Production, purification and characterization of a minor form of xylanase from Aspergillus versicolor

Process Biochemistry 40 (2005) 359–364

Production, purification and characterization of a minor form of xylanase from Aspergillus versicolor Eleonora Cano Carmona∗ , Mauricio Batista Fialho, Érika Bicalho Buchgnani, Glauciane Danusa Coelho, Márcia Regina Brocheto-Braga, João At´ılio Jorge Departamento de Bioqu´ımica e Microbiologia, Instituto de Biociˆencias, UNESP, Caixa Postal 199, 13.506-900, Rio Claro, SP, Brazil Received 17 June 2003; received in revised form 12 November 2003; accepted 3 January 2004

Abstract A strain of Aspergillus versicolor produces a xylanolytic complex containing two components, the minor component being designated xylanase II. The highest production of xylanase II was observed in cultures grown for 5 days in 1% wheat bran as carbon source, at pH 6.5. Xylanase II was purified 28-fold by DEAE-Sephadex and HPLC GF-510 gel filtration. Xylanase II was a monomeric glycoprotein, exhibiting a molecular mass of 32 kDa with 14.1% of carbohydrate content. Optimal pH and temperature values for the enzyme activity were about 6.0–7.0 and 55 ◦ C, respectively. Xylanase II thermoinactivation at 50 ◦ C showed a biphasic curve. The ions Hg2+ , Cu2+ and the detergent SDS were strong inhibitors, while Mn2+ ions and dithiothreitol were stimulators of the enzyme activity. The enzyme was specific for xylans, showing higher specific activity on birchwood xylan. The Michaelis–Menten constant (Km ) for birchwood xylan was estimated to be 2.3 mg ml−1 while maximal velocity (Vmax ) was 233.1 ␮mol mg−1 min−1 of protein. The hydrolysis of oat spelt xylan released only xylooligosaccharides. Published by Elsevier Ltd. Keywords: Aspergillus versicolor; Xylanase; Endoxylanase; Enzyme purification

1. Introduction The primary component of hemicellulose is xylan, a heteropolysaccharide formed by a main chain of linked ␤-1,4 xylose residues, and a variety of substituents, differing from plant to plant. It may constitute up to 30% cellular walls in hard wood types and annual plants. Therefore, considerable amounts of xylan are found in solid agricultural and agroindustrial residues, as well as in effluents released during wood processing, which, due to frequent inappropriate discard, cause great damage to the ecosystem [1,2]. Xylan biodegradation is performed by a xylanolytic complex, which is primarily produced by fungi and bacteria. Hydrolysis of the main chain is accomplished by the action of endo-1,4-␤-xylanases (1,4-␤-d-xylan xylanohydrolases, EC 3.2.1.8), which release xylooligosaccharides of different sizes [1,3,4]. Basic and applied research on microbial hemicellulases has not only produced significant scientific knowledge, but also revealed their enormous biotechnological potential. Xy-

lanases, associated or not with other enzymes, can be used in the food industry in order to enhance the digestibility of animal feeding, as well as in the textile industry, among other applications. Special attention has been given to their use in the pulp and paper industry for bleaching purposes, resulting in a decrease of chlorine utilization and consequently lowering environmental impact [4–6]. A strain of Aspergillus versicolor produces high levels of xylanolytic activity associated with low cellulolytic activity, an essential characteristic for certain industrial applications [7]. The xylanolytic complex of this fungus consists of two enzymes, the more abundant of which, xylanase I, has been purified and characterized [8]. The aim of this work was the establishment of optimal conditions for the production, purification and biochemical characterization of xylanase II, the minor component of A. versicolor xylanolytic complex.

2. Materials and methods 2.1. Organism and growth

∗

Corresponding author. Tel.: +55-19-3526-4175; fax: +55-19-3526-4176. E-mail address: [email protected] (E.C. Carmona). 0032-9592/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.procbio.2004.01.010

Conidia of A. versicolor were obtained from cultures in Vogel solid medium [9] containing 1.5% (w/v) sucrose.

