Biochimica et Biophysica Acta 1347 Ž1997. 75–81 Oxydation of oleic acid to ž E/ -10-hydroperoxy-8-octadecenoic and ž E/ -10-hydroxy-8-octadecenoic acids by Pseudomonas sp. 42A2 Angel Guerrero a , Isidre Casals b, Montse Busquets c , Yolanda Leon d , Angeles Manresa d,) a Departament de Quımica Organica Biologica, C.I.D. (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain ´ ` ` Laboratori de Cromatografia, SerÕeis Cientıfic-Tecnics, UniÕersitat de Barcelona, 08028 Barcelona, Spain ´ ` Departament de Bioquimica i Biologia Molecular, Facultat de Quımica, UniÕersitat de Barcelona, 08028 Barcelona, Spain ´ d Laboratori de Microbiologia, Facultat de Farmacia, UniÕersitat de Barcelona, 08028 Barcelona, Spain ` b c Received 2 January 1997; revised 1 April 1997; accepted 3 April 1997 Abstract Biotransformation of oleic acid with Pseudomonas sp. 42A2 has been found to produceŽE.-10-hydroxy-8-octadecenoic acid Ž2a., ŽE.-10-hydroperoxy-8-octadecenoic acid Ž3a., and ŽE.-7,10-dihydroxy-8-octadecenoic acid Ž4a.. Structures of the metabolites were fully characterized by infrared and 1 H and 13 C NMR spectra of the acids, by fast atom bombardment ŽFAB. and electron impact ŽEI. and chemical ionization ŽCI. mass spectrometry of the corresponding methyl esters. This is the first time that the two former compounds of trans stereochemistry have been described to have originated from a Pseudomonas sp. cell culture. Time course of products accumulation showed that biotransformation started with bacterial growth, the amount of products 2a Ž5.58 grl. and 4a Ž2.63 grl. being optimum after 24 h of incubation while hydroperoxide 3a Ž1.15 grl. reached its maximum after 16 h of the biotransformation process. Experiments conducted to ascertain whether the conversion enzymeŽs. was cell-bound or extracellular, showed that the enzymeŽs. is cell bound, located in the periplasmic space and has lipoxygenase activity. q 1997 Elsevier Science B.V. Keywords: Biotransformation; Fatty acid; Oleic acid; Hydroxy fatty acid; Ž Pseudomonas. 1. Introduction Microorganisms and their enzymes are being increasingly used as biocatalysts to obtain compounds of potential interest w1,2x. In this context, biotransformations of fatty acids are receiving particular attention since the new chemicals can be used as lubricants w3x, additives in coating and paint industries w4x or surfactants w5x, among others. ) Corresponding author. Fax: q34 3-4021886. E-mail: [email protected] Microbial transformations of oleic acid have been extensively studied. They include hydroxylation to ricinoleic acid by a soil bacterium w4x or to 10-hydroxy-8Z-octadecenoic acid and 7,10-dihydroxy-8Eoctadecenoic acid Ž 4a. by Pseudomonas sp. w6–9x, hydration of the double bond of oleic acid to 7,10-hydroxyoctadecanoic acid by Pseudomonas sp. w10x or to 10-hydroxyoctadecanoic acid by Nocardia cholesterolicum and Rhodococcus sp. w11x or the formation of 10-oxo-octadecanoic acid by Saccharomyces sp., Candida sp. and other microorganisms w12x, and voxidation to 1,18-octa-9-decenedioic acid by Candida tropicalis w13x. 0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 5 6 - 8 76 A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 Fig. 1. Microbial transformation of oleic acid Ž1a. by Pseudomonas sp. 42a2 leading to ŽE.-10-hydroxy-8-octadecenoic acid Ž2a., ŽE.-10-hydroperoxy-8-octadecenoic acid Ž3a. and ŽE.