土曲霉来源的β-葡萄糖苷酶的酶学特性及其对大豆异黄酮的水解

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones # # 土曲霉来源的β-葡萄糖苷酶的酶学特性及其对大豆异黄酮的水解 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:lcqian@zju.edu.cn †E-mail:lcqian@zju.edu.cn lcqian@zju.edu.cn The Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China The Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0–8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters K m (apparent Michaelis-Menten constant) and V max (maximal reaction velocity) for p -nitrophenyl-β- D -glucopyranoside ( p NPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The K m and V max for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products. 目的 2 分离纯化土曲霉来源的β-葡萄糖苷酶（At-Bgl），探究其酶学特性及其对大豆异黄酮的水解效果。 创新点 2 成功将At-Bgl分离纯化出来，并首次将分离纯化后的At-Bgl应用于水解大豆异黄酮糖苷。 方法 2 利用超滤、透析、阴离子交换柱层析和聚丙烯酰氨凝胶电泳（SDS-PAGE）等手段分离纯化土曲霉来源的β-葡萄糖苷酶（表1），并用解析电离串联飞行时间质谱技术（MALDI-TOF/TOF MS）鉴定蛋白条带。以对硝基苯基β-D-葡萄糖苷（ p NPG）为底物进行酶学特性研究；以 p NPG和纤维二糖为底物，进行酶动力学参数研究 （表2）；以胃蛋白酶和胰蛋白酶模拟动物胃肠道酸性环境，进行酸耐受性研究。通过高效液相色谱（HPLC）检测At-Bgl对大豆异黄酮糖苷的水解效果（表3）。 结论 2 At-Bgl属糖苷水解酶第三家族（GH3），分子量约为120 kDa（图1），最适酶解条件为pH 5.0和65 °C，具有良好的热稳定性和pH稳定性（图2），且胃蛋白酶和胰蛋白酶耐受性强（图3）。At-Bgl可将大豆异黄酮糖苷高效转化为异黄酮苷元 （图4）。综上所述，At-Bgl在增强动物胃肠道对大豆异黄酮的水解方面具有重要应用价值。 β-glucosidase Aspergillus terreus Characterization Hydrolysis Soybean isoflavones β-葡萄糖苷酶 土曲霉 酶学特性 水解 大豆异黄酮 Introduction 1 Isoflavones, a class of nonsteroidal estrogens, are diphenolic secondary metabolites of plants and are frequently abounded in soybeans ( Glycine max ). They have attracted considerable attention for their pharmacological effects, such as preventing certain cancers (breast, prostate, and colon) (Ravindranath et al., 2004 2004 ; Ullah et al., 2016 2016 ), decreasing the risk of cardiovascular diseases (Imai, 2015 2015 ), improving bone health (Alekel et al., 2015 2015 ), and preventing menopausal symptoms (Ko, 2013 2013 ). Moreover, studies revealed that isoflavone aglycones, such as daidzein and genistein, are superior to isoflavone glycosides in various bioactivities. However, most of the isoflavones in natural food exist as isoflavone glycosides and are difficult to absorb in the intestines (Izumi et al., 2000 2000 ). Thus, to obtain good bioavailability and pharmacological effects, isoflavone glycosides need to be hydrolyzed to aglycones. β-Glucosidases (β- D -glucoside glucohydrolase, EC 3.2.1.21) mainly hydrolyze the β-1,4-glucosidic linkage in various disaccharides, oligosaccharides, alkyl-and aryl-β- D -glucosides (Kudou et al., 1991 1991 ). They have attracted considerable interest due to their important roles in a variety of fundamental biological processes, such as the hydrolysis in glucosides of isoflavones (Matsuura and Obata, 1993 1993 ), bio-ethanol production (Coughlan, 1985 1985 ), pharmaceutical (Zhou et al., 2012 2012 ) and flavor production (Gueguen et al., 1996 1996 ). According to the amino acid sequences, β-glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, GH30, and GH116 ( http://www.cazy.org http://www.cazy.org ). The majority of fungal β-glucosidases belong to GH1 and GH3. The nutritional availability of foods and feeds can be improved for the release of vitamins, antioxidants, aglycones, and other beneficial compounds from their glycosides by adding β-glucosidases. Therefore, animal feeds are often treated with crude β-glucosidases (Cairns and Esen, 2010 2010 ). Desired features which are important to the animal feed applications of β-glucosidases include high hydrolysis activity, good thermostability and pH stability, and high pepsin and trypsin tolerance. The possibilities for different industrial applications of β-glucosidases justify the incessant search for new sources of microbial enzymes that can be produced with high structural stability in low economic value fermentation media (de Cassia Pereira et al., 2015 2015 ). Fungal strains are known to be efficient