2012 lam

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Characterization of an ethanol-tolerant 1,4-b-xylosidase produced by Pichia membranifaciens A.M. Romero, J.J. Mateo and S. Maicas Departament de Microbiologia i Ecologia, Universitat de Vale`ncia, Burjassot, Spain

Keywords 1,4-b-xylosidase, characterization, ethanol tolerant, Pichia membranifaciens, purification.

Correspondence Sergi Maicas, Departament de Microbiologia i Ecologia, Universitat de Vale`ncia, Dr. Moliner 50, Burjassot E-46100, Spain.

E-mail: sergi.maicas@uv.es

2012 ⁄ 0470: received 14 March 2012, revised 7 August 2012 and accepted 7 August 2012 doi:10.1111/j.1472-765X.2012.03297.x

Abstract Aims: The purification and biochemical properties of the 1,4-b-xylosidase of an oenological yeast were investigated.

Methods and Results: An ethanol-tolerant 1,4-b-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28 C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS-PAGE. The activity of 1,4-b-xylosidase was optimum at pH 6Æ0 and at 35 C. The activity had a Km of 0Æ48 ± 0Æ06 mmol l)1 and a Vmax of 7Æ4 ± 0Æ1 lmol min)1 mg)1 protein for p-nitrophenyl-b-d-xylopyranoside.

Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol.

Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4-b-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Introduction Pichia membranifaciens is a film-forming yeast causing off-aroma and flavour in table wine (Esteve et al. 2001).

Other members of this genus have also been isolated from diverse sources: flowering plants, fruit skins, insect intestinal tracts, human tissue and faeces, dairy and baked food products, contaminated oil, wastewaters, tree exudates, salted foods and from the marine environment (El-Sharoud et al. 2009; Sinigaglia et al. 2010). This represents a wider range of habitats in nature compared with other common yeasts as Saccharomyces cerevisiae. Furthermore, Pichia sp. exhibits great diversity with regard to its natural habitat, growth morphology, metabolism, stress tolerance and antimicrobial properties (Walker 2011). Other potential biotechnological applications of Pichia isolates include environmental bioremediation, biopharmaceuticals and biofuels. b-1,4-Xylan is a heteroglycan with a backbone of b-(1 fi 4)-linked d-xylopyranose residues that can be substituted with l-arabinofuranose, d-glucuronic acid and ⁄ or 4-O-methyl-d-glucuronic acid (Puls and Schuseil 354

1993; Bhat and Hazlewood 2001). It constitutes the major component of hemicelluloses found in the cell walls of monocots and hard woods and represents one of the most abundant biomass resources. Recently, xylanolytic enzymes of microbial origin have received great attention because of their possible industrial applications for sustainable fuel ethanol production from xylan. Two key reactions proceed during hydrolysis of the xylan backbone; endo-1,4-b-xylanases (1,4-b-d-xylan xylanohydrolase) hydrolyse internal b-(1 fi 4)-xylosidic linkages in the insoluble xylan backbone to yield soluble xylooligosaccharides, while 1,4-b-xylosidases are exoglycosidases that cleave terminal xylose monomers from the nonreducing end of short-chain xylooligosaccharides (Polizeli et al.

2005). Additional enzyme activities, such as a-l-arabinofuranosidase, a-d-glucuronidase and acetyl xylan esterase, remove side-chain substituents. 1,4-b-Xylosidase is important in xylan degradation, considering that xylans are not completely hydrolysed by xylanases alone. Many micro-organisms can produce 1,4-b-xylosidase; however, only few yeast species and limited number of strains can produce it (Linden and Hahn-Hagerdal 1989). The use of

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yeasts, rather than filamentous fungi, which are a very important source of enzymes including 1,4-b-xylosidase, can avoid the induction of allergenic effects, undesirable odours and oxidation artefacts during the hydrolysis of glycosides (Sefton and Williams 1991; Mateo and Jime´nez 2000). By the other way, industrial xylose-fermenting strains capable of complete and efficient d-xylose consumption (Fonseca et al. 2011) and xylanase supplementation strategies (Alvira et al. 2011) are now under research, and food-grade enzymes obtained from yeasts are required to be used for simultaneous saccharification and co-fermentation (SSCF) (Jin et al. 2012).

