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Preparation of radioactive diacetyl, acetoin, and 2,3-butylene glycol
ANALYTICAL
118, 405-409
BIOCHEMISTRY
Preparation
( 198 1)
of Radioactive
Diacetyl,
R. A. SPECKMAN’ Department
of Food
Science
Acetoin, and 2,3-Butylene
Glycol
AND E. B. COLLINS
and Technology,
University
of California,
Davis,
California
95616
Received September 14, 198 1 Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[ 3-‘%]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 X lo4 cpm/pmol and acetoin with specific activity of 6.8 X lo4 cpm/pmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [ ‘%]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 X lo4 cpm/ pmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.
Radioactive isotopes of acetoin and diacetyl have facilitated resolution of the mechanism of diacetyl biosynthesis by lactic acid bacteria and other microorganisms ( l-3). Such isotopes, which are commercially unavailable, should be useful also in studies of the 2,3-butylene glycol cycle of aerobic sporeformers (45) riboflavin biosynthesis by various fungi (6,7), studies of faulty pyruvate metabolism (8- 1 1 ), reversibility and specificity of the diacetyl reductase reaction (2,12- 14) and investigations of the initiation of dental caries by acidogenic bacteria ( 15). This paper reports an economical procedure for the preparation of radioactive diacetyl, acetoin, and 2,3-butylene glycol. MATERIALS
AND METHODS
Oregon State University, Corvallis, Oregon. The organisms were grown in a complex medium (glucose 2%, Bacto-peptone l%, Bacto-yeast extract 1.5%, KH,PO, 0.05%, MgS04 0.02%, Na-acetate 0.21%, water 95.22%; pH adjusted to 6.5; autoclaved at 12 1“C for 15 min). Sodium citrate (2%) was added to the medium for growing S. diacetilactis. Bacto-peptone and yeast extract were from Difco Laboratories, Detroit, Michigan. Separation and collection of metabolites.
Diacetyl, acetoin, and 2,3-butylene glycol were separated with a column salting-out chromatographic procedure ( 16). Column effluents were collected and distributed into test tubes by a Radi-Rak fraction collector, Stockholm, Sweden. Fraction sizes were determined by a drop counter synchronized with the collector.
Organisms and media. Enterobacter aerogenes (ATCC 15038), Serratia marcescens Chemicals and analytical procedures. (ATCC 4003), and Bacillus subtilis (ATCC Diacetyl and acetoin were from Eastman
82) were from the culture collection of E. B. Collins. Streptococcus diacetilactis 18- 16 (ATCC 15346) was from W. E. Sandine, ’ Present address: Department of Food Science, University of Illinois, Urbana, Il. 61801. 405
Organic Chemicals, Rochester, New York; 2,3-butylene glycol was from K&K Laboratories, Jamaica, New York. Acetoin was washed with ether to remove any diacetyl, and the crystalline dimer of acetoin was used to prepare standard solutions. 2,3-Butylene 0003-2697/81/180405-05$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
406
SPECKMAN
glycol was distilled, and the fraction at 184°C was used to prepare standard solutions. Acetoin and diacetyl were determined calorimetrically by the Westerfeld procedure (17). 2,3-Butylene glycol was determined by the method of Desneulle and Naudet (18). Recrystallized cY-napthol was obtained from Fisher Chemical Company. Creatine was obtained from Cal Biochem, La Jolla, California. Radioactive Na-pyruvate was obtained from Calatomic, Los Angeles, California. Radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer with 0.3% 2,4-diphenyloxazole and 0.01% 1,4-bis-2-( 5 phenyl-oxazolyl)benzene in toluene with 20 ml as the total volume. All inorganic chemicals were reagent grade. Preparation of resting cells and toluenized cells. Cells of S. diacetilactis 18-16
were grown for 24 h at 32°C in the complex medium plus 2% (w/v) citrate. Cells of the other organisms were grown for 47 h at 32°C in the complex medium without citrate. Cells were harvested by centrifugation, washed three times with cold phosphate buffer (0.5 M; pH 6.2) and resuspended in 35 ml of the buffer (cell density ca. 50 mg/ml). A IO-ml portion of this resting cell suspension was shaken with 10 ml of cold (4°C) toluene for 5 min ( 19) and the cells were allowed to settle in the cold (4°C) for 1 h. The cell layer was carefully removed with a Pasteur pipet. Preparation of cell-free extracts. Preparative operations were performed in a refrigerated room maintained at 4°C except where indicated otherwise. Cells were disrupted by ballistic disintegration with a Mickle tissue disintegrator operated at maximal amplitude for 45 min. After centrifugation for 30 min in a refrigerated centrifuge (4°C) at 15,OOOg, the cell-free supernatant fluid was dialyzed for 24 h against 100 vol of 1 N Tris (hydroxymethyl) aminoethane - cysteine (Tris-cysteine) buffer at pH 7.3 (0.001 M with respect to L-cysteine). This crude cellfree extract was either used immediately or stored at -20°C.
