Vol. 51, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1986, p. 532-5380099-2240/86/030532-07$02.00/0

Biotransformation of Unsaturated Long-Chain Fatty Acids byEubacterium lentum

A. VERHULST,* G. PARMENTIER, G. JANSSEN, S. ASSELBERGHS, AND H. EYSSEN

Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Louvain, Belgium

Received 27 September 1985/Accepted 17 Decemnber 1985

Eubacterium lentum (33 strains) isomerized the 12-cis double bond of C18 fatty acids with cis double bondsat C-9 and C-12 into an 11-trans double bond before reduction of the 9-cis double bond. The 14-cis double bondof homo-y-linolenic acid was isomerized by 29 strains into a 13-trans double bond. The same strains isomerizedthe 14-cis double bond of arachidonic acid into a 13-trans double bond and then isomerized the 8-cis doublebond into a 7-trans double bond; the 13-cis double bond of 10-cis,13-cis-nonadecadienoic acid was isomerizedinto a 12-trans double bond. None of these isomerization products was further reduced. Studies with restingcells showed optimal isomerization velocity at a linoleic acid concentration of 373S ,uM; higher concentrationswere Inhibitory. The pH optimum for isomerization was 7.5 to 8.5. The isomerase was inhibited by thesulfhydryl reagents iodoacetamide, bromoacetate, and N-ethylmaleimide and by the chelators EDTA and1,10-phenanthroline.

The intestinal microflora of the rat is capable of hydroge-nating unsaturated long-chain fatty acids (1). In earlierstudies (3), Eubacterium lentum Rega was isolated fromconventional rat feces, and it was shown that this strainhydrogcnated linoleic acid (18:2; 9-cis, 12-cis) into trans-vaccenic acid (18:1; 11-trans). More recently, 32 other E.lentum strains, which, based on their bile acid transforma-tions, were divided into five groups (A, B, C, D, and E),were investigated, and it was shown that hydrogenation oflinoleic acid into trans-vaccenic acid via a 9,11-octadecad-ienoic acid was performed by all the strains tested (4).Hence, this transformation is a new characteristic of E.lentum. In the present investigations we determined themechanism of this reaction in more detail, analyzing 33 E.lentum strains for their ability to transform polyunsaturatedfatty acids which are structurally related to linoleic acid.Furthermore, we studied the growth-inhibiting effect ofsome of these long-chain fatty acids on E. lentum.

MATERIALS AND METHODSBacteria, media, and incubation techniques. All strains were

identified and maintained on medium T+ as describedpreviously (4). Medium L+ was T+ plus 7 mg of freeze-driedbeef brain per ml. Medium BHI+ was brain heart infusionbroth (BBL Microbiology Systems, Cockeysville, Md.)supplemented with 0.5% yeast extract (Difco Laboratories,Detroit, Mich.), 0.0005% hemin (Nutritional BiochemicalsCorp., Cleveland, Ohio), 0.0001% vitamin K1 (Konakion;Roche, Basel, Switzerland), 0.05% cysteine (E. Merck AG,Darmstadt, Federal Republic of Germany), and 0.25%arginine (Merck). Inoculations and incubations were carriedout at 37°C in an anaerobic glove box under 90%nitrogen-10% hydrogen as described elsewhere (4).

Substrates and reagents. All substrates and reagents wereof the highest purity. Margaric acid (17:0), trans-vaccenicacid (11-trans-octadecenoic acid), cis-vaccenic acid (11-cis-octadecenoic acid), oleic acid (9-cis-octadecenoic acid),

* Corresponding author.

elaidic acid (9-trans-octadecenoic acid), linoleic acid (9-cis,12-cis-octadecadienoic acid), linolenic acid (9-cis,12-cis,15-cis-octadecatrienoic acid), -y-linolenic acid (6-cis,9-cis,12-cis-octadecatrienoic acid), methyl linoleate, ethyllinoleate, linoleyl alcohol (9-cis,12-cis-octadecadien-1-ol),linoleyl acetate, linolelaidic acid (9-trans,12-trans-octadecadienoic acid), trilinolein (triglyceride of linoleicacid), and arachidonic acid (5-cis,8-cis,11-cis,14-cis-eicosatetraenoic acid) were from Sigma Chemical Co. (St.Louis, Mo.) or Serva (Heidelberg, Federal Republic ofGermany). 11-cis,14-cis-eicosadienoic acid, homo-y-linolenic acid (8-cis,11-cis,14-cis-eicosatrienoic acid), and10-cis,13-cis-nonadecadienoic acid were from Nu CheckPrep (Elysian, Minn.). J3ovine serum albumin was fromPoviet (Amsterdam, The Netherlands). Chemical reductionof flavin mononucleotide and flavin adenine dinucleotide wasperformed as described by Feighner and Hylemon (5) bymixing, in 0.05 M potassium phosphate buffer (pH 6.2),equal volumes of anaerobic stock solutions of Na2S204 (8mM) and flavin adenine dinucleotide (2 mM) or flavinmononucleotide (2 mM). Reduction of the flavines wasconsidered complete after decoloration of the reaction mix-ture.

