- Email: [email protected]
On the mechanism of enzyme action. XLVIII. Investigation of the fat of Fusarium lini Bolley by means of urea adducts
On the Mechanism of Enzyme Action. XLVIII. Investigation of the Fat of Fusarium Zini Bolley by Means of Urea Adducts J. A. Maselli From the Department
and F. F. Nord
of Organic Chemistry and Enzymology,1
Fordham University,
New York 68, New York
Received December 3, 1951
Members of the genus Pusurium are both par excellence nonphosphorylating alcoholic fermenters and fat-forming organisms (1). These molds, therefore, have long served as test organisms for the study of the mechanism of carbohydrate -+ fat conversion. Moreover, in the field of fats and oils (2,3) application has been made of the observation that urea gives precipitable adducts with long-chain hydrocarbons (4). Accordingly, it was thought that these findings could be extended to develop analytical methods for the elucidation of the composition of fats formed by microorganisms. EXPERIMENTAL A methanol solution saturated with urea (c.P.) was used in all experiments for adduct formation. Two procedures were employed to obtain urea adducts. In the first case, fat solvents were employed; namely, isoiictane and ethyl ether. However, since these solvents tend to precipitate urea from the saturated methanol solution, an attempt was made to overcome this difficulty. Twenty ml. of isoiictane was thoroughly shaken with 299 ml. of saturated urea-methanol solution. After all the precipitated urea had been removed, the isoiictane layer was separated from the resulting mixture. In this solution was dissolved 2 g. of stearic acid. The solution so obtained was mixed with the urea-methanol layer. Adduct formation then resulted. However, more consistent results were obtained when no fat solvent was employed. In this procedure, the solid fatty acids were first melted, and then the urea-methanol solution was added. r Communication No. 252. This work was carried out under the aegis of the Office of Naval Research and was aided by a grant from the F. G. Cottrell Fund of the Research Corporation. 377
378
J. A. MASELLI
AND
F. F. NORD
The crystallization of the adducts from methanol was performed at 0°C. by cooling in an ice bath a solution of 2 g. of the adduct in 15 ml. of methanol. After 30 min. standing, the mixture was filtered, using a filter stick which had been cooled to the same temperature. The residue was washed once with 5 ml. of methanol and recrystallized in the same manner. Decomposition of the adducts was brought about by shaking with cold water. The fatty acids were then extracted with ether. The washed ether extract was dried over anhydrous NaaS04 and, with a stream of nitrogen bubbling through, the solution was evaporated in vacua. The mold Fusarium Mini Bolley (FIB) was grown for 21 days on a Raulin-Thorn medium of the following composition: Tartaric acid. Magnesium carbonate.. Ammonium tartrate.. Potassium carbonate. Ammonium sulfate. Zincsulfate............................................ Ferroussulfate......................................... Ammonium phosphate dibasic. Glucose............................................... Tapwater.............................................to301.
So”‘.0 8.0 80.0 12 .O 5.0 1.4 1.4 12.0 750.0
One liter of the above solution was used as. the medium for each flask. The methods of fat extraction and isolation are described elsewhere (5). Erlenmeyer flasks, containing the following materials were used in the dehydrogenation experiments: 3 g. ground, defatted FIB mycelia 2 ml. substrate [15 g. stearic acid emulsified with 500 ml. polyvinyl tion (6)] 3 ml. phosphate buffer (pH 8.0) 1 ml. muscle adenylic acid (1 mg./ml.) 10 ml. water.
alcohol solu-
To destroy enzyme activity, the control flasks were immediately autoclaved at 23 lb. pressure for 5 min. Then, both the control and the experimental flasks were shaken for 24 hr. in a thermostat. The filtrates from the flasks were shaken (after acidification) with three RJO-ml. portions of low-boiling petroleum ether (30-50”). The obtained solutions were combined and used to extract the dried mycelia from the Basks in a Soxhlet apparatus. The final contents were washed, dried over N&SO,, and evaporated in vacua under a stream of nitrogen. All iodine values were determined by the Hanus method. RESULTS
In our experiments, which were directed mainly to a study of the potentialities of the formation of urea adducts with the fatty acids
ENZYME
ACTION.
