I Zeitschrift fur Allg. Mikrobiologie I 9 I 6 I 1969 I 443-448 I

(Institute of Applied Microbiology, The University of Tokyo, Tokyo)

Oxidation of d 5-3-Hy droxy -ster ols to 44-3 -Ket o-steroids by hydrocarbon-growing Corynebucterium hydrocarboclastus

H. IIZUKA, M. IIDA, S. TESHIMA~) and Y. MIXEMURA

(Eingegangen am 14. 4.1969)

It has been known that the oxidation of other sterols except stigmasterol to A4-3-Keto-steroids, took place during fermentation with different microorga- nisms (IIZUKA et al. 1967, CHANEY et al. 1967). Many investigators have report,- ed the transformation of steroids by microorganisms, which use non-hydrocarbon materials as a sole source of carbon.

Since 1956, we have isolated many hydrocarbon-assimilating microorganisms from soil samples of oil fields and gas fields (IIZUKA et al. 1964) and studied the microbial metabolism of hydrocarbons (IIZUKA et al. 1966, 1968). On the other hand, we are studying the microbial transformation of sterols by hydrocarbon- growing cells as one of the utilization of hydrocarbons.

The present study describes the oxidation of several sterols by the hydrocarbon- growing Corynebacteriurn hydrocarboclastus strain 272 -9.

Haterial and methods

Cultivation: Hydrocarbon-assimilating microorganisnis isolated from the various gas fields and oil brain and from aircraft fuel were investigated for the screening of sterol oxi- dation.

Corynebacterium hydrocarboclastus strain 272-9 (JIZUKA et al. 1964) was the most appro- priate microorganism for our purpose. The strain was grown on glucose medium (1.00,b malt extract, 0.4% yeast extract, and 0.4% glucose, p H 7 . 2 ) , two loops of the cells were transferred to 80 ml of sterile mineral medium (pH 7.2) containing 1.0% of kerosene (KANTO Chemical Co., Ltd.) as the sole carbon source. The composition of mineral medium was as follows: 0.2% NH4Cl,0.1% Na,HPO,, 0.05% KH,PO,, 0.05y0 RlgSO,, 0 .2~oNaCI,0.02~omalt extract, and 0.02y0 yeast extract. The culture was incubated a t 30 "C for 48 hrs. on a rotary shaker (210 rpm). The large scale fermentation was carried out in 5-1-ERLENMEYER flasks containing 800 ml of the same medium. At the log-phase of growth, 40 mg of sterol in 8 nil of N,N-dimethylformamide were added to the culture.

Substrates used: p-sitosterol was obtained from Tokyo Chemical Co., Ltd. and determined to be the mixture of 8-sitosterol and campesterol (1.1 : 1.0, v/v) by gas-liquid chromatography (QLC). Cholesterol, stigmasterol, 19-hydroxy-p-sitosterol, and 19-hydroxy-cholesterol were kindly supplied by SANKYO Co., Ltd., Japan.

Isolation of oxidation product: After additional 72 hrs. of incubation on a rotary shaker a t 30 "C, each reaction mixture was acidified to p H 1.0 with dil. sulfuric acid, extracted twice with equal volumes of ethyl acetate, washed with deionized water, dehydrated over sodium sulfate (anhydrous), and finally concentrated under reduced pressure. The extract dissolved in hexane was chromatographed on a column of Kieselgel (MERCR, 0.05-0.20 mm) with hexane-acetone as the eluent. The fraction containing the oxidation product wasapplied t o preparative thin-layer chromatography on a Kieselgel (GF254, 100 ,u) using two developing

l ) Present address : Faculty of Fisheries, The University of Kagoshima, Kagoshima, Japan.

444 H. IIZUEA, 31. IIDA, S. TESHIMA and T. MISEXCRA

solvent systems: cycloliesane-ethyl acetate (5: 1). and hcsiuie-acetone (3: 1). The oxidation product was locatecl under L'V-light (253 nip) and eluted with inethanol from the UV-ab- sorbing zone. The crystallizable product obtained was recrystallized from ethanol and methanol.

