Production of Daidzein and Genistein from Seed and Root Extracts of Korean Wild Soybean (Glycine soja) by Thermostable β-Galactosidase from Thermoproteus uzoniensis

Featured Application

The β-galactosidase enzyme identified in this study can be used to convert isoflavone glycosides to aglycones in wild soybeans.

Abstract

Isoflavone glycosides are commonly biotransformed into isoflavone aglycones due to the superior biological activities of the latter. Wild soybeans contain a higher isoflavone content than domesticated soybeans due to their high level of genetic diversity. In this study, we cloned and characterized a thermostable β-galactosidase from the extreme thermophile Thermoproteus uzoniensis for potential application in isoflavone conversion in Korean wild soybeans. The purified recombinant enzyme exhibited a maximum specific activity of 1103 μmol/min/mg at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme exhibited the highest activity for p-nitrophenyl (pNP)-β-D-galactopyranoside among aryl glycosides and it hydrolyzed isoflavone glycosides in the order genistin > daidzin > ononin > glycitin. The enzyme completely hydrolyzed 2.77 mM daidzin and 3.85 mM genistin in the seed extract of wild soybean after 80 and 70 min with productivities of 1.86 and 3.30 mM/h, respectively, and 9.89 mM daidzin and 1.67 mM genistin in the root extract after 180 and 30 min, with the highest productivities of 3.30 and 3.36 mM/h, respectively, compared to other glycosidases. Our results will contribute to the industrial production of isoflavone aglycone using wild soybean and this is the first report on the enzymatic production of isoflavone aglycones from isoflavone glycosides in wild soybeans.

1. Introduction

Isoflavones, one of the most abundant phytochemicals in leguminous plants, have attracted attention because of their activity against cancer [1], cardiovascular disease [2], osteoporosis [3], and menopausal symptoms [4]. The biological activity of isoflavones is improved by their deglycosylation into aglycones, which are more quickly absorbed in the gut because of their lower molecular weight and better hydrophobicity [5,6]. However, isoflavone aglycones exist at low concentrations in leguminous plants. Therefore, many studies have focused on the biotransformation of isoflavone glycosides such as daidzin, genistin, and glycitin into their corresponding isoflavone aglycones; daidzein, genistein, and glycitein. These isoflavone aglycones have been obtained through the hydrolytic activity of microorganisms, including Bacillus subtilis, Enterococcus durans, Lactobacillus paraplantarum, Rhizopus oligosporus, Streptococcus salivarius, and Weissella confusa [7,8,9]. However, microbial hydrolysis has several disadvantages, including low productivity, poor yield, and the production of by-products. Therefore, enzymes have been commonly used for the hydrolysis of isoflavone glycosides because of their high specificity, productivity, and yield. Although various microbial and plant-derived glycosidases have been used, thermostable glycosidases from thermophilic microorganisms, including Dictyoglomus turgidum [10], Microbulbifer thermotolerans [11], Neosartorya fischeri [12], Pyrococcus furiosus [13], Sulfolobus solfataricus [14], Talaromyces leycettanus [15], Thermofilum pendens [16], and Thermotoga maritima [17] have exhibited high hydrolytic activity toward isoflavone glycosides. In order to use thermostable glycosidases from thermophilic microorganisms, it is more efficient to use recombinant enzymes expressed in E. coli through cloning, because the growth conditions of thermophilic microorganisms are generally harsh.

The extreme thermophile Thermoproteus uzoniensis is a rod-shaped archaebacterium found in hot springs and soil of the Uzon caldera (SW of Kamchatka pen.) [18]. Glycosidase from this strain is a powerful candidate for application in the hydrolysis of isoflavone glycosides because it showed high hydrolytic activity on ginsenosides (ginseng saponins) in a previous study [19]. However, this enzyme has not yet been characterized.

Soybean (Glycine max (L.) Merr.), a representative leguminous plant, contains a high abundance of isoflavones. However, significant differences have been noted in the total isoflavone content of soybean depending on the country or region of cultivation [20]. Wild soybean (Glycine soja Siebold & Zucc.), the ancestor of the domesticated soybean, was found to harbor a high level of genetic diversity that was abolished during the process of domestication [21]. Therefore, it has been previously suggested that the isoflavone content of domesticated soybean is not as high as that of wild soybean due to unintentional selection [22] and Korean wild soybeans have been reported to contain a high amount of isoflavones [23,24,25].

