title: 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 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.
authors.affiliation: Department of Biochemistry, Kurukshetra University , Kurukshetra , India.
authors.forename: Ashwani
authors.initials: A
authors.lastname: Sanghi
authors.forename: Neelam
authors.initials: N
authors.lastname: Garg
authors.forename: V K
authors.initials: VK
authors.lastname: Gupta
authors.forename: Ashwani
authors.initials: A
authors.lastname: Mittal
authors.forename: R C
authors.initials: RC
authors.lastname: Kuhad
keywords: Alkalophilic; Bacillus subtilis; Purification; Xylanase
references.citation: Appl Environ Microbiol. 1999 Feb;65(2):694-7
references.pmid: 9925602
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Braz J Microbiol. 2008 Jul;39(3):602-4
references.pmid: 24031273
references.citation: Appl Biochem Biotechnol. 2008 Jun;149(3):229-43
references.pmid: 18500584
references.citation: Biosci Biotechnol Biochem. 1994 Jan;58(1):78-81
references.pmid: 27315708
references.citation: Biosci Biotechnol Biochem. 2000 Apr;64(4):887-90
references.pmid: 10830514
references.citation: Enzyme Microb Technol. 2000 Jul 1;27(1-2):95-99
references.pmid: 10862907
references.citation: Bioresour Technol. 2005 Aug;96(12):1370-9
references.pmid: 15792585
references.citation: Braz J Microbiol. 2008 Jul;39(3):535-41
references.pmid: 24031262
references.citation: Bioresour Technol. 2008 Mar;99(5):1252-8
references.pmid: 17408951
references.citation: Appl Environ Microbiol. 1993 Jun;59(6):1725-30
references.pmid: 8328796
references.citation: World J Microbiol Biotechnol. 1992 Jul;8(4):353-68
references.pmid: 24425504
references.citation: Bioresour Technol. 2007 Oct;98(14):2727-33
references.pmid: 17123815
references.citation: J Appl Microbiol. 2000 Feb;88(2):325-34
references.pmid: 10736002
references.citation: Appl Environ Microbiol. 1995 Dec;61(12):4468-70
references.pmid: 8534112
references.citation: Enzyme Microb Technol. 2000 Feb 1;26(2-4):187-192
references.pmid: 10689076
references.citation: Enzyme Microb Technol. 2001 Mar 8;28(4-5):397-403
references.pmid: 11240197
references.citation: Appl Environ Microbiol. 1993 Jul;59(7):2311-6
references.pmid: 8292206
references.citation: J Biol Chem. 1951 Nov;193(1):265-75
references.pmid: 14907713
references.citation: Crit Rev Biotechnol. 1997;17(1):39-67
references.pmid: 9118232