producers of β-glucosidases (Souza et al., 2010 2010 ). There have been several reports (Workman and Day, 1982 1982 ; Rodionova et al., 1987 1987 ; Nazir et al., 2008 2008 ; Elshafei et al., 2014 2014 ) about the purification of β-glucosidase from Aspergillus terreus ; however, there have not been any investigations about their potential industrial applications. In this study, the newly isolated strain of fungus, A. terreus , can produce extracellular β-glucosidases with outstanding pepsin and trypsin tolerance, which indicates the potential applications in animal feeds. The absence of reports in scientific literature of this fact encouraged us to study the physicochemical characteristics and the industrial applicabilities of the A. terreus β-glucosidases (At-Bgls). Materials and methods 1 Materials 2 p -Nitrophenyl-β- D -glucopyranoside ( p NPG) and cellobiose were purchased from the Sigma-Aldrich Corp. (St. Louis, MO, USA). Pepsin (2500 U/mg) and trypsin (2500 U/mg) were purchased from the Shanghai Songon Company (Shanghai, China). Soybeans (Zhongdou-27) were provided by the Center of Soybean Research, Zhejiang Agricultural Academy (Hangzhou, China). Isoflavone glycosidic standards (daidzin, genistin, and glycitin) and isoflavone aglycone standards (daidzein, genistein, and glycitein) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were analytical-grade reagents unless otherwise stated. Organism and culture condition 2 A. terreus (Trichocomaceae, Eurotiales, Ascomycota) was isolated from composting soil of the Jinhua region, located in Zhejiang Province, China, and was identified by its 18S DNA sequence. The strain was maintained in potato dextrose agar (PDA; 200 g potato, 20 g glucose, 20 g agar, 1000 ml distilled water) slant at 4 °C. For the preparation of inoculum, an agar plug of 5 mm diameter cut out from the PDA containing mycelium was incubated into a 20-ml basal medium, which had been autoclaved at 121 °C for 20 min, containing 2 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 0.4 g/L MgSO 4 ·7H 2 O, and 0.5 g/L CaCl 2 . The culture was then incubated on a rotary shaker at 35 °C for 24 h. After incubation, the mycelium was stained with methylene blue, rinsed, and examined under a microscope to prove its purity, and was then broken into pieces using a homogenizer. An aliquot (1 ml) of the homogenate was incubated into 100 ml culture media, which had been autoclaved at 121 °C for 20 min, containing 30 g/L wheat bran, 4 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 0.4 g/L MgSO 4 ·7H 2 O, and 0.5 g/L CaCl 2 . Submerged fermentation was carried out in 250-ml flasks under continuous shaking (180 r/min) at 35 °C for 5 d with the flask sealed with sterile gauze and a cap of kraft paper in order to avoid excessive water evaporation and microorganism contamination. The culture suspension was squeezed through sterile absorbent gauze and centrifuged at 8000 r/min for 30 min at 4 °C. Thus, the cell-free culture supernatant was used for enzyme activity measurements and for the purification of β-glucosidase. Enzyme purification and molecular mass determination 2 The cell-free culture supernatant was concentrated at 4 °C using Amicon Ultra-15 30K filter devices (Millipore, USA) and then dialyzed against a 20 mmol/L Tris-HCl buffer (pH 7.5). The dialyzed enzyme solution was loaded onto a HiTrap Q HP column (1.6 cm×2.5 cm, Amersham Pharmacia, Uppsala, Sweden) which was pre-equilibrated with a 20 mmol/L Tris-HCl buffer (pH 7.5). The column was first eluted with an equilibration buffer (two bed volumes) followed by a linear gradient of 1 mol/L NaCl at 2.0 ml/min. Then the fractions with high specific activity were pooled and concentrated using an Amicon Ultra-0.5 centrifugal Filter Unit with Ultracel-3 membrane (Millipore, USA). The apparent molecular mass of the denatured protein was