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Oxoester oxidoreductase activities in new isolates of Pichia anomala from apple, grape and cane juices
FEMS Yeast Research 5 (2005) 685–690 www.fems-microbiology.org
Oxoester oxidoreductase activities in new isolates of Pichia anomala from apple, grape and cane juices Francisco Pe´rez-Mendoza a, Francisco Ruiz-Tera´n b, Blanca Escudero Abarca a, Arturo Navarro-Ocan˜a b, Guadalupe Aguilar-Uscanga a, Gerardo Valerio-Alfaro a,* b
a Instituto Tecnolo´gico de Veracruz-UNIDA, Av. Miguel A. de Quevedo #2779, Veracruz, Ver. 91897, Me´xico Facultad de Quı´mica, Edificio E, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior, Ciudad Universitaria, Coyoaca´n, C.P. 04510, D.F. Me´xico, Me´xico
Received 13 July 2004; received in revised form 23 November 2004; accepted 29 November 2004 First published online 24 December 2004
Abstract Thirty-nine yeasts isolated from apple, grape and cane juices were screened for their oxidoreductase activity. The two strains of Pichia, one isolated from apple and one from cane juices, appear to be promising strains for oxidoreductase activity on a-oxoesters. They showed similar high yields in converting ethyl pyruvate to ethyl lactate as Saccharomyces spp. (86.6% and 85.3% versus 86.6%), and higher yields in the reduction of a-oxocarboxylic esters (ketopantolactone to pantolactone: 74% and 73.3%, respectively) compared to Saccharomyces spp. (yield 60%). 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Saccharomyces spp.; Pichia anomala; a-Oxoesters; Oxidoreductase activity
1. Introduction It is becoming increasingly important to find new biocatalysts with stereoselectivity, regioselectivity, and substrate specificity that work under mild conditions for asymmetric synthesis of organic compounds. Some of these new biocatalysts including entire microbial cells [1–3], microbial cells with overexpressed enzymes [4], enzymes from animal tissues [5], cultured [6] and noncultured plant cells [7], have been used to transform and synthesize chiral compounds more selectively and under milder reaction conditions than conventional chemical catalysts. This will contribute to broadening the ‘‘green’’ routes for catalytic biotransformation of organic compounds in the fields of chemistry, biochemistry, and medicine. *
Corresponding author. E-mail address: [email protected] (G. Valerio-Alfaro).
In the case of the synthesis of optically active alcohols, new biocatalysts, such as immobilized dehydrogenase NAD-(P) from plant cells, food proteins isolated from pea, soybean and wheat are alternatives to bakerÕs yeast [8]. It has also been reported that other nonSaccharomyces yeast genera are able to compete with S. cerevisiae, which is the more common and widely accepted biocatalyst as a source of oxidoreductases [9,10]. There are reports where c- and d-ketoacids and their corresponding esters were reduced with yeasts such as Pichia fermentans, P. glucozyma, P. etchellsii, Candida boidinii, C. utilis and Kluyveromyces marxianus with high enantiomeric excess [11]. In contrast, methanolotrophic yeasts of the genera Pichia, Candida, Hansenula and Torulopsis showed alcohol oxidase activity with numerous primary alcohols that makes them potential catalysts for organic synthesis [12]. In the present work the oxidoreductase activity of several yeast strains isolated from different natural sources was first evaluated
1567-1356/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsyr.2004.11.010
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on a positive/negative basis, and thereafter those with better biotransformation yields than Saccharomyces spp. were selected. This opens up new possibilities for the synthesis of optically active compounds, and an alternative source of oxidoreductases from non-Saccharomyces strains.
2. Materials and methods 2.1. Chemicals Ethyl pyruvate, (R) and (S) ethyl lactates, ethyl benzoyl formate, (R) and (S) ethyl mandelate, ketopantolactone and (R)-pantolactone were obtained from Sigma–Aldrich (Milwaukee, WI, USA); sodium chloride, peptone and sucrose were obtained from J.T. Baker (Mexico). A commercial bakerÕs yeast ‘‘Instant Yeast Nevada’’ was acquired from a local supermarket. Precoated TLC plates of silica gel 60 GF254, and column chromatography silica gel grade 60, 70–230 mesh were used, both from Merck KgaA (Darmstadt, Germany). 2.2. Microorganisms Two commercial dried active Saccharomyces spp. cultures, BYC (bakerÕs yeast cultured) and BYNC (bakerÕs yeast not cultured), were used as reference strains. 39 Yeast strains were isolated from natural sources. These were labeled M1–M14 (isolated from apple juice), UV1–UV13 (isolated from grape juice), and CA1–CA12 (isolated from cane juice), followed by serial numbers. An additional five strains, labeled WBY1–WBY5, were obtained from bakeries in Veracruz City. 2.3. Culture and maintenance media: isolation medium Yeasts strains were isolated from 150 ml fruit juice supplemented with 1 g l1 (NH4)SO4, 5 g l1 glucose and 90 m g l1 of ampicillin, and initial pH was adjusted to 4.5 with orthophosphoric acid (10% w/w). The culture was incubated for 24 h at 30 C, 150 rpm on an orbital shaker. The samples were diluted with sterile water and spread on agar plates containing (g l1): glucose 10, agar 10 and yeast extract 1. The plates were incubated at 30 C for 2 d and then the pure cultures were spread on agar slants, stored at 4 C and subcultured at regular intervals (each 2 months). 2.4. Isolation medium for oxidoreductase activity Yeasts strains were suspended in 100 ml of medium containing 20 mg of substrate (ethyl pyruvate, ethyl benzoyl formate or ketopantolactone), 0.1 g yeast extract with the initial pH adjusted to 6.0. Media were sterilized
