Soybean Isoflavone Glycosides Research Articles

Characterization of a Novel Hyperthermophilic GH1 β-Glucosidase from Acidilobus sp. and Its Application in the Hydrolysis of Soybean Isoflavone Glycosides.

A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, Km, Vmax, and Kcat/Km of BglAc toward pNPG were 357.62 U mg-1, 3.41 mM, 474.0 μmol min-1·mg-1, and 122.7 s-1mM-1. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.

Characterization of a novel cold-adapted GH1 β-glucosidase from Psychrobacillus glaciei and its application in the hydrolysis of soybean isoflavone glycosides

The novel β-glucosidase gene (pgbgl1) of glycoside hydrolase (GH) family 1 from the psychrotrophic bacterium Psychrobacillus glaciei sp. PB01 was successfully expressed in Escherichia coli BL21 (DE3). The deduced PgBgl1 contained 447 amino acid residues with a calculated molecular mass of 51.4 kDa. PgBgl1 showed its maximum activity at pH 7.0 and 40 °C, and still retained over 10% activity at 0 °C, suggesting that the recombinant PgBgl1 is a cold-adapted enzyme. The substrate specificity, Km, Vmax, and Kcat/Km for the p-Nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate were 1063.89 U/mg, 0.36 mM, 1208.31 U/mg and 3871.92/s, respectively. Furthermore, PgBgl1 demonstrated remarkable stimulation of monosaccharides such as glucose, xylose, and galactose, as well as NaCl. PgBgl1 also demonstrated a high capacity to convert the primary soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their respective aglycones. Overall, PgBgl1 exhibited high catalytic activity towards aryl glycosides, suggesting promising application prospects in the food, animal feed, and pharmaceutical industries.

Effect of pulsed electric field on soybean isoflavone glycosides hydrolysis by β-glucosidase: Investigation on enzyme characteristics and assisted reaction

This work aimed to investigate how pulsed electric field (PEF) technology as an alternative to enhance the enzymatic hydrolysis of soybean isoflavone glycosides (SIG). To achieve it, the effect of PEF treatment on the activity, kinetics, thermodynamics and structure of β-glucosidase (β-GLU) were evaluated. The parameters for PEF-assisted hydrolysis of soybean isoflavone glycosides were optimized by response surface methodology. The results showed that PEF treatment increased the relative activity and catalytic efficiency of β-GLU with moderate electric field intensity. Furthermore, PEF treatment induced the secondary and tertiary structural change of β-GLU, the α-helix content increased by 4.23% and the β-fold content decreased by 3.70%. The optimum conditions for PEF treatment were established as the highest yield of isoflavone aglycones achieved 94.58%. Therefore, these results indicated that PEF treatment could be used as an efficient process to improve the β-GLU properties, converting soybean isoflavone glycoside to their aglycones form.

Neuroprotective Potency of Tofu Bio-Processed Using Actinomucor elegans against Hypoxic Injury Induced by Cobalt Chloride in PC12 Cells

Fermented soybean products have attracted great attention due to their health benefits. In the present study, the hypoxia-injured PC12 cells induced by cobalt chloride (CoCl2) were used to evaluate the neuroprotective potency of tofu fermented by Actinomucor elegans (FT). Results indicated that FT exhibited higher phenolic content and antioxidant activity than tofu. Moreover, most soybean isoflavone glycosides were hydrolyzed into their corresponding aglycones during fermentation. FT demonstrated a significant protective effect on PC12 cells against hypoxic injury by maintaining cell viability, reducing lactic dehydrogenase leakage, and inhibiting oxidative stress. The cell apoptosis was significantly attenuated by the FT through down-regulation of caspase-3, caspases-8, caspase-9, and Bax, and up-regulation of Bcl-2 and Bcl-xL. S-phase cell arrest was significantly inhibited by the FT through increasing cyclin A and decreasing the p21 protein level. Furthermore, treatment with the FT activated autophagy, indicating that autophagy possibly acted as a survival mechanism against CoCl2-induced injury. Overall, FT offered a potential protective effect on nerve cells in vitro against hypoxic damage.

