A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homo geneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metal-chelating agents and metal ions such as Hg_??_, Pb_??_, Cu_??_ and Fe_??_ markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity.

The neutral protease of Bacillus amylosacchariticus was inactivated by low concentra tions of several metal-chelating agents and the inactivated enzyme with EDTA restored its activity almost completely by the addition of Zn++ or Co++ and partially by Fell or Mn++, if these metal ions were added shortly after the EDTA-treatment. The native enzyme was found to contain 0.19% of zinc together with a significant amount of calcium. Parallel increase in specific activity and zinc content of enzyme preparation was observed throughout the purification procedure. The elution pattern of enzyme activity on a CM-cellulose column chromatography also completely coincided with that of protein-bound zinc. A zinc-free inactive enzyme was also reactivated by the addition of zinc or cobalt ions, clearly showing that the neutral protease of B. amylosacchariticus is a zinc metalloenzyme.

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Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coef ficient were estimated to be 22,700 and 2.89s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was com posed of 220 residues of amino acid: lyre, hiss, arg3, asp20, thr14, ser37, glu12, prol0, g1Y25, ala27, val20, met3, isoleu12, leu12, tyr9, phe2, try3 and amide ammonia16. The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN'

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic a-amy lase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to lIon twenty hour incubation at 30 a C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg++. Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compoundsglycyl-L-prolyl-L-alanine and Cbz-L-tyrosyl-glycinamide, were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -L-valyl-L-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.