Purification and Biochemical Characterization of Xylanases from Bacillus Pumilus and their Potential for Hydrolysis of Polysaccharides

Extracellular xylanases free of cellulase produced by the alkalophilic bacteria Bacillus pumilus was purified to homogeneity throughout the precipitation with (NH4)2SO4, Q-Sepharose chromatography and characterized. The purified xylanases were proteins, with molecular mass ~14 kDa (Xyl 1), ~ 35 kDa (Xyl 2) and ~ 60 kDa (Xyl 3) as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 50oC and 7 respectively. They exhibited thermal stability over a range of 20 to 40oC at pH-7 and has retained 85 % at 60oC. The activity strongly inhibited by 10 mm of Hg2+, SDS and Fe2+. The xylanase exhibited Km and Vmax values were 4.0 mg/ ml, 5000 μmol/ min/ mg protein (Xyl 1) as well as 3.5 mg /ml, 3448 μmol/ min/ mg of protein (Xyl 2) for oatspelt xylan.


Introduction
Hemicelluloses which act as the cementing material between cellulose and lignin, it consists of pectin, arbinoxylan and xylan. Xylan is the principal component with β-1, 4-linked-D-xylopyranosyl residues as backbone and different substitute groups in the side chain. Several enzymes are required for complete hydrolysis and assimilation of xylans, including β-endoxylanase, β-xylosidase, and xylose isomerase of which β-endoxylanase received the broader attention among these enzymes. Xylanases have potential applications in food, feed, chemical, pharmaceutical and paper industries. These enzymes produced by several bacterial and fungal species. There was reports related endoxylanase production by Bacillus, with different molecular masses [1]. Solid-state cultivation (SSC) on wheat bran as a substrate is an effective procedure for xylanase production. However, SSC has gained renewed interest in recent years for the production of many enzymes due to lower operation costs and energy requirements [2]. The thermotolerant alkalophilic bacteria B. pumilus has attracted attention as a rich source of xylanolytic enzymes. B. pumilus was reported previously as good producer of cellulase-free endoxylanase [3]. This paper focuses on the purification and biochemically characterize extracellular xylanases produced by B. pumilus grown on SSC using wheat bran. Until now there were no reports related to low molecular weight xylanases from this bacterial species by SSC. Of the three isoforms purified Xyl-1 and Xyl-2 exhibited high activity against hydrolysis of plant polysaccharides.

Materials and Methods
Microorganism and culture conditions B. pumilus was isolated from forest soil of Kerala and maintained at 4 o C on Xylan agar slants [3]. For xylanase production B. pumilus was cultivated in 250 ml Erlenmeyer flasks containing 10 g of fresh wheat bran as solid substrate. It was moistened with basal medium of composition (g/l) KH 2 PO 4 , 2 and MgSO 4 .7H 2 O, 0.4, in the ratio 1: 2.5 and autoclaved at 15 lbs for 45 min. The cooled medium was inoculated with 18 h grown culture spores and incubated at 35 o C. After fermentation process, the protein content of the solid-state culture flasks was extracted with distilled water. For extraction it was placed on a rotary shaker (150 rpm) at room temperature for 1 h. Content filtered through a muslin cloth and filtrate was centrifuged at 10,000 rpm for 20 min at 4ºC. The supernatant used as crude enzyme sources for further studies.

Xylanase purification
The purification steps were all performed at room temperature since previous experiments showed no difference in enzyme stability at 4 or 25 o C. Firstly, proteins were precipitated between 20 and 70% of ammonium sulphate saturation and centrifuged at 10,000×g for 20 min. The pellet was resuspended in 50 mM sodium phosphate buffer, pH 7 and dialyzed for 18 h against this buffer. Q-Sepharose F column (2.5 × 12 cm) which was pre-equilibrated with 10 mM Tris -HCl buffer (pH 8.0). The column was washed with 200 ml of the respective buffers at a flow rate of 3.0 ml /min followed by the sample elution with 5-bedvolume of NaCl salt in gradient (0-1.0 M) in the equilibrating buffers. The proteins were eluted with a NaCl gradient (0 -1.0 M) in the same buffer. The active xylanases fractions were collected and concentrated by lyophilisation. One milliliter fractions were collected and used to measure absorbance at 280 nm and xylanase activity. Three peaks with xylanase activity were eluted and pooled, dialyzed against sodium phosphate buffer and used for biochemical characterization.