Here, we describe the purification and properties of the first xylan 1,4-b-xylosidase from P. membranifaciens isolated from an oenological environment. The enzyme exhibited a combined tolerance to ethanol, pH and temperature, which offers a good candidate to be used in SSCF processes for biofuel production.

Materials and methods Screening of 1,4-b-xylosidase-producing strain Samples of wine fermentations from wineries of Requena (Spain) were diluted and inoculated onto plates containing lysine agar (Heard and Fleet 1986). A total of 164 isolates of non-Saccharomyces yeasts isolated were screened for 1,4-b-xylosidase activity. Plate screening was carried out on agar plates containing per litre: 0Æ67 YNB (Yeast Nitrogen base without aminoacids; Difco, Franklin Lakes, NJ) and 20 g agar. Then, 4-methylumbelliferyl-b-d-xyloside (Sigma, St Louis, MO) was spread onto the surface of the agar plates, yeasts were inoculated, and the plates were incubated at 28 C for 48 h. The hydrolysis of 4-methylumbelliferyl-b-d-xyloside could be visualized under UV lamp as fluorescent halos surrounding yeast growth.

Culture conditions For routine maintenance, yeasts were cultured on YPD agar (1% yeast extract, 2% mycological peptone, 2% glucose, 2% agar) slants at 4 C and subcultured every month at 28 C. The seed culture was grown in YPD liquid medium. Cells were counted in a Thoma chamber and inoculated (1Æ00 · 106 CFU ml)1) in a 250-ml shake flask containing 100 ml of induction medium, 1Æ0% (w ⁄ v) mycological peptone (Pronadisa, Madrid, Spain), 0Æ1% (w ⁄ v) yeast extract (Pronadisa) and 1Æ0% (w ⁄ v) beechwood xylan (Sigma) on a rotary shaker (200 rev min)1 at 30 C for 3 days, unless otherwise stated). The effect of xylan on 1,4-b-xylosidase activities were determined by varying the concentration of this compound in the induction medium from 0 to 5% (w ⁄ v).

P. membranifaciens 1,4-b-xylosidase

Enzyme purification All purification procedures were conducted at 4 C. Yeasts were harvested from the induction culture by centrifugation at 6000 g for 15 min and washed with 20 mmol l)1 Tris–HCl buffer (pH = 7Æ0) and 2 mmol l)1 phenyl methyl sulphonyl fluoride (PMSF). The debris cells (0Æ6 g) were added of with 0Æ5 g of glass beads per tube (425–600 mm from Sigma) and vigorously shaken using a Fast Prep Instrument (Bio 101, Inc., Vista, CA, USA) and processed in seven cycles of 30 s to achieve the complete breakage of the cells. Between the cycles, samples were placed in ice for 60 s to decrease the temperature of cellular suspensions. After the last cycle, tubes were removed from the instrument, placed 5 min in ice and the resulting lysate was centrifuged at 12 000 g for 10 min to eliminate DNA and cellular debris, and the supernatant solution (2 ml) was used as starter material for chromatography. Purification of enzyme was performed on an AKTA-FPLC General Electric Healthcare system (Uppsala, Sweden) as shown later.

