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Fermentability of maltose
Fermentability of Maltose Mary Grace Blair
Prom
the Biochemistry
Department, dfedical-Dental Schools, Alabama, Birmingham, Alabama Received
February
17n~icersitg
of
9, 1953
In a previous paper (l), the fermentation of n-glucose by yeast was studied from the standpoint of the factors affecting the yield of alcohol for conditions that might be useful in the removal or determination of fermentable sugars. The present paper is an ext’ension of the former work and describes some of the factors affecting alcohol yields from maltose? an important component of starch hydrolyzates. Earlier work with this sugar, the fermentation (2) of which requires more specialized conditons than for D-glucose, has been concerned principally with rates of carbon dioxide evolution. EXPERIMENTAL
Method All fermentations and determinations of yields of alcohol were carried out b> the “standard procedure” of t,he previous paper (1)) unless otherwise indicat,ed. Known amounts of buffered sugar solution (usually 15 g. sugar in 100 ml.) were fermented with large amounts of yeast (3 g. moist yeast) for 3 or 5 days at 30”. The alcohol was then removed by distillation and determined densimetrically. The fermentation efficiency (F.E.) is the percentage of the theoretical yield based on the Gay-Lussac equation. The reported yields do not include the alcohol lost in the vapors, an amount shown to equal 1.3% of the theoretical in the case of D-glucose. Because of the earlier observations (3-5) that maltose will be fermented prop erly only in a restricted region of acidity, all work was carried out, in t,he presence
17
of ammonium and potassium phosphates and citrates as before, at, a pH of about 5. Blank studies for yeast-S showed that no alcohol was produced in the absence of the sugar under these conditions. The range of concentration which is permissible is indicated by the following fermentation efficiencies for commercial maltose fermented for 5 days and with 5% of the indicated amount of maltose substituted by n-glucose: 5% maltose, F.E. 86.5; lo%, F.E. 89.0; 150/o,F.E. 88.8; 17.5%, F.E. 38.5. The presence or absence of nutrients (yeast extract) made no difference in alcohol yields; hence, this substance was omitted when other measurements were to be made or where special precautions were desired to insure the absence of extraneous material as in the study of the effect of the addition of glucose. In the latter case, the yeast was washed as an additional precaution against the introduction of traces of sugar. Five commercial compressed yeasts, previously described, were used. Unless otherwise specified, the yeast was yeast-S (Fleischmann’s compressed baker’s yeast).
Maltose “Technical maltose” is a crude commercially available mixture of maltose, n-glucose, and dextrins. The “commercial maltose” used in this work was a fairly good grade of maltose, “c.P. Maltose” of the Pfanstiehl Chemical Company. Its fermentation characteristics indicate that it contained small amounts of n-glucose. This product could be purified sufficiently by two recrystallizations from methanol. The pure product, which ia the monohydrate and which is referred to as purified maltose, had a rotation [a]:’ = $130.6 (c = 4, water) at equilibrium. All sugars were dried in vucuo at 60’ over calcium chloride.
