Stereospecific microbial production of isoflavanones ... - Springer Link

Appl Microbiol Biotechnol (2011) 91:1173–1181 DOI 10.1007/s00253-011-3310-7

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Stereospecific microbial production of isoflavanones from isoflavones and isoflavone glucosides Hye-Yeon Park & Mihyang Kim & Jaehong Han

Received: 17 March 2011 / Revised: 1 April 2011 / Accepted: 5 April 2011 / Published online: 12 May 2011 # Springer-Verlag 2011

Abstract A Gram-negative anaerobic microorganism, MRG-1, isolated from human intestine showed high activities of deglycosylation and reduction of daidzin, based on rapid TLC analysis. A rod-shaped strain MRG1was identified as a new species showing 91.0% homology to Coprobacillus species, based on 16S rRNA sequence analysis. The strain MRG-1 showed β-glucosidase activity toward daidzin and genistin, and daidzein and genistein were produced, respectively. However, the strain MRG-1 did not react with flavone glycosides, flavanone glycosides, and isoflavone C-glucoside. Besides, MRG-1 showed stereoselective reductase activity to isoflavone, daidzein, genistein, 7-hydroxyisoflavone, and formononetin, resulting in the formation of corresponding R-isoflavanone enantiomers. The new isoflavanones of 7-hydroxyisoflavanone and dihydroformononetin were characterized by NMR, and the absolute configurations of the enantiomers were determined with CD spectroscopy. The kinetic study of the anaerobic biotransformation showed both activities were exceptionally fast compared to the reported conversion by other anaerobic bacteria. Keywords Biotransformation . Daidzein . Equol . Isoflavonoids . Stereospecificity

Electronic supplementary material The online version of this article (doi:10.1007/s00253-011-3310-7) contains supplementary material, which is available to authorized users. H.-Y. Park : M. Kim : J. Han (*) Metalloenzyme Research Group and Department of Biotechnology, Chung-Ang University, Anseong 456-756, South Korea e-mail: [email protected]

Introduction There are growing interests in human gut microorganism interactions with dietary polyphenolic compounds due to the potential implication to health-related benefits (Laparra and Sanz 2010; van Duynhoven et al. 2010). For examples, plant lignans are converted by intestinal microbiota to enterodiol and enterolactone (Jin et al. 2007), and ellagic acid is transformed to urolithins A and B (Larrosa et al. 2006). Enterolactone was reported to induce apoptosis and inhibit human colon cancer cells (Chen et al. 2007), and the urolithins were shown to inhibit the growth of human breast cancer cells (Larrosa et al. 2006). Soy isoflavone daidzein is converted to equol, found to inhibit the growth of benign and malignant prostate epithelial cells in vitro at the concentration obtained from the dietary soy consumption (Hedlund et al. 2003). Along with anti-androgenic activity (Lund et al. 2004), equol was reported to show estrogenic (Hwang et al. 2006), anticancer (Lee et al. 1991), cardioprotective (Lampe et al. 1998), and antioxidant activities (Miyase et al. 1999). While chemical synthesis of equol from daidzein produces racemic mixture of S-equol and R-equol, bacterial biosynthesis is known to produce exclusively S-equol, a biologically relevant isoflavonoid. Because S-equol shows different biological activities from its enantiomer R-equol (Muthyala et al. 2004), we have studied stereochemistry of S-equol biosynthesis (Kim et al. 2008; Kim et al. 2009) and established the biosynthetic pathway of the unusual (3R,4S)-tetrahydrodaidzein to (3S)-equol conversion by Eggerthella species Julong 732 isolated from the human intestine (Kim et al. 2010a). Although the biotransformation of DHD (dihydrodaidzein) to equol has been indisputably established from Julong 732, the stereochemistry of the preceding reaction,

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daidzein to DHD conversion, has never been studied. Previously, Hur et al (2000) reported bacterial strain HGH6 could produce DHD from daidzein in 7 days, and later, Tamura et al (2007) reported DHD production from daidzin by the Chlorstridium-like anaerobe. The report on Niu-O16 from bovine rumen by Wang et al (2005) is the only study to discuss about the stereochemistry of DHD racemate produced from daidzein. In the search of anaerobic microorganisms metabolizing isoflavones and their glucosides to the isoflavanones, a new anaerobic bacterium, MRG-1, exhibiting exceptionally high conversion rate of isoflavanone production was isolated from human intestine. MRG-1 converted various isoflavones and their glucosides to isoflavanones in a stereospecific manner. To the best of our knowledge, the stereospecificity of bacterial isoflavanone formation has never been studied due to the relatively fast enol tautomerization of the product. The reaction by MRG-1 was much faster than the isomerization, and stereospecificity of isoflavone reductase was able to be studied. Here, we report the substrate specificity and biotransformation kinetics of MRG-1 for isoflavonoids. The stereochemical property of the isoflavone reductase from MRG-1 was also studied by means of CD spectroscopy.

