β-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

FEMS Microbiology Letters 240 (2004) 137–143 www.fems-microbiology.org

b-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose Fabiana Fonseca Zanoelo, Maria de Lourdes Teixeira de Moraes Polizeli, He´ctor Francisco Terenzi, Joa˜o Atı´lio Jorge * Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜oPreto, Sa˜o Paulo, Brasil Received 15 June 2004; received in revised form 4 August 2004; accepted 17 September 2004 First published online 29 September 2004 Edited by B.A. Prior

Abstract An inducible mycelial b-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60
C and 6.5, respectively. The enzyme was stable up to 1 h at 50
C and exhibited a half-life of 20 min at 55
C. The enzyme hydrolyzed p-nitrophenyl-b-D -glucopyranoside, p-nitrophenyl-b-D -xylopyranoside, o-nitrophenyl-b-D -galactopyranoside, p-nitrophenyl-a-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. b-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum b-glucosidase is a novel characteristic which distinguish this enzyme from all other b-glucosidases so far described.  2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: b-Glucosidase; b-Glycosidase; Cellobiase; Glucose-exhanced b-glucosidase; Xylose-exhanced b-glucosidase; Scytalidium thermophilum

1. Introduction b-Glucosidases, under physiological conditions, catalyze the hydrolysis of alkyl- and aryl-b-glucosides, as well diglucosides and oligosaccharides. These enzymes are widespread in nature, occurring in all the living kingdoms and perform varied functions. The initial interest in studying b-glucosidases arose in the 1950s, due to its involvement in the biological conversion of cellulose. The enzymatic hydrolysis of cellulosic substrates by microorganisms usually involves the syner*

Corresponding author. Tel.: + 55 16 6023709; fax: +55 16 6331758. E-mail address: [email protected] (J.A. Jorge).

gistic action of endo-1,4-b-glucanase (EC 3.2.1.4), exo-1,4-b-glucanase (EC 3.2.1.91) and 1,4-b-glucosidase (EC 3.2.1.21) [1]. Only the first two enzymes act directly, depolymerizing the cellulose fiber and releasing as final products oligosaccharides of different sizes, and cellobiose. b-Glucosidase hydrolyses cellobiose, an inhibitor of the depolymerizing activities, releasing glucose [2]. Thus, for most cellulolytic systems b-glucosidase is the rate-limiting factor, and it is also very sensitive to glucose inhibition [2–5]. The search for bglucosidases insensitive to product inhibition, and of high thermal stability, has increased recently. Enzymes with these characteristics would improve the process of saccharification of lignocellulosic materials [5,6]. Moreover, a few microbial b-glucosidases are known to be

0378-1097/$22.00  2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.09.021

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activated by glucose [7,8]. Thermophilic filamentous fungi are good producers of b-glucosidases with high thermal stability [9,10], and among these Scytalidium thermophilum is an efficient consumer of cellulosic materials [11]. This report describes the properties of a glucose- and xylose-activated b-glucosidase from this fungus.

2. Materials and methods 2.1. Organism and growth conditions Scytalidium thermophilum strain (CBS 619.91) was a gift of Dr. G. Straastma from the Mushroom Experimental Station, The Netherlands. The fungus was maintained at 40
C on slants of solid medium containing 4% oatmeal baby food (Quaker). Conidia from 10-day-old cultures were inoculated into liquid medium containing 0.2% NaNO3, 0.1% KH2PO4, 0.1% KCl, 0.05% MgSO4 Æ 7H20, 0.01% FeSO4 Æ 7H20, 0.03% ZnSO4 Æ 7H2O, 0.8% yeast extract, a carbon source at 1% concentration, and pH adjusted to 6.0. The cultures were incubated at 40
C for 48 h, in a rotary shaker at 140 rpm.

b-glucopyranoside (PNP-glu) at final concentration of 2 mg ml 1. The reaction was stopped at different time intervals by adding two volumes of saturated sodium tetraborate solution, and the absorbance was read at 410 nm. b-Xylosidase, b-galactosidase and a-arabinofuranosidase activities were assayed under the same conditions, except that p-nitrophenyl-b-D -xylopyranoside (PNP-xyl), o-nitrophenyl-b-D -galactopyranoside (ONPG) and p-nitrophenyl-a-L -arabinopyranoside (PNP-ara) were the respective substrates. Cellobiase and lactase activities were measured as described above, using 10 mg ml 1 of substrate and the glucose released was detected by the glucose oxidase method [14], or by HPLC. Activity against the polymeric substrates avicel, CM-cellulose and xylan was assayed using the dinitrosalycilic acid method [15]. Transglycosylation activity was assayed under the same conditions used to assay cellobiase activity, except that the substrate was cellobiose 250 mM, and the reaction products were detected by thin layer chromatography (TLC). One enzyme unit (U) was defined as the amount of enzyme that releases one lmol of product per minute. Specific activity was expressed as U mg protein 1.

