The purification and characterization studies on the neutral protease from Bacillus subtilis

Abstract

A neutral protease from Bacillus subtilis was first isolated and partially characterized by Fukumoto, et al. (Nippon Nogei Kazaku Kaishi, 32, 230, 233, and 375 (1958). A new purification method starting with a crude commercial enzyme preparation has been developed employing calcium-equilibrated CM-cellulose which gives an enzyme with a higher specific activity than the neutral protease reported by Fukumoto, et al. (Nippon Nogei Kazaku Kaishi, 33, 9 (1959). The enzyme is shown to be homogeneous by the criteria of ultra-centrifugation, electrophoresis, amino terminal amino acid analysis, and rechromatography. Physical studies on the neutral protease show that the enzyme is stabilized by calcium ions. Molecular weight determinations by the Archibald procedure yield a value of 44,700. Electrophoretic studies give an isoelectric point of about 9.0. The enzyme is relatively stable in the pH range of 6.0 to 10.0. (0.002 M calcium acetate is present from pH 6.0 to 9.0). Unlike other known B. subtilis proteases, the neutral protease is a zinc metalloenzyme. The zinc content is about 2.3 ug zinc per mg protein. At the present time, the data indicate that there are 2 moles of zinc per mole of enzyme. This metalloenzyme was found to be inactivated by several chelating agents and heavy metals but not by diisopropylphosphorofluoridate. The amino acid analysis of the neutral protease shows that the enzyme contains no cysteine. The total sulfur is accounted for in the methionine content. Preliminary investigations into the substrate specificity of the enzyme show that the enzyme can hydrolyze certain dipeptides. if their amino and carboxyl terminal groups are blocked. The most sensitive synthetic substrate found to date is hippuryl-L-leucinamide. The protease showed no esterase activity against N-acetyl tyrosine ethyl ester or compounds such as methyl butyrate. and amyl acetate. Detailed kinetic studies with hippuryl-L-leucinamide as substrate show class IV kinetics (Laidler. K. J., Trans. Faraday Soc. 52, 497 (1955) and give a Michaelis constant of 4.7 x 10^-2 M at 30°C. Certain thermodynamic parameters and inhibition studies are also reported.