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Biotransformation of monoterpenoid ketones by yeasts and yeast-like fungi
Journal of Molecular Catalysis B: Enzymatic 5 Ž1998. 149–154
Biotransformation of monoterpenoid ketones by yeasts and yeast-like fungi M.S. van Dyk ) , E. van Rensburg, I.P.B. Rensburg, N. Moleleki Department of Microbiology and Biochemistry, UniÕersity of the Orange Free State, Bloemfontein, South Africa Received 26 September 1997; accepted 24 November 1997
Abstract A large number of yeasts were screened for the biotransformation of Žy.-piperitone, Žq.- and Žy.-carvone, Žy.menthone, Žq.-pulegone and Žy.-verbenone. A relatively small number of yeasts gave hydroxylation products of Žy.-piperitone. Products obtained from Žy.-piperitone were 7-hydroxy-piperitone, cis-6-hydroxy-piperitone, trans-6-hydroxy-piperitone and 2-isopropyl-5-methyl-hydroquinone. Yields for the hydroxylation reactions varied between 8% and 60%, corresponding to product concentrations of 0.04 grl to 0.3 grl. Not one of the yeasts tested reduced Žy.-piperitone. In contrast, almost all the yeasts tested gave reduction of carvone, although the enzyme activity varied. Reduction of Žy.-carvone was often much faster than reduction of Žq.-carvone. Some yeasts only reduced the carbon5carbon double bond to yield the dihydrocarvone isomers with the stereochemistry at C-1 always R, while others also reduced the ketone to give the dihydrocarveols with the stereochemistry at C-2 always S for Žy.-carvone, but sometimes S and sometimes R for Žq.-carvone. In the case of Žy.-carvone yields of up to 90% were obtained within 2 h. Only one organism, a Hormonema isolate ŽUOFS Y-0067., quantitatively reduced Žy.-menthone and Žq.-pulegone to Žq.-neomenthol. This same organism reduced Ž4S .-isopiperitenone to Ž3 R,4S .-isopiperitenol, a precursor of Žy.-menthol. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Monoterpenoid; Piperitone; Carvone; Menthone; Pulegone; Verbenone; Menthol; Yeast; Reduction; Hydroxylation
1. Introduction Monoterpenoids like carvone and menthol are very important to the flavour and fragrance industry. The organoleptic properties of these compounds are determined by their stereochemistry. Stereoselective reduction of these com-
) Corresponding author. Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa. Fax: q27-51-4482004; E-mail: [email protected]
pounds can yield the desired monoterpenoid products, i.e. Ž y. -menthol from Žy.-menthone. We are interested in the biotransformation of monoterpenes and monoterpenoids by unicellular fungi. These include conventional yeasts, non-conventional yeasts and yeast-like fungi. According to Faber w1x and Holland w2x Saccharomyces cereÕisiae Žbaker’s yeast. has been extensively studied for the bioreduction of carbonyls and carbon5carbon double bonds. Only monoterpenoids are suitable for bioreductions, since isolated carbon5carbon double bonds without an allylic alcohol, carbonyl or other
1381-1177r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 1 - 1 1 7 7 Ž 9 8 . 0 0 0 2 4 - 1
150
M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
omyces, Dekkera, Eremothecium, Exophiala, Geotrichum, Hanseniaspora, Hormonema, Kloeckera, KluyÕerom yces, Lipom yces, Metschnikowia, Pachytichospora, Pichia, Rhodotorula, Saccharomyces, Schwanniomyces, Sporodiobolus, Torulaspora, Trichosporon, Yarrowia, Zygozyma. 2.2. Chemicals Scheme 1. Monoterpenoid ketones tested for biotransformations: Ž1. Ž4S .-isopiperitenone, Ž2. Ž4 R .-Žy.-piperitone, Ž3. Ž1 R,4S .Žy.-menthone, Ž4. Ž1 R .-Žq.-pulegone, Ž5. Žy.-verbenone, Ž6. Ž4 R .-Žy.-carvone and Ž7. Ž4S .-Žq.-carvone.
‘activating’ substituent are only rarely reduced by microorganisms w1x. We had previously obtained isopiperitenone Ž 1 . as a hydroxylation product from Žq.limonene w3x. Reduction of this product can yield Žy.-menthol. In this study we were thus interested in the reduction of 3-keto monoterpenoids, i.e., Žy.-piperitone Ž2., Žy.-menthone Ž3., Ž q.-pulegone Ž4. and Žy.-verbenone Ž5. ŽScheme 1.. The two carvone enantiomers Ž6 and 7; Scheme 1. were included as positive controls, since the reduction of Žq.-carvone Ž7. by Rhodotorula mucilaginosa had previously been described by Mironowicz et al. w4x. Biotransformations were carried out using two sets of conditions. The first set of conditions involved biotransformation by whole cultures in early stationary phase, while the second set of conditions involved biotransformation by harvested cells in a glucose containing phosphate buffer. The latter conditions were initially regarded as selective for bioreductions.