at 121 C for 15 min and incubated at 30 C on an orbital shaker at 250 rpm and screened from 8 to 30 h. 2.5. Screening of yeasts for oxidoreductase activity In a preliminary screening of oxidoreductase activity, shaken-flask assays were performed in duplicate. Previously isolated yeast strains [M1–M14, UV1–UV13, CA1–CA12, one non-cultured (BYNC) and one cultured (BYC) commercial strain of bakerÕs yeast] were incubated with 100 ml of medium. Inoculum was added to attain 3 · 106 viable cells per ml. Biotransformations were monitored by TLC (hexane:ethyl acetate, 7:3 v/v) at different times (8, 12, 24, 30 h). 2.6. Yeast identification The selected strains (5) were grown for 24 h at 30 C in 10 ml YM broth (3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g glucose per liter of distilled water) on a rotary shaker at 150 rpm and harvested by centrifugation at 13,000 rpm for 20 min. The cells were washed twice and resuspended with 1 ml of sterile distilled water in 1.5 ml Eppendorf tubes. Cells were mechanically broken down, using 1 ml of extraction buffer, pH 6.5 (200 mM Tris–HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS ) and a small capful of 0.5 mm glass beads. The DNA was extracted using the phenol–chloroform method [12,13]. Yeast identification was made according to Kurtzman and Robnett [14], sequencing the D1/D2 motif of the 26S-rDNA gene. PCR amplification was performed with primers NL1 (5 0 GCA TAT CAA TAA GCG GAG GAA AAG and NL4 5 0 GGT CCG TGT TTC AAG ACG G 3 0 , and nucleotide sequences were obtained with an ABIPRISM 310 Automated Sequencer (Perkin–Elmer, USA). Search and comparison of the partial sequence obtained (26S rDNA) in the GenBank were performed using the Blast-Program [15]. 2.7. Preparative biotransformations A sterile 180 ml broth was prepared in 250-ml flasks and an inoculum of 3 · 106 cells viable ml1 (determined by methylene blue staining method) [16] with 150 mg of substrate was added. The biotransformation was carried out at 30 C and 250 rpm on an orbital shaker until the reaction was complete (no more substrate was detected by TLC monitoring). The broth was saturated with NaCl, filtered over a Celite bed and then washed with 100 ml ethyl acetate. Additional extractions with the same solvent (100 · 3) were achieved over the recovered filtrate. The combined organic phases were dried over Na2SO4 and concentrated in a rotatory evaporator. The product of the reaction mixture was purified by column chromatography (hexane ethyl acetate, 7:3) until purified products were obtained.
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2.8. Instrumental analysis Optical rotations were measured using a Perkin– Elmer 343 polarimeter. IR spectra were recorded on a Paragon 500 FT Infrared spectrometer adapted with HATR accessory. Chromatographic analyses and mass spectra were performed on a Shimadzu QP 5050 gas chromatograph– mass spectrometer using an electronic impact procedure (75 eV) and a quadrupole analyzer. The column used in the chiral analyses was Chiraldex B-PM (a permethylated b-cyclodextrin column, 50 m long and 0.25 mm i.d.) obtained from Alltech Associates, Inc USA. Temperature conditions were determined for each substrate: ethyl pyruvate–lactate system, injector and detector 170 C, and a program column temperature of 50–180 C at 10 C min1; ketopantolactone–pantolactone system, injector and detector 180 C, a program column temperature 100–150 C at 10 C min1; ethyl benzoyl formate– ethyl mandelate system, injector and detector 180 C, a program column of temperature 100–140 C at 2.5 C min1. The column was able to detect the enantiomers without derivatization, and the elution times varied from 15 to 60 min for the various reactants and products. The enantiomeric excess (ee) for the reactions studied with substrates 1a–c (Fig. 1) was determined by gas chromatography chiral analyses and optical rotation comparing with pure chiral standards. The ee values were determined by ee = ([S] [R])/([S] + [R]) or ([R] [S])/([S] + [R]) for S and R products, respectively.
3. Results The substrates chosen in this study were ketoesters, such as natural short-chain ethyl pyruvate (1a), cyclic oxoester ketopantolactone (1b), and aromatic ethyl benzoyl formate (1c) (Fig. 1). In general, the ability to convert ketopantoyl lactone to pantoyl lactone is widely O
OH
Yeast O
R
oxidoreductases
O
R
O
O
1a, R= Me 1c, R= Ph
2a, R= Me 2c, R= Ph OH
O Yeast O O 1b
O oxidoreductases
O 2b
Fig. 1. Bioreduction reactions of a-oxoesters: (1a) ethyl pyruvate, (1b) ketopantolactone and (1c) benzoyl formic acid ethyl ester, mediated by native cultured yeasts to the corresponding a-hydroxyesters: (2a) ethyl lactate, (2b) pantolactone and (2c) ethyl mandelate.