Results Fermentation of Maltose in Relation to the Requirement of D-Glucose When highly purified maltose was fermented 3-5 days under the standard conditions (15 y0 sugar solution with 3 g. of pressed yeast-S), the yields of alcohol were low and exceedingly erratic. For six such experiments, they were distributed over the range 32.7-67.0 % of the theoretical amount. With the C.P. commercial maltose, similar variation was noted but the average yields were higher (52.6-84.4 ~~0).Four other brands of commercial pressed yeasts behaved in the same fashion. Where a small amount of n-glucose was added to the purified maltose before the fermentation period, the alcohol yields were very close to those previously observed for n-glucose (88.7 %). The amount of D-ghcase required for complete fermentation is established by the following data which show the effect of the addition of various amounts t,o 15 g. of purified maltose. The yields of alcohol after 3 and 5 days fermentation by washed compressed yeast-S were: 0.025 g. n-glucose, 62.9 % (3 days)
and 8’2.7 y0 (5 days); 0.075 g. D-glucose, 72.5 % (3 days) and 88.6 % (5 days); 0.150 g. n-glucose, 89.7 % (3 and 5 days); 0.45 g. n-glucose, 89.8 % (3 days) and 90.4 % (5 days) ; 0.75 g. n-glucose, 88.8 % (5 days). These results show t’hat when the malt’ose contains about 1 % of D-gluwse, practically the same alcohol yields will be obt’ained as fol n-glucose alone. The four other compressed yeasts gave closely similar results, although the fermentation was freyue&y less complete in the shorter time period. Possibly some of the differences ill rate may have a,risen from differences in the ages of the yeasts, since maltose fermentations are k~lown to be more susceptible than glucose ferment.ations to age variations (5-7). Complete fermentation of maltose was also obtained by performing the ferment#ations in the presence of enzyme preparations ront’aining maltase, including Aspergillw oryzae emulsin (0.2 g. of Clarase 970 of Takamine Laboratories; F.E., 86.9 y0 for commercial maltose) and R malt extract (0.5 g. of a Wallerstein concentrate; F.E. 87.1 y0 for commercial maltose). It should he noted that the fermentation of enzymaticallp convert,ed starch occurs completely only in the presence of similar enzymes (8). Nature of the “Glucose lS$‘ect” The results of this investigation show that D-glucose has an important role in determining the extent of fermentation of maltose, a role more significant than solely an initial acceleration, such as has been noted previously in the studies of the rate of rarbon dioxide evolution. Srhult,z, Atkin, and Frey (3, 9, 10) considered that the effect on the rate could be ascribed to an effect on the permeahilit,y of the cell wall of the yeast. Leibowitz and Hestrin (4j, who observed that, n-fructose and o-mannose but not wgalactose showed the same effect, cont,ended that variations of permeability could not be the sole explanatioll. In the present work, additional experiments were performed with the purpose of further elucidating the “glucose effect.” In one set of experiments, fresh yeast (3 g.), with and without D-glucose, was added to 15 y0 solutions of purified maltose which had been fermented previously for 3 days. The alcohol yields after 3 or 5 days additional time with the fresh yeast, even with the added glucose, fell in the range 74.2-76.7 % of the theory. Evidently, the addition of the glucose must take place prior t#othe initial fermentation, and t,he lacbk of complete frrment,atioll is not simply 8 loss in activit>y of the yeast.
20
MARY
GRACE
BLAIR
AND
WARD
PIGMAN
On the other hand, the still residues left from the alcohol determinations could be fermented further by the addition of fresh portions of yeast (3 g.) to an extent that the total alcohol yields fell in the range 85-90 % of the theoretical. As an example, recrystallized maltose (15 g.) when fermented with yeast-S (3 g.) showed at the end of 5 days fermentation efficiencies of only 61.6 and 33.2 %, respectively, in two experiments. The still residues from these solutions when diluted and fermented 3-5 days more with fresh yeast-S produced enough additional alcohol to bring the total yields to 85.9 and 86.2 %. Reducing sugar determinations (as maltose) were made on the centrifuged still residues, by means of the Scales method as modified by Isbell, Pigman, and Frush (11). For this work 15 g. of purified maltose was fermented for 3-5 days with yeast-S, and the alcohol was removed and determined in the usual manner by distillation. The residual solution was analyzed