2. Experimental 2.1. Yeasts Yeasts tested belonged to the genera Arxula, Brettanomyces, Bullera, Candida, Debary-
Žq.-Carvone, Žy.-carvone, Žy.-menthone, Žq. -pulegone, Ž y. -verbenone and Žy.-menthol were obtained from Fluka. Žy. -Piperitone was a generous gift from AECI, South Africa. Ž4S . Isopiperitenone was prepared from Ž q . limonene with the use of Trichosporon sp. UOFS Y-2041 w3x. 2.3. Screening method A Yeasts were cultivated in yeast extractrmalt extract broth containing 10 g glucose, 20 g malt extract ŽDifco., 10 g peptone ŽBiolab. and 3 g yeast extract ŽBiolab. per liter distilled water with pH unadjusted. Conical flasks Ž100 ml. containing 20 ml yeast extractrmalt extract broth were inoculated from a 48 h old starter culture and propagated on a rotary shaker Ž150 rpm. at 258C. After 48 h, the monoterpenoid substrate was added Ž0.5 mlrl.. Samples were taken after 24 h, 48 h and 120 h. 2.4. Screening method B Yeasts were cultivated in yeast extractrmalt extract broth containing 40 g glucose, 20 g malt extract ŽDifco., 10 g peptone ŽBiolab. and 3 g yeast extract ŽBiolab. per liter distilled water with pH unadjusted. At early stationary phase cells were harvested by centrifugation Ž 5000 rpm, 10 min, 108C., washed twice and resuspended Ž20% wrv. in sterile phosphate buffer Ž10 mM, pH 6.8. containing glucose Ž7.5% wrv. . The resuspended cells were divided in 1
M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
151
3. Results and discussion Žy.-Piperitone was initially included in a general screen for monoterpene biotransformations using method A w3x. This screen involved 100 isolates of yeasts and yeast-like fungi isolated from monoterpene-rich environments as well as 27 classified yeast strains. In a second screen, using method B, we screened specifically for the reduction of Žy.-piperitone Ž2. , Žy. - and Ž q. -carvone Ž6 and 7., Ž y. -menthone Ž3., Ž q.-pulegone Ž 4. and Ž y.-verbenone Ž 5. . This screen involved yeast strains from 23 genera. During the initial screen with Žy.-piperitone Ž2. only hydroxylation products were observed ŽScheme 2. . The hydroxylation products Ž 8, 9 and 10. obtained with non-conventional yeasts from the genera Arxula, Candida, Yarrowia and Trichosporon have recently been described w3x Ž Scheme 2.. A previously undescribed Žy.piperitone product, cis-6-hydroxy-piperitone Ž11. ŽScheme 2., was obtained as a major product from a Hormonema sp. UOFS Y-0067. The 1 H-NMR and mass spectrometry Ž MS. data for the Ž y.-piperitone products are given in Tables 1 and 2. The data for the previously described products are also included for comparison. Products 8 and 9 had also been described as biotransformation products of 2 formed by filamentous fungi w5,6x.
Scheme 2. Hydroxylation products of Žy.-piperitone Ž2.: Ž8. Ž4 R,6S .-trans-6-hydroxy-piperitone, Ž9. Ž4 R .-7-hydroxypiperitone, Ž10. 2-isopropyl-5-methyl hydroquinone, and Ž11. Ž4 R,6 R .cis-6-hydroxy-piperitone.
ml aliquots in 4 ml vials and monoterpenoids Ž1 m l. added. Reaction mixtures were incubated at 308C and extracted periodically. Extraction, analysis and characterization of biotransformation products were done as previously described w3x.
Table 1 1 H-NMR data of identified Žy.piperitone biotransformation products Ža. Chemical shifts Žppm. of specific protons H-2
H-3a
H-3e
H-4
H-5a
H-5e
H-6a
H-6e
H-8
H-7
H-9
H-10
8 9a 10 a 11
5.77 6.05 6.61 5.79
y y y y
y y y y
2.34 2.06 y 2.1
2.12 1.81 y 1.69
2.01 2.00 6.52 2.43
y 2.20 y 4.43
4.33 2.29 y y
2.22 2.32 3.11 2.48
2.00 4.20 2.15 2.00
0.86 0.82 1.19 0.79
0.90 0.91 1.21 0.92
8 9 10 11
Žb. Coupling constants ŽHz. J5ay4 s 9.0; J5ey4 s 6.0; J5ay5e s 14.0; J5ay6e s 4.5; J5ey6e s 4.5; J4y8 s 5.0; J8y9 s J8y10 s 7.0 J5ay4 s 10.8; J5ey4 s 5.0; J5ay5e s 13.5; J5ay6a s 8.5; J5ay6e s J5ay6a s J5ey6e s J4y8 s 5.0; J6ay6e s 17.5; J4y8 s J4y8 s 7.0 J8y 9 s J8y10 s 7.0 J5ay4 s 13.5; J5ey4 s 4.5; J5ay5e s 11.5; J5ey6a s 4.5; J5ay6a s 10.5; J4y8 s 3.0; J8y9 s J8y10 s 7.0
a
a1
H-NMR data taken from Ref. w3x.
