CONVERSION OF ALCOHOLIC FERMENTATION TO GLYCEROL FERMENTATION BY p-benzoquinone SABURO FUKUI Department of Industrial Chemistry, School of Engineering, University of Kyoto, Sakyo-ku, Kyoto (Received October 20, 1955) In normal alcoholic fermentation, acetaldehyde, a decarboxylated product of pyruvic acid by yeast carboxylase, is reduced to alcohol by dihydrocozy mase. However, if the aldehyde is removed from the reaction system by aldehyde-fixing agents, e.g., NaHSO3 or dimedone, or when the aldehyde formation is retarded by some possible means, reduction of triosephosphate by dihydrocozymase to glycerol takes place, a reaction which is usually scarcely observed due to its extremely low reaction rate. Assuming that the inhibition of yeast carboxylase by applying thiamine-antagonist may lead to the occurrence of glycerol fermentation, sulfathiazole (2) was tested and thus the view of the author was proved to be correct (3). In this paper, an experiment will be reported in which p-benzoquinone (BQ) was used as a carboxylase-inhibitant. Many works have been reported on the antibacterial activity of quinone derivatives. Thus, Kensler et al. (4) observed that the oxidation products of N, N Œ-dimethylaminobenzene have an inhibitory effect on yeast carboxylae and diphosphopyridine nucleotide (DPN) system, and Kuhn et al. (5) identified the inhibiting substance as BQ. Akabori and Uehara (6) carried out in vitro experiments with BQ. EXPERIMENTAL AND RESULTS Effect of p-benzoquinone on Carbon Dioxide Evolution. As a preliminary experiment, the effect of the amount of BQ on CO2 evolution was tested as in the previous report. One g of compressed cake of Saccharomyces sake containing 12ƒÁ of thiamine or about 18ƒÁ of cocarboxy lase, was suspended in 50ml of 10 per cent glucose solution containing 1 per cent of KH2PO4 and NH4H2PO4. After incubating for 3 hours, the CO2 formed was measured and compared with that of a control. Fig. 1 shows the ratios of the CO2 formed in the main experiment/the CO2 formed in a control versus the amount of BQ used. It will be seen from Fig. 1, that 2mg of BQ inhibited the CO2 evolution to one half, and more than 3mg inhibited it remarkably. According to the in vitro experiments of Akabori et al. using carboxylase, 18
Vol. 2 CONVERSION OF ALCOHOLIC FERMENTATION 19 FIG. 1 Effect of BQ on CO2 Production. ca. 10ƒÁ of BQ was supposed to be enough to inhibit the activity of the carboxylase in the yeast cells used in this experiment. However, far more BQ was found to be needed in the case of living cells. As for the mechanism of carboxylase inhibition by BQ, Kuhn et al. assumed that BQ did not act as an antivitamin but as an inhib itor of apoenzyme and not observed the resumption of the activity by cysteine or glutathione. The requirement of a large amount of BQ in the case of living cells may be ascribed to be the presence of these sulfhydryl substances in yeast. The inhibitory activity of BQ fell gradually as illustrated in Fig. 2. In the case of 4mg BQ, fermentation started after 5 hours. When 100ml of the 10 per cent sugar medium containing 36mg BQ and 2g yeast was employed, fermentation was completely arrested for several days; thereafter a gradual fermentation started lasting for a few days. Some cells were found to be dead, but the majority of them were alive, forming spores. The use of a large amount of BQ caused the death of all cells. The lethal doses of BQ against yeast were found to be dependent on species, age, freshness, etc. The reason for the retarded start of fermen tation by BQ may be considered to be as follows: Quinone added was gradually inac tivated by the mechanism to be described later and at the arrival at a certain low con centration, fermentation took place. Hydro FIG. 2 Inhibition of Cocarboxylase quinone in such a concentration as was used in this experiment failed to reveal any inhib itory action on fermentation. Glycerol Formation after Adding p-benzoquinone. 1. Effect of Culture Duration. by BQ. A, BQ 4mg. B, 2mg. C, Control. Experiments were carried out to trace the fermentation process in the presence of BQ. Eight grams of the compressed cake of Saccharomyces sake was added to 500ml of the 10 per cent sugar medium containing 90mg of BQ, and the whole was incubated at 30. Fermentation began slowly on the third day lasting until the 8th day. Table T indicates the amounts of sugar, alcohol and glycerol measured after 1, 3, 5, and 7 days respectively. Glycerol was determined by Zeisel Fanto-Stritar's method, glucose by Bertrand's method, and alcohol by the oxidation method. This Table shows that the sugar was not consumed,