Biotransformation of unsaturated long-chain fatty acids bygrowing cultures. Biotransformation was tested by incubat-ing cultures in 7 ml of medium L+ containing 150 ,ug of thesubstrate tested per ml and 7 mg of freeze-dried beef brainper ml to reduce the toxicity of the fatty acids. Afterincubation, the cultures were acidified with 2 N HCI to pH 2.To determine the amount of bacterially transformed fattyacids, linoleyl alcohol, or linoleyl esters, 1 ml of acidifiedculture medium was diluted with 1 ml of water and 1 ml ofmethanol. This sample was mixed with 50 ,ig of margaricacid (17:0) as an internal standard, and the fatty acids or fattyacid derivatives were extracted twice with diethyl ether.After the ether extract was evaporated to dryness, fattyacids were esterified with diazomethane and analyzed bygas-liquid chromatography (GLC) on a column (1.52 m by 4mm [inside diameter]) packed with 15% Hi Eff 2BP at 166°C.

Biohydrogenation of linoleic acid by resting cells. E. lentum

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TABLE 1. Time course of the biohydrogenation of linoleic acid, linolenic acid, and y-linolenic acid by growing cultures ofE. lentum Rega

% Indicated transformation product of:

Linoleic acid Linolenic acid y-Linolenic acidTime(h) 18:2 (9-cis, 18:2 (9-cis, 18:1 C18:3 (9-cis, 18:3 (9-cis. 18:2 (11-trans. yl8:3 18:3 (6-cis, 18:2 (6-cis

12-cis) 11-trans) (11-trans) 11-trans, 12-cis) 15-cis) (6-1s9,9-), 11-rans) 11-trans)

0 100 0 0 100 0 0 100 0 06 64 36 0 64 36 0 93 7 012 45 55 0 47 53 0 85 15 024 2 26 72 7 19 74 25 73 248 3 9 88 0 6 94 0 42 5872 5 7 88 0 3 97 0 16 84120 0 8 92 0 5 95 0 8 92

Rega cells harvested by centrifugation of an early-exponentional-phase culture on medium BHI+ were washedtwice in potassium phosphate buffer (pH 7, 0.1 M) andresuspended in the same solution to a turbidity between 0.4and 0.6 at 620 nm and a protein concentration between 400and 600 ,ug/ml. Turbidity was measured with a Bausch andLomb Spectronic 20 photometer without use of a specialfilter. The path length of the tube was 10 mm. The proteinconcentration of the cell suspensions was determined by themethod of Lowry et al. (12). Broken-cell suspensions wereprepared by anaerobic sonication of a twice-frozen-and-thawed washed-cell suspension in a Heat System-Ultrasonics sonicator model W-225R. The broken-cell sus-pension was then diluted in 0.1 M potassium phosphatebuffer (pH 7) to a turbidity between 0.08 and 0.12 at 620 nmand a protein concentration between 400 and 600 ,ug/ml ofcell suspension. All steps of this procedure were carried outin an anaerobic glove box, except the sonication and cen-trifugation, which were carried out in airtight polypropylenetubes. All buffers used were steam sterilized and prereducedfor at least 5 days before use. Reaction mixtures (4 ml, finalvolume) were prepared by adding the cell suspension to testtubes containing 10 ,ul of a solution of linoleic acid inmethanol and other reagents in known concentrations. Sam-ples were mixed thoroughly and incubated at 37°C. Afterincubation, the reaction was stopped by addition of HCI topH 2, and fatty acids were analyzed by GLC.