A Simple Method for Beta-glucosidase Immobilization and Its Application in Soybean Isoflavone Glycosides Hydrolysis

In this study, a simple, inexpensive and fast β-glucosidase immobilization system was constructed and evaluated in isoflavone glycosides hydrolysis. A β-glucosidase gene from Thermoascus aurantiacus IFO9748 was recombinantly expressed in Pichia pastoris KM71H and immobilized on regenerated amorphous cellulose (RAC) by fused cellulose binding module 3. Through simple mixing cellulose and crude enzyme for 15 min under room temperature, 96.04% β-glucosidase was immobilized onto RAC. The optimum temperature for β-glucosidase activity was increased by 5oC after immobilization. The half-life (t½) of heat inactivation of immobilized enzyme at 60oC was improved over 8 folds. After 30 rounds recycled at 40oC, 96.9% daidzin and 98.9% genistin could still be hydrolyzed. A continuous hydrolysis system was also constructed, and at the flow rate of 0.2 mL/min after 30 h hydrolysis, 95.6% genistin and 90.2% daidzin can still be hydrolyzed. Combined the simple and high efficient enzyme immobilization procedure and inexpensive cellulose, this scalable and practical system may have broad prospects for industrial utilization.

Carbohydrate-binding module assisted purification and immobilization of β-glucosidase onto cellulose and application in hydrolysis of soybean isoflavone glycosides

Complicated purification steps, together with the fact that β-glucosidase has to be tolerant to ethanol restricts the application of β-glucosidase in isoflavone aglycone hydrolyzing process. β-Glucosidase Bgl1A(A24S/F297Y) is a promising enzyme in hydrolyzing isoflavones. In this work, six different carbohydrate-binding modules (CBMs), which were from 3 families, were fused to the C-terminal of Bgl1A(A24S/F297Y), respectively, to simplify the enzyme preparation process. The fusion proteins were expressed in Escherichia coli and adsorbed onto cellulose. The Bgl-CBM24 was found to have the highest immobilization efficiency at room temperature within 1h adsorption. Notably, 1-g cellulose absorbs up to 254.9±5.7 U of Bgl-CBM24. Interestingly, the immobilized Bgl-CBM24 showed improved ethanol tolerance ability, with the IC50 of 35% (v/v) ethanol. Bgl-CBM24 effectively hydrolyze soybean isoflavone glycosides. The hydrolysis rate of daidzin and gemistin was 85.22±3.24% and 82.14±3.82% within 10min, with the concentrations of daidzein and genistein increased by 6.36±0.18mM and 3.98±0.22mM, respectively. In the repetitive hydrolytic cycles, the concentrations of daidzein and genistein still increased by 3.07±0.24mM and 1.94±0.34mM in the fourth cycle with 20% (v/v) ethanol. These results suggest that the immobilized Bgl-CBM24 has excellent potential in the preparation of isoflavone aglycones.

Acceleration of Aglycone Isoflavone and γ-Aminobutyric Acid Production from Doenjang Using Whole-Cell Biocatalysis Accompanied by Protease Treatment.

Recently, soybean isoflavone aglycones (i.e., daidzein and genistein) and γ-aminobutyric acid (GABA) have begun to receive considerable consumer attention owing to their potential as nutraceuticals. To produce these ingredients, multiple microorganisms and their enzymes are commonly used for catalysis in the nutraceutical industry. In this work, we introduce a novel fermentation process that uses whole-cell biocatalysis to accelerate GABA and isoflavone aglycone production in doenjang (a traditional Korean soybean paste). Microbial enzymes transform soybean isoflavone glycosides (i.e., daidzin and genistin) and monosodium glutamate into soybean isoflavone aglycones and GABA. Lactobacillus brevis GABA 100 and Aspergillus oryzae KACC 40250 significantly reduced the production time with the aid of a protease. The resulting levels of GABA and daidzein were higher, and genistein production resembled the levels in traditional doenjang fermented for over a year. Concentrations of GABA, daidzein, and genistein were measured as 7,162, 60, and 59 μg/g, respectively on the seventh day of fermentation. Our results demonstrate that the administration of whole-cell L. brevis GABA 100 and A. oryzae KACC 40250 paired with a protease treatment is an effective method to accelerate GABA, daidzein, and genistein production in doenjang.