2.5. Purification of b-glucosidase 2.2. Cellular distribution of b-glucosidase The total enzymatic fraction bound at the mycelium surface was estimated by assaying a sample of intact mycelium rinsed with one equivalent culture volume of chilled water. A part of this enzymatic fraction (loosely bound enzyme) could be released by extensively washing the mycelium with 20 equivalent culture volumes of chilled water. The residual activity (tightly bound enzyme) was totally destroyed by treating the intact extensively washed mycelium with HCl at 0
C, according to Mandels [12]. The intracellular enzyme fraction was estimated in the supernatant fraction of a portion of the acid-treated mycelium ground with glass beads, as described in the next section. 2.3. Preparation of crude enzyme The mycelium was harvested by filtration, rinsed with distilled water, blotted, frozen, and ground in a mortar with glass beads. Disrupted cells were extracted with distilled water, the slurry was centrifuged at 10,000g for 15 min at 4
C and the supernatant was used as the source of crude enzyme. 2.4. Enzyme assay b-Glucosidase was routinely assayed in McIlvaine buffer [13], pH 6.5, at 60
C, using p-nitrophenyl-

The crude enzyme, obtained from mycelia grown with 1% avicel for 48 h, was heated at 50
C for 20 min, and cooled in an ice bath. Denatured protein was spun down (10,000g) and solid ammonium sulfate was added to achieve 75% saturation. The suspension was stored overnight at 4
C. Precipitated protein was separated by centrifugation at 12,000g for 20 min, dissolved in a small volume of 100 mM sodium phosphate buffer, pH 6.0, and dialyzed exhaustively against the same buffer. The dialyzed sample was applied to a Sephadex G-100 column (87 · 2 cm), equilibrated and eluted at a flow rate of 20 ml h 1 with 100 mM sodium phosphate, pH 6.0. Active fractions were pooled, dialyzed overnight against 100 mM sodium phosphate buffer, pH 6.8, applied to a DEAE–cellulose column (13.0 · 1.5 cm) equilibrated with the same buffer, and eluted at a flow rate of 50 ml h 1 with a sodium chloride linear gradient from 0 to 0.5 M, in the same buffer. Active fractions were pooled, dialyzed against distilled water, lyophilized and dissolved in a small volume of distilled water.

2.6. Polyacrylamide gel electrophoresis Nondenaturing electrophoresis was carried out in rod gels according to Davis [16], using 8% acrylamide. SDS– PAGE was carried out according to Laemmli [17], using 15% acrylamide. Isoelectric focusing using Pharmalyte pH 5–8 was carried out in rod gels, according to O
Far-

F.F. Zanoelo et al. / FEMS Microbiology Letters 240 (2004) 137–143

rel et al. [18]. The 6% gels (0.6 cm · 13.0 cm) contained 5% (v/v) carrier ampholyte and 25 lg of purified enzyme. After isoelectric focusing at 500 V for 6 h, the pH gradient was measured by slicing one gel in 5 mm thick slices, and extracting each slice with 2.5 ml of 25 mM KCl. Protein was stained with Coomassie blue. Visualization of enzymatic activity (PAGE or IEF– PAGE) was performed as described by Peralta et al. [9], using 6-bromo-2-naphtyl-b-D -glucopyranoside as substrate. 2.7. Gel filtration The native molecular weight of the purified b-glucosidase was estimated by filtration in Sephadex G100 (95 · 2 cm) equilibrated and eluted with 100 mM sodium phosphate, pH 6.0, containing 100 mM NaCl. Elution was performed at a rate of 20 ml h 1 and fractions of 1.5 ml were collected and analyzed for absorbance (280 nm) and b-glucosidase activity. The void volume of the column was determined using blue dextran, and the following proteins were used for column calibration: alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase and cytochrome C.