XLVIII
379
present in FlB fat, linseed oil fatty acids and corn oil fatty acids were also investigated for the purpose of comparison. First of all, it was necessary to determine whether adducts with saturated fatty acids and unsaturated fatty acids differ in composition. Our measurements substantiate the observation (2) that no appreciable differences. are noted. In the present case, it was found that the stearic acid adduct contained 13.0 moles of urea, and that the oleic acid adduct contained 13.4 moles of urea per mole of fatty acid. Therefore, it can be concluded that the molar ratios of the respective adducts do not provide a suitable basis for a method of analysis. Furthermore, we have found that glycerides of fatty acids, e.g., in the form of corn oil, do not form adducts under the conditions used. Corn oil itself, when mixed with a saturated solution of urea in methanol, failed to give a precipitate even after long standing. A 50 : 50 mixture of corn oil with corn oil fatty acids resulted in adduct formation, but a layer of corn oil remained in the uncombined state. When 2 g. of corn oil fatty acids was added to 80 ml. of urea solution, the fatty acids obtained from the adduct were found to have an iodine value of 81. With 2 g. of fatty acids and 200 ml. of urea solution, the resulting fatty acids had an iodine value of 97. Since it is known that saturated fatty acids form adducts more readily than unsaturated fatty acids, it is obvious that the latter method should be given preference, since the results indicate that adduct formation is more complete. When the solution of corn oil fatty acids and urea was placed in a refrigerator overnight, 8.8 g. of adduct was formed from 2 g. of fatty acids. Only 6.4 g. was obtained when the solution was allowed to remain at room temperature for the same time. However, in the first case, only 0.72 g. of fatty acids having an iodine value of 91.0 was obtained from 5 g. of the filtered adduct, while in the latter case, 5 g. of adduct yielded 0.91 g. of fatty acids with an iodine value of 97.6. Hence, room temperature was found to be more favorable. Apparently, at the lower temperature, increased precipitation of urea from the saturated solution and less adduct formation result. The relative degrees of unsaturation of the fatty acids after adduct formation were determined as outlined above. The results found are listed in Table I, together with the weights of the various fractions obtained. About twenty solvents were used to determine the preferential solubilities of the stearic and oleic acid adducts. These measurements
380
J.
A.
MASELLI
AND
TABLE
F.
F.
NORD
I
Data on Adduct Fractions Source of fatty
acids
Iodine values of fatty acids Weights of adduct per 2 g. of fatty acids, g. Weights of filtrate oil per 2 g. of fatty acids, g. Weights of oil in 5 g. of adduct, g. Iodine values of oil from adduct Iodine values of oil from filtrate
Corn oil
Linseed oil
Oil of FIB
129.7 6.40 0.62 0.91 97.6 163.9
175.0 2.65 1.22 1.04 102.9 204.8
130.0 4.01 1.03 0.95 80.9 189.1
were undertaken at 20,0, and - 15°C. As a result of these comparisons, methanol was chosen as the most satisfactory solvent, since it was found that at 0°C. the oleic acid adduct remained in solution while the stearic acid adduct was completely precipitated. Hence, in all fractionations, these conditions of solvent and temperature were employed. In Table II are listed the data obtained from the fractionation studies which were carried out in the above manner. TABLE Adduct Fractions Recrystallized Source of fatty
acids
Fatty acid residue per 10 g. of adduct, g. Filtrate fatty acids per 10 g. of adduct, g. Iodine values of residual fatty acids Iodine values of filtrate fatty acids
II from Methanol
at 0°C.