AND COLLINS
RESULTS AND DISCUSSION Resolution of analytical problems. Since we were preparing a radioactive isotope of a volatile compound (diacetyl), we studied the effects of storage time and container closure on diacetyl recovery. An aqueous solution of diacetyl (20 pg/ml) was prepared, added to unstoppered, Parafilm-capped, and glass-stoppered test tubes ( 15 ml/tube), and the solutions were analyzed immediately for diacetyl. The samples then were stored at room temperature, analyzed after various storage times, and the results were compared to determine any losses of diacetyl that resulted from storage or the different closures. Diacetyl values for the solution stored in glass-stoppered and Parafilm-capped tubes were similar, but those for the solution stored in unstoppered tubes were different (Fig. 1). There were significant losses of diacetyl in 5 h with the tubes unstoppered and in 24 h with the tubes stoppered. After storage for 72 h, only 20% (unstoppered) or 59% (stoppered) of the diacetyl had not evaporated.
‘1L
HOURS OF STORAGE
FIG. I. Influence of storage time and container closure on evaporation of diacetyl. Test tubes: 0, stoppered, 0, unstoppered.
PREPARATION
OF
DIACETYL,
ACETOIN,
It thus became apparent that diacetyl preparations must be assayed and used immediately or stored frozen in capped containers. Selection of organisms. The organisms studied were S. diacetilactis, S. marcescens, E. aerogenes, and B. subtilis. Each was grown at 32°C in the complex medium (inoculum, 1%) and the cells were removed (by centrifugation) after 24 h for S. diacetilactis, or after 48 h for the other bacteria. A portion of each spent medium was fractionated by salting-out chromatography, and the fractions were analyzed for acetoin, diacetyl, and 2,3-butylene glycol. Based on the results (Table 1), S. diacetilactis was chosen as the organism for producing radioactive acetoin and diacetyl, and E. aerogenes was chosen for producing radioactive 2,3-butylene glycol. Selection of cell preparation. Cell-free extracts, resting cell preparations and toluenized cells were made from washed cells of the selected organisms and placed ( 10 ml) in separate 125ml flasks. To each flask were added 100 pmol of MgS04. These mixtures (total volume, 15 ml) were incubated in a thermostatically controlled water bath 1.O h at 30°C. A portion of each reaction mixture was separated by salting-out chromatography and analyzed for acetoin, diacetyl,
AND
2,3-BUTYLENE
GLYCOL
407
and 2,3-butylene glycol. The results are in Table 2. As had been found earlier with growing cells (Table l), these cell preparations of S. diacetilactis produced diacetyl and acetoin without 2,3-butylene glycol. Both toluenetreated cells and cell-free extracts produced much larger amounts than resting cells, which showed that destruction of the cell permeability barrier is desirable, but the amounts of diacetyl and acetoin produced by toluene-treated cells were almost as large as those produced by cell-free extracts. Based on these results we selected the simpler procedure (toluene treatment) for the preparation of radioactive diacetyl and acetoin with S. diacetilactis. Under the experimental conditions of Table 1, which involved growing E. aerogenes for 48 h, the organism obviously converted the acetoin produced to 2,3-butylene glycol, and only the glycol was found upon analysis of the spent medium. However, these cell preparations of E. aerogenes produced considerable acetoin in addition to 2,3-butylene glycol, and the amounts of the glycol were not appreciably greater than had been found with growing cells. Thus, we decided to use growing cells of E. aerogenes for producing radioactive 2,3-butylene glycol. Preparation of radioactive acetoin, diacetyl, and 2,3-butylene glycol. For producing
