Fatty Acid Metabolism in Microorganisms. Part I. Production of Pimelic Acid from Azelaic Acid* By Koichi OGATA, Tatsurokuro TOCHIKURA,

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1 [Agr. Biol. Chem., Vol. 30, No. 2, p. 176 `180, 1966] Fatty Acid Metabolism in Microorganisms Part I. Production of Pimelic Acid from Azelaic Acid* By Koichi OGATA, Tatsurokuro TOCHIKURA, Masahiro OSUGI, and Shojiro IWAHARA Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto. Received August 30, 1965 The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as n sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physico - chemical and biological methods. From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp. INTRODUCTION It has been known that pimelic acid serves as a metabolic precursor for the biosynthesis of biotin-vitamers.l `5) Azelaic acid is also found to serve as a precursor for the biosyn thesis of biotin-vitamers.l,5) It is probable that pimelic acid may be a degradation pro duct of azelaic acid. However, little has been informed about the formation of pimelic acid from azelaic acid. The present investigation was undertaken to throw light upon the production of pimelic acid from azelaic acid by microorganisms as a series of investigations on fatty acid metabolism. Recently, Janota-Bassalik and Wrights6,7) * This report was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Tokyo, Apr. 3, ) L. D. Wright, E. L. Cresson and C. A. Driscoll, Proc. Soc. Exp. Biol. Med., 89, 234 (1955). 2) D. S. Genghof, Arch. Biochem. Biophys., 62, 63 (1956). 3) M. A. Eisenberg, J. Bacteriol., 86, 673 (1963). 4) A. Lezius, E. Ringelmann and F. Lynen, Biochem. Z., 336, 510 (1963). 5) K. Ogata, T. Tochikura, S. Iwahara, K. Ikushima, S. Takasawa, M. Kikuchi and A. Nishimura, This Journal, 29, 889 (1965). 6) L. Janota-Bassalik 36, 405 (1964). and L. D. Wright, J. Gen. Microbiol., preliminarily reported the isolation of pimelic acid as a by-product of azelaic acid degrada tion using bacteria belonging to Pseudomonas sp., and explained the role of azelaic acid in the biosynthesis of biotin. Independently of their investigation, we found that a bacterial strain A133 which utilized azelaic acid as a sole carbon source produced a large quantity of pimelic acid from azelaic acid, in the culture fluid during the cultivation as preliminarily reported. 8)In this paper, we will report the isolation of bacterial strains which produce pimelic acid from azelaic acid, and the identification of pimelic acid produced during the cultiva tion of the isolate strain A133. The morpho logical and physiological characteristics of this microorganism are also presented. EXPERIMENTAL Materials and Methods Microorganisms The bacterial strains used in the 7) L. Janota-Bassalik and L. D. Wright, Nature, 204, 501 (1964). 8) M. Osugi, S. Iwahara, T. Tochikura and K. Ogata, Mem. Res. Ins. Food Sci., Kyoto Univ., 26, 18 (1965).