Protein concentrations were measured by the method of Bradford (1976), using BSA (Sigma) as an external standard. The concentrate was loaded onto on a DEAE Sepharose Fast Flow (10 mm · 40 mm; GE Healthcare) column equilibrated with 20 mmol l)1 Tris–HCl buffer (pH 8Æ0). The adsorbed proteins were eluted with a linear gradient of 0–1Æ0 mol l)1 NaCl in the same buffer at a flow rate of 1Æ0 ml min)1. The fractions exhibiting the enzyme activity were pooled and further loaded onto a Sephadex G-100 column (16 mm · 50 cm; GE Healthcare) equilibrated with 20 mmol l)1 Tris–HCl buffer (pH 7Æ0) containing 0Æ15 mol l)1NaCl. The enzyme was eluted with the same buffer at a flow rate of 0Æ5 ml min)1. 1,4-b-Xylosidase was finally eluted as a single peak of protein that coincided with that of enzyme activity. The fractions exhibiting the enzyme activity were pooled and desalted by Sephadex G-25 Superfine (GE Healthcare) column (1Æ6 · 2Æ5 cm) with 20 mmol l)1 phosphate buffer (pH 6Æ0) at a flow rate of 3Æ0 ml min)1.

Determination of enzymatic activity 1,4-b-Xylosidase activity was basically assayed using 4-nitrophenol-b-d-xylopyranoside (Sigma) as the substrate (Mateo et al. 2011). The reaction mixture consisting of 2Æ0 ml of appropriately diluted enzyme solution, 0Æ1 ml of 5 mmol l)1 pNP-b-d-xylopyranoside in deionized water and 0Æ7 ml of 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer (pH 6Æ0) was incubated at 35 C for 90 min. The reaction was stopped by adding 1Æ0 ml of 0Æ2 mol l)1 Na2CO3, and absorbance at 404 nm was measured. One

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unit (U) of 1,4-b-xylosidase activity was defined as the amount of enzyme that liberated 1 lmol of p-nitrophenol per min from pNP-b-d-xylopyranoside.

1,4-b-Xylosidase characterization The purified 1,4-b-xylosidase was subjected to SDS-PAGE for the determination of its molecular mass (Laemmli 1970). After electrophoresis, the gel was stained with Coomassie Brilliant Blue R250 solution, and the molecular mass of the purified 1,4-b-xylosidase was determined by comparison with commercial molecular mass protein marker (Invitrogen, Carlsbad, CA). The pH optimum was determined in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer covering a pH range from 3Æ0 to 8Æ0, at 35 C for 90 min. The pH stability of the enzyme was assessed by pre-incubating the 1,4-b-xylosidase in the same buffer from pH 3Æ0 to 8Æ0 for 25 min, and the residual activities were determined. The temperature optimum was measured from 20 to 70 C for 90 min of incubation in the same buffer, at pH 6Æ0. The thermal stability of the enzyme was assessed by pre-incubating the 1,4-b-xylosidase in the same buffer from 20 to 60 C for 25 min, and the residual activities were determined. The apparent Michaelis–Menten constant (Km) and maximum velocity (Vmax) for pNP-b-d-xylopyranoside were determined according to Lineweaver and Burk (1934). Initial hydrolysis rates during the first 15 min of the reaction at 35 C were measured as a function of pNP-b-d-xylopyranoside concentrations ranging from 0Æ3 to 3Æ0 mmol l)1. The effect of various metal ions and reagents at 5 mmol l)1 on 1,4-b-xylosidase activity was determined by pre-incubating

the enzyme with the individual reagents in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer (pH = 6Æ0) at 35 C for 25 min. Activities were then measured at 35 C for 90 min in the presence of the metal ions or reagents.

The activity assayed in the absence of metal ions or reagents was recorded as 100%. Carbohydrate effects were also determined following this scheme. The effect of ethanol on 1,4-b-xylosidase activity was determined by pre-incubating the enzyme in 0–20% ethanol (v ⁄ v) for 25 min, and the residual activities were determined.

Control assays, without enzyme, were performed for every experiment.

Results and discussion The commercial uses of 1,4-b-xylosidase include processing wood pulp (Tsujibo et al. 2001), improving bread dough baking and nutritional quality (Dornez et al.

