Fatty Acid Oxidation by Spores of Penicillium roqueforti

Transcription

1 Fatty Acid Oxidation by Spores of Penicillium roqueforti Department of Bacteriology, R. F. GEHRIG AND S. G. KNIGHT University of Wisconsin, Madison, Wisconsin Received for publication 7 December 1962 ABSTRACT GEHRIG, R. F. (University of Wisconsin, Madison) AND S. G. KNIGHT. Fatty acid oxidation by spores of Penicillium roqueforti. Appl. Microbiol. 11: When 1,AM sodium octanoate was the substrate for spores, most of the molecule was recovered as CO2 and no ketone was produced. However, when larger concentrations (20 um) were used as substrate, part of the molecule was converted to methyl ketone and part was completely oxidized. Optimal conditions for the production of 2-heptanone were determined because of the importance of this compound in giving aroma and flavor to mold-ripened cheeses. Optimal ketone formation was not dependent upon the temperature and length of time at which the spores were stored. The spore suspensions were stored for over 36 months at 4 C without losing their ability to convert octanoic acid to 2-heptanone. The oxidation of octanoic acid was inhibited by cyanide, carbon monoxide, mercury, 2, 3-dimercapto-1-propanol, and a, a-dipyridyl. No ketone was produced under anaerobic conditions. Although no intermediates of fatty acid oxidation were isolated, since an active cell-free preparation could not be obtained, this investigation has yielded some evidence for the beta oxidation of the fatty acids by spores of Penicillium roqueforti. Stairkle (1924) attributed the characteristic odor and flavor of Roquefort cheese to methyl ketones. He distilled Roquefort cheese and obtained material that had an intensive and characteristic odor of 2-heptanone and 2- nonanone; however, the amount was too small to permit chemical separation and identification. He also found that Aspergillus niger and A. fumigatus, as well as Penicillium roqueforti,' formed methyl ketones when grown for several weeks in pure culture with individual fatty acids as the carbon source. Later, Stokoe (1928) found that P. palitans and P. glaucum produced methyl ketones from coconut oil and postulated that the fatty acids therein had a narcotic effect, which caused a decarboxylation of the,b-keto acid rather than continued oxidation. More important to the present work is the fact that he found that the nonsporeforming fungus, Oidium lactis, did not form ketones. More recently, Gehrig and Knight (1958) found that the spores of P. roqueforti, and not the vegetative cells, were responsible for the formation of methyl ketones from 166 fatty acids. While extending this work, Gehrig and Knight (1961) also found that the spores of a number of penicillia and aspergilli and a few related molds also produced, 2-heptanone from octanoic acid. This paper reports on the ketone formation by spores of P. roqueforti, with the conversion of octanoic acid to 2-heptanone as the model system. MATERIALS AND METHODS P. roqueforti Minnesota strain P 12-1 was used throughout these studies. Stock cultures were maintained in soil at room temperature (Greene and Fred, 1934). The sporulation medium consisted of 100 ml of sterile 5 % agar and 100 ml of sterile tomato juice, mixed and slanted in sterile Roux bottles. Soil from the soil stocks was spread over the surface of the agar and the cultures were incubated at 25 C until the mold sporulated, usually in 3 to 4 days. To harvest the spores, 100 ml of sterile distilled water were added to each Roux bottle, and the spores were suspended in the water as they were scraped from the surface of the mycelium with a sterile needle. The spore suspension usually was stored at 4 C. Two media were used to obtain hyphal cells devoid of spores for controls, Peterson's nonsynthetic medium (Peterson et al., 1952) and Meyers' synthetic medium (Meyers and Knight, 1958). The mold was grown at 25 C in submerged culture on a rotary shaker rotating at 200 rev/min and describing a radius of 0.5 in. At the end of the growth period of 2 to 5 days, the cells were removed from the growth medium by filtration on a Buchner funnel. To wash free of nutrients, the cells were suspended three or four times in distilled water and refiltered. The hyphal mat