Sixty bacterial strains isolated from the soils sample in the presence of organic solvent were screened for xylanase production. Among them, strain RSPP-15 showed the highest xylanase activity which was identified as
Xylanase (endo-1,4-
Recently, interest in xylanase has evidently increased due its broad variety of biotechnological purposes such as prebleaching of pulp, improving the digestibility of animal feed stocks, alteration of cereal-based stuffs, bioconversion of lignocellulosic material and agrowastes to fermentable products, clarification of fruit juices, and degumming of plant fibers [
The soil samples were collected aseptically from different sites of pulp and paper industry of Faizabad to isolate xylanase producing bacteria. One-gram soil was suspended in 9.0 mL sterile distilled water, agitated for a minute. Then 0.1 mL suspension was spread over birch wood xylan agar plates (pH 7.0) containing 1.0% xylan (birchwood); 0.5% ammonium sulphate, and 2% agar. The inoculated plates were overlaid with 7.0 mL of organic solvents (ethanol, propanol, cyclohexane, toluene, butanol, methanol, and isopropanol) and incubated at 55°C, till sufficient growth appeared. After sufficient growth incubated plates were overlaid with Congo-Red solution (0.1%) for 10 min and then washed with 1 N sodium chloride solution for destaining. If a strain was xylanolytic, it started hydrolyzing the xylan present in the surrounding and in the zone degradation there was no red color formation. Selection was done as per colonies with and without clear and transparent zone as xylanase producing and xylanase nonproducing strain, respectively. Bacterial colonies showing clear zones were selected, streaked twice on xylan agar plates for purification, and maintained as pure culture over xylan agar slants (pH 7.0, 4°C). The isolate having maximum clearance zone was selected for further studies. The selected bacterial isolate RSPP-15 was identified by morphological and biochemical characterization as per Bergey’s Manual of Systematic Bacteriology [
The culture was grown in a 150 mL Erlenmeyer flask that contained 50 mL of basal medium containing 2.0% xylan and 0.5% ammonium sulphate. The pH of the medium was adjusted to 7.0 prior to sterilization. The flask was inoculated and incubated at 55°C for 24 h for sufficient growth. The crude enzyme was filtered and centrifuged at 12000 rpm for 10 min and enzyme assay was carried out. Xylanase was assayed by measuring the reducing sugar released by reaction on birchwood xylan. Xylanase assay was done by Nelson [
Bacterial cells in broth were harvested by centrifugation (10000 rpm for 10 min at 4°C), washed with distilled water, and dried in an oven at 80°C until reaching a constant weight. The biomass was reported in the form of dry cell mass (g/L).
The various process parameters influencing xylanase production were optimized individually and independently of the others. The optimized conditions were subsequently used in all the experiments in sequential order. For the optimization, the basal medium was inoculated and incubated at different temperatures, namely, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80°C under the standard assay conditions. The samples were withdrawn at every 8 h interval up to 72 h to study the effect of incubation period. The influence of pH on the enzyme activity was determined by measuring the enzyme activity at varying pH values ranging from 4.0 to 11.0 at 55°C using different suitable buffers at concentration of 100 mM citrate buffer (pH 4.0–6.0, 1 M), phosphate buffer (7.0-8.0), Tris-HCl buffer (pH 8.0-9.0), and glycine-NaOH (10–11.0) under standard assay conditions. The growth medium was supplemented with different carbon sources, namely, fructose, glucose, lactose, soluble starch, sucrose, birchwood xylan, sugarcane bagasse, wheat bran, rice bran, rice husk, and maize bran (at the level of 2%, w/v). Different organic nitrogen sources (beef extract, gelatin, casein, malt extract, peptone, and yeast extract, 0.5% w/v) and inorganic nitrogen sources (sodium nitrate, ammonium nitrate, ammonium chloride, potassium nitrate, ammonium sulphate, and urea, 0.5% w/v) were also used for enzyme production. Thereafter, optimized carbon and nitrogen sources were further optimized at different concentrations.
The effect of various metal ions on enzyme activity was investigated by using FeSO4, CaCl2, NaCl, MgCl2, MnCl2, ZnSO4, CuSO4, CoCl2, HgCl2, and NiCl2 at a final concentration of 5 mM and 10 mM. The enzyme was incubated with different metals at 55°C for 1 h to study metal ion stability of the enzyme and assayed under standard assay conditions. The enzyme activity was measured by conducting the reaction at temperature 55°C and pH 7.0. The activity of the enzyme was considered as 100% under standard assay conditions.
Cell free supernatant having maximum xylanase activity was filtered with nitrocellulose membrane (pore size 0.22
The influence of temperature on activity of xylanase was studied by incubating the reaction mixture at different temperatures (35–100°C). The enzyme was incubated at different temperatures, 35–100°C, for 1 h to study the stability of the enzyme. The residual xylanase activity was determined by performing the reaction at temperature 55°C and pH 7.0. The activity of the enzyme was considered as 100% under standard assay conditions.