2007), hydrolysis of bitter xylosylated compounds from grape juice during extraction and liberation of aroma derived from xylosylated compounds of grapes during wine making (Manzanares et al. 1999) and hydrolysis of xylan to d-xylose residues for subsequent reduction to xylitol (Polizeli et al. 2005). Currently, 1,4-b-xylosidase is prepared from fungal and bacterial sources such as Aspergillus niger, Trichoderma reesei and Bacillus sp. among others (Jordan and Wagschal 2010) (Table 1). The discovery of cost-efficient 1,4-b-xylosidase from yeasts, which includes positive enzyme attributes such as high kcat and kcat ⁄ Km, low affinity for monosaccharide inhibitors and good stabilities to pH, temperature or ethanol, would improve the opportunities to produce this enzyme under

Table 1 Comparative properties of some fungal 1,4-b-xylosidases Micro-organism

Mw (kDa)

Optimum pH

Optimum T ( C)

References

Aspergillus carbonarius Aspergillus fumigatus Aspergillus niger Aspergillus phoenicis Aspergillus pulverulentus Aureobasidium pullulans Emericella nidulans Fusarium verticilloides Humicola grisea Neocallimastrix patriciarum Neomallimastix frontalis Penicillium wortmanni Pichia membranifaciens Pichia stipitis Talaromyces emersonii Trichoderma reesei Trichoderma viride

100Æ0 360Æ0 122Æ0 132Æ0 65Æ0 88Æ5 116Æ0 94Æ5 43Æ0 39Æ5–150Æ0 150Æ0 110Æ0–210Æ0 50Æ0 NR 181Æ0 100Æ0 102Æ0

4Æ0 4Æ5 3Æ8–4Æ0 4Æ0–4Æ5 4Æ0–5Æ0 3Æ5 4Æ5–5Æ0 4Æ5 6Æ0 6Æ0–6Æ4 6Æ4 3Æ0–4Æ5 6Æ0 4Æ8–5Æ0 2Æ5 4Æ0 4Æ5

60 75 70 75 60 70 55 65 50 37–50 37 55–65 35 50 60 60 55

Kiss and Kiss (2000) Kitpreechavanich et al. (1986) Rodionova et al. (1983) Rizzatti et al. (2001) Sulistyo et al. (1995) Ohta et al. (2010) Matsuo and Yasui (1984) Saha (2001) Almeida et al. (1995) Zhu et al. (1994) Garcia-Campayo and Wood (1993) Matsuo et al. (1987) This work Basaran and Ozcan (2008) Tuohy et al. (1993) Poutanen and Puls (1988) Matsuo and Yasui (1984)

NR, not reported.

356

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P. membranifaciens 1,4-b-xylosidase

fermenter controlled conditions. Moreover, undesirable odours and oxidation artefacts detected when fungal enzymatic preparations are used are not reported by using yeasts (Mateo and Jime´nez 2000).

Table 2 Purification of 1,4-b-D-xylosidase from a 3-day-old culture of Pichia membranifaciens

Step

Identification, screening, selection of 1,4-b-xylosidaseproducing yeasts and culture condition optimization

Total Total protein units (mg) (U)

Crude extract 63Æ0 DEAE cellulose 5Æ0 Sephadex G-100 0Æ2

We have identified several Pichia strains with 1,4-b-xylosidase activity in our laboratory (unpublished data). The yeast Pichia sp. C105 displayed the strongest 1,4-b-xylosidase activity among the 164 isolates of non-Saccharomyces yeasts isolated from local wineries of Requena (Spain) used in this study. Halos detected in agar plates were confirmed by enzymatic assays using pNP-b-d-xylopyranoside substrate. The effect of xylan concentration on activity was also evaluated. The highest values were with 1–2% (w ⁄ v) of this compound, and no activity was detected at low xylan concentrations, lower than 0Æ5% (w ⁄ v). The optimum time for crude extract preparation, leading to a specific activity of 24 U (mg protein))1, was determined in a flask culture (Fig. 1).