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observed in a variety of organisms, including yeasts [17]. In the first screening of the thirty nine different non-Saccharomyces yeast species isolated from fruit juices, plus one cultured and one non-cultured strain of bakerÕs yeast, seventeen yielded pantoyl lactone (2b) from (1b) in 8–30 h. An important ability to convert (1b) to (2b) was mainly found in yeast strains from cane (six) and apple (six), although three strains from grape converted the ketolactone (1b) into (2b). In the case of substrate (1a) (Table 1), we found 15 non-Saccharomyces strains plus both cultured and non-cultured strains of bakerÕs yeast producing (2a) within 8–30 h. In particular, yeast strains from apple showed a higher reductase activity to this substrate than to a-ketolactone (1b) in contrast with yeast strains from cane. Neither yeast strains from apple nor from cane expressed any reductase activity to ethyl benzoylformate (1c). On the other hand, out of all the juice strains only strain UV9 (from grape) converted benzoylformate (1c) to ethyl mandelate (2c). In contrast, ethyl pyruvate (1a) was reduced to ethyl lactate (2a) within 8–30 h by UV9, without being able to biotransform the a-ketopantolactone (1b) to the corresponding a-hydroxyl lactone (2b). Although reductase activities of the strains tested almost showed no relation to their source, in the case of both cultured and non-cultured strains of bakerÕs yeast, a notable ability to reduce all the a-ketoesters to the a-hydroxyesters within 8 and 30 h was found, in contrast to WBY1–WBY5 which were not able to reduce the a-oxoesters tested. Once the enzymatic activities of the isolates had been established, five of the isolates that exhibited pronounced enzymatic activity and two commercial yeast strain isolates were selected to test for a-oxidoreductase enantioselectivity. Even though oxoester reductase activities were observed in other isolates from apple, grape and cane juices, we decided to select strains M1, M7, CA10, CA11 and UV9 and commercial BYC and BYNC strains to continue screening studies, based on the criteria that these strains showed positive activity from 8 h onwards, except strain CA10 which showed excellent growth (Table 2). The biotransformations were scaled up to 150 mg and carried out with the same substrate biocatalyst (cell count) ratio (Table 2), including the cultured commercial strain of bakerÕs yeast. In the case of M1 and M7, CA10 and CA11, and BYC and BYNC, the products (2a) and (2b) were observed in 8–30 h. UV9 reduced (1a), but was not able to biotransform (1b). It is interesting to note that, although with low yields (9.5%), UV9 reduced (1c)–(2c). With the exception of BYC (8.6%) and BYNC (9.2%), this biotransformation was not observed in the other cultured yeast strains. The selected strains (Table 2) gave better yields in conversion of pantolactone (2b) compared with strains
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Table 1 Reactions for (a) ethyl pyruvate to ethyl lactate and (b) ketopantolactone to pantolactone mediated by isolated yeast strains and Saccharomyces spp. at different times Strain
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 UV1 UV2 UV3 UV4 UV5 UV6 UV7 UV8 UV9 UV10 UV11 UV12 CA1 CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 WBY1 WBY2 WBY3 WBY4 WBY5 BYC BYNC
Time conversion ethyl pyruvate (1a) to ethyl lactate (2a) (h)
Time conversion ketopantolactone (1b) to pantolactone (2b) (h)
8
12
24
30
8
12
24
30
+ + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
BYC and BYNC. In the second screening with the five cultured strains, the (S)-enantiomer ethyl lactate (2a) was the only detectable product (the chromatogrammass spectra indicated the presence of only one peak), and the enantioselectivity was >99% (in a racemic standard of (2a), two enantiomeric peaks with the same mass pattern but different retention times were resolved) (Table 3). Compared with ethyl pyruvate (1a), the reaction of ketopantolactone (1b) showed a mixture (1:1) of two enantiomeric peaks for the
product (2b), indicating the non-enantioselectivity of the process. Finally, with the third substrate, (R)-ethyl mandelate (1c), (2c) was the only product observed, indicating an enantioselectivity of the process of >99% ee. In addition, the (S)-(2a) and (R)-(2b) configurations were assigned based on the comparison of the sign of their specific rotations with those previously reported. In spite of biochemical differences among the isolates (data not shown), the five selected yeast (CA10, CA11,
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Table 2 Bioreductions of a-ketoesters with P. anomala and Saccharomyces spp. Straina
a-Hydroxy esterb
Yield (%)
a-Hydroxy ester
Yield (%)
a-Hydroxy ester
Yield (%)
M1 M7 CA10 CA11 UV9 BYC BYNC
2a 2a 2a 2a 2a 2a 2a
86.6 ± 0.71 84.0 ± 0.61 85.3 ± 0.62 85.3 ± 0.55 85.0 ± 0.44 86.6 ± 0.05 86.6 ± 0.05
2b 2b 2b 2b 2b 2b 2b
74.0 ± 0.48 66.6 ± 0.55 73.3 ± 0.33 64.0 ± 0.42 ND 60.00.36 60.00.29
2c 2c 2c 2c 2c 2c 2c
– – – – 9.5 ± 0.71 8.6 ± 0.59 9.2 ± 0.66
a b
M1 to UV9 are isolated strains of P. anomala and BYC and BYNC are cultured and non-cultured strains of bakerÕs yeast. The values represent the mean ± SD of three measurements.
Table 3 Enantiomeric excess by polarimetry and GC–MS chiral Strains
Product
[a] Exp.a
eeb (%)
eec (%)
Configuration
Ethyl lactate M1 2a M7 2a CA 10 2a CA 11 2a BYC 2a BYNC 2a
9.8 10.0 10.1 10.1 9.9 9.8
97 100 100 100 98.7 98.4
>99 >9 >9 >9 >9 >9
S S S S S S
Pantolactone M1 2b M7 2b CA 10 2b CA 11 2b BYC 2b BYNC 2b
0.6 0.5 0.5 0.5 0.4 0.4
0.01 0.01 0.01 0.01 0.01 0.01
0.7 0.4 0.4 0.3 0.2 0.2
n.d.d n.d. n.d. n.d. n.d. n.d.
