Xylanase SMXL1 from Stenocarpella maydis: Purification and biochemical characterization

Abstract

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

Full Article

Xylanase SMXL1 from Stenocarpella maydis: Purification and Biochemical Characterization

Edna M. Hernández-Domínguez,^a Jorge Álvarez-Cervantes,^a Pedro Gersain Lucio-Ávila,^a Gerardo Díaz-Godínez,^b and Yuridia Mercado-Flores ^a,*

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5 0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (K_m) value was 2.61 mg/mL and the V_max was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn²⁺ and by Fe³⁺ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

Keywords: Xylanolytic activity; Phytopathogenic fungus; Enzyme; Ascomycete; Xylan substrate; Xylan degradation

Contact information: a: Universidad Politécnica de Pachuca, Zempoala Hidalgo, Carretera Pachuca – Cd. Sahagún Km 20 Ex-Hacienda de Santa Bárbara, C.P. 43830, Zempoala Hidalgo, México; b: Laboratory of Biotechnology, Research Center for Biological Sciences, Universidad Autónoma de Tlaxcala, Carretera San Martin Texmelucan S/N Km 10.5, C.P. 90120, Ixtacuixtla de Mariano Matamoros, Tlaxcala, México; * Corresponding author: yuridiamercado@upp.edu.mx

INTRODUCTION

Xylan, after cellulose, is an abundant polysaccharide in nature. It is found in hemicellulose and constituted by a chain of β-D-xylose molecules linked by bonds β,1-4; it is a polymer present in the amorphous matrix in the vegetal cell wall (Kulkarni et al. 1999; Collins et al. 2005; Sharma and Kumar 2013). Xylan can be degraded by microorganisms, such as bacteria and fungi, which have enzymatic systems to complete the hydrolysis of xylan. Due to the heterogeneity of xylan as a substrate, different enzymes are required, each with a defined and specific role for the complete degradation of this polymer (Biely 1985; Coughlan and Hazlewood 1993).

Degrading enzymes of xylan are classified into two groups, the endo-xylanases and exo-xylanases; these groups act inside and at the end of the xylan molecule, respectively (Coughlan and Hazlewood 1993). The β-1,4-xylanases (EC 3.2.1.8) are known as endo-xylanases, which hydrolyze the β (1-4) bonds, producing xylooligosaccharides of different sizes (Biely 1985).

There is increasing interest in the study of xylanases due to their applications in different industries, such as pulp and paper, textile, food, and pharmaceutical products, as well as fuel manufacturing, besides in lignocellulosic residues degradation (Subramaniyan and Prema 2002; Techapun et al. 2003; Dhiman et al. 2008; Deutschmann and Dekker 2012; dos Santos et al. 2018; Souza et al, 2018; Marques et al. 2018, 2019; de Almeida et al. 2020). Although xylanase production of microbial origin is well documented, their role in phytopathogenic fungi has received little attention even though it is known that xylanase has potential to degrade polymers in the plant cell wall. For example, the xylanase S2 of Sclerotinia sclerotiorum has been partially purified and characterized (Ellouze et al. 2007). Fusarium oxysporum f. sp. melonis produces an extracellular endo-1,4-beta-xylanase in medium with oat spelt xylan and glucose, in which these components are the carbon source (Alconada and Martinez 1994). In F. oxysporum f. sp. licopersici the gene xyl5 encodes for a xylanase of the family 11. Its transcript is present in tomato roots at the beginning of the infection process, but it has no impact on virulence (Gómez-Gómez et al. 2002). F. oxysporum f. sp. cucerus presented xylanolytic activity using the cell walls of chickpea and xylan as the carbon source (Inmaculada et al. 2005). Myrothecium verrucaria produced xylanase in submerged fermentation with glucose and xylan (Moreira et al. 2005). Álvarez-Cervantes et al. (2013) purified and characterized one xylanase (SRXL1) produced by Sporisorium reilianum grown in both liquid and solid-state fermentations using xylan as a carbon source. In addition, it is known that the number of genes coding for xylanases in industrially important microorganisms, such as Trichoderma reesei, is less than in the phytopathogenic fungi. With this antecedent, the use of the microorganisms able to infect plants to search for new enzymes is very important to develop effective processes used in the enzymatic hydrolysis of polysaccharides present in lignocellulosic materials. This knowledge can then be used to obtain biotechnological products and biofuels (Martinez et al. 2008).

