Purification and characterization of a membrane bound serine protease of Bacillus subtilis IFO 3027.

A novel serine protease was purified from the crude membranefraction of Bacillus subtilis IFO 3027 by a procedure involving extraction with «-butanol, precipitation in a solution of low ionic strength, washing with organic solvents, Phenyl Sepharose CL-4B column chromatography and Sephacryl S-300 gel filtration.The purified enzyme was homogeneous on SDS-polyacrylamide gel electrophoresis.The molecular weight of the native enzyme and its subunit were determined to be 540,000 and 62,000, respectively.The enzyme was precipitated in the low ionic strength buffer.The enzyme was a serine protease and inhibited by PMSFand certain microbial serine protease inhibitors such as MAPIand chymostatin.The enzyme showed a broad substrate specificity against peptides with arginine and tyrosine bonds.The Km for Z-L-Ala-L-Ala-L-Leu-/?NA was calculated to be 1.2 x 10~4m.Casein hydrolytic activity of the enzyme was maximumat pH ll and at 55°C. Intracellular proteolytic activity is generallynegligible in cells of Bacillus grown exponentially in a liquid medium but increases significantly during the sporulation phase.Intracellular serine proteases have been isolated and purified from sporulating cells of Bacillus subtilis,1>2) Bacillus cereus^Bacillus megaterium,4' 5) and Bacillus thuringiensis.6)Although a variety of functions are attributed to these proteases during sporulation, i.e. protein turnover,3'7'8) modification of DNAdependent RNApolymerase,6) processing of precursors of spore coat proteins9) and scavenger action,10) our knowledge of their chemical and physiological features is rather limited.One approach is to study the effect of serine protease inhibitors on the sporulation.