130.1 130.4 124.0
97.0 97.4 92.5
>99 >99 >99
R R R
Ethyl mandelate UV9 2c BYC 2c BYNC 2c a b c d
Experimental optical rotation. Enantiomeric excess by polarimetry. Enantiomeric excess by GC–MS. n.d., Not determinated.
UV9, M1, M7) were identified as Pichia anomala strains using the partial sequence 26S rADN.
4. Discussion Among the many types of carbonyl-containing compounds that have been reduced using bakerÕs yeast, the main focus has been on enantioselective reductions of b-ketoesters and ketocarboxylic acids. To a lesser extent, a-ketoesters have also been investigated, along with c- and d-ketoesters, lactones, and diketonic compounds. However, with some substrates low chemical yields and ee are reported. For instance, ketopantoyl lactone (1b) was reduced to pantoyl lactone (2b) by a bakerÕs yeast culture with a chemical yield of 42%, and an ee of 50% [17]. The non-enantioselectivity of ketopantoyl lactone (1b) bioreduction shown by all the isolates suggests that
this substrate is utilized by different competitive ketoreductases with different stereoselectivities. Although the ability to convert (1b)–(2b) is widely encountered in a variety of microorganisms, there is a variety of stereospecificity in the ratio of D -()-2b and L -(+)-2b isomers yielded by molds, yeasts, basidiomycetous fungi and bacterial strains which bear no relation to the genera or sources [18]. Nakamura et al. [19] have found a-ketoesters with from 3 to 8 carbon-atoms to be reduced, and they compared the results of incubation with normal bakerÕs yeast, immobilized bakerÕs yeast in water, and immobilized bakerÕs yeast in hexane [17]. The enantiomeric excess ranged from 30% to 94% and decreased with increasing alkyl chain size. Similar results were obtained in stereoselective reduction of ethyl pyruvate (1a) to (S)-ethyl lactate (2a) with fermenting bakerÕs yeast. The differences in stereoselective reduction of the substrates (1a), (1b) and (1c), with isolates from apple, grape and cane juices, and with BYC and BYNC, could follow from differences in reduction rate of ketoester reductases acting simultaneously with opposite stereochemical substrate specifities, as has been observed with bakerÕs yeast oxido-reductases [20]. In the case of openchain a-oxoesters, enzymatic reductions can provide high enantioselectivity. On the other hand, the diminished enantioselectivity was found in hydroxyl ester products when a substrate is accepted by multiple enzymes has been attributed either to the fact that some of these reductases possess overlapping substrate specificities but with opposite enantioselectivities, or that a single reductase with relatively low enantioselectivity can deliver hydrides to both Re/Si faces in the substrate [21]. With respect to chiral results, similar enantiomeric excess was observed by polarimetry and GC–MS (Table 3, columns 4 and 5). These results show that the reductions by our P. anomala strains obey PrelogÕs rule in cases (2a) and (2c). From the yeasts examined, P. anomala was found to be the most versatile strain in cane, apple and grape juices acting on a-oxoesters. The enantioselectivity was
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consistently high in bioreduction of ethyl pyruvate, nonenantioselective with ketopantolactone, although with good yields in both cases. In the case of benzoyl formic acid ethyl ester, one strain of P. anomala (UV9) was found to reduce this substrate with a low yield and excellent enantioselectivity. Non-cultured and cultured commercial strains of bakerÕs yeast also yielded similar biotransformation results. The relevance of this study is that these biotransformations are an alternative to those with well-known Saccharomyces species, whereby the catabolic activities observed in native yeasts contribute to a broadening of the bioreduction spectrum.
Acknowledgements The authors thank Patricia M. Hayward Jones and Dulce Ma. Barradas Dermitz for a critical reading of the manuscript.
References [1] Nakamura, K. and Matuda, T. (1998) Asymmetric reduction of ketones by the acetone powder of Geotrichum candidum. J. Org. Chem. 63, 8957–8964. [2] Seebach, D., Gipovannini, F. and Lamatsch, B. (1985) Preparative asymmetric reduction of 3-keto butyrate and -valerate by suspended cells of thermophilic bacteria (Thermoanaerobium brockii) in ordinary laboratory equipment. Helv. Chim. Acta 68, 958–960. [3] Maconi, E. and Aragozzini, F. (1989) Stereoselective reduction of noncyclic ketones with lactic acid bacteria. Appl. Microbiol. Biotechnol. 31, 29–31. [4] Kataoka, M., Yamamoto, K., Kawabata, H., Wada, M. and Shimizu, S. (1999) Stereoselective reduction of ethyl 4-chloro-3oxobutanoate by Escherichia coli tranformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes. Appl. Microbiol. Biotechnol. 51, 486–490. [5] Matsumoto, K., Kitajima, H. and Nakata, T. (1995) Enantioselectivity promoting factor in enzyme-mediated asymmetric hydrolysis of enol esters. J. Mol. Catal. B: Enzym. 1, 17–21.