Isolation and identification of the reaction products. Reac-tion products were isolated by preparative thin-layer chro-matography (TLC) of the methyl esters on silica gel impreg-nated with silver nitrate. Benzene-diethyl ether (99:1) wasused as the solvent system to analyze the reaction productsof diunsaturated fatty acids; reaction products of unsatur-ated fatty acids with more double bonds were chromato-graphed in benzene-ethylacetate (90:10). Elution was per-formed with diethyl ether. Identification of these compoundsand subsequent location of the double bond(s) was achievedby mass spectrometry as described elsewhere (6, 7). Todetermine the geometry of the double bonds, the polyunsat-urated fatty acid methyl esters were hydrolyzed to the freefatty acids and subjected to partial reduction with hydrazinein the presence of oxygen (13). The geometry of the resultingoctadecenoic acids in the reduced reaction mixture wasdetermined by comparison of their methyl ester derivativeswith reference compounds on AgNO3 TLC. Afterwards, thechromatographed octadecenoic acids were isolated andagain subjected to mass spectrometry to confirm the positionof the double bonds.

RESULTS

Biohydrogenation of linoleic acid, linolenic acid, and -y-linolenic acid. After 12 h of incubation of E. lentum Rega inmedium L+, ca. 55% of the linoleic acid (tR, 2.16) wasisomerized into an octadecadienoic acid with a tR of 3.00 onGLC. Mass spectrometry showed that the intermediate wasa 9,11-octadecadienoic acid. After 24 h of incubation, 72% ofthe substrate was hydrogenated into trans-vaccenic acid (tR,1.52), and this increased to 92% after 120 h of incubation(Table 1). Partial reduction of the double bonds of theintermediate followed by separation on AgNO3 TLC yieldeda trans-octadecenoic acid (Rf, 0.78) and a cis-octadecenoicacid (Rf, 0.59). These fatty acids were shown by massspectrometry to be 11-trans-octadecenoic acid and 9-cis-octadecenoic acid.

Linolenic acid (tR, 2.64) was transformed by E. lentumRega at a rate similar to that of linoleic acid. After 12 h ofincubation, 53% was transformed into an intermediate acid(R, 3.44); after 24 h, 74% of linolenic acid was transformedinto an end product (OR, 2.00) which amounted to about 95%after 120 h (Table 1). Mass spectrometry showed that theintermediate was a 9,11,15-octadecatrienoic acid and thatthe end product was an 11,15-octadecadienoic acid. Partialreduction of the double bonds of the intermediate, followedby separation on TLC, yielded cis-octadecenoic acid(s) (Rf,0.59) and trans-octadecenoic acid(s) (Rf, 0.78) as 18:1 fattyacids. These fatty acids were defined by mass spectrometryas 9-cis-octadecenoic acid, 15-cis-octadecenoic acid, and11-trans-octadecenoic acid. Hence, the intermediate pro-duced from linolenic acid was 9-cis,1l-trans,15-cis-octadecatrienoic acid. Partial reduction of the double bondsin the end product, followed by separation on TLC, showeda cis-octadecenoic acid (Rf, 0.60) and a trans-octadecenoicacid (Rf, 0.78). Mass spectrometry showed that these 18:1fatty acids were 15-cis-octadecenoic acid and 11-trans-octadecenoic acid. The end product of linolenic acid wasthus 11-trans,15-cis-octadecadienoic acid.

y-Linolenic acid was transformed more slowly thanlinoleic acid or linolenic acid (Table 1). After 12 h ofincubation, only 15% of y-linolenic acid (tR, 2.36) wastransformed into an intermediate acid (tR, 3.20), and after 24h, only 2% was converted into the end product (tR, 1.76).However, after 120 h, 92% of y-linolenic acid was convertedinto the end product. Mass spectrometry showed that theintermediate was a 6,9,11-octadecatrienoic acid and that theend product was a 6,11-octadecadienoic acid. Partial reduc-tion of the double bonds of the intermediate, followed by

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TABLE 2. Transformation of unsaturated long-chain fatty acids by growing E. lentum cultures"No. of

Substrate Type of transformation (%) positivestrains

Linoleic acid (18:2; 9-cis, 12-cis) A12cis,Al 'trans isomerization + .9cis hydrogenation (>70) 33Linolenic acid (18:3; 9-cis, 12-cis, 15-cis) A12cis,1l'trans isomerization + A9cis hydrogenation (>70) 33-y-Linolenic acid (18:3; 6-cis, 9-cis, 12-cis) A2cis,Alltrans isomerization + A9cis hydrogenation (>70) 33Methyl linoleate None 0Ethyl linoleate None 0Linoleyl alcohol None 0Linoleyl acetate None 0Trilinolein None 0Linolelaidic acid (18:2; 9-trans, 12-trans) None 0Homo-y-linolenic acid (20:3; 8-cis, 11-cis, 14-cis) A'4cis,A13trans isomerization (>50) 29Arachidonic acid (20:4; 5-cis, 8-cis, 11-cis, 14-cis) M14cis,A13trans isomerization (>50); A8cis,,A7trans 29