Purification of 1,4-b-xylosidase Results for 1,4-b-xylosidase purification are summarized in Table 2. The enzymatic extract was eluted in a DEAE Sepharose column with approximately 0Æ2 mol l)1 NaCl together with other proteins >40 kDa. The active fractions were applied to a Sephadex G-100 column, and active fractions were desalted and subsequently used for biochemical characterization. These chromatography methods resulted in a 32-fold purification of 1,4-b-xylosidase with a recovery of about 10%. The enzyme produced

Specific Purification Yield activity U (%) (mg protein))1 (-fold)

149Æ2 2Æ4 108Æ1 21Æ6 15Æ2 76Æ6

1Æ0 9Æ1 32Æ1

100Æ0 72Æ5 10Æ2

an apparent single 50-kDa band (Fig. 2), similar to the enzyme reported in the yeast Candida utilis (58 kDa) (Yanai and Sato 2001), and significantly lower to fungal enzymes, Penicillium wortmanni (100 kDa) and Aspergillus spp. (90–132 kDa) (Kitpreechavanich et al. 1986; Kurakake et al. 1997; Rizzatti et al. 2001).

Enzymatic characterization To determine the pH optimum, 0Æ2 mol l)1 citrate– 0Æ1 mol l)1 phosphate buffer ranging from pH 3Æ0 to 8Æ0 at intervals of 1Æ0 were applied. 1,4-b-Xylosidase of P. membranifaciens exhibited high pH stability between pH 6Æ0 and 8Æ0, retaining more than 90% residual activity in vitro, with an optimum value around pH 6Æ0 (Fig. 3a).

Yanai and Sato (2001) reported similar behaviour in pH

KDa

1

2

120

Relative activity (%)

100

85

80

60 60

40

20 0

2

4 Time (days)

6

8

Figure 1 Time course of 1,4-b-xylosidase production by Pichia membranifaciens. Data represent means ± standard deviations from at least triplicate assays.

Figure 2 SDS-PAGE of the purified b-d-xylosidase from Pichia membranifaciens after protein staining with Coomassie Brilliant Blue solution. Lane 1: molecular weight standard [the positions of protein sizes (kDa) are shown on the left]; lane 2: purified b-D-xylosidase.

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(a) 100

100

80

80

Relative activity (%)

Relative activity (%)

(a)

60 40

60 40 20

20

0

0 4

3

5

6

7

20

8

30

40

pH

50

60

70

15

20

25

T (°C)

(b)

(b)

100

Residual activity (%)

Residual activity (%)

100 95 90 85 80 75

60 40 20 0

0

5

10

15

20

25

Time (min)

0

5

10

Time (min)

Figure 3 Effect of pH on activity. (a) pH (d) b-D-xylosidase was incubated for 90 min at 35 C at the indicated pH values in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer. The maximum value was 100%. (b) pH inactivation. b-D-Xylosidase was pre-incubated for 25 min at 35 C at the indicated pH values in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer: pH 3 (m), pH 4 (4), pH 5 (d), pH 6 (s), pH 7 (n) and pH 8 (h). Samples were withdrawn and activities were determined. Data represent means ± standard deviations from at least triplicate assays.

assays with C. utilis. The activity of other 1,4-b-xylosidases from fungal origin at pH > 6Æ0 is very low and cannot be industrially considered under these conditions (Rizzatti et al. 2001). Pichia stipitis and P. membranifaciens 1,4b-xylosidases are stable at pH 7Æ0 (Basaran and Ozcan 2008 and this work). Stability at different values of pH ranging from 3Æ0 to 8Æ0 were also assayed by pre-incubating the enzyme for 25 min. Despite of the low residual activity detected at pH range from 3Æ0 to 5Æ0, activity was higher than 80% for 25 min. The 0-min value was 100% (Fig. 3b).

The enzymatic activity did not vary with temperature around to 25–40 C (Fig. 4a), with an optimum of 35 C; the P. membranifaciens enzyme has 50% activity at 50 C, which was similar to other yeast proteins (Basaran and Ozcan 2008). However, 1,4-b-xylosidase activity decreased 358

80

Figure 4 Effect of temperature on activity. (a) Temperature. b-D-Xylosidase was added to 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer (pH 6Æ0) and incubated for 90 min at different temperatures. (b) Thermal inactivation. b-D-Xylosidase was pre-incubated for 25 min at pH 6Æ0 in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer: 20 C (m), 30 C (4), 40 C (d), 50 C (s) and 60 C (n). Samples were withdrawn and activities were determined. Data represent means ± standard deviations from at least triplicate assays.

at 20 C. At higher temperature (70 C), thermal denaturalization probably occurs. Candida utilis activity at this temperature is zero, and P. stipitis mutant is 20% (Yanai and Sato 2001; Basaran and Ozcan 2008).