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M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
Table 2 Mass spectra data of identified biotransformation products mre with relative intensities and assignment in brackets 8c 9c 10 c 11 12 13 14 15 16 17 18 19 20 21 22
168Ž1.wMxq;153Ž2.wM-15xq;150Ž1.wM-18xq;98Ž100. a ;126Ž63. b ;135Ž5.;111Ž49.;69Ž73. 168Ž18.wMxq;153Ž13.wM-15xq;97Ž100. a ;98Ž99. a ;126Ž93.;140Ž14.;107Ž24.;69Ž55.;67Ž52. 166Ž28.wMxq;151Ž100.wM-15xq;123Ž18.;107Ž17.;105Ž16.;95Ž33.;91Ž27.;77Ž55. 168Ž6.wMxq;153Ž2.wM-15xq;150Ž1.wM-18xq;98Ž100. a ;126Ž38. b ;135Ž6.;111Ž28.;70Ž49.69Ž73. 152Ž13.wMxq;137Ž10.wM-15xq;109Ž31.;95Ž71.;82Ž44.;81Ž42.;67Ž100.;55Ž35.;41Ž55. 154Ž1.wMxq;136Ž56.wM-18xq;121Ž75.wM-18-15xq;107Ž100.;93Ž78.;79Ž97.;67Ž62.;55Ž55.;41Ž83. 152Ž30.wMxq;137Ž12.wM-15xq;109Ž19.;95Ž89.;82Ž42.; 67Ž100.;55Ž38.;41Ž58. 154Ž3.wMxq;136Ž27.wM-18xq;121Ž67.wM-18-15xq;107Ž77.;93Ž100.;79Ž68.;67Ž67.;55Ž67.;41Ž88. 154Ž8.wMxq;136Ž53.wM-18xq;121Ž68.wM-18-15xq;107Ž99.;93Ž79.;82Ž96.;79Ž96.;67Ž77.;55Ž78.;41Ž100. 170Ž3.wMxq;152Ž1.wM-18xq;128Ž5.;126Ž10.;99Ž38.;86Ž17.;81Ž21.;71Ž21.;57Ž13.;55Ž17.;43Ž100.;41Ž43. 170Ž4.wMxq;152Ž1.wM-18xq;127Ž3.;123Ž2.;109Ž12.;95Ž6.;84Ž65.;71Ž25.;69Ž26.;57Ž49.;43Ž60.;41Ž100. 170Ž10.wMxq;152Ž2.wM-18xq;137Ž3. wM-18-15xq;109Ž13.;84Ž100.;71Ž27.;57Ž35.;43Ž29.;41Ž47. 138Ž9.wM-18xq;123Ž9.wM-18-15xq;109Ž4.;95Ž33.;81Ž30.;71Ž70.;67Ž25.;57Ž33.;55Ž47.;43Ž72.;41Ž100. 152Ž13. wMxq;134Ž13. wM-18xq;84Ž100. a 138Ž13.wM-18xq;123Ž28.wM-18-15xq;109Ž10.;95Ž38.;81Ž74.;71Ž100.; 57Ž29.;55Ž43.;43Ž45.;41Ž80.
a
Fragments resulting from retro-Diels–Alder type reactions w5x. Fragments resulting from McLafferty rearrangements w5x. c Mass spectra data taken from Ref. w3x. b
During the second screen only reductions of Žy. - and Žq. -carvone Ž6 and 7. were observed. With the exception of L. starkeyi all the yeasts tested had the ability to reduce 6 and 7, although with some yeasts the yields were quite low. Mironowicz et al. w4x described the reduction of Žq.-carvone Ž7. by R. mucilaginosa and found that it was reduced exclusively to Žy.neoisodihydrocarveol Ž15. ŽScheme 3. . Hirata et al. w7x studied the reduction of 6 and 7 by cell
Scheme 3. Bioreduction products of Žy.- and Žq.-carvone Ž6 and 7.: Ž12. Ž1 R,4 R .-Žq.-dihydrocarvone, Ž13. Ž1 R,2 S,4 R .-Žq.neodihydrocarveol, Ž14. Ž1 R,4S .-Žy.-sodihydrocarvone, Ž15. Ž1 R,2 S,4S .-Žy.-neoisodihydro-carveol, Ž16. Ž1 R,2 R,4S .-Žy.isodihydrocarveol.
suspensions of Nicotiana tabacum. They found that Ž4 R .-Žy.-carvone Ž6. was reduced to Ž 1 R,2 S,4 R . -Ž q. -neodihydrocarveol Ž 13. via Ž1 R,4 R .-Žq.-dihydrocarvone Ž12., while Ž4S .Žq. -carvone Ž7. was reduced to Ž1 R,4S .-Žy. isodihydrocarvone Ž14. and Ž1 R,2 S,4S .-Žy.neoisodihydrocarveol Ž15. as main products, and Ž1 R,2 R,4S .-Žy.-isodihydrocarveol Ž16. as a minor product ŽScheme 3. . Comparison of mass spectra ŽTable 2. showed that we obtained the same products from 6 and 7. Noma and Asakawa w8x investigated the oxidationrreduction of the four carveol isomers by an alga Euglena gracilis Z. The alga yielded the same dihydrocarveol products obtained with plant cells and with yeasts. The selectivity and yields obtained with yeasts were, however, remarkable Ž Fig. 1. . With the plant cells yields were in the order of 10–15% after three weeks incubation. Reduction of 6 by yeasts gave single products in yields of 70%–90% after 2 h incubation ŽFig. 2., while reduction of 7 gave similar results after 24 h incubation. Some yeasts even gave 16 as a major product Ž Fig. 1. . The yeasts that gave hydroxylation of 2 were tested again for the biotransformation of the other monoterpenoid ketones, using whole cul-
M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
153
Fig. 2. Time course of the reduction of Žy.-carvone Ž6. by: Ža. H. guilliermondii CSIR Y894 T and Žb. Y. lipolytica KBP 3364.