[6] Chai, W., Sakamaki, H., Kitanaka, S. and Horiuchi, A. (2003) Biotransformation of cycloalkanediones by Caragana chamlagu. Bull. Chem. Soc. Jpn. 76, 177–182. [7] Bruni, R., Fantin, G., Medici, A., Pedrini, P. and Sacchetti, G. (2002) Plants in organic synthesis, an alternative to bakerÕs yeast. Tetrahedron Lett. 43, 3377–3379. [8] Nagaoka, H. (2003) Chiral resolution function with immobilized food proteins. Biotechnol. Prog. 19, 1149–1155. [9] Faber, K. (2000) Biotransformations in Organic Compounds. Springer, Berlin. [10] Csuk, R. and Gla¨nzer, G. (1991) BakerÕs yeast-mediated transformations in organic chemistry. Chem. Rev. 91, 49–97. [11] Forzato, C., Gandolfi, R., Molinari, F., Nitti, P., Pitaccoa, G. and Valentina, E. (2001) Microbial bioreductions of c- and dketoacids and their esters. Tetrahedron: Asymm. 12, 1039–1046. ˇ itavicˇius, D. and [12] Dienys, G., Jarmalavicˇius, S., Budrien_e, S., C Sereikait_e, J. (2003) Alcohol oxidase from the yeast Pichia pastoris – a potential catalyst for organic synthesis. J. Mol. Catal. B: Enzym., 47–49. [13] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, New York, pp. 2.118–2.120. [14] Kurtzman, C.P. and Robnett, C.J. (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear largesubunit (26S) ribosomal partial sequences. Antonie van Leeuwenhoek. 73, 331–371. [15] Altschul, S.G., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. [16] Bonora, A. and Mares, D. (1982) A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms. Curr. Microbiol. 7, 217–222. [17] Lee, S.S., Robinson, F.M. and Wang, H.Y. (1981) Rapid determination of yeast viability. Biotechnol. Bioeng. Symp. 11, 641–649. [18] Shimizu, S., Hattori, S., Hata, H. and Yamada, Y. (1987) Synthesis of (R)-pantolactone via enantioespecific reduction of ketopantolactone by a broad variety of microorganisms. Agric. Biol. Chem. 53, 500–519. [19] Nakamura, K., Inoue, K., Ushio, K., Oka, S. and Ohno, A.Y. (1988) Stereochemical control on yeast reduction of a-keto esters. Reduction by immobilized bakersÕ yeast in hexane. J. Org. Chem. 53, 2589–2593. [20] Servi, S. (1990) BakerÕs yeast as a reagent in organic synthesis. Synthesis 1990, 1–25. [21] Kayser, M.M., Mihovilovic, M.D., Kearns, J., Feicht, A. and Stewart, J.D. (1999) BakerÕs yeast-mediated reductions of a-keto esters and an a-keto-b-lactam. Two routes to the paclitaxel side chain. J. Org. Chem. 64, 6603–6608.
Oxoester oxidoreductase activities in new isolates of Pichia anomala from apple, grape and cane juices Francisco Pe´rez-Mendoza a, Francisco Ruiz-Tera´n b, Blanca Escudero Abarca a, Arturo Navarro-Ocan˜a b, Guadalupe Aguilar-Uscanga a, Gerardo Valerio-Alfaro a,* b
a Instituto Tecnolo´gico de Veracruz-UNIDA, Av. Miguel A. de Quevedo #2779, Veracruz, Ver. 91897, Me´xico Facultad de Quı´mica, Edificio E, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior, Ciudad Universitaria, Coyoaca´n, C.P. 04510, D.F. Me´xico, Me´xico
Received 13 July 2004; received in revised form 23 November 2004; accepted 29 November 2004 First published online 24 December 2004
Abstract Thirty-nine yeasts isolated from apple, grape and cane juices were screened for their oxidoreductase activity. The two strains of Pichia, one isolated from apple and one from cane juices, appear to be promising strains for oxidoreductase activity on a-oxoesters. They showed similar high yields in converting ethyl pyruvate to ethyl lactate as Saccharomyces spp. (86.6% and 85.3% versus 86.6%), and higher yields in the reduction of a-oxocarboxylic esters (ketopantolactone to pantolactone: 74% and 73.3%, respectively) compared to Saccharomyces spp. (yield 60%). 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Saccharomyces spp.; Pichia anomala; a-Oxoesters; Oxidoreductase activity
1. Introduction It is becoming increasingly important to find new biocatalysts with stereoselectivity, regioselectivity, and substrate specificity that work under mild conditions for asymmetric synthesis of organic compounds. Some of these new biocatalysts including entire microbial cells [1–3], microbial cells with overexpressed enzymes [4], enzymes from animal tissues [5], cultured [6] and noncultured plant cells [7], have been used to transform and synthesize chiral compounds more selectively and under milder reaction conditions than conventional chemical catalysts. This will contribute to broadening the ‘‘green’’ routes for catalytic biotransformation of organic compounds in the fields of chemistry, biochemistry, and medicine. *
Corresponding author. E-mail address: [email protected] (G. Valerio-Alfaro).
In the case of the synthesis of optically active alcohols, new biocatalysts, such as immobilized dehydrogenase NAD-(P) from plant cells, food proteins isolated from pea, soybean and wheat are alternatives to bakerÕs yeast [8]. It has also been reported that other nonSaccharomyces yeast genera are able to compete with S. cerevisiae, which is the more common and widely accepted biocatalyst as a source of oxidoreductases [9,10]. There are reports where c- and d-ketoacids and their corresponding esters were reduced with yeasts such as Pichia fermentans, P. glucozyma, P. etchellsii, Candida boidinii, C. utilis and Kluyveromyces marxianus with high enantiomeric excess [11]. In contrast, methanolotrophic yeasts of the genera Pichia, Candida, Hansenula and Torulopsis showed alcohol oxidase activity with numerous primary alcohols that makes them potential catalysts for organic synthesis [12]. In the present work the oxidoreductase activity of several yeast strains isolated from different natural sources was first evaluated
1567-1356/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsyr.2004.11.010
686
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on a positive/negative basis, and thereafter those with better biotransformation yields than Saccharomyces spp. were selected. This opens up new possibilities for the synthesis of optically active compounds, and an alternative source of oxidoreductases from non-Saccharomyces strains.