for reducing sugar. Unfortunately, no data were obtained as to the reducing power of the solution before distillation. Two typical results are: F.E., 52.4 % for purified maltose; apparent reducing sugar remaining, 5.27 g. F.E., 82.9 ‘% for commercial maltose; reducing sugar 0.45 g. For fermented n-glucose solutions, this residue contained amounts in the order of 0.06 g. apparent n-glucose from 15 g. of sugar. When the amounts of residual reducing sugar in the still residues were added to the amounts of maltose corresponding to the alcohol formed (with the assumption of an F.E. of 88.7 %), the totals corresponded to 94.7-98.3 % of the initially added maltose. The discrepancy indicates that the use of a maltose factor is not correct and that higher oligosaccharides were probably present. DISCUSSION
The results obtained in the present work are consistent with the interpretation that maltose in the absence of sufficient n-glucose is partially converted, to the extent of 10 % or more, into a nonfermentable form. This conversion product, if stored intracellularly, is liberated when the yeast cells are boiled. Although the boiled material is fermentable, the low reducing power indicates that it is not all in the form of maltose and/or glucose. Previously it has been shown by Pigman (8) that certain mold and malt enzymes will synthesize unfermentable sugars from maltose although not from n-glucose. Guillemet (12) also reported the conversion of t,he fermentation residue from maltose, but not from glucose, to ferment,able sugars by treatment with acid. An unfermentable product synthesized by Aspergillus niger enzymes was
isolated by Pan and co-workers (13) and was shown by Wolfrom, Thompson, and Galkowski (14) to be =4-a-isomaltopyranosyl-D-glucose. &lany of the observed facts of maltose fermentation by yeasts can be explained by the “t’ransglycosidase” scheme, which has been demonstrated in other connections by Hehre (15), Torriani and Rionod (l(i), I)oudoroff and Hasxid (17), and others (18), if this rea&ion is compctitive lvit,h a more rapid fermentation of ho01 glucose and mahose. 7~Glucosyl glucose 7=t (anhydroglucose
unit,s), + 11glucose
If the glucose concentration is kept low by rapid fermentation, t,hc polymer should accumulate. But, if enough glucose is added initially to maintain a sufficiently high concentration throughout the reaction, the polymerative reaction could be reversed and all the carbohydrate would be fermented. The lack of the effect of added glucose after the initial period is more difficult to explain. Possibly the polysaccharide is bound to the yeast cell, or the reverse react,ion is slow for long-chain compounds. An alternative role for the initially added D-glucose is that it is necessary for the production of a sufficient amount of a,11enzyme \\hich is essential in limiting the extent of polymerizatjion. Yeast, exhausted 1)~ storage to a point of inability t’o ferment, maltose is cluickly restored it1 this respect by incubation with D-glucose (19, XI), a behavior sometimes at,tributed to mahase formation. The question of direct versus indirect fermentation of maltose is still unsettled (19, 21-23). Hestrin, a strong advocate of direct fermentation, has proposed polymerative cleavage as the probable route (19). The evidence of t#his paper st)rongly supports the idea of a polymerative react,ioll as one of a series of competitive reactions. The sensitivity of the fermentation reaction to small amounts of ~-glucose is additional evidenye for the cluest,ionat)ilit,y of direct maltasc action in the fermentation of maltose. &KNO\VLEDGMENT This work was begun while the authors were employed at the National Bureau of Standards. The courtesy of E. U. Condon in releasing this material is great13 appreciated.
SUMMARY Pure maltose is fermented
incompletely, but maltose with at, least can be fermented to the same extent, as D-gh~cosc. The still residues from incompletely fcrment,ed maltose solu1 yO D-glncosc
Ktially
preselxt
22
MARY
GRdCE
BLdIR
AND
WARD
PIGMAN