Fig. 1. Reduction of Žy.-carvone Ž6. and Žq.-carvone Ž7. by different yeasts. Yeast strains: H. guilliermondii CSIR Y894 T; G. candidum G 400; Y. lipolytica KBP 3364; K. lindnerii G 392; P. transÕaalensis CBS 2186 T; S. occidentalis KBP 2866; P. onychis G 387; S. cereÕisiae CBS 3093.
tures in early stationary phase Ž method A. . Even under these conditions only reduction products were obtained as major products from 6 and 7. Under these conditions the Trichosporon sp. UOFS Y-2041 formed three products from Žy. -menthone Ž3.. It was evident from the mass spectra ŽTable 2, entries 16, 17 and 18. that a single oxygen had been incorporated. These products were difficult to separate and the positions of oxygen incorporation were not determined. Under the above mentioned conditions the Hormonema sp. UOFS Y-0067 reduced Žy.menthone Ž 3. and Ž q.-pulegone Ž 4. to Žq.neomenthol Ž20. ŽScheme 4.. This product was identified as 20 because its GC retention time and MS spectrum ŽTable 2. differed from that
of Žy.-menthol Ž22. but were the same as that of the second product obtained from the chemical reduction of 3 with NaBH 4 . Mironowicz and Siewinski w9x found that R. mucilaginosa also reduced 3 to 20. Žy.-Menthone Ž3. was reduced to 22 by an NADH dependant hydroxysteroid dehydrogenase from Cellulomonas turbata w10x.
Scheme 4. Reduction products of Žy.-menthone Ž3., Žq.-pulegone Ž4. and Ž4S .-isopiperitenone Ž1.: Ž20. Ž1 R,3S,4S .-Žq.neomenthol, Ž21. Ž3 R,4S .-isopiperitenol, Ž22. Ž1 R,3 R,4S .-Žy.menthol.
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M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
Only the black yeast Hormonema sp. UOFS Y-0067 was tested for the biotransformation of Ž4S .-isopiperitenone Ž 1., obtained from the hydroxylation of Ž q. -limonene by the Trichosporon sp. UOFS Y-2041 w3x. A single product was formed. Surprisingly, platinum oxide catalyzed hydrogenation of this product Ž21. yielded Ž y.-menthol Ž22. ŽScheme 4.. The alcohol Ž 21. obtained from 1 thus has the opposite stereochemistry Ž R-configuration. than the alcohol Ž20. obtained from 3 Ž S-configuration. , even though the stereochemistries of the adjacent carbons ŽC-4. are the same.
4. Conclusions The substrate specificity of the yeast enzymes responsible for the biotransformation of the monoterpenoid ketones is remarkable. It is possible that the positions of the methyl and isopropyl groups relative to the carbonyls determine the orientation and fit of the molecules in the catalytic sites of the monooxygenases, hydrogenases and reductases involved. In fungi, the enzymes responsible for the hydroxylation of alkanes and benzoate were shown to be membrane bound cytochrome P-450 monooxygenases w11,12x. Alcohol dehydrogenases are involved in the reduction of carbonyls, while enoate reductases are responsible for the reduction of ‘activated’ carbon5carbon double bonds w1x. The enzymes responsible for the reduction of the carvone enantiomers are apparently present in many yeasts and fungi, as well as in plants and algae. The dehydrogenases responsible for the formation of the S-alcohols from 6 and 7 and from 3 follow Prelog’s rule w1x.
Dehydrogenases following Prelog’s rule are apparently more abundant in nature w1x. The yeast enzymes involved in these biotransformations of the monoterpenoid ketones deserve further characterization. Acknowledgements This study was funded by AECI and the South African Foundation for Research Development. The authors are grateful to Mr. P.J. Botes for assistance with GC and GC-MS analyses, Prof. V. Brandt and Prof. P. Wessels for NMR analyses and Rudi Cronje and Andri van Wyk for technical assistance. The authors thank Dr. Vu Nguyen Thanh for the use of cultures from his personal collection. References w1x K. Faber, Biotransformations in Organic Chemistry, Springer-Verlag, Heidelberg, 1992. w2x H.L. Holland, Organic Synthesis with Oxidative Enzymes, VCH Publishers, New York, 1992. w3x E. van Renburg, N. Moleleki, J.P. van der Walt, P.J. Botes, M.S. van Dyk, Biotechnol. Lett. 19 Ž1997. 779. w4x A. Mironowicz, J. Raczkowska, A. Siewinski, J. Szykula, A. Zabsa, Pol. J. Chem. 56 Ž1982. 735. w5x E.V. Lassak, J.T. Pinhey, B.J. Ralph, T. Sheldon, J.J.H. Simes, Aust. J. Chem. 26 Ž1973. 845. w6x M. Miyazawa, H. Kakita, M. Hyakumachi, H. Kameoka, Chem. Express 8 Ž1993. 61. w7x T. Hirata, H. Hamada, T. Aoki, T. Suga, Phytochemistry 21 Ž1982. 2209. w8x Y. Noma, Y. Asakawa, Phytochemistry 31 Ž1992. 2009. w9x A. Mironowicz, A. Siewinski, Acta Biotechnol. 6 Ž1986. 141. w10x S. Kise, M. Hayashida, J. Biotechnol. 14 Ž1990. 221. w11x T. Zimmer, M. Ohkuma, A. Ohta, M. Takagi, W.-H. Schunck, Biochem. Biophys. Res. Commun. 224 Ž1996. 784. w12x J.M. van den Brink, C.A.M.J.J. van den Hondel, R.F.M. van Gorcom, Appl. Microbiol. Biotechnol. 46 Ž1996. 360.