then was pressed between paper towels to remove excess moisture, and was weighed. The amount of cells required was suspended in distilled water and blended in a chilled Waring Blendor cup for 10 sec; this suspension then could be added directly to Warburg vessels or to Erlenmeyer flasks, as the experiment indicated. To obtain large quantities of spores, the sporulation medium was used as described. Here, it was more convenient to use a flattened metal rod to remove the spores from the mat. The spores were suspended in a solution (1:10,000) of Tween 80 (polyoxyethylene sorbitan monooleate) to aid in obtaining even dispersion. To separate spores from cells, the suspension then was filtered through several layers of cheesecloth; this procedure was very efficient in that the spores filtered through the cloth, while

2 VOL. 1 1, 1963 IV1ATTY ACID OXIDATION BY P. ROQUEFORTI SPORES 167 the hyphal cells and bits of agar were retained. After harvesting, the spores were washed several times with distilled water and finally resuspended in distilled water. The spore suspension was standardized in a hemacytometer to a concentration of 1 X 109 spores per ml and was stored at either 4 or -5 C. The mineral salts medium of Chiang and Knight (1959) was used for the basal carbon-free medium. In some of the flasks, sodium octanoate was added directly to the liquid medium; however, in the solidified medium, the octanoate was layered on the glass surface of the Roux bottle opposite to the agar. The sodium salts of the fatty acids were pre - pared by neutralizing the acids with sodium hydroxide to ph 7.0 and making to volume. Standard manometric techniques (Umbreit, Burris, and Stauffer, 1957) were used throughout this investigation. Ketone production was determined by the method of Haidle and Knight (1960). To further identify the ketones, phenylhydrazone derivatives of the known and unknown ketones were prepared by the method of Girolami and Knight (1955). The analysis of the reaction mixture of spore suspension and sodium octanoate was accomplished by first steamdistilling the mixture to obtain the neutral fraction. The fraction was extracted with ether, which was dried over anhydrous calcium sulfate. The ether solution was evaporated to 0.5) ml at room temperature under a gentle stream of nitrogen and stored in a tightly capped test tube at 4 C. The volatile acids were recovered from the aqueous residue by adjusting the ph to 1.0 with 18 N H2S04 and distilling until 50 ml were collected. The distillate was neutralized with NaOH and evaporated to dryness in vacuo. This residue was again acidified to ph 1.0 with 18 N H2S04 and again extracted with ether. The solution was methylated with diazomethane and stored in a tightly capped test tube at 4 C. The neutral and methyl ester fractions were analyzed by Frank M. Strong of the Biochemistry Department in a gas-liquid chromatography apparatus similar to that described by Coffman, Smith, and Andrews (1960). All radioactive samples, except sodium octanoate-3-ci4, were counted as infinitely thin films deposited on stainlesssteel planchets by the evaporation of aqueous solutions under an infrared lamp. The films never exceeded 1 mg on a 2.5 cm2 planchet, and, therefore, no self-absorption corrections were necessary. The sodium octanoate-3-cl4 was counted as an infinitely thick film. Since only the comparative amount, not the absolute amount, of radioactivity in the samples was being determined, self-absorption was not considered significant. The radioactivity was measured in an automatic Mylar window gas-flow counter obtained from Tracerlab, Inc., Waltham, Mass. The radioactive compounds were obtained from the California Corporation for Biochemical Research, except the sodium octanoate- 3-C14, which was prepared by Gerald S. Brenner in the Department of Chemistry. RESULTS AND DISCUSSION It seemed appropriate to grow the mold in media that that would not support sporulation and to determine if indeed the hyphal cells lack the enzymes necessary for the oxidation of the fatty acids and ketone formation. Mleyers' synthetic medium, used in earlier studies of P. roqueforti, was found to be one of the media that gave excellent yields of hyphal cells and no sporulation. In repeated