TABLE
1
END PRODUCTS FORMED BY SELECTED BACTERIA GROWNONGLUCOSE
Products (mmol/ 100 mm01 glucose fermented) Organism Streptococcus diacetilactis Serratia marcescens Enterobacter aerogenes Bacillus subtilis
2,3-Butylene glycol
Diacetyl
Acetoin
0.5
15
0
3
45
0
0
21
0
2
53
0
radioactive acetoin and diacetyl with toluene-treated cells of S. diacetilactis, the reaction mixture was the same as that given in the preceding section except that 100 wmol of Na-[3-‘4C]pyruvate (sp act, 4.6 X lo4 cpm/pmol) was substituted for unlabeled pyruvate. For producing radioactive 2,3-butylene glycol with growing cells of E. aerogenes in the complex medium, uniformly labeled [ 14C]glucose (sp act, 15.6 X lo4 cpmlpmol) was substituted for unlabeled glucose. Reaction mixtures (15 ml) containing toluene-treated cells of S. diacetilactis were incubated 1.0 h at 30°C and acidified with 1.8 N H2S04 (1 drop/ml) to decarboxylate (to acetoin) any a-acetolactic
408
SPECKMAN
AND
COLLINS
TABLE
2
ENDPRODUCTSFORMEDFROMPYRUVATEBYCELLPREPARATIONSOF Streptococcus diacetilactis AND Enterobacter Products
Organism
Cell preparation
Streptococcus diacetilactis
Enterobacter aerogenes
2,3-Butylene glycol
12.5 39.1 41.8
0 0 0
Resting Toluene Cell-free
cells treated extract
0 0 0
13.1 18.5 23.3
15.0 16.4 20.1
CULTURE1 $ dwcetiacilo
I IOml Tube-of
E. oerogW!eS
Glywl Fractions 1
Acetoin
Na-pyruvate)
1.1 3.9 4.2
1 LYOPHILIZED
--j-$jf~‘g”pc
Diacetyl
Fmol
cells treated extract
,846
Litmus Milk 2daysai3oc ,(,“,I T” b B of Broth 1 3 daily transfers at 30C I L Flask of A~,opne+@ Broth f “Gglucoss preserd for E. aeropsn*s ) r HARVEST CElLSl
collect
(~mol/JOO
Resting Toluene Cell-free
acid that might be present (20). Flasks containing 500 ml of the complex medium and inoculated with E. aerogenes (1%) were incubated 48 h at 3O”C, and the bacterial cells were removed by centrifugation. End products were separated by salting-out chromatography, tested for radioactivity, and assayed with the Westerfeld test ( 17). To ensure radiochemical purity of each separated compound, only the fraction at the
~.oerogsner SUPERNATANT
aerogenes
--S diacetibcte CELLS ---jz$;~,2 Washed Cell Suspensiwr 1 tohmne treat parmsdbobzed Cells I react at 3oc
FIG. 2. Summary of preparation of radioactive diacetyl, acetoin, and 2,3-butylene glycol. (Boxes represent possible stopping places.)
apex of the separation curve was used for metabolic studies. The specific activities of acetoin and diacetyl made with S. diacetila&is were 6.1 X lo4 and 6.8 X lo4 cpm/ pmol, respectively. The specific activity of the 2,3-butylene glycol made with E. aerogenes was 10.8 X lo4 cpm/pmol. Each of the three compounds was found to be chromatographically homogeneous. The preparations were apportioned according to expected use, labeled, and stored frozen. The procedures for preparing the compounds are summarized in Fig. 2. REFERENCES 1. Chuang, L. F., and Collins, E. B. (1968) J. Eacteriol. 95, 2083-2089. 2. Speckman, R. A., and Collins, E. B. (1968) J. Bacteriol. 95, 114- 180. 3. Speckman, R. A., and Collins, E. B. (1973) Appl. Microbial. 26, 144-146. 4. Gottschalk, G. (1979) Bacterial Metabolism, Springer-Verlag, New York/Berlin. 5. Juni, E., and Heym, G. A. (1956) J. Bacterial. 71, 425-432. 6. Goodwin, T. W., and Treble, D. H. ( 1958) Biochem. J. 70, 14P. 7. Masuda, T. (1957) Pharm. Bull. 5, 136. 8. Bigler, F., Tholen, H., and Staub, H. (1961) Helv. Physiol. Pharmacol. Acta 19, Cl l-C13. 9. Bigler, F., Tholen, H., and Staub, H. (1962) He/v. Physiol. Pharmacol. Acta 20, 368-372. 10. Freese, E., and Fortnagel, U. (1969) J. Bacterial. 65, 581-586.
