CHARACTERIZATION OF A CHELATOR-RESISTANT PROTEINASE FROM THERMUS STRAIN RT4A2

The Thermus isolate Rt4A2 was found to produce an extracellular chelat or-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtrati on chromatography, and weak anion-exchange chromatography. The Rt4A2 p roteinase was found to have properties typical of an alkaline serine p roteinase. It had a pH optimum of 9.0 and was specifically inhibited b y phenylmethanesulphonyl fluoride. Its isoelectric point was greater t han 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity wi th other serine proteinases from Thermus species. The proteinase hydro lysed a number of substrates including fibrin, casein, haemoglobin, co llagen, albumin and the synthetic chromogenic peptide substrate Suc-Al a-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition wa s observed above an azocasein concentration of 0.05% (w/v). Esterase a ctivity was directed mainly towards those substrates containing the al iphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70-degrees-C, 43 h at 80-degrees-C and 90 min at 90-degrees-C were found in the presence of 5 mM CaCl2. At 90-degrees-C increasing the Ca Cl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increas e in the half-life of the enzyme form 30 to 130 min. Half-lives of 19. 4 min at 100-degrees-C and 4.4 min at 105-degrees-C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activator s such as Ca2+, Mg2+ and Zn2+.