experiments using only young hyphal cells (ca. 40 hr of growth) and a wide range of both ph (4.0 to 8.0) and substrate (1 to 10 Am), no exogenous oxygen was consumed during manometric studies, and no ketone was detectable. To confirm this, Peterson's nonsynthetic medium was used to grow hyphal cells and the results were the same. It was noted that substrate concentrations over 5,uM were decidedly inhibitory toward endogenous respiration. It therefore seemed that hyphal cells did not have the ability to oxidize the fatty acid, regardless of the type of medium in which they were grown. A second approach was used to study the inability of hyphal cells to produce ketone. A 2-ml amount of spore suspension (1 X 109) was used to inoculate eight 50-ml Erlenmeyer flasks containing 20 ml of Meyers' synthetic medium. These were placed on a rotary shaker at 25 C. At 0, 16, 24, and 48 hr, duplicate flasks were removed from the shaker, and the cells were removed by centrifugation at 5,000 X g for 30 min. The preparations were waslhed several times by centrifugation in distilled water, and, at the last washing, were resuspended to a volume of 2.0 ml. The mnold loses the ability to oxidize and to form ketone from octanoate as the spores form vegetative cells (Table 1). These results offer strong evidence that the spore is the active site for the metabolism of the fatty acid to the ketone. Early in these investigations, attempts were made to obtain an active, i.e., ketone-forming, cell-free extract from the spores, since cell-free extracts offer many advantages in metabolic studies. However, none of the extracts prepared was active when tested for ability to take up oxygen, or to produce ketone, from octanoate. The following were added either alone or in various combinations in an attempt to restore activity: adenosine triphosphate, di- TABLE 1. Amount of ketone formed from octanoate and oxygen consumed with spore germination of Penicillium roqueforti* Age of 2-Heptanone Oxygen culture No. of spores/ml formed consumedt hr M X ca. 1.2 X < * Each Warburg vessel contained 1.0 ml of cell suspension, 20,uM sodium octanoate, 1.0 ml of 0.5 M phosphate buffer (ph 6.5), and distilled water to 3.0 ml. Gas phase, air. Temperature, 25 C. Incubation period, 2 hr. t All figures were corrected for endogenouis respiration.

3 168 GEHRIG AND KNIGHT APPL. MICROBIOL. phosphopyridine nucleotide, flavin adenine dinucleotide, coenzyme A, M\4g, cytochrome c, acetate, and glucose. None of the additions reactivated the extracts. Other additives were also tried. For example, reduced glutathione was added to the spore suspension prior to grinding or disruption by sonic vibrations in an attempt to maintain reduced conditions. Substrate was added to the Raytheon cup to protect the enzymes (Hugo, 1954), and nicotinamide was added to protect the system against any disphosphopyridine nucleosidase that may have been present (Hochster and Quasted, 1951). Separately or together, none of these procedures was effective in restoring activity to the extracts. When 100 % disruption of the spores was approached, no enzymatic activity remained in the debris. The supernatant when tested alone, as well as a mixture of spore debris and supernatant, was inactive. Since it was not possible to obtain a cell-free extract that would metabolize the substrate, it was necessary to employ other methods for examining the mechanism by which the spores formed ketones from fatty acids. The first experiments with labeled octanoate, using sodium octanoate-1-c14 (10 4M), were done to determine whether the fatty acid was indeed being decarboxylated to yield the methyl ketone as originally suggested by Stokoe (1928). The assumption by Starkle (1924) and Stokoe (1928) that the 3-keto acid is first formed, and that this is decarboxylated to form the methyl ketone, is probably correct, since our hydrazone derivatives of the ketone were not labeled and the CO2 trapped in the KOH was labeled. Because ketone was not formed by heat-killed or nonviable spores, it must be assumed that the reaction is at least in part enzymatic. Furthermore, the amount of oxygen consumed by viable spores in the presence of octanoate was approximately 100,uliters over endogenous respiration for a 