PREPARATION
OF DIACETYL,
ACETOIN,
11. Steckerl, R., and Deppel, H. (1959) Arch. Biochem. Biophys.
79, 393-396.
12. Silber, P., Chung, H., Gargiluo, P., and Schulz, H. (1974) J. Bucferiol. 118, 919-927. 13. Stormer, F., Bryn, K., and Hetland, 0. (197 1) J. Biol. Chem. 242, 1756-1759. 14. Thompson, J. W., Shovers, J., Sandine, W. E., and Elliker, P. R. (1970) Appl. Microbial. 19, 883889. 15. Harris, R. S. (1968) Art and Science of Dental Caries Research, Academic Press, New York.
AND 2,3-BUTYLENE
GLYCOL
409
16. Speckman, R. A., and Collins, E. B. (1968) Anal. Biochem. 22, 154- 160. 17. Westerfeld, W. W. (1945) J. Biol. Chem. 161,495502. 18. Desnuelle, P., and Naudet, M. (1945) Bull. Sot. Chim.
Fr.
12, 871-875.
19. Gerhardt, P., McGregor, D. R., Marr, A. G., Olsen, C. B., and Wilson, J. B. (1953) J. Bacterial. 65, 581-586. 20. Collins, E. B., and Speckman, R. A. (1974) Cunad. J. Microbial. 20, 805-8 11.
118, 405-409
BIOCHEMISTRY
Preparation
( 198 1)
of Radioactive
Diacetyl,
R. A. SPECKMAN’ Department
of Food
Science
Acetoin, and 2,3-Butylene
Glycol
AND E. B. COLLINS
and Technology,
University
of California,
Davis,
California
95616
Received September 14, 198 1 Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[ 3-‘%]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 X lo4 cpm/pmol and acetoin with specific activity of 6.8 X lo4 cpm/pmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [ ‘%]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 X lo4 cpm/ pmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.
Radioactive isotopes of acetoin and diacetyl have facilitated resolution of the mechanism of diacetyl biosynthesis by lactic acid bacteria and other microorganisms ( l-3). Such isotopes, which are commercially unavailable, should be useful also in studies of the 2,3-butylene glycol cycle of aerobic sporeformers (45) riboflavin biosynthesis by various fungi (6,7), studies of faulty pyruvate metabolism (8- 1 1 ), reversibility and specificity of the diacetyl reductase reaction (2,12- 14) and investigations of the initiation of dental caries by acidogenic bacteria ( 15). This paper reports an economical procedure for the preparation of radioactive diacetyl, acetoin, and 2,3-butylene glycol. MATERIALS
AND METHODS
Oregon State University, Corvallis, Oregon. The organisms were grown in a complex medium (glucose 2%, Bacto-peptone l%, Bacto-yeast extract 1.5%, KH,PO, 0.05%, MgS04 0.02%, Na-acetate 0.21%, water 95.22%; pH adjusted to 6.5; autoclaved at 12 1“C for 15 min). Sodium citrate (2%) was added to the medium for growing S. diacetilactis. Bacto-peptone and yeast extract were from Difco Laboratories, Detroit, Michigan. Separation and collection of metabolites.
Diacetyl, acetoin, and 2,3-butylene glycol were separated with a column salting-out chromatographic procedure ( 16). Column effluents were collected and distributed into test tubes by a Radi-Rak fraction collector, Stockholm, Sweden. Fraction sizes were determined by a drop counter synchronized with the collector.