2 mƒê ~103. Fatty Acid Metabolism in Microorganisms. Part I. 177 present investigation were isolated from soil and other natural materials. Medium Unless otherwise stated, the medium used throughout was as follows: azelaic acid 1.0%; (NH4)2SO4 0.5%; K2HPO4 0.2%; KH2PO4 0.1%; MgSO4 E7H2O 0.05% and tap water. The initial ph of the medium was adjusted to 7.0. Isolation Method of Microorganisms utilizing Azelaic Acid The isolation of bacterial strains, which utilized azelaic acid as a sole carbon source, was carried out by agar plate culture technique using the medium mentioned above. The bacteria isolated were maintained on agar slopes of the azelaic acid medium described above. Culture Conditions The bacteria were grown in 500ml of the medium contained in a 2,000ml shak ing flask on a reciprocal shaker (130 reciprocations per minute) at 28 Ž for 3 days. Theinoculum consist ed of 5ml of the bacterial cultures preincubated in the same medium at 28 Ž for 18 hours on a shaker (280 reciprocations per minute). Growth Measurements The growth of the bacteria was routinely estimated by measuring the turbidity using a Hitachi photometer at the wave length of 470mƒÊ. Paper Chromatography Ascending paper chromato graphy was carried out on Toyo No. 53 filter paper with phenol-water (4:1 by volume) as the develop ing solvent. The acids on the paper-chromatograms were detected by spraying the bromphenol blue in citric acid solution.9) RF values of azelaic acid and pimelic acid were 0.53 and 0.40, respectively. Both acids could be detected beyond the concentration cell system of Bacillus sphaericus.l0) The reaction mixture contained 22.5mg cell (as dry weight) of Bacillus sphaericus (18 hours culture), 50ƒÊmoles of alanine, 0.01 `0.5ƒÊmoles of pimelic acid, 100ƒÊmoles of tris buffer, ph 7.5, in a final volume of 1ml. The reaction mixture was incubated at 28 Ž for 2 hours with continuous shaking (340 reciprocations per minute) and the reaction was arrested by heating at 100 Ž for one minute. The cells were removed by centrifugation. The biotin-vitamers in the supernatant were quantitatively determined by microbiological assay with Saccharomyces cerevisiae.11) RESULTS AND DISCUSSION Effect of Carbon Source on Growth of Micro organisms isolated The bacteria (105 strains) which utilized azelaic acid as a sole carbon source were isolated from soil and other natural materials. In order to select a strain favorable for the production of pimelic acid from aze laic acid, the bacteria were incubated on the media containing azelaic acid, pimelic acid or acetic acid as a sole carbon source, respec tively. It was found that the majority of the bacteria tested utilized pimelic acid as well as azelaic acid. However, as shown in Table TABLE I. EFFECT OF CARBON SOURCE ON GROWTH OF ISOLATED BACTERIA. range of 10 `20ƒÊg per spot. Determination of Acids The quantitative deter minations of azelaic acid and pimelic acid were car ried out by paper chromatographic method. The bands on the paper corresponding to the azelaic acid and pimelic acid were cut out and the pieces of paper thus obtained were eluted with 5ml of 0.01N sulfuric acid solution and then extracted with 10 ml of n-butyl acetate. The extract was then titrated with 0.01N methanolic sodium hydroxide solution using phenol phthalein as the indicator. Biological Activity Test The biological activity of the pimelic acid isolated as precursor for the biosynthesis of biotin-vitamers was tested by resting * After the growth for 40 hours, optical density at 470 9) E. P. Kennedy and H. A. Barker, Anal. Chem., 23, 1033 (1951). 10) S. Iwahara, T. Tochikura and K. Ogata, This Journal, 29, 262 (1965). 11) E. E. Snell, R. E. Eakin and R. J. Williams, J. Am. Chem. Soc., 62, 175 (1940).

3 178 Koichi OGATA. Tatsurokuro TOCHIKURA, Masahiro OSUGI and Shojiro IWAHARA I, some strains showed heavy growth in both azelaic acid and acetic acid media, but feeble growth in the pimelic acid medium. Production of Pimelic Acid from Azelaic Acid In order to illustrate the characteristics of the degradation product of azelaic acid, organic acids in the culture fluids were ex amined by paper chromatography. Typical data are shown in Fig. 1. In the culture FIG. 2. Production of Pimelic Acid from Azelaic Acid by Strain A133. Culture conditions are cited in the text. \ Growth - - Azelaic acid - œ- Pimelic acid in the medium was completely vanished and a large amount of pimelic acid was produced. In the successive cultivation, pimelic acid accumulated in the medium was gradually decreased along with elapsing time and almost disappeared after 50 hours, at the stationary FIG. 1. Paper Chromatogram of Culture Fluid of Strain A133. S: Authentic azelaic acid (a) and pimelic acid (b). fluid at 10 and 14 hours cultivation, two spots were detected. Of these, one of RF 0.40 could be considered corresponding to pimelic acid produced and the other of RF 0.53 re sidual azelaic acid. From the results mentioned above, it was possible that the degradation product of azelaic acid might be a pimelic acid. Other organic acid was hardly detected. Subsequ ently, residual azelaic acid and pimelic acid produced in the culture fluid were quantita tively determined by paper chromatographic method at various stages of growth. The re sults are shown in Fig. 2, from which it was found that azelaic acid degradation and pime lic acid production were almost stoichiometri cally proceeded. In the 22 hours' culture fluid corresponding to the medial stage of logarithmic phase of growth, the azelaic acid phase of growth. The same experiment were performed with other strains such as A131, A301, A305 and A307 which were almost un able to utilize pimelic acid. The quantity of pimelic acid produced was more or less at variance with the strain tested, but nearly similar to the results from the experiments with strain A133. From the results described above, it is pro bably considered that the growth of strain A133 is depending on the utilization of acetic acid which may be produced by the oxidative degradation of azelaic acid. Isolation of Pimelic Acid from Culture Fluid Strain A133 was grown on the azelaic acid medium at 28 Ž on the reciprocating shaker. Cultivation was carried out for 41 hours, dur ing which azelaic acid in the medium was completely consumed. Yield of cells was ap proximately 1.5g (as dry weight) per 1,000ml. After the growth, the culture fluid was cen trifuged to remove the cells and about 1,000ml of the supernatant was obtained. About 39 of active carbon was added to the supernatant