Enzyme assay and protein determination
Xylanase activity was assayed using 0.5 % (w/v) of oat spelt xylan as substrate. Reaction mixtures contained 0.1 ml enzyme and 0.9 ml substrate in 100 mM sodium phosphate buffer pH 7. The mixture was incubated at 50 o C, and after 10 min the released reducing sugars were estimated with 3-5-dinitrosalicylic acid (DNS), using xylose as standard [2]. One unit (U) of xylanase activity defined as the amount of enzyme that released 1µmol of reducing sugar equivalent to xylose / minute, under the standard assay conditions. Endoxylanase production by SSC expressed as U/g DBB (dry bacterial bran). Total soluble protein was estimated using bovine serum albumin as standard and also by direct method where the absorbance taken at 280 nm, the free Tyrosine (Tyr) and Tryptophan (Trp), uric acid, and bilirubin interfere at 280 nm [4].

Characterization of xylanase
Optimal temperature for endoxylanase activity was determined between 30-65 o C. Thermal stability estimated by incubation the enzyme at different temperatures ranging 25-65 o C for 90 min and measuring the residual activity. Optimal pH was determined by using different buffers ranging between 5 and 11 (citrate phosphate buffer, pH 5 to 7, phosphate buffer 6 to 8 and Glycine -NaOH buffer, 9 to11 at 100 mM). pH stability determined by incubating equal volume of enzyme solution with different buffers ranging between 5 and 11 at 50 o C for 90 min residual activity estimated. The effect of various compounds on xylanase activity was determined by incubating the purified enzyme in the presence of 10 mM in 100 mM sodium phosphate buffer pH 7 of each compound for 30 min before the reaction with the substrate. Residual activity was expressed as the percentage of the activity observed by the standard assay described above.

Kinetic parameters
The effect of substrate concentration, ranging from 2.0 to 20 mgmL −1 , on xylanase activity was evaluated under optimal assay conditions (50 o C and pH 7.0). Xylan presents low solubility, so higher concentrations could not be used. The kinetic parameters of Michaelis-Menten constant, Km, and maximal reaction velocity, Vmax, were estimated by linear regression according to the Lineweaver and Burk double-reciprocal plot [6].

Hydrolysis of plant polymeric substrate
In order to assess the enzyme ability to hydrolyze several different agro-industrial residues, reaction mixtures containing purified enzyme solution and 50 mg dry weight of selected agro-industrial residues (rice bran, rice straw, wheat bran, oat spelt xylan, brich wood xylan, beech wood xylan, carboxymethyl cellulose and filter paper) suspended in 100 mM sodium phosphate buffer pH 7.0 were incubated for 1 h at 60 o C and 100 rpm. The reaction was stopped by placing the mixture in boiling water for 5 min, and then centrifuged at 8000 x g for 10 min. Reducing sugars liberated by hydrolysis of these substrates were quantified by the dinitrosalicylic acid method.