Different studies have demonstrated that phytopathogenic fungi produces high xylanolytic activity against xylan and untreated biomass. The substrate preference of these enzymes depends on whether the host plant is monocotyledonous or dicotyledonous, so that these enzymatic characteristics could be used to design a specific process to degrade the lignocellulosic materials (Martinez et al. 2008). Phytopathogenic fungi that cause rot of plant tissues have a high potential for the production of extracellular hydrolytic enzymes, such is the case of Stenocarpella maydis, the causative agent of white rot of stalk and cob in maize (Bensch and Van Staden 1992; Álvarez-Cervantes et al. 2016b). In this ascomycete, an extracellular aspartyl protease was purified and biochemically characterized. Moreover, at least two xylanase isoforms were produced in liquid and solid-state fermentations with different carbon sources, and three xylanase isoforms in solid-state fermentation using natural supports from maize were reported (Hernández-Domínguez et al. 2014; Mandujano-González et al. 2016).

In this work, the purification method of a xylanase of S. maydis that has been called SMXL1 and its biochemical characterization were performed; these results could inform about potential biotechnological applications and its possible association with the life cycle of the fungus.

EXPERIMENTAL

Microorganism and Culture Media

The S. maydis strain was donated by Dr. Dan Jeffers (International Maize and Wheat Improvement Center, CIMMYT, Texcoco City, Mexico). The fungus was conserved at 4 °C in a tube with potato dextrose agar (BD Bixon^TM, Mexico City, Mexico).

The culture media for enzyme production was prepared with the following components (g/L): K₂HPO₄, 0.4; ZnSO₄ 7H₂O, 0.001; KH₂PO₄, 0.6; FeSO₄ 7H₂O, 0.05; MnSO₄ H₂O, 0.05; MgSO₄ 7H₂O, 0.5; Birch xylan, 5.0, and yeast extract, 9.0 (Hernández-Domínguez et al. 2014).

Culture Conditions for Xylanase Production

The production of the xylanase SMXL1 was carried out by culture of S. maydis in 1 L Erlenmeyer flasks with 250 mL of production medium. Each flask was inoculated with five mycelium plugs (5 mm diameter), they were incubated at 28 ° C for 7 days in orbital agitation at 160 rpm. The biomass was separated through filtration and the broth obtained with xylanase activity was considered the crude enzyme extract (ECE). The cultivation was done in triplicate. (Hernández-Domínguez et al. 2014).

Xylanase Assay and Protein Determination

The xylanase activity was determined by the release of reducing sugars using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). A mixture of 475 L of birch xylan (0.5%) was prepared in 0.1 M acetate buffer at pH 5.3 and 25 L of ECE, which was incubated at 50 °C. The amount of enzyme that liberated 1 mol of xylose per minute was one unit of xylanase. The Bradford method (Bradford 1976) was used to determine the protein content, using bovine serum albumin as a standard.

Purification and Biochemical Characterization

Isoelectric focusing separation was completed with 58 mL of ECE using the Rotofor preparative chamber with 2 mL of ampholytes (pH 3 to 10). The enzymatic activity and protein concentration were determined in all fractions obtained. Those with the higher xylanase activity values were mixed and desalted through a Bio-ScaleTM Mini Bio-Gel® P-6 desalting cartridge equilibrated with 20 mM of phosphate buffer at pH 8.2. This sample was eluted at 2 mL/min and the fractions collected with protein were pooled and introduced in an ion exchange column Bio-Scale Mini UNOsphere Q Cartridge, coupled to an FPLC (Fast Protein Liquid Chromatography, model 2110; BioLogic System, Hercules, CAUSE). The protein separation was done using a linear gradient 0 to 1 M NaCl in phosphate buffer with pH 8.2 at 0.5mL / min. The purification procedure was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10% (Laemmli 1970). Gels were stained with the silver technique (Silver Stain kit). To estimate the molecular mass, protein markers ranging from 10 to 250 kDa was used (Precision Plus Protein TM Standards, Hercules, CA, USA) was used.