4 Fatty Acid Metabolism in Microorganisms. Part I. 179 (1,000ml) in order to decolorize the medium and was removed through filtration. The filtrate was adjusted to ph 2 with sulfuric acid and concentrated to about 50ml at 40 Ž under the reduced pressure. Pimelic acid in the concentrate was extracted three times with 150ml of n-butyl acetate. The crystal (2.6g) of pimelic acid was obtained after evapora tion of the solvent. After recrystaillization from benzene, 2.4g of pure crystal which melted at `105.0 Ž was obtained. The melting point of this crystal was not depress ed on admixture with an authentic specimen. Identification of Pimelic Acid Paper chromatography of this crystal gave only one spot corresponding to pimelic acid. The elemental analysis was found as follows: Anal. Found: C, H, 7.63 Calcd. for C7H12O4: C, 52.49; H, 7.55%. The infrared spectrum of the crystal was identical with that of the authentic pimelic acid as shown in Fig. 3. The biological activity of the crystal as pre cursor for the biosynthesis of biotin-vitamers was tested using the resting cell system of Bacillus sphaericus. As shown in Fig. 4, the crystal obtained served as a precursor for the biosynthesis of biotin-vitamers as the authentic FIG. 4. Biological Activity of the Crystal as a Precursor for Biosynthesis of Biotin-Vitamers. - - Authentic pimelic acid - œ- Crystal - œ- Azelaic acid firmed that the degradation product of azelaic acid is a pimelic acid. Taxonomical Properties of Strain A133 Morphological Properties (1) Microscopic observation: Cells are spheri cal, and occur singly, or in two or three. Size of cells, 0.6 to 1.0 microns in diameter. No capsules nor endospores observed. Non motile, and gram-negative (Fig. 5). pimelic acid, to the same extent. From the results stated above, it was con- FIG. 5. Electron Micrograph of Strain A133. (magnification ~ 10,000) FIG. 3. The Infrared Absorption Spectra of the Crystal and Authentic Pimelic Acid (Nujol Mull). A: The crystal isolated B: Authentic pimelic acid (2) Gelatin stab: Slow liquefaction. (3) Agar colony: Circular or spindle. Entire, capitate, translucent and nacreous. (4) Agar slant: Spreading, entire and capi-

5 180 Koichi OGATA, Tatsurokuro TOCHIKURA, Masahiro OSUGI and Shojiro IWAHARA tate. Moderately heavy growth, smooth, and nacreous. (5) Agar stab: Surface growth. (6) Nutrient broth: Slightly turbid, with slight white ring and sediment. Physiological Properties. (1) Milk: Coagulate after 3 days and slowly redissolved with acid reaction. (2) Indole not produced. (3) Nitrites produced from nitrates after 2 days. (4) Hydrogen sulfide is slightly produced. (5) Ammonia is produced after 5 days. (6) Voges-Proskauer test, negative. (7) Methyl red test, negative. (8) Production of acid from carbohydrate in peptone media. (a) Acid from xylose (4.0*), glucose (1.5*) mannose (1.5*) galactose (1.5*). (* decrease of ph value during 7 days incubation) b) No acid from fructose, arabinose, sucrose, lactose mal tose, trehalose, raffinose, mannitol, gly cerin, dextrin, starch, inulin, glycogen, sorbitol. (9) No gas was produced from any of the above cited carbohydrates in peptone media. (10) Starch hydrolysis, negative. (11) Thermal death point, at 70 Ž for 10 minutes. (12) ph range: Optimum ph 6.0 to 9.0. (13) Catalase, positive. Strain A133 is considered to be a Micrococcus sp., because the characteristics of this strain were found to fall under the head of Micrococcus after they were referred to Bergey's Manual of Determinative Bacteriology, 7th Edition. Acknowledgement The authors wish to ex press their thanks to Prof. T. Mitsui, Kyoto University, for the microanalysis, Prof. M. Nakajima, Kyoto University, for the infrared spectral analysis and Prof. K. Onodera, Kyoto University, for having the electron micrograph taken. The authors are indebted to Dr. H. Yamada, the Institute for Food Science, Kyoto Univer sity, for his interest during the course of this work.