2. Materials and methods 2.1. Chemicals Ethyl pyruvate, (R) and (S) ethyl lactates, ethyl benzoyl formate, (R) and (S) ethyl mandelate, ketopantolactone and (R)-pantolactone were obtained from Sigma–Aldrich (Milwaukee, WI, USA); sodium chloride, peptone and sucrose were obtained from J.T. Baker (Mexico). A commercial bakerÕs yeast ‘‘Instant Yeast Nevada’’ was acquired from a local supermarket. Precoated TLC plates of silica gel 60 GF254, and column chromatography silica gel grade 60, 70–230 mesh were used, both from Merck KgaA (Darmstadt, Germany). 2.2. Microorganisms Two commercial dried active Saccharomyces spp. cultures, BYC (bakerÕs yeast cultured) and BYNC (bakerÕs yeast not cultured), were used as reference strains. 39 Yeast strains were isolated from natural sources. These were labeled M1–M14 (isolated from apple juice), UV1–UV13 (isolated from grape juice), and CA1–CA12 (isolated from cane juice), followed by serial numbers. An additional five strains, labeled WBY1–WBY5, were obtained from bakeries in Veracruz City. 2.3. Culture and maintenance media: isolation medium Yeasts strains were isolated from 150 ml fruit juice supplemented with 1 g l1 (NH4)SO4, 5 g l1 glucose and 90 m g l1 of ampicillin, and initial pH was adjusted to 4.5 with orthophosphoric acid (10% w/w). The culture was incubated for 24 h at 30 C, 150 rpm on an orbital shaker. The samples were diluted with sterile water and spread on agar plates containing (g l1): glucose 10, agar 10 and yeast extract 1. The plates were incubated at 30 C for 2 d and then the pure cultures were spread on agar slants, stored at 4 C and subcultured at regular intervals (each 2 months). 2.4. Isolation medium for oxidoreductase activity Yeasts strains were suspended in 100 ml of medium containing 20 mg of substrate (ethyl pyruvate, ethyl benzoyl formate or ketopantolactone), 0.1 g yeast extract with the initial pH adjusted to 6.0. Media were sterilized
at 121 C for 15 min and incubated at 30 C on an orbital shaker at 250 rpm and screened from 8 to 30 h. 2.5. Screening of yeasts for oxidoreductase activity In a preliminary screening of oxidoreductase activity, shaken-flask assays were performed in duplicate. Previously isolated yeast strains [M1–M14, UV1–UV13, CA1–CA12, one non-cultured (BYNC) and one cultured (BYC) commercial strain of bakerÕs yeast] were incubated with 100 ml of medium. Inoculum was added to attain 3 · 106 viable cells per ml. Biotransformations were monitored by TLC (hexane:ethyl acetate, 7:3 v/v) at different times (8, 12, 24, 30 h). 2.6. Yeast identification The selected strains (5) were grown for 24 h at 30 C in 10 ml YM broth (3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g glucose per liter of distilled water) on a rotary shaker at 150 rpm and harvested by centrifugation at 13,000 rpm for 20 min. The cells were washed twice and resuspended with 1 ml of sterile distilled water in 1.5 ml Eppendorf tubes. Cells were mechanically broken down, using 1 ml of extraction buffer, pH 6.5 (200 mM Tris–HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS ) and a small capful of 0.5 mm glass beads. The DNA was extracted using the phenol–chloroform method [12,13]. Yeast identification was made according to Kurtzman and Robnett [14], sequencing the D1/D2 motif of the 26S-rDNA gene. PCR amplification was performed with primers NL1 (5 0 GCA TAT CAA TAA GCG GAG GAA AAG and NL4 5 0 GGT CCG TGT TTC AAG ACG G 3 0 , and nucleotide sequences were obtained with an ABIPRISM 310 Automated Sequencer (Perkin–Elmer, USA). Search and comparison of the partial sequence obtained (26S rDNA) in the GenBank were performed using the Blast-Program [15]. 2.7. Preparative biotransformations A sterile 180 ml broth was prepared in 250-ml flasks and an inoculum of 3 · 106 cells viable ml1 (determined by methylene blue staining method) [16] with 150 mg of substrate was added. The biotransformation was carried out at 30 C and 250 rpm on an orbital shaker until the reaction was complete (no more substrate was detected by TLC monitoring). The broth was saturated with NaCl, filtered over a Celite bed and then washed with 100 ml ethyl acetate. Additional extractions with the same solvent (100 · 3) were achieved over the recovered filtrate. The combined organic phases were dried over Na2SO4 and concentrated in a rotatory evaporator. The product of the reaction mixture was purified by column chromatography (hexane ethyl acetate, 7:3) until purified products were obtained.
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2.8. Instrumental analysis Optical rotations were measured using a Perkin– Elmer 343 polarimeter. IR spectra were recorded on a Paragon 500 FT Infrared spectrometer adapted with HATR accessory. Chromatographic analyses and mass spectra were performed on a Shimadzu QP 5050 gas chromatograph– mass spectrometer using an electronic impact procedure (75 eV) and a quadrupole analyzer. The column used in the chiral analyses was Chiraldex B-PM (a permethylated b-cyclodextrin column, 50 m long and 0.25 mm i.d.) obtained from Alltech Associates, Inc USA. Temperature conditions were determined for each substrate: ethyl pyruvate–lactate system, injector and detector 170 C, and a program column temperature of 50–180 C at 10 C min1; ketopantolactone–pantolactone system, injector and detector 180 C, a program column temperature 100–150 C at 10 C min1; ethyl benzoyl formate– ethyl mandelate system, injector and detector 180 C, a program column of temperature 100–140 C at 2.5 C min1. The column was able to detect the enantiomers without derivatization, and the elution times varied from 15 to 60 min for the various reactants and products. The enantiomeric excess (ee) for the reactions studied with substrates 1a–c (Fig. 1) was determined by gas chromatography chiral analyses and optical rotation comparing with pure chiral standards. The ee values were determined by ee = ([S] [R])/([S] + [R]) or ([R] [S])/([S] + [R]) for S and R products, respectively.