tions can be further fermented to produce the expected quantity of alcohol, but their reducing power is lower than that calculated for the unfermented maltose. The results obtained are consistent with the occurrence of a polymerization reaction as one of a series of competing reactions involved in the fermentation of maltose. REFERENCES 1. BLAIR, M. G., AND PIGMAX, W., Arch. Biochem. and Biophys. 42, 278 (1953). 2. See NORD, F. F., AND WEISS, S., in SURZNER and MYRB~CK, eds., The Enzymes, Vo12, pp. 715-716. Academic Press, New York, 1951. 3. SCHULTZ, A. S., AND ATKIN, L., J. Am. Chem. Sot. 61, 291 (1939). 4. LEIBOWITZ, J., AND HESTRIN, S., Biochenz. J. (London) 36, 772 (1942). 5. STARK, I. E., AND SOMOGYI, M., J. Biol. Chem. 143, 579 (1942). 6. HARDING, V. J., AND NICHOLSON, T. F., Biochem. J. (London) 27, 1082 (1933). 7. SANDSTEDT, R. M., AND BLISH, M. J., Cereal Chem. 11, 368 (1934); J. Biol. Chem. 116, 765 (1937). 8. PIGMAN, W. W., J. Research Natl. Bur. Standards 33, 105 (1944). 9. SCHULTZ, A. S., AND ATKIN, L., U. S. Patent 2,202,356, May 28, 1940. 10. SCHULTZ, A. S., ATKIN, L., AND FREY, C. N., J. Am. Chem. Sot. 62,227l (1940). 11. ISBELL, H. S., PIGMAN, W. W., ASI) FRUSH, H., J. Research Natl. Bur. Standards 24, 241 (1940). 12. GIJILLEMET, R., Con@. rend, sot. biol. 130, 1402 (1939). 13. PAN, S. C., ANDREASEX, A. A., AND KoL.~CHOV, P., Science 112, 115 (1950); PAN, S. C., NICHOLSON, L. W., AND KOLACHOV, P., J. Am. Chem. Sot. 73, 2547 (1951). 14. WOLFROM, M. L., THOMPSON, A., .~ND GALKOWSKI,.T. T., J. Am. Chem. Sot. 73, 4093 (1951). 15. HEHRE, E. J., J. Biol. Chem. 192, 161 (1951). 16. TORRIANI, A. M., ASD MONOD, J., Compt. rend. 226, 718 (1949); MONOD, J., AND TORRIANI, A. M., Compt. rend. 227, 240 (1949). 17. DOUDOROFF, M., HASSID, W. Z., ASD BARKER, H. A., J. Biol. Chem. 166, 725 (1947); DOUDOROFF, M., HASHII), W. Z., PUTMAN, E. W., POTTER, A. L., AND LEDERBERG, S., J. Biol. Chem. 179, 921 (1949). 18. See also: BARKER, H. A., AND HASSID, W. Z., Degradation and Synthesis of Complex Carbohydrates, in WERKXAN, C. H., and WILSOS, 1’. W., eds., Bacterial Physiology, pp. 548-65. Academic Press, New York, 1951. 19. HESTRIN, S., Wallerstein Labs. Communs. 11, 193 (1948); ibid. 12, 45 (1949). 20. LEIBOWITZ, J., AND HESTRIN, S., Advances in Enzymol. 6, 87 (1945). 21. LEIBOWITZ, J., AND HESTRIN, S., Enzymologia 6, 15 (1939). 22. GOTTSCHALK, A., Wallerstein Labs. Communs. 12, 55 (1949). 23. VIRTANEN, A. I., Szcomen Kemistilehti 19B. 60 (1946).
Prom
the Biochemistry
Department, dfedical-Dental Schools, Alabama, Birmingham, Alabama Received
February
17n~icersitg
of
9, 1953
In a previous paper (l), the fermentation of n-glucose by yeast was studied from the standpoint of the factors affecting the yield of alcohol for conditions that might be useful in the removal or determination of fermentable sugars. The present paper is an ext’ension of the former work and describes some of the factors affecting alcohol yields from maltose? an important component of starch hydrolyzates. Earlier work with this sugar, the fermentation (2) of which requires more specialized conditons than for D-glucose, has been concerned principally with rates of carbon dioxide evolution. EXPERIMENTAL
Method All fermentations and determinations of yields of alcohol were carried out b> the “standard procedure” of t,he previous paper (1)) unless otherwise indicat,ed. Known amounts of buffered sugar solution (usually 15 g. sugar in 100 ml.) were fermented with large amounts of yeast (3 g. moist yeast) for 3 or 5 days at 30”. The alcohol was then removed by distillation and determined densimetrically. The fermentation efficiency (F.E.) is the percentage of the theoretical yield based on the Gay-Lussac equation. The reported yields do not include the alcohol lost in the vapors, an amount shown to equal 1.3% of the theoretical in the case of D-glucose. Because of the earlier observations (3-5) that maltose will be fermented prop erly only in a restricted region of acidity, all work was carried out, in t,he presence
17
of ammonium and potassium phosphates and citrates as before, at, a pH of about 5. Blank studies for yeast-S showed that no alcohol was produced in the absence of the sugar under these conditions. The range of concentration which is permissible is indicated by the following fermentation efficiencies for commercial maltose fermented for 5 days and with 5% of the indicated amount of maltose substituted by n-glucose: 5% maltose, F.E. 86.5; lo%, F.E. 89.0; 150/o,F.E. 88.8; 17.5%, F.E. 38.5. The presence or absence of nutrients (yeast extract) made no difference in alcohol yields; hence, this substance was omitted when other measurements were to be made or where special precautions were desired to insure the absence of extraneous material as in the study of the effect of the addition of glucose. In the latter case, the yeast was washed as an additional precaution against the introduction of traces of sugar. Five commercial compressed yeasts, previously described, were used. Unless otherwise specified, the yeast was yeast-S (Fleischmann’s compressed baker’s yeast).
