Agric. Biol Chem., 49 (7), 1993-2002, 1985 1993

Microbial Oxidation of Isoprenoid , , Norpristane and Farnesane1 Kenji Nakajima, Akio Sato, Yoshimasa Takahara and Takeo Iida* Fermentation Research Institute, Yatabe-machi, Tsukuba, Ibaraki 305, Japan *The Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan Received September 10, 1984

Rhodococcus sp. BPM 1613, a pristane oxidizing microorganism, grows on isoprenoid hydrocarbons such as phytane (2,6, 10, 14-tetramethylhexadecane), norpristane (2,6, 10-trimethyl- pentadecane) and farnesane (2,6,10-trimethyldodecane) as the sole carbon source, resulting in accumulation of oxidation products in the culture broth. The oxidation products of phytane, norpristane and farnesane in the respective culture broth were isolated and purified by the use of silica gel column chromatography. Their chemical structures were determined by instrumental analyses such as IR, NMRand mass spectrometry. The oxidation products of phytane were identified as 2,6, 10, 14-tetramethyl- l -hexadecanol and 2,6, 1 0, 14-tetramethylhexadecanoic acid, the product of norpristane as 2,6,10-trimethyl-l-pentadecanol, and that of farnesane as 2,6,10- trimethyl-1-dodecanol. All these oxidation products were either monoalcohols or monocarboxylic acids derived through oxidation of the isopropyl terminus of each . In addition, the relationship between the terminal structure of isoprenoid hydrocarbons and microbial oxidation was explored on the basis of these results.

The presence of isoprenoid hydrocarbons trimethyltetradecane as the sole carbon such as phytane (2,6,10,14-tetramethylhexa- source, monocarboxylic acids were derived decane), pristane (2,6, 10, 14-tetramethylpenta- through terminal oxidation and further /?- decane), norpristane (2,6,10-trimethylpenta- oxidation of the oxidation products, which decane) and farnesane (2,6,10-trimethyldo- were identified by GLC-MS. decane) in shale oil,1>2) crude oil3) and sedi- In our previous work,8'13) Rhodococcus sp. mentary rocks4) has been demonstrated. BPM 1613 was grown on pristane, and its Amongthem, phytane and pristane have been monoterminal oxidation products including reported to occur widely in nature, for exam- pristanol, , pristyl pristanate and ple, in the tissues5) of men and animals. pristyl aldehyde, and other pristane-derived There have been several reports6~n) on metabolites were isolated from the culture microbial oxidation of pristane. Cox et al12) broth, and their chemical structures were iden- reported the microbial oxidation of phytane, tified. In the present study, Rhodococcus sp. norpristane and related compounds, but BPM1613 was cultured on phytane, norpris- nothing has been reported on the microbial tane and farnesane as the sole carbon source oxidation of farnesane. According to Cox for identification of their microbial oxidation et al., whenMycobacterium fortuitum was products. As a result, all the alkanes afforded grown on each of 2,6,10,14-tetramethyl- products oxidized at the isopropyl terminus; heptadecane, phytane, norpristane and 2,6,10- phytane was oxidized to 2,6, 10, 14-tetramethyl-

f Microbial Oxidation of Isoprenoid Hydrocarbons. Part V. This paper was presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, April 2, 1978. 1994 K. Nakajima et al.

