Chiang Mai Journal of Science

Extracellular protease was produced by a thermophilic bacterium strain TLS33 isolated from Thephanom hot spring in Chiang Mai, Thailand. The bacterium was grown and secreted high protease activity when cultured in a medium containing 0.1% w/v yeast extract and 0.25% w/v skim milk in 80% v/v base mixture pH 7.2 at 65^oC for 48 h. The protease has been characterized as neutral protease and its optimum pH and temperature were 7.0 and 70-80^oC respectively. It was stable up to 75^oC after preincubated over a range of temperature for 1 h and its thermostability was assessed at 80^oC in the presence of 60 mM CaCl₂. Thermostability of the protease was markedly increased by the addition of CaCl₂. The protease could hydrolyse a number of substrates including casein, soybean, hemoglobin, BSA, lysozyme, skim milk, ovalbumin and the azocasein (synthetic substrate). It was completely inactivated by 40 mM EDTA which could be classified as a neutral metalloprotease. The inhibition effects of PMSF and thiol reagents suggest that the hydroxy and thiol groups also have a role in enzume integrity. Thus, the metal ion composed in the protease molecule was studied using the holoenzyme, apoenzyme, and Zn²⁺-apoenzyme preparation. The apoenzyme containing no metal ions had low residual activity (33.12%) and its activity was restored up to 65% both by the addition of 1 mM ZnCl₂ in the assay medium and the dialysis of apoenzyme against 0.02 M Tris-HCl buffer pH 7.0 containing 5 mM ZnCl₂ which name as Zn²⁺-apoenzyme. Regarding to stabilization, 1 mM CaCl₂ imparted the stability on the apoenzyme and Zn²⁺-apoenzyme over the 90 min. pre-incubation period at 70^oC. The results indicated that Zn²⁺ enhanced the protease activity whereas the stability of the enzyme was increased by Ca²⁺. The enzyme could be characterized as a thermostable metalloprotease containing Zn²⁺ in its molecule.