Organisms and media. Enterobacter aerogenes (ATCC 15038), Serratia marcescens Chemicals and analytical procedures. (ATCC 4003), and Bacillus subtilis (ATCC Diacetyl and acetoin were from Eastman
82) were from the culture collection of E. B. Collins. Streptococcus diacetilactis 18- 16 (ATCC 15346) was from W. E. Sandine, ’ Present address: Department of Food Science, University of Illinois, Urbana, Il. 61801. 405
Organic Chemicals, Rochester, New York; 2,3-butylene glycol was from K&K Laboratories, Jamaica, New York. Acetoin was washed with ether to remove any diacetyl, and the crystalline dimer of acetoin was used to prepare standard solutions. 2,3-Butylene 0003-2697/81/180405-05$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
406
SPECKMAN
glycol was distilled, and the fraction at 184°C was used to prepare standard solutions. Acetoin and diacetyl were determined calorimetrically by the Westerfeld procedure (17). 2,3-Butylene glycol was determined by the method of Desneulle and Naudet (18). Recrystallized cY-napthol was obtained from Fisher Chemical Company. Creatine was obtained from Cal Biochem, La Jolla, California. Radioactive Na-pyruvate was obtained from Calatomic, Los Angeles, California. Radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer with 0.3% 2,4-diphenyloxazole and 0.01% 1,4-bis-2-( 5 phenyl-oxazolyl)benzene in toluene with 20 ml as the total volume. All inorganic chemicals were reagent grade. Preparation of resting cells and toluenized cells. Cells of S. diacetilactis 18-16
were grown for 24 h at 32°C in the complex medium plus 2% (w/v) citrate. Cells of the other organisms were grown for 47 h at 32°C in the complex medium without citrate. Cells were harvested by centrifugation, washed three times with cold phosphate buffer (0.5 M; pH 6.2) and resuspended in 35 ml of the buffer (cell density ca. 50 mg/ml). A IO-ml portion of this resting cell suspension was shaken with 10 ml of cold (4°C) toluene for 5 min ( 19) and the cells were allowed to settle in the cold (4°C) for 1 h. The cell layer was carefully removed with a Pasteur pipet. Preparation of cell-free extracts. Preparative operations were performed in a refrigerated room maintained at 4°C except where indicated otherwise. Cells were disrupted by ballistic disintegration with a Mickle tissue disintegrator operated at maximal amplitude for 45 min. After centrifugation for 30 min in a refrigerated centrifuge (4°C) at 15,OOOg, the cell-free supernatant fluid was dialyzed for 24 h against 100 vol of 1 N Tris (hydroxymethyl) aminoethane - cysteine (Tris-cysteine) buffer at pH 7.3 (0.001 M with respect to L-cysteine). This crude cellfree extract was either used immediately or stored at -20°C.
AND COLLINS
RESULTS AND DISCUSSION Resolution of analytical problems. Since we were preparing a radioactive isotope of a volatile compound (diacetyl), we studied the effects of storage time and container closure on diacetyl recovery. An aqueous solution of diacetyl (20 pg/ml) was prepared, added to unstoppered, Parafilm-capped, and glass-stoppered test tubes ( 15 ml/tube), and the solutions were analyzed immediately for diacetyl. The samples then were stored at room temperature, analyzed after various storage times, and the results were compared to determine any losses of diacetyl that resulted from storage or the different closures. Diacetyl values for the solution stored in glass-stoppered and Parafilm-capped tubes were similar, but those for the solution stored in unstoppered tubes were different (Fig. 1). There were significant losses of diacetyl in 5 h with the tubes unstoppered and in 24 h with the tubes stoppered. After storage for 72 h, only 20% (unstoppered) or 59% (stoppered) of the diacetyl had not evaporated.
‘1L
HOURS OF STORAGE
FIG. I. Influence of storage time and container closure on evaporation of diacetyl. Test tubes: 0, stoppered, 0, unstoppered.