3. Results The substrates chosen in this study were ketoesters, such as natural short-chain ethyl pyruvate (1a), cyclic oxoester ketopantolactone (1b), and aromatic ethyl benzoyl formate (1c) (Fig. 1). In general, the ability to convert ketopantoyl lactone to pantoyl lactone is widely O
OH
Yeast O
R
oxidoreductases
O
R
O
O
1a, R= Me 1c, R= Ph
2a, R= Me 2c, R= Ph OH
O Yeast O O 1b
O oxidoreductases
O 2b
Fig. 1. Bioreduction reactions of a-oxoesters: (1a) ethyl pyruvate, (1b) ketopantolactone and (1c) benzoyl formic acid ethyl ester, mediated by native cultured yeasts to the corresponding a-hydroxyesters: (2a) ethyl lactate, (2b) pantolactone and (2c) ethyl mandelate.
687
observed in a variety of organisms, including yeasts [17]. In the first screening of the thirty nine different non-Saccharomyces yeast species isolated from fruit juices, plus one cultured and one non-cultured strain of bakerÕs yeast, seventeen yielded pantoyl lactone (2b) from (1b) in 8–30 h. An important ability to convert (1b) to (2b) was mainly found in yeast strains from cane (six) and apple (six), although three strains from grape converted the ketolactone (1b) into (2b). In the case of substrate (1a) (Table 1), we found 15 non-Saccharomyces strains plus both cultured and non-cultured strains of bakerÕs yeast producing (2a) within 8–30 h. In particular, yeast strains from apple showed a higher reductase activity to this substrate than to a-ketolactone (1b) in contrast with yeast strains from cane. Neither yeast strains from apple nor from cane expressed any reductase activity to ethyl benzoylformate (1c). On the other hand, out of all the juice strains only strain UV9 (from grape) converted benzoylformate (1c) to ethyl mandelate (2c). In contrast, ethyl pyruvate (1a) was reduced to ethyl lactate (2a) within 8–30 h by UV9, without being able to biotransform the a-ketopantolactone (1b) to the corresponding a-hydroxyl lactone (2b). Although reductase activities of the strains tested almost showed no relation to their source, in the case of both cultured and non-cultured strains of bakerÕs yeast, a notable ability to reduce all the a-ketoesters to the a-hydroxyesters within 8 and 30 h was found, in contrast to WBY1–WBY5 which were not able to reduce the a-oxoesters tested. Once the enzymatic activities of the isolates had been established, five of the isolates that exhibited pronounced enzymatic activity and two commercial yeast strain isolates were selected to test for a-oxidoreductase enantioselectivity. Even though oxoester reductase activities were observed in other isolates from apple, grape and cane juices, we decided to select strains M1, M7, CA10, CA11 and UV9 and commercial BYC and BYNC strains to continue screening studies, based on the criteria that these strains showed positive activity from 8 h onwards, except strain CA10 which showed excellent growth (Table 2). The biotransformations were scaled up to 150 mg and carried out with the same substrate biocatalyst (cell count) ratio (Table 2), including the cultured commercial strain of bakerÕs yeast. In the case of M1 and M7, CA10 and CA11, and BYC and BYNC, the products (2a) and (2b) were observed in 8–30 h. UV9 reduced (1a), but was not able to biotransform (1b). It is interesting to note that, although with low yields (9.5%), UV9 reduced (1c)–(2c). With the exception of BYC (8.6%) and BYNC (9.2%), this biotransformation was not observed in the other cultured yeast strains. The selected strains (Table 2) gave better yields in conversion of pantolactone (2b) compared with strains
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Table 1 Reactions for (a) ethyl pyruvate to ethyl lactate and (b) ketopantolactone to pantolactone mediated by isolated yeast strains and Saccharomyces spp. at different times Strain
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 UV1 UV2 UV3 UV4 UV5 UV6 UV7 UV8 UV9 UV10 UV11 UV12 CA1 CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 WBY1 WBY2 WBY3 WBY4 WBY5 BYC BYNC
Time conversion ethyl pyruvate (1a) to ethyl lactate (2a) (h)
Time conversion ketopantolactone (1b) to pantolactone (2b) (h)
8
12
24
30
8
12
24
30
+ + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
BYC and BYNC. In the second screening with the five cultured strains, the (S)-enantiomer ethyl lactate (2a) was the only detectable product (the chromatogrammass spectra indicated the presence of only one peak), and the enantioselectivity was >99% (in a racemic standard of (2a), two enantiomeric peaks with the same mass pattern but different retention times were resolved) (Table 3). Compared with ethyl pyruvate (1a), the reaction of ketopantolactone (1b) showed a mixture (1:1) of two enantiomeric peaks for the
product (2b), indicating the non-enantioselectivity of the process. Finally, with the third substrate, (R)-ethyl mandelate (1c), (2c) was the only product observed, indicating an enantioselectivity of the process of >99% ee. In addition, the (S)-(2a) and (R)-(2b) configurations were assigned based on the comparison of the sign of their specific rotations with those previously reported. In spite of biochemical differences among the isolates (data not shown), the five selected yeast (CA10, CA11,
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Table 2 Bioreductions of a-ketoesters with P. anomala and Saccharomyces spp. Straina
a-Hydroxy esterb
Yield (%)
a-Hydroxy ester
Yield (%)
a-Hydroxy ester
Yield (%)
M1 M7 CA10 CA11 UV9 BYC BYNC
2a 2a 2a 2a 2a 2a 2a
86.6 ± 0.71 84.0 ± 0.61 85.3 ± 0.62 85.3 ± 0.55 85.0 ± 0.44 86.6 ± 0.05 86.6 ± 0.05
2b 2b 2b 2b 2b 2b 2b
74.0 ± 0.48 66.6 ± 0.55 73.3 ± 0.33 64.0 ± 0.42 ND 60.00.36 60.00.29
2c 2c 2c 2c 2c 2c 2c
– – – – 9.5 ± 0.71 8.6 ± 0.59 9.2 ± 0.66
a b
M1 to UV9 are isolated strains of P. anomala and BYC and BYNC are cultured and non-cultured strains of bakerÕs yeast. The values represent the mean ± SD of three measurements.