Analysis of oxidation product : (.:as-liquid chroniatographic analysis was performed with SEIIXADZI- Nodel GC-IB clirortiatoprap2iic unit. using nitrogen (2.0 kg/cm2) as the carrier gas and SE-30 (1.5:,) on 1.3 :i 4 n i n i cohiinn. lnfrared absorption spectrnm was obtained with a XIITOX B t - S I i O DS-301 i;pectropliotometer in chloroforiii or in l<Br pellet. Ultra- violet absorption spectrum \\as measured in ethanol or methanol wing SH~NAUZU SV-BOA spectrophotometer. Xass spectra analysis \\-as nieasnred on the HITACHI RPIIU-GD instru- ment (chaniber voltage, $0 eV). Suclear magnetic resonance deterinination (NMR) was made \r-ith HITACHI spectrophotometer niotlified to be a GO-megacycle instrument. Samples in dentrated chloroform were analyzed b - using tetrnmetliylsilaiie as an internal standard t o determine chemical shifts.

Oxidation of fi-sitohtrrd (I) The oxidation product. after recrystallization from ethanol, p roducd 5i nig froin 188 ing of (1) : nip 80-82 "C; JbE:zJL 242 mp; vk5L13 1665 (d4-3-CO). 1617 (C-C) e n r l Tlic inass spectrmn of product (Fig. 1)

124

C n u

z

+ c

f:

-

0 100 200 300 400 m/e

Fig. 1. Mass spectrnni of J-sitost-4-en-3-one and campest-4-en-3-01ie produced from p-sito- sterol by C. 1iUdiocarboclrtstuF strain 372-9

sl ioi~ed prominant peakb a t niie 412 (31;). 398 (&I;), 397 (&I:-CH,), 383 (31;-CH,). 3iO(JI;-CH,CO),356(31,-CH,CO). 2 i l (Rlt-K or?rl$-R',R orR'=alkyl side chain of stcrol). and 124. The peaks 124 and 370 or 356 are characteristic of .14-3-Keto-steroids (BTDZIKIEWICZ et a l . 1964, SHAPIRO e t al. 1963). The com- pound (1) XI as oxidized by tlie method of OPPESACER to p-sitostenone and cam- pestenone (EASTHAM et n l . 1955). Tlic two coniponents of product were identical with tha t of authentic p-sitostenone (Rt = 28.3 min) and campestenone (Rt = 22.9 niin). respectively on GLC. 011 the basis of the above data , the product was assigned the mixture of p-sitostenone a i d campestenone.

Oxidation of 19-hydroxy-P-sitosterol (11). The product, after recrystalliza- tion from methanol, procluccd 26 nig from 200 nig of (11): nip 168-170 "C; "ha, ''IeoH 244 mu (E max 16000). v:5:'3 1665 (J4-3-C0). 1617 (C=C) cm-l; M W (mass spectrum) 428. Thc mass spectrum of product (Fig. 2 ) showed prominant peaks a t ni/e 428 (AIL). 410 (JI--H,O), 398 (31--CH,O), 287 (M+-R, R=CI0Hz1, the alkyl sicle chain of sterol). 383 (X-CH,O-CH,), 257 (&I+-CH,O-R), 215 (JI+-CH,O- (R + 42)). XJlK (Fig. 3) . T 6.02 (singlet, proton on CH,OH at C-19). z 4.05 (vinylic proton a t C-4).

On the basis of the aborc data, tlie product nas assigned the structure of 19-hydroxy-/3-sitostenone.

Oxidation of sterol by hydrocarbon-growing bacteria 445

Fig. 2. Mass spectrum of 19-hydroxy-/3-sitost-4-en-3-one produced from 19-hydroxy-P- sitosterol by C . hydrocarboclastus strain 272 -9

Oxidation of cholesterol (111). The product, after crystallization from etha- nol, produced 4.5 mg from 80 mg of (111) : mp 76-83 "C; MW (mass spectrum) 384: 242 mp (E max 17000); v ~ ~ ~ l s 1668 (d4-3-C0), 1618 (C=C) cm-l. The mass spectrum of product (Fig. 4) showed prominant peaks at m/e 384 (M+), 369 (M+-CH,), 342 (M+-CH,CO), and 124. The peaks a t m/e 124 and 342 are characteristic of A4-3-Keto-steroids. On the basis of the above data, the pro- duct was assigned the structure of cholestenone.