The global Crop Wild Relatives (CWR) project, launched in 2011, aims to adapt agriculture to climate change. The wild relatives of crops, which have evolved to survive tough conditions, can often contain useful traits, the analysis of which could help develop crops that are more resilient to climate change. Soybeans are one of the main target species for this project and wild soybean is an important CWR of soybean. Thus, considering the global importance of the soybean crop, the enzymatic conversion of isoflavones in wild soybeans to their aglycones is of great interest.

In this study, we cloned a thermostable β-galactosidase from T. uzoniensis and characterized its properties, such as molecular weight, pH, temperature, thermostability, and substrate specificity. The enzyme was used to produce daizein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybeans.

2. Materials and Methods

2.1. Materials

The aryl-glycosides, p-nitrophenyl (pNP)-β-D-glucopyranoside, pNP-β-D-galactopyranoside, pNP-β-D-xylopyranoside, pNP-α-L-arabinopyranoside, pNP-α-L-rhamnopyranoside, oNP-β-D-glucopyranoside, and oNP-β-D-galactopyranoside; and isoflavones, including daidzin, daidzein, genistin, genistein, glycitin, and glycitein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Korean wild soybeans were provided by the Baekdudagan Wild Plant Seed Bank. All enzymes used for gene cloning were purchased from Thermo Fisher Scientific (Waltham, MA, USA) or New England Biolabs (Hertfordshire, UK).

2.2. Bacterial Strains, Plasmids, and Gene Cloning

The DNA template and expression vector for the β-galactosidase gene (GenBank accession no. AEA12778) cloning and host strain were T. uzoniensis DSM 5263 (DSMZ, Braunschweig, Germany), pET-29b (+) vector (Novagen, Darmstadt, Germany), and Escherichia coli BL21 (DE3), respectively. The β-galactosidase gene was cloned using the one-step isothermal DNA assembly method [26]. For the amplification of DNA fragments, the following primers were used: forward primer for β-galactosidase gene, AGC AGC GAA AAC CTG TAT TTT CAG GGA CAT ATG CGG AAG TTC CCA AGC GGA TTC AGA TGG; reverse primer for β-galactosidase gene, ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TTA AAG CGA TCT GAG GCC CCT CAA GAC CTC; forward primer for vector, GAG GTC TTG AGG GGC CTC AGA TCG CTT TAA CTC GAG CAC CAC CAC CAC CAC CAC TGA GAT; and reverse primer for vector, CCA TCT GAA TCC GCT TGG GAA CTT CCG CAT ATG TCC CTG AAA ATA CAG GTT TTC GCT GCT. The DNA fragments of the β-galactosidase gene and vector were synthesized at an annealing temperature of 55 °C using the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) by PCR. The synthesized DNA fragments, which consisted of amplicons with 60 bp of overhangs for the gene (1539 bp) and vector (5299 bp), were ligated using Master Mix for Gibson Assembly (New England Biolabs, Ipswich, MA, USA), and the ligated DNA was transformed into E. coli BL21.

2.3. Preparation of β-Galactosidase

E. coli cells containing β-galactosidase from T. uzoniensis were cultivated in a 2 L flask containing 500 mL of Luria-Bertani medium containing 50 µg/mL kanamycin at 37 °C, with 200 rpm shaking. When the optical density at 600 nm of the recombinant E. coli cells reached 0.6, expression of β-galactosidase from T. uzoniensis was induced by adding 100 mM IPTG and the cells were further incubated at 16 °C with 150 rpm shaking for 14 h.

The cells were harvested by centrifugation at 10,000 × g, suspended in a Ni-NTA lysis buffer containing 1 mg/mL lysozyme and disrupted by sonication on ice for 20 min. Cell debris and undisrupted cells were removed by centrifugation at 13,000× g for 20 min at 4 °C and the supernatant was used as a crude extract. The crude enzyme solution was applied to a His-Trap HP affinity column (Amersham Biosciences, Uppsala, Sweden) on a fast protein liquid chromatography system (Bio-Rad, Hercules, CA, USA) [27]. The bound protein was subsequently eluted with 20 mM phosphate buffer containing 250 mM imidazole at a flow rate of 1 mL/min. The collected fraction with hydrolytic activity was dialyzed against 50 mM citrate/phosphate buffer (CPB) at pH 5.0 for 12 h at 4 °C. The resulting solution was used as the purified β-galactosidase.