Materials and methods Chemicals Flavonoids were purchased from Indofine Chemical Company (Hillsborough, NJ, USA). 1,4-Naphthoquinone was purchased from Sigma-Aldrich Korea (Yongin. Korea). DHD and DHG (dihydrogenistein) were synthesized according to the published method (Kim et al. 2009). GAM (Gifu Anaerobic Medium) used for the isolation and growth media was purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). Acetonitrile, ethyl acetate, and methanol (HPLC grade) were purchased from Fisher (Pittsburgh, PA, USA). Thin layer chromatography (TLC) silica gel 60 F254 plates were obtained from Merck (Merck, Darmstadt, Germany). General methods To monitor the product formation, a Finnigan Surveyor Plus HPLC system (Thermo Scientific, Waltham, MA) equipped with a photodiode array detector (PDA Plus) and a C18 reversed-phase column (Hypersil GOLD 5 μm, 4.6 by 100 mm; Thermo Scientific, Waltham, MA) was employed and monitored at 291 nm. The flow rate was 1 ml/min. 1H and 13 C nuclear magnetic resonance (NMR) spectra of the compounds in dimethyl

Appl Microbiol Biotechnol (2011) 91:1173–1181

sulfoxide (d6) were respectively obtained at 600 and 150 MHz on an Avance 600 NMR spectrometer (Bruker, Germany) at 296 K. The CD spectra of the enantiomers in ethanol were measured using a J-715 CD spectropolarimeter (Jasco Corp., Tokyo, Japan). Bacterial isolation and culture conditions Fecal sample in a healthy female was taken in the sterilized vial and placed immediately in anaerobic chamber (CO2 5%, H2 10%, N2 85%) at 37°C. Using cotton swap, the sample was diluted in saline solution, and the solution was filtered through sterile cheesecloth for the isolation. For broth, GAM (5.9 g) in 100 mL of distilled water was used, and 1.5 g of agar was added for plate. For dilution of sample, 1 mL of original filtrate was diluted to 10 mL in GAM broth to spread on the plate. The diluted broth (0.1 mL) was spread on the zone-marked plate for the isolation. Single colonies in each zone were taken and inoculated to the GAM broth media (1 mL) containing daidzin (200 μL/mL) and anaerobically incubated in the anaerobic chamber for 2 days at 37°C. For the activity check, 1 mL of ethyl acetate was added, and the mixture was vortexed for 20 s. After centrifugation, 800 μL of supernatant was taken to the dryness under vacuum. For the thin layer chromatography (TLC) analysis, 2 μL out of 10 μL of each methanol extract were applied with reference compounds of daidzein, dihydrodaidzein (DHD), tetrahydrodaidzein (THD), and Sequol on silica gel TLC plate. The developing solvent was toluene:acetone=2:1. Although S-equol producing activity was observed, only one bacterial strain, named MRG-1, producing DHD from daidzin was isolated. The isolated strain MRG-1 was deposited in the Korean Collection for Type Cultures (KCTC) under accession number KCTC11894BP. 16S rRNA gene sequence analysis was performed for the identification of strain MRG-1. Strain MRG-1 was grown in the 5 mL of GAM broth media in an anaerobic chamber for 1 day. The 16S rRNA gene was amplified by PCR using the universal bacteria primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′), 1492R (5′-GGY TA C C T T G T TA C G A C T T- 3 ′ ) a n d 7 8 3 R ( 5 ′ ACCMGGGTATCTAATCCKG-3′). Amplification reactions, PCR product purification, and sequencing of partial 16S rDNA sequences (1,263 bp) were performed by Solgent Inc. (Daejeon, Korea) Sequencing service. The sequence of 16S rDNA was deposited in GenBank under accession number HQ687764.The data of 16S rDNA gene sequencing were analyzed using EzTaxon server (http://www.eztaxon.org/) for sequence similarity (Chun et al. 2007). The phylogenetic tree was constructed by the neighbor-joining method using MEGA 4 program (Saitou and Nei 1987). The neighborjoining tree topologies were evaluated by bootstrap analyses based on 1,000 resampled datasets (Felsenstein 1985).