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Liquid cultures were prepared in the same medium with 1% (w/v) wheat bran as carbon source and pH was adjusted to 6.5 or 5.8 as indicated in the text and legends. Erlenmeyer flasks (250 ml) containing 50 ml of medium were inoculated with 2 ml of the spore suspension (107 spores/ml) and incubated at 30 ◦ C for 5 or 9 days, or as indicated in the text and legends. The mycelium was removed by vacuum filtration and the filtrate was used as crude enzyme extract. 2.2. Enzymic assay Xylanase activity was assayed at 55 ◦ C using 2% (w/v) birchwood xylan or oat spelt xylan in 100 mM sodium phosphate buffer pH 6.0. The reducing sugars released were quantified by the dinitrosalicylic acid method [10], using xylose as standard. One enzymic activity unit was defined as the enzyme amount that releases 1 ␮mol of reducing sugar per minute. Specific activity was expressed as unit per milligram of protein. 2.3. Determination of neutral carbohydrates and protein Total carbohydrate content was determined by phenol sulphuric method [11] with glucose as standard. Protein concentration was determined by the method of Lowry et al. [12] using bovine serum albumin as standard. 2.4. Separation of the xylanolytic complex components The culture filtrate obtained after different cultivation conditions was dialyzed against 50 mM Tris–HCl buffer pH 7.0, and submitted to a batch assay with DEAE-Sephadex A-50 equilibrated in the same buffer. Xylanase I, which does not bind to the resin under these conditions, was removed by centrifugation at 6000 × g for 15 min at 4 ◦ C and the resin was exhaustively washed with the same buffer. The xylanase I-rich supernatants were pooled to estimate xylanolytic activity. The resin-bound components (including xylanase II) were eluted by three washes with 50 mM Tris–HCl buffer pH 7.0, containing 0.7 M NaCl, followed by centrifugation at the conditions described above. The pooled supernatants were also assayed for xylanolytic activity. Total xylanolytic activity corresponded to the sum of the activities of both supernatant pools. 2.5. Purification of xylanase II The culture filtrate (140 ml) was dialyzed against 50 mM Tris–HCl buffer pH 8.8 and chromatographed in a DEAE-Sephadex A-50 column (1.0 cm × 13.8 cm) pre-equilibrated with the buffer at a flow rate of 20 ml/h. The bound proteins were eluted with a NaCl gradient (0.0–0.6 M) in the same buffer. The fractions with higher activity were pooled and concentrated approximately 10 times through reverse osmosis using polyethylene glycol (MW 15,000–20,000). The concentrated sample was dialyzed

against 50 mM ammonium acetate buffer pH 6.8, and submitted to a gel filtration in HPLC Shimadzu CLASS-LC10, using a Shodex Asahipak GF-510 HQ column equilibrated in the same buffer. 2.6. Electrophoresis SDS-PAGE was carried out in an acrylamide gradient (5–20%) as described by Hames and Rickwood [13]. Molecular weight standards were: phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and ␣-lactalbumin. Proteins were stained with Coomassie brilliant blue R-250. 2.7. Optimal temperature and pH, and thermal stability Optimal assay temperature was determined by performing a standard activity assay in a temperature range from 25 to 70 ◦ C. In order to determine optimal pH, the enzymic assay was carried out at different pH values, at 55 ◦ C. McIlvaine buffer was used for pH 4.0–8.0, Tris–HCl buffer for pH 8.0–9.0 and glycine–NaOH buffer for pH 9.0 and 9.5. 2.8. Effect of potential inhibitors and activators on purified xylanase II activity The effect of various compounds on xylanase activity was determined by performing the enzyme assay in 50 mM sodium acetate buffer pH 5.5, with birchwood xylan as substrate at final concentrations of 2 and 10 mM. Residual activity was expressed as the percentage of the activity observed in the absence of any compound. 2.9. Kinetic parameters The effect of oat spelt xylan concentration, ranging from 2.0 to 40 mg ml−1 , on xylanase activity was evaluated under optimal assay conditions. The kinetic parameters (Michaelis–Menten constant, Km , and maximal reaction velocity, Vmax ) were estimated by linear regression from double-reciprocal plots according to Lineweaver and Burk [14]. The initial velocities (V0 ) were expressed in ␮mol ml−1 min−1 and the maximum velocity was converted into ␮mol mg−1 min−1 of protein. 2.10. Analysis of xylan hydrolysis products The products of oat spelt xylan hydrolysis were analyzed by thin-layer chromatography on silica gel G-60 with ethyl acetate/acetic acid/formic acid/distilled water (9:3:1:4, v/v/v/v) as the mobile phase. Xylose and xylobiose were used as standards, and thymol blue as a front indicator. Development was performed with 0.2% (w/v) orcinol in sulphuric acid/methanol (1:9, v/v) [15].

E.C. Carmona et al. / Process Biochemistry 40 (2005) 359–364 100

20

3 mA280

Total xylanase activity ( U ) .

25

361

15

50

2 4

1

10

5 0

5

0

10

20

30

40

50

Time (min)

0 5.8/5

6.5/5 5.8/9 pH/period of culture (days) xylanase I

6.5/9

Fig. 3. Gel filtration of the xylanase II from A. versicolor in HPLC. The Shodex Asahipak GF-510 HQ column was equilibrated and eluted with 50 mM ammonium acetate buffer, pH 6.8.

xylanase II

Fig. 1. Effect of pH and time of cultivation on the proportion of the components from A. versicolor xylanolytic system. The fungus was grown at pH 5.8 or 6.5 for 5 or 9 days, in liquid medium with 1% (w/v) wheat bran.