Maltose “Technical maltose” is a crude commercially available mixture of maltose, n-glucose, and dextrins. The “commercial maltose” used in this work was a fairly good grade of maltose, “c.P. Maltose” of the Pfanstiehl Chemical Company. Its fermentation characteristics indicate that it contained small amounts of n-glucose. This product could be purified sufficiently by two recrystallizations from methanol. The pure product, which ia the monohydrate and which is referred to as purified maltose, had a rotation [a]:’ = $130.6 (c = 4, water) at equilibrium. All sugars were dried in vucuo at 60’ over calcium chloride.
Results Fermentation of Maltose in Relation to the Requirement of D-Glucose When highly purified maltose was fermented 3-5 days under the standard conditions (15 y0 sugar solution with 3 g. of pressed yeast-S), the yields of alcohol were low and exceedingly erratic. For six such experiments, they were distributed over the range 32.7-67.0 % of the theoretical amount. With the C.P. commercial maltose, similar variation was noted but the average yields were higher (52.6-84.4 ~~0).Four other brands of commercial pressed yeasts behaved in the same fashion. Where a small amount of n-glucose was added to the purified maltose before the fermentation period, the alcohol yields were very close to those previously observed for n-glucose (88.7 %). The amount of D-ghcase required for complete fermentation is established by the following data which show the effect of the addition of various amounts t,o 15 g. of purified maltose. The yields of alcohol after 3 and 5 days fermentation by washed compressed yeast-S were: 0.025 g. n-glucose, 62.9 % (3 days)
and 8’2.7 y0 (5 days); 0.075 g. D-glucose, 72.5 % (3 days) and 88.6 % (5 days); 0.150 g. n-glucose, 89.7 % (3 and 5 days); 0.45 g. n-glucose, 89.8 % (3 days) and 90.4 % (5 days) ; 0.75 g. n-glucose, 88.8 % (5 days). These results show t’hat when the malt’ose contains about 1 % of D-gluwse, practically the same alcohol yields will be obt’ained as fol n-glucose alone. The four other compressed yeasts gave closely similar results, although the fermentation was freyue&y less complete in the shorter time period. Possibly some of the differences ill rate may have a,risen from differences in the ages of the yeasts, since maltose fermentations are k~lown to be more susceptible than glucose ferment.ations to age variations (5-7). Complete fermentation of maltose was also obtained by performing the ferment#ations in the presence of enzyme preparations ront’aining maltase, including Aspergillw oryzae emulsin (0.2 g. of Clarase 970 of Takamine Laboratories; F.E., 86.9 y0 for commercial maltose) and R malt extract (0.5 g. of a Wallerstein concentrate; F.E. 87.1 y0 for commercial maltose). It should he noted that the fermentation of enzymaticallp convert,ed starch occurs completely only in the presence of similar enzymes (8). Nature of the “Glucose lS$‘ect” The results of this investigation show that D-glucose has an important role in determining the extent of fermentation of maltose, a role more significant than solely an initial acceleration, such as has been noted previously in the studies of the rate of rarbon dioxide evolution. Srhult,z, Atkin, and Frey (3, 9, 10) considered that the effect on the rate could be ascribed to an effect on the permeahilit,y of the cell wall of the yeast. Leibowitz and Hestrin (4j, who observed that, n-fructose and o-mannose but not wgalactose showed the same effect, cont,ended that variations of permeability could not be the sole explanatioll. In the present work, additional experiments were performed with the purpose of further elucidating the “glucose effect.” In one set of experiments, fresh yeast (3 g.), with and without D-glucose, was added to 15 y0 solutions of purified maltose which had been fermented previously for 3 days. The alcohol yields after 3 or 5 days additional time with the fresh yeast, even with the added glucose, fell in the range 74.2-76.7 % of the theory. Evidently, the addition of the glucose must take place prior t#othe initial fermentation, and t,he lacbk of complete frrment,atioll is not simply 8 loss in activit>y of the yeast.