Biotransformation of isoflavonoids For the substrate specificity experiments, strain MRG-1 was grown to OD 0.2 at 600 nm in GAM broth media containing 100 μM of the tested substrate. After 1 day, 1 mL of ethyl acetate was added, and the mixture was vortexed for 20 s. After centrifugation, 800 μL of supernatant was taken to dryness under vacuum. For the HPLC analysis, 10 μL of 100 μL methanol extract were used for injection. Finnigan Surveyor Plus HPLC system (Thermo Scientific, Waltham, MA) equipped with a photodiode array detector (PDA Plus) and a C18 reversed-phase column (Hypersil GOLD 5 μm, 4.6 by 100 mm; Thermo Scientific, Waltham, MA) was employed and monitored at 291 nm. The mobile phase was composed of 10% acetonitrile in 0.1% acetic acid (A) and 90% acetonitrile in 0.1% acetic acid (B). The elution profile started with an A/B ratio at 80:20 (v/v) for 3 min and linearly to 20:80 (v/v) for 12 min. Biotransformation kinetics For the biotransformation kinetics, isoflavonoids of daidzein, genistein, daidzin, genistin, formononetin, and 7hydroxyisoflavone were used. Strain MRG-1 was grown to OD 0.5 at 600 nm in GAM broth media containing 100 μM of each substrate. The bacterial culture (100 μL) was taken and allocated for the reaction. In every 10 min, 1 mL of ethyl acetate was added to the reaction mixture and vortexed for 20 s. After centrifugation, 800 μL of supernatant was taken to dryness under vacuum. For the HPLC analysis, 10 μL of 100 μL methanol extract were used for injection. Finnigan Surveyor Plus HPLC system (Thermo Scientific, Waltham, MA) equipped with a photodiode array detector (PDA Plus) and a Sumi-chiral OA-7000 column (5 μm, 4.6 by 250 mm) was employed and monitored at 291 nm. The mobile phase was composed of 10% acetonitrile in 0.1% acetic acid (A) and 90% acetonitrile in 0.1% acetic acid (B). The elution profile was same as above.

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Chiral OA-7000 column (5 μm, 8 by 250 mm) chromatography. Metabolite 1 (7.1 mg) and metabolite 2 (7.7 mg), as well as formononetin (5.5 mg) were isolated after solvent removal. For 7-hydroxyisoflavone, 300 μL of 7-hydroxyisoflavone (100 mM) was added to the cell culture three times in every hour, and the solution was reacted for 24 h. Ethyl acetate (300 mL X 5) was added to the reaction mixture to extract the products. The isolation method of the products was same as the one for formononetin. All the substrate was biotransformed and metabolite 1 (2.9 mg) and metabolite 2 (9.9 mg) were isolated after solvent removal.

Results Isolation and identification of strain MRG-1 The activity-guided screening of daidzein-metabolizing bacteria was carried out by TLC. The activity check by TLC was much faster than conventional HPLC analysis. Although TLC analysis was semi-quantitative regarding product formation, it was enough for the screening purpose. TLC analyses found that the multi-colony culture converted daidzein (Rf =0.28) to DHD (Rf =0.35) (Fig. 1S, panel A) to DHD. The selected multi-colony cultures (Fig. 1S, panel A, lanes 5, 6, 7, and 11) were further separated to the single colony cultures and individually incubated with daidzein to isolate the bacterial colonies responsible for the conversion of daidzein (Fig. 1S, panel B, lanes 5–2, 6–1, 7–1, and 11– 1). Finally, a rod-shaped, Gram-negative strict anaerobic bacterium, named MRG-1, was cultured in GAM medium and its 16S rRNA was sequenced for the identification. The 16S rDNA partial sequence (1,263 bp) showed 91.04% similarity to that of Coprobacillus cateniformis JCM 10604T (accession no. AB030219) (Fig. 1). The growth curve of strain MRG-1 in GAM medium under anaerobic conditions at 37°C showed that the cell growth reached stationary phase in 7 h after 3-h incubation period (Fig. 2S). Substrate range of strain MRG-1