1-hexadecanol and 2,6, 10, 14-tetramethylhexa- Preparation offarnesane. Farnesane was prepared from decanoic acid, norpristane to 2,6,10-trimethyl- farnesol in a mannersimilar to in the case of phytane. Farnesol was hydrogenated with Pd-carbon to farnesanol 1-pentadecanol, and farnesane to 2,6,10- (3,7,1 1-trimethyl-l-dodecanol) and the farnesanol was trimethyl-1-dodecanol. Among these oxida- tosylated. The tosylated farnesanol was reduced with tion products, the former three alcohols are LiAlH4 to farnesane. The purified farnesane gave a sin- newcompounds. gle peak on GLCand the chemical structure was deter- Based on the results, the relationship be- mined by IR, 13C-NMRand mass spectrometry. tween the terminal structure of these iso- Preparation of norpristane. Phytone (6,10,14-tri- prenoid hydrocarbons and microbial oxida- methylpentadecan-2-one) was prepared from by tion was discussed. the methodreported by Kates et al.15) Phytol was oxidized with potassium permanganate in glacial acetic acid and MATERIALS AND METHODS the oxidation product wasextracted into hexaneand then concentrated. Thecrude product was then purified on a Microorganism. Rhodococcus sp. BPM 1613,8) a mi- silica gel column to isolate phytone. The phytone (6.3g) thus obtained was reduced with croorganism isolated from soil and able to utilize hy- LiAlH4 in absolute ethanol to 6,10,14-trimethyl-2- drocarbons such as ^-paraffin and pristane, was used. pentadecanol (6.5g). The alcohol was heated with a Medium and cultivation. The medium employed for mixture of 3g iodine and 3 g red phosphorous in a boiling water bath. The iodide compound thus obtained was KH2PO4precultivation1.5g, wasNa2HPO4prepared 1.5g,by dissolvingMgSO4-7H2ONaNO35.0g,0.5g, purified on a silica gel column (30 x 144mm, eluted with hexane). The purified iodide (7.2g) was transferred to a FeSO4à" 7H2O 0.01 g, CaCl2à"2H2O 0.01 g and yeast extract glass tube. Heating at 100°C for 8 hours with sodium 0.2g in distilled water, the final volume was 1 liter, and ethylate in the sealed tube afforded an olefin. A silica gel the pH was adjusted to 7.2. To the solution was added 0.5% (v/v) of mixed «-paraffin14) as the carbon source. column (30 x 280mm, eluted with hexane) was used for the The mixed n-paraffin was replaced in the mediumfor ac- purification of the olefin (3.6g). Using Raney nickel as a cumulation of the oxidation products by 3% (v/v) each catalyst, the olefin was hydrogenated at 1 10°C and 70 atm to a saturated hydrocarbon, norpristane, which was pu- of phytane, norpristane and farnesane, and the amount rifed on a silica gel column (30x 155mm, eluted with of yeast extract added was increased to 2.0 g. hexane); yield, 2.5 g. The mass spectrum showed MSm/z: A 50ml portion of the precultivation medium in a 254 (M+), 183 (M+-C5Hn), and 113 (M+-C10H21). 500 ml shaking flask was sterilized and inoculated from an agar slant culture. The inoculated mediumwas incubated Furthermore, the product was identified as norpristane by at 30°C for 2 days on a reciprocal shaker (120rpm). This IR and 13C-NMRspectrometry. seed cell suspension was added at 8%(v/v) to shaking Chromatography. The purity of phytane, norpristane flasks containing 50ml of the accumulation mediumfol- lowed by incubation at 30°C for 5 days as described and farnesane thus prepared was examined by gas liquid chromatography (GLC). The oxidation products were for the preliminary cultivation. fractionated on a silica gel column with monitering by TLC on silica gel 60 F254 (Merck). The purity of each Chemicals. Phytol (3,7,ll,15-tetramethyl-2-hexa- product was examined by TLC and GLC. For TLC, decen-1-ol) and farnesol (3,7,1l-trimethyl-2,6,10-do- development was mainly with a mixture of - decatrien-1-ol) were products of Tokyo Kasei Kogyo acetone (20 : 1, v/v). Alcohols were detected by spraying Co., Ltd. Phytane, norpristane and farnesane were synthe- sized from these compounds by the following methods. 