Improve ethanol tolerance of β-glucosidase Bgl1A by semi-rational engineering for the hydrolysis of soybean isoflavone glycosides

A β-glucosidase Bgl1A variant (A24S/F297Y) with improved ethanol tolerance was obtained by semi-rational engineering. At 30–40°C, IC50 values (the amount required for inhibiting 50% enzyme activity) of the variant for ethanol were 17–30% (v/v), 1.4- to 2.4-fold of Bgl1A. When incubating in 15% (v/v) ethanol at 30°C, the half-life of A24S/F297Y was 13min; whereas Bgl1A lost all enzyme activity within 5min. A24S/F297Y was more stable at pH 7.5 than at pH 6.5, and more than 50% of the original activity remained after incubation at 30°C for 10h. At 35°C and pH 7.5, the half-life of A24S/F297Y was 80min, 4.3 times longer than that of Bgl1A. When converting isoflavone glycosides to aglycones using A24S/F297Y as catalyst, the hydrolysis rates were 99% for daidzin and 98% for genistin. The concentrations of daidzein and genistein rapidly increased by 7.02mM and 4.35mM within 10min, respectively. These results showed that A24S/F297Y was a promising candidate for the enzymatic hydrolysis of soybean isoflavone glycosides.

Heterologous expression of a GH3 β-glucosidase from Neurospora crassa in Pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides

Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 β-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60 °C, and had its maximum activity against p-nitrophenyl-β-d-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-β-d-cellobioside (pNPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 μM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries.

Characterization of a novel β-glucosidase from Gongronella sp. W5 and its application in the hydrolysis of soybean isoflavone glycosides.

A novel β-glucosidase named BglW5 from Gongronella sp. was isolated, purified, and characterized for the first time. Under solid state fermentation, the yield of BglW5 was 49.9 U/g fermented medium. BglW5 was stable over a wide pH range of 3.0-8.5 and retained more than 50% of its maximal activity after incubation at 25 °C for 96 h. The half-lives of BglW5 were 20 h at 60 °C, and 1 h at 70 °C. The activity of BglW5 was stimulated by xylose and fructose at concentrations up to 500 mM, with maximal stimulatory effect of 1.6-fold and 2.2-fold, respectively. BglW5 converted isoflavone glycosides to aglycones, with a hydrolysis rate of 96.2% for daidzin and 96.7% for genistin. The productivities were 1.5 mmol L(-1) h(-1) for daidzein and 1.23 mmol L(-1) h(-1) for genistein, respectively. These features suggest that BglW5 has great application potential in the hydrolysis of soybean isoflavone glycosides.

Molecular characterization of a highly-active thermophilic β-glucosidase from Neosartorya fischeri P1 and its application in the hydrolysis of soybean isoflavone glycosides.

Isoflavone occurs abundantly in leguminous seeds in the form of glycoside and aglycone. However, isoflavone glycoside has anti-nutritional effect and only the free type is beneficial to human health. In the present study we identified a β-glucosidase from thermophilic Neosartorya fischeri P1, termed NfBGL1, capable of efficiently converting isoflavone glycosides into free isoflavones. The gene, belonging to glycoside hydrolase family 3, was successfully overexpressed in Pichia pastoris at high cell density in a 3.7-l fermentor. Purified recombinant NfBGL1 had higher specific activity (2189±1.7 U/mg) and temperature optimum (80°C) than other fungal counterparts when using p-nitrophenyl β-d-glucopyranoside as the substrate. It retained stable at temperatures up to 70°C and over a broad pH range of 3.0−10.0. NfBGL1 had broad substrate specificity including glucosidase, cellobiase, xylanase and glucanase activities, and displayed preference for hydrolysis of β-1,2 glycosidic bond rather than β-1,3, β-1,4, β-1,6 bonds. The enzyme showed high bioconversion ability for major soybean isoflavone glycosides (daidin, gensitin and glycitin) into free forms. These properties make NfBGL1 potential for the wide use in the food, feed, pharmacy and biofuel industries.