3-hr period; no oxygen was consumed by the killed spores. These and similar experiments suggested that more CO2 was being evolved than the amount theoretically needed for the complete conversion of the fatty acid to the ketone. This is contrary to the assumption of Starkle (1924) and Stokoe (1928), who maintained that a considerable portion of the fatty acid was converted to ketone. Furthermore, in TABLE 2. Comparison of the amouint of C402 forimed from sodium butyrate-1-c14, sodium butyrate-2-c14, and sodium butyrate- 34C14 by spores of Penicillium roqueforti* Substrate C02 (counts/min) Butyrate-1-C14 32,450 Butyrate-2-C14 10,620 Butyrate-3-CI4 27,970 * Each Warburg vessel contained 1.0 ml of spore suspension (1 X 109/ml); substrate (0.5 jsm) with specific activity of 50,000 counts per min per 0.5 sm; 1.0 ml of 0.5 M phosphate buffer (ph 6.5), and distilled water to 3.0 ml. The CO2 was trapped in 0.2 ml of KOH (20%) in the center well. Gas phase, air. Temperature, 25 C. Incubation period, 4 hr. Background was less than 20 counts per mmn per sample. only 3 hr, using 1,uM sodium octanoate-1-c'4, one-third of the C1402 was trapped in the KOH, and no ketone was detected. There may be several explanations for this lack of ketone formation at the end of 3 hr. Perhaps a critical level of keto acid, or another product, must be formed before ketone is formed; the compound at a critical level might be converted into ketone (detoxication), or it might stimulate the ketone-forming system. Another possibility is that the ketone-forming system is only inducible, and a certain amount of energy must be expended for the formation or "unmasking" of the protein involved. It was found that the RQ with endogenous respiration subtracted was 0.77 to 1.2, depending upon the length of the experiment and the amount of substrate used. When the reaction was allowed to go to completion, using 1,uM substrate, the RQ approximated that for the complete oxidation of 1,umole of the fatty acid. To obtain more evidence that the fatty acids can be completely oxidized to C02, the relative amount of C'402 produced from sodium butyrate-l-c14, -2-C'4, and -3-C14 was determined (Table 2). The carboxyl group from butyrate-1-c'4 was rapidly released as C02, whereas the release as C14 from butyrate-2-c'4 was retarded, and the C14 from butyrate-3-c'4 was released almost as fast as that from butyrate-1-c14. Although there was a rapid conversion of the butyrate molecule to C02, it is possible that a short-chain fatty acid is more completely oxidized than a long-chain fatty acid. To test this possibility, C1402 produced from sodium octanoate-7-c'4 was determined by the same procedure. Only a small amount of this compound was available but it was found that the molecule was degraded to the extent that C1402 was formed from the seventh carbon. Using 1 Mm sodium octanoate-3-c14, it was found that, after complete oxidation of the molecule, approximately 60 % of the C1402 could be recovered from the KOH of the center well of the Warburg vessel; 30 % of the C14 was obtained from a hot-water extract of the washed spores. Although all of the C14 was not recovered, none was found as ketone when the contents of replicate Warburg vessels were examined. However, when larger concentrations of labeled octanoate (20,uM) were used, the ketone distilled from the contents of the Warburg vessels was found to be labeled. The ability of P. roqueforti spores to oxidize other fatty acids is shown in Table 3. The amount of oxygen consumed increased in a stepwise manner as the length of the carbon chain increased. This might indicate that there was little difference in the manner by which the acids were metabolized. The variation in the amount of oxygen consumed could be attributed to permeability factors or additional metabolic pathways or both. It could be assumed, also, that a portion of the substrate that was not oxidized had been assimilated, as suggested by Stout and Koffler (1951). MVIany attempts were made to use octanoic acid as a carbon source in a growth medium. The salt of the acid as

