Purification and characterization of thermostable protease

Abstract

Bacteria produced thermostable protease were isolated from soil of hot spring at Amphor Hui Sai Kwao, Chiang Rai province. It was identified by biochemical tests and 16S rRNA gene sequence as Bacillus mycoides. The protease production was conducted on optimum condition which consisted of peptone medium at initial pH 8.0, inoculum size of 1.0%, initial nitrogen content of 1.4%, at 37 ํC and peptone was replaced by tryptone as a nitrogen source. This thermostable protease had optimum activity at 60 ํC and pH 8.0 in 10mM Tris -HCl buffer. The thermostable protease was purified by 50% acetone precipitation and Sephadex G-75 column chromatography with 0.46% yield and specific activity of 30.97 units/mg protein. The results from SDS-PAGE and Sephadex G-75 column chromatography indicated that the molecular mass of thermostable protease was 27,600 Da. The loss of activity in the presence of PMSF and EDTA indicated the significant of serine in active site of protease and metal ion had an important on protease activity. Thermostable protease exhibited substrate specificity with the K[subscript m] on casein and azocasein 125.00 mg/ml and 26.13 mg/ml while the V[subscript max] on casein and azocasein were 3.80 Units/mg protein and 17.00 Units/mg protein. The application of this protease towards protein wastes from tuna canned industry was studied. The highest digested product with 3.9% nitrogen content was obtained by protease digestion of the mixture of viscera and blood