PREPARATION
OF
DIACETYL,
ACETOIN,
It thus became apparent that diacetyl preparations must be assayed and used immediately or stored frozen in capped containers. Selection of organisms. The organisms studied were S. diacetilactis, S. marcescens, E. aerogenes, and B. subtilis. Each was grown at 32°C in the complex medium (inoculum, 1%) and the cells were removed (by centrifugation) after 24 h for S. diacetilactis, or after 48 h for the other bacteria. A portion of each spent medium was fractionated by salting-out chromatography, and the fractions were analyzed for acetoin, diacetyl, and 2,3-butylene glycol. Based on the results (Table 1), S. diacetilactis was chosen as the organism for producing radioactive acetoin and diacetyl, and E. aerogenes was chosen for producing radioactive 2,3-butylene glycol. Selection of cell preparation. Cell-free extracts, resting cell preparations and toluenized cells were made from washed cells of the selected organisms and placed ( 10 ml) in separate 125ml flasks. To each flask were added 100 pmol of MgS04. These mixtures (total volume, 15 ml) were incubated in a thermostatically controlled water bath 1.O h at 30°C. A portion of each reaction mixture was separated by salting-out chromatography and analyzed for acetoin, diacetyl,
AND
2,3-BUTYLENE
GLYCOL
407
and 2,3-butylene glycol. The results are in Table 2. As had been found earlier with growing cells (Table l), these cell preparations of S. diacetilactis produced diacetyl and acetoin without 2,3-butylene glycol. Both toluenetreated cells and cell-free extracts produced much larger amounts than resting cells, which showed that destruction of the cell permeability barrier is desirable, but the amounts of diacetyl and acetoin produced by toluene-treated cells were almost as large as those produced by cell-free extracts. Based on these results we selected the simpler procedure (toluene treatment) for the preparation of radioactive diacetyl and acetoin with S. diacetilactis. Under the experimental conditions of Table 1, which involved growing E. aerogenes for 48 h, the organism obviously converted the acetoin produced to 2,3-butylene glycol, and only the glycol was found upon analysis of the spent medium. However, these cell preparations of E. aerogenes produced considerable acetoin in addition to 2,3-butylene glycol, and the amounts of the glycol were not appreciably greater than had been found with growing cells. Thus, we decided to use growing cells of E. aerogenes for producing radioactive 2,3-butylene glycol. Preparation of radioactive acetoin, diacetyl, and 2,3-butylene glycol. For producing
TABLE
1
END PRODUCTS FORMED BY SELECTED BACTERIA GROWNONGLUCOSE
Products (mmol/ 100 mm01 glucose fermented) Organism Streptococcus diacetilactis Serratia marcescens Enterobacter aerogenes Bacillus subtilis
2,3-Butylene glycol
Diacetyl
Acetoin
0.5
15
0
3
45
0
0
21
0
2
53
0
radioactive acetoin and diacetyl with toluene-treated cells of S. diacetilactis, the reaction mixture was the same as that given in the preceding section except that 100 wmol of Na-[3-‘4C]pyruvate (sp act, 4.6 X lo4 cpm/pmol) was substituted for unlabeled pyruvate. For producing radioactive 2,3-butylene glycol with growing cells of E. aerogenes in the complex medium, uniformly labeled [ 14C]glucose (sp act, 15.6 X lo4 cpmlpmol) was substituted for unlabeled glucose. Reaction mixtures (15 ml) containing toluene-treated cells of S. diacetilactis were incubated 1.0 h at 30°C and acidified with 1.8 N H2S04 (1 drop/ml) to decarboxylate (to acetoin) any a-acetolactic
408
SPECKMAN
AND
COLLINS
TABLE
2
ENDPRODUCTSFORMEDFROMPYRUVATEBYCELLPREPARATIONSOF Streptococcus diacetilactis AND Enterobacter Products
Organism
Cell preparation
Streptococcus diacetilactis
Enterobacter aerogenes
2,3-Butylene glycol
12.5 39.1 41.8
0 0 0
Resting Toluene Cell-free
cells treated extract
0 0 0
13.1 18.5 23.3
15.0 16.4 20.1
CULTURE1 $ dwcetiacilo
I IOml Tube-of
E. oerogW!eS
Glywl Fractions 1
Acetoin
Na-pyruvate)
1.1 3.9 4.2
1 LYOPHILIZED
--j-$jf~‘g”pc
Diacetyl
Fmol
cells treated extract
,846
Litmus Milk 2daysai3oc ,(,“,I T” b B of Broth 1 3 daily transfers at 30C I L Flask of A~,opne+@ Broth f “Gglucoss preserd for E. aeropsn*s ) r HARVEST CElLSl
collect
(~mol/JOO
Resting Toluene Cell-free
acid that might be present (20). Flasks containing 500 ml of the complex medium and inoculated with E. aerogenes (1%) were incubated 48 h at 3O”C, and the bacterial cells were removed by centrifugation. End products were separated by salting-out chromatography, tested for radioactivity, and assayed with the Westerfeld test ( 17). To ensure radiochemical purity of each separated compound, only the fraction at the
~.oerogsner SUPERNATANT
aerogenes
--S diacetibcte CELLS ---jz$;~,2 Washed Cell Suspensiwr 1 tohmne treat parmsdbobzed Cells I react at 3oc
FIG. 2. Summary of preparation of radioactive diacetyl, acetoin, and 2,3-butylene glycol. (Boxes represent possible stopping places.)