60% sulfuric acid solution followed by heating, and car- boxylic acids by spraying 0.04% BCGsolution in 95% Preparation of phytane. Phytane was prepared from ethanol. The gas chromatograph used was a Shimadzu phytol by the method reported by Avigan et al.5) as GC-5A, equipped with a glass column, 0.3 (i.d.) x 200cm, packed with 1.5% OV-17 on Chromosorb W AW follows: Phytol was hydrogenated at 140 atm in the (80~100 mesh) and a FID. The flow rate ofN2 gas was presence of Pd-carbon powder in 99.5% ethanol to pro- 40 ml/min. duce phytanol (3,7, 1 1, 1 5-tetramethyl-1-hexadecanol). The phytanol was tosylated and the tosylated phytanol was Instrumental analyses. The infrared absorption (IR) purified on a silica gel column, and then reduced with spectra were recorded by a thin-film technique with a LiAlH4 to phytane. The product purified on a silica gel column was shown to be virtually one component by JASCOModel IR-G, and the proton nuclear magnetic resonance (*H-NMR) spectra with a JEOL Model JNM- GLC. The chemical structure was determined by IR, 13C- MH-60 (60MHz, CC14) or a Hitachi Model R-24 NMRand mass spectrometry. (60MHz, CDC13). A JEOL Model JNM-FX-60 (15 MHz, Microbial Oxidation of Phytane, Norpristane and Farnesane 1995 CDC13) was used to record carbon nuclear magnetic after dehydration over anhydrous sodium resonance (13C-NMR) spectra with tetramethylsilane sulfate. The concentrate (3.18g) was dis- (TMS) as an internal standard. Mass spectrometry was solved in a small portion of hexane, trans- carried out with a JEOL Model JMS-D 300 (GLC-MS) comprising a GLCwith a glass column, 0.3 (i.d.) x 100cm, ferred to a silica gel column (40x260mm), packed with 2% OV-1 (He 0.5kg/cm2, and 120~200°C), and eluted with hexane and then benzene. and a MS: ionization potential, 20eV, and ion source Phytane (2.50g) and product A (0.62g) were temperature, 250°C. eluted in the hexane and benzene fractions, respectively. TLC and GLC of product A RESULTS showedessentially one component. The potas- Isolation of oxidation products from phytane sium carbonate solution containing product Apart of the culture broth after microbial B was acidified with sulfuric acid and ex- growthon phytaneas the sole carbonsource tracted with , and 0.37g of an was acidified and extracted with diethyl ether. oily substance was obtained after dehydration The extract was spotted on a thin layer plate. and concentration. The oily substance was Color development of the chromatogram was spotted on a preparative thin layer plate and carried out by the method described above. developed twice with hexane and three times Thechromatogramshowedtwo spots of prod- with benzene. After air-drying, the spot of ucts A and B as shown in Fig. 1. Product A product Bon the plate was scraped off, ground was distinguished from phytanol, a hydro- and transferred to an Erlenmeyer flask. To the genated phytol, by the Rfvalue on TLC and flask were added 0.1 n sulfuric acid solution, the retention time on GLC.Product B was until the whole of the silica gel powder in the considered to be an acidic substance based flask was moistened, and diethyl ether. on the coloration with BCG. Product B was extracted into the diethyl ether A 200ml portion of the culture broth was by stirring the mixture with a stirrer. The adjusted to pH 2 with sulfuric acid and ex- extraction was repeated 3 times and 0.01 g of tracted with diethyl ether. The acidic sub- product B wasobtained. stance (product B) in the ether was extracted into 5% potassium carbonate solution. The Identification of products A and B remaining ether layer containing substrate The chemical structures of products A and B phytane and product A' was concentrated were investigated by instrumental analyses.