estimated by sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970 1970 ) with 5% (w/v) stacking polyacrylamide gel and 10% (w/v) separating gel which run at 120 V. The protein content in the fractions was determined using Lowry’s method. Enzyme activity assay 2 The β-glucosidase activity was determined using p NPG as substrate. The reaction mixture contained 100 μl of appropriately diluted enzyme, 100 μl of p NPG (10 mmol/L), and 300 μl of McIlvaine buffer (100 mmol/L, pH 5.0). After incubation at 50 °C for 10 min, the reaction was stopped by adding 500 μl of 0.5 mol/L Na 2 CO 3 and the absorbance was read at 405 nm for the amount of p -nitrophenol released. One unit of β-glucosidase activity was defined as the amount of enzyme that produced 1 μmol of p -nitrophenol per minute under the conditions of the assay. The experiments were performed in triplicate. Effects of pH and temperature 2 The optimum pH of the purified β-glucosidase was determined at 50 °C for 10 min in the range from pH 3.0 to 8.0 using a 100 mmol/L McIlvaine buffer containing 10 mmol/L p NPG. For the pH stability study, the enzyme was pre-incubated in the above buffer systems for 60 min at 25 °C. The residual activities at different pH levels were determined under standard assay conditions after the incubation. The optimal temperature of the enzyme was measured at the temperature range from 35 to 85 °C for 10 min in a McIlvaine buffer (100 mmol/L, pH 5.0) containing 10 mmol/L p NPG. For the thermal stability study, the purified enzyme was pre-incubated at different temperatures (50, 60, and 70 °C) in the absence of substrate. After incubation for different intervals (5–60 min), the residual activity was evaluated. All experiments were performed in triplicate. Kinetic parameters 2 The apparent Michaelis-Menten constant ( K m ) and the maximal reaction velocity ( V max ) for the purified β-glucosidase were assessed from Lineweaver-Burk plots using p NPG or cellobiose as substrates. The enzyme was incubated in McIlvaine buffer (100 mmol/L, pH 5.0) with the substrate in concentrations ranging from 1.25 to 18.00 mmol/L of p NPG or 1.25–20.00 mmol/L of cellobiose at 65 °C for 10 min. Tolerance to pepsin and trypsin 2 Glycine-HCl buffer (100 mmol/L, pH 3.0) containing 0.1% (1 g/L) pepsin and Tris-HCl buffer (100 mmol/L, pH 7.0) containing 0.1% (1 g/L) trypsin were prepared. To evaluate the tolerance to pepsin, the purified β-glucosidase (1 mg/ml) was incubated in glycine-HCl buffer solution for different intervals (30, 60, 120 min) with a β-glucosidase/pepsin mass ratio of 1:1 and 1:0.2, respectively. Similarly, the purified enzyme (1 mg/ml) was incubated in Tris-HCl buffer solution with a β-glucosidase/trypsin mass ratio of 1:0.1 and 1:0.02, respectively. After incubation, the residual activities were measured under standard assay conditions. Protein identification by a matrix-assisted laser desorption ionization tandem time using a flight mass spectrometry 2 The band of glucosidase was manually excised from SDS-PAGE stained with Coomassie Brilliant Blue (CBB), and digested in gel overnight with trypsin as described by Oda et al. ( 2006 2006 ). The gel piece was washed with Millipore pure water three times, destained twice with 50% acetonitrile in 25 mmol/L ammonium bicarbonate (Ambic) until the CBB disappeared, then reduced with 10 mmol/L dithiothreitol (DTT) in 25 mmol/L Ambic, alkylated with 55 mmol/L iodoacetamide in 25 mmol/L Ambic, dried twice with 100% acetonitrile, and digested overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI, USA) in 25 mmol/L Ambic. The peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile. Samples were then spotted onto a freshly cleaned target plate and after air-drying, the crystallized spots were analyzed. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis was performed using an ABI Sciex 5800 MALDI-TOF/TOF MS Analyzer (AB SCIEX, Framingham, MA, USA). Thereafter, a database search was conducted using the MASCOT program online ( http://www.matrixscience.com http://www.matrixscience.com ) against National Center for Biotechnology Information nonredundant (NCBInr) databases. The searching parameters were set as follows: tryptic digestion, one missed cleavage allowed, carbamidomethylation of cysteines as fixed modification, methionine oxidation as variable modification, all peptides monoisotopic, peptide mass tolerance of 100 ppm (10 −6 ), and fragment mass tolerance of 0.3 Da. Only significant hits, as defined by the MASCOT probability analysis ( P < 0.05), were accepted. Isoflavone extraction from soybean flour and enzymatic hydrolysis 2 Soybeans were ground to powder using a coffee grinder and then defatted using petroleum ether, with a Soxhlet extractor. The defatted soybean flour (1 g) was extracted with 5 ml 80% ethanol at 70 °C for 2 h. The slurry was then centrifuged at 12 000 r/min for 15 min, and the supernatant was filtered through a 0.45-μm filter and stored at 4 °C for further study. To evaluate the potential application of At-Bgl in our study, the commonly used commercial β-glucosidase Novozyme 188 was used to hydrolyze the isoflavone glycosides. A 250-μl reaction mixture containing 50 μl soybean isoflavone extract was hydrolyzed with 0.025, 0.050, and 0.075 U of individual β-glucosidase, respectively, in McIlvaine buffer (pH 5.0) in a thermostatically controlled incubator at 50 °C for 10 min. The reaction was stopped by boiling for 5 min. A control reaction of extract samples without enzyme was set up in the same manner. The determination of isoflavone in hydrolyzed samples was determined by high performance liquid chromatography (HPLC) as previously described (Li et al., 2012 2012 ). HPLC analyses were performed using a Waters HP1100 HPLC system (Milford, MA, USA) equipped with a Diamonsil C18 column (5 μm, 250 mm×4.6 mm; Dima Co., Ltd., Orlando, FL, USA), and six types of high purity soybean isoflavones, daidzin, glycitin, genistin, daidzein, glycitein, and genistein, were used as standards. The productivity (mmol/(L·h)) of isoflavone aglycones was defined as the increase in the concentration produced in 1 h (Yeom et al., 2012 2012 ). The hydrolysis effect was calculated by the following equation (Song et al., 2011 2011 ): hydrolysis rate=100%×(isoflavone glycosides in control samples−isoflavone glycosides in hydrolyzed samples)/isoflavone glycosides in the control samples. Statistical analysis 2 Statistical analysis was performed using the one-way analysis of variance (ANOVA) procedure of the SPSS 19.0 statistical software package (IBM, Armonk, New York, USA). Treatment means were compared using Duncan’s multiple range tests. It was declared significant when P < 0.05. Results and discussion 1 Purification of A. terreus β-glucosidase 2 The A. terreus culture mycelium was proved purity (Fig. S1 S1 ) and the procedures to purify β-glucosidase from the crude extract of A. terreus are presented in Table 1 1 . β-Glucosidase was purified from the culture supernatant of A. terreus by ultrafiltration and anion exchange, and the enzyme was 11.45-fold purified to a specific activity of 25.73 U/mg with a yield of 7.62%. The results suggest that β-glucosidase may be a minor component in the culture supernatant, and the production may be related to the production medium (Nazir et al., 2008 2008 ). After purification, the enzyme showed a single band in SDS-PAGE, with the apparent molecular mass of about 120 kDa (Fig. 1 1 ). Thus the enzyme was within the broad range of fungal β-glucosidase (35 to 250 kDa) (Song et al., 2011 2011 ). Data represent the mean±SD ( n =3) Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification (fold) Recovery (%)  Crude extract 115.14±1.82 258.83±4.12 