Purification of endoxylanase
High levels of xylanolytic activity (about 159.96 U/ml), but less than 0.0017 U/ml of cellulolytic activities was exhibited by crude filtrates of cultures from B. pumilus grown on wheat bran [3]. The xylanase from B. pumilus precipitated using ammonium sulphate (0-70 % saturation) to yield an active pellet. The active pellet was dialyzed and used as starting material for further purification using Q-Sepharose fast flow column. The profile of elution from this culture filtrate exhibited three peaks showing xylanase activities (Figure 1), these fractions were designated as Xyl 1 and Xyl 2 and Xyl 3. A summary of a representative purification protocol is shown in Table 1.
The three purified enzyme preparations appeared homogeneous, since they migrated as a single protein band under non-denaturing  condition (PAGE). The xylanases were also assayed for activity on the gel, after PAGE, and the activities were synchronized with the protein band ( Figure 2 A and B). The purified xylanases preparation was represented in the form of a single band on SDS -PAGE (Figure 3). The molecular mass of the purified xylanases was estimated to be approximately 14, 35 and 60 kDa for Xyl 1, Xyl 2 and Xyl 3 respectively, using SDS-PAGE. Purification and characterization are the most important fundamentals for thriving biotechnological application of enzymes. Purification of xylanase from microorganisms normally needs precipitation of the active protein followed by two to three chromatographic steps. Xylanase with molecular mass of 40 kDa purified to homogeneity by (NH 4 ) 2 SO 4 fractionation and anion-exchange chromatography from thermo alkalophilic Bacillus sp. strain TAR-l [7]. Xylanase II from A. versicolor was purified 28 -fold with DEAE-Sephadex and HPLC GF-510 gel filtration [8]. Crude xylanase (80 %) precipitated by acetone from wild strain of A. nidulans was subjected to SDS-PAGE, which revealed the occurrence of four isoxylanases with molecular weights of 50, 43, 20 and 18 kDa [9]. Only few reports were there related to low molecular weight xylanase that has application in paper pulp industry, due to its high penetration in the Kraft pulp that help in the easy removal of reprecipitated xylan. In the present study ammonium sulphate precipitated xylanase B. pumilus was purified to homogeneity following single step anion exchange chromatography. Ion exchange chromatography can easily scale up in comparison to other filtration or precipitation techniques related to procedure. Current procedure intended for high through put purification of xylanases for commercial application.
The molecular weights of purified xylanases of B. pumilus were observed to be 14, 35 and 60 kDa and are within the range of molecular weight for xylanases i.e. 11-85 kDa [10,11]. Comparative analysis of molecular weight of Xyl 1 and its pI-8 (data not shown) of purified xylanases from B. pumilus indicated that it follows dichotomous pattern and fall in Group 1 xylanases of low molecular and basic protein based on classification suggested by Wong [10]. Two xylanases from B. subtilis purified by native PAGE and homogenization extraction analyzed using HPLC. Both xylanase were endo acting, designated xyl I and xyl II (activity range pH 5.0 to 9.0 at 50 o C with optimum activities at pH -7 and 50 o C) [4]. A. versicolor produces a xylanolytic complex with two components, the minor component designated xylanase II (5 days in 1 % wheat bran) a monomeric glycoprotein with molecular mass of 32 kDa with 14.1 % of carbohydrate content. Both xylanase isolated reported to have optimum activity at pH 6 -7 and temperature 55 o C [8].

Characterization of xylanases
Important external factors that influence enzyme activity were temperature and pH. The purified xylanase, Xyl 1 and 2 exhibited maximum activity at temperature 50 o C and retained 93 % of activity at 55 o C where as Xyl 3 active at 55 o C at pH 7. Temperature and pH stability of the enzyme are very important factors when we intend to study the industrial importance of the enzyme. The isoforms were stable at 20 to 40 o C and retained 90 % activity at 50 o C and 85 % at 60 o C. The purified xylanase Xyl 1 was active at pH 7 in phosphate buffer and Xyl 2 and 3 at pH 6 with citrate phosphate and phosphate buffer respectively. Xyl 1 also exhibited activity at pH 10 and 11 while Xyl 2 and 3 were inactive at this pH. Xyl 1 is stable at pH 7 and retained 90 % at pH 6, 8 and 9 in addition to it has retained 82 % at pH 10 and 11. Xyl 2 stable at pH 6, 7 and 8, retained 85 % activity at 9 and 10 additionally 80 % activity at pH 5 and 11. Xyl 3 is steady at pH 7 and retained 62 % activity at pH 8, inactive at pH 11 and very low activity at other pHs. Xylanase from Streptomyces. sp AMT-3 was active over a range of temperature 55 -60 o C and pH -6 [12]. Endoxylanase from Rhizopus oryzae have optimum pH and temperature at 4.5 and 55°C, respectively [13]. Xylanases (Xyl Ia and Ib) from Myceliophthora sp. IMI 387099 were optimally active at 75 o C and at pH 6.0 and stable at pH 9.2 at 60 o C for 2 h, but varying at pH 6.0 and above 50 o C [14]. Difference in pH and temperature tolerance for xylanase excreted might be due to the effect of different enzymes mixtures excreted, and / or the post -translation modifications in xylanase excretion process, such as glycosylation, that improve stability in more extreme pH and temperature conditions [15].
The effects of potential inhibitors or activators on purified xylanases were shown in Figure 4. The results demonstrated some differences between Xyl 1, Xyl 2 and Xyl 3. Although both enzymes suffered inhibition by almost all the ions tested, activity of Xyl 1 was   more sensitive than Xyl 2 and 3. Addition of Hg 2+ drastically inhibited xylanase activities in all three, suggesting the existence of thiol groups at the active site of the enzymes. Hg 2+ is known to react with protein sulphydryl groups as well as histidine and tryptophan residues as well as presences of cysteine residue in or close to the active site of the enzyme [16]. Addition of Fe 2+ , SDS also completely inhibited xylanase activity. EDTA did not considerably affect the activities, signifying that metal ions were not required for enzyme activities. The enzyme activities were stimulated by addition of Mg 2+ in Xyl 1 and Mn 2+ in other two fractions [17]. These ions seem to affect enzyme activity by altering the conformation of the polynucleotide complexes.