2.4. SDS-PAGE and Gel Filtration Chromatography

The molecular mass of the β-galactosidase subunit was examined by SDS-PAGE using a prestained protein ladder (MBI Fermentas, Hanover, MD, Canada) as a reference. Cell debris, crude extract, and purified enzyme were used at a concentration of 1 mg/mL to evaluate the degree of purification. All protein bands were stained with Coomassie blue dye for visualization. Gel-filtration chromatography was performed using a HiPrep^TM 16/60 Sephacryl S-300 HR column (GE Healthcare, Chicago, IL, USA) to determine the total molecular mass of the recombinant β-galactosidase. The recombinant β-galactosidase was transferred through the column and the enzyme-bound column was eluted with 50 mM CPB (pH 5.0) containing 150 mM NaCl at a flow rate of 0.5 mL/min. The retention time (migration length) of β-galactosidase from T. uzoniensis was measured during the elution process using reference proteins (Amersham Biosciences) such as ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). The total molecular mass of the β-galactosidase from T. uzoniensis was calculated by comparing its retention time (migration length) with those of the reference proteins.

2.5. Preparation of Wild Soybean Extracts

Korean wild soybean cultivated in the Baekdudaegan National Arboretum was used to prepare its seed, stem, and root extracts. The extract method was modified on the basis of the extraction method for soy flour [17]. Each part of the Korean wild soybean was ground using an electric grinder (BioloMix, Guangzhou, China). A fine powder (100 g) was extracted using 1 L of methanol at 40 °C for 24 h. Each extract was filtered, the methanol was completely removed using a rotary evaporator, and the residue was dissolved in 2 L of distilled water. To prevent the Maillard reaction of the β-galactosidase from T. uzoniensis with free sugars at temperatures above 70 °C, the sugars in the extract were removed using a column packed with Diaion HP20 resin (Sigma-Aldrich). Each extract was loaded onto the column and the isoflavone-adsorbed resin was washed with distilled water. The absorbed isoflavone was then eluted with 2 L of methanol at a flow rate of 0.5 mL/min. The methanol in the eluent was completely evaporated, and the residue was redissolved in 1 L of distilled water. The resulting extracts were used for the analysis of isoflavone content and the production of daidzein and genistein.

2.6. Hydrolytic Activity

Unless otherwise mentioned, the enzyme reaction was performed with 0.005 mg/mL of β-galactosidase from T. uzoniensis and 1 mM pNP-β-D-glucopyranoside for 10 min. The activity was determined by the increase in absorbance at 405 nm due to the release of nitrophenol. To assess the effects of pH and temperature on the hydrolytic activity of β-galactosidase from T. uzoniensis, the pH was varied from 4.0 to 7.0 using 50 mM CPB at 70 °C and the temperature was varied from 65 to 95 °C at pH 5.0. The effect of temperature on the stability of β-galactosidase from T. uzoniensis was monitored as a function of incubation time by incubating the enzyme solution in 50 mM CPB (pH 5.0) at different temperatures (75, 80, 85, 90, and 95 °C). After incubation, the enzyme solutions were assayed in 50 mM CPB (pH 5.0) containing 1 mM pNP-β-D-glucopyranoside at 90 °C for 10 min.

The specific activity of β-galactosidase from T. uzoniensis was determined using aryl-glycosides, including pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, pNP-β-D-xylopyranoside, pNP-α-L-arabinopyranoside, pNP-α-L-rhamnopyranoside, oNP-β-D-glucopyranoside, and oNP-β-D-galactopyranoside, and isoflavones such as daidzin, genistin, and glycitin. The reactions were performed in 50 mM CPB (pH 5.0) containing 0.005−0.05 mg/mL β-galactosidase from T. uzoniensis and 1 mM aryl-glycoside or 0.2 mM isoflavone at 90 °C for 10 min.

2.7. Biotransformation of Isoflavones

Time-course reactions for the biotransformation of isoflavones by β-galactosidase from T. uzoniensis were assessed using reagent grade daidzin and genistin, and seed and root extracts of Korean wild soybean as substrates. The biotransformation of reagent grade daidzin or genistin into daidzein or genistein, respectively, was performed in 50 mM CPB (pH 5.0) containing 0.2 mg/mL enzyme and 3 mM isoflavone at 90 °C for 100 or 90 min, respectively. Daidzein and genistein were produced from daidzin and genistin, respectively, in seed or root extracts of Korean wild soybean, in 50 mM CPB (pH 5.0) containing 0.4 mg/mL enzyme and 20% (w/v) seed or root extract at 90 °C for 100 or 200 min, respectively.

