Freeman, S.A., Peek, K., Prescott, M. & Daniel, R.M. (1993). Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2. Biochemical Journal, 295(2), 463-469.
Permanent Research Commons link: http://hdl.handle.net/10289/4483
The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 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 with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic 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 CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 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 activators such as Ca2+, Mg2+ and Zn2+.