Table 3 Enantiomeric excess by polarimetry and GC–MS chiral Strains
Product
[a] Exp.a
eeb (%)
eec (%)
Configuration
Ethyl lactate M1 2a M7 2a CA 10 2a CA 11 2a BYC 2a BYNC 2a
9.8 10.0 10.1 10.1 9.9 9.8
97 100 100 100 98.7 98.4
>99 >9 >9 >9 >9 >9
S S S S S S
Pantolactone M1 2b M7 2b CA 10 2b CA 11 2b BYC 2b BYNC 2b
0.6 0.5 0.5 0.5 0.4 0.4
0.01 0.01 0.01 0.01 0.01 0.01
0.7 0.4 0.4 0.3 0.2 0.2
n.d.d n.d. n.d. n.d. n.d. n.d.
130.1 130.4 124.0
97.0 97.4 92.5
>99 >99 >99
R R R
Ethyl mandelate UV9 2c BYC 2c BYNC 2c a b c d
Experimental optical rotation. Enantiomeric excess by polarimetry. Enantiomeric excess by GC–MS. n.d., Not determinated.
UV9, M1, M7) were identified as Pichia anomala strains using the partial sequence 26S rADN.
4. Discussion Among the many types of carbonyl-containing compounds that have been reduced using bakerÕs yeast, the main focus has been on enantioselective reductions of b-ketoesters and ketocarboxylic acids. To a lesser extent, a-ketoesters have also been investigated, along with c- and d-ketoesters, lactones, and diketonic compounds. However, with some substrates low chemical yields and ee are reported. For instance, ketopantoyl lactone (1b) was reduced to pantoyl lactone (2b) by a bakerÕs yeast culture with a chemical yield of 42%, and an ee of 50% [17]. The non-enantioselectivity of ketopantoyl lactone (1b) bioreduction shown by all the isolates suggests that
this substrate is utilized by different competitive ketoreductases with different stereoselectivities. Although the ability to convert (1b)–(2b) is widely encountered in a variety of microorganisms, there is a variety of stereospecificity in the ratio of D -()-2b and L -(+)-2b isomers yielded by molds, yeasts, basidiomycetous fungi and bacterial strains which bear no relation to the genera or sources [18]. Nakamura et al. [19] have found a-ketoesters with from 3 to 8 carbon-atoms to be reduced, and they compared the results of incubation with normal bakerÕs yeast, immobilized bakerÕs yeast in water, and immobilized bakerÕs yeast in hexane [17]. The enantiomeric excess ranged from 30% to 94% and decreased with increasing alkyl chain size. Similar results were obtained in stereoselective reduction of ethyl pyruvate (1a) to (S)-ethyl lactate (2a) with fermenting bakerÕs yeast. The differences in stereoselective reduction of the substrates (1a), (1b) and (1c), with isolates from apple, grape and cane juices, and with BYC and BYNC, could follow from differences in reduction rate of ketoester reductases acting simultaneously with opposite stereochemical substrate specifities, as has been observed with bakerÕs yeast oxido-reductases [20]. In the case of openchain a-oxoesters, enzymatic reductions can provide high enantioselectivity. On the other hand, the diminished enantioselectivity was found in hydroxyl ester products when a substrate is accepted by multiple enzymes has been attributed either to the fact that some of these reductases possess overlapping substrate specificities but with opposite enantioselectivities, or that a single reductase with relatively low enantioselectivity can deliver hydrides to both Re/Si faces in the substrate [21]. With respect to chiral results, similar enantiomeric excess was observed by polarimetry and GC–MS (Table 3, columns 4 and 5). These results show that the reductions by our P. anomala strains obey PrelogÕs rule in cases (2a) and (2c). From the yeasts examined, P. anomala was found to be the most versatile strain in cane, apple and grape juices acting on a-oxoesters. The enantioselectivity was
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consistently high in bioreduction of ethyl pyruvate, nonenantioselective with ketopantolactone, although with good yields in both cases. In the case of benzoyl formic acid ethyl ester, one strain of P. anomala (UV9) was found to reduce this substrate with a low yield and excellent enantioselectivity. Non-cultured and cultured commercial strains of bakerÕs yeast also yielded similar biotransformation results. The relevance of this study is that these biotransformations are an alternative to those with well-known Saccharomyces species, whereby the catabolic activities observed in native yeasts contribute to a broadening of the bioreduction spectrum.
Acknowledgements The authors thank Patricia M. Hayward Jones and Dulce Ma. Barradas Dermitz for a critical reading of the manuscript.
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