-7,10 dihydroxy-8-octadecenoic acid Ž4a.. In our investigation on microbial transformations of natural and available resources such as vegetable oils, we have isolated a bacterial strain, Pseudomonas sp. 42A2 ŽNCIMB 40044. , which produces compound 4a when incubated in olive oil w9x. This compound presents surface active properties w14x. Continuing our work with this strain, we now want to report on isolation and characterization of ŽE.-10-hydroxy-8-octadecenoic acid Ž 2a. and ŽE.-10-hydroperoxy-8-octa-decenoic acid Ž 3a., as well as the already known compound 4a ŽFig. 1.. This is the first time that the two former compounds of trans stereochemistry are reported to be formed from a Pseudomonas sp. cell culture. We also describe the time course of production of the new chemicals as well as experiments conducted to localize the enzymeŽ s. responsible for the biotransformation. 2. Material and methods 2.1. Organism and growth conditions Pseudomonas sp. 42A2 Ž NCIMB 40044. was originally isolated in our laboratory from an oil-contaminated water sample and maintained on Trypticase Soy Agar ŽTSA. ŽADSA, Barcelona, Spain.. Cells were grown in a mineral salt medium containing 3.5 grl of sodium nitrate as nitrogen source and 20 grl of oleic acid as carbon source w15x. The initial pH of the medium was adjusted to 6.8. A 2% Ž vrv. cell suspension ŽOD 2.0. on saline serum of an overnight culture on TSA was used as inoculum. 2.2. Chemicals Oleic acid Ž98% pure by GC. was a gift from Sandoz Quımica SAE Ž Barcelona, Spain. . Ricinoleic ´ acid and Soybeen lipoxygenase were purchased from Sigma Chem. Co ŽSt. Louis, USA. . All chemicals and solvents were ACS grade and used as received. Analytical TLC plates ŽKieselgel G60. were obtained from Merck ŽDarmstadt, Germany.. 2.3. Instrumentation Optical rotations were measured on a Perkin Elmer 141 polarimeter. Infrared spectra were recorded on a NICOLET 510 with Fourier transform spectrometer ŽNicolet, Madison, USA.. 1 H and 13 C NMR spectra were obtained in CDCl 3 solutions on a Varian Unity spectrometer Ž Varian Assoc., Palo Alto, USA. , operating at 300 MHz for 1 H and 75 MHz for 13 C and the values are expressed in d scale relative to internal tetramethylsilane. Electron impact-low resolution mass spectra ŽEI-MS. were run on the trimethylsilyl ethers of the methyl ester derivatives w16x using a Fisons MD 800 mass spectrometer ŽFisons Instruments, Milan, Italy. coupled to a Fisons GC 8000 gas chromatograph. The GC was equipped with a HP-5, 25 m = 0.20 mm ID fused silica capillary column and the helium flow was 0.8 mlrmin. The oven temperature was kept at 1008C for 3 min followed by a ramp of 78Crmin to 2508C and held at this temperature for 10 min. Sample injections were in splitless mode at 2508C. Chemical ionization mass spectra ŽCI-MS. of the methyl esters, using CH 4 as ionization gas, were A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 recorded on a HP-5890 Ser. II Gas Chromatograph ŽHewlett-Packard, Palo Alto, USA. coupled to a HP5989A mass spectrometer, equipped with a HP-5, 30 m = 0.20 mm ID fused silica capillary columm using helium as the effluent gas. The oven temperature was kept constant at 2008C. Fast atom bombardment spectra ŽFAB-MS. were recorded on a VG AutoSpec-Q ŽFisons Instruments, Milano, Italy., equipped with caesium atom gun and working with an accelerating