2.25±0.07 1.00±0.02 100.00±0.82  Ultra-filtraction 58.06±1.11 194.84±2.74 3.36±0.53 1.49±0.03 75.28±0.75  HiTrap Q HP 0.77±0.02 19.72±0.88 25.73±0.62 11.45±0.53 7.62±0.18 M, protein molecular weight marker (Thermo Fisher Scientific, Inc.); Lane 1, purified A. terreus β-glucosidase Optimal pH and temperature, and stability 2 The pH optima of most fungal β-glucosidases have a range between pH 4.0 and 7.5 (Cairns and Esen, 2010 2010 ). In this study, the purified β-glucosidase exhibited the highest activity at pH 5.0 and was sensitive to pH, while the activity decreased rapidly below pH 5.0 and above pH 7.0 (Fig. 2b 2b ). Based on the relative activity of 100% for the β-glucosidase at pH 5.0, only 23.6% and 64.5% of the maximum activity remained at pH 4.0 and 6.0, respectively. The optimal pH of the enzyme is similar to that of enzyme from A. terreus (Nazir et al., 2008 2008 ), A. niger (Fujita et al., 2015 2015 ), Penicillium piceum (Gao et al., 2013 2013 ), and Rhizomucor miehei NRRL 5282 (Krisch et al., 2012 2012 ). However, the enzyme was found to be very stable in the pH range from 3.0 to 8.0, remaining above 90% of the maximum activity, though it showed a significant decrease with pH above or below 5.0 ( P < 0.05; Fig. 2d 2d ). Before reaching the distal part of the intestinal tract and exerting their catalytic effects, enzymes must retain activity during transport through the stomach and the upper part of the intestinal tract (Peng et al., 2014 2014 ). The mean pH (fed ad libitum) values of the porcine stomach and small intestine are 4.4 and 6.1–6.7, respectively (Merchant et al., 2011 2011 ). Therefore, the At-Bgl should have high potential for use as catalysts in animal feeds. (a) Effect of temperature on A. terreus β-glucosidase activities. The activity was measured in McIlvaine buffer (100 mmol/L, pH 5.0) using p NPG as the substrate. (b) Effect of pH on A. terreus β-glucosidase activities. The enzyme activities at various pH values were measured at 50 °C for 10 min using p NPG as the substrate. (c) Thermostability of A. terreus β-glucosidase. The residual activity was measured using p NPG as the substrate after pre-incubation without substrate at 50, 60, and 70 °C for different periods of time. (d) pH stability of A. terreus β-glucosidase. The residual activities were measured at optimal conditions against p NPG after incubation at various pH values, 25 °C for 1 h. The error bars represent the standard deviation (SD), with n =3 The purified β-glucosidase had an optimal temperature of 65 °C (Fig. 2a 2a ), which was slightly higher than the previous reports (Li et al., 2012 2012 ; 2013 2013 ; Fujita et al., 2015 2015 ; Kaur and Chadha, 2015 2015 ; Zhao et al., 2015 2015 ). In addition, the enzyme showed more than 97% relative activity at 75 °C, and showed no significant difference from the activity at 65 °C ( P > 0.05), while other fungal counterparts were generally inactivated at 70 °C (Li et al., 2013 2013 ; Fujita et al., 2015 2015 ; Kaur and Chadha, 2015 2015 ; Zhao et al., 2015 2015 ). Moreover, the At-Bgl showed good thermostability (Fig. 2c 2c ), maintaining 90.0% and 65.1% of the initial activity after incubation at 50 and 60 °C for 1 h, respectively, though it decreased significantly after incubation at 50, 60, or 70 °C for 5 min ( P < 0.05). These superior properties make this purified enzyme more advantageous for industrial processes, since most applications of β-glucosidase require high temperatures (50 °C or above) (Li et al., 2012 2012 ). Specific activity and kinetic parameters 2 Kinetic parameters of the purified At-Bgl for p NPG and cellobiose at pH 5.0 and 65 °C were estimated, respectively. The K m and V max values of the enzyme are shown in Table 2 2 . The results indicate that the enzyme has greater affinity towards p NPG than cellobiose, and the hydrolysis of p NPG