The activity of the purified xylanase was also detected in situ through zymogram (Raghukumar et al. 2004), using a native PAGE, with birchwood xylan at 0.5% as substrate in the gel. Once the electrophoretic run was finished, the gel was incubated in agitation at 40 rpm at 50 ºC for 40 min in acetates buffer 0.1 mM at pH 5.3. The enzymatic activity was visualized placing the gel in a Congo red solution at 2% and incubating at room temperature for 30 min. Posteriorly, washes were made with 1 M NaCl until the white bands were observed.

To determine the optimal pH of the purified enzyme, 100 mM buffers of McIlvaine, Tris-HCl and glycine-NaOH were used for pH 2.0 to 8.0, 8.0 to 9.0, and 9.0 to 11, respectively. Xylan was used as a substrate and the reaction mixtures were incubated at 50 °C. The stability of the purified xylanase to the pH was made by pre-incubating the enzyme for 12 h at 4 °C in each buffer mentioned above, after which the enzyme activity was determined.

The optimum temperature of the enzyme was determined performing the assays at 5, 25, 30, 35, 40, 45, 50, 55, 60, 70, and 80 °C, at its optimal pH. To determine the thermal stability, the purified xylanase was incubated for 35 min at each of the temperatures mentioned above and subsequently its activity was quantified.

The effect of different metal ions at 2 and 10 mM (Zn²⁺, Mg²⁺, Fe³⁺, Mn²⁺, Cu²⁺, Ca²⁺, and K⁺) was evaluated on the purified xylanase and reported as a percentage of activity, considering 100% at the activity value obtained in the absence of any ion or chemical compound. The salts used were ZnCl₂, MgCl₂, FeCl₃, MnCl₂, CuCl₂, CaCl₂ and KCl. The data obtained were compared by means of a two-way ANOVA test. The level of significance was established as P < 0.05.

The Michaelis-Menten constant (K_m) and V_max were estimated using birch xylan as substrate at 1 to 10 mg/mL, using the Michaelis-Menten equation. The data were analyzed with the non-linear least square-fitting program “Solver” (Excel Microsoft 2016, Redmond, WA, USA). The experiments were conducted in triplicate.

Birch xylan hydrolysis by the purified xylanase

For the enzymatic hydrolysis of xylan, the reaction mixture consisted of 0.5% birch xylan (5 mg/mL in 0.1 M acetate buffer at pH 4.0) with different units of the purified xylanase (0.1- 0.1 U/mL) in 1 mL, and was incubated at 55 °C for 12 h. The tubes were placed in boiling water to stop the reaction. The high-performance liquid chromatography (HPLC; Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the products of hydrolyses, with a 300 × 7.7 mm 8μ HyperREZ XP Carbohydrate H column. Water HPLC grade was applied as eluent at 6 mL/min. The standards xylooligosaccharides of 1, 2, 3, 4, and 5 monomers were used (Pérez-Rodríguez et al. 2019).

RESULTS AND DISCUSSION

The enzyme purified to homogeneity in two steps was called SMXL1. The fractions with activity obtained in the isoelectric focusing showed an isoelectric point (pI) around 2.5 (Fig. 1). In the second step of purification (ion exchange chromatography), fractions 17 and 18 had activity and were eluted at around 0.21 and 0.23 mol/L of NaCl (Fig. 2).

The enzyme was purified 4.2-fold with a yield of 6.5% (Table 1). Figure 3 shows the purified protein (SDS-PAGE) and its xylanase activity (zymogram). The molecular weight of the enzyme estimated by SDS-PAGE (denaturing conditions) was 55 kDa.

