Characterization of a β-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei

Bioresource Technology 114 (2012) 555–560

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Characterization of a b-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei Judit Krisch a, Ottó Bencsik b, Tamás Papp b, Csaba Vágvölgyi b, Miklós Takó b,⇑ a b

Institute of Food Engineering, Faculty of Engineering, University of Szeged, H-6725 Szeged, Mars tér 7, Hungary Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary

a r t i c l e

i n f o

Article history: Received 14 September 2011 Received in revised form 10 February 2012 Accepted 24 February 2012 Available online 3 March 2012 Keywords: Rhizomucor miehei Extracellular b-glucosidase Transglucosylation Transgalactosylation Phenolic antioxidants

a b s t r a c t An extracellular b-glucosidase from the zygomycete Rhizomucor miehei NRRL 5282 cultivated in a wheat bran-based solid state fermentation system was characterized. The purified enzyme exhibited an optimum temperature of 68–70 °C and pH of 5.0. It efficiently hydrolyzed oligosaccharides having b-(1?4) glycosidic linkages and exhibited some b- and a-galactosidase activity. The Vmax for p-nitrophenyl-b-dglucopyranoside and cellobiose was 468.2 and 115.5 U/mg, respectively, while the Km was 0.12 mM for both substrates. The enzyme had transglucosylation and transgalactosylation activities resulting in the formation of glycosides from cellobiose, lactose and ethanol. The enzyme increased the amounts of free phenolic antioxidants in sour cherry pomace indicating that its hydrolyzing activity could potentially be applicable to improve the bioavailability of these compounds. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction b-Glucosidases (b-glucoside glucohydrolases; EC 3.2.1.21) are ubiquitous and biologically important enzymes catalyzing the hydrolysis of alkyl- and aryl-b-glycosides as well as di- and oligosaccharides. They have an active role in many biological processes, such as degradation of structural and storage polysaccharides, host–pathogen interactions, cellular signaling and oncogenesis (Bhatia et al., 2002). Their hydrolyzing activity is utilized in various applications, such as fuel ethanol production from cellulosic agroindustrial residues (Harnpicharnchai et al., 2009; Ng et al., 2010), or liberation of aroma compounds from plant-derived products (Su et al., 2010). b-Glucosidases can also be used to liberate phenolic aglycons from their glycosidic bonds, and can therefore be used to increase the amount and nutraceutical activity of phenolic antioxidants (Pham and Shah, 2009). Under certain conditions, b-glucosidases also have synthetic activity, and are able to transfer glycosyl groups to saccharides and alcohols resulting in the formation of oligosaccharides, alkyl-glycosides, and different glycoconjugates, which can be used as therapeutic agents, diagnostic tools and growth promoting agents for probiotic bacteria (Smaali et al., 2007). Enzymatic synthesis of these compounds by transglycosylation or reverse hydrolysis can be achieved in one step instead of several protection/de-protection steps required during chemical synthesis. ⇑ Corresponding author. Tel.: +36 62 544005; fax: +36 62 544823. E-mail address: [email protected] (M. Takó). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.117

In combination with endoglucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), fungal b-glucosidases have an important role in cellulose degrading enzyme systems, wherein they split short chain oligosaccharides and cellobiose into glucose monomers, preventing inhibition of the other enzymes by cellobiose. The arising glucose, however, is generally a strong inhibitor of b-glucosidases (Eyzaguirre et al., 2005). Due to the fact that high hydrolyzing activity and glucose, alcohol, heat and acid tolerance are important features of enzymes that are potentially applicable in biotechnological and industrial processes, screening for good b-glucosidase producing fungal strains has mainly focused on these parameters. Due to increasing interest in compounds synthesized by glucosidases in the pharmaceutical and food industry, characterization of the transglucosylation activity of fungal b-glucosidases is also an intensively studied area (Christakopoulos et al., 1994; Smaali et al., 2007). Filamentous fungi are known to be good producers of b-glucosidases and numerous fungal enzymes have been isolated and analyzed (Eyzaguirre et al., 2005); however, only a few b-glucosidases have been purified and characterized from zygomycetes (Yoshioka and Hayashida, 1980, 1981; Borgia and Mehnert, 1982; Petruccioli et al., 1999; Takii et al., 2005). Additionally, synthetic activity of b-glucosidase from zygomycetes has not been described, and only a few reports are available on the liberation of free phenolic antioxidants due to b-glucosidase activity by this fungal group (Vattem and Shetty, 2002; Correia et al., 2004). The thermophilic fungus Rhizomucor miehei is particularly interesting because of its effective extracellular enzyme production,