20 FUKUI 1956 TABLE T Analysis of Fermentation Fluid. throughout the entire period without fermentation. This may be ascribed to the inhibitory action of BQ not only on carboxylase but also on DPN system. 2. Influence of BQ Amount on Glycerol Formation. Two grams of the compressed cake of Saccharomyces sake was suspended in 100ml of the 10 per cent sugar medium containing 8 to 18mg of BQ, and the mash was incubated at 30. The results is shown in Table U. TABLE U Effect of BQ on Glycerol Formation. After adding 16 and 18mg of BQ respectively, fermentation did not occur for 1-2 days. Generally speaking, the more BQ in the medium, the longer the duration of fermentation, but it did not exceed 3-5 days. When more than 12mg of BQ was used, alcohol formation remarkably decreased. No significant difference in glycerol production was detected after adding 12-18mg of BQ. The reason may be as follows: Fermentation did not take place until BQ fell to a certain level as a result of inactivation by reduction, etc. Subsequently, the amount of BQ which inhibited fermentation, may remain practically almost within a constant level. When BQ exceeded a definite limit, fermentation was completely arrested. Repeated experiments with various strains of yeast, regardless of age, showed that a concentration of BQ above 40mg/100ml was approximately sufficient to inhibit fermenta tion completely. 3. Effect of Fractional Addition of BQ. A further experiment was conducted to determine the amount of alcohol and glycerol formed after additions of BQ in successive small portions.
Vol. 2 CONVERSION OF ALCOHOLIC FERMENTATION 21 Various amounts of BQ were added to 100ml of the 10 per cent sugar medium, and the fermentation products were determined after 10 days. When 24, and 36mg of BQ, respectively, added at one time, fermentation did not occur for the first 2 and 3 days respectively, followed by a gradual fermentation lasting for 5 to 7 days. In the fractional use of 24mg BQ, wherein it was inter mittently added every day, the fermentation took place, though slowly from the start. After adding 36mg in fractional portions, the first addition of 18mg suppressed the fermentation for almost one day but it started on the following day. 9mg was therefore added on the third day, and further, 9mg on the 4th day. The result is given in Table V. As can be seen from the Table, the fractional addition of BQ at such a rate that fermentation TABLE V Effect of Fractional Addition of BQ. may take place very slowly, led to the rise of glycerol formation. Compared with the result of the previous experiment (1) using sulfathiazole, the pre sent result is inferior in glycerol formation. More addition of BQ may not be practical, since it caused a complete inhibition of glycerol formation. SUMMARY 1. The conversion of alcoholic fermentation to glycerol fermentation was achieved by the use of p-benzoquinone as an anticarboxylase substance. 2. After a fractional addition of 20 to 30mg of p-benzoquinone to the cultu re of 2g yeast in 100ml of 10 per cent glucose medium, a slow fermentation took place lasting for about one week, producing ca. 3g of alcohol and 1.5g of glycerol. ACKNOWLEDGEMENT The author wishes to express his hearty thanks to Professor Ryohei Takata for kind guidance throughout the experiment.
22 FUKUI 1956 REFERENCES 1. Negelein, E., and Bromel, H., Biochem. Z. 303, 231 (1939). 2. Sevag, M. G., Shelburne, M., and Mudd, S., J. Bact. 49, 65 (1945). 3. Fukui, S., and Mohara, K., J. Ferm. Technology (Japan) 29, 198 (1951). 4. Kensler, C. J., Young, N. F., and Rhoads, C. P., J. Biol, Chem. 143, 465 (1942). 5. Kuhn, R., and Beinert, H., Ber. 76B, 904 (1944). 6. Akabori, S., and Uehara, K., Proc. Vitamin B Res. Comm. 8 (1946).