20
MARY
GRACE
BLAIR
AND
WARD
PIGMAN
On the other hand, the still residues left from the alcohol determinations could be fermented further by the addition of fresh portions of yeast (3 g.) to an extent that the total alcohol yields fell in the range 85-90 % of the theoretical. As an example, recrystallized maltose (15 g.) when fermented with yeast-S (3 g.) showed at the end of 5 days fermentation efficiencies of only 61.6 and 33.2 %, respectively, in two experiments. The still residues from these solutions when diluted and fermented 3-5 days more with fresh yeast-S produced enough additional alcohol to bring the total yields to 85.9 and 86.2 %. Reducing sugar determinations (as maltose) were made on the centrifuged still residues, by means of the Scales method as modified by Isbell, Pigman, and Frush (11). For this work 15 g. of purified maltose was fermented for 3-5 days with yeast-S, and the alcohol was removed and determined in the usual manner by distillation. The residual solution was analyzed for reducing sugar. Unfortunately, no data were obtained as to the reducing power of the solution before distillation. Two typical results are: F.E., 52.4 % for purified maltose; apparent reducing sugar remaining, 5.27 g. F.E., 82.9 ‘% for commercial maltose; reducing sugar 0.45 g. For fermented n-glucose solutions, this residue contained amounts in the order of 0.06 g. apparent n-glucose from 15 g. of sugar. When the amounts of residual reducing sugar in the still residues were added to the amounts of maltose corresponding to the alcohol formed (with the assumption of an F.E. of 88.7 %), the totals corresponded to 94.7-98.3 % of the initially added maltose. The discrepancy indicates that the use of a maltose factor is not correct and that higher oligosaccharides were probably present. DISCUSSION
The results obtained in the present work are consistent with the interpretation that maltose in the absence of sufficient n-glucose is partially converted, to the extent of 10 % or more, into a nonfermentable form. This conversion product, if stored intracellularly, is liberated when the yeast cells are boiled. Although the boiled material is fermentable, the low reducing power indicates that it is not all in the form of maltose and/or glucose. Previously it has been shown by Pigman (8) that certain mold and malt enzymes will synthesize unfermentable sugars from maltose although not from n-glucose. Guillemet (12) also reported the conversion of t,he fermentation residue from maltose, but not from glucose, to ferment,able sugars by treatment with acid. An unfermentable product synthesized by Aspergillus niger enzymes was
isolated by Pan and co-workers (13) and was shown by Wolfrom, Thompson, and Galkowski (14) to be =4-a-isomaltopyranosyl-D-glucose. &lany of the observed facts of maltose fermentation by yeasts can be explained by the “t’ransglycosidase” scheme, which has been demonstrated in other connections by Hehre (15), Torriani and Rionod (l(i), I)oudoroff and Hasxid (17), and others (18), if this rea&ion is compctitive lvit,h a more rapid fermentation of ho01 glucose and mahose. 7~Glucosyl glucose 7=t (anhydroglucose
unit,s), + 11glucose
If the glucose concentration is kept low by rapid fermentation, t,hc polymer should accumulate. But, if enough glucose is added initially to maintain a sufficiently high concentration throughout the reaction, the polymerative reaction could be reversed and all the carbohydrate would be fermented. The lack of the effect of added glucose after the initial period is more difficult to explain. Possibly the polysaccharide is bound to the yeast cell, or the reverse react,ion is slow for long-chain compounds. An alternative role for the initially added D-glucose is that it is necessary for the production of a sufficient amount of a,11enzyme \\hich is essential in limiting the extent of polymerizatjion. Yeast, exhausted 1)~ storage to a point of inability t’o ferment, maltose is cluickly restored it1 this respect by incubation with D-glucose (19, XI), a behavior sometimes at,tributed to mahase formation. The question of direct versus indirect fermentation of maltose is still unsettled (19, 21-23). Hestrin, a strong advocate of direct fermentation, has proposed polymerative cleavage as the probable route (19). The evidence of t#his paper st)rongly supports the idea of a polymerative react,ioll as one of a series of competitive reactions. The sensitivity of the fermentation reaction to small amounts of ~-glucose is additional evidenye for the cluest,ionat)ilit,y of direct maltasc action in the fermentation of maltose. &KNO\VLEDGMENT This work was begun while the authors were employed at the National Bureau of Standards. The courtesy of E. U. Condon in releasing this material is great13 appreciated.