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e. g. aspartic protease and lipase (Rao et al., 1998; Rodrigues and Fernandez-Lafuente, 2010). In a recent study, b-glucosidase activity of several zygomycetes grown in liquid and solid media was measured and some R. miehei strains showed intensive extracellular enzyme activity on wheat bran (Takó et al., 2010a). Based on these results, the b-glucosidase coding gene from the R. miehei NRRL 5282 was cloned and characterized and the corresponding enzyme was purified (Takó et al., 2010b). The present paper presents additional data on the purification of the b-glucosidase from the thermophilic R. miehei NRRL 5282 grown on wheat bran; analysis of several biochemical properties and oligosaccharide production are also reported.

2. Methods 2.1. Organism and solid-state fermentation Spores (106 spores/mL) of R. miehei NRRL 5282 were inoculated into a 3 L Erlenmeyer flask that contained 130 g of wheat bran and 130 mL distilled water. The culture was incubated at 40 °C for six days. To maintain humidity, 60 mL sterile distilled water was added to the Erlenmeyer flasks every second day.

2.2. Purification of the extracellular b-glucosidase Details of the R. miehei b-glucosidase isolation procedure and the LC–MS analysis were described previously by Takó et al. (2010b).

2.3. Estimation of the protein concentration Protein content in gel chromatography fractions was monitored by measuring the absorbance at 280 nm. After each purification step, total protein content was determined using a Qubit Fluorometer (Invitrogen) and the Quant-iT Protein Assay Kit (Invitrogen) according to the instructions of the manufacturer.

2.4. Assay of b-glucosidase activity The b-glucosidase activity was determined by using p-nitrophenyl-b-d-glucopyranoside (pNPG; Sigma) as substrate in a reaction mixture containing 180 lL diluted enzyme solution and 20 lL of 7 mM pNPG. The reaction was carried out at 50 °C for 30 min and was stopped by adding 50 lL of 0.1 M Na2CO3. Para-nitrophenol release was monitored at 405 nm in 96-well microtiter plates using an ASYS Jupiter HD microplate reader (ASYS Hitech). One unit of b-glucosidase activity corresponded to the release of 1 lM p-nitrophenol per minute under the conditions of the assay. Enzyme activities were determined in three independent experiments.

2.5. Isoelectric focusing Isoelectric focusing (IEF) was performed by using Novex IEF gels (Invitrogen) containing 5% polyacrylamide and 2% ampholytes (pH range 3.0–10.0). Running conditions were set up according to the manufacturer’s instructions. The gel was fixed in 12% (w/v) trichloroacetic acid containing 3.5% (w/v) sulfosalicylic acid for 30 min, and was stained with 0.0025% (w/v) Coomassie Brilliant Blue R250. The pI of the purified b-glucosidase was determined by using an IEF standard marker mix (Sigma) containing proteins with pI values from pH 3.6 to 9.3.