voltage of 20 kV. The matrix used was glycerol and the spectra were recorded in positive-ion mode. Quantitative analyses were conducted on a Shimadzu GC-14A gas chromatograph Ž Shimadzu, Kyoto, Japan. equipped with a split-splitless injector and a flame ionization detector Ž FID.. A Supelco SPB-1, 30 m = 0.25 mm ID fused silica capillary column at an oven temperature of 2008C Žisothermal. and with a helium flow rate of 0.57 mlrmin, was used. The split ratio was 1:100 and integration of peaks was carried out on a Shimadzu C-R4A Chromatopac Integrator ŽShimadzu, Kyoto, Japan. using methyl ricinoleate of known concentration as standard. High Performance Liquid Chromatography ŽHPLC. was performed on a LKB 2150 gradient system ŽPharmacia, Uppsala, Sweden. equipped with a Spherisorb ODS-2 column 250 = 4.6 mm ŽTeknokroma, St. Cugat, Spain. , and a Promis automatic injector ŽSpark, Holland.. The absorbance of the eluate was monitored by a UV-Vis Waters 486 absorbance detector ŽWaters, Mildford, USA. and a Sedere evaporative light scattering detector ŽS.E.D.E.R.E., Orleans, France.. Compounds eluting from the column were collected on a Heli-Frac fraction collector Ž Pharmacia Biotech, St. Cugat, Spain. and chromatographic data recorded on a Nelson double channel interface ŽPerkin Elmer, Norwalk, USA.. 2.4. BioconÕersion and isolation of products After inoculation, biotransformation was carried out in 1000 ml baffled Erlenmeyer flasks containing 100 ml of mineral medium and incubated on a rotary shaker at 308C and 200 rpm for 24 h. Cell growth was monitored by the protein content following the method of Lowry w17x. To establish the extent of conversion, cultures were sampled at various time intervals by taking 5 ml of the medium and centrifuged at 5500 = g for 15 min in a Centrikon T-124 77 centrifuge ŽKontron, Milano, Italy.. The cell free culture was acidified with 6 N HCl to pH 2 and the supernatant was extracted twice with an equal volume of chloroform. The organic phases were collected and dried with anhydrous Na 2 SO4 , the solvent removed to dryness by rotary evaporation. Isolation of products was carried out by injecting 200-m l aliquots of the crude extract dissolved in methanol in HPLC. Separation was achieved at 358C by a linear gradient from 50% to 100% of 0.1% acetic acid in water Žsolvent A. and 0.1% acetic acid in acetonitrile Ž solvent B. with a flow of 2.5 mlrmin. 2.5. Lipoxygenase actiÕity Lipoxygenase ŽSigma Chem., St. Louis, SA. activity was measured following the indication of the supplier, using oleic acid as substrate. 2.6. Analyses of products Qualitative analyses of samples were carried out by TLC on 0.25 mm silica gel G60 plates using a mixture of chloroform:methanol:acetic acid 65:25:4 vrvrv as developing solvent. Plates were visualized by spraying with a-naphthol sulfuric acid solution and heating at 1008C. Quantitative analyses were carried out by gas chromatography on the methyl esters of the extract. Samples were methylated with diazomethane w18x, diluted with ether and injected on a Shimadzu GC-14A, as described previously. 2.7. Characterization of products The biotransformation products were characterized in base to their spectroscopic and chromatographic features as follows: 2a: IR Žfilm. Õ 3400, 3300, 2923, 1711, 1466, 1412, 1290, 1225, 1088, 968 Ž trans alkene. cmy1. 