Biotransformation of monoterpenoid ketones by yeasts and yeast-like fungi M.S. van Dyk ) , E. van Rensburg, I.P.B. Rensburg, N. Moleleki Department of Microbiology and Biochemistry, UniÕersity of the Orange Free State, Bloemfontein, South Africa Received 26 September 1997; accepted 24 November 1997
Abstract A large number of yeasts were screened for the biotransformation of Žy.-piperitone, Žq.- and Žy.-carvone, Žy.menthone, Žq.-pulegone and Žy.-verbenone. A relatively small number of yeasts gave hydroxylation products of Žy.-piperitone. Products obtained from Žy.-piperitone were 7-hydroxy-piperitone, cis-6-hydroxy-piperitone, trans-6-hydroxy-piperitone and 2-isopropyl-5-methyl-hydroquinone. Yields for the hydroxylation reactions varied between 8% and 60%, corresponding to product concentrations of 0.04 grl to 0.3 grl. Not one of the yeasts tested reduced Žy.-piperitone. In contrast, almost all the yeasts tested gave reduction of carvone, although the enzyme activity varied. Reduction of Žy.-carvone was often much faster than reduction of Žq.-carvone. Some yeasts only reduced the carbon5carbon double bond to yield the dihydrocarvone isomers with the stereochemistry at C-1 always R, while others also reduced the ketone to give the dihydrocarveols with the stereochemistry at C-2 always S for Žy.-carvone, but sometimes S and sometimes R for Žq.-carvone. In the case of Žy.-carvone yields of up to 90% were obtained within 2 h. Only one organism, a Hormonema isolate ŽUOFS Y-0067., quantitatively reduced Žy.-menthone and Žq.-pulegone to Žq.-neomenthol. This same organism reduced Ž4S .-isopiperitenone to Ž3 R,4S .-isopiperitenol, a precursor of Žy.-menthol. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Monoterpenoid; Piperitone; Carvone; Menthone; Pulegone; Verbenone; Menthol; Yeast; Reduction; Hydroxylation
1. Introduction Monoterpenoids like carvone and menthol are very important to the flavour and fragrance industry. The organoleptic properties of these compounds are determined by their stereochemistry. Stereoselective reduction of these com-
) Corresponding author. Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa. Fax: q27-51-4482004; E-mail: [email protected]
pounds can yield the desired monoterpenoid products, i.e. Ž y. -menthol from Žy.-menthone. We are interested in the biotransformation of monoterpenes and monoterpenoids by unicellular fungi. These include conventional yeasts, non-conventional yeasts and yeast-like fungi. According to Faber w1x and Holland w2x Saccharomyces cereÕisiae Žbaker’s yeast. has been extensively studied for the bioreduction of carbonyls and carbon5carbon double bonds. Only monoterpenoids are suitable for bioreductions, since isolated carbon5carbon double bonds without an allylic alcohol, carbonyl or other
1381-1177r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 1 - 1 1 7 7 Ž 9 8 . 0 0 0 2 4 - 1
150
M.S. Õan Dyk et al.r Journal of Molecular Catalysis B: Enzymatic 5 (1998) 149–154
omyces, Dekkera, Eremothecium, Exophiala, Geotrichum, Hanseniaspora, Hormonema, Kloeckera, KluyÕerom yces, Lipom yces, Metschnikowia, Pachytichospora, Pichia, Rhodotorula, Saccharomyces, Schwanniomyces, Sporodiobolus, Torulaspora, Trichosporon, Yarrowia, Zygozyma. 2.2. Chemicals Scheme 1. Monoterpenoid ketones tested for biotransformations: Ž1. Ž4S .-isopiperitenone, Ž2. Ž4 R .-Žy.-piperitone, Ž3. Ž1 R,4S .Žy.-menthone, Ž4. Ž1 R .-Žq.-pulegone, Ž5. Žy.-verbenone, Ž6. Ž4 R .-Žy.-carvone and Ž7. Ž4S .-Žq.-carvone.
‘activating’ substituent are only rarely reduced by microorganisms w1x. We had previously obtained isopiperitenone Ž 1 . as a hydroxylation product from Žq.limonene w3x. Reduction of this product can yield Žy.-menthol. In this study we were thus interested in the reduction of 3-keto monoterpenoids, i.e., Žy.-piperitone Ž2., Žy.-menthone Ž3., Ž q.-pulegone Ž4. and Žy.-verbenone Ž5. ŽScheme 1.. The two carvone enantiomers Ž6 and 7; Scheme 1. were included as positive controls, since the reduction of Žq.-carvone Ž7. by Rhodotorula mucilaginosa had previously been described by Mironowicz et al. w4x. Biotransformations were carried out using two sets of conditions. The first set of conditions involved biotransformation by whole cultures in early stationary phase, while the second set of conditions involved biotransformation by harvested cells in a glucose containing phosphate buffer. The latter conditions were initially regarded as selective for bioreductions.