2.6. Characterization of the b-glucosidase 2.6.1. Effect of pH and temperature The pH optimum of the purified b-glucosidase activity was determined at 50 °C for 30 min in the range from pH 2.2 to 8.0 by using 50 mM McIlvaine buffer supplemented with 0.7 mM pNPG. For the pH stability studies, the enzyme was pre-incubated in the same buffer for 24 h at 4 °C, and then the residual activity was evaluated by incubation for 30 min at 50 °C using 0.7 mM pNPG as a substrate. Optimum temperature for the activity was determined by incubating the purified enzyme in the range from 20 to 80 °C for 30 min in 0.1 M acetate buffer (pH 5.0) containing 0.7 mM pNPG. Thermal stability was established by incubating the enzyme for 4 h at the desired temperature, and then the residual activity was estimated at 50 °C for 30 min using 0.7 mM pNPG. Thermal inactivation was followed at 70, 75 and 80 °C by measuring the residual activity at different time intervals in the presence and absence of cellobiose. Half-life (t(1/2)) was determined for each temperature. 2.6.2. Substrate specificity assays Substrate specificity of the b-glucosidase was estimated by incubating the purified enzyme (0.1 U/mL) in 0.1 M acetate buffer (pH 5.0) containing 0.7 mM aryl-glycosides (pNP-b-d-glucopyranoside, pNP-a-d-glucopyranoside, pNP-N-acetyl-b-d-glucosaminide, pNPa-d-maltohexaoside, pNP-b-d-cellobioside, pNP-a-d-galactopyranoside, pNP-b-d-galactopyranoside, pNP-a-d-mannopyranoside, oNP-b-d-glucopyranoside and pNP-b-d-xylopyranoside; Sigma) or 0.2% (w/v) saccharides (cellobiose, sucrose, salicin, trehalose, amygdalin, lactose, cellulose, sophorose, laminaribiose, maltose and laminarin; Sigma) at 50 °C for 30 min. The activities were monitored by measuring the liberated p-nitrophenol and reducing sugars (Miller, 1959), or quantifying the released glucose using glucose oxidase/ peroxidase reagent (Sigma) according to the instructions of the manufacturer. The relative rate of hydrolysis on aryl-glycosides and saccharides was determined as percentages of the initial rate of hydrolysis obtained with pNPG and cellobiose, respectively. 2.6.3. Determination of kinetic parameters The apparent Michaelis–Menten constants (Km) and maximum velocities (Vmax) for the purified b-glucosidase were assessed from Lineweaver–Burk plots by using pNPG or cellobiose as substrates. The enzyme was incubated in acetate buffer (0.1 M, pH 5.0) with the substrates in concentrations ranging from 0.2 to 1.2 mM of pNPG or 0.07–1.02 mM of cellobiose at 50 °C for 30 min. The inhibition constant (Ki) for glucose inhibition was determined by the intersections of the lines on Dixon plots. The b-glucosidase was incubated with 1.2 and 1.6 mM pNPG by adding glucose at concentrations up to 55 mM. 2.6.4. Determination of the effect of metal ions and chemical reagents Enzyme activity was measured under standard assay conditions in the presence of 5 mM CoCl2, HgCl2, CuSO4, ZnCl2, MnCl2, CaCl2, MgSO4, NaCl and KCl, and 10 mM N-bromosuccinimide (NBS), dimethyl sulfoxide (DMSO), diethylpyrocarbonate (DEPC), ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS). 2.6.5. Effect of various sugars and ethanol The effect of mono- and disaccharides on the enzyme activity was determined by using standard enzyme assay conditions in the presence of fructose, xylose, galactose, arabinose, lactose and sucrose at concentrations ranging from 5% to 20% (w/v). The effect of ethanol on the pNPG hydrolyzing activity was studied in the range from 5% to 25% (v/v).

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in the range observed for other fungal b-glucosidases (Yoshioka and Hayashida, 1981; Pitson et al., 1997; Riou et al., 1998).

2.7. Enzymatic synthesis assays To investigate the production of oligosaccharides and ethyl-glucosides by purified b-glucosidase, 1 U/mL enzyme was incubated with 35% w/v of cellobiose or lactose or with 30% (v/v) ethanol and 10% (w/v) cellobiose at pH 5.0 and 50 °C. For transgalactosylation assays, reaction mixtures comprising 1 U/mL b-glucosidase, 90 mM pNP-b-d-galactopyranoside and 10% (w/v) sucrose were incubated at the abovementioned conditions. In each case, reactions were stopped by boiling for 5 min, and samples were analyzed by isocratic HPLC on YMC-Pack Polyamine II column (250  4.6 mm, S 5 lm, 12 nm; YMC) at a flow rate of 1 mL/min using acetonitrile (Merck) and distilled water at a ratio of 60:40 as mobile phase. Compounds in the eluate were detected by a Shimadzu differential refractometer (RID-10A). Glucose, galactose, lactose, cellobiose, cellotriose, cellotetraose and ethanol (Sigma) were used to calibrate standard curves. The percentage of the products was evaluated from the area of the chromatogram peaks.