2.8. HPLC Analysis

Isoflavones were analyzed using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV detector at 254 nm and a C18 column with dimensions of 4.6 × 150 mm (YMC, Kyoto, Japan). The column was eluted at 35 °C for 40 min at a flow rate of 1.0 mL/min with the following gradient of solvent A (3% acetic acid in water)/solvent B (3% acetic acid in acetonitrile) (v/v): 90/10 to 85/15 for 5 min; 85/15 to 80/20 for 5 min; 80/20 to 75/25 for 5 min; 75/25 to 70/30 for 5 min; 70/30 to 65/35 for 5 min; 65/35 to 10/90 for 5 min; and 10/90 to 90/10 for 10 min.

The retention times of daidzin, daidzein, genistin, genistein, glycitin, and glycitein were 6.2, 14.0, 9.3, 18.5, 7.5, and 15.8 min, respectively. The isoflavones in the reaction samples were confirmed to have the same retention times as those of the isoflavone standards. The quantitative analysis of isoflavones in Korean wild soybean extracts and reaction samples was performed using linear calibration curves relating the peak areas to the concentrations of isoflavone standards [28].

3. Results

3.1. Cloning, Purification, and Molecular Mass Determination of β-Galactosidase from T. uzoniensis

A glycoside hydrolase family 1 (GH1) protein from T. uzoniensis showed high hydrolytic activity on glucosides such as ginsenosides [19], but it has not yet been characterized and applied to hydrolyzed isoflavone. Therefore, the 1482 bp gene encoding the protein from T. uzoniensis DSM 5263 with the same sequence in GenBank (Accession No., AEA12778; gene name, TUZN_RS06265) was cloned and expressed in E. coli BL21 (DE3) in a soluble form. The amino acid sequence of the expressed enzyme showed a 62.1%, 61.9%, 56.9%, and 55.2% identity with those of the GH1 glycosidases from Sulfolobus shibatae (AAA79030) [29], S. solfataricus (AAA72843) [30], Sulfolobus acidocaldarius (AAY81155) [31], and P. furiosus (AAL80197) [32], respectively.

β-Galactosidase from T. uzoniensis was purified using HisTrap affinity chromatography into a soluble protein with a 10.5-fold purification, a final yield of 21%, and a specific activity for pNP-β-D-galactopyranoside of 1103 μmol/min/mg. The molecular mass of the purified enzyme was approximately 57 kDa on SDS-PAGE (Figure 1a), which was consistent with the calculated value of 57,131 Da based on 493 amino acids combined with six histidine residues. The native enzyme was determined to be a homodimer with a molecular mass of 113 kDa by gel-filtration chromatography (Figure 1b). The GH1 glycosidases from S. shibatae, S. solfataricus, S. acidocaldarius, and P. furiosus with high sequence identity (>50%) to the T. uzoniensis β-galactosidase are tetramers, whereas those from Caldicellulosiruptor owensensis [27], Caldicellulosiruptor saccharolyticus [33], and Thermotoga petrophila [34] with low sequence identity (<30%) exist as dimers. These results indicate that within the GH1 family, the level of sequence identity is not critical in determining the association form.

3.2. Effects of pH and Temperature on the Hydrolytic Activity of β-Galactosidase from T. uzoniensis

The hydrolytic activity of β-galactosidase from T. uzoniensis was examined in the pH range 4.0 to 7.0 and the maximal activity was observed at pH 5.0 (Figure 2a). At pH 4.5 and 6.0, the hydrolytic activity was approximately 70% of the maximum. The temperature was varied from 65 to 95 °C at pH 5.0, where the maximal activity was observed, to investigate its effect on the hydrolytic activity of β-galactosidase from T. uzoniensis, and the activity was maximal at 90 °C (Figure 2b). Thermophilic β-glucosidases from M. thermotolerans [11], T. leycettanus [15], N. fischeri [12], D. turgidum [10], T. pendens [16], S. solfataricus [14], and P. furiosus [13], which were applied to the hydrolysis of isoflavone glycosides, showed maximal activities at 50, 65, 80, 85, 90, 90, and 95 °C, respectively. The thermal stability of the enzyme was examined in the temperature range of 75–95 °C. First-order kinetics were displayed for thermal inactivation and the half-lives of β-galactosidase from T. uzoniensis were 235, 122, 68, 46, and 26 h at 75, 80, 85, 90, and 95 °C, respectively (Figure 3). Among the enzymes applied to the hydrolysis of isoflavone glycosides, β-glucosidases from P. furiosus [13] and S. solfataricus [14] had the highest thermal stabilities, respectively, displaying half-lives of 476, 307, 181, 96, and 59 h and 80, 51, 31, 18, and 10 h, at 75, 80, 85, 90, and 95 °C, respectively. At 90 °C, the half-lives of β-galactosidase from T. uzoniensis were 2.1-fold lower and 2.6-fold higher than those of β-glucosidases from P. furiosus and S. solfataricus, respectively.