isomerization (>50)Eicosadienoic acid (20:2; 11-cis, 14-cis) None 010-cis,13-cis-Nonadecadienoic acid A13cis,,A2trans isomerization (>50) 29Oleic acid (18:1; 9-cis) None 09-trans,ll-trans-Octadecadienoic acid None 0

a A total of 33 E. lentum strains were tested. Group A, strains 9104A, 8135A, 3162P, 9130, 8662C, 6701K2, 6598A, 6718E, and 8902; group B, strains ATCC25559, 3995, 3999, 8330TA, 5438, 8371B, 8123, 8114, 11450C, 7725, 6266C, and 9758; group C, strains 3197 and 9066; group D, strains 3060D, 9262, 9391B, 7616B,11602, and 9489B; group E, strains Rl, 7E, Rega, and 116. Strains of groups A, B, C, and D and strain 116 were kindly provided by I. A. Macdonald (DalhousieUniversity, Halifax, Nova Scotia, Canada) and L. V. Holdeman (Virginia Polytechnic Institute and State University, Blacksburg). Strains Rl, 7E, and Rega wereisolated at the Rega Institute from conventional rat feces.

separation on TLC, yielded cis-octadecenoic acid(s) (Rf,0.59) and trans-octadecenoic acid(s) (Rf, 0.78). Mass spec-trometry identified the three 18:1 fatty acids as 6-cis-octadecenoic acid, 9-cis-octadecenoic acid, and 11-trans-octadecenoic acid. The intermediate fatty acid was thus6-cis,9-cis,11-trans-octadecatrienoic acid. Partial reductionof the double bonds of the end product of -y-linolenic acid,followed by separation on TLC, yielded a cis-octadecenoicacid (Rf, 0.59) and a trans-octadecenoic acid (Rf, 0.78). Massspectrometry showed that these C18:1 fatty acids were 6-cis-octadecenoic acid and 11-trans-octadecenoic acid. Hence,the end product of the biohydrogenation of -y-linolenic acidwas 6-cis,11-trans-octadecenoic acid. From these results weconclude that E. lentum Rega hydrogenates linolenic acidand y-linolenic acid via the same pathway as linolenic acid:first an isomerization of the 12-cis double bond into an11-trans double bond and then reduction of the 9-cis doublebond.

After 1 week of incubation in medium L+ containing 150,ug of the fatty acid tested per ml, all strains converted morethan 70% of linolenic acid and -y-linolenic acid intooctadecadienoic acids with the same tRS as the respectiveend products produced by E. lentum Rega (group E) (Table2). Similarly, all strains performed these biohydrogenationreactions via intermediates with the same tRS as the respec-tive ones produced by E. lentum Rega. Analysis by TLC andmass spectrometry of the reaction products of linolenic acidand y-linolenic acid produced by one representative strain ofeach of the four other groups of E. lentum (strain 9104A fromgroup A, strain ATCC 25559 from group B, strain 3197 fromgroup C, and strain 7616B from group D) showed the samereaction products as those produced by E. lentum Rega.These results strongly suggest that the strains of the fivegroups hydrogenate linolenic acid and y-linolenic acid in thesame way as linoleic acid. Methyl linoleate, ethyl linoleate,linoleyl alcohol, linoleyl acetate, trilinolein, linolelaidic acid,9-trans,11-trans-octadecadienoic acid, and oleic acid werenot transformed by any of the 33 strains.

Isomerization of homo-y-linolenic acid, 10-cis, 13-cis-nonadecadienoic acid, and arachidonic acid. All strains were

incubated for 1 week in medium L' containing 150 p.g of thesubstrate tested per ml (Table 2). Homo--y-linolenic acid,arachidonic acid, and 10-cis-,13-cis-nonadecadienoic acidwere transformed by 29 strains (groups A, B, C, and D).These fatty acids were unaffected by the strains of group E.After 1 week of incubation the 29 positive strains trans-formed more than 50% of homo--y-linolenic acid (tR, 4.85; Rf,0.38) into a fatty acid with a tR of 5.90 on GLC and an Rf of0.70 on TLC. Mass spectrometry further showed that theisomerization product of the four type strains was an8,11,13-eicosatrienoic acid. Partial reduction of the doublebonds of the conjugated eicosatrienoic acid, followed byseparation on TLC, showed cis-eicosadecenoic acid(s) (Rf,0.57 and 0.65) and trans-eicosadecenoic acid(s) (Rf, 0.80).The eicosadecenoic acids were determined by mass spec-trometry as 8-cis-, 11-cis-, and 13-trans-eicosadecenoic acid,respectively. We conclude that the 29 strains transformhomo--y-linolenic acid into 8-cis,11-cis,13-trans-eicosatrienoic acid. The isomer produced was not furtherreduced.