Corn oil
Linseed oil
Oil of FIB
0.82 0.95 43.8 137.8
0.50 1.41 50.2 117.4
0.85 1.21 10.4 97.4
In the procedure adopted for the isolation of stearic acid from FlB fat, the residue obtained by crystallizing the FlB fatty acids adduct at 0°C. in methanol was washed once more with 5 ml. of methanol and again cooled to 0°C. and filtered. After its final decomposition, there was obtained a solid white material which melted at 5268°C. The yield was 1 g. from 12 g. of adduct. This solid mixture was assumed to contain mainly palmitic and stearic acids. After four recrystallizations from methanol, a product which melted at 67-69°C. was isolated. A mixed melting point with an authentic sample of stearic acid showed no depression. A mixed melting point with an authentic sample of palmitic acid showed a depression of 11°C. This result indicated that FlB is capable of forming, from carbohydrate, fat containing stearic acid. An in vitro experiment, using ground, defatted mycelia from FlB added further evidence that this organism is able to dehydrogenate
ENZYME
ACTION.
XLVIII
381
stearic acid. A polyvinyl alcohol emulsion of stearic acid was placed in contact with a suspension of FlB in the presence of a small amount of adenylic acid. After shaking for 24 hr. at 28”C., the contents of the previously autoclaved control flasks and of the experimental flasks were extracted with petroleum ether. The fatty acids obtained from the controls had an iodine value of 5.3, while those from the experiments proper had an iodine value of 10.7, further indicating the presence of a fatty a,cid dehydrogenase in FlB. DISCUSSION
It has been demonstrated that urea adds preferentially to saturated fatty acids, although there is a substantial combination with the unsaturated fatty acids. On the basis of these findings, it was thought that a fractionation procedure could be developed. It is evident from the results obtained with corn oil that urea adduct formation has but little place in studies with unsaponified fats. However, in the case of fatty acids, several applications have been found. As previously indicated (2)) and as reported in this paper, urea adducts provide a useful means for isolating fatty acids of varying degrees of saturation. Linseed fatty acids, which are the most unsaturated of the three mixtures used, have the least tendency to form an adduct, as shown in Table I by the relatively small amount of adduct obtained and by the relatively large amount of oil that remained uncombined. That there is a higher yield of adduct with corn oil fatty acids than with fatty acids of FlB may be attributable to the fact that FlB fat contains linolenic acid, while corn oil does not. The amount of fatty acids which combined with urea to form 5 g. of adduct, was approximately the same in all cases. This is further substantiation of the observation that the molar ratio of urea to fatty acids seems not to vary with the degree of unsaturation. The preference for adduct formation with the saturated components of the fatty acid mixtures is again strikingly demonstrated by the large differences between the iodine values of the adduct fatty acids and those of the filtrate. This preference served as the basis for the attempted fractionations. An extension of the procedure of crystallizing the adducts from methanol at 0°C. has enabled us to obtain, more completely, saturated fatty acids from the adducts, as is borne out in Table II. The data on the adducts and filtrates resulting from this crystallization point to the
382
J.
A.
MASELLI
AND
F.
F.
NORD
fact that the adduct formed with the linseed oil fatty acids had the highest iodine value and resulted in the lowest residual weight as expected. It can be seen by a consideration of the iodine values of the residual fatty acids and the filtrate fatty acids, that crystallizations at 0°C. cause a definite removal of unsaturated fatty acids, especially in the case of FlB fatty acids. Our method of removing unsaturated fatty acids possesses the advantage that it is unnecessary to apply temperatures as low as -3O”C., as is customary in low-temperature crystallization procedures. Furthermore, the bulk of the fatty acids is increased by the urea present. This latter fact is helpful, particularly when working with mold fats, since one of the major obstacles in the study of the enzymatic carbohydrate -+ fat conversion is the small amount of fat which is generally available for quantitative study. In the case of FlB fatty acids adduct, the results of the methanol crystallization at 0°C. were sufficiently satisfactory to prompt an attempt to isolate and to identify stearic acid from the final solid fraction obtained. This relatively simple method obviates the employment of very low temperatures and the use of a complicated fractionating apparatus. In a study of the quantitative composition of FlB fat, it was shown that saturated acids amounted to about 23% of the total quantity of fatty acids (7). Of this, at least 60% was palmitic acid. Since it had been demonstrated that Fusaria are capable of converting stearic acid to unsaturated fatty acids, the identification of it in the fat from FlB is of interest, since this acid could be a starting material upon which a fatty acid dehydrogenase might act in the living organism to form the mono-, di-, and triethenoid fatty acids known to be present. This finding, together with the fact that FIB is capable of dehydrogenating stearic acid both in viva and in vitro, provides a susbtantial indication for the enzymatic formation of unsaturated fatty acids in FlB by means of the action of a fatty acid dehydrogenase system. Additional corroboration of this point is offered by the fact that in the presence of small amounts of naphthoquinones, a more unsaturated fat has been shown to be formed by FlB (8). These naphthoquinones are thought to interact in the normal equilibrium of the hydrogen transport carried out by the fatty acid dehydrogenases, either with the interlocking codehydrogenases or in the cytochrome system.