2. Experimental 2.1. Yeasts Yeasts tested belonged to the genera Arxula, Brettanomyces, Bullera, Candida, Debary-
Žq.-Carvone, Žy.-carvone, Žy.-menthone, Žq. -pulegone, Ž y. -verbenone and Žy.-menthol were obtained from Fluka. Žy. -Piperitone was a generous gift from AECI, South Africa. Ž4S . Isopiperitenone was prepared from Ž q . limonene with the use of Trichosporon sp. UOFS Y-2041 w3x. 2.3. Screening method A Yeasts were cultivated in yeast extractrmalt extract broth containing 10 g glucose, 20 g malt extract ŽDifco., 10 g peptone ŽBiolab. and 3 g yeast extract ŽBiolab. per liter distilled water with pH unadjusted. Conical flasks Ž100 ml. containing 20 ml yeast extractrmalt extract broth were inoculated from a 48 h old starter culture and propagated on a rotary shaker Ž150 rpm. at 258C. After 48 h, the monoterpenoid substrate was added Ž0.5 mlrl.. Samples were taken after 24 h, 48 h and 120 h. 2.4. Screening method B Yeasts were cultivated in yeast extractrmalt extract broth containing 40 g glucose, 20 g malt extract ŽDifco., 10 g peptone ŽBiolab. and 3 g yeast extract ŽBiolab. per liter distilled water with pH unadjusted. At early stationary phase cells were harvested by centrifugation Ž 5000 rpm, 10 min, 108C., washed twice and resuspended Ž20% wrv. in sterile phosphate buffer Ž10 mM, pH 6.8. containing glucose Ž7.5% wrv. . The resuspended cells were divided in 1
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3. Results and discussion Žy.-Piperitone was initially included in a general screen for monoterpene biotransformations using method A w3x. This screen involved 100 isolates of yeasts and yeast-like fungi isolated from monoterpene-rich environments as well as 27 classified yeast strains. In a second screen, using method B, we screened specifically for the reduction of Žy.-piperitone Ž2. , Žy. - and Ž q. -carvone Ž6 and 7., Ž y. -menthone Ž3., Ž q.-pulegone Ž 4. and Ž y.-verbenone Ž 5. . This screen involved yeast strains from 23 genera. During the initial screen with Žy.-piperitone Ž2. only hydroxylation products were observed ŽScheme 2. . The hydroxylation products Ž 8, 9 and 10. obtained with non-conventional yeasts from the genera Arxula, Candida, Yarrowia and Trichosporon have recently been described w3x Ž Scheme 2.. A previously undescribed Žy.piperitone product, cis-6-hydroxy-piperitone Ž11. ŽScheme 2., was obtained as a major product from a Hormonema sp. UOFS Y-0067. The 1 H-NMR and mass spectrometry Ž MS. data for the Ž y.-piperitone products are given in Tables 1 and 2. The data for the previously described products are also included for comparison. Products 8 and 9 had also been described as biotransformation products of 2 formed by filamentous fungi w5,6x.
Scheme 2. Hydroxylation products of Žy.-piperitone Ž2.: Ž8. Ž4 R,6S .-trans-6-hydroxy-piperitone, Ž9. Ž4 R .-7-hydroxypiperitone, Ž10. 2-isopropyl-5-methyl hydroquinone, and Ž11. Ž4 R,6 R .cis-6-hydroxy-piperitone.
ml aliquots in 4 ml vials and monoterpenoids Ž1 m l. added. Reaction mixtures were incubated at 308C and extracted periodically. Extraction, analysis and characterization of biotransformation products were done as previously described w3x.
Table 1 1 H-NMR data of identified Žy.piperitone biotransformation products Ža. Chemical shifts Žppm. of specific protons H-2
H-3a
H-3e
H-4
H-5a
H-5e
H-6a
H-6e
H-8
H-7
H-9
H-10
8 9a 10 a 11
5.77 6.05 6.61 5.79
y y y y
y y y y
2.34 2.06 y 2.1
2.12 1.81 y 1.69
2.01 2.00 6.52 2.43
y 2.20 y 4.43
4.33 2.29 y y
2.22 2.32 3.11 2.48
2.00 4.20 2.15 2.00
0.86 0.82 1.19 0.79
0.90 0.91 1.21 0.92
8 9 10 11
Žb. Coupling constants ŽHz. J5ay4 s 9.0; J5ey4 s 6.0; J5ay5e s 14.0; J5ay6e s 4.5; J5ey6e s 4.5; J4y8 s 5.0; J8y9 s J8y10 s 7.0 J5ay4 s 10.8; J5ey4 s 5.0; J5ay5e s 13.5; J5ay6a s 8.5; J5ay6e s J5ay6a s J5ey6e s J4y8 s 5.0; J6ay6e s 17.5; J4y8 s J4y8 s 7.0 J8y 9 s J8y10 s 7.0 J5ay4 s 13.5; J5ey4 s 4.5; J5ay5e s 11.5; J5ey6a s 4.5; J5ay6a s 10.5; J4y8 s 3.0; J8y9 s J8y10 s 7.0
a
a1
H-NMR data taken from Ref. w3x.