4 VOL. 11, 1963 FATTY ACID OXIDATION BY P. ROQUEFORTI SPORES 169 the sole carbon source failed to support growth in either solid or liquid culture. However, growth did occur after 4 days at 25 C, usinig Chiang's medium with 5 to 10 ml of 0.1 M sodium octanoate placed on the glass surface opposite the solidified medium in Roux bottles. It is initeresting to niote that there was nio ketone produced when the mold was girowii onl the vapor of octanoic acid. However, when the bottles were reversed and the octanioate allowed to come into contact with the sporulating culture, ketonie was produced within a few hours. This substantiates the foregoing observations that the spores will use the acid as a source of carbon when the substrate is added in small quantities. However, when larger amounts of substrate are present, the spore converts at least part of the acid to the ketone. To see whether a series of ketonies or other products were produced as the fatty acid was oxidized, 10 ml of spore suspension (1 X 109/ml), 10 ml of 0.5 M phosphate buffer (ph 6.5), 200 jam sodium octanoate, and distilled water to 30 ml were added to a 125-ml Erlenmeyer flask, and the mixture was incubated at 25 C on a rotary shaker. At the end of 4 hr, it was prepared for gas-chromatographic anialysis as inidicated in Mlaterials and Methods. The same reaction mixture was prepared and distilled immediately to give a zero-hour control. The only component to appear uponi anialysis of the mixture incubated for 4 hr was 2--heptanone. Cyanide (1.0 Mm), azide (5,M), a,a-dipyridyl (2.0 Mm), carbon monioxide (CO:O, 1:1), and 2,3-dimercapto-1- propanol (BAL; 0.8,AM), tested separately by addition to the standard reaction mixture, caused 100 % inhibition of ketonie formation. The cyanide, azide, aind BAI also caused 100 % inhibition of the oxygen uptake. In contrast, a, a-dipyridyl, at concentrations which effected 100 %/' inhibition of 2-heptanone formation, caused onily 30 % inhibitioni of the oxygen uptake. Figure 1 shows the effect of ph and temperature on the ketonie productioni and oxygen consumption by a spore suspeinsioii of 1'. roqueforti, with sodium octanoate as a TABi,E 3. Anmoutnt of oxygen consnined during the oxidation of various fatty acids by spores of Penicillium roqueforti* Oxygen uptake Fatty acid Theoretical Observed Per cent of theoretical /AM Acetic Propionic Butyric Valeric Caproic Heptanoic ()ctanoic * Each Warbturg vessel contained 1.0 ml of spore suspension (1 X 109/ml), 1.0 Mm substrate, 1.0 ml of 0.5 M phosphate buffer (ph 6.5), and distilled water to 3.0 ml. Gas phase, air. Temperature, 25 C. Incubation period, 8 hr. All figures were corrected for endogenous respiration. JAM substrate. These results show that the oxygen uptake by the mold was not greatly affected by temperature; however, ketone production at 37 C was practically abolished. The combination of these effects would seem to indicate a series of reactioiis. In most of the experiments, phosphate buffer, at ph 6.5 and a temperature of 25 C, was used because it provided optimal ketone production and oxygen consumption. The effect on ketone formation of freezing the spore suspensions was pronounced. Storage at -5 C almost doubled the amount of ketone formed by the spores; probably a permeability factor would account for this phenomenon, or the spores might be better protected in the frozen state than at normal refrigeration temperatures. Spores, in contrast to hyphal cells, are extremely durable in their resistance to autolysis. For this reason, they can be used as veritable "factories" for the production of methyl ketonies and other products (Schleg and Knight, unpublished data). In fact, spores that have been stored at 4 C for a period of 36 months still produce 2-heptanone when sodium octanoate is used as a substrate. The effect of oxygen tension on octanioate oxidation by spores showed that the productioni of 2-heptaiione increased inl direct proportioni to the amount of oxygen 370 C i w z 30-C 4IN. 25s C w Ix N ph ;00 31ioo x 900 Gi z c 600 X --4 X 300 m r_ FIG. 1. Effect of ph and temperature on the oxidation of sodium octanoate. Each Warburg vessel contained 1.0 ml of spore suspension (1 X 101), 20 Mm sodium octanoate, 1.0 ml of 0.5 M phosphate buffer with ph as indicated, and distilled water to 3.0 ml. Gas phase, air. Temperature as indicated. Incubation period, 3 hr. (0) Oxygen uptake; (*) 2-heptanone formed.