Enzyme kinetics
The xylanases exhibited Km and Vmax values of 4.0 mg/ ml, 5000 µmol/ min/ mg protein for Xyl 1 and 3.5 mg/ ml and 3448 µmol/ min/ mg of protein for Xyl 2. Vmax / Km for Xyl 1 -1250 and Xyl 2 -985. Xyl 1 activity exhibited a higher Vmax than Xyl 2, and showed more affinity for the substrate. Because of the high Km the endoxylanases could not be saturated with substrate, and thus it is not known if substrate inhibition kinetics also occurs with the substrate. Xylanase from Trichoderma sp. reported to have similar high specific activity with wide range of Km (0.5 -12.5 mg/ ml) and Vmax values (4,025 U / mg protein). These differences could be to a certain extent, attributed to different xylans or temperatures used in the xylanase assay and methods used in determination of sugar concentration [22]. Similar Km for birch wood xylan reported as 2.3 mg/ ml while Vmax was 233.1µmol/ mg/ min of protein from A. versicolor [8].

Substrate specificity
Purified enzyme fractions were subjected to hydrolysis experiment on several agro industrial by-products, and results explained in Table 2. Both Xyl 1and Xyl 2 were mainly active on oat spelt xylan. The isoforms also hydrolysis natural sources like; wheat bran and rice bran. These sugar conversion abilities are very important since inexpensive agro industrial by-products, rich in cellulose and hemicelluloses, are the main potential substrates for the production of useful biomolecules, including chemicals and organic solvents. The increasing interest in biotechnological processes employing lignocellulosic residues is quite justifiable because these materials are cheap, renewable and a widespread source of sugars [18].

Conclusion
The biochemical property like optimum of temperature, pH and molecular mass of all isoforms were in the range of other Bacillus xylanases. According to these properties, the xylanases produced by Bacillus pumilus fit into the categories xylanases proposed by Wong and coworkers [11]. Xylanases from Bacillus, Aspergillus, Clostridium,  Streptomyces and Trichoderma, species suggested having multiplicity. The existence of a evolutionarily conserved relationship between their molecular weight and pI, in which low MW (< 22.000) xylanases are basic proteins whereas high MW (> 43.000) xylanases are acidic proteins. Many commercially available fungal xylanases are acidic, but that from bacteria such as Bacillus are more promising. Reducing sugars were released from all the agro industrial residues, making this enzyme a promising candidate with application in the recovery of fermentable sugars from hemicelluloses. The characteristics of xylanase from B. pumilus enhance its industrial potential and can effectively used in clarification of juice and baking industry, in paper and pulp industry, as well as feed industry.