The enzyme exhibited apparent Km and Vmax values of 0Æ48 ± 0Æ06 mmol l)1 and 7Æ4 ± 0Æ1 lmol min)1 mg)1 protein, respectively, for pNP-b-d-xylopyranoside. These values are in accord with the apparent results published for other 1,4-b-xylosidases (Rizzatti et al. 2001; Basaran and Ozcan 2008).

The influence of metal ions, a metal chelator (EDTA) and various other compounds on 1,4-b-xylosidase activity was also assayed (Table 3). Ca2+, Fe2+ and K+ had little effect on the activity. Mg2+, Cu2+ and Zn2+ had a moderate inhibitory effect on activity. The activity was increased

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P. membranifaciens 1,4-b-xylosidase

No addition KCl ZnSO4 FeSO4 MgCl2 CuSO4 CaCl2 HgCl2 b-Mercaptoethanol EDTA SDS Methanol

Concentration

Residual activity (%)*

0 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 5 mmol l)1 0Æ25% (v ⁄ v) 0Æ25% (v ⁄ v) 5Æ00% (v ⁄ v)

100Æ0 102Æ9 84Æ6 95Æ6 88Æ2 67Æ6 98Æ1 ND 153Æ1 78Æ3 30Æ0 147Æ5

± ± ± ± ± ± ±

3Æ0 3Æ2 4Æ1 3Æ3 3Æ0 0Æ3 4Æ7

± ± ± ±

6Æ5 5Æ1 3Æ2 0Æ6

ND, not detected. *Each value represents the mean of triplicate measurements.

by 50% with b-mercaptoethanol. This result suggests that sulphydryl compounds keep the active enzyme in a reduced state. The enzyme activity was completely inhibited by a sulphydryl oxidant metal (Hg2+), supporting the importance of a sulphydryl group for the expression of the enzyme activity (Yanai and Sato 2001). EDTA is employed in many biochemical tests to decrease the action of proteases in crude extracts because of its ability to chelate metallic ions. Nevertheless, its use can be a problem if the enzyme studied is metallic ion dependent.

The addition of standard concentration (5 mmol l)1) slightly inhibited the activity. This result allowed us to conclude that the active site of these enzymes could be dependent on divalent cations for enzyme activation, as previously reported (Mateo and Di Stefano 1997). The

enzyme exhibited a high degree of sensitivity to SDS probably due to protein denaturation. Methanol effects can be explained by a change in polarity of the medium, as is discussed further for ethanol assays.

The influence of sugars and ethanol at concentrations that are normally present in wines and musts was also examined. Glucose, fructose and sucrose inhibition tests were carried out with pNP-b-d-xylopyranoside as the substrate. The enzyme retained more than 50% of its maximal activity even in the presence of 0Æ2 mol l)1 glucose, and only a moderate inhibitory effect was detected in the presence of oenological quantities of fructose or sucrose (Fig. 5). These results are in agreement with other Pichia species (Manzanares et al. 1999) regarding their high tolerance to glucose. Kamble and Jadhav (2012) have recently reported that xylanase is inhibited by low concentration of glucose. On the other hand, ethanol had (a) 180

Relative activity (%)

Table 3 Effects of metal ions and chemical reagents on the activity of 1,4-b-xylosidase purified from Pichia membranifaciens

160

140

120

100 0

5

10

15

20

Ethanol % (v/v) (b)

110 Relative activity (%)

Relative activity (%)

100 90 80 70 60

105

100

95

50

90 40

0

50

100

150

200

0

5

10

15

20

25

Time (min)

Sugar concentration (mol l–1) Figure 5 Effects of glucose (m), fructose (4) and sucrose (d) concentrations on b-d-xylosidase. Enzyme activities were measured in the presence of various amounts of glucose, fructose or sucrose under the standard assay conditions. Data represent means ± standard deviations from triplicate assays.

