One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash
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 ºC. 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 Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. 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 one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.
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Sociedade Brasileira de Microbiologia
2010
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oai:scielo:S1517-838220100002000292010-04-16One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ashSanghi,AshwaniGarg,NeelamGupta,V.K.Mittal,AshwaniKuhad,R.C. Alkalophilic Bacillus subtilis Purification Xylanase 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 ºC. 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 Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. 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 one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.info:eu-repo/semantics/openAccessSociedade Brasileira de MicrobiologiaBrazilian Journal of Microbiology v.41 n.2 20102010-06-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000200029en10.1590/S1517-83822010000200029 |
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Sanghi,Ashwani Garg,Neelam Gupta,V.K. Mittal,Ashwani Kuhad,R.C. |
spellingShingle |
Sanghi,Ashwani Garg,Neelam Gupta,V.K. Mittal,Ashwani Kuhad,R.C. One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
author_facet |
Sanghi,Ashwani Garg,Neelam Gupta,V.K. Mittal,Ashwani Kuhad,R.C. |
author_sort |
Sanghi,Ashwani |
title |
One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
title_short |
One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
title_full |
One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
title_fullStr |
One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
title_full_unstemmed |
One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash |
title_sort |
one-step purification and characterization of cellulase-free xylanase produced by alkalophilic bacillus subtilis ash |
description |
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 ºC. 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 Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. 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 one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications. |
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Sociedade Brasileira de Microbiologia |
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2010 |
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http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000200029 |
work_keys_str_mv |
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1756424925650878464 |