An alkaline xylanase secreted by Paenibacillus macquariensis RC 1819 has been purified using ammonium sulfate fractionation, ion exchange chromatography using DEAE-cellulose and gel filtration chromatography over Sephadex G-200 and Sephadex G-100. The purified enzyme had the specific activity, 25.2 units/mg protein with birchwood xylan as a substrate. The purified enzyme showed a single protein band over sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme has been found to be 31,000 ± 2000 as determined by using Sephadex G-200 gel filtration chromatography. The subunit molecular weight has also been found to be ~31,000 as determined using SDS-PAGE indicating monomeric enzyme. The enzyme showed optimum activity at pH 8.6 and temperature, 50°C. The Michaelis constant (Km) of the enzyme for birchwood xylan was 2.2 mg/ml as determined using velocity saturation plot. The metal ions viz. Co+2 and Mn+2 stimulated xylanase enzyme activity whereas Hg+2 inhibited the enzyme activity.
Hemicellulose is the second most abundant component in plant cell wall and xylan is the major component of hemicellulose which is found in solid agricultural and agroindustrial residues, as well as in effluents released during wood processing [1,2]. Xylan is a heterogeneous carbohydrate consisting of a backbone of β-1, 4 linked D-xylopyranosyl units and short chain branches consisting of O-acetyl, α-L-arabinofuranosyl and α-D-glucuronyl residues [
Xylanase is produced by many bacteria and fungi [6,7]. It has been exploited for a range of industrial and environmental applications. Xylanase is commercially important enzyme used in the pulp and paper industries to increase the brightness of pulp without the use of bleach [8,9]. Besides, xylanase is also used in number of other industries viz. food, beverage, textile and animal feed industries [
Xylanases are also used to convert the polymeric xylan into fermentable sugars for the production of ethanol and xylitol from plant biomass [11,12]. Xylanases can also be used for tailor designing of drugs and modifying the properties of food. Xylanases have also been used in animal feed to improve the digestibility of animal feed for better feed utilization [13,14].
Earlier, we showed that Paenibacillus macquariensis secretes extra-cellular xylanase and gets induced in the presence of xylan. We also optimized the conditions for its secretion. The Paenibacillus macquariensis is a gram positive bacteria. It showed optimum growth at 37˚C. However, the growth is much poor at 42˚C. The bacteria showed good growth in the pH range 5.2 to 10 indicating that this bacteria has much tolerance of pH change and has growth in acidic to highly alkaline range. This bacteria secreted xylanase enzyme as tested by Congo red dye staining method [
A soil bacterium Paenibacillus macquariensis was isolated in pure form from petroleum containing soil collected from the vicinity of a petrol pump at Indore as described earlier [
Xylanase enzyme was assayed by measuring the release of the reducing sugars from birchwood xylan following the dinitrosalicylic acid (DNS) method [
Protein estimation was carried out according to the procedure of Lowry et al. [
The Paenibacillus macquariensis culture (500 ml) in Emmerson medium with 2% xylan after growth for 48 hours at 37˚C was centrifuged at 10,000 ×g for 10 minutes at 0˚C to 4˚C in a cooling centrifuge. The supernatant was used as crude enzyme preparation. Thereafter, following procedure was carried out at 0˚C - 4˚C.
To the crude enzyme preparation (500 ml), finely ground solid ammonium sulfate was added slowly with constant stirring maintaining the pH at 9.0 by the addition of 1% (v/v) ammonia to get 0% - 30% saturation. After storage for 3 hours in the cold condition, the suspension was centrifuged at 10,000 ×g for 30 minutes and the supernatant was further subjected to 30% - 60% saturation by adding finely ground ammonium sulfate. After storage for 3 hours in the cold condition, the suspension was centrifuged at 10,000 ×g for 30 minutes. The pellet (30% to 60% pellet) was dissolved in 50 mM potassium phosphate buffer, pH 8.6 (buffer A). It was a turbid suspension, therefore, it was centrifuged at 10,000 ×g for 10 minutes to get clear supernatant which was desalted by passing through a column of Sephadex G-25.