was about eight times faster than that determined on cellobiose. It is worth mentioning that the K m constant was slightly lower than those estimated for the At-Bgl in other reports, with a K m value of 2.5 mmol/L (Elshafei et al., 2014 2014 ) and 14.2, 4.34, 11.1 mmol/L (Nazir et al., 2008 2008 ), respectively. To our best knowledge, there are reports about another two At-Bgls with lower K m values of 0.78 mmol/L (Workman and Day, 1982 1982 ) and 1.25 mmol/L (Rodionova et al., 1987 1987 ), respectively. However, one of important drawbacks of those two enzymes is their low reaction temperatures, with optimal conditions of 50 °C (Workman and Day, 1982 1982 ) and 60 °C (Rodionova et al., 1987 1987 ), respectively. Therefore, those At-Bgls are still not enough for satisfying biotechnological applications, and the purified At-Bgl in our study displayed high potential for industrial applications. Data represent the mean±SD ( n =3) Substrate V max (U/mg) K m (mmol/L)  Cellobiose 5.70±0.17 4.11±0.16   p NPG 42.37±0.74 1.73±0.04 Tolerance to pepsin and trypsin 2 The tolerance to the gastrointestinal microenvironment is important for the application of feed additives (Peng et al., 2014 2014 ). As shown in Fig. 3 3 , the purified β-glucosidase maintained high activity after the treatment with pepsin or trypsin, indicating its high tolerance towards the animal digestive tract. The enzyme maintained more than 90% of the initial activity after the incubation with pepsin (in both 1:1 and 1:0.2 ratios) for 120 min. Moreover, the tolerance to trypsin is slightly lower than that to pepsin. The purified β-glucosidase retained 81.5% of the initial activity after incubating with trypsin (1:0.1) for 120 min and kept 87.4% of the initial activity when incubated with trypsin at a 1:0.02 ratio for 120 min. (a) The tolerance of A. terreus β-glucosidase to pepsin. The enzyme activity was measured at 65 °C, pH 5.0 (McIlvaine buffer, 100 mmol/L) against p NPG after being pre-incubated in glycine-HCl buffer (100 mmol/L, pH 3.0) containing 0.1% pepsin for different intervals (30, 60, and 120 min). (b) The tolerance of A. terreus β-glucosidase to trypsin. The enzyme activity was measured at 65 °C, pH 5.0 (McIlvaine buffer, 100 mmol/L) against p NPG after being pre-incubated in Tris-HCl buffer (100 mmol/L, pH 7.0) containing 0.1% trypsin for different intervals. The error bars represent the SD ( n =3) and bars with different alphabets differ significantly ( P < 0.05) Peptide sequence determination and protein identification 2 The purified samples of β-glucosidase were denatured and analyzed by SDS-PAGE and subjected to MALDI-TOF/TOF MS analysis. The results indicated that the inner peptide sequence of purified protein in the present showed 100% identity with a β-glucosidase from A. terreus NIH2624 (GenBank Accession No. X_P001212225), which belonged to the GH3. The matched fragments were HYILNE QEHFR, DEYGFAHFFPSEGAYER, VNEFVNVQR, and VEDASSYLYPEGLKR. Hydrolysis of soybean isoflavone by A. terreus β-glucosidase 2 Enzymatic hydrolysis of β-glucosidase towards soybean isoflavone is an important application in the food and feed industry, which has been investigated by direct use of β-glucosidase from plants and microorganisms (Chuankhayan et al., 2007 2007 ; Kaya et al., 2008 2008 ; Yang et al., 2009 2009 ; Song et al., 2011 2011 ; Kim et al., 2012 2012 ; Fang et al., 2014 2014 ; Fujita et al., 2015 2015 ). However, low solubility of isoflavones reduces the productivity of isoflavone aglycones in industrial production (Horii et al., 2009 2009 ). To test the reactivity of At-Bgl toward isoflavone, the hydrolysis rate and the productivity were measured by HPLC using isoflavone flour extract as the substrate. The peaks of daidzin, genistin, glycitin, daidzein, genistein, and glycitein in soybean isoflavone flour extract were identified