139

3. Results and discussion 3.1. Effect of the carbon source on mycelial b-glucosidase production The largest enzyme activity was obtained when the fungus was grown for 48 h on avicel (0.49 ± 0.06 U mg protein 1) or cellobiose (0.43 ± 0.04 U mg protein 1) as source of carbon. Addition of glucose to avicel or cellobiose culture media drastically repressed the production of enzyme. Addition of cycloheximide (50 mg ml 1) also inhibited the synthesis of b-glucosidase, suggesting a demand for de novo protein synthesis. 3.2. Cellular distribution of b-glucosidase About 87% of the b-D -glucosidase activity in mycelia induced with cellobiose or avicel was surface-bound (e.g. it could be detected by assaying intact cells). Repeated washings with water could extract part of the enzyme. Forty percent of the enzyme still remained adsorbed to the cell surface, while about 13% was detected only after cell disruption, representing the intracellular enzyme fraction. With the aging of the mycelium the enzyme was partially released into the culture medium.

2.8. Analytical methods

3.3. Purification of b-glucosidase

The stimulatory effect of glucose on the b-glucosidase activity was tested using the reaction mixture described in Section 2.4, using 10 mM cellobiose as substrate and 100 mM glucose as activator. The reaction was stopped at different times by boiling, and was diluted 50–100-fold in MilliQ water. The glucose released was determined by HPLC using a Nucleosil 100-5NH2 (4.6 · 250 mm) column (Macherey-Nagel Co.) with acetonitrile/water (80:20, v/v), and running at 40
C. The control sample contained all the constituents, except the enzyme. Total neutral carbohydrate was estimated by the method of Dubois et al. [19], using mannose as standard. Protein was estimated by the method of Lowry et al. [20], using bovine serum albumin as standard. Products of transglycosylating activity were analyzed by TLC on silica gel G-60, using butanol/pyridine/water (6:4:3, v/v/v) as the mobile phase system. The cellooligosacharides were detected with 0.2% (w/v) orcinol in sulfuric acid/methanol (10–90, v/v).

After the DEAE–cellulose step, the specific activity of b-glucosidase was about 9.1 ± 1.3 U mg protein 1 and 23-fold purification was achieved with a yield of 25%. The purified enzyme, when run on PAGE, SDS–PAGE and isoelectric focusing (IEF–PAGE), produced a single band when stained with Coomasie blue, indicating the homogeneity of the preparation (Fig. 1). Also, duplicate gels (PAGE or IEF–PAGE), when stained for activity, produced bands coincident with those stained for protein with Coomasie blue (data not shown).

2.9. Determination of kinetic parameters Maximum activity (Vm), Michaelis–Menten constant (Km) and the apparent affinity constants (K0.5) were calculated using the Sigraf software [21] which uses a sum of two Hill equations to fit experimental data, using a non-linear regression method.

3.4. Molecular properties SDS–PAGE analysis showed that the purified bglucosidase had a molecular mass of 42 ± 3 kDa (Fig. 1(b)). Molecular sieving of the purified enzyme in Sephadex G-100 indicated an apparent molecular mass of 39 ± 4 kDa (data not shown). Together these data suggested that the enzyme was a monomeric protein. This value was similar to that reported for b-glucosidases from Humicola grisea [9,10], which is considered a synonym of S. thermophilum [11]. Isoelectric focusing (IEF–PAGE) of the purified b-glucosidase indicated a pI of about 6.5, higher than that of most microbial b-glucosidases, which are acidic proteins with pI values ranging from 3.5 to 5.5 [22]. The carbohydrate content of the purified enzyme was estimated to be 15%. Thermophilic fungi

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Residual Activity (%)

100

80

60

40

20

15

30

45

60

Time (min) Fig. 2. Thermal inactivation at 50
C (closed symbols) and at 55
C (open symbols) of purified b-glucosidase. Fig. 1. Analysis of purified b-glucosidase in PAGE (a), SDS–PAGE (b) and IEF–PAGE (c). Lane 1, molecular weight standards; lane 2, 15 lg of purified b-glucosidase; F, front marker. Gels were stained with Coomassie blue.

usually produce richly glycosylated b-glucosidases [9,10]. 3.5. Biochemical characterization 3.5.1. Effect of temperature and pH At temperatures ranging from 40 to 60
C the enzyme activity increased steadily, achieving 90% of its maximum at 55
C. Maximum activity was observed at 60
C, but at 65
C the detected activity dropped to only 10% of the maximal value. Most fungal b-glucosidases exhibit temperature optima ranging from 40 to 50
C [5]. Maximum activity was found at pH 6.5. The purified enzyme was stable up to 60 min at 25
C in a pH range of 5.0 to 8.0. At pH 4.0 or pH 8.5 the enzyme lost about 46% of its initial activity. Most fungal b-glucosidases exhibit pH optima ranging from 5.0 to 6.5 [5]. The enzyme was thermostable up to 60 min when incubated at 50
C in McIlvaine buffer [13], pH 6.5, and exhibited a half-life of 20 min when incubated at 55
C (Fig. 2). The thermoinactivation profile at 55
C was monophasic, suggesting that the enzyme had a unique state of stable conformation, and absence of isoenzymes. The thermostability of the purified enzyme was higher than that of H. grisea bglucosidase [9]. 3.5.2. Effect of metals ions While Hg2+ and Ag2+ ions were strong inhibitors, most of other metals ions, such as Mg2+, Ca2+, Al3+, Co2+, Zn2+, Cu2+, tested at 1 mM concentration, or EDTA, were without effect (data not shown).