Isolation and characterization of biotransformation products For the identification of formononetin and 7-hydroxyisoflavone biotransformation product, preparative scale biotransformation was carried out. To the cell culture (OD600 =0.2, 220 mL), 220 μL of formononetin (100 mM) was added four times in every hour, and the solution was reacted for 190 h. Ethyl acetate (200 mL X 3) was added to the reaction mixture to extract the products. The ethyl acetate extract was dried and dissolved in DMF (1 mL) for the preparative HPLC. Each injection was 50 μL and two metabolites were isolated from semi-preparative Sumi-

Strain MRG-1 produced daidzein and DHD from daidzin biotransformation (Fig. 2). Since reduction product of daidzin (DHD glucoside) was not found, it was concluded that only daidzein underwent reduction to DHD. Daidzein reduction was completed within an hour, and there was no substrate inhibition up to the concentration of 2 mM daidzein. Interestingly, preferential R-DHD formation was observed from the chiral column HPLC analysis, which was identified by comparison with the prepared authentic compounds (Won et al. 2008). It is well known that racemization of DHD in medium, through tautomerization, is so fast that often both

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Fig. 1 Neighbor-joining phylogenetic tree of strain MRG-1 based on 16S rDNA gene sequences. Bootstrap percentages greater than 50% (based on neighbor-joining analyses of 1,000 resampled data sets) are shown at nodes. Bar, 0.02 nucleotide substitutions per position. The sequence of 16S rDNA was deposited in GenBank under accession number HQ687764

enantiomers are observed from biotransformation. However, the microbial reduction of daidzein by MRG-1 was much faster than the isomerization, and the stereospecific R-DHD production was confirmed (Fig. 3). Because strain MRG-1 showed two different activities of β-glucosidase and reductase, substrate specificity of strain MRG-1 was tested using a variety of flavonoids, rutin, nariginin, and hesperidin, the flavone and flavanone glycosides with rutinosyl groupi at C3 and C7. β-glucosidase activity was not observed with all tested flavonoid substrates. Reductase activity was not found with flavone, 7-hydroxyflavone, and 4 ,7-dihydroxyflavone.

Fig. 2 Daidzin biotransformation by strain MRG-1

On the contrary, strain MRG-1 showed reactivity for the various isoflavonoids (Table 1). First, it hydrolyzed β-glucoside linkage at C7 of isoflavone and produced daidzein and genistein from daidzin and genistin, respectively. But C-glucoside of puerarin was not metabolized. Methoxy group on C7 was not hydrolyzed, either. Second, it catalyzed stereospecific reduction of isoflavone C2–C3 double bond. Isoflavone, 7-hydroxyisoflavone, daidzein, formononetin, and genistein produced the corresponding R-isoflavanones (vide infra), although they were isomerized to the racemic mixture in the medium eventually.


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Fig. 3 HPLC chromatograms using chiral column showing the time-course of strain MRG-1 biotransformation of daidzein

of other isoflavone in which only 80% conversion was observed in 3 h incubation (Fig. 4d).

Biotransformation kinetics Strain MRG-1 transformed 0.1 mM of daidzin and genistin completely within 100 min (Fig. 4a, b). The deglycosylated metabolites, daidzein and genistein, were first produced from both substrates in 10 min. The reduction of daidzein and genistein was observed after 10 min, and the reaction was much faster than the deglycosylation reaction. Reduction of daidzein, genistein, and 7-hydroxyisoflavone (Fig. 4c) was completed in 30 min. Reduction of formononetin by strain MRG-1 was found different from those

Table 1 Substrate specificity of MRG-1 R3 8

R2

O

7

2

6

3

2' 3'

4

5 R1

4'