The desalted ammonium sulfate fraction was loaded onto a DEAE-cellulose column (2.5 × 40 cm), previously equilibrated with buffer A. After washing with buffer A, enzyme was eluted by using a linear NaCl gradient (0 to 1 M) and volume of the gradient was 1000 ml (500 ml buffer A in each mixing chamber and reservoir). Fractions of 10 ml were collected at a flow rate of 2 ml/min. The fractions having xylanase enzyme activity constituting a single peak were pooled. To this, solid ammonium sulfate was added to get 90% saturation and the suspension was kept for 5 hours for complete precipitation. The precipitate was collected by centrifugation at 10,000 ×g for 10 minutes and dissolved in buffer A. The turbid suspension was centrifuged at 10,000 ×g for 10 minutes to get clear supernatant
A Sephadex G-200 column (1.5 × 60 cm) was equilibrated with buffer A. The concentrated xylanase fraction after DEAE-cellulose chromatography was loaded onto the column and subsequently chromatographed using buffer A. Fractions of 2 ml were collected at a flow rate of 10 ml/hour and the active fractions pooled. To the pooled fraction, solid ammonium sulfate was added to get 90% saturation and the suspension was kept for 5 hours for complete precipitation. The precipitate was collected by centrifugation at 10,000 ×g for 10 minutes and dissolved in buffer A. The turbid suspension was centrifuged at 10,000 ×g for 10 minutes to get clear supernatant.
A Sephadex G-100 column (1.5 × 50 cm) was equilibrated with buffer A. The concentrated xylanase fraction after Sephadex G-200 chromatography was loaded onto the column and subsequently chromatographed using buffer A. Fractions of 2 ml were collected at a flow rate of 12 ml/hour and the active fractions pooled.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the procedure of Laemmli [
Native molecular weight of xylanase was determined using gel filtration chromatography over Sephadex G- 200 according to the procedure of Whitaker [
The relative xylanase activity at different pHs was determined using 1% birchwood xylan as substrate. The pH range used was from 4 to 11. Three different buffers were used viz. 0.1 M citrate buffer for pH range 4 to 6, 0.1 M sodium phosphate buffer for pH range 6 to 8, and 0.1 M glycine NaOH buffer for pH range 8 to 11. The substrate (1% birchwood xylan) was prepared in buffers of different pHs and pre-incubated at 50˚C for 10 minutes. To this, 0.1 ml enzyme was added and incubated for 15 minutes. The reaction was stopped by adding 1.5 ml of DNS solution and the tubes were put in a boiling water bath for 15 minutes. The further procedure was same as given under the enzyme assay.
For pH stability, xylanase enzyme was incubated with different buffers viz. 0.1 M citrate buffer for pH range 4 to 6, 0.1 M sodium phosphate buffer for pH range 6 to 8, and 0.1 M glycine NaOH buffer for pH range 8 to 11 at room temperature (25˚C) for 30 minutes. Thereafter, enzyme activity was determined at pH 8.6 by using the enzyme assay as described above.
The apparent optimum temperature for the purified xylanase was determined by assaying the enzyme activity at different temperatures ranging from 30˚C to 70˚C. At each temperature, 0.9 ml of 1% birchwood xylan dissolved in 50 mM glycine-NaOH buffer, pH 8.6 was preincubated for 10 minutes. To this, 0.1 ml enzyme was added and incubated for 15 minutes. The reaction was stopped by adding 1.5 ml of DNS solution and the tubes were put in a boiling water bath for 15 minutes. The further procedure was same as given under the enzyme assay.
For thermo-stability, xylanase enzyme was pre-incubated at different temperatures ranging from 30˚C to 100˚C in a water bath. After every 30 minutes, the sample tubes were removed from the water bath and stored on ice prior to enzyme assay. Thereafter, enzyme activity was determined at 50˚C as described above.
Xylanase activity was determined using cellulose, birchwood xylan, xylobiose as substrates.
Xylanase aliquots (0.5 ml) were incubated at the room temperature (25˚C) with 1 mM metal ions viz. Ca+2, Mg+2, Hg+2, Fe+2, Cu+2, Mn+2, Co+2, Zn+2, As+3, Mo+2 for 2 hours. Thereafter, enzyme activity was determined using 0.1 ml of the incubated enzyme with 0.9 ml of 1% birchwood xylan dissolved in 50 mM glycine-NaOH buffer, pH 8.6 and incubated at 50˚C for 15 min.
Birchwood xylan hydrolysis rates were determined at all substrate concentrations ranging from 0.5% to 8% in 50 mM glycine-NaOH buffer at pH 8.6.