ENZYME
ACTION.
XLVIII
383
SUMMARY
The usefulness of urea adducts for the fractionation of natural fatty acids has been demonstrated. Stearic acid has been isolated and identified from the fat of Fusarium Eini Bolley by purification of the solid fraction obtained from the urea adduct of its fatty acids. The conversion of stearic acid to fatty material with a higher iodine value has provided further evidence for the enzymatic formation of unsaturated fatty acids in Fusarium Zini Bolley by means of the action of a fatty acid dehydrogenase system. REFERENCES
1. NORD, F. F., AND WEISS, S., in SUMNER, J. B., AND MYRBHCK, K., Ed., The
Enzymes, Vol. 2, part 1, p. 740. New York, Academic Press, 1951. 2. SCHLENK, H., AND HOLMAN, R. T., J. Am. Chem. Sot. 73, 5001 (1950). 3. NEWEY, H.A., SHOKAL, E.C., MUELLER, A.C., BRADLEY, T.F., ANDFETTERLY, L. C., Ind. Eng, Chem. 42, 2538 (1950). 4. SCHLENK, W., JR., Ann. 666, 204 (1949). 5. FIORE, J. V., Arch. Biochem. 16, 161 (1948). 6. FIORE, J. V., AND NORD, F. F., Arch. Biochem. 23, 473 (1949). 7. MULL, R. P., AND NORD, F. F., Arch. Biochem. 6, 283 (1944). 8. MASELLI, J. A., AND NORD, F. F., Arch. Biochem. 24, 235 (1949).
and F. F. Nord
of Organic Chemistry and Enzymology,1
Fordham University,
New York 68, New York
Received December 3, 1951
Members of the genus Pusurium are both par excellence nonphosphorylating alcoholic fermenters and fat-forming organisms (1). These molds, therefore, have long served as test organisms for the study of the mechanism of carbohydrate -+ fat conversion. Moreover, in the field of fats and oils (2,3) application has been made of the observation that urea gives precipitable adducts with long-chain hydrocarbons (4). Accordingly, it was thought that these findings could be extended to develop analytical methods for the elucidation of the composition of fats formed by microorganisms. EXPERIMENTAL A methanol solution saturated with urea (c.P.) was used in all experiments for adduct formation. Two procedures were employed to obtain urea adducts. In the first case, fat solvents were employed; namely, isoiictane and ethyl ether. However, since these solvents tend to precipitate urea from the saturated methanol solution, an attempt was made to overcome this difficulty. Twenty ml. of isoiictane was thoroughly shaken with 299 ml. of saturated urea-methanol solution. After all the precipitated urea had been removed, the isoiictane layer was separated from the resulting mixture. In this solution was dissolved 2 g. of stearic acid. The solution so obtained was mixed with the urea-methanol layer. Adduct formation then resulted. However, more consistent results were obtained when no fat solvent was employed. In this procedure, the solid fatty acids were first melted, and then the urea-methanol solution was added. r Communication No. 252. This work was carried out under the aegis of the Office of Naval Research and was aided by a grant from the F. G. Cottrell Fund of the Research Corporation. 377
378
J. A. MASELLI
AND
F. F. NORD
The crystallization of the adducts from methanol was performed at 0°C. by cooling in an ice bath a solution of 2 g. of the adduct in 15 ml. of methanol. After 30 min. standing, the mixture was filtered, using a filter stick which had been cooled to the same temperature. The residue was washed once with 5 ml. of methanol and recrystallized in the same manner. Decomposition of the adducts was brought about by shaking with cold water. The fatty acids were then extracted with ether. The washed ether extract was dried over anhydrous NaaS04 and, with a stream of nitrogen bubbling through, the solution was evaporated in vacua. The mold Fusarium Mini Bolley (FIB) was grown for 21 days on a Raulin-Thorn medium of the following composition: Tartaric acid. Magnesium carbonate.. Ammonium tartrate.. Potassium carbonate. Ammonium sulfate. Zincsulfate............................................ Ferroussulfate......................................... Ammonium phosphate dibasic. Glucose............................................... Tapwater.............................................to301.