Arachidonic acid (tR, 5.45; Rf, 0.12) was first convertedinto a fatty acid with a tR of 7.63 on GLC and an Rf of 0.34on TLC. Mass spectrometry showed that the transformationproduct of the four type strains was a 5,8,11,13-eicosatetraenoic acid. Partial reduction of the double bondsof the conjugated eicosatetraenoic acid, followed by separa-tion on TLC, showed cis-eicosadecenoic acid(s) (Rf, 0.57and 0.70) and trans-eicosadecenoic acid(s) (Rf, 0.87). Massspectrometry showed that the eicosadecenoic acids were5-cis-, 8-cis-, 11-cis-, and 13-trans-eicosadecenoic acid, re-spectively. The isomer was thus 5-cis,8-cis,11-cis,13-trans-eicosatetraenoic acid. Prolonged incubation resulted in afurther transformation of this product into a fatty acid with atR of 13.91 and an Rf of 0.66. Mass spectrometry showed thatthis fatty acid was a 5,7,11,13-eicosatetraenoic acid. Partialreduction of the double bonds of this isomer, followed byseparation on TLC, showed cis-eicosadecenoic acids (Rf,0.57 and 0.70) and trans-eicosadecenoic acids (Rf 0.77 and0.87). Mass spectrometry showed that the eicosadecenoicacids were 5-cis-, 11-cis-, 7-trans-, and 13-transeico-

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8.0-

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0 18 36 54 72 144 360 pM

ConcentrationFIG. 1. Influence of fatty acids on the growth of E. lentum Rega. After 72 h of incubation on medium T+ containing the fatty acid tested,

the pH of the cultures was measured as an indication of growth. Values are the means of four determinations. *, Cultures containingtrilinolein (cultures with stearic acid, elaidic acid, methyl linoleate, or ethyl linoleate showed a similar inhibition curve), O, cultures containingoleic acid (cultures with linolelaidic acid showed a similar inhibition curve); 0, cultures containing 9-cis-11-trans-octadecadienoic acid; 0,cultures containing linoleic acid (cultures with linolenic acid or y-linolenic acid showed a similar curve), x, cultures containing arachidonicacid; -----, uninoculated culture medium.

sadecenoic acid, respectively. Hence, the 29 strains isomer-ized the 14-cis double bond of arachidonic acid into a13-trans double bond and then isomerized the 8-cis doublebond into a 7-trans double bond. The conjugated fatty acidwas not further hydrogenated.

After 1 week of incubation more than 50% of 10-cis,13-cis-nonadecadienoic acid (tR, 2.88; Rf, 0.50) was isomerizedby the 29 strains into a fatty acid with a tR of 3.37 and an Rfof 0.76. Mass spectrometry showed that 10-cis,13-cis-nonadecadienoic acid was isomerized into a conjugated10,12-nonadecadienoic acid. Partial reduction of the doublebonds of the conjugated nonadecadienoic acid, followed byseparation of the reaction products on TLC, showed thepresence of a cis-nonadecenoic acid (Rf, 0.68) and a trans-nonadecenoic acid (Rf, 0.82). These acids were defined bymass spectrometry as 10-cis-nonadecadecenoic acid and12-trans-nonadecenoic acid. Hence, the 29 strains trans-formed 10-cis,13-cis-nonadecadienoic acid into 10-cis,12-trans-nonadecadienoic acid. This isomer was reduced at arate of 0 to 5% into a 12-trans-nonadecadienoic acid.

Inhibition of growth ofE. Ientum Rega by certain long-chainfatty acids and their derivatives. The influence of long-chainfatty acids on the growth of E. lentum Rega in medium T+ isshown in Fig. 1. Since these culture media were not opticallyclear and since E. lentum alkalizes culture media containingarginine, the pH of the culture medium after 72 h of

incubation can be used to measure growth (4). Linoleic acid,linolenic acid, and -y-linolenic acid inhibited the growth of E.lentum Rega in a similar way: inhibition started at concen-trations higher than 18 ,uM and was complete at a concen-tration of 144 ,uM. Arachidonic acid was even more inhibi-

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FIG. 2. Relationship of substrate concentration to isomeraseactivity. Test tubes contained 4 ml of whole-cell suspension in 0.1 Mpotassium phosphate buffer (pH 7) and linoleic acid in the indicatedconcentrations.

