Partial Purification and Characterization of Endoxylanase from a fungus, Leohumicola incrustata

Xylanases are glycoside hydrolases (GH) that degrade β-1,4-xylan, a linear polysaccharide found as hemicellulose in cell wall of plants. Endoxylanase (Endo-1,4-β-xylanase, EC 3.2.1.8) randomly catalyses xylan to produce varying short xylooligosaccharides (XOS). This study aimed to determine the characteristics of a partially purified endoxylanase from Leohumicola incrustata. Enzyme production was carried out using beechwood (BW) xylan, after which the cell-free crude filtrate was concentrated using the ammonium sulphate precipitation method. The hydrolysed products were analysed by thin-layer chromatography (TLC) and zymography. The result showed that the enzyme produced varying smallersized linear xylooligosaccharides with Rf values corresponding to those of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers. The endoxylanase had a molecular mass of 72 kDa. The enzyme is stable in the presence of K+, Na+, Ca2+, Fe2+, Mg2+, Zn2+, Co2+, pH of 5.0 and temperature of 37oC. However, the activity gradually decreased after 60 min at 50oC and retained over 69% activity after 120 min, while at 60 and 70oC, the enzyme activity sharply decreased (pre-incubation periods). Endoxylanase from L. incrustata is comparable to those of other microorganisms and should be considered an attractive candidate for future industrial applications.


INTRODUCTION
Xylan is an essential component of hemicellulosic polysaccharide in cell walls of most plants, making up to 7-12% and 15-30% of the total dry weight in softwood from gymnosperms and hardwood from angiosperms, respectively (Saha, 2003). Hardwood hemicellulose mainly consists of O-acetyl-L-4-O-methyl-glucuronic acid xylan, e.g., the content of hemicellulose in birchwood is approximately 35% (Chen, 2014;Sakthiselvan et al., 2014). Xylan hemicellulose in softwoods is 4-O-methyl-glucuronic acid arabinose-xylan with almost no acetyl (Chen, 2014). Beechwood (BW) xylan from agricultural residues is an inexpensive and abundant raw material that could be used for oligosaccharide production. BW xylan consists of a backbone of p-1,4-linked D-xylopyranose residues, with side chains of 4-O-methylglucuronic acid attached to the C-2 position of xylose and O-acetyl groups at C-2 or C-3 positions (Freixo and De Pinho, 2002).  Corral and Villaseñor-Ortega, 2006).
Their catalytic ability is lower than those of enzymes in the GH10 class. The action of the GH11 family enzymes is solely on D-xylose containing substrates, and they cannot cleave cellulose or aryl β-D-cellobiosides (Chakdar et al., 2016). The GH11 family cleaves unsubstituted regions of the backbone because they cannot attack the xylosidic linkage towards the nonreducing end (next to a branched xylose). Most bacterial xylanases belong to the GH10 family while fungal xylanases majorly belong to the GH11 family (Liu et al., 2012). Interest in carbohydrate-active enzymes has increased over the years because of their potential application. Xylanases are used in food, feed, bleaching (pulp and paper) industries, and could also be utilised in improving the effectiveness of detergent in cleaning, biochemical and biofuel production (Juturu and Wu, 2011;Yun et al., 2015). This study aimed to determine the characteristics of a partially purified endoxylanase from L. incrustata.

Culture
Leohumicola incrustata (Isolate code ChemRU330 / Genbank Accession Number MF374380 / The South African National Collection of Fungi Accession Number PPRI 17268) was obtained from Mycorrhizal Research Laboratory, Rhodes University, Grahamstown. The isolate was preserved on PDA at a temperature of 4 o C throughout the study period.