2.8. Liberation of phenolics Five grams of lyophilized sour cherry pomace (prepared at the Institute of Food Engineering, University of Szeged, Szeged, Hungary) was extracted with 60 mL distilled water, and 5 mL from this extract was treated with 1 U purified b-glucosidase at 50 °C for 5 h. At intervals, samples were taken and analyzed for free phenolics by using a modified linear gradient HPLC procedure based on the method of Tüzen and Özdemir (2003). Samples were subjected to chromatography on a Prodigy ODS 3 C18 column (100 Å, 5 l, 250  4.6 mm; Phenomenex); detection was performed at 220 nm with a SPD-10AVP UV–vis (Shimadzu) detector. A mixture of solvent A (acetic acid:distilled water, 2:98) and solvent B (acetic acid:acetonitrile:distilled water, 2:30:68) was used as mobile phase at a flow rate of 1 mL/min. For linear gradient elution, solvent A was decreased from 90% to 0% and solvent B was increased from 10% to 100% in 30 min, and this condition was maintained for 10 min. Phenolic acids (gallic acid, vanillic acid, syringic acid, pcoumaric acid, cinnamic acid) and quercetin were purchased from Sigma and used to calibrate standard curves.

3. Results and discussion 3.1. Production and purification of b-glucosidase In a previous study testing extracellular b-glucosidase activities of several zygomycetes using wheat bran as substrate, R. miehei NRRL 5282 showed an outstanding yield of activity (229.8 U/g substrate) (Takó et al., 2010a). Moreover, b-glucosidase from this strain gave the highest residual activity in the heat tolerance test at 75 °C; furthermore, in the presence of a protective cellobiose substrate, this enzyme proved to be thermo-tolerant (Takó et al., 2010a). The purified enzyme (Table 1) has a nominal mass of 82.293 kDa as determined by mass spectrometry analysis (data not shown). The purified enzyme has an acidic pI about 4.6, which

3.2. Characterization of the purified b-glucosidase 3.2.1. Temperature and pH optimum and stability The pNPG hydrolysis by the R. miehei b-glucosidase was maximal at 65–70 °C as the enzyme retained over 80% of its activity in the temperature range from 55 to 75 °C (Fig. 1A). The optimal temperature for the enzyme activity was slightly higher than those reported for most fungal b-glucosidase, which generally have their optimum at temperatures below 65 °C (Bhatia et al., 2002; Eyzaguirre et al., 2005). The enzyme was stable from 20 to 50 °C for 4 h but at higher temperatures, a dramatic loss of stability was observed. Half-life (t(1/2)) of the enzyme was 35, 4 and 3 min at 70, 75 and 80 °C, respectively; however, the presence of cellobiose prolonged the t(1/2) to 90, 9 and 4 min at the same temperatures, respectively, presumably due to binding of cellobiose to the active site (Harnpicharnchai et al., 2009). The optimum pH for the action of the R. miehei b-glucosidase was 5.0 (Fig. 1B), which is similar to the optimal condition of b-glucosidases purified from Rhizopus oryzae MIBA 348 (Takii et al., 2005), Aspergillus oryzae (Riou et al., 1998) and Sclerotinia sclerotiorum (Smaali et al., 2004). The b-glucosidase from Mucor miehei YH10 had a pH optimum of 8.0 (Yoshioka and Hayashida, 1980), which is significantly different from the value measured in the present case. Since the b-glucosidase of R. miehei NRRL 5282 retained 20% of its initial activity at pH 3.0, it can be considered as an acido-tolerant enzyme. The purified enzyme had a relatively narrow pH stability range, and retained 80–100% of its original activity only between pH 4.0 and 6.0. This range is similar to those of the most fungal b-glucosidases (Eyzaguirre et al., 2005). Since the R. miehei b-glucosidase was highly stable at 50 °C and pH 5.0, further analyses of the enzyme were performed under these conditions. 3.2.2. Substrate specificity The enzyme hydrolyzed oligosaccharides having b- and a(1?4) and b-(1?2) glycosidic linkages, such as cellobiose, lactose, maltose and sophorose (Table 2), respectively, but had no activity on cellulose, laminaribiose, sucrose, salicin, amygdalin, laminarin and trehalose. No hydrolysis was detected on soluble polysaccharides with b-(1?3) glycosidic linkages (amygdalin and laminarin), which could be due to the exotype nature of the enzyme (Riou et al., 1998). The R. miehei b-glucosidase proved to be the most active against cellobiose and pNPG. Furthermore, the purified enzyme was able to efficiently hydrolyze pNP-b-d-galactopyranoside, while it showed moderate a-galactosidase and N-acetylglucosaminidase activities on pNP-a-d-galactopyranoside and pNP-N-acetyl-b-d-glucosaminide, respectively. Several b-glucosidases with b-galactosidase or with a-galactosidase activities have been documented from various fungal sources, such as Sporobolomyces singularis (Ishikawa et al., 2005) and Penicillium citrinum (Ng et al., 2010). It is also known that several members of the family 3 glycoside hydrolases exhibit a combination of different activities, thus the b-glucosidase