So”‘.0 8.0 80.0 12 .O 5.0 1.4 1.4 12.0 750.0
One liter of the above solution was used as. the medium for each flask. The methods of fat extraction and isolation are described elsewhere (5). Erlenmeyer flasks, containing the following materials were used in the dehydrogenation experiments: 3 g. ground, defatted FIB mycelia 2 ml. substrate [15 g. stearic acid emulsified with 500 ml. polyvinyl tion (6)] 3 ml. phosphate buffer (pH 8.0) 1 ml. muscle adenylic acid (1 mg./ml.) 10 ml. water.
alcohol solu-
To destroy enzyme activity, the control flasks were immediately autoclaved at 23 lb. pressure for 5 min. Then, both the control and the experimental flasks were shaken for 24 hr. in a thermostat. The filtrates from the flasks were shaken (after acidification) with three RJO-ml. portions of low-boiling petroleum ether (30-50”). The obtained solutions were combined and used to extract the dried mycelia from the Basks in a Soxhlet apparatus. The final contents were washed, dried over N&SO,, and evaporated in vacua under a stream of nitrogen. All iodine values were determined by the Hanus method. RESULTS
In our experiments, which were directed mainly to a study of the potentialities of the formation of urea adducts with the fatty acids
ENZYME
ACTION.
XLVIII
379
present in FlB fat, linseed oil fatty acids and corn oil fatty acids were also investigated for the purpose of comparison. First of all, it was necessary to determine whether adducts with saturated fatty acids and unsaturated fatty acids differ in composition. Our measurements substantiate the observation (2) that no appreciable differences. are noted. In the present case, it was found that the stearic acid adduct contained 13.0 moles of urea, and that the oleic acid adduct contained 13.4 moles of urea per mole of fatty acid. Therefore, it can be concluded that the molar ratios of the respective adducts do not provide a suitable basis for a method of analysis. Furthermore, we have found that glycerides of fatty acids, e.g., in the form of corn oil, do not form adducts under the conditions used. Corn oil itself, when mixed with a saturated solution of urea in methanol, failed to give a precipitate even after long standing. A 50 : 50 mixture of corn oil with corn oil fatty acids resulted in adduct formation, but a layer of corn oil remained in the uncombined state. When 2 g. of corn oil fatty acids was added to 80 ml. of urea solution, the fatty acids obtained from the adduct were found to have an iodine value of 81. With 2 g. of fatty acids and 200 ml. of urea solution, the resulting fatty acids had an iodine value of 97. Since it is known that saturated fatty acids form adducts more readily than unsaturated fatty acids, it is obvious that the latter method should be given preference, since the results indicate that adduct formation is more complete. When the solution of corn oil fatty acids and urea was placed in a refrigerator overnight, 8.8 g. of adduct was formed from 2 g. of fatty acids. Only 6.4 g. was obtained when the solution was allowed to remain at room temperature for the same time. However, in the first case, only 0.72 g. of fatty acids having an iodine value of 91.0 was obtained from 5 g. of the filtered adduct, while in the latter case, 5 g. of adduct yielded 0.91 g. of fatty acids with an iodine value of 97.6. Hence, room temperature was found to be more favorable. Apparently, at the lower temperature, increased precipitation of urea from the saturated solution and less adduct formation result. The relative degrees of unsaturation of the fatty acids after adduct formation were determined as outlined above. The results found are listed in Table I, together with the weights of the various fractions obtained. About twenty solvents were used to determine the preferential solubilities of the stearic and oleic acid adducts. These measurements
380
J.
