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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Main Authors: Sanghi,Ashwani, Garg,Neelam, Gupta,V.K., Mittal,Ashwani, Kuhad,R.C.
Format: Digital revista
Language:English
Published: Sociedade Brasileira de Microbiologia 2010
Online Access:http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000200029
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spelling 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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country Brasil
countrycode BR
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libraryname SciELO
language English
format Digital
author 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.
publisher Sociedade Brasileira de Microbiologia
publishDate 2010
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000200029
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