Fig. 1. Isoelectric focusing profile of ECE with xylanase activity of S. maydis grow in from liquid culture: pH (solid line) and xylanase activity (dashed line)

Fig. 2. Ion exchange chromatography of xylanase SMXL1 using an FPLC equipment; Proteins at 280 nm (dashed line), xylanase activity (solid line), and NaCl gradient (dotted line)

Table 1. Purification Steps of Xylanase SMXL1 from S. maydis

The enzyme was stable for 12 h at 4 °C across a wide range of pH values. At pH 3 there was 40% residual activity, while at pH values of 4 to 6 and 7 to 10, the activity values were 70 and 80 to 100%, respectively. The optimum pH for activity of SMXL1 was 4.0 (Fig. 4A), and was stable at temperature values between 45 and 60 °C for 35 min, where it maintained 70 to 100% activity. The optimum temperature for this xylanase was 55 °C (Fig. 4B).

Fig. 3. Electrophoretic pattern (SDS-PAGE) of xylanase SMXL1: Lane 1- molecular weight markers (kDa); lane 2- ECE; lane 3- fraction of Isoelectric focusing; lane 4- fraction obtained of Ion exchange chromatography; and lane 5- zymogram

Fig. 4. The effect of pH and temperature on the activity of the xylanase SMXL1: (a) Optimal pH (), pH stability (); (b) Optimal temperature (), temperature stability ()

Such as is observed in the Fig. 5, the activity was inhibited predominantly by Mn²⁺ at 2 and 10 mM. Inhibition was likewise observed for Cu²⁺ but only at 10 mM; at 2 mM this cation increased the enzymatic activity (P < 0.001). The Fe³⁺ showed a slight inhibition effect at the two concentrations assayed, while for the K⁺ and the Zn²⁺ only at 10 mM this same effect was observed (P < 0.05). The Mg²⁺ and the Ca²⁺ did not have effect.

The purified enzyme degraded the birch xylan, in which the principal product was xylobiose; following this was xylose and after xylotriose when 1 to 7 U/mL were used and from 8 to 10 U/mL the amount of xylose was smaller than that of xylotriose (Fig. 6). Xylopentose and xylotetrose were not found in the hydrolysis products.

The K_m value was 2.61 mg/mL and the V_max was 3.02 µmol/mL/min (Fig. 7).

Fig. 5. Metal ions effect on the activity of xylanase SMXL1 of S. maydis. * P < 0.05, ** P < 0.001

Fig. 6. Hydrolysis products of birch xylan by the xylanase SMXL1 xylanase from S. maydis

Fig. 7. Effect of xylan concentration on the initial velocity of the xylanase SMXL1 from S. maydis

DISCUSSION

In this study, a xylanase called SMXL1 was produced by S. maydis grown in liquid culture using a medium previously reported (Hernández-Domínguez et al. 2014). The purification process described was simple and involved only two steps. However, yields were low. This can be attributed to the presence of more isoforms in the ECE, because in recent work, using the same culture conditions and the same fungus, two xylanase isoforms by zymograms were observed (Hernández-Domínguez et al. 2014). The purified xylanase showed a pI of 2.5. However, other fractions of isoelectric focusing had a reported activity at pH of 2.1, which is probably due to the presence of another isoform.

It is reported that F. oxysporum f. sp. ciceris produced extracellular xylanases that were classified into basic, neutral, and acidic depending on their pI values (10, 8, and 4, respectively). Production of the first two is favored by the cell walls of chickpea, while acidic occurred in the presence of xylan as a carbon source (Inmaculada et al. 2005). The presence of xylanase isoforms could favor the infection of the plants by this phytopathogenic fungus, although the environmental conditions change (Álvarez-Cervantes et al. 2016a). The xylanase SMXL1 showed a pI value lower than those reported for fungal xylanases, which are in the range 3.4 to 10.2 (Álvarez-Cervantes et al. 2016a).

In general, xylanases show molecular weights ranging from 15 to 145 kDa; basic xylanases present a maximum weight of 30 kDa; and acidic xylanases show higher weight than 30 kDa (Álvarez-Cervantes et al. 2016a). The xylanase SMXL1 is acidic with a molecular weight of 55 kDa. It has been suggested that the ability to produce different xylanases is conditioned by both environmental factors and producer microorganisms (Inmaculada et al. 2005; Hernández-Domínguez et al. 2014; Álvarez-Cervantes et al. 2016a).