O < phytane * CH}

product A->q s~\ <_- phytanol product A --» O q\_ phytanol (^^^^ product B >() productB->K

broth authentic broth authentic solvent: benzene-acetone solvent: benzene (20:1) development: 1 run development: 5 runs Fig. 1. TLC of Culture Broth with Phytane. 1996 K. Nakajima et al.

100 I å -å ~ ~~

50- " I 1035 3340 H

I i I I 1 1 1 1 1 4000 2000 1 600 1 200 800 400

Wave number (cm )

Fig. 2. IRSpectrumof ProductA.

5 4 ^ 3 2 1 0

6 (ppm) Fig. 3. !H-NMRSpectrum of Product A.

The IR spectrum of product A is shown in Fig. CH3 2. Product A was determined to be an a- (2H, d, -CH-CH2OH). 13C-NMR spectrom- branched primary alcohol based on the ab- etry16) (Fig. 4) revealed NMR5g2gf: ll.4 (d, sorption bands at 3340 and 1035cm"1. The ab- CH3), 16.6 (a, CH3), 19.2 (c, CH3), 19.7 (b, sorption bands at 1375 and 1365cm"1 charac- CH3), 24.5 (C-4, 8, 12), 29.6 (C-15), 32.9 (C- teristic of the isopropyl group of the substrate 6, 10), 33.5 (C-3), 34.4 (C-14), 35.8 (C-2), 37.0 phytane disappeared and a new absorption (C-13), 37.4 (C-5, 7, 9, ll), and 68.3 (C-l). band of a methyl group appeared at Mass spectrometry of product A (Fig. 5) re- 1370cm"1, suggesting that product A is an vealed m/z\ 280 (M+-H2O) as the peak for alcohol derived on oxidation of the isopropyl an ion with the maximum mass. Anal. termimus of phytane. ^-NMRspectrometry (Found: C, 80.45; H, 14.06, Calcd. for (Fig. 3) revealed NMR 8%?;si: 0.7~ 1.0[15H, C20H42O: C, 80.40; H, 14.18%). Product A (CH3)5], 1.0~1.7[24H, (~CH2-)10 and was identified as 2,6,10,14-tetramethyl-1- CH3 hexadecanol, which was formed on oxida- (-CH-)4], 1.82 (1H, s, -CH2OH), and 3.39 tion of the isopropyl terminus of phytane. Microbial Oxidation of Phytane, Norpristane and Farnesane 1997

5,7,9,ll

c b b a

' 15 v4-i^k^vjv^^.H13 ll 9 7 5 3 ^H20H 0H 4,8,12b á"S TM,

6,10 13 14J

1 1 nil 1 Mill1 1 I1 100 80 60 40 20 0

6(ppm) Fig. 4. 13C-NMRSpectrum of Product A.

£> 100.

£ 50" 280(M-H20)

150 250 300 m/z Fig. 5. Mass Spectrum of Product A.

The IR spectrum of product B revealed dissolved in a small portion of hexane and absorption bands at 2500-3300cm"1 (OH) transferred to a silica gel column (30x and 1710cm"1 (C=O), suggesting the pres- 300mm). The column was eluted with hexane ence of carboxylic acid. Esterification of prod- and then benzene. Substrate norpristane uct B with diazomethane followed by re- (0.63 g) was eluted in the hexane fraction, and duction with LiAlH4 gave rise to an alcohol product C (16mg) in the benzene fraction. The which was coincident with product A on TLC purity of product C thus obtained was found developed 5 times with benzene and GLCwith to be satisfactory by TLC and GLC. the following conditions: 3 mglass column of 1.5% OV-17 on Shimalite W; temperature, Identification of product C 180°C; carrier gas, 40ml N2/min; and a FID. The IR spectrum of product C is shownin Accordingly, product B was identified as Fig. 6. The absorption bands at 3340 and 2,6, 10, 14-tetramethylhexadecanoic acid form- 1035cm"1 suggest the presence of an a- ed on further oxidation of product A. branched primary alcohol. The absorption band at 1375cm"1 for a methyl group ap- Isolation of the oxidation product from peared to be coupled with the disappearance norpristane of absorption bands at 1375 and 1365cm"1, A 50ml portion of the norpristane culture characteristic of the isopropyl group in the broth was acidified with sulfuric acid and norpristane molecule. This suggests that extracted with diethyl ether. TLC of the ether product C is an alcohol formed when nor- extract revealed, apart from one spot of nor- pristane undergoes terminal isopropyl oxi- pristane addedas the substrate, essentially one dation. After further investigation by 1H- spot of an oxidation product (product C). The NMR(Fig. 7) and mass spectrometry (Fig. ether layer was evaporated to dryness after 8), product C was identified as 2,6,10-tri- dehydration. The oily residue (0.71 g) was methyl-1-pentadecanol formed on oxidation 1998 K. Nakajima et ah

