title: Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101.
abstract: An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.
authors.affiliation: Department of Biochemistry , Gulbarga University, Gulbarga-585106 , India.
authors.forename: Malashetty
authors.initials: M
authors.lastname: Vidyasagar
authors.forename: S
authors.initials: S
authors.lastname: Prakash
authors.forename: Vineet
authors.initials: V
authors.lastname: Mahajan
authors.forename: Yogesh S
authors.initials: YS
authors.lastname: Shouche
authors.forename: K
authors.initials: K
authors.lastname: Sreeramulu
keywords: Chromohalobacter sp. TVSP101; halothermophilic protease; organic solvents; osmolytes; purification
references.citation: Curr Opin Chem Biol. 1999 Feb;3(1):39-46
references.pmid: 10021406
references.citation: Biosci Biotechnol Biochem. 2006 Jun;70(6):1395-401
references.pmid: 16794319
references.citation: J Basic Microbiol. 2001;41(6):375-83
references.pmid: 11802548
references.citation: Int J Syst Evol Microbiol. 2001 Jul;51(Pt 4):1457-1462
references.pmid: 11491346
references.citation: J Bacteriol. 1983 Aug;155(2):826-30
references.pmid: 6348027
references.citation: Extremophiles. 2000 Jun;4(3):181-8
references.pmid: 10879563
references.citation: Bioinformatics. 2001 Dec;17(12):1244-5
references.pmid: 11751241
references.citation: J Biol Chem. 1951 Nov;193(1):265-75
references.pmid: 14907713
references.citation: J Chromatogr A. 2005 May 20;1075(1-2):103-8
references.pmid: 15974123
references.citation: J Exp Biol. 2005 Aug;208(Pt 15):2819-30
references.pmid: 16043587
references.citation: Extremophiles. 2001 Apr;5(2):73-83
references.pmid: 11354458
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Am J Trop Med Hyg. 2004 Jun;70(6):597-603
references.pmid: 15210998
references.citation: J Ind Microbiol Biotechnol. 2007 Feb;34(2):111-5
references.pmid: 17024426
references.citation: J Bacteriol. 1992 Jun;174(11):3439-44
references.pmid: 1592801
references.citation: Biotechnol Bioeng. 1997 Aug 5;55(3):471-9
references.pmid: 18636512
references.citation: World J Microbiol Biotechnol. 1995 Jan;11(1):85-94
references.pmid: 24414413
references.citation: Biochem Mol Biol Int. 1994 Jul;33(4):785-92
references.pmid: 7981666
references.citation: Biochem Cell Biol. 1990 Jan;68(1):352-9
references.pmid: 2112401
references.citation: Appl Environ Microbiol. 1982 Sep;44(3):561-9
references.pmid: 6753744
references.citation: Enzyme Microb Technol. 1994 Apr;16(4):266-75
references.pmid: 7764632
references.citation: Adv Biochem Eng Biotechnol. 1998;61:117-53
references.pmid: 9670799