O

R4

a

Isoflavonoids

R1

R2

R3

R4

Product

Isoflavone

H

H

H

H

R-isoflavanone

7-Hydroxyisoflavone

H

OH

H

H

R-7-hydroxyisoflavanone

7-Methoxyisoflavonea

H

OCH3 H

H

No reaction

Daidzein

H

OH

H

OH

R-DHD

Daidzin

OH OGlc

H

OH

Daidzein, R-DHD

Formononetin

H

OH

H

OCH3

R-DHF

Genistein

OH OH

H

OH

R-DHG

Genistin

OH OGlc

H

OH

Genistein, R-DHG

No reaction in 3 days

Isolation and characterization of dihydroformononetin (DHF) and 7-hydroxyisoflavanone enantiomers The reduction products of formononetin and 7hydroxyisoflavanone were isolated from semi-preparative Sumi-chiral HPLC. The structures of isolated products were characterized with 1H and 13C NMR spectroscopy (Table 2, Fig. 3S), and the absolute configuration was determined by CD spcectroscopy. Compared to the isoflavone substrate, the reduction products showed characteristic peaks corresponding to H-2 and H-3 in the upfild of 1H NMR spectrum. In fact, the C-ring double bond hydrogen atoms of all the reduction products showed very similar 1H NMR peaks at the range between 4.0 and 4.6 ppm. While dihydroformononetin showed characteristic methoxy peak at 3.72 ppm, 7-hydroxyisoflavanone shows multiplets of Bring protons at the region between 7.26 and 7.32 ppm. The reduction of substrate also resulted in typical UV– Vis spectral changes, and dihydroformononetin showed absorption bands at 231, 276, and 313 nm. The formononetin showed two absorption bands at 249 and 302 nm. Similary, UV-absoprtion bands of 7-hydroxyisoflavane at 247 and 301 nm changed to 235, 276, and 312 nm after reduction to 7-hydroxyisoflavanone. For the absolute configuration determination of two enantiomers separated from chiral column chromatography (Fig. 4S), CD spectra in ethanol were measured (Fig. 5). The spectra features were similar to those of reported DHD and DHG (Wang et al. 2005). The lonest wavelength CD transitions found at the region of 320 and 350 nm are due to n→π* transition. It was reported that R-isoflavanone and S-isoflavanone showed positive and negative Cotton-effect in this region, respectively (Kim et al. 2010b; Won et al. 2008).

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a

b 1.0

Conversion rate (291nm)


1.0 0.8 0.6 0.4 0.2 0.0

0.6 0.4 0.2 0.0

0

20

40

Dzin Dz DHD

60

80

100

120

0

Time (min)

20

Gntin Gn DHG

c

40

60

80

100

120

Time (min)

d 1.0


1.0


0.8

0.8 0.6 0.4 0.2 0.0

0.8 0.6 0.4 0.2 0.0

0

10

20

30

40

50

Time (min) Dz DHD Gn DHG 7-Hydroxyisoflavone 7-Hydroxyisoflavanone

0

10

FM DHF

20

30

40

50

Time (min)

Fig. 4 Biotransformation kinetics of strain MRG-1 with daidzin (A), genistin (B), daidzein, genistein, 7-hydroxyisoflavone (C), and formononetin (D)

Acoordingly, DHF1 and DHF2, first and second fraction from the chiral separation, were assigned as S-DHF and RDHF2, respectively (Fig. 5a). The absolute configuration of 7-hydroxyisoflavanone enantiomers was also determined analogously (Fig. 5b).

Discussion Due to the various beneficial biological activities of flavonoids as neutraceuticals, growing numbers of reports on flavonoids, especially related to microbial biotechnology, are emerging. For example, deglycosylation (Raimondi et al. 2009), glycosylation (Jiang et al. 2008), and oxidation

(Seo et al. 2010a, 2010b and 2011) of flavonoids, along with metabolic engineering of Escherichia coli for the biosynthesis of certain flavonoids (Du et al, 2010), have been reported. However, stereospecific microbial reduction of flavonoids is rare and only a few, related to S-equol biosynthesis, are reported (Kim et al. 2008). A rod-shaped Gram-negative MRG-1 strain was isolated from human intestine by means of activity-guided screening. Comparison of partial 16S rDNA (1,263bp) of strain MRG-1 showed low similarity to the known species (Fig. 1b). However, high sequence similarity (99%) was found with strain TM-40, which was reported by Tamura et al (2007). Even though TM-40 was reported as Clostridiumlike bacterium, we suggest strain MRG-1 belongs to

Appl Microbiol Biotechnol (2011) 91:1173–1181 Table 2

1

H (400 MHz) and

1179

13

C (100 MHz) NMR spectral data of dihydroformononetin ((CD3)2SO) and 7-hydroxyisoflavanone (CDCl3)