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BFIG. 3. Effect of pH on linoleic acid 12-cis,11-trans isomerization. The reaction was started by adding 4 ml of whole-cell suspension

prepared in 0.1 M phosphate buffer (0) or in 0.1 M glycine-NaOH buffer (O) to a test tube containing linoleic acid in a final concentrationof 25 puM. Isomerization was tested in a buffer system without (A) or with (B) 0.003% Triton X-100.

tory and completely suppressed growth at a concentration of72 p.M. The 9-cis,11-trans-octadecadienoic acid had an in-hibitory effect between that of the c is-polyunsaturated fattyacids and that of oleic acid and linolelaidic acid, which were

only weakly inhibitory. Trilinolein, ethyl linoleate, methyllinoleate, stearic acid, and elaidic acid had virtually no

influence on bacterial growth in concentrations up to 360p.M.Relationship of linoleic acid concentration to isomerase

activity of resting cells. The isomerase activity of whole cellsin 0.1 M phosphate buffer (pH 7) increased with risingsubstrate concentrations: half-maximal velocity was reachedat a substrate concentration of 15.5 puM, and the isomeraseactivity was optimal at a concentration of 37.5 p.M linoleicacid; at this concentration the isomerase transformed 1.90.5 nmol of linoleic acid acid per min per mg of protein (Fig.2). Higher substrate concentrations were inhibitory. At a

concentration of 50 p.M linoleic acid, the isomerase activitydropped to 1.5 + 0.3 nmol/min per mg of protein; at a

concentration of 75 p.M linoleic acid, only 1 ± 0.3 nmol oflinoleic acid per min per mg of protein was isomerized. Afteraddition of 0.003% Triton X-100 to the buffer system, thesame relationship between substrate concentration andisomerase activity was found.pH optimum. The optimal pH of the isomerase activity of

whole-cell suspensions was determined in 0.1 M phosphatebuffer or in glycine-NaOH buffer with and without 0.003%Triton X-100. Optimal isomerization in a phosphate buffersystem without Triton X-100 occurred between pH 8 and 8.5(Fig. 3A). At pH 8.5 the preparation isomerized 1.5 ± 0.4nmol of linoleic acid per min per mg of protein in thephosphate buffer system and 2 ± 0.4 nmol/min per mg ofprotein in the glycine-NaOH buffer. Half-maximalisomerization was observed at pH 7 and at pH 9.2. When0.003% Triton X-100 was added to the buffer system (Fig.3B), the pH optimum was situated at pH 7.5; at this pH theisomerase transformed 1.6 + 0.4 nmol of linoleic acid per

min per mg of protein. Figure 3 also shows that addition of0.003% Triton X-100 to the test system resulted in a higherisomerization potential at lower pH values.

Cofactor requirements. We also tested the effect on the

isomerase activity of ATP, NAD+, NADH2, NADP+, coen-

zyme A, flavin mononucleotide, reduced flavin mono-

nucleotide, flavin adenine dinucleotide, reduced flavin ade-nine dinucleotide. FeSO4 plus glutathione, and Fe2SO4 plus

cysteine in concentrations of 0.1 mM. Glutathione andcysteine were tested in concentrations of 0.025 mM, 0.05mM, and 0.1 mM. Besides the usual control without addedcofactor(s), the effect of 0.4 mM Na2S204 was also deter-mined. A broken-cell suspension (3.6 ml) was added to tubescontaining 400 pul of a 1 mM solution of the tested cofactor(s)in 0.05 M potassium phosphate buffer (pH 6.2) and linoleicacid in a final concentration of 25 puM. After incubation at37°C, the reaction was stopped by addition of HCl to a pH of<2. It was found that the reaction mixtures with cofactor(s),as well as the controls without added cofactor(s), isomerized1.20 to 1.40 nmol of linoleic acid per min per mg of protein.Thus, none of the tested agents or combinations of agentssignificantly influenced the isomerase activity.

Inhibition by sulfhydryl reagents and by metal chelators.The effect of sulfhydryl reagents and metal chelators was

tested by adding 3.6 ml of broken-cell suspension in 0.1 Mphosphate buffer (pH 7) to test tubes containing 400 pL. of a

solution of the sulfhydryl reagent or metal chelator in a

known concentration and 25 p.M linoleic acid. After incuba-tion at 370C, the reaction was stopped by addition of HCl toa pH of <2. It appeared that the isomerase was stronglyinhibited by sulfhydryl reagents; 0.1 mM iodoacetamidecaused 25% inhibition of the isomerase activity, whereasbromoacetate and N-ethylmaleimide in the same concentra-tions caused 50 and 100% inhibition, respectively. In a

concentration of 10 mM, the metal chelators EDTA and1,10-phenanthroline caused 40 and 52% inhibition, respec-tively.

