Production, Partial Purification and Biochemical Characterization of Thermostable Xylanase from Bacillus brevis

Additional to industrial use of xylanase in kraft pulp production, thermostable xylanase has wider application in fishery, piggery, cattle food and human food. In this research, 1-4-β-Dendoxylanase was isolated from liquid state cultures of Bacillus brevis containing wheat straw as carbon source. Xylanase was purified to apparent homogeneity by gel filtration and ion exchange chromatography. The enzyme was optimally active at 55 °C and pH 7.0. The molecular weight of xylanase (determined through SDS-PAGE) was 23 kDa. The partially purified xylanase was found active in gel when birchwood xylan was used as substrate in zymogram analysis. The enzyme showed good thermal stability with half life of 3 h at 55 °C and 2 h at 70 °C, respectively. Key wordsxylanase, thermostable, Bacillus brevis, hemicellulose, β-1, 4linked-D-xylopyranosyl residues, arabinofuranosyl residues, bleaching agents but for that the enzyme should be active at alkaline pH and high temperature. To meet the industrial requirements especially for kraft biobleaching the bacterial xylanases are preferred as compare to fungal xylanase because mostly fungal xylanase has celulase activity, which may adversely decrease the quality of paper10-11. Additional to industrial use of xylanase in kraft pulp production, thermostable xylanase has wider application in fishery, piggery, cattle food and human food12. The present study describes partial purification and characterization of a termostable xylanase from Bacillus brevis grown on wheat straw as substrate. Figure 1 shows production of xylanase using different substrates. To our knowledge, this is the first report describing the production of xylanase by Bacillusbevis using agrowaste for xylanase production and the enzyme was stable at higher temperature. 436 GOSwAMI et al., Biomed. & Pharmacol. J., Vol. 6(2), 435-440 (2013) pH shown in figure 3 was determined by measuring the activity at 55 °C. 50mM sodium acetate (pH 3.0-6.0); 50 mM sodium phosphate (pH 6.5-8.0) and Tris buffer (pH 8.5-9.5) were used for different pH ranges. The temperature stability of xylanase was determined by pre-incubating the enzyme at 55 °C and 60 °C and removing aliquots at intervals to measure the xylanase activity as described earlier. Protein concentration was measured by [14] using BSA as the standard. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDSPAGE, 12%) was carried out as prescribed [15]. After electrophoresis, the protein bands were silver stained, following standard protocol. Zymogram was developed by Congo reg solution (1%) for 30 min at 37 °C, 1M NaCl was used for distaining while 0.5% acetic acid was used as fixative16.