The effect of pH on xylanase activity was measured in the pH range of 4 to 10, using the appropriate buffers at concentration of 100 mM (4.0–6.0, sodium acetate; 6.0–8.0, sodium phosphate; 8.0–10.0, Tris-HCl) under standard assay conditions. To evaluate the stability as a function of pH, 100
Each experiment was performed thrice in triplicate, and mean standard deviation for each experimental result was calculated using the Microsoft Excel.
Sixty (60) bacterial isolates producing variable xylanolytic zones on birchwood xylan agar plates stained with Congo-Red solution followed with sodium chloride solution were studied. The zones of clearance by isolates reflect their extent to xylanolytic activity. Those having clearance zone greater than >1.0 cm were considered as significant isolates. Among 60 bacterial isolates, 35 bacterial isolates exhibited good xylanase activities which were reassessed by loading their culture broth in the wells on birchwood xylan agar plates which stained with Congo-Red solution followed with sodium chloride solution (pH 7.0). The culture broth having good xylanase activity cleared more than >1.0 cm zone within 4-5 h of incubation at 55°C, thereby indicating an extracellular nature of the xylanase. The isolate RSPP-15, showing maximum clearance zone diameter, was selected for further studies.
The efficient strain RSPP-15 was rod-shaped, Gram-positive, motile, aerobe, and facultative in nature. It gave positive results for acetylmethylcarbinol, catalase, and oxidase test. It grew over a wide range of pH (4.0–11), temperatures (10–85°C), and sodium chloride concentrations (0.0–12%) and was able to hydrolyze gelatin, casein, starch, and Tween 20, 40, and 80. It produced acid (acetic and lactic acid) from glucose, xylose, mannitol, and arabinose. It gave positive test for citrate utilization and nitrate reduction. The strain was halotolerant as it grew in the presence of 0.0–12% sodium chloride. On account of morphological and biochemical characteristics, it was identified as
Phylogenetic tree showing relation between strain RSPP-15 and other
Influence of temperature on xylanase production in submerged fermentation is one of the important parameters. Figure
Effect of temperature on xylanase production. The flasks were inoculated with culture in the medium and were incubated at different temperatures (35–80°C) for 48 h at pH 7.0. For enzyme activity reaction mixture was incubated at 55°C for 15 min and reaction was conducted as standard assay method. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
Just after optimization of temperature for xylanase production in the liquid medium, incubation period was optimized for enzyme production. The results clearly indicated that
Effect of incubation periods on xylanase production. The flasks were inoculated with culture and were incubated at different incubation periods (8–72 h) at initial pH 7.0, 55°C. For enzyme activity the reaction was assayed at respective incubation periods at 55°C for 15 min. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
Initial pH of the medium is playing a vital role in enzyme production. To study the effect of initial pH on xylanase production, medium was adjusted using different buffers. It was observed that the maximum xylanase production (756.9 U/mL) with 2.5 g/L biomass production by strain
Effect of pH on xylanase production. The flasks were inoculated with culture and were incubated at different pH (4–11) for 48 h at 55°C. For enzyme activity the reaction was assayed at respective pH with buffers (100 mM) at 55°C for 15 min. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
Various carbon sources, namely, starch, sugarcane bagasse, birchwood xylan, wheat bran, rice bran, rice husk, glucose, fructose, lactose, maltose, and sucrose, at a concentration of 2.0% (w/v) were individually tested in the basal medium at their optimal temperature, incubation period, and pH to observe the effect on enzyme production by
Effect of different carbon sources on xylanase production. Test flasks contained different carbon sources in the medium at a level of 2% (w/v). The flasks were inoculated with culture and incubated at 55°C for 48 h at pH 7.0. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
In another set of the experiment, different concentrations of birchwood xylan in the medium were tested for xylanase production at the same growth conditions at which carbon sources were evaluated.
Effect of different concentrations of birch wood xylan on xylanase production. Test flasks contained different concentrations of birch wood xylan (0.5–4.0%, w/v) in the medium. The flasks were inoculated with culture and incubated at 55°C for 48 h at pH 7.0. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
Inorganic and organic nitrogen sources, namely, peptone, beef extract, yeast extract, malt extract, gelatin, casein, urea, sodium nitrate, ammonium nitrate, potassium nitrate, ammonium sulphate, and ammonium chloride, at the rate of 0.5% (w/v) were used in the basal medium for xylanase production (Figure
Effect of different nitrogen sources on xylanase production. The control flask does not contain any nitrogen sources. Test flasks contained different nitrogen sources in the medium at a level of 0.5% (w/v). The flasks were inoculated with culture and incubated at 55°C for 48 h at pH 7.0 with 1.0% birch wood xylan. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
Different concentrations of beef extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 7.0, 8.0, 9.0, and 1.0%, w/v) in the medium were also tested for xylanase production at the same growth condition at which nitrogen sources were evaluated.
Effect of different concentrations of beef extract on xylanase production. Test flasks contained different concentrations of beef extract (0.1–1.0%, w/v) in the medium. The flasks were inoculated with culture and incubated at 55°C for 48 h at pH 7.0. Error bars presented mean values of ± standard deviation of triplicates of three independent experiments.