Table 1 Purification of b-glucosidase from R. miehei NRRL 5282. Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Recovery (%)

Purification (fold)

Crude extract Ammonium sulfate (75–85%) Sephadex G-100 Anion exchange Sephacryl S200HR

965 51.2 7.8 0.7 0.2

1436.5 408.3 141.3 40.7 10.7

1.5 8 18.1 54.9 62.2

100 28.4 9.8 2.8 0.7

1 5.3 12.1 36.6 41.5

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Fig. 1. Effect of temperature (A) and pH (B) on the activity (-j-) and stability (-h-) of the b-glucosidase purified from R. miehei NRRL 5282. Values are averages counted from three independent measures; error bars represent standard deviation.

Table 2 Relative initial rates of hydrolysis of various substrates by the b-glucosidase of R. miehei NRRL 5282. Hydrolysis was undetectable on cellulose, laminaribiose, sucrose, salicin, amygdalin, laminarin, and trehalose, and aryl-glycosides such as pNP-a-d-maltohexaoside and pNP-a-d-mannopyranoside. Substrate Saccharides (2 mg/mL) Cellobiose Lactose Sophorose Maltose Aryl-glycosides (0.7 mM) pNPG pNP-a-dglucopyranoside pNP-b-d-cellobioside pNP-b-dgalactopyranoside pNP-b-dxylopyranoside oNP-b-dglucopyranoside pNP-N-acetyl-b-dglucosaminide pNP-a-dgalactopyranoside

Linkage of glycosyl group

Relative initial rate of hydrolysis (%)

b-(1?4)Glc b-(1?4)Gal b-(1?2)Glc a-(1?4)Glc

100 22.8 6 13.5

b Glc a Glc

100 14

b Cel b Gal

11.6 69.1

b Xyl

1.4

b Glc

20.4

b Glc

13.7

a Gal

17.1

enzymes may also possess some N-acetylglucosaminidase activity (Mayer et al., 2006). 3.2.3. Kinetic parameters The estimated Km value was 0.12 mM for both pNPG and cellobiose, but Vmax was 468.2 U/mg for the former and 115.5 U/mg for the latter substrate (Fig. S1). These results show that the enzyme has equal affinity to pNPG and cellobiose, but the hydrolysis of pNPG was about four times faster than to that determined on cellobiose. The calculated Km values for pNPG are in the same range as those of the majority of the extracellular b-glucosidases from other filamentous fungi (Eyzaguirre et al., 2005). It is worth mentioning that the Km constants were considerably lower than those estimated for the b-glucosidase from M. miehei YH-10 of 0.58 mM and 1.04 mM for pNPG and cellobiose, respectively (Yoshioka and Hayashida, 1981). The calculated Ki towards glucose was 8 mM indicating that the R. miehei enzyme, like most fungal b-glucosidases, is sensitive to product inhibition. In the case of B-glucosidases from filamentous fungi, the inhibition constant for glucose generally varies between 0.5 and 100 mM (Eyzaguirre et al., 2005). 3.2.4. Effect of metal ions and reagents Significant inactivation was observed by adding Cu2+, Hg2+, Zn2+ and SDS. The effect of Hg2+ suggests that thiol groups are required