Oxidation of 19-hydroxy-cholesterol (IV). The product, after recrystallizatioii from methanol, produced 18 mg from 200 mg of (IV): mp 139-142 "C; N W (mass spectrum) 400; AZ::" 244mp (E max 14000); ~g::~3 1665 (d4-3-C0), 1617 (C=C) cm-l. The mass spectrum of product (Fig. 5) showed prominant peaks a t m/e 400 (M+), 382 (M+-H,O), 370 (M+-CH,O), 355 (M+-CH,O-CH,), 287 (M+-R, R=C,H,,, alkyl side chain of sterol), 357 (M+-CH,O-R), and 215 (M+-CH,O- (R + 42)); NMR, z 6.02 (siglet, proton on CH,OH at C-19), 4.05

2 3 4 5 6 7 8 9 v I I 1 I r

Fig. 3. Nuclear magnetic resonance spectrum of 19-hydroxy-p-sitost-4-en-3-one produced from 19-hydroxy-p-sitosterol by C. hydrocarboclastus strain 272 -9

i i 124

I

m/e Pig. 4. Mass spectrum of cholebt-4-en-3-one proclnced from cholesterol by C . hydrocnrbnclas-

tiis strain 2 i 2 - 9

L & A J L # I I A . . . . i 365

215 257 287

0 100 200 30 0

3 7 0

M'

400 m/e

Fig. 5. Mass spectrum of 19-h~dros~--cIiolest-4-en-3-one produced from 19-hydroxy-choles- terol b - C. 1 1 1 ~ ~ 1 ~ 0 ~ 0 ~ boclnstus strain 272-9

(vinylic proton a t C-4). On the basis of tlw a h o w data, the product u as assigned the structure of 19-1iydrox;v-cholrste~ioiic.

Oxidation of stigmasterol (V). (I-) I\ as oxidized by glucose medium containing 1 Ooio krroscne gron ing cells. The product. aftrr crystallization from ethanol, produced 39.1 mg from 80 mg of I-: nip 121-123 "C. 2%;:" 243 mp (E max 18000): ~&::~3 1660 (d4-3-C0). 1617 (C=C) c1ii-l: MIY (mass spectrum) 410. The mass spectrum of product (Fig. 6) sliov ecl proininant peaks a t m/e 410 (&I+), 39.5 (If+-CH,), 368 (,\rT-CH,CO), 229 ()I-- (R + 42), R=C,,H,,) and 124. The peaks a t in:c 124 and 368 arr characteristic of J4-3-Kcto-steroids. On the basis of the above data, the product 11-as assigned the structure of stigmastenonc. The product obtained from Immwic~-gron ing cells \\ as identical with that of stigmastenone on GLC. The product ha. not yet bccn demonstrayed in micro- bial transformation

Relationship betn een itigina tc.iiont1 formation ant1 culture medium is sum- niarizcci in TaIdc 1. Tlic oxidat on of .trio1 to stenonc could be performed not

2 g 10 C

L ._

h

100 2 00 300 400

Fig. 6. 3Iass spectrum of stig.inast-4-en-3-onr produced from stigmasterol by C . hydrocarbo- clnsius strain 272-Y

Oxidation of sterol by hydrocarbon-growing bacteria 447

Table 1 Stigmastenone formation from stigmasterol in kero- sene and glucose medium by C. hydrocarboclastus. C . hydrocarboclastus was incubated with 80 ml of each medium a t 30 "C. At the log-phase, 4.0 mg of stig- masterol in 0.8 ml N,N-dimethylformamide were added and the culture was incubated further 72 hrs. Stigmastenone formed was estimated by GLC using

cholesterol as an internal standard - .- ~ -

Medium Stigmastenone formed (C-source) (mg)

0.31 0.21 1.86

Kerosene Glucose Glucose + kerosene ,

only by hydrocarbon-fermentation but also by glucose-fermentation to some extend. As a typical example, the stenone formation from stigmasterol was investigated using both fermentation methods. I n glucose-fermentation, the formation of stigmastenone scarcely took place, and i t was highly accelerated by the addition of kerosene to the substrate.