3.3. Substrate Specificity of β-Galactosidase from T. uzoniensis for Aryl Glycosides and Isoflavone Glycosides

The specific activity of β-galactosidase from T. uzoniensis was investigated using aryl glycosides and isoflavone glycosides. The enzyme exhibited specific activities on aryl-glycosides as substrates in the order oNP-β-D-galactopyranoside > pNP-β-D-galactopyranoside > pNP-β-D-glucopyranoside > oNP-β-D-glucopyranoside > pNP-β-D-xylopyranoside (

Table 1. Substrate specificity of β-galactosidase from T. Uuzoniensis ^a.

Substrate	Specific Activity (μmol/min/mg)
pNP-β-D-glucopyranoside	1021 ± 10
pNP-β-D-galactopyranoside	1103 ± 13
pNP-β-D-xylopyranoside	105 ± 2.7
pNP-α-L-arabinopyranoside	ND
pNP-α-L-rhamnopyranoside	ND
oNP-β-D-glucopyranoside	830 ± 8.1
oNP-β-D-galactopyranoside	1208 ± 16
daidzin	304 ± 9.6
genistin	405 ± 5.5
ononin	197 ± 3.5
glycitin	96 ± 1.3

^a Data are expressed as the means of triplicate experiments ± standard deviation. ND, not detected.

). However, no activity was observed on pNP-α-L-arabinopyranoside and pNP-α-L-rhamnopyranoside. These results indicated that this enzyme is a β-galactosidase with higher specificity for the β-1,2 linkage than for the β-1,4 linkage. The substrate specificity of β-galactosidase from T. uzoniensis for isoflavone glycosides followed the order: genistin > daidzin > ononin > glycitin (Table 1). Most other glycosidases from Dalbergia cohinchinensis [35], D. turgidum [10], P. furiosus [13], Paecilomyces thermophila [36], T. leycettanus [15], and T. maritima [17] also showed a higher specific activity for genistin than for daidzin as a substrate. Conversely, the substrate specificity of β-glucosidase from N. fischeri [12] was higher for daidzin than for genistin.

Among the enzymes used for the hydrolysis of isoflavone glycosides, β-glucosidase from N. fischeri showed the highest specific activity (2189 μmol/min/mg) for pNP-β-D-glucopyranoside [12], followed by β-glucosidase from P. furiosus with a specific activity of 912 μmol/min/mg [13], which was 2.14-fold higher and 1.12-fold lower than that of β-galactosidase from T. uzoniensis, respectively. Nevertheless, the specific activities of β-galactosidase from T. uzoniensis were highest for daidzin and genistin and were 1.69- and 1.23-fold higher, respectively, than those of β-glucosidase from P. furiosus, with the highest activity so far [13]. These results indicate that β-galactosidase from T. uzoniensis is an enzyme suitable for the hydrolysis of isoflavone glycosides.

3.4. Conversion of Reagent Grade Isoflavone Glycosides into Isoflavone Aglycones by β-Galactosidase from T. uzoniensis

The time-course reactions for the conversion of daidzin and genistin, isoflavone glycosides abundantly expressed in soybeans, into daidzein and genistein were carried out under the conditions of pH 5.0, 90 °C, 2 mM substrate, and 0.2 mg/mL β-galactosidase from T. uzoniensis. Conversions were investigated by varying the enzyme concentration from 0.05 to 1 mg/mL for 2 mM reagent grade daidzin or genistin for 60 min. The production of daidzein or genistein increased with increasing enzyme concentrations up to 0.2 mg/mL and reached a plateau above that concentration (data not shown). Therefore, the optimal enzyme concentration for 2 mM reagent grade daidzein or genistein was 0.2 mg/mL.

