One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

Abstract

The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH.Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 °C. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent K m 3.33 mg/ml and V max 100 IU/ml. The enzyme was strongly inhibited by Hg 2+ and Cu 2+ while enhanced by Co 2+ and Mn 2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by onestep procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

Immobilization of xylanase purified from Bacillus pumilus VLK-1 and its application in enrichment of orange and grape juices

“…Xylanase was purified to apparent homogeneity in one step from Bacillus subtilis ASH by 10.5-fold with a recovery of 43 % (Sanghi et al 2010) and from Bacillus pumilus 85S by 25.3-fold with 63.2 % recovery (Nagar et al 2012a). On the other hand, two forms of alkaliphilic xylanase have been reported from purified from Bacillus licheniformis P11(C) (Bajaj and Manhas 2012).…”

Investigation on lignocellulosic saccharification and characterization of haloalkaline solvent tolerant endo-1,4 β-d-xylanase from Halomonas meridiana APCMST-KS4

“…Hpl-002. Sanghi et al (2010) have also reported that xylanase by B. subtilis ASH required 55°C for optimum activity.…”

“…MX47 (Chi et al, 2012), Paenibacillus sp. Hpl-002 , Bacillus halodurans (Kumar and Satyanarayana, 2011) B. pumilus GESF-1 (Menon et al, 2010), B. subtilis ASH (Sanghi et al, 2010), Chromohalobacter sp. TPSV (Prakash et al, 2009), Bacillus sp.…”

Applications of Microbial Enzymes in Food Industry

“…and Pseudomonas sp. (104)(105)(106). Those produced by bacteria and actinomycetes are effective in a broader range of pH (5.0-9.0), with the optimum temperature for xylanase activity between 35 and 60 °C.…”