Purification of xylanase produced by Bacillus pumilus

* Corresponding author (arva26arva@yahoo.com) Abstract: This study was aimed at purifying xylanase produced by Bacillus pumilus. The spent medium contained 27.9 UmL xylanase activity and 1.5 mgmL protein. The highest specific activity (33.7 Umg protein) was achieved with 50 % (NH 4 ) 2 SO 4 saturation and the xylanase recovery was 94.8 %. The dialyzed and DEAE-Sepharose purified enzyme showed 6.7-fold increase in specific activity with a yield of 84.2 %. Molecular weight of the purified xylanase was 55.4 KDa. Thus B. pumilus xylanase can be purified by precipitating with 50 % (NH 4 ) 2 SO 4 saturation and DEAE-Sepharose ion exchange chromatography.


INTRODUCTION
Xylanase deconstructs xylan into xylose (Nakamura et al., 1995). Many bacterial and fungal species can produce xylanase. Xylanases, which are active under alkaline and thermostable conditions are widely used in paper industry to bleach craft pulp and to increase the brightness (Zamost et al., 1991), to improve the digestibility of animal feed and to clarify juices in food industry (Nakamura et al., 1995). As the xylanases produced by different microorganisms vary in physiochemical characteristics (McCarthy, 1987), it is important to optimize the procedures adopted for the purification of xylanases of different microbial sources. Usually in the purification studies more than one method is adopted. Thus the purification of thermostable alkaline xylanase (Kapilan & Arasaratnam, 2011a) produced by Bacillus pumilus (Kapilan & Arasaratnam, 2010) was studied.

METHODS AND MATERIALS
DEAE-Sepharose (diethyl amino ethyl Sepharose fast flow) was purchased from Pharmacia, Uppsala, Sweden. All the other chemicals used were from standard sources. Xylanase was produced by solid state fermentation of Bacillus pumillus (Kapilan & Arasaratnam, 2010;2011b). The spent medium was centrifuged at 3000 rpm for 30 min and the supernatant was used for purification studies.
The xylanase activity was measured in terms of reducing sugar produced by its action on xylan (Kapilan & Arasaratnam, 2010). Protein concentration was determined by Lowry's method (Lowry et al., 1951). One unit of xylanase activity is defined as the amount of enzyme that produces 1 µmole of reducing sugar in 1 min at pH 9.0 and 60 °C with 20 gL -1 of xylan.
Solid (NH 4 ) 2 SO 4 was added to crude xylanase to bring different saturation values (Dawson et al., 1969), mixed for 2 hrs, allowed to settle, and centrifuged (8000 rpm at 4 °C , 30 min). The precipitate was dissolved

December 2014
Journal of the National Science Foundation of Sri Lanka 42 (4) in distilled water and dialyzed overnight against distilled water. The xylanase activity and the protein content were measured.
DEAE-Sepharose was activated with 0.1 M NaOH and HCl, and equilibrated with 0.01 M Tris buffer (pH 8.0). The precipitated and dialyzed xylanase was added and mixed (100 rpm, 30 min) at room temperature. The unbound enzyme was removed by centrifugation and the residue was re-suspended in Tris buffer (pH 8.0) to wash the unbound enzyme. The DEAE-Sepharose bound xylanase was eluted with different concentrations of sodium chloride. The bound and unbound xylanase activities and protein contents were measured.
Xylanase, which was precipitated with (NH 4 ) 2 SO 4 and dialyzed overnight, was loaded to the activated and equilibrated DEAE-Sepharose (with 0.01 M Tris buffer, pH 8.0) containing column [7 × 1 cm (bed volume 5.5 mL)]. Unbound proteins were removed with 10 bed volumes of 0.01 M Tris buffer (pH 8.0) at a flow rate of 1 mLmin -1 . Bound proteins were eluted with optimized NaCl -0.01 M Tris buffer, (pH 8.0) at a flow rate of 1 mLmin -1 . The xylanase activity and the protein contents were analyzed. The pooled purified xylanase sample was subjected to SDS acrylamide gel electrophoresis (Koseki et al., 1997) and molecular weight of the purified xylanase was calculated (Weber & Osborn, 1969).

RESULTS AND DISCUSSION
The culture supernatant used for the purification contained 27.9 UmL -1 xylanase activity and 1.5 mgmL -1 protein. The precipitation of protein increased with the (NH 4 ) 2 SO 4 saturation percentage while the enzyme activity increased up to 50 % of (NH 4 ) 2 SO 4 (33.7 Umg -1 protein) (Table 1) and the specific activity of xylanase increased by 1.8 times.
The enzyme (2 mL) having 46.8 UmL -1 of xylanase activity and 1.4 mgmL -1 protein was used for the purification. When the concentration of NaCl in Tris buffer varied, the xylanase activity and protein eluted increased up to 0.8 M NaCl (80 %) ( Table 1) with the recovery of protein up to 32 %. Therefore 0.8 M NaCl was selected.
The (NH 4 ) 2 SO 4 precipitated sample (2 mL -specific activity 33.2 Umg -1 , total protein content 4.7 mg, total xylanase activity 156.8 U) was loaded to the DEAE-Sepharose column. Fractions from 5 to 9 contained the proteins without any xylanase activity (Figure 1). The fractions from 16 to 22 (7 fractions) contained proteins with xylanase activity, fraction 18 having the highest. Pooled fraction (16 to 22) showed 18.9 UmL -1 xylanase activity with 0.09 mgmL -1 protein. Thus the specific activity of xylanase was increased from 33.2 to 223.7 Umg -1 protein, which was 6.7 fold higher than that of the crude xylanase with 84.2 % yield ( Table 2).
The distance travelled by the molecular markers and purified xylanase were measured when the purified xylanase was subjected to gel electrophoretic separation ( Figure 2) and the molecular weight of the purified xylanase was estimated to be 55.4 KDa. The molecular weight of this xylanase closely resembled that of Micrococcus sp. AR-135 xylanase (Gessesse & Mamo, 1999) while those of B. thermantarcticus (Bataillan et al., 2000) and B. amyloliquefaciens (Breccia et al., 1997) had lower values, and that of Bacillus sp. strain SPS -O had a higher value (Bataillan et al., 2000). (