A.
MASELLI
AND
TABLE
F.
F.
NORD
I
Data on Adduct Fractions Source of fatty
acids
Iodine values of fatty acids Weights of adduct per 2 g. of fatty acids, g. Weights of filtrate oil per 2 g. of fatty acids, g. Weights of oil in 5 g. of adduct, g. Iodine values of oil from adduct Iodine values of oil from filtrate
Corn oil
Linseed oil
Oil of FIB
129.7 6.40 0.62 0.91 97.6 163.9
175.0 2.65 1.22 1.04 102.9 204.8
130.0 4.01 1.03 0.95 80.9 189.1
were undertaken at 20,0, and - 15°C. As a result of these comparisons, methanol was chosen as the most satisfactory solvent, since it was found that at 0°C. the oleic acid adduct remained in solution while the stearic acid adduct was completely precipitated. Hence, in all fractionations, these conditions of solvent and temperature were employed. In Table II are listed the data obtained from the fractionation studies which were carried out in the above manner. TABLE Adduct Fractions Recrystallized Source of fatty
acids
Fatty acid residue per 10 g. of adduct, g. Filtrate fatty acids per 10 g. of adduct, g. Iodine values of residual fatty acids Iodine values of filtrate fatty acids
II from Methanol
at 0°C.
Corn oil
Linseed oil
Oil of FIB
0.82 0.95 43.8 137.8
0.50 1.41 50.2 117.4
0.85 1.21 10.4 97.4
In the procedure adopted for the isolation of stearic acid from FlB fat, the residue obtained by crystallizing the FlB fatty acids adduct at 0°C. in methanol was washed once more with 5 ml. of methanol and again cooled to 0°C. and filtered. After its final decomposition, there was obtained a solid white material which melted at 5268°C. The yield was 1 g. from 12 g. of adduct. This solid mixture was assumed to contain mainly palmitic and stearic acids. After four recrystallizations from methanol, a product which melted at 67-69°C. was isolated. A mixed melting point with an authentic sample of stearic acid showed no depression. A mixed melting point with an authentic sample of palmitic acid showed a depression of 11°C. This result indicated that FlB is capable of forming, from carbohydrate, fat containing stearic acid. An in vitro experiment, using ground, defatted mycelia from FlB added further evidence that this organism is able to dehydrogenate
ENZYME
ACTION.
XLVIII
381
stearic acid. A polyvinyl alcohol emulsion of stearic acid was placed in contact with a suspension of FlB in the presence of a small amount of adenylic acid. After shaking for 24 hr. at 28”C., the contents of the previously autoclaved control flasks and of the experimental flasks were extracted with petroleum ether. The fatty acids obtained from the controls had an iodine value of 5.3, while those from the experiments proper had an iodine value of 10.7, further indicating the presence of a fatty a,cid dehydrogenase in FlB. DISCUSSION
It has been demonstrated that urea adds preferentially to saturated fatty acids, although there is a substantial combination with the unsaturated fatty acids. On the basis of these findings, it was thought that a fractionation procedure could be developed. It is evident from the results obtained with corn oil that urea adduct formation has but little place in studies with unsaponified fats. However, in the case of fatty acids, several applications have been found. As previously indicated (2)) and as reported in this paper, urea adducts provide a useful means for isolating fatty acids of varying degrees of saturation. Linseed fatty acids, which are the most unsaturated of the three mixtures used, have the least tendency to form an adduct, as shown in Table I by the relatively small amount of adduct obtained and by the relatively large amount of oil that remained uncombined. That there is a higher yield of adduct with corn oil fatty acids than with fatty acids of FlB may be attributable to the fact that FlB fat contains linolenic acid, while corn oil does not. The amount of fatty acids which combined with urea to form 5 g. of adduct, was approximately the same in all cases. This is further substantiation of the observation that the molar ratio of urea to fatty acids seems not to vary with the degree of unsaturation. The preference for adduct formation with the saturated components of the fatty acid mixtures is again strikingly demonstrated by the large differences between the iodine values of the adduct fatty acids and those of the filtrate. This preference served as the basis for the attempted fractionations. An extension of the procedure of crystallizing the adducts from methanol at 0°C. has enabled us to obtain, more completely, saturated fatty acids from the adducts, as is borne out in Table II. The data on the adducts and filtrates resulting from this crystallization point to the
382
J.