TOOi ,

y 1035

4000 2000 1 600 1 200 800 400 Wave number (cm" )

Fig.6. IRSpectrumofProductC.

farnesol. A 300ml portion of the farnesane-culture broth after cultivation was acidified with sul- furic acid and extracted with diethyl ether. The ether layer was concentrated following dehy- dration. The concentrated oily substance (4.51g) was dissolved in a small portion of hexane and passed through a silica gel column (30 x 285mm)with hexane and then benzene.

I I I I I I_ The substrate farnesane (3.42 g) was recovered 5 4 3 2 1 0 from the column in the hexane fraction and 6(ppm) then product D (0.87g) in the benzene frac- tion. TLCand GLCshowed that product D is Fig. 7. ^-NMRSpectrum of Product C. a single component. of the isopropyl terminus of norpristane: Identification of product D NMR 5g2Sf: 0.7-1.0 [12H, (CH3)J, 1.0-1.6 The IR spectrum of product D is shown in CH3 Fig. 10. On the basis of the absorption bands [23H, (-CH2-)10 and (-CH-)3], 1.45 (1H, s, at 3320 and 1035cm"1, product D is con- CH3 sidered to be an a-branched primary alcohol. -CH2OH), and 3.40 (2H, d, -CH-CH2OH), The absorption band for a methyl group at and MS m/z: 252 (M+-H2O), 183 (M+- 1368cm"1 appeared to be coupled with the CH3 disappearance of absorption bands at 1376 CH2CH2CHCH2OH), and 169 (M+- and 1365cm"1 which are characteristic of the CH3 isopropyl group in the farnesane molecule, CH2CH2CH2CHCH2OH). suggesting that product Dis an alcohol formed when farnesane is oxidized at the isopropyl Isolation of products on oxidation offarnesane terminus. The ^-NMRspectrum of product Figure 9 shows TLC of a diethyl ether D (Fig. ll) revealed NMR 8%?;Si: 0.7-1.08 extract of the acidified farnesane-culture [12H, (CH3)4], 1.08- 1.55 [17H, (-CH2-)7 and broth. The TLC shows, besides a spot of CH3 substrate farnesane, another spot whose Rf (-CH-)3], 1.63 (1H, s, -CH2OH), and 3.34 value is different from that of authentic far- CH3 nesanol resulting from hydrogenation of (2H, d, -CH-CH2OH). The 13C-NMR spec- Microbial Oxidation of Phytane, Norpristane and Farnesane 1999

169

100 150

252(M) KM 200 m/z Fig. 8. Mass Spectrum of Product C.

Farnesane -*O O*-Farnesane -*CZD CD+- Farnesane

Product D-^O O*- Faá"esan01 --*O O"~"Pr0duct D

broth authentic authentic broth solvent: benzene-acetone solvent: benzene (20:1) development: 1 run development: 5 runs Fig. 9. TLC of Culture Broth with Farnesane.

1001 -

3320 1 1035

4000 2000 1 600 1 200 800 400 Wave number (cm" )