3.5.3. Enzyme specificity The enzyme exhibited a broad specificity (Table 1) and hydrolysed PNP-glu, ONPG, PNP-xyl, PNP-ara, cellobiose, laminaribiose and lactose, however, PNPglu and cellobiose were the preferred substrates. The enzyme had no activity on sucrose, maltose, trehalose, xylobiose, avicel, CM-cellulose or xylan. The enzyme could also hydrolyze cellotriose and cellotetraose as detected by TLC (data not shown). Studies with equimolar mixtures of PNP-glu with ONPG, PNP-xyl or PNP-ara

Table 1 Substrate specificity of purified b-glucosidasea Substrateb

Specific activity (U mg protein 1)c

p-Nitrophenyl-b-D -glucopyranoside p-Nitrophenyl-b-D -xylopyranoside o-Nitrophenyl-b-D - galactopyranoside p-Nitrophenyl-a-L -arabinopyranoside p-Nitrophenyl-b-D -manopyranoside p-Nitrophenyl-a-D -glucopyranoside Cellobiose Lactose Laminaribiose Sucrose Maltose Trehalose Xylobiose Avicel CM-cellulose Xylan

8.9 ± 0.9 1.4 ± 0.2 1.8 ± 0.3 0.7 ± 0.1 ND ND 3.8 ± 0.5 0.9 ± 0.2 1.6 ± 0.2 ND ND ND ND ND ND ND

ND – Aactivity not detected up to 2 h of enzyme reaction. a The different substrates were tested in McIlvaine buffer pH 5.5 at 60
C. b Artificial substrates were used at final concentration of 2 mg ml 1 and natural substrates at 10 mg ml 1. c Values are means ± SD of six different experiments.

F.F. Zanoelo et al. / FEMS Microbiology Letters 240 (2004) 137–143

Table 2 Effect of carbohydrates on the purified b-glucosidase Carbohydrate (50 mM)

Enzyme activity (%)

Control Glucose Xylose Mannose Fructose Ribose Galactose L-Sorbose Arabinose Lactose Cellobiose Maltose Sucrose

100 204 ± 12 191 ± 14 100 ± 9 111 ± 11 100 ± 12 123 ± 11 95 ± 8 97 ± 6 92 ± 6 24 ± 3 113 ± 9 105 ± 7

The effect of carbohydrates on the enzyme activity were determined using PNP-glu as substrate. Values are means ± SD of six experiments.

ß-Glucosidase (U mg protein-1)

3.5.4. Effect of carbohydrates on b-glucosidase activity Several carbohydrates were tested using PNP-glu as substrate (Table 2). Surprisingly, b-glucosidase activity increased 2.0-fold and 1.9-fold with 50 mM glucose or 50 mM xylose, respectively. Although rare, there are precedents of the enhancement of b-glucosidase activity by glucose, for enzymes purified from bacteria [8,22], or very high tolerance to glucose for fungal b-glucosidases [7,23,24]. However, to our knowledge, this is the first report of a b-glucosidase simultaneously activated by glucose or xylose. Fig. 3(a) shows the effect of increasing glucose or xylose concentrations on the b-glucosidase activity. Glucose or xylose exhibited maximal effects on the enzyme activity at about 150 mM concentration, increasing the rate of PNP-glu hydrolysis by 2.6-fold and 2.4-fold, respectively. In the presence of 500 and 700 mM, respectively, of xylose or glucose, the b-glucosidase activity against PNP-glu was identical to the enzymatic reactions tested in absence of activators (data not shown). The mixture of xylose and glucose exhibited no additive effect on the enzyme activity, suggesting that xylose and glucose may act at the same site of the S. thermophilum b-glucosidase (Fig. 3(a)). Mannose, fructose, ribose, galactose, sorbose, arabinose, maltose and sucrose were without effect (Table 2). The effect of xylose on the cellobiase activity was tested using the glucose

24

20

16

12

8

4 0

50

100

150

200

Effector (mM)

(a) 7 .5

Cellobiase (U mg protein-1)

demonstrated inhibition for the hydrolysis of PNP-glu, suggesting that the broad specificity exhibited by the purified enzyme against synthetic substrates was not due to minor contaminants (data not shown). Mixture of lactose with PNP-glu also showed inhibition for the hydrolysis of the synthetic substrate (data not shown). b-Glucosidases of thermophilic fungi [9,10] and other microbial sources are also characterized by a broad substrate specificity [5]. When cellobiose was used as substrate at 250 mM concentration, the purified enzyme exhibited transglycosylation activity, producing cellotriose, cellotetraose and cellopentaose (data not shown).