Dihydroformononetin

7-Hydroxyisoflavanone

δ of

δ of

13

71.2 50.2 190.6 129.1 110.8 163.2 102.4 113.6 164.7 128.0 129.8 113.9 158.5 55.1

C

δ of 1H

4:55 dd; JH2a;2b ¼ 11:3 Hz; JH2a;3 ¼ 4:2Hz; 1H; H 2a 4:61 dd; JH2a;2b ¼ 11:3Hz; JH2b;3 ¼ 8:8Hz; 1H; H 2b 3:96 dd; JH2a;3 ¼ 4:2Hz; JH2b;3 ¼ 8:8Hz 7.66 6.51 3.16 6.34

(d, JH5,6 =8.8 Hz) (dd, JH5,6 =8.8 Hz, J (s, OH) (d, JH6,8 =2.1 Hz)

H6,8 =2.1 Hz)

7:17 d; JH20 ;30 ¼ 8:8Hz; 2H 6:89 d; JH20 ;30 ¼ 8:8Hz; 2H 3.72 (s, 3H)

Fig. 5 CD spectra of dihydroformononetin and 7hydroxyisoflavanone enantiomers

13

71.8 51.9 190.9 130.1 110.6 162.5 103.1 115.3 163.6 135.1 128.8 128.6 127.7

C

Assignment

δ of 1H 4.62 (m, 2H)

2

3:91 dd; JH2a;3 ¼ 2:8Hz; JH2b;3 ¼ 6:1Hz 7.87 (d, JH5,6 =8.8 Hz) 6.51 (dd, JH5,6 =8.8 Hz, J 6.40 (d, JH6,8 =2.1 Hz)

7.26–7.32 (m) 7.26–7.32 (m) 7.26–7.32 (m)

H6,8 =2.1

Hz)

3 4 5 6 7 8 4a 8a 1′ 2′,6′ 3′,5′ 4′ 4′-OMe

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Coprobacillus species in the same Clostridiaceae family according to the argument by Kageyama and Benno (2000). The strict anaerobic bacterium MRG-1 showed βglucosidase activity for daidzin and genistin and reductase activity for isoflavones including daidzein. Although βglucosidase activity from intestinal microorganisms is common, isoflavone reductase activity is unusual and of great interest due to the stereoselective product formation. HGH6 (Hur et al. 2000), TM-40 (Tamura et al. 2007), and Niu-16 (Wang et al. 2005) have been reported to have similar activity. Under the reported conditions, bacterial strain HGH6 and TM-40 converted daidzin to DHD incompletely. Strain Niu-O16 was reported to produce both R- and S-DHD enantiomers. On the contrary, strain MRG-1 completely converted substrates and produced only R-DHD from daidzin within 2 h and from daidzein within 40 min, respectively (Fig. 3). From the kinetics study (Fig. 4), it was clear that slower β-glucosidase activity of MRG-1 was the rate limiting step of DHD production from daidzin. From the substrate specificity study at Table 1, MRG-1 could not reduce flavones but it can reduce isoflavones in a stereospecific manner. Isoflavone, 7-hydroxyisoflavone, daidzein, formononetin, and genistein were reduced to the corresponding R-isoflavanones. It appears that the substrate range for reductase is relatively broad in isoflavone compounds, regardless of location of hydroxyl groups. It could not reduce any flavone compounds either 1,4naphthoquinone. Therefore, it was concluded the reductase requires aromatic B-ring at C3 position for the activity. The stereochemistry of isoflavones reduction products, including DHD, was also studied. All the isoflavone substrates, isoflavone, 7-hydroxyisoflavone, daidzein, genistein, and formononetin, metabolized by strain MRG-1 produced corresponding reduction product R-isoflavanones. Especially, 7-hydroxyisoflavanone and dihydroformononetin have never been characterized before to the best of our knowledge. In conclusion, new anaerobic bacterium MRG-1 was isolated, and the biotransformation property has been characterized. The exceptionally high conversion rate of isoflavone reduction was stereoselective, and the possible biotechnological applications of the enantiopure isoflavanone production are manifested. Acknowledgements The authors thank Ms. Jae Eun Hwang for her excellent NMR measurements. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 331-2008-1-F00014).

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