3. Results and discussion The effect of initial culture pH on the production of A. versicolor xylanolytic complex and on the proportion of its components, xylanase I and II, was studied in cultures grown in 1% wheat bran for 5 and 9 days (Fig. 1). At pH 5.8 the

levels of xylanase II were lower than at pH 6.5, independently of the culturing time. At pH 6.5 xylanase II levels represented about 27 and 24% of the total xylanase activity in the culture filtrates after 5 and 9 days of culturing, respectively. Based on these results, the fungus was cultivated for 5 days at pH 6.5 in order to maximize xylanase II production, and to standardize purification protocols. The elution profile of xylanase II activity in DEAESephadex A-50 at pH 8.8 is shown in Fig. 2. The fractions showing xylanase activity were pooled and submitted to gel filtration in HPLC (Fig. 3). Xylanase activity was detected

2

6

5

1

3

2

mM NaCl.

A280

4

xylanase activity (U/mL)

0.6

1

0

0 1

6

11

16

21 Fraction nº.

26

31

0.0

36

Fig. 2. DEAE-Sephadex A-50 chromatography of the xylanase II from A. versicolor. The column was equilibrated with 50 mM Tris–HCl buffer pH 8.8. The enzyme was eluted with a linear salt gradient in the same buffer. The flow rate and fraction size were 20 ml/h and 3.0 ml, respectively. (䉱) Xylanase activity, (䊏) A280 and (—) NaCl concentration. Table 1 Purification of xylanase II from A. versicolor Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg protein)

Recovery (%)

Purification (fold)

Crude extract Dialyzed extract DEAE-Sephadex A-50 pH 8.8 HPLC GF-510 HQ pH 6.8

79.9 48.7 0.3 0.09

863.4 947.8 68.6 27.1

10.8 19.5 210.6 302.1

100.0 109.8 7.9 3.1

1.0 1.8 19.5 28.0

Data are the mean of three different preparations.

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Relative xylanase activity (%)

100

80

60

40

20

0 3

4

5

Relative xylanase activity (%)

(a)

6

7

8

9

10

pH

100

80

60

40

20

at peak 3, corresponding to 81% of the total protein amount, with a retention time of 23 min. Ion exchange chromatography followed by gel filtration was an efficient strategy to purify the enzyme (28-fold purification), resulting in a xylanase II preparation with high specific activity (302 U/mg), although with a low yield (3.1%). The results of xylanase II purification are summarized in Table 1. SDS-PAGE of the purified enzyme revealed a single protein band, suggesting that xylanase II was formed by a single polypeptide chain with a molecular mass of about 32 kDa (Fig. 4). The carbohydrate content of the purified enzyme was estimated to be 14.1%, in contrast with xylanase I, that exhibited 71% of total carbohydrate [8]. Maximal activity of purified enzyme was achieved between pH 6.0–7.0, and the optimum of temperature was 55 ◦ C (Fig. 5). The pH profile was slightly different from that reported for xylanase I, since xylanase II maintained more than 50% of activity in the pH range of 7.5–9.0. [8]. The thermoinactivation profile for xylanase II from A. versicolor at 50 ◦ C was biphasic, with an initial fast phase followed by a second slower phase (Fig. 6). It has been suggested that a biphasic thermoinactivation curve for enzymes might be due to the existence of different isoenzymes which are denaturated at different rates [16–18], or to reflect the

20

30

(b)

40 50 60 Temperature (ºC)

70

80

Fig. 5. Influence of pH and temperature on the xylanase II activity from A. versicolor. (a) The activity was assayed at 50 ◦ C with birchwood xylan in McIlvaine buffer for pH 4.0–8.0, Tris–HCl buffer for pH 8.0–9.0 and glycine–NaOH buffer, pH 9.0 and 9.5. (b) The activity was assayed in 100 mM sodium phosphate buffer, pH 6.0, containing birchwood xylan as substrate.

100

Residual activity (%) .

Fig. 4. Gradient SDS-PAGE (5–20%) of purified xylanase II from A. versicolor. Lane 1, standard molecular weight: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and ␣-lactoalbumin (14.4 kDa); Lane 2, 50 ␮g of purified enzyme.