141

6 .0

4 .5

3 .0 0

(b)

50

10 0

15 0

200

Xylose (mM)

Fig. 3. Effect of glucose or xylose on b-glucosidase activity. Activity was measured using PNP-glu (a) or cellobiose (b) as substrate. PNPglu was used at final concentration of 6.6 mM. Symbols: d, glucose; s, xylose; j, equimolar mixture of glucose and xylose, containing each sugar at concentration equal to the points described in the abscissa.

oxidase method in order to detect the glucose released (Fig. 3(b)). Cellobiase activity increased 2.1-fold at xylose concentration of 100–200 mM and did not become inhibited until xylose concentration reached 500 mM (data not shown). The positive effect of glucose on cellobiase activity (2.3-fold) was also detected using HPLC (data not shown). 3.5.5. Kinetic parameters Km and Vmax values (Table 3) were 1.61 ± 0.23 mM, 4.12 ± 0.55 U mg protein 1 for cellobiose and 0.29 ± 0.03 mM and 13.27 ± 1.32 U mg protein 1 for PNP-glu. The efficiency of substrate utilization was estimated on the basis of Vmax/Km ratio, indicating that the

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Table 3 Kinetic parameters of the purified b-glucosidase from S. thermophilum Substrate

Addition

Km (mM)

Vmax (U mg protein 1)

Vmax/Km

KI (mM)

PNP-glu (0.15–10 mM) Cellobiose (0.5–15 mM) PNP-glu (0.15–15 mM) PNP-glu (0.15–15 mM) PNPglu (0.15–3.0 mM)

– – Glucose (80 mM) Xylose (100 mM) Cellobiose (0.5–15 mM)

0.29 ± 0.03 1.61 ± 0.23 1.26 ± 0.15 1.33 ± 0.13 –

13.27 ± 1.32 4.12 ± 0.55 40.04 ± 4.80 30.49 ± 3.01 –

45.76 ± 4.73 2.56 ± 0.33 31.78 ± 3.81 22.92 ± 2.26 –

– – – – 1.32 ± 0.13

Data are the means ± SD of at least four different experiments.

PNP-glu was the best substrate. The presence of xylose or glucose on the reaction media, containing PNP-glu as substrate, increased both of Km and Vmax values (Table 3). The apparent affinity constants (K0.5) for glucose and xylose were 36.69 ± 3.71 and 43.24 ± 5.98 mM, respectively (Fig. 3(a)). These data are consistent with the hypothesis that xylose and glucose compete by the same site on the enzyme. The mechanism of the activation by glucose or xylose of the S. thermophilum b-glucosidase is not yet fully understood, and deserves further studies. The presence of cellobiose inhibited competitively the hydrolysis of PNP-glu with a KI of 1.32 ± 0.13, suggesting that both, natural and synthetic substrate, are hydrolyzed at the same catalytic site (Table 3). Competitive inhibition by glucose is a common characteristic of fungal b-glucosidases, that limits their use for the enzymatic hydrolysis of plant products [2,23]. Most microbial b-glucosidases have a glucose inhibition constant (KI) ranging from 0.5 mM to no more than 100 mM [23,25]. For example, some b-glucosidases from Aspergillus species exhibit KI values ranging from 3 to 14 mM [26,27]. Besides the good thermal stability, the positive effect of glucose and xylose on the S. thermophilum b-glucosidase is certainly a property that distinguish this enzyme from all other b-glucosidases described, and qualifies it for application in the hydrolysis of cellulosic materials. Acknowledgements This work was supported by a grant from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). H.F.T., J.A.J., M.L.T.M.P. are research Fellows of CNPq. F.F.Z. is a fellowship recipient from FAPESP. This work is part of Doctoral Thesis of F.F.Z. (Departamento de Biologia, Programa de Biologia Comparada, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo). References [1] Wood, M.T. and Garcia-Campayo, V. (1990) Enzymology of cellulose degradation. Biodegradation 1, 147–161.

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