Figure 6 (a) Effects of different concentrations of ethanol (%): 0% (m), 5% (4), 10% (d) and 20% (s) on b-d-xylosidase. (b) Ethanol inactivation. b-d-xylosidase was pre-incubated for 25 min at pH 6Æ0 at 35 C in 0Æ2 mol l)1 citrate–0Æ1 mol l)1 phosphate buffer supplemented with ethanol (%); 0% (4), 5% (s), 10% (m) and 20% (d).

Data represent means ± standard deviations from triplicate assays.

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not a negative effect on the enzyme activity at concentration up to 20% (v ⁄ v) (Fig. 6a). Moreover, ethanol at 5% (v ⁄ v) was found to be an activator for the enzyme activity (increase of 70%), and at 10% (v ⁄ v) yielding an increase of 40% with respect to the control series without ethanol.

The stability on ethanol was also studied. The purified 1,4-b-xylosidase was highly stable and maintained approximately 100% activity when incubated in the presence of 20% ethanol for 25 min (Fig. 6b). We have also observed an increase in enzyme activity after 5 min of incubation, which decreases suddenly and reaches a value lower than that obtained at higher ethanol concentrations after 25 min of incubation. In the case of higher ethanol concentrations, the enzyme activity decreased in 5 min and then increased. The ethanol tolerance assays (ranging from 0 to 20%) showed stability under these conditions.

The change in polarity of the medium induced by ethanol could stabilize enzyme conformation (Mateo et al. 2011).

The inhibition of the enzyme activity caused by Hg2+ indicates that it is dependent of cysteine residues. Most of the reported 1,4-b-xylosidases show a similar effect as noncompetitive inhibitor for enzyme function (Matsuo and Yasui 1984).

The low inhibition rate by glucose and ethanol show this enzyme is a good candidate to be used in many biotechnological processes, including xylan degradation, where combined stability is appreciated. There are very few yeast species able to metabolize this substrate for biofuel production processes (Linden and Hahn-Hagerdal 1989), although fungal enzymes are extensively described (John et al. 1979; Rodionova et al. 1983; Sulistyo et al.

1995; Kumar and Ramo´n 1996; Yinbo et al. 1996; Kitamoto et al. 1999).

In conclusion, a combined pH, temperature and ethanol, stable 1,4-b-xylosidase has been isolated from P. membranifaciens. The 1,4-b-xylosidase also displays resistance to common inhibitors. The successful purification and characterization has been conducted, and the enzyme will be used for further immobilization studies.

Acknowledgements This work was supported by grants from RM2007-0000100-00 ⁄ INIA and INV-AE112-66049 ⁄ UV. This research has been performed within the Programme VLC ⁄ Campus, Microcluster IViSoCa. References Almeida, E.M.D., Lourdes, M.D., Polizeli, T.M., Terenzi, H.F. and Jorge, J.A. (1995) Purification and biochemical characterization of b-xylosidase from Humicola grisea var. thermoidea. FEMS Microbiol Lett 130, 171–176.

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Basaran, P. and Ozcan, M. (2008) Characterization of b-xylosidase enzyme from a Pichia stipitis mutant. Bioresour Technol 99, 38–43.

Bhat, M.K. and Hazlewood, G.P. (2001) Enzymology and other characteristics of cellulases and xylanases. In Enzymes in Farm Animal Nutrition ed. Bedford, M. and Partridge, G. pp. 11–57. Oxford, UK: CAB International.

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Dornez, E., Gebruers, K., Cuyvers, S., Delcour, J.A. and Courtin, C.M. (2007) Impact of wheat flour-associated endoxylanases on arabinoxylan in dough after mixing and resting. J Agric Food Chem 55, 7149–7155.

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