for the adequate function of the enzyme (Table 3). Thiol groups may be essential to maintain the structure of active b-glucosidases, but could also be important for catalysis (Riou et al., 1998; Wallecha and Mishra, 2003). Inhibition by Cu2+ and Zn2+ suggests that basic (Arg, Lys, His) and acidic (Asp, Glu) amino acids may have important roles in the active site (Ng et al., 2010). This finding corresponds well with the prediction of Asp254 being situated in the conserved motif SDW as the catalytic nucleophile (Takó et al., 2010b). The enzyme activity was completely abolished by NBS in the presence of pNPG substrate, which suggests that tryptophan may be involved in substrate binding (Riou et al., 1998; Bhatia et al., 2002). Although the His177 has been proposed to be the putative H+ donor (Takó et al., 2010b), DEPC treatment caused no significant change in the activity (Table 3). The cations Ca2+, Co2+, Mn2+ and Na+ had no effect on enzyme activity, but activity was stimulated by Mg2+ and K+, which may affect the structural stability of the enzyme. EDTA treatment slightly decreased the enzyme activity, supporting the role of metal ions in enzyme stability. 3.2.5. Effect of various sugars and ethanol on enzyme activity Arabinose and galactose inhibited activity in a dose-dependent manner, resulting in more than 80% reduction at the highest concentration (Fig. 2). Other monosaccharides such as fructose and xylose retained or slightly increased activity, followed by a reduction at the highest concentrations. Among disaccharides, lactose decreased activity in a dose-independent manner. At concentrations of 5–15% (w/v), it reduced the activity to 40%, which was most likely caused by glucose released during lactose hydrolysis. It is also interesting that the enzyme activity was improved by up to 160% in the presence of 15% (w/v) sucrose. In light of these results, it can be hypothesized that the disaccharides acted as glucose-acceptors in

Table 3 Effects of metal ions (5 mM) and reagents (10 mM) on the activity of b-glucosidase from R. miehei NRRL 5282. The activity measured without any additive (control) was considered to be 100%. Metal salt or reagent

Relative initial rate of hydrolysis (%)