1 H NMR d 5.612 Ždt J s 15.3, JX s 6.6 Hz, 1H, CH s CHCHOH., 5.439 Žddt J s 15.3, JX s 6.6, JY s 1.2 Hz, 1H, CH s CHCHOH., 4.035 Ždt J s 6.6, JX s 6.3 Hz, 1H, CHOH., 2.346 Žt J s 7.5 Hz, 2H, CH2 CO 2 H., 2.033 Žm, 2H, CH2 C s C., 1.637 Žm, 2H, CH2 CHOH., 1.60–1.18 Žc, 20H, 10CH 2 ., 0.875 Žt J s 6.9 Hz, 3H, CH 3 .. 13 C NMR d 178.2 Ž CO., 133.14, 131.97 ŽC-8, C-9., 73.27 ŽC-10., 37.30 ŽC11., 33.69 ŽC-7., 32.0, 31.87 ŽC-16, C-2., 29.55– 28.55 ŽC-13, C-14, C-15, C-4, C-5, C-6., 25.48 ŽC-3., 78 A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 24.56 ŽC-12., 22.66 Ž C-17. , 14.11 Ž CH 3 . . EI-MS of the TMS derivative of the methyl ester 2b mrz Ž%. : 271 Ž 100. , 241 Ž 12., 149 Ž21., 129 Ž26., 121 Ž17., 75 Ž32., 73 Ž85.. CI-MS of the TMS derivative of the methyl ester 2b ŽCH 4 . mrz Ž%. : 271 Ž62., 263 Ž33., 75 Ž75. , 73 Ž100.. CI-MS of the methyl ester Ž CH 4 . 2b mrz Ž%. : 295 Ž M-H 2 O q Hq,32., 263 Ž97., 245 Ž58., 199 Ž54., 179 Ž44., 167 Ž82., 141 Ž60., 139 Ž62., 121 Ž64. , 111 Ž 65. , 109 Ž 66. , 97 Ž 92. , 95 Ž 85. , 83 Ž100. . FABq-MS ŽGlycerol. mrz Ž %.: 281 ŽMH 2 O q Hq, 90., 280 Ž71., 279 Ž64., 265 Ž85., 263 ŽM-2H 2 O q Hq, 100.. Specific rotation of the acid: w a x 25 Ž . D s q41 c s 0.315, MeOH . Specific rotation 25 w x of the methyl ester 2b: a D s q101 Žc s 0.25, EtOH.. 3a: IR Žfilm. Õ 3300, 2929, 2855, 1711, 1464, 1288, 968 Ž trans alkene. cmy1. UV l s 231 nm ŽCH 3 CN.. 1 H NMR d 5.749 Ždt J s 15.6, JX s 6.9 Hz, 1H, CHsCHCHCOOH., 5.373 Žddt J s 15.6, JX s 6.9, JY s 1.5 Hz, 1H, CH s CHCOOH. , 4.264 Ždt J s 6.9, JX s 6.0 Hz, 1H, CHOOH., 2.352 Žt J s 7.5 Hz, 2H, CH2 CO 2 H., 2.080 Žm, 2H, CH2 C s C., 1.628 Žm, 4H, CH2 CH 2 COOH and CH2 CHOOH., 1.55–1.15 Ž c, 20H, 10CH 2 . , 0.876 Ž t J s 7.0 Hz, 3H, CH 3 .. 13 C NMR d 177.8 ŽCO., 136.78 ŽC-9., 128.82 ŽC-8., 87.05 ŽC-10., 33.53 ŽC-7., 32.43, 32.09 ŽC-16, C-2., 31.08 ŽC-11., 29.52–28.50 ŽC-13, C-14, C-15, C-4, C-5, C-6. , 25.34 ŽC-3. , 24.54 ŽC-12., 22.65 ŽC-17., 14.09 Ž CH 3 .. CI-MS of the methyl ester ŽCH 4 . 3b mrz Ž%.: 339 ŽMqq 29-H 2 O, 20., 311 ŽMqq 1-H 2 O, 32., 309 ŽMq-1-H 2 O, 23., 279 Ž Mqq 1-H 2 O-CH 3 OH, 47., 183 Ž55., 141 Ž100.. FABq-MS ŽGlycerol. mrz Ž %.: 297 ŽM-H 2 O q Hq, 99., 281 Ž69., 279 ŽM-2H 2 O q Hq, 48., 263 Ž41.. Specific Ž . rotation: w a x 25 D s q6.81 c s 0.136, MeOH . 4a: IR ŽKBr. Õ 3407, 2927, 2851, 1696, 960 Ž trans alkene. cm y 1. 1 H NMR d 5.68 Žm, 2H, CHsCH., 4.12 Žm, 2H, 2CHOH., 2.36 Žt, J s 6.0 Hz, 2H, CH 2 CO 2 H., 1.66 Žm, 4H, 2CH2 CHOH., 1.53 Žm, 2H, CH2 CH 2 CO 2 H. , 1.45–1.15 Ž c, 16H, 8CH 2 ., 0.88 Žt, J s 6.0 Hz, 3H, CH 3 .. 13 C NMR d 177.5 ŽCO., 133.81, 133.55 ŽC-8, C-9., 72.41, 72.27 ŽC-7, C-10. , 37.24, 36.86 Ž C-6, C-11. , 33.47, 31.85 ŽC-12, C-16. , 29.53–28.84 Ž C-13, C-14, C-15, C-4. , 25.41–24.51 ŽC-12, C-3, C-5., 22.65 ŽC-17., 14.10 ŽCH 3 .. EI-MS of the TMS methyl ester 4b mrz Ž%.: 343 Ž10., 269 Ž 14., 253 Ž19., 231 Ž7., 215 Ž5., 155 Ž9. , 147 Ž14., 119 Ž 10. , 75 Ž 16. , 73 Ž 100. . CI-MS of the methyl ester 4b ŽCH 4 . mrz Ž%.: 311 Ž17., 293 Ž15., 279 Ž37., 261 Ž21., 233 Ž13., 183 Ž14., 157 Ž36., 141 Ž100.. FABq-MS ŽGlycerol. mrz Ž%. : 297 ŽM-H 2 O q Hq, 61., 279 ŽM-2H 2 O q Hq, 100.. SpeŽ . cific rotation: w a x 25 D s q13.31 c s 0.103, MeOH . 