80

60

40

20

0 0

20

40

60

80

100

120

Time (min) Fig. 6. Thermal stability of xylanase II from A. versicolor. The enzyme was incubated at 50 ◦ C (䊏) and 60 ◦ C (䉱) in 100 mM sodium phosphate buffer, pH 6.0. Aliquots withdrawn at different time intervals were cooled before assaying for residual enzyme activity at 55 ◦ C.

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Table 2 Effect of potential inhibitors and activators on the xylanase II activity from A. versicolor

0.40 0.35 1/Vo (µmol/ml.min)-1

363

Substance

0.30

Relative xylanase activity (%) 2 mM

10 mM

87 ND 96 88 ND 89 100 95 56 22 62 53 122 92 125

79 ND 62 74 ND 51 98 59 27 14 10 10 225 65 185

0.25 0.20 0.15 0.10 0.05 0.00 - 0.5 -0.4 -0.3 -0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

1/[S] (mg/ml)-1

Fig. 7. Lineweaver–Burk plot of initial velocity data for A. versicolor xylanase II. The enzyme activity was measured in 100 mM sodium phosphate, pH 6.0 at 55 ◦ C. The date are the mean of three different experiments. Symbols: V0 in ␮mol ml−1 min−1 ; [S]: oat spelt xylan concentration in mg ml−1 .

ability of the enzyme to exist in more than a stable conformational state [18]. In addition, the purified xylanase II exhibited a half-life of 1.7 min at 60 ◦ C (Fig. 6), whereas the half-life for xylanase I from the same fungus, at 60 ◦ C, was around 15 min [8]. The lower thermal stability exhibited by xylanase II could be attributed to its lower sugar content. The rate dependence of the enzymic reaction on oat spelt xylan concentration at pH 6.0 and 55 ◦ C followed Michaelis–Menten kinetics. Reciprocal plots (Fig. 7) showed apparent Km and Vmax values respectively of 2.3 mg ml−1 and 5.6 ␮mol ml−1 min−1 . The last value corresponded to 233 ␮mol mg−1 min−1 of protein. Xylanase I from A. versicolor exhibited a Km of 6.5 mg ml−1 with a Vmax of 1.440 ␮mol mg−1 min−1 protein−1 for oat spelt xylan [8]. Thus, in spite of the higher specific activity of xylanase I, xylanase II had a higher affinity for oat spelt xylan. The effect of potential inhibitors or activators on purified xylanase II is shown in Table 2. While Hg2+ and Cu2+ ions, as well as SDS were strong inhibitors of the enzyme activity, either at 2 and 10 mM, EDTA and the ions Zn2+ and Pb2+ slightly inhibited the activity at 2 mM, but this effect was remarkable at 10 mM. Otherwise, PMSF, Ca2+ , Co2+ and Fe2+ ions showed inhibitory effect only at 10 mM. PMSF reversibly inhibited xylanase II from A. versicolor, indicating the presence of serine and/or cysteine residues important for the catalytic activity. In addition, NH4 + and Ba2+ ions slightly inhibited the enzymic activity at both concentrations, but Mg2+ ions had no effect, and Mn2+ ions and DTT significantly stimulated xylan hydrolysis. Activation by a thiol group-containing reagent, such as DTT confirmed the presence of a reduced thiol group of cysteine in the enzyme [19]. Xylanase II was highly specific for xylans, showing a two-fold higher specific activity on birchwood xylan, com-

NH4 Cl HgCl2 CaCl2 BaCl2 CuCl2 CoCl2 MgSO4 FeSO4 ZnSO4 SDS EDTA Pb(H3 C2 O2 )2 MnSO4 PMSF DTT

ND: not detectable by the method in the assayed conditions.

pared to that on oat spelt xylan. Xylanase I from A. versicolor also presented high specificity for xylans, but with higher specific activity on oat spelts xylan than on birchwood xylan [8]. The proportions of xylose, glucose, galactose and arabinose in the molecules of these water-soluble xylans are: 52.5, 15.7, 9.5 and 22.3% on that of oat spelt and 94.1, 1.4, 4.5 and 0.0% on that of birchwood, respectively [20]. Thus, xylanase II, in contrast to xylanase I, exhibits higher affinity for more homogeneous substrates, with lower branching degree. Thin-layer chromatography analysis of hydrolysis products from oat spelt xylan showed that xylanase II did not release xylose or xylobiose, but only xylooligosaccharides larger than xylobiose, such as xylotriose and xylotetrose. Also, sugars usually present in the branching of xylan main chain were not released. Therefore, xylanase II can be classified as an endoxylanase, releasing the same products as does xylanase I from A. versicolor [8].

Acknowledgements This work was supported by grants from FAPESP and FUNDUNESP. We thank CNPq, FAPESP and CAPES for the scholarships awarded to the second, third and fourth authors, respectively.

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