Fig. 10. IR Spectrum of Product D. trum16) (Fig. 12) showed NMRSggsf: UA 78.80; H, 13.19. Calcd. for C15H32O: C, (d, CH3), 16.6 (a, CH3), 19.2 (c, CH3), 19.7 78.88; H, 14.12%). Accordingly, product D (b, CH3), 25.5 (C-4, 8), 29.6 (C-ll), 32.9 was identified as 2,6, 10-trimethyl-l-dodecanol (C-6), 33.5(C-3), 34.4(C-10), 35.8(C-2), 37.0(C- derived on oxidation of farnesane at the iso- 9), 37.4 (C-5, 7), and 68.3 (C-l). Mass propyl terminus. spectrometry (Fig. 13) showed MS m/z: DISCUSSION 210 (M+-H2O), and 127 (M+- ^H3 In our previous reports, isolation of mono- CH2CH2CHCH2OH). Anal. (Found: C, terminal oxidation products of pristane (pris- 2000 K. Nakajima et al. tanol, pristanic acid, pristyl pristanate and Rhodococcus sp. BPM1613 grown on pristane pristyl aldehyde)8) and of three dicarboxylic and their identification were described. In the acid metabolites13* from culture broth of present work, the sameorganismwasused for TMS metabolic conversion of phytane, norpristane and farnesane. The oxidation products of these three substrates were identified as 2,6,10,14- tetramethyl-1-hexadecanol and 2,6,10,14- tetramethylhexadecanoic acid, 2,6,10-tri- methyl-1-pentadecanol, and 2,6,10-trimeth- yl- 1 -dodecanol, respectively. Microbial oxidation of phytane, norpristane and related compoundshas been reported only by Cox et al.i2) They grew Mycobacterium fortuitum on2,6, 1 0, 14-tetramethylheptadecane, phytane, norpristane and 2,6,10-trimethyl- tetradecane as the sole carbon source, and isolated the oxidation products of these sub- I I I I I strates. The products were identified as mono- 5 4 3 2 1 0 carboxylic acids derived on monoterminal 6(ppm) oxidation and as further oxidized monocar- Fig. ll. ^-NMR Spectrum of Product D. boxylic acids via the ^-oxidation process. The

5,7

c b a d i l l ll 9 7 5 3 CH9OH 1l

ho bc d á"S 1 2 6 I .i a coci3 "

1 1 1 1 1 1 T OO 80 60 40 20 0 6(ppm) Fig. 12. 13C-NMRSpectrum of Product D.

~ 100

50-

2 ° Ml 1 II, I 210(M-H20) 100 m/z Fig. 13. Mass Spectrum of Product D. Microbial Oxidation of Phytane, Norpristane and Farnesane 2001

Table I. Growth of Rhodococcus sp. BPM1613 on Norpristane and Pristane, and Production of Alcohols and Carboxylic Acids on Terminal Oxidation The following medium was used: substrate (norpristane or pristane) 1% (v/v), NaNO30.5%, KH2PO4 0.15%, Na2HPO40.15%, MgSO4-7H2O 0.05%, FeSO4-7H2O 0.001%, CaCl2-2H2O 0.001%, and yeast extract 0.2% (pH 7.2). Cultivation was carried out at 30°C.

o i, t t Cells Residual substrate Monoalcohol Monocarboxylic acid (g/liter) (g/liter) (g/liter) (g/liter)