2.8. Reduction of 3a with NaBH4 A similar procedure to that described by Bartolotleazzi and Pizzale w19x was applied. Thus, compound 3a Ž50 mg. was freed from solvent, taken up in methanol Ž1 ml. and treated with a catalytic amount of NaBH 4 . When evolution of hydrogen ceased Žca. 30 min., the reaction mixture was acidified with 2–3 drops of 6 M HCl and concentrated under a gentle stream of nitrogen. Water Ž0.5 ml. was then added, extracted with diethyl ether Ž3 = 0.5 ml. and dried ŽNa 2 SO4 .. After filtration, the solvent was evaporated to dryness and the residue taken up in anhydrous ether. The resulting hydroxy acid presented identical spectroscopic and chromatographic features Ž 1 H NMR, GC capillary column. than 2a. Mass spectrum of the TMS derivative of the methyl ester of the reduced compound was also identical to that of 2b, including the diagnostic peak for the position of the hydroxyl group at mrz 241. 3. Results and discussion 3.1. Biotransformation and isomerization of the substrate Biosynthesis of structural fatty acids by bacteria usually leads to cis monounsaturated fatty acids and only recently the presence of trans unsaturated fatty acids was reported w20x. The first occurrence of trans fatty acids in aerobic bacteria was described in 1978 and since then several other anaerobic and aerobic bacteria have been reported to contain this type of fatty acids as components of their membrane lipids w21x. By contrast, it has long been known that bacterial transformations of fatty acids often lead to trans isomers, which may be due to the presence of cistrans isomerases w22–25x. We have previously found that incubation of Pseudomonas sp. 42A2 in olive oil produced compound 4a in 20% yield w9x. Following our investigation on Pseudomonas sp. 42A2, we have now incubated the strain in oleic acid and isolated and charac- A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 terized two new trans unsaturated fatty acids, ŽE.10-hydroxy-8-octadecenoic acid Ž2a. and ŽE.-10-hydroperoxy-8-octadecenoic acid Ž3a. in addition to the previously reported 4a. 3.2. Time-dependent oleic acid transformation Time course of the biotransformation is shown in Fig. 2. After incubation of cells in oleic acid at 308C, aliquots were taken and esterified with diazomethane. GC analyses of the methyl esters showed four main peaks of retention times 11.57, 21.6, 25.6 and 32.6 min, which were characterized as unreacted methyl oleate Ž1b. and the methyl esters 2b, 3b and 4b, respectively ŽFig. 1.. The three bioconversion compounds appeared from the beginning of the bacterial growth and their amounts increased with time. Maximum accumulation of 3a Ž1.15 grl. was obtained at the end of the logarithmic phase of cell growth Ž16 h.. After bacterial growth ceased production of 2a and 4a increased up to a maximum production of 5.58 and 2.63 grl, respectively. 3.3. Isolation and identification of products Isolation of the main compounds was achieved without derivatization of the products by HPLC using an evaporative light-scattering detector. The high sensitivity of this detector is particularly useful for com- Fig. 2. Time course of cell growth and biotransformation of oleic acid by Pseudomonas sp. 42A2 incubated in liquid mineral medium. ^: Protein; =: Oleic acid Ž1a.; e: ŽE.-10-hydroxy-8octadecenoic acid Ž2a.; I: ŽE.-10-hydroperoxy-8-octadecenoic acid Ž3a.; v: ŽE.