The enzyme completely hydrolyzed 2 mM reagent grade daidzin and genistin to daidzein and genistein within 90 min and 80 min, respectively (Figure 4a,b). The HPLC profiles for the conversion of daidzin and genistin into daidzein and genistein are shown in Figure 4c. The productivities for daidzein and genistein corresponded to 1.33 and 1.50 mM/h, which were 3.1- and 2.5-fold higher than those achieved with β-glucosidase from P. furiosus for isoflavone aglycones from soybean flour extract [13]. Therefore, β-galactosidase from T. uzoniensis was further applied to the production of isoflavone aglycones from extracts of Korean wild soybean.

3.5. Production of Ioflavone Aglycones from Isoflavone Glycosides in Seed and Root Extracts of Korean Wild Soybeans by β-Galactosidase from T. uzoniensis

Isoflavones were extracted from the seeds, stems, and roots of Korean wild soybeans and the amount of isoflavones was measured (

Table 2. Isoflavone contents in 10% (w/v) seed, stem, and root extracts of Korean wild soybean ^a.

Isoflavone	Content (mg/g)
	Seed	Stem	Root
daidzin	5760 ± 63	ND	20,580 ± 135
daidzein	330 ± 0.9	210 ± 7.8	7560 ± 171
genistin	8310 ± 99	1320 ± 33	3600 ± 45
genistein	360 ± 2.7	ND	1770 ± 33
glycitin	ND	ND	ND
glycitein	510 ± 3.0	ND	ND

^a Data are expressed as the means of three experiments ± standard deviation. ND, not detected.

). In Table 2, statistical steps through parametric or non-parametric tests were not performed because it was only to indicate the content of isoflavones required for biotransformation. Large amounts of daidzin (20,580 mg/g) and genistin (3600 mg/g) were found in the root and seed extracts, whereas no glycitin was found in any of the extracts. The root extract not only contained a 1.7-fold higher isoflavone glycoside content than that of the seed extract but also contained a high content of isoflavone aglycones, including 7560 mg/g of daidzein and 1770 mg/g of genistein. In contrast, the stem extract contained only genistin as an isoflavone glycoside without daidzin, which was only about 16% of the genistin contained in the seed extract. Therefore, we applied β-galactosidase from T. uzoniensis to the production of daidzein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybeans. The production was investigated by varying the enzyme concentration from 0.1 to 2 mg/mL for 20% (w/v) seed or root extracts for 60 or 90 min. The daidzein or genistein yield increased with increasing enzyme concentrations up to 0.4 mg/mL and decreased considerably at higher enzyme concentrations (data not shown), indicating that the optimal enzyme concentration for 20% (w/v) seed or root extracts was 0.4 mg/mL.

The time-course reactions for the production of daidzein and genistein from daidzin and genistin in the extracts were carried out at pH 5.0 and 90 °C with 20% (w/v) seed or root extract, and 0.4 mg/mL β-galactosidase from T. uzoniensis. The seed and root extracts at 20% (w/v) contained 6.62 mM and 11.56 mM isoflavone glycosides (2.77 mM daidzin and 3.85 mM genistin, and 9.89 mM daidzin and 1.67 mM genistin) as substrates and 0.53 mM and 6.08 mM of the corresponding aglycones (0.26 mM daidzein and 0.27 mM genistein, and 5.95 mM daidzein and 0.13 mM genistein), respectively. Daidzin and genistin in the seed extract were completely hydrolyzed to daidzein and genistein within 80 and 70 min, corresponding to the productivities of 1.86 and 3.30 mM/h, respectively. Daidzein and genistein were produced from daidzin and genistin in the root extract with higher productivities of 3.30 and 3.36 mM/h, respectively, and molar yields of 100%, after 180 min and 30 min, respectively (Figure 5).

The production of daidzein and genistein by glycosidases from complex substrates is summarized in

Table 3. Production of daidzein and genistein from complex substrates by glycosidases.