3 days Norpristane 2.85 2.29 0. 14 - Pristane 1.07 3.20 1.09 0. 14

5 days Norpristane 4.32 0. 1 1 - - Pristane 1.50 0.64 1.49 0. 17 terminus to be oxidized by the bacterium, dimethyloctane ( \A/\As) with an iso- however, varies among the substrates. 2, 6, propyl group as one terminus and an ethyl 10,14-Tetramethylheptadecane and phytane, group as the opposite terminus, but unable to both of which have an isopropyl group as one utilize 3,6-dimethyloctane ( n/vn/v.) with terminus, their other termini being «-propyl two ethyl groups as the two termini. In the case and ethyl moieties, respectively, underwent of norpristane with isopropyl and «-pentyl oxidation only at the isopropyl terminus. termini, only a small quantity of the monoal- Norpristane with isopropyl and n-pentyl ter- cohol was formed on terminal isopropyl oxi- mini was predominantly oxidized at the iso- dation and accumulated without accumula- propyl terminus, but also partly at the oppo- tion of the oxidation product at the rc-pentyl site n-pentyl terminus. In addition, 2,6,10-tri- terminus. The poor accumulation of the methyltetradecane with isopropyl and rc-butyl oxidation product was not due to the in- termini underwent monoterminal oxidation at ability of the organism to use norpristane, both ends, resulting in accumulation of two but attributable to oxidative degradation different carboxylic acids in comparable starting from the relatively lengthy n-pentyl proportions. chain. Utilization of pristane and norpristane In our previous study,13) pristane having by this organism and formation of their oxida- isopropyl groups as both termini was found to tion products are compared in Table I. The be oxidized by Rhodococcus sp. BPM 1613 comparison also supports this view. through two metabolic pathways: (1) pristane The results described above combined with -åº pristanic acid -åº /^-oxidation, and (2) pris- the data presented by Coxet al. suggest that tanic acid -åº pristanedioic acid (co-oxidation) branched alkanes having termini comprising -åº/^-oxidation. The present report describes methyl, ethyl and propyl groups are more that phytane having isopropyl and ethyl ter- insusceptible to microbial oxidation than those mini is oxidized at the isopropyl terminus, having termini of longer alkyl groups such as resulting in a monoalcohol and a monocarbo- butyl, pentyl and hexyl groups. xylic acid, and farnesane with a terminal struc- Aiming at the profitable utilization of un- ture similar to that of phytane is oxidized at touched resources such as isoprenoid hydro- the isopropyl terminus to a monoalcohol. The carbons contained relatively abundantly in results well agree with those obtained in the shale oil, microbial oxidation of phytane, pris- preliminary experiments in which the same tane, norpristane and farnesane wasinvesti- organism was shown to be able to utilize 2,7- gated in the present experiments. Rhodococcus dimethyloctane ( yv\A ) with two iso- sp. BPM 1613 utilizes all of these isoprenoid propyl groups as the two termini and 2,6- hydrocarbons, resulting in accumulation of 2002 K. Nakajima et al. their oxidation products in the culture broth. 7) E. J. McKenna and R. E. Kallio, Proc. Natl. Acad. Further studies on microbial oxidation of iso- Set U.S.A., 68, 1552 (1971). prenoid hydrocarbons contained in shale oil 8) K. Nakajima, A. Sato, T. Misono, T, Iida and K. Nagayasu, Agric. Biol. Chem., 38, 1859 (1974). by this organism and utilization of the oxida- 9) M. P. Pirnik, R. M. Atlas and R. Bartha, /. tion products are scheduled. Bacterioi, 119, 868 (1974). 10) R. E. Cox,J. R. Maxwell, R. G. Ackmanand S. H. REFERENCES Hooper, Biochim. Biophys. Acta, 360, 166 (1974). ll) T. Hagihara, M. Mishina, A. Tanaka and S. Fukui, 1) J. J. Cummins and W. E. Robinson, J. Chem. Eng. Agric. Biol Chem., 41, 1745 (1977). Data, 9, 304 (1964). 12) R. E. Cox, J. R. Maxwell and R. N. Myers, Lipids, 2) T. Iida, E. Kitatsuji and S. Hayashi, Yakugaku ll, 72 (1976). Zasshi, 96, 769 (1976). 13) K. Nakajima and A. Sato, Nippon Nogeikagaku 3) M. Omokawa,Report of the Technology Research Kaishi, 57, 299 (1983). Center, J. P. D. C. No. 4, 17 (1977). 14) K. Nakajima, A. Sato, Y. Takahara, T. Hosaka and 4) V. E. Modzeleski, W. D. MacLeod, Jr., and B. Nagy, M. Taniguchi, Nippon Nogeikagaku Kaishi, 54, 27 Anal Chem., 40, 987 (1968). (1980). 5) J. Avigan, G. W. A. Milneand R. J. Highet, Biochim. 15) M. Kates, C. N. Joo, B. Palameta and T. Shier, Biophys. Ada, 144, 127 (1967). Biochemistry, 6, 3329 (1967). 6) D. F. Jones, J. Chem. Soc. (Q, 2809 (1968). 16) Y. Takeuchi, Chemistry, 33, 475 (1978).