In this experiment, maximum xylanase production was reported in the presence of Co2+ (10 mM) followed by Ca2+, Mg+2, Zn+2, and Fe+3. In this experiment, maximum enzyme activity (3768 U/mL) considered 100% xylanase activity. Results suggest that xylanase showed maximum relative activity (181.5, 153.7, 147.2, 133.6, and 127.9%) and stability (138.2, 119.3, 113.9, 109, and 104.5%) in the presence of Co2+, Ca2+, Mg+2, Zn+2, and Fe+3 ions, respectively. Some other researchers also reported that Co2+, Ca2+, Mg+2, Zn+2, and Fe+3 ions strongly stimulated xylanase activity [
Effect of metal ions on enzyme activity and stability.
Metal ions | Concentration (mM) | Residual activity (%) | |
---|---|---|---|
Activity | Stability | ||
Control | 100.0 | 100.0 | |
|
|||
CaCl2 | 5 |
104.4 |
135.2 |
|
|||
NiCl2 | 5 |
101.8 |
99.1 |
|
|||
FeSO4 | 5 |
112.2 |
126.3 |
|
|||
MgCl2 | 5 |
110.9 |
125.6 |
|
|||
CuSO4 | 5 |
62.4 |
53.5 |
|
|||
HgCl2 | 5 |
41 |
47.7 |
|
|||
MnCl2 | 5 |
90.9 |
83.9 |
|
|||
NaCl | 5 |
106.2 |
101.3 |
|
|||
ZnSO4 | 5 |
102.9 |
128.4 |
|
|||
CoCl2 | 5 |
125.4 |
107.8 |
Enzyme activity was determined at 55°C in the presence of metal ions in the reaction mixture directly and for stability enzyme was preincubated with different metal ions at 55°C for 1 h and assayed as standard assay method. The enzyme activity without incubation with metal ions was taken as 100%. Mean standard deviation for all the values is <±5.0%.
In another approach, the effect of various organic solvents (30%, v/v) on xylanase stability was also investigated for 7 days, and the results are depicted in Table
Stability of xylanase in the presence of various organic solvents.
Organic solvents (30%) |
|
Residual activity (%) | |||||||
---|---|---|---|---|---|---|---|---|---|
1 h | 24 h | 48 h | 72 h | 96 h | 120 h | 144 h | 168 h | ||
Methanol | −0.76 | 100 | 119.2 | 131.1 | 118.5 | 110.3 | 101.9 | 94.5 | 88.4 |
Isopropanol | −0.28 | 89 | 95.7 | 100 | 90.6 | 87 | 80 | 77.9 | 73.8 |
Ethanol | −0.24 | 89 | 93.6 | 97.9 | 90.2 | 89.2 | 85.5 | 80.8 | 78.3 |
Benzene | 2.13 | 90 | 93 | 100 | 100 | 98 | 94 | 90 | 85.8 |
Cyclohexane | 3.3 | 90 | 95 | 100 | 111.6 | 101.5 | 93 | 92 | 91.6 |
Acetone | −0.23 | 95.3 | 100.4 | 100.5 | 95.4 | 90 | 90 | 83 | 80 |
Butanol | −0.80 | 90.6 | 179.3 | 154.7 | 124.5 | 112.5 | 100.8 | 96.7 | 90 |
Toluene | 2.5 | 96.7 | 190.5 | 170.6 | 149.9 | 100.7 | 99.0 | 90.6 | 90 |
Isooctane | 2.9 | 97.5 | 137.7 | 116.8 | 102.5 | 100.6 | 92.7 | 90.5 | 87.9 |
Xylene | 3.1 | 90 | 100 | 107 | 103 | 100 | 96 | 90 | 86 |
Hexane | 3.6 | 98 | 179 | 194.7 | 179.8 | 147.5 | 119.9 | 104 | 95 |
|
5.6 | 99.8 | 189.3 | 219.8 | 208.4 | 160.9 | 137.8 | 107 | 90.0 |
|
6.0 | 100.8 | 207.5 | 230.8 | 219.5 | 200.6 | 160.9 | 140.0 | 100.0 |
Enzyme was preincubated with different organic solvents at a concentration of 30% (v/v) at 55°C for different time periods and assayed as standard assay method. The enzyme activity without incubation with organic solvent was taken as 100%. Mean standard deviation for all the values is <±5.0%.
Influence of temperature on xylanase activity is one of the important parameters. Figure
Effect of temperature on enzyme activity. For enzyme activity reaction mixture was incubated at different temperatures (35–105°C) for 1 h and reaction was conducted as standard assay method.
The effect of pH on enzyme activity was examined by evaluating the enzyme activity at varying pH values ranging from 4.0 to 10.0 using different suitable buffers. The crude enzyme of
Effect of different pH on enzyme activity. For enzyme activity the reaction was assayed at respective pH and enzyme was preincubated with buffers (100 mM, in ratio 1 : 1) of different pH (4–10) at 55°C for 1 h and assayed by standard assay method.
A thermosolvent stable xylanase is produced by a novel isolate
The authors declare that they have no conflict of interests.
Financial assistance by Council of Science and Technology, UP, India, is greatly acknowledged by Rajeeva Gaur, Soni Tiwari, Priyanka Rai, and Versha Srivastava.