Substrate	Strain	Substrate (mM)	Product (mM)	Molar Yield (%)	Productivity (mM/h)	Ref.
Soybean meal	Alicyclobacillus sp.	daidzin (NR a)	daidzein (0.21)	NR	0.10	[39]
		genistin (NR)	genistein (0.32)	NR	0.16
Soybean milk	Soybean	daidzin (0.15)	daidzein (0.04)	27	0.01	[40]
		genistin (NR)	genistein (NR)	17	NR
Soybean flour	Aspergillus niger	daidzin (NR)	daidzein (0.77 b)	NR	NR	[41]
		genistin (NR)	genistein (0.36 b)	NR	NR
	T. maritima	daidzin (NR)	daidzein (0.35 b)	86	NR	[17]
		genistin (NR)	genistein (0.38 b)	94	NR
Soybean flour extract	Almond	daidzin (NR)	daidzein (NR)	81	NR	[42]
		genistin (NR)	genistein (NR)	79	NR
	A. niger	genistin (NR)	genistein (9.38 b)	NR	NR	[43]
	A. oryzae	daidzin (2.50)	daidzein (1.80)	73	0.61	[44]
		genistin (1.20)	genistein (0.54)	45	0.11
	A. oryzae	daidzin (10.00)	daidzein (8.40)	84	0.17	[37]
		genistin (5.00)	genistein (4.09)	76	0.08
	Aspergillus terreus	daidzin (0.21)	daidzein (0.07)	33	0.41	[45]
		genistin (0.14)	genistein (0.04)	28	0.24
	Gongronella sp.	daidzin (5.00)	daidzein (4.50)	90	1.50	[38]
		genistin (4.10)	genistein (3.70)	86	1.23
	P. thermophila	daidzin (NR)	daidzein (NR)	98	NR	[36]
		genistin (NR)	genistein (NR)	95	NR
	P. fusiosus	genistin (1.00)	genistein (1.00)	100	1.00	[13]
	Unculturable microbes	daidzin (NR)	daidzein (1.74)	NR	0.87	[46]
		genistin (NR)	genistein (1.18)	NR	0.59
Soy molasses	Debaryomyces hansenii	daidzin (NR)	daidzein (0.07 b)	NR	NR	[47]
		genistin (NR)	genistein (6.73 b)	NR	NR
Spent coffee ground extract	D. turgidum	daidzin (1.20)	daidzein (1.20)	100	0.60	[10]
Wild soybean seed extract	T. uzoniensis	daidzin (2.76)	daidzein (2.76)	100	1.86	this study
		genistin (3.84)	genistein (3.84)	100	3.30
Wild soybean root extract	T. uzoniensis	daidzin (9.90)	daidzein (9.90)	100	3.30	this study
		genistin (1.68)	genistein (1.68)	100	3.36

^a NR, not reported. ^b Daidzein or genistein (mg)/soybean flour (g).

. The previous highest concentrations of the produced daidzein and genistein were 8.40 and 4.09 mM, respectively, using β-glucosidase from Aspergillus oryzae with soybean flour extract [37]. These values were 1.18-fold lower and 1.07-fold higher than those of daidzein and genistein from root and seed extracts of Korean wild soybean, respectively. However, the levels of daidzein and genistein produced using β-glucosidase from A. oryzae were 19- and 42-fold lower, respectively, than those produced using β-galactosidase from T. uzoniensis with the root extract of Korean wild soybean. The previous highest productivities obtained for daidzein and genistein were 1.50 and 1.23 mM/h, respectively, using β-glucosidase from Gongronella sp. with soybean flour extract [38]. These values are 2.2- and 2.7-fold lower, respectively, than those obtained using β-galactosidase from T. uzoniensis with the root extract of Korean wild soybean in this study. β-Galactosidase from T. uzoniensis completely hydrolyzed daidzin and genistin in seed and root extracts of Korean wild soybean to daidzein and genistein and the productivities were the highest reported to date. Thus, β-galactosidase from T. uzoniensis is a potent enzyme for the production of isoflavone aglycones.

4. Conclusions

In conclusion, a thermostable β-galactosidase from T. uzoniensis was cloned and characterized as a purified recombinant enzyme, which exhibited a maximum specific activity of 1103 μmol/min/mg for pNP-β-D-galactopyranoside at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme produced daidzein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybean with productivities of 1.86 and 3.30 mM/h, respectively. To the best of our knowledge, this enzyme exhibits the highest productivity for both daidzein and genistein of any enzyme reported to date. These results indicate that β-galactosidase from T. uzoniensis may be industrially useful for the hydrolysis of isoflavone glycosides and that wild soybeans are valuable materials for isoflavone aglycone production.

5. Patents

This section is not mandatory but may be added if there are patents resulting from the work reported in this manuscript.