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). V_max and K_m 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.