apex of the separation curve was used for metabolic studies. The specific activities of acetoin and diacetyl made with S. diacetila&is were 6.1 X lo4 and 6.8 X lo4 cpm/ pmol, respectively. The specific activity of the 2,3-butylene glycol made with E. aerogenes was 10.8 X lo4 cpm/pmol. Each of the three compounds was found to be chromatographically homogeneous. The preparations were apportioned according to expected use, labeled, and stored frozen. The procedures for preparing the compounds are summarized in Fig. 2. REFERENCES 1. Chuang, L. F., and Collins, E. B. (1968) J. Eacteriol. 95, 2083-2089. 2. Speckman, R. A., and Collins, E. B. (1968) J. Bacteriol. 95, 114- 180. 3. Speckman, R. A., and Collins, E. B. (1973) Appl. Microbial. 26, 144-146. 4. Gottschalk, G. (1979) Bacterial Metabolism, Springer-Verlag, New York/Berlin. 5. Juni, E., and Heym, G. A. (1956) J. Bacterial. 71, 425-432. 6. Goodwin, T. W., and Treble, D. H. ( 1958) Biochem. J. 70, 14P. 7. Masuda, T. (1957) Pharm. Bull. 5, 136. 8. Bigler, F., Tholen, H., and Staub, H. (1961) Helv. Physiol. Pharmacol. Acta 19, Cl l-C13. 9. Bigler, F., Tholen, H., and Staub, H. (1962) He/v. Physiol. Pharmacol. Acta 20, 368-372. 10. Freese, E., and Fortnagel, U. (1969) J. Bacterial. 65, 581-586.
PREPARATION
OF DIACETYL,
ACETOIN,
11. Steckerl, R., and Deppel, H. (1959) Arch. Biochem. Biophys.
79, 393-396.
12. Silber, P., Chung, H., Gargiluo, P., and Schulz, H. (1974) J. Bucferiol. 118, 919-927. 13. Stormer, F., Bryn, K., and Hetland, 0. (197 1) J. Biol. Chem. 242, 1756-1759. 14. Thompson, J. W., Shovers, J., Sandine, W. E., and Elliker, P. R. (1970) Appl. Microbial. 19, 883889. 15. Harris, R. S. (1968) Art and Science of Dental Caries Research, Academic Press, New York.
AND 2,3-BUTYLENE
GLYCOL
409
16. Speckman, R. A., and Collins, E. B. (1968) Anal. Biochem. 22, 154- 160. 17. Westerfeld, W. W. (1945) J. Biol. Chem. 161,495502. 18. Desnuelle, P., and Naudet, M. (1945) Bull. Sot. Chim.
Fr.
12, 871-875.
19. Gerhardt, P., McGregor, D. R., Marr, A. G., Olsen, C. B., and Wilson, J. B. (1953) J. Bacterial. 65, 581-586. 20. Collins, E. B., and Speckman, R. A. (1974) Cunad. J. Microbial. 20, 805-8 11.