Xylanases show activity at pH of 4 to 6.5, temperatures from 40 to 80 °C, and K_m values between 1.3 and 348 mg/mL (Polizeli et al. 2005; Belien et al. 2006; Pollet et al. 2009; Álvarez-Cervantes et al. 2016a), and the xylanase SMXL1 had these characteristics. The xylanase SMXL1 was not stable at low temperatures, however, the xylanases of Aspergillus niger and Rhizopus sp. showed stability at those same conditions (dos Santos et al. 2018).

It is considered that xylanases have an important role in the pathogenic process, allowing them to attack the plant through the rupture of the cell wall. An example of this has been previously reported by Diebold et al. (2004) and Kubicek et al. (2014), in which the endo-xylanases have the function of promoting the virulence of phytopathogenic microorganisms, thereby inducing a defense response of plants. This could be a function of xylanase SMXL1 in S. maydis.

Microbial enzyme systems currently have a major biotechnological potential applicable in numerous industries in the field of so-called clean technologies or sustainable technologies (Collins et al. 2005; Belien et al. 2006; St. John et al. 2006; Kubicek et al. 2014; Álvarez-Cervantes et al. 2016a; Kaushik and Malik 2016; Kumar et al. 2016). In this sense, the xylanase SMXL1 presented biochemical characteristics for possible biotechnological use. It showed activity and stability in acidic pH ranges and temperatures between 45 and 60 °C. The xylanolytic activity is affected by the presence of metal ions. Specifically, Ca²⁺ and Mg²⁺ are activators, while Co⁺², Hg⁺², Fe⁺², Mn⁺², and Cu⁺² are inhibitors of these enzymes (Álvarez-Cervantes et al. 2016a). Regarding the xylanase SMXL1, the Cu²⁺ at 2 mM was an activator, and at 10 mM a strong inhibitory effect was observed, while to the xylanase produced by Penicillium roqueforti ATCC 10110 strain in where this cation had an activator effect on the enzyme at 1M (Marques et al. 2018). In other hand, the Mn²⁺ was an inhibitor of the enzyme purified, and the Ca²⁺ and Mg²⁺ did not show an activator effect.

The endo-xylanases catalyze the hydrolysis of xylan or xylo-oligomers with different degrees of polymerization to xylooligosaccharides but they cannot hydrolyze the xylobiose (Wong et al. 1988; Álvarez-Cervantes et al. 2016a). The results showed that the xylanase SMXL1 was an enzyme that degraded the birch xylan producing xylotriose and xylobiose. The presence of xylose could have been due to the xylostriose hydrolysis of the enzyme.

This work is the first report where the purification and biochemical characterization was made of a xylanase of S. maydis called SMXL1.

CONCLUSIONS

1. The xylanase SMXL1 from S. maydis was purified using the Rotofor preparative chamber and through ion exchange chromatography.
2. The purified enzyme had a molecular weight of 55 kDa and a Km and V_max of 2.61 mg/mL and 3.02 µmol/mL/min, respectively.
3. The optimal values of pH and temperature of SMXL1 were 4.0 and 55 °C, respectively, and was stable at pH of 4 to 10 and temperatures of 45 to 60 °C.
4. The xylanase activity was inhibited by Mn²⁺ and by Fe³⁺ at 2 and 10 mM, while that for the cations K⁺ and Zn²⁺ only a slightly effect was observed at 10 mM.
5. The Mg²⁺ and the Ca²⁺, do not have effect on the purified enzyme.
6. This enzyme hydrolyzed birch xylan at 55 °C and a pH value of 4, where the principal product was the xylobiose.

ACKNOWLEDGMENTS

Edna Ma. Hernández-Domínguez was a fellow of CONACyT. This work was supported by the Fund Basic Science SEP-CONACyT (Project No. 167459).

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Article submitted: August 15, 2019; Peer review completed: December 14, 2019; Revisions accepted: February 12, 2020; Published: March 13, 2020.

DOI: 10.15376/biores.15.2.2947-2960