African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12182

Full Length Research Paper

Purification and characterization of protease enzyme from Burkholderia stabilis

Moon-Hyun Park, Buddhi Charana Walpola and  Min-Ho Yoon*
Department of Bio-Environmental Chemistry, College of Agriculture and LifeSciences, Chungnam National University, Daejeon 305-764, Korea.
Email: [email protected]

  •  Accepted: 31 October 2012
  •  Published: 31 March 2013

Abstract

 

Burkholderia stabilis, a protease producing organism was isolated from green house soils in Chungchugnam-do province, Gongju-Gun area in South Korea. Optimum protease activity of 116.4 U/ml was observed in the growth medium containing 0.7% KH2PO4, 0.2% K2HPO4, 0.01% MgSO4.7H2O, 0.05% citric acid dehydrate, 0.1% yeast extract and 0.2% casein. The protease production was found to be optimized in 1: 5 cultivation volume with 1% inoculum, shaken at 150 rpm. The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ and Mn2+. The maximum enzyme activity was displayed with casein as the substrate followed by egg albumin, gelatin and bovine serum albumin (BSA). Vmax and Km values were 89.28 U/ml and 0.82 mg/ml, respectively when casein was a substrate. The protease was purified to homogeneity by a combination of ammonium sulphate precipitation and gel filtration chromatography. The molecular weight of the enzyme was recorded as 45 kDa by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis.

 

Key words: Purification, characterization, protease, Burkholderia stabilis.

Abbreviation

BSA, Bovine serum albumin; PMSF, phenymethylsulfonyl fluoride;EDTA, tetra-sodium ethylene-diamine tetra acetate; DTNB, dithionitrobenzoic acid; β-ME, β-mercaptoethanol; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

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