-7,10-dihydroxy-8-octadecenoic acid Ž4a.. 79 pounds devoid of chromophores and allowed us to detect and isolate the bioconversion products 2a-4a. The retention times of the compounds eluting from the column were: 4a: 10.09; 3a: 16.49; 2a: 17.33; 1a: 22.04 min. Structures of compounds 2a, 3a and 4a were established by 1 H NMR, 13 C NMR and mass spectral analyses. Trans stereochemistry of the isolated products was ascertained by the presence of a 960–968 cmy1 band in the IR spectra, as well as by the vinylic vicinal coupling constant JHa-Hb s 15.3–15.6 Hz in the doublet of doublet of triplets absorption of the olefinic protons at d 5.612 and 5.439 ppm in compound 2a and at 5.749 and 5.373 ppm in compound 3a. In acid 4a the absorption of the olefinic protons appeared as a multiplet at d 5.68 ppm. Other characteristic features of compound 2a are the doublet of triplets at d 4.035 corresponding to an allylic hydroxyl group in the 1 H NMR, as well as the fragments of mrz 271, 241 and 129 corresponding to wMe 3 SiOCHCH s CHŽ CH 2 . 6 CO 2 CH 3 xq, wCH s CHCH Ž OSiM e 3 .Ž CH 2 . 7 CH 3 x q and w CH 2 s CHCHOSiMe 3 xq, respectively, in the MS of the TMS derivative of the methyl ester 2b. Compound 2a is a fungitoxic chemical isolated from the timothy plant Epichloe typhina w26x and later characterized as one of the fatty alcohols arising from methyl oleate autoxidation w27x. In the case of compound 3a, particularly noticeable is the profound effect induced by the hydroperoxy group on the chemical shifts of the protons and carbons located in the proximity of the group. Thus, the olefinic protons in b and g positions resonate at 5.749 and 5.373 ppm Ž Dd .0.4., respectively, while the corresponding carbon signals appear at 136.78 and 128.82 ppm Ž Dd .8., respectively. Moreover, the adjacent proton to the hydroperoxy group is deshielded y0.23 ppm in comparison to that of the parent alcohol 2a, while the corresponding carbon shifts downfield by a remarkable 14 ppm. This is in agreement with the data reported for methyl hydroperoxy octadecenoates w14x. Position determination of the hydroperoxy group was carried out by the diagnostic ions at mrz 271 and 241 in the EI mass spectrum of the ions derivatives of the NaBHa reduction product. Likewise the CI-MS of 36 showed fragmentation ions at mrs 141 and 183 corresponding, respectively, to a McLafferty rearrangement and 80 A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 loss of methanol of the mrz 215, the a scission fragment to the hydroperoxy group. Moreover the NaBH 4 reduction compound of 3a displayed identical spectographic and chromatographic features Ž 1 H NMR, GC capillar column. to 2a. Compound 4a was readily identified by its spectroscopic data and localization of the hydroxy groups determined by GC-MS of the trimethylsilyl ether of a methylated sample 4b as previously reported w14x. Our results suggest that oxidation of oleic acid with our bacterial strain occurs through a different pathway to that proposed by Hou and Bagby w7x with Pseudomonas PR3. These authors postulate oxidation of oleic acid to cis-2a as the first step, followed by isomerization to trans and oxidation to compound 4a. In our case, we postulate that the first stage would be oxidation of the fatty acid at C-10 and cis-trans isomerization to produce hydroperoxide 3a. This oxidation may be due to the activity of a lipoxygenase type enzyme. Lipoxygenase-like enzymes have been reported by Shimahara et al. in Pseudomonas aeruginosa w28x and by Iny et al. in Thermoactinomyces Õulgaris w29x to produce hydroperoxides in linoleic, arachidonic acid methyl oleate and methyl linoleate. Compound 3a would be a ready precursor of alcohol trans-2a, which would finally oxidize at C-7 to give rise to compound 4a. The trans stereochemistry of the products isolated in our study would be originated, consequently, in one of the first stages of the conversion pathway. 