Control CaCl2 CoCl2 HgCl2 KCl MnCl2 MgSO4 NaCl ZnCl2 DEPC DMSO EDTA NBS SDS

100 101 103 3.4 123 100 148 96.5 10 91.8 98 89.1 0.9 3

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a transglucosylation reaction; moreover, in the case of lactose, development of a hydrolysis–reverse hydrolysis equilibrium between the released and bound glucose can also be proposed. It was previously ascertained that the specific activity of the R. miehei b-glucosidase was enhanced by adding ethanol up to 15% (v/v) concentration to crude extract (Takó et al., 2010a). Analyses of the purified enzyme also showed that ethanol stimulate enzyme activity (130% in the presence of 15% (v/v) ethanol). Similar stimulative effects of ethanol have been described for b-glucosidases from A. oryzae (Riou et al., 1998) and Melanocarpus sp. (Kaur et al., 2007). In addition, the R. miehei b-glucosidase proved to be more tolerant to glucose in reaction mixtures containing 10% (v/v) ethanol or 10 mg/mL sucrose (Fig. 3). In the presence of 10 mg/mL glucose, sucrose and ethanol increased the residual activity by 45% and 60%, respectively. A cause of this effect could be the transglucosylation activity of the R. miehei b-glucosidase. To prove this suggestion, synthesis studies using cellobiose, lactose and ethanol were also carried out. 3.3. Enzymatic synthesis 3.3.1. Synthesis of gluco-oligosaccharides Since previous studies showed that formation of cello-oligosaccharides and product yield were positively influenced by high initial substrate concentration (Smaali et al., 2007; Saibi et al., 2007), 35% (w/v) of cellobiose was added to the reaction mixtures. The main product of the synthetic reaction was cellotriose (Fig. S2A), but formation of cellotetraose was also detected after a 24 h incubation period. This result is similar to findings reported for the enzyme of Stachybotrys microbispora (Saibi et al., 2007). The concentration of cellotriose increased continuously during incubation and reached up to 22% of total saccharides after 24 h. When the reaction time was prolonged to 168 h (data not shown), maximum concentration of the products (86 mg/mL for cellotriose and 4.1 mg/mL for cellotetraose) was achieved after 72 h. 3.3.2. Synthesis of ethyl-glucosides In the presence of alcohols, b-glucosidase may catalyze the synthesis of alkyl-glucosides through transglucosylation by using different sugar donors such as cellobiose and pNPG (Smaali et al., 2007; Ito et al., 2007). HPLC analysis showed the presence of a new synthetic compound at a retention time of 4.2 min (Fig. S2B), suggesting that the R. miehei b-glucosidase is able to synthesize alkyl-glucosides. Based on literature data, it can be as-

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Fig. 3. Inhibitory effect of glucose in the presence of 10% (v/v) ethanol (-j-) or 100 mg/mL sucrose (-N-). Control reaction (--) was performed without ethanol and sucrose. Activities were measured under standard assay conditions by using 0.7 mM pNPG as substrate.

sumed that the product is an ethyl-glucoside (Christakopoulos et al., 1994). Since cellobiose was used as glucose donor, low amounts of cellotriose were also detected in the reaction mixture. The alkyl-glucoside synthesis may also proceed through reverse hydrolysis by using glucose and various alcohols as substrate and acceptor, respectively (van Rantwijk et al., 1999); however, no product was detected under these conditions.

3.3.3. Synthesis on lactose It is known that some b-glucosidases are able to synthesize oligosaccharides through transglucosylation or transgalactosylation of lactose; however, enzymes from fungal sources have been rarely investigated in this regard. HPLC analysis detected, besides the peaks corresponding to glucose/galactose (6.3 min) and lactose (7.6 min), that of a new, probably synthetic oligosaccharide product having a retention time of 9.3 min (Fig. S2C). Glucose and galactose could not be separated by the applied HPLC method. It is possible that the oligosaccharide is a trisaccharide, which represented 18.4% of the total saccharides in the reaction mixtures. Transgalactosylation activity of the R. miehei b-glucosidase was identified in the presence of 10% (w/v) sucrose and 90 mM pNP-bd-galactopyranoside. After incubation at 50 °C for 24 h, a new oligosaccharide was detected by HPLC with a retention time of 8.5 min (Fig. S2D). As sucrose (6.3 min) is not hydrolyzed by the enzyme (see the Section 3.2.2), and only galactose monosaccharide can be released by the hydrolysis of pNP-b-d-galactopyranoside, the oligosaccharide could be a galactose–glucose–fructose trisaccharide formed as a result of transgalactosidase activity of the R. miehei b-glucosidase. Such transgalactosidase activity has previously been observed for various b-galactosidases and b-galactosidase-like b-glucosidases (Martínez-Villaluenga et al., 2008; Ishikawa et al., 2005).

3.4. Liberation of phenolic antioxidants by the b-glucosidase

Fig. 2. Effect of mono- (-N- arabinose, -h- fructose, -e- galactose, -⁄- xilose) and disaccharides (-j- lactose, -d- sucrose) on the pNPG hydrolysis by the purified enzyme. Values are averages counted from three independent measures; error bars represent standard deviation.

