Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

Summary

An extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

• References

• 1 Bachmann F, Fletcher A. P, Alkjaersig N, Sherry S. Partial purification and properties of the plasminogen activator from pig heart. Biochemistry 1964; 3: 1578
• 2 Cohen J. A, Oosterbaan R. A, Berends F. Organophosphorus compounds. In Hirs C. H. W. (ed) Methods in Enzymology . 1967. 11 686
• 3 Davis B. J. Disc electrophoresis – II. Method and application to human serum proteins. Annals of New York Academy of Sciences 1964; 121: 404
• 4 Fletcher A. P, Alkjaersig N, Sherry S. The maintenance of sustained thrombolytic state in man 1. Induction and effects. Journal of Clinical Investigation 1959; 38: 1096
• 5 Güntelberg A. V. A method for the production of the plakalbumin-forming proteinase from Bacillus Subtilis. Comptes Rendus des Travaux der Laboratoire Carlsberg, Sér. Chim 1954; 29: 27
• 6 Janik B. A, Papaioannou S. E. A comparative study of plasminogen activators and proteases for fibrinolytic activity and side effects in rabbits. Thrombosis and Haemostasis 1977; 37: 154
• 7 Johnson A. J, Kline D. L, Alkjaersig N. Assay methods and standard preparations for plasmin, plasminogen and urokinase in purified systems, 1967-1968. Thrombosis et Diathesis Haemorrhagica 1969; 21: 259
• 8 Jürgens J. H, Eriksson A. F. V, Svärd P. O. Aspergilicpeptidase for use in therapy and process of preparation thereof. U. S. Patent. 1970. 3,509,024
• 9 Lowry O. H, Rosebrough M. J, Farr A. C, Randall A. R. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 1951; 193: 265
• 10 Morihara K, Tsuzuki H. Comparative study of various neutral proteinases from microorganisms: Specificity with oligspeptides. Archives of Biochemistry and Biophysics 1971; 146: 291
• 11 Ploug J, Kjeldgaard N. O. Urokinase, An activator of plasminogen from human urine. Isolation and properties. Biochimica et Biophysica Acta (Amst) 1957; 24: 278
• 12 Prentice C. R. M, Rogers K. M, McNicol G. P. Evaluation of a new preparation of urokinase. Thrombosis et Diathesis Haemorrhagica (Stuttg.) 1973; 30: 114
• 13 Schaeffer P. Sporulation and production of antibiotics, exoenzymes, and exotoxins. Bacteriological Reviews 1969; 33: 48
• 14 Stefanini M, Adamis B. M, Soardi F, Marin H. M, Mele R. H. Purification of Aspergillin O. Lancet 1969; 2: 443