The summary of purification of xylanase from Paenibacillus macquariensis has been shown in
The molecular weight of xylanase was found to be 31,000 ± 2000 as determined by using Sephadex G-200 gel filtration chromatography according to the procedure of Whitaker [
Enzyme assay was carried out in the pH range varying from pH 4 to 11 to find out the optimum pH at which the enzyme shows maximum activity. The purified xylanase showed maximum activity (optimum pH) at pH 8.6 and half maximum activity at pH 5.8 and pH 10.6 (
The pH stability profile showed that xylanase is more stable in the pH range 8 to 9 (
Enzyme assay was carried out in the temperature range of 30˚C to 70˚C to find out the apparent optimum temperature at which the enzyme shows maximum activity. The purified xylanase showed maximum activity (optimum temperature) at 50˚C and half maximum activity at 27˚C and 67˚C (
The thermo-stability studies showed that xylanase is stable up to 60˚C. At 50˚C, enzyme had a half life of two hours whereas at 60˚C, it showed a half life of one hour only (
The enzyme hydrolyzed β-1,4-xylan (birchwood xylan). It also hydrolyzed cellulose (CM-cellulose) but activity was nearly 10% as compared to xylan. The enzyme showed no activity with xylobiose.
Purified xylanase aliquots were incubated at the room temperature (25˚C) with 1 mM metal ions viz. Ca+2, Mg+2, Hg+2, Fe+2, Cu+2, Mn+2, Co+2, Zn+2, As+3, Mo+2 for 2 hours. Enzyme activity got stimulated upon incubation with Co+2 and Mn+2. However, metal ions viz. Ca+2, Fe+2, Mo+2, Cu+2, Mg+2, Zn+2, As+3 showed no considerable effect on xylanase activity. In contrast, 1 mM Hg+2 showed 35% inhibition of xylanase activity. The purified xylanase was also incubated without any metal ion which was taken as a control (
Xylan saturation curve was found to be rectangular hyperbolic indicating that the enzyme obeyed classical
Michaelis kinetics. The Km value of xylanase for birchwood xylan was calculated to be 2.2 ± 0.2 mg/ml.
Microorganisms of extreme environments have attracted much attention owing to their habitat-related adaptive properties. For example, alkaline xylanases have been isolated from microbes and plants of alkaline or neutral environments.
To obtain an alkaline xylanase we selected the soil containing petroleum as the source material for microorganism isolation. The enzyme production was maximum on birchwood xylan. Alkaline xylanases are of much industrial importance especially in paper and pulp Industries.
Earlier, from our laboratory, Mahatman et al. [
Xylanase from Paenibacillus macquariensis showed maximum activity at pH 8.6. However, it exhibited enzyme activity over a broad pH range of pH 4 - 11, Earlier, from our laboratory, Mahatman et al. [
The present enzyme may be exploited as chicken feed additive since physiological pH of chicken digestive tract has been reported to be ranging from pH 4.5 to 7.5 [
Xylanase from Paenibacillus macquariensis showed maximum (optimum) activity at 50˚C. The thermo-stability of the enzyme was also tested after pre-incubation of the enzyme at 30˚C to 100˚C for 30 minutes. The enzyme had half life of two hours at 50˚C whereas it had a half life of one hour at 60˚C. At high temperatures, enzyme gets partly unfolded. Indeed, this point of maximum activity is the result of the intersection of an exponential curve corresponding to the increase of the activity as a function of temperature and of an unfolding curve due to deleterious effect of heat. Dheeran et al. [
In the present case, xylanase activity was stimulated in the presence of Co+2 and Mn+2 ions. However, xylanase activity got inhibited in the presence of Hg+2 ions. There are reports of similar results [37-39]. Inkyung and Jaiesoon [
In the present case, Km value of the enzyme for birchwood xylan has been calculated to be 2.2 mg/ml. The Km value of the enzyme from Arthrobacter for wheat bran has been reported to be 0.9 mg/ml. Similarly, Km value of the enzyme from Bacillus subtilis and Bacillus sp. strain TAR-1 has been reported in the range of 0.9 mg/ml [42,36]. Therefore, in the present case, Km value is more compared to other reported values indicating lower affinity for the substrate. However, here we used birchwood xylan and in the other reports, it was wheat bran.
The molecular weight of the enzyme in the present case has been found ~31,000. Ratanakhanokchai and Tanticharoen [
The authors acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, India for its facilities under M.Sc. Biotechnology program and the Bioinformatics Sub Centre.