DISCUSSIONAll 33 strains of E. lentlim were shown to hydrogenate

linolenic acid and -y-linolenic acid via the same pathway as

linoleic acid. First the 12-cis double bond was isomerizedinto an 11-trans-double bond, and then the 9-cis double bondwas reduced. Since none of the 33 strains tested transformedlinolelaidic acid, trilinolein, linoleyl alcohol, linoleyl acetate,

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BIOTRANSFORMATION OF FATTY ACIDS BY E. LENTUM

or the methyl or ethyl ester of linoleic acid, we suggest thattwo cis double bonds and a free carboxyl group are neces-sary for isomerization at C-12 and reduction at C-9. -y-Linolenic acid was isomerized more slowly than linoleic acidand linolenic acid; the 6-cis double bond of -y-linolenic acidseems to inhibit the isomerization reaction. Arachidonicacid, homo--y-linolenic acid, and 10-cis,13-cis-nonadec-adienoic acid were isomerized by 29 of the 33 strains. Theseresults show that for most of the E. lentum strains the two cisdouble bonds did not have to be strictly located at C-9 andC-12; the double bonds may also be located farther from orcloser to the carboxyl group, as long as they are separated by5 or 11 carbon atoms from the terminal methyl group.Because the 14-cis double bond of 11-cis,14-cis-

eicosadienoic acid and the 8-cis double bond of homo--y-linolenic acid are not isomerized, a double bond between thecarboxyl group and the double bond to be isomerized seemsto be essential for biotransformation. Most probably, thisdouble bond, just like the carboxyl group, interacts with theenzyme in binding the substrate correctly. Except for 9-cis ,11-trans-octadecadienoic acid, 9-cis,1 1-trans-,15-cis-octadecatrienoic acid, and 6-cis,9-cis ,11-trans-octadec-atrienoic acid, none of the substrates was reduced at the9-cis position. Hence, a conjugated double bond system anda 9-cis configuration are necessary for reduction of thisdouble bond.

In an earlier study (4) we classified the 33 strains into fivegroups (A, B, C, D, and E) and distinguished, using bile acidtransformations as the most important marker, steroid-active strains (groups A, B,C, and E) and steroid-inactivestrains (group D). We have now shown that isomerization offatty acids with double bonds located at C-11 and C-14 or atC-10 and C-13 is also characteristic of certain of these groupsof E. lentum, the strains of groups A, B, C, and D arecapable of isomerizing these fatty acids, while group Estrains are not.

In his study of cis,trans isomerizations, Seltzer (14) dis-tinguished two classes of enzymatic cis,trans isomerases: (i)the isomerases which catalyze cis,trans isomerization with-out double bond migration and (ii) the cis,trans isomeraseswhich catalyze a geometrical isomerization with migration ofthe double bonds. Until now, all the isomerases of the firstclass appeared to require one or more functional sulfhydrylgroups for enzymatic activity, and in most cases this sulfhy-dryl group is provided by glutathione. The linoleic acidisomerase of E. lentum is a member of the second group.Although our results with sulfhydryl reagents suggest thatthe linoleic acid isomerase of E. lentum contains a functionalsulfhydryl group, stimulation by agents containing functionalsulfhydryl groups, such as glutathione and cysteine, was notobserved. Seltzer (14) further suggested that iron(II),complexed with two enzyme-bound thiolate groups, mighthave properties similar to iron(O) complexes, which areknown to catalyze the isomerization of nonconjugateddienes. In our studies, inhibition of the linoleic acidisomerase of E. lentum by 10 mM EDTA and 10 mM1,10-phenanthroline was observed, but stimulation of theisomerase activity by 0.1 mM FeSO4 or by FeSO4 in com-bination with glutathione or cysteine never occurred. Thelinoleic acid isomerase of Butyrivibriofibrisolvens, a rumenbacterium which hydrogenates linoleic acid into trans-vaccenic acid via the same pathway as E. lentum, is inhibitedby the same sulfhydryl reagents and metal chelators as E.lentum but the effects in this bacterium of agents containingfunctional sulfhydryl groups, either free or complexed withmetal ions, were not studied (11). It was further observed