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Table 2 Mass spectra data of identified biotransformation products mre with relative intensities and assignment in brackets 8c 9c 10 c 11 12 13 14 15 16 17 18 19 20 21 22
168Ž1.wMxq;153Ž2.wM-15xq;150Ž1.wM-18xq;98Ž100. a ;126Ž63. b ;135Ž5.;111Ž49.;69Ž73. 168Ž18.wMxq;153Ž13.wM-15xq;97Ž100. a ;98Ž99. a ;126Ž93.;140Ž14.;107Ž24.;69Ž55.;67Ž52. 166Ž28.wMxq;151Ž100.wM-15xq;123Ž18.;107Ž17.;105Ž16.;95Ž33.;91Ž27.;77Ž55. 168Ž6.wMxq;153Ž2.wM-15xq;150Ž1.wM-18xq;98Ž100. a ;126Ž38. b ;135Ž6.;111Ž28.;70Ž49.69Ž73. 152Ž13.wMxq;137Ž10.wM-15xq;109Ž31.;95Ž71.;82Ž44.;81Ž42.;67Ž100.;55Ž35.;41Ž55. 154Ž1.wMxq;136Ž56.wM-18xq;121Ž75.wM-18-15xq;107Ž100.;93Ž78.;79Ž97.;67Ž62.;55Ž55.;41Ž83. 152Ž30.wMxq;137Ž12.wM-15xq;109Ž19.;95Ž89.;82Ž42.; 67Ž100.;55Ž38.;41Ž58. 154Ž3.wMxq;136Ž27.wM-18xq;121Ž67.wM-18-15xq;107Ž77.;93Ž100.;79Ž68.;67Ž67.;55Ž67.;41Ž88. 154Ž8.wMxq;136Ž53.wM-18xq;121Ž68.wM-18-15xq;107Ž99.;93Ž79.;82Ž96.;79Ž96.;67Ž77.;55Ž78.;41Ž100. 170Ž3.wMxq;152Ž1.wM-18xq;128Ž5.;126Ž10.;99Ž38.;86Ž17.;81Ž21.;71Ž21.;57Ž13.;55Ž17.;43Ž100.;41Ž43. 170Ž4.wMxq;152Ž1.wM-18xq;127Ž3.;123Ž2.;109Ž12.;95Ž6.;84Ž65.;71Ž25.;69Ž26.;57Ž49.;43Ž60.;41Ž100. 170Ž10.wMxq;152Ž2.wM-18xq;137Ž3. wM-18-15xq;109Ž13.;84Ž100.;71Ž27.;57Ž35.;43Ž29.;41Ž47. 138Ž9.wM-18xq;123Ž9.wM-18-15xq;109Ž4.;95Ž33.;81Ž30.;71Ž70.;67Ž25.;57Ž33.;55Ž47.;43Ž72.;41Ž100. 152Ž13. wMxq;134Ž13. wM-18xq;84Ž100. a 138Ž13.wM-18xq;123Ž28.wM-18-15xq;109Ž10.;95Ž38.;81Ž74.;71Ž100.; 57Ž29.;55Ž43.;43Ž45.;41Ž80.
a
Fragments resulting from retro-Diels–Alder type reactions w5x. Fragments resulting from McLafferty rearrangements w5x. c Mass spectra data taken from Ref. w3x. b
During the second screen only reductions of Žy. - and Žq. -carvone Ž6 and 7. were observed. With the exception of L. starkeyi all the yeasts tested had the ability to reduce 6 and 7, although with some yeasts the yields were quite low. Mironowicz et al. w4x described the reduction of Žq.-carvone Ž7. by R. mucilaginosa and found that it was reduced exclusively to Žy.neoisodihydrocarveol Ž15. ŽScheme 3. . Hirata et al. w7x studied the reduction of 6 and 7 by cell
Scheme 3. Bioreduction products of Žy.- and Žq.-carvone Ž6 and 7.: Ž12. Ž1 R,4 R .-Žq.-dihydrocarvone, Ž13. Ž1 R,2 S,4 R .-Žq.neodihydrocarveol, Ž14. Ž1 R,4S .-Žy.-sodihydrocarvone, Ž15. Ž1 R,2 S,4S .-Žy.-neoisodihydro-carveol, Ž16. Ž1 R,2 R,4S .-Žy.isodihydrocarveol.
suspensions of Nicotiana tabacum. They found that Ž4 R .-Žy.-carvone Ž6. was reduced to Ž 1 R,2 S,4 R . -Ž q. -neodihydrocarveol Ž 13. via Ž1 R,4 R .-Žq.-dihydrocarvone Ž12., while Ž4S .Žq. -carvone Ž7. was reduced to Ž1 R,4S .-Žy. isodihydrocarvone Ž14. and Ž1 R,2 S,4S .-Žy.neoisodihydrocarveol Ž15. as main products, and Ž1 R,2 R,4S .-Žy.-isodihydrocarveol Ž16. as a minor product ŽScheme 3. . Comparison of mass spectra ŽTable 2. showed that we obtained the same products from 6 and 7. Noma and Asakawa w8x investigated the oxidationrreduction of the four carveol isomers by an alga Euglena gracilis Z. The alga yielded the same dihydrocarveol products obtained with plant cells and with yeasts. The selectivity and yields obtained with yeasts were, however, remarkable Ž Fig. 1. . With the plant cells yields were in the order of 10–15% after three weeks incubation. Reduction of 6 by yeasts gave single products in yields of 70%–90% after 2 h incubation ŽFig. 2., while reduction of 7 gave similar results after 24 h incubation. Some yeasts even gave 16 as a major product Ž Fig. 1. . The yeasts that gave hydroxylation of 2 were tested again for the biotransformation of the other monoterpenoid ketones, using whole cul-
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Fig. 2. Time course of the reduction of Žy.-carvone Ž6. by: Ža. H. guilliermondii CSIR Y894 T and Žb. Y. lipolytica KBP 3364.