From these results, it was concluded that the formation of A4-3-Keto-steroids occurs when the substrates are incubated with Corynebacterium hydrocarboclastus strain 272-9. This phenomenon suggests that sterols are oxidized to stenonc by a co-oxidation system (FOSTER 1962) of kerosene and sterol, and that the enzyme system is possible to be induced by hydrocarbons. Therefore, this approach will be considered as an important new technique in the microbial transformation of steroids.

Summary

I n this study, the oxidation of several sterols was performed by hydrocarbon-growing Corynebacterium hydrocarboclastus strain 272 -9. This strain oxidizes p-sitosterol, 19- hydroxy-P-sitosterol, cholesterol, 19-hydroxy-cholesterol, campesterol, and stigmasterol to p-sitost-4-en-3-one, 19-hydroxy-/3-sitost-4-en-3-one, cholest-4-en-3-one, 19-hydroxy-cholest- 4-en-3-one, campest-4-en-3-one, and stigmast-$-en-3-one, respectively.

Acknowledgements

Elementary analysis was performed by the members of the laboratory for microanalysis of the Institute of Applied Microbiology, The University of Tokyo, to whom the authors wish to express their thanks. They are also indebted to Mr. KOBAYASHI for measurement of infrared spectra, and Mr. SHIDA for measurement of mass spectra, and Mr. ITO for measu- rement of nuclear magnetic resonance spectra.

R e f e r e n c e s

BUDZIKIEWJCZ, H., DJTERASSI, C. and WILLIAMS, D. H., 1964. Structure Elucidation of

CHARNEY, W. and HERZOG, H. L., 1967. Microbial Transformation of Steroids. Academic

DORBMANN, L., 1953. Ultraviolet absorption of steroids. Chem. Reviews, 68, 47 -144. EASTHAM, J. p. and TERANISHI, R., 1955. 4-Cholesten-3-one. org. Syntheses, 36, 39-42. FOSTER, J. W., 1962. Hydrocarbon as substrates for microorganisms. Antonie van Leeu-

Natural Product by Mass Spectrometry, Vol. 2. Holden Day, San Francisco.

Press, New Pork and London.

wenhoek, 28, 241 -274. 31 Zeitschrift f. Allg. Dlikrobiologie, Bd. 9, H. 6

IIZL-KA, H. and I<ox~uaT.&, I<.. 1Y64. Microbiological studies on pet.roleuni and nnt,ural gas. I. Determination of hydrocarbon-ntiliziiiR bacteria. J. gen. appl. JIicrobiol., 10, '207-231.

IIZTKA. H. and SAITQ -4.. 1965. Microbial transformation of steroids and alkaroids. Univ. of Tokyo Press and Unir . Park Press. p. 215-235.

I I Z c l i A k , H.. 11u.i. 31. and USAXI, T., 19(i(i. Jlicrobiological studies on petroleum and natnral gas. IS. Candidal oxidation of decane. J. gen. appl. Microbial., 11, 119-126.

IIZL-KA? H., IID.~. 31.. Usam, Y. and HOSHISO, T.. 1968. 12-Decane dehydrogenation by a cell-free extract of Ca)trZidu rugoscr. Z. Allg. 3tilcrobiol., S, 145-149.

SHIPIRO. K. H., ~VILSOS, J. 31. and D.JIERASSI. C., 1963. Mass spectrometry in structural and stereochemical problems. S S I I I . Observations on the mass spectral fragmentation of steroid l-and-3-cn-3-ones. Steroids, 1, 1-6.