that the isomerase activity of E. lentutm is inhibited bylinoleic acid concentrations higher than 37.5 ,uM, while theisomerase activity of B. fibrisolvens is inhibited by linoleicacid concentrations higher than 50 ,uM (10). Hence, thereseems to be a similarity between the isomerases of these twospecies.We further observed that cis-unsaturated fatty acids inhib-

ited growth of E. lentum. The inhibition by cis-polyunsat-urated fatty acids was much stronger than the inhibition bycis-monounsaturated fatty acids. trans-Polyunsaturatedfatty acids seemed to have only a slightly inhibitory effect.trans-Monounsaturated fatty acids, cis-polyunsaturatedfatty acid esters, or saturated fatty acids had no noticeablegrowth-inhibiting effect. We also observed the phenomenonof toxicity of cis-polyunsaturated fatty acids with severalother anaerobic intestinal bacteria, including both hydroge-nating and nonhydrogenating bacteria (A. Verhulst et al.,unpublished data). Kemp and Lander (8, 9) suggested thatmicrobial hydrogenation of polyunsaturated fatty acids has asurvival value in the rumen by removing potentially toxicfatty acids. Dawson and Kemp (2) showed that linoleic andlinolenic acids which are more bacteriostatic than oleic acidfor rumen organisms are indeed hydrogenated at a faster ratethan oleic acid. Although our results did not allow us torelate biohydrogenation rates with toxicity of different typesof long-chain fatty acids, we found that trans fatty acidswere not toxic until they reached a concentration of 360 ,uM.One might speculate that the high amount of trans monoenesin rumen contents and feces of animals and humans can bepartly explained by microbial hydrogenation of polyunsatu-rated fatty acids into nontoxic trans fatty acids.

ACKNOWLEDGMENT

We thank G. Vandenbulcke for technical assistance.

LITERATURE CITED1. Coates, M. E. 1984. Fatty acids, p. 285-289. In M. E. Coates and

B. E. Gustaffson (ed.), The germ-free animal in biomedialresearch. Laboratory Animals, Ltd., London.

2. Dawson, R. M. C., and P. Kemp. 1970. Biohydrogenation ofdietary fats in ruminants, p. 504-518. In A. T. Phillipson (ed.),Physiology of digestion and metabolism in the ruminant. OrielPress, Newcastle upon Tyne, England.

3. Eyssen, H. J., and G. G. Parmentier. 1979. Influence of themicroflora of the rat on the metabolism of fatty acids, sterolsand bile salts in the intestinal tract. Zentralbl. Bakteriol.Parasitenkd. Infektionskr. Hyg. Abt. 1 Suppl. 7:39-44.

4. Eyssen, H., and A. Verhulst. 1984. Biotransformation of linoleicacid and bile acids by Eubacterium lentum. Appl. Environ.Microbiol. 47:39-43.

5. Feighner, S. D., and P. B. Hylemon. 1980. Characterization of a

corticoid 21-dehydroxylase from the intestinal anaerobic bacte-rium, Eubacterium lentm. J. Lipid Res. 21:585-592.

6. Janssen, G., and G. Parmentier. 1978. Determination of doublebonds in fatty acids with conjugated double bonds. Biomed.Mass. Spectrom. 5:439-443.

7. Janssen, G., G. Parmentier, A. Verhulst, and H. Eyssen. 1984.Location of the double bond positions in microbial isomeriza-tion and hydrogenation products of a- and y-linolenic acids.Biomed. Mass Spectrom. 12:134-138.

8. Kemp, P., and D. J. Lander. 1984. The hydrogenation of somecis- and trans-octadecenoic acids to stearic acid by a rumen

Fusocillus sp. Br. J. Nutr. 52:165-170.9. Kemp, P., and D. J. Lander. 1984. The hydrogenation of the

series of methylene-interrupted cis,cis-octadecadienoic acids bypure cultures of six rumen bacteria. Br. J. Nutr. 52:171-177.

10. Kepler, C. R., and S. B. Tove. 1967. Biohydrogenation ofunsaturated fatty acids. J. Biol. Chem. 242:5686-5692.

VOL. 51, 1986 537

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APPL. ENVIRON. MICROBIOL.

11. Kepler, C. R., W. P. Tucker, and S. B. Tove. 1970. Bio-hydrogenation of unsaturated fatty acids. J. Biol. Chem.245:3612-3620.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

13. Privett, 0. S., and E. C. Nickell. 1966. Determination of thespecific positions of cis and trans double bonds in polyenes.Lipids 1:98-103.

14. Seltzer, S. 170. Cis-t(alns isomerization, p. 381-406. In P. D.Boyer (ed.), 3rd ed., vol. 6. Academic Press, Inc., NewYork.

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