High production of succinyl isoflavone glycosides by Bacillus licheniformis ZSP01 resting cells in aqueous miscible organic medium.

To achieve efficient production of succinyldaidzin and succinylgenistin, resting cells of a solvent-stable strain Bacillus licheniformis ZSP01 were used to react with pure isoflavones or soybean flour extract in a reaction medium with 10% dimethyl sulfoxide. Strikingly, 0.8 mM daidzein, 0.8 mM genistein, 2.0 mM daidzin, and 2.0 mM genistin were transformed to succinyl isoflavone glycosides in 27 H (yield >90%). The soybean flour extract (6.1%, w/v) contained 0.32 mM daidzein, 0.84 mM daidzin, 0.38 mM genistein, and 1.04 mM genistin. Over 95% of total isoflavones (daidzein, daidzin, genistein, and genistin) in the soybean flour extract were converted to succinyl isoflavone glycosides after 27 H. Strain ZSP01 shows both high glycosylation and succinylation activities. These results suggest that B. licheniformis ZSP01 could be useful for the efficient production of succinyl soybean isoflavone glycosides.

Molecular cloning and characterization of a novel β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library

A novel β-glucosidase (Bgl1269) was identified from a metagenomic library of mangrove soil by activity-based functional screening. Sequence analysis revealed that Bgl1269 encodes a protein of 422 amino acids. After being overexpressed in Escherichia coli and purified, the enzymatic properties of Bgl1269 were investigated. The recombinant enzyme displayed a pH optimum of 6.0 and a temperature optimum of 40°C, and the addition of most common metal ions (1 or 10mM) increased the enzymatic activity evidently. In addition, the enzyme showed high hydrolyzing ability for soybean isoflavone glycosides, and 0.8unit of enzyme could completely converted daidzin and genistin (0.5mg/mL) to daidzein and genistein at 40°C for 0.5h. Interestingly, Bgl1269 also exhibited a very high glucose-tolerance, with the highest inhibition constant Ki (4.28M) among β-glucosidases reported so far. These properties make it a good candidate in the production of soybean isoflavone aglycones after further study.

The antioxidant effect of genistein on the in vitro metal-mediated formation of free radicals

Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 β-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60 °C, and had its maximum activity against p-nitrophenyl-β-d-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-β-d-cellobioside (pNPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 μM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries.

Comparison of Three Thermostable β-Glucosidases for Application in the Hydrolysis of Soybean Isoflavone Glycosides

A novel thermostable β-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two β-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-β-d-glucopyranoside (pNPG) at 80 °C and pH 7.0, was stable for a pH range of 4.6-7.8 and at 65 °C for 3 h, and had the lowest K(m) for the natural glycoside salicin and the highest relative substrate specificity (k(cat)/K(m))((salicin))/(k(cat)/K(m))((pNPG)) among the three enzymes. It converted isoflavone glycosides, including malonyl glycosides, in soybean flour to their aglycons more efficiently than Tm-BglA and Tm-BglB. After 3 h of incubation at 65 °C, Te-BglA produced complete hydrolysis of four isoflavone glycosides (namely, daidzin, genistin and their malonylated forms), exhibiting higher productivity of genistein and daidzein than the other two β-glucosidases. Our results suggest that Te-BglA is preferable to Tm-BglA and Tm-BglB, but all three enzymes have great potential applications in converting isoflavone glycosides into their aglycons.