A.
MASELLI
AND
F.
F.
NORD
fact that the adduct formed with the linseed oil fatty acids had the highest iodine value and resulted in the lowest residual weight as expected. It can be seen by a consideration of the iodine values of the residual fatty acids and the filtrate fatty acids, that crystallizations at 0°C. cause a definite removal of unsaturated fatty acids, especially in the case of FlB fatty acids. Our method of removing unsaturated fatty acids possesses the advantage that it is unnecessary to apply temperatures as low as -3O”C., as is customary in low-temperature crystallization procedures. Furthermore, the bulk of the fatty acids is increased by the urea present. This latter fact is helpful, particularly when working with mold fats, since one of the major obstacles in the study of the enzymatic carbohydrate -+ fat conversion is the small amount of fat which is generally available for quantitative study. In the case of FlB fatty acids adduct, the results of the methanol crystallization at 0°C. were sufficiently satisfactory to prompt an attempt to isolate and to identify stearic acid from the final solid fraction obtained. This relatively simple method obviates the employment of very low temperatures and the use of a complicated fractionating apparatus. In a study of the quantitative composition of FlB fat, it was shown that saturated acids amounted to about 23% of the total quantity of fatty acids (7). Of this, at least 60% was palmitic acid. Since it had been demonstrated that Fusaria are capable of converting stearic acid to unsaturated fatty acids, the identification of it in the fat from FlB is of interest, since this acid could be a starting material upon which a fatty acid dehydrogenase might act in the living organism to form the mono-, di-, and triethenoid fatty acids known to be present. This finding, together with the fact that FIB is capable of dehydrogenating stearic acid both in viva and in vitro, provides a susbtantial indication for the enzymatic formation of unsaturated fatty acids in FlB by means of the action of a fatty acid dehydrogenase system. Additional corroboration of this point is offered by the fact that in the presence of small amounts of naphthoquinones, a more unsaturated fat has been shown to be formed by FlB (8). These naphthoquinones are thought to interact in the normal equilibrium of the hydrogen transport carried out by the fatty acid dehydrogenases, either with the interlocking codehydrogenases or in the cytochrome system.
ENZYME
ACTION.
XLVIII
383
SUMMARY
The usefulness of urea adducts for the fractionation of natural fatty acids has been demonstrated. Stearic acid has been isolated and identified from the fat of Fusarium Eini Bolley by purification of the solid fraction obtained from the urea adduct of its fatty acids. The conversion of stearic acid to fatty material with a higher iodine value has provided further evidence for the enzymatic formation of unsaturated fatty acids in Fusarium Zini Bolley by means of the action of a fatty acid dehydrogenase system. REFERENCES
1. NORD, F. F., AND WEISS, S., in SUMNER, J. B., AND MYRBHCK, K., Ed., The
Enzymes, Vol. 2, part 1, p. 740. New York, Academic Press, 1951. 2. SCHLENK, H., AND HOLMAN, R. T., J. Am. Chem. Sot. 73, 5001 (1950). 3. NEWEY, H.A., SHOKAL, E.C., MUELLER, A.C., BRADLEY, T.F., ANDFETTERLY, L. C., Ind. Eng, Chem. 42, 2538 (1950). 4. SCHLENK, W., JR., Ann. 666, 204 (1949). 5. FIORE, J. V., Arch. Biochem. 16, 161 (1948). 6. FIORE, J. V., AND NORD, F. F., Arch. Biochem. 23, 473 (1949). 7. MULL, R. P., AND NORD, F. F., Arch. Biochem. 6, 283 (1944). 8. MASELLI, J. A., AND NORD, F. F., Arch. Biochem. 24, 235 (1949).