To investigate the production of free phenolic antioxidants by the hydrolyzing activity of the purified R. miehei b-glucosidase, sour cherry pomace, which has high antioxidant capacity and contains large quantities of phenolics (Chaovanalikit and Wrolstad, 2004), was used. The crude extract of lyophilized sour cherry pomace was treated with the purified enzyme and the liberated phenolic compounds were determined by HPLC analysis. The amount of the different phenolic antioxidants increased during the incubation period (Table 4). The quantity of 4-hydroxybenzoic acid, vanillic acid and syringic acid increased considerably and p-coumaric acid and cinnamic acid was also detected after 5 h. These results showed that the health promoting effect of food industry products could be increased by adding R. miehei b-glucosidase.

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Table 4 Liberation of free phenolics (lg/mL) from lyophilized sour cherry pomace treated with R. miehei NRRL 5282 b-glucosidase. Analysis was carried out by using 1 U enzyme at 50 °C. Time (h)

Gallic acid

4-hydroxybenzoic acid

Vanillic acid

Syringic acid

p-coumaric acid

Quercetin

Cinnamic acid

1 2 5

0 2.38 6.29

0.55 8.45 29.48

0 3.62 12.71

0.24 3.57 12.61

0 0 1.23

0 1.07 2.03

0 0 8.17

4. Conclusion The R. miehei NRRL 5282 b-glucosidase is suitable for various biotechnological applications since it has short-term stability at 60 °C, is stable in acidic environments, and has a good hydrolyzing activity against glycosylated phenolics. The enzyme is capable of synthesizing various oligosaccharides and alkyl-glucosides. Besides its good transglucosylation activity, the enzyme is able to catalyze the transgalactosylation reaction; a property that has not yet been described for b-glucosidases from filamentous fungi. Identification of the synthetic compounds formed in the presence of lactose and ethanol and analysis of the parameters affecting transglucosylation and transgalactosylation are in progress. Acknowledgements This work was supported by the grants of the KTIA-OTKA (CK 80188) and the Hungarian–French Intergovernmental S&T Cooperation Programme (TÉT_10-1-2011-0747). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.117. References Bhatia, Y., Mishra, S., Bisaria, V.S., 2002. Microbial b-glucosidases: cloning, properties, and applications. Crit. Rev. Biotechnol. 22, 375–407. Borgia, I.P., Mehnert, W.D., 1982. Purification of a soluble and a wall-bound form of b-glucosidase from Mucor racemosus. J. Bacteriol. 149, 512–522. Chaovanalikit, A., Wrolstad, R.E., 2004. Anthocyanin and polyphenolic composition of fresh and processed cherries. J. Food Sci. 69, 73–83. Christakopoulos, P., Goodenough, P.W., Kekos, D., Macris, B.J., Claeyssens, M., Bhat, M.K., 1994. Purification and characterization of an extracellular b-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur. J. Biochem. 224, 379–385. Correia, R.T.P., McCue, P., Magalhaes, M.M.A., Macedo, G.R., Shetty, K., 2004. Production of phenolic antioxidants by the solid-state bioconversion of pineapple waste mixed with soy flour using Rhizopus oligosporus. Process Biochem. 39, 2167–2172. Eyzaguirre, J., Hidalgo, M., Leschot, A., 2005. b-Glucosidases from filamentous fungi. In: Yarema, K.J. (Ed.), Handbook of carbohydrate engineering. CRC Press, Taylor and Francis Group, Boca Raton, Florida, pp. 645–686. Harnpicharnchai, P., Champreda, V., Sornlake, W., Eurwilaichitr, L., 2009. A thermotolerant b-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr. Purif. 67, 61– 69. Ishikawa, E., Sakai, T., Ikemura, H., Matsumoto, K., Abe, H., 2005. Identification, cloning, and characterization of a Sporobolomyces singularis b-galactosidase-like enzyme involved in galacto-oligosaccharide production. J. Biosci. Bioeng. 99, 331–339. Ito, J., Ebe, T., Shibasaki, S., Fukuda, H., Kondo, A., 2007. Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus bglucosidase 1. J. Mol. Catal. B: Enzym. 49, 92–97. Kaur, J., Chadha, B.S., Kumar, B.A., Kaur, G.S., Saini, H.S., 2007. Purification and characterization of b-glucosidase from Melanocarpus sp. MTCC 3922. Electron. J. Biotechnol. 10, 260–270.

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