Enzyme production
Beechwood (BW) xylan (1%, w/v, Lot # 141202, Megazyme, Bray, Ireland) was weighed and added to a salt solution composed of: (g L -1 ): malt extract 3.0; (NH4)2HPO4 0.25; MgSO4.7H2O 0.15; CaCl2 0.05; NaCl 0.025; ZnSO4.7H2O 0.003; thiamine-HCl 100 μg L -1 and 1.2 mL of FeCl3 (1%, w/v). Production medium of 100 mL was distributed into each of 150 mL Erlenmeyer flasks; the contents were thoroughly mixed and sterilized at 121 o C for 15 min. The sterilized medium was inoculated with two discs of 5 mm mycelial plugs of the fungus. A non-inoculated medium was used as a control. Growth was allowed to proceed at 28 o C in the dark for three weeks in a rotary incubator shaker at 150 rpm. After incubation, cultures were homogenized using IKA's ULTRA-TURRAX homogenizer (20,000 rpm), and centrifugation was performed at 10,000 x g for 15 min to get crude enzyme filtrates (supernatant) using Beckman Coulter Avanti-J high-speed centrifuge. The pellet containing the mycelia was re-suspended in 10 mL water and filtered using vacuum suction filtering system to recoup the mycelia on a filter paper (Adeoyo et al., 2018).

Ammonium sulphate precipitation and dialysis of crude enzyme extract
Cell-free crude filtrate was concentrated and optimised using the ammonium sulphate precipitation method (Kamble and Jadhav, 2012). The precipitation was carried out by diluting 120 mL crude enzyme extract into (NH4)2SO4 with a concentration of 80% (w/v). The pellet and filtrate were separated by centrifugation at 6000 x g for 15 min at 4 o C. The precipitated enzyme in the pellet was diluted with 10 mL acetate buffer (pH 5.0). Dialysis of the partially purified enzyme was performed using a pre-treated dialysis bag (10 kDa cut-off). The partially purified enzyme (10 mL) was dialysed against 0.1 M acetate buffer (pH 5.0) at 4ºC with three changes of buffer according to the method described by Kusuda et al. (2004).

Zymography
Zymogram analysis was performed using a modified zymographic method (Ratanakhanokchai et al., 1999). The culture supernatant in the sample application buffer was boiled for 2 min at 95 o C and was followed by electrophoresis on a 10% sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) gel containing 1% BW xylan. After electrophoresis, the gel was soaked in 2.5% (v/v) Triton X-100 with gentle shaking which removed the SDS and renatured the proteins in the gel for 45 min at 4 o C. The gel was then washed with 0.01 M acetate buffer (pH 5.0) and incubated for 1 h at 37°C. The gel was soaked in 0.1% Congo red solution for 30 min at room temperature and washed with 1 M NaCl until the excess dye was removed from the active band. A Bio-Rad ChemiDoc X-Ray Spectrometer (XRS) system was used to capture photographic images.

Enzyme assay
A dinitrosalicylic acid (DNS) assay (Miller, 1959) was conducted by adding 1% (w/v) BW xylan to a volume of 10 mL sodium acetate buffer (pH 5.0) in a Schott bottle and boiled for 30 s. A volume of 100 μL of crude enzyme and uninoculated control was added to 300 μL of BW xylan-containing medium in triplicate, while the blank contained 400 μL buffer. All samples were incubated at 37°C for 1 h, followed by centrifugation at 6000×g for 2 min. A 300 μL aliquot of DNS was added to 150 μL of each supernatant sample. This was followed by boiling on a heating block at 100°C for 5 min after which it was cooled on ice for 5 min. A volume of 250 μL of each sample was placed into each well of a 96-well plate and read with the aid of a spectrophotometer (BioTek's Synergy Mx) at a wavelength of 540 nm. The supernatant was taken to determine the reducing sugar using DNS assay with xylose as a standard. Enzyme activity was measured using 1% BW xylan in acetate buffer (pH 5.0) for 1 h at 37 o C. The reducing sugars released were assayed using the DNS method of Miller (1959).

Protein estimation
Protein content was estimated according to the method described by Bradford (1976) using bovine serum albumin (BSA, Lot # A9647, Sigma-Aldrich) as a standard.

