PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE PROTEINASE ISOLATED FROM THERMUS SP STRAIN RT41A

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25 - 10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7 % carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75 % of this activity was retained in the pH range 7.0 - 1O.O. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0 - 10.0 and maximum activity, in a 10-min assay, was observed at 90-degrees-C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70-degrees-C and the half-lives at 80-degrees-C and 90-degrees-C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60-degrees-C and the half-life at 70-degrees-C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt4lA proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.