Purification and characterization of a β-glucosidase capable of hydrolyzing soybean isoflavone glycosides from Pichia guilliermondii K123-1

A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45°c and it decreased dramatically above 50°c. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4°C. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.

Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomyces thermophila

Abstract The β -glucosidase from Paecilomyces thermophila J18 was found to be capable of hydrolysing daidzin and genistin in a previous study. This report further evaluated the thermostability and hydrolysis of soybean isoflavone glycosides. The enzyme was found to be very stable at 50 °C, and retained more than 95% of its initial activity after 8 h at 50 °C. It converted isoflavone glycosides, in soybean flour extract and soybean embryo extract, to their aglycones, resulting in more than 93% of hydrolysis of three isoflavone glycosides (namely, daidzin, genistin and glycitin) after 4 h of incubation. Also, addition of the β -glucosidase greatly increased the contents of isoflavone aglycones in the suspended soybean flour and soymilk. The results indicate that the thermostable β -glucosidase may be used to increase the isoflavone aglycones in soy products. This is the first report on the potential application of fungal β -glucosidases for converting isoflavone glycosides to their aglycones in soy products.

Biotransformation of soybean isoflavones by a marine Streptomyces sp. 060524 and cytotoxicity of the products

A marine Streptomyces sp. 060524 capable of hydrolyzing the glycosidic bond of isoflavone glycosides, was isolated by detecting its β-glucosidase activity. 5 isoflavone aglycones were isolated from culture filtrates in soybean meal glucose medium. They were identified as genistein (1), glycitein (2), daidzein (3), 3′,4′,5,7-tetrahydroxyisoflavone (4), and 3′,4′,7-trihydroxyisoflavone (5), based on UV, NMR and mass spectral analysis. The Streptomyces can selectively hydroxylate at the 3′-position in the daidzein and genistein to generate 3′-hydroxydaidzein and 3′-hydroxygenistein, respectively. The Strain biotransformed more than 90% of soybean isoflavone glycosides into their aglycones within 108 h. 3′-hydroxydaidzein and 3′-hydroxygenistein exhibited stronger cytotoxicity against K562 human chronic leukemia than daidzein and genistein.

Brönsted acidic ionic liquids as novel catalysts for the hydrolyzation of soybean isoflavone glycosides

In this work, the hydrolyzation of soybean isoflavone glycosides using Bronsted acidic ionic liquids (ILs) as novel catalysts was studied. The experimental results show that those ILs have good catalytic activities to the hydrolyzation reactions, and their activities are mainly dependent on the types of their anions. The SO3H-functional ILs with HSO4- as anion have the best catalytic activities which are similar to that of sulfuric acid, with the conversion of glycitin more than 90%.

Hydrolysis of black soybean isoflavone glycosides by Bacillus subtilis natto

Hydrolysis of isoflavone glycosides by Bacillus subtilis natto NTU-18 in black soymilk is reported. At the concentration of 3-5% (w/v), black soymilk in flask cultures, the isoflavones, daidzin, and genistin were highly deglycosylated within 24 h. Deglycosylation of isoflavones was further carried out in a 7-l fermenter with 5% black soymilk. During the fermentation, viable cells increased from 10(3) to 10(9) CFU ml(-1) in 15 h, and the activity of beta-glucosidase appeared at 8 h after inoculation and reached a maximum (3.3 U/ml) at 12 h, then decreased rapidly. Deglycosylation of isoflavone glycosides was observed at the same period, the deglycosylation rate of daidzin and genistin at 24 h was 100 and 75%, respectively. It is significantly higher than the previous reports of fermentation with lactic acid bacteria. In accordance with the deglycosylation of isoflavone glycosides, the estrogenic activity of the 24 h fermented black soymilk for ERbeta estrogen receptor increased to threefold; meanwhile, the fermented broth activated ERalpha estrogen receptor to a less extent than ERbeta. These results suggest that this fermentation effectively hydrolyzed the glycosides from isoflavone in black soymilk and the fermented black soymilk has the potential to be applied to selective estrogen receptor modulator products.