SUMMARY Pure maltose is fermented
incompletely, but maltose with at, least can be fermented to the same extent, as D-gh~cosc. The still residues from incompletely fcrment,ed maltose solu1 yO D-glncosc
Ktially
preselxt
22
MARY
GRdCE
BLdIR
AND
WARD
PIGMAN
tions can be further fermented to produce the expected quantity of alcohol, but their reducing power is lower than that calculated for the unfermented maltose. The results obtained are consistent with the occurrence of a polymerization reaction as one of a series of competing reactions involved in the fermentation of maltose. REFERENCES 1. BLAIR, M. G., AND PIGMAX, W., Arch. Biochem. and Biophys. 42, 278 (1953). 2. See NORD, F. F., AND WEISS, S., in SURZNER and MYRB~CK, eds., The Enzymes, Vo12, pp. 715-716. Academic Press, New York, 1951. 3. SCHULTZ, A. S., AND ATKIN, L., J. Am. Chem. Sot. 61, 291 (1939). 4. LEIBOWITZ, J., AND HESTRIN, S., Biochenz. J. (London) 36, 772 (1942). 5. STARK, I. E., AND SOMOGYI, M., J. Biol. Chem. 143, 579 (1942). 6. HARDING, V. J., AND NICHOLSON, T. F., Biochem. J. (London) 27, 1082 (1933). 7. SANDSTEDT, R. M., AND BLISH, M. J., Cereal Chem. 11, 368 (1934); J. Biol. Chem. 116, 765 (1937). 8. PIGMAN, W. W., J. Research Natl. Bur. Standards 33, 105 (1944). 9. SCHULTZ, A. S., AND ATKIN, L., U. S. Patent 2,202,356, May 28, 1940. 10. SCHULTZ, A. S., ATKIN, L., AND FREY, C. N., J. Am. Chem. Sot. 62,227l (1940). 11. ISBELL, H. S., PIGMAN, W. W., ASI) FRUSH, H., J. Research Natl. Bur. Standards 24, 241 (1940). 12. GIJILLEMET, R., Con@. rend, sot. biol. 130, 1402 (1939). 13. PAN, S. C., ANDREASEX, A. A., AND KoL.~CHOV, P., Science 112, 115 (1950); PAN, S. C., NICHOLSON, L. W., AND KOLACHOV, P., J. Am. Chem. Sot. 73, 2547 (1951). 14. WOLFROM, M. L., THOMPSON, A., .~ND GALKOWSKI,.T. T., J. Am. Chem. Sot. 73, 4093 (1951). 15. HEHRE, E. J., J. Biol. Chem. 192, 161 (1951). 16. TORRIANI, A. M., ASD MONOD, J., Compt. rend. 226, 718 (1949); MONOD, J., AND TORRIANI, A. M., Compt. rend. 227, 240 (1949). 17. DOUDOROFF, M., HASSID, W. Z., ASD BARKER, H. A., J. Biol. Chem. 166, 725 (1947); DOUDOROFF, M., HASHII), W. Z., PUTMAN, E. W., POTTER, A. L., AND LEDERBERG, S., J. Biol. Chem. 179, 921 (1949). 18. See also: BARKER, H. A., AND HASSID, W. Z., Degradation and Synthesis of Complex Carbohydrates, in WERKXAN, C. H., and WILSOS, 1’. W., eds., Bacterial Physiology, pp. 548-65. Academic Press, New York, 1951. 19. HESTRIN, S., Wallerstein Labs. Communs. 11, 193 (1948); ibid. 12, 45 (1949). 20. LEIBOWITZ, J., AND HESTRIN, S., Advances in Enzymol. 6, 87 (1945). 21. LEIBOWITZ, J., AND HESTRIN, S., Enzymologia 6, 15 (1939). 22. GOTTSCHALK, A., Wallerstein Labs. Communs. 12, 55 (1949). 23. VIRTANEN, A. I., Szcomen Kemistilehti 19B. 60 (1946).