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Braz J Microbiol. 2010 Apr;41(2):467-76. doi: 10.1590/S1517-838220100002000029. Epub 2010 Jun 1.

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

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1
Department of Biochemistry, Kurukshetra University , Kurukshetra , India.

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 Km 3.33 mg/ml and Vmax 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 one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

KEYWORDS:

Alkalophilic; Bacillus subtilis; Purification; Xylanase

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