Effect pH of endoxylanase activity and stability
The optimum pH was obtained by assaying the partially purified enzyme in buffer at different pH (1.0-9.0) prepared in 0.1 M buffer having pH values of 1.0, 2.0 (hydrochloric acid-potassium chloride); 3.0, 4.0, and 5.0 (citrate-phosphate); 6.0, 7.0 (phosphate); 8.0 and 9.0 (Tris-HCl). For enzyme stability, the enzyme was preincubated in acetate buffer (pH 5.0) at 37 o C. The optimum pH was determined by assaying enzyme activity at 37 o C for 1 h using the DNS method (Miller, 1959).

Effect of temperature on endoxylanase activity and stability
The optimum temperature was obtained by incubating the enzyme under different temperatures (4, 20, 30, 40, 50, 60, 70, and 80 o C, while stability was tested by pre-incubating the enzyme at 37, 50, 60, and 70 o C. The enzyme activity was determined every 6 h for 30 h.

Substrate specificity
A 10 mg/ml each of either BW xylan, CMC, starch, glycogen, microcrystalline cellulose (Avicel) or chitin was used to determine the substrate specificity of the xylanase. Each substrate was incubated with the xylanase extract at 50 o C for 1 h (pH 5.0). Activity was determined as previously described.

Statistical analysis
All experiments were conducted in triplicate and analysed using one-way ANOVA. Error bars were represented as the standard errors of the means (±SEM). Table 2 shows partial purification table, where the protein was precipitated with 80% ammonium sulphate, dialysed against acetate buffer and concentrated using an Amicon ultrafiltration unit. The enzyme was partially purified to 49.6 fold with a specific activity of 1.57 U/mg protein and a recovery yield of 77%. Figure 2 shows the SDS-PAGE and zymogram of the crude extract from Leohumicola incrustata. The partially purified sample gave a single band of yellow against the red colour of the Congo red used for gel staining. The molecular weight of the endoxylanase was estimated by plotting a relative migration distance (Rf) graph based on the electrophoretic mobilities of endoxylanase and of the reference standard (Rf) on SDS-PAGE with the corresponding zymographic position. The partially purified endoxylanase was observed to have a molecular weight of 72 kDa.

Thin layer chromatographic analysis of the xylanase
Beechwood xylan (1%) was incubated with the enzyme for 24 h to assure maximum hydrolysis. The mixtures were analysed by thin layer chromatography (TLC), and the hydrolytic products were compared to those of the standards. The result showed that the tested enzyme liberated varying smaller-sized linear xylooligosaccharides with Rf values corresponding to those of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers (Figure 3).

The effect of pH on endo-1,4-β-xylanase activity and stability
The pH of any medium plays a crucial role in influencing enzyme production and activity. Figure 4 shows that pH 5.0 had the highest activity of 1.11 U/mg protein. Low enzyme activity was found at a pH of 2.0 and 8.0 while the activity at the pH of 1.0 was significantly low. The optimum pH for the endoxylanase activity from L. incrustata was 5.0, and the activity gradually decreased below this pH. Also, it was observed that the enzyme activity was stable at pH of 5.0 for 6 h ( Figure 5), after which the activity started to decrease gradually in a similar faction to the activity at the other pH. After 24 h of preincubation, the enzyme retained over 70% of its activity at a pH of 5.0 at 37 o C. Figure 6 shows that the optimal temperature was obtained between 60 o C (1.69 U/mg protein). The xylanase was stable at 37 o C. However, the activity of endoxylanase gradually decreased after 1 h at 50 o C and retained over 69% activity after 120 min, while at 60 and 70 o C, the enzyme activity sharply decreased (pre-incubation periods) (Figure 7).

Effect of metal ions and chemicals on the activity of the endo-1,4-β-xylanase
Metal ions such as Cu 2+ , Al 3+ , Hg 2+ and Cd 2+ inhibited enzyme activity significantly at a concentration of either 1, 5 or 10 mM, while Cobalt had an effect only at a concentration of 10 mM ( Figure 8). Also, for most chemicals used in this experiment (Figure 9), only SDS showed an inhibitory effect on endoglucanase activity at 1, 5 and 10 mM concentrations.