5 170 GEHRIG AND KNIGHT APPL. MICROBIOL. present in the reaction mixture. No ketone was formed when the spores were incubated in either 100 % nitrogen or 100 % carbon dioxide. This is considered adequate proof of the requirement of oxygen for ketone production but still does not answer the question as to whether the oxygen of the ketone is atmospheric or aquatic in origin. Unfortunately, tritiated water could not be used because of the strong quenching action of the ketones. It was found that the spores were unable to oxidize the ketone, and 2-heptanone neither stimulated nor inhibited the oxidation of the fatty acid. ACKNOWLEDGMENT This investigation was supported in part by research grants from the Wisconsin Alumni Research Foundation. LITERATURE CITED CHIANG, C., AND S. G. KNIGHT D-Xylose metabolism by cellfree extracts of Penicillium chrysogenum. Biochim. Biophys. Acta 35: COFFMAN, J. R., D. E. SMITH, AND J. S. ANDREWS Analysis of volatile food flavors by gas-liquid chromatography. I. The volatile components from dry Blue Cheese and dry Romano cheese. Food Research 25: GEHRIG, R. F., AND S. G. KNIGHT Formation of ketones from fatty acids by spores of Penicillium roqueforti. Nature 182:1937. GEHRIG, R. F., AND S. G. KNIGHT Formation of 2-heptanone from caprylic acid by spores of various filamentous fungi. Nature 192:1185. GIROLAMI, R. L., AND S. G. KNIGHT Fatty acid oxidation by Penicillium roqueforti. Appl. Microbiol. 3: GREENE, H. C., AND E. B. FRED Maintenance of vigorous mold stock cultures. Ind. Eng. Chem. 26: HAIDLE, C. W., AND S. G. KNIGHT A colorimetric assay for 2-heptanone and other ketones. Biochim. Biophys. Acta 39: HOCHSTER, R. M., AND J. H. QUASTEL The effect of nicotinamide on fermentations by fresh and by acetone-dried powders of cell-free extract. Arch. Biochem. Biophys. 31: HUGO, W. B The preparation of cell-free enzymes from microorganisms. Bacteriol. Rev. 18: MEYERS, E., AND S. G. KNIGHT Studies on the nutrition of Penicillium roqueforti. Appl. Microbiol. 6: PETERSON, D. H., H. C. MURRAY, S. H. EPPSTEIN, L. H. REINEKE,; A. WEINTRAUB, P. D. MEISTER, AND H. M. LEIGH Microbiological transformations of steroids. 1. Introduction of oxygen at carbon-11 of progesterone. J. Am. Chem. Soc. 74: STARKLE, M Die Methylketone im oxydativen Abbau einiger Triglyceride (bzw. Fettauren) durch Schimmelpilze unter Berucksichtigung der besonderen Ranziditat des Kokofettes. I. Die Bedeutung der Methylketone im Biochemismus der Butterranziditat. II. Uber die Entstehung und Bedetung der Methylketone als Aromastoffe im Roquefortkase. Biochem. Z. 151: STOKOE, W. N The rancidity of coconut oil produced by mold action. Biochem. J. 22: STOUJT, H. A., AND H. KOFFLER Biochemistry of filamentous fungi. I. Oxidative metabolism of gluicose by Penicillium chrysogenum. J. Bacteriol. 62: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUFFER Manometric techniques and tissue metabolism, 2nd ed. Burgess Publishing Co., Minneapolis.