by comparison with commercial standards. The results showed that daidzin was the predominant isoflavones in soybean flour extract. However, after 10 min hydrolysis of At-Bgl, the peaks of daidzin, genistin, and glycitin were greatly decreased and the peaks of daidzein, genistein, and glycitein increased remarkably (Fig. 4b 4b ), which suggested that most of the glycosidic soybean isoflavones were converted to corresponding aglycones. As shown in Table 3 3 , the At-Bgl exhibited higher hydrolytic activity than the commercial β-glucosidase cellobiase (Novozyme 188) toward daidzin, glycitin, and genistin (95.78% vs. 59.16%, 72.08% vs. 23.07%, and 86.74% vs. 54.15%, respectively). Thus, the At-Bgl offers a better advantage for the hydrolysis of isoflavone β-glycoside (daidzin, glycitin, and genistin) than Novozyme 188. (a) Isoflavones in soybean flour extract; (b) Soybean flour extract hydrolysed with 0.075 U of A. terreus β-glucosidase at 50 °C for 10 min; (c) Soybean flour extract hydrolysed with 0.075 U of commercial β-glucosidase cellobiase Novozyme 188 at 50 °C for 10 min. 1, daidzin; 2, glycitin; 3, genistin; 4, daidzein; 5, glycitein; 6, genistein The reactions were performed in McIlvaine buffer (100 mmol/L, pH 5.0) containing 50 μl soybean isoflavone flour extract and 0.025, 0.050, or 0.075 U β-glucosidase. All reactions were performed at 50 °C for 10 min. At-Bgl: β-glucosidase from Aspergillus terreus . Nov 188: commercial β-glucosidase cellobiase Novozyme 188. Data (mean±SD ( n =3)) with different superscripts within a row differ significantly ( P < 0.05) 2 Amount (U) 2 Enzyme 3 Hydrolysis rate (%) 3 Productivity (mmol/(L·h)) Daidzin Glycitin Genistin Daidzein Glycitein Genistein 2 0.025 At-Bgl 32.76±0.28 A 27.58±0.10 B 28.78±0.13 B 0.41±0.02 a 0.08±0.01 c 0.24±0.02 b Nov 188 10.31±0.03 C 12.38±0.02 A 11.97±0.03 B 0.15±0.02 a 0.03±0.01 c 0.08±0.01 b 2 0.050 At-Bgl 95.46±0.22 A 64.25±0.29 C 84.68±0.38 B 1.06±0.06 a 0.18±0.03 c 0.68±0.02 b Nov 188 34.04±0.04 A 20.58±0.28 C 28.52±0.29 B 0.57±0.02 a 0.07±0.01 c 0.19±0.02 b 2 0.075 At-Bgl 95.78±0.20 A 72.08±0.08 C 86.74±0.04 B 1.14±0.01 a 0.19±0.01 c 0.72±0.03 b Nov 188 59.16±0.16 A 23.07±0.04 C 54.15±0.16 B 0.84±0.04 b 0.13±0.02 a 0.40±0.02 c The results indicate that At-Bgl also showed better productivity than Novozyme 188 after incubation for 10 min with 0.075 U of At-Bgl. By comparison, the At-Bgl had the second highest productivity for daidzein (1.14 mmol/(L·h)) and genistein (0.72 mmol/(L·h)), much higher than that of β-glucosidase from A.oryzae (Horii et al., 2009 2009 ), soybean okara (Chiou et al., 2010 2010 ), and Pyrococcus furiosus (Yeom et al., 2012 2012 ). In addition, the previously reported highest productivities for daidzein and genistein were 1.50 and 1.23 mmol/(L·h), respectively, produced by a β-glucosidase from Gongronella sp. (Fang et al., 2014 2014 ). To the best of our knowledge, this is the first report of an At-Bgl hydrolyzing soybean isoflavones and its high hydrolysis rates of daidzin and genistin, and the second highest productivity for daidzein and genistein makes it a good candidate for hydrolysis of soybean isoflavones. 4 Conclusions 2 The purified β-glucosidase from A. terreus , a member of GH3, is a promising enzyme to be used as feed additives. The purified enzyme exhibited maximal activity at pH 5.0 and 65 °C. 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Enhancement of amygdalin activated with β-D-glucosidase on HepG2 cells proliferation and apoptosis Compliance with ethics guidelines Feng-ying YAN, Wei XIA, Xiao-xu ZHANG, Sha CHEN, Xin-zheng NIE, and Li-chun QIAN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500317 Project supported by the Innovation Team Program of Zhejiang Province (No. 2011R50025-12), China 2015-12-15 2016-05-12 2016-05-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 17