Substrate specificity
The substrate (1%) of either BW xylan, CMC, starch, glycogen, Avicel or chitin was used to determine specificity. Figure 10 shows that endo-1,4-β-xylanase was specific for two substrates -BW xylan and CMC with values of 1.61 and 0.42 U/mg protein, respectively.

DISCUSSION
The study revealed a single protein band with a molecular weight (MW) of 72 kDa (Figure 2). This corroborates a report on an endo-1,4-β-xylanase from some fungi that displayed a MW ranging from 8.5 to 85 kDa (Polizeli et al., 2005). The capacity of endo-1,4-β-xylanase to degrade the substrates (BW xylan) was demonstrated by analysing the hydrolysates through a TLC method. The qualitative identification showed different XOS (xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers as products of hydrolysis) while no XOS was produced where the enzyme was not included. The hydrolytic profile observed in the L. incrustata was similar to that of Aspergillus niger (Takahashi et al., 2013) and Penicillium oxalicum (Liao et al., 2015).
Xylanases obtained from fungal sources are known to be active and stable in the acidic range of pH (Burke and Cairney, 1997a). The pH study indicated that pH 5.0 was the optimum while optimum stability was observed at a pH between 5.0 and 7.0. Also at this pH, the enzyme retained 70% of its activity after incubation for 24 h at 37 o C. This is in contrast to a report that an endo-1,4-β-xylanase from the ericoid mycorrhizal fungus (Hymenoscyphus ericae) had an optimum pH of 4.5 and was stable between pH 3.5-4.0 (Burke and Cairney, 1997a). According to Burke and Cairney (1997a), one of the environmental factors regulating enzyme activity at the mycorrhizal-host root interface is pH, which tends to reduce enzyme activity and growth when outside the optimum or permissible range. Also, endoxylanase of L. incrustata had optimal activity at a temperature of 60 o C (pH 5.0), was more stable at 50 o C, retaining about 69% activity after 2 h. These results confirmed that the endoxylanase from L. incrustata compared well with those of commonly used Trichoderma (Abbas et al., 2012) and Aspergillus species (Subramaniyan and Prema, 2002).
The effects of metals, detergents, and other chemicals were evaluated, and the partially purified enzyme showed strong stability in the presence of most metals used (K + , Na + , Ca 2+ , Fe 2+ , Mg 2+ , Zn 2+ , Co 2+ and Mn 2+ ) except for Cu 2+ Al 3+ , Hg 2+ and Cd 2+ (Figure 8). The study was at variance with a report that endoxylanase was inhibited in the presence of Co 2+ and Mn 2+ (Chen et al., 2006;Hmida-Sayari et al., 2012). Also, substrate specificity is a key indicator to determine the efficiency and products of hydrolysis because not all of the xylosidic linkages in the heteroxylans are readily accessible to a particular xylanase (Gírio et al., 2010). The degradation profile of BW xylan by xylanase monitored by the TLC analysis revealed that the hydrolytic products were longer xylooligomers. This result revealed that the xylanase produced from L. incrustata had similar catalytic properties to those in the GH11 family, a characteristic feature of fungal xylanases (Liu et al., 2012). The efficient utilisation of xylanases relies on a proper understanding of their substrate specificity and the complex structures of heteroxylans. A good number of studies on the three-dimensional structures of xylanases from different GH families in complex with the substrate provide insight into the various mechanisms through which xylanases bind and hydrolyse structurally different heteroxylans and xylooligosaccharides (Pollet et al., 2010).

CONCLUSION
In conclusion, endoxylanase from L. incrustata compared well with those reported for those of other fungal endoxylanase. Further purification of this enzyme to homogeneity would show far more accurate results. Finally, the unique properties exhibited by xylanase from L. incrustata had placed the fungus among the attractive candidates for future industrial applications. For example, it can be used to produce xylanase for the conversion of agricultural residues into other useful bio-based products such as ethanol.