than about 4 hours. Maltose has a distinct retarding effect on the fermentation of all zymohexoses, glucose, fructose, and mannose alike. It is of interest to note, that the abolishing C0,7-
" !" ° ~ L ~ . ~ o
-
"
!2
CO n
,
,
7-:f/:i t " to
'
•
/
/ O f +o ) + l
/e
$0 0
t
l
/
I+ / 2
$
dh,1
/
./
. / ®i~°+z <<.~
Q
Fig. i. Effect of small a d d i t i o n s of z y m o h e x o s e s on t h e i n d u c t i o n period of m a l t o s e f e r m e n t a t i o n b y c o m m e r c i a l b a k e r ' s yeast. I n t e r v a r i a t i o n s in t h e f e r m e n t a t i o n r a t e s of z y m o h e x o s e s are p a r t l y d u e to e x p e r i m e n t series being r u n with different lots of yeast.
Re/erences p. 322.
INTERACTION OF ZYMOHEXOSES AND MALTOSE
VOL. 20 (I956)
321
co,,
" .......... /t
~/ucose ferrnentst~n
Is
• --
• $~
Fructon fermentation
,.
o - - o
0.80Xfr~elom
o--o
t~
-
......... /
"
Mannose fermentation
o --
Is
*0.ao~
o oJo~neee
•--o
s~
.
~ojo~
.
t
04"7
$
/"/
,
,
Fig. 2. Fermentation rates of small amounts of zymohexoses by commercial baker's yeast as such and in maltose solution. effect of zymohexoses on the induction period of maltose fermentation and the retarding effect of maltose on the fermentation of zymohexoses are inversely proportional. Thus, the effect of maltose on the mannose fermentation is most retarding and the effect of mannose on the maltose fermentationleast activating. The retarding effect of maltose being detectable also with dry yeast (Fig. 3) it cannot be thought that the entrance of sugar molecules into the cells is prevented by the effect of unfermentable s u g a r - - i . e . maltose during its induction p e r i o d - - a s SOBOTKA, HOLZMANAND REINERs assumed with regard to the "pentose effect" they noted with brewer's yeast. There is presumably a competition for a certain factor connected with the fermentation enzyme complex. If maltose were fermented by baker's yeast after preceding hydrolysis to glucose, this ought to be detectable in the fermentation solution unless the activity of aglucosidase (maltase) were the limiting factor for fermentation. It has not been possible, however, to prove the appearance of glucose in the fermentation solution of maltose fermentation b y baker's yeast as has been done with brewer's yeast n. If again, the a-glucosidatic activity of baker's yeast were the limiting factor of maltose fermentation, glucose added to the fermentation solution ought to disappear rather quickly, its rate of fermentation being greater than the rate of formation. Yet, if maltose i t s e l f - - a s has been s h o w n - - a c t s as an inhibitor for glucose fermentation, maltose might also retard its own fermentation, especially with increasing coneen-
°-°
i$
-• --
....
• 9 ~ u ~ n o x *o35~.gluc • 3x ,..O,30X
dlfJnncwe fwment~'/on +
/
+ o~g
4D O--o
9x
.
*0.30~'.
/-°-//
//2" ..... o
!
~
j
d~
Fig. 3- Fermentation rates of small amounts of zymohexoses by dried commercial baker's yeast as such and in maltose solution. Re/erences p. 32z.
322
H. SUOMALAINEN, E. AXELSON, E. OURA
VOL. 2 0
(1956)
trations. It can at least be assumed that the more concentrated the maltose solution, the more effective the competitive effect in respect to glucose fermentation. LEIBOWITZ AND HESTRIN3 reported that high concentrations of maltose may inhibit the rate at which maltose is fermented by aged baker's yeast. We have noted that high concentrations of maltose retard its own fermentation by f r e s h - - a n d d r y - baker's yeast, whereas glucose fermentation in glucose solutions of even twice the same molar concentration is not notably retarded 1.. Yet, after glucose which followed maltose as an impurity had fermented away we were unable to show by paper chromatography the presence of glucose in the mMtose fermentation solution, to say nothing of its enrichment. The problem of the inhibitory effect of maltose on its own fermentation by baker's yeast will be returned to in another paper. Since the presence of maltose, as already stated, retards the fermentation of hexoses, the absence of glucose from the fermentation solution suggests that maltose fermentation is retarded for some other reason than the depressing action of maltose on the fermentation of glucose formed through hydrolysis or phosphorolysis. SUMMARY Zymohexoses activate maltose fermentation by commercial baker's yeast, glucose in the highest degree, m a n n o s e in the least. Maltose on the other hand, retards the f e r m e n t a t i o n of small a m o u n t s of zymohexoses to the extent t h a t the rate of carbon dioxide formation is distinctly higher when zymohexoses are present alone in the solution t h a n w h e n maltose is also present. The p h e n o m e n o n is m o s t pronounced with regard to mannose, least so with glucose. I t is also detectable with dried yeast. The retarding effect of maltose on the fermentation of zymohexoses has also been proved by paper c h r o m a t o g r a p h y . Higher concentrations of maltose retard its own fermentation b u t it has not been possible to show the occurrence of glucose in the fermentation solutions by p a p e r c h r o m a t o g r a p h y . This suggests t h a t the retardation would be ascribable to soine other reason t h a n the depressing effect of maltose on the fermentation of glucose formed either by hydrolysis or phosphorolysis.
REFERENCES 1 N. ALBON AND D. GROSS, Analyst, 75 (195 o) 454. 2 j LEIBOWITZ AND S. HESTRIN, Enzymologia, 6 (1939) I5. 3 j. LEIBOWITZ AND S. HESTRIN, Biochem. J., 36 (1942) 772. 4 K. MYRB~,CK AND S. RENVALL, Arkiv Kemi, Mineral. Geol., 23A, No. 6 (1946). 5 A. S. SCHULTZ AND L. ATKIN, J. Am. Chem. Soc., 61 (1939) 291. e A. S. SCHULTZ, L. ATKIN AND C. N. FREY, Ind. Eng. Chem., Anal. Ed., 14 (I942) 35. A. SLATOR, J. Chem. Soc., 93 (19o8) 2i 7. 8 H. SOBOTKA, M. HOLZMAN AND M. REINER, Biochem. J., 3° (1936) 933. 9 S. SPI/~GELMAN, J. M. REINER AND R. COHNBERG, J. Gen. Physiol., 31 (i948) 27. 10 H. SUOMALAINEN ANn E. AXELSON, Biochim. Biophys. Acta, 2o (I956) 315. ll H. SUOMALAINEN,The Role of a-Glucosidase in the F e r m e n t a t i o n of Oligosaccharides b y Living Yeast. Dissertation, Helsinki, 1948. li H. SUOMALAINEN AND E. OURA, unpublished.
Received August 3rd, 1955