3.4. Location of the enzyme(s) In order to determine whether the oleic acid conversion enzymeŽs. was cell-bound or extracellular, the strain 42a2 was grown for 24 h and cells separated by centrifugation at 6000 = g for 15 min at 48C. The supernatant was filtered with a microfiltration unit to remove any remaining cells and the extracellular proteins precipitated with 80% saturation of ŽNH 4 . 2 SO4 .aq. soln. These proteins, previously dialized against phosphate buffer Ž pH 7.5., were incubated with oleic acid in the mineral salt medium at 308C for 30 h. After extraction and derivatization, no products were found by GC analysis, suggesting that the biotransformation enzymeŽs. was cell-bound. To determine more precisely which subcellular fraction contained the enzymatic activity, the cell pellet was washed twice with phosphate buffer, resuspended in the minimum amount of the same buffer and fractionated with lysozyme, following the methods of Filip w30x, Dennis w31x and Nieto w32x. After subsequent centrifugation steps, five different fractions of the cell were isolated, i.e., outer membrane, proteins loosely bound to the outer membrane, periplasm, cytoplasmic membrane and cytoplasm Fig. 3. Gas chromatogram of the methyl derivatives of the products 1b: 11.57 min; 2b: 21.6 min and b4: 32.6 min, obtained from the enzymatic activity of the different cell fractions compared with a commercial lipoxygenase. Cytoplasm chromatogram was considered control. A. Guerrero et al.r Biochimica et Biophysica Acta 1347 (1997) 75–81 components. These extracts were separately incubated under the standard conditions, and the organic material extracted with chloroform. After esterification, methyl esters were analyzed by GC. The enzymatic acitivity of the biotransformation found in the periplasmic fraction was similar to that of a commercial lipoxygenase used as control, suggesting that we are dealing with a lipoxygenase-like enzymeŽs. ŽFig. 3.. No other fraction of the cell gave clues about the presence of the oxidation compounds, pointing out that the enzymeŽs. responsible for the biotransformation is located in the periplasmic space. In summary, we have shown that microbial oxidation of oleic acid with Pseudomonas sp. 42a2 yields ŽE.-10-hydroxy-8-octa-decenoic acid Ž2a., ŽE.-10-hydroperoxy-8-octadecenoic acid Ž3a., and ŽE.-7,10-dihydroxy-8-octadecenoic acid Ž4a.. The two former compounds are being reported to be originated from a Pseudomonas sp. cell culture for the first time. The enzyme responsible of the first step of the bioconversion has been found to be located in the periplasm. Studies are currently under way in this laboratory to assess the complete pathway and the isolation and characterization of the enzymeŽs. involved in the biotransformation. Acknowledgements We gratefully acknowledge CICYT ŽAMB 9-0691, AMB 96-1429, PB 93-0185 and AGF 95-0185. for financial support and MEC for a predoctoral fellowship to Y.L. 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