Fig. 1. Reduction of Žy.-carvone Ž6. and Žq.-carvone Ž7. by different yeasts. Yeast strains: H. guilliermondii CSIR Y894 T; G. candidum G 400; Y. lipolytica KBP 3364; K. lindnerii G 392; P. transÕaalensis CBS 2186 T; S. occidentalis KBP 2866; P. onychis G 387; S. cereÕisiae CBS 3093.
tures in early stationary phase Ž method A. . Even under these conditions only reduction products were obtained as major products from 6 and 7. Under these conditions the Trichosporon sp. UOFS Y-2041 formed three products from Žy. -menthone Ž3.. It was evident from the mass spectra ŽTable 2, entries 16, 17 and 18. that a single oxygen had been incorporated. These products were difficult to separate and the positions of oxygen incorporation were not determined. Under the above mentioned conditions the Hormonema sp. UOFS Y-0067 reduced Žy.menthone Ž 3. and Ž q.-pulegone Ž 4. to Žq.neomenthol Ž20. ŽScheme 4.. This product was identified as 20 because its GC retention time and MS spectrum ŽTable 2. differed from that
of Žy.-menthol Ž22. but were the same as that of the second product obtained from the chemical reduction of 3 with NaBH 4 . Mironowicz and Siewinski w9x found that R. mucilaginosa also reduced 3 to 20. Žy.-Menthone Ž3. was reduced to 22 by an NADH dependant hydroxysteroid dehydrogenase from Cellulomonas turbata w10x.
Scheme 4. Reduction products of Žy.-menthone Ž3., Žq.-pulegone Ž4. and Ž4S .-isopiperitenone Ž1.: Ž20. Ž1 R,3S,4S .-Žq.neomenthol, Ž21. Ž3 R,4S .-isopiperitenol, Ž22. Ž1 R,3 R,4S .-Žy.menthol.
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Only the black yeast Hormonema sp. UOFS Y-0067 was tested for the biotransformation of Ž4S .-isopiperitenone Ž 1., obtained from the hydroxylation of Ž q. -limonene by the Trichosporon sp. UOFS Y-2041 w3x. A single product was formed. Surprisingly, platinum oxide catalyzed hydrogenation of this product Ž21. yielded Ž y.-menthol Ž22. ŽScheme 4.. The alcohol Ž 21. obtained from 1 thus has the opposite stereochemistry Ž R-configuration. than the alcohol Ž20. obtained from 3 Ž S-configuration. , even though the stereochemistries of the adjacent carbons ŽC-4. are the same.
4. Conclusions The substrate specificity of the yeast enzymes responsible for the biotransformation of the monoterpenoid ketones is remarkable. It is possible that the positions of the methyl and isopropyl groups relative to the carbonyls determine the orientation and fit of the molecules in the catalytic sites of the monooxygenases, hydrogenases and reductases involved. In fungi, the enzymes responsible for the hydroxylation of alkanes and benzoate were shown to be membrane bound cytochrome P-450 monooxygenases w11,12x. Alcohol dehydrogenases are involved in the reduction of carbonyls, while enoate reductases are responsible for the reduction of ‘activated’ carbon5carbon double bonds w1x. The enzymes responsible for the reduction of the carvone enantiomers are apparently present in many yeasts and fungi, as well as in plants and algae. The dehydrogenases responsible for the formation of the S-alcohols from 6 and 7 and from 3 follow Prelog’s rule w1x.
Dehydrogenases following Prelog’s rule are apparently more abundant in nature w1x. The yeast enzymes involved in these biotransformations of the monoterpenoid ketones deserve further characterization. Acknowledgements This study was funded by AECI and the South African Foundation for Research Development. The authors are grateful to Mr. P.J. Botes for assistance with GC and GC-MS analyses, Prof. V. Brandt and Prof. P. Wessels for NMR analyses and Rudi Cronje and Andri van Wyk for technical assistance. The authors thank Dr. Vu Nguyen Thanh for the use of cultures from his personal collection. References w1x K. Faber, Biotransformations in Organic Chemistry, Springer-Verlag, Heidelberg, 1992. w2x H.L. Holland, Organic Synthesis with Oxidative Enzymes, VCH Publishers, New York, 1992. w3x E. van Renburg, N. Moleleki, J.P. van der Walt, P.J. Botes, M.S. van Dyk, Biotechnol. Lett. 19 Ž1997. 779. w4x A. Mironowicz, J. Raczkowska, A. Siewinski, J. Szykula, A. Zabsa, Pol. J. Chem. 56 Ž1982. 735. w5x E.V. Lassak, J.T. Pinhey, B.J. Ralph, T. Sheldon, J.J.H. Simes, Aust. J. Chem. 26 Ž1973. 845. w6x M. Miyazawa, H. Kakita, M. Hyakumachi, H. Kameoka, Chem. Express 8 Ž1993. 61. w7x T. Hirata, H. Hamada, T. Aoki, T. Suga, Phytochemistry 21 Ž1982. 2209. w8x Y. Noma, Y. Asakawa, Phytochemistry 31 Ž1992. 2009. w9x A. Mironowicz, A. Siewinski, Acta Biotechnol. 6 Ž1986. 141. w10x S. Kise, M. Hayashida, J. Biotechnol. 14 Ž1990. 221. w11x T. Zimmer, M. Ohkuma, A. Ohta, M. Takagi, W.-H. Schunck, Biochem. Biophys. Res. Commun. 224 Ž1996. 784. w12x J.M. van den Brink, C.A.M.J.J. van den Hondel, R.F.M. van Gorcom, Appl. Microbiol. Biotechnol. 46 Ž1996. 360.