B. subtdis NRRL B3411 neutral protease has been extensively purified by solvent and salt fractionation, pigment removal with DEAE-cellulose followed by chromatography on hydroxylapatite, and a final passage through a Sephadex G-100 column. The neutral protease was shown to be homogeneous by disc gel and cellulose acetate electrophoresis, gel filtration chromatography, and ultracentrifugation. The molecular weight w&s determined by osmometry and ultracentrifugation to be about 3842,000 and the amino acid composition and zinc content determined. The general properties of the enzyme, pH-activity relationship, stability, effect of inhibitors, and specificity are discussed. Comparative studies were carried out on the B. subtilas NRRL B3411 and B. sublilis vur. umylosacchuriticus neutral proteases and these enzymes were found to be indistinguishable by the methods used, but quite distinct from the thermostable enzyme thermolysin from B. thermoproteolyticus. ABBREVLATIONS FAGLA-Furylacryloylglycyl Gleucine amide CBZ -Carbobenzoxy DFP -DiisopropyHuorophosphate DEAE -Diethylaminoethyl CM-carboxymethyl P N E -p-nitrophenyl ester TMED -tetramethylethylenediamine 179 possessing similar specificities with respect to the peptide bonds cleaved2J; and (c) alkaline proteases having maximum activity at pH 9.0-11.0, cleaving a wide range of peptide bonds, possessing esterase activity, and unaffected by metal-chelating agents but inhibited by DFP.4,5Microbial enzymes have recently found widespread application in the field of detergents and so far all of the enzymes in use are produced by organisms of the genus Bacillus. The neutral protease may not play as significant a role in detergent applications of the enzymes as the alkaline protease because of the high pH in detergent solutions as well as the relatively poor stability of the neutral protease.6 B. subtilis enzymes have also been shown to reduce dental plaque in humans? and the neutral protease is the more effective agent in this application.The neutral proteases have been less extensively studied than the alkaline proteases. The neutral protease of B. subtilis var. amylo-~a c c h a r i t i c u s~.~ is claimed to be different from the neutral protease of a strain of B subtilis (now registered as strain S R R L B3411)6J0J1 in certain properties. The protease of B. megaterium shows similar general properties and specificity to the B. subtilis neutral protease.12J3The other Bacillus neutral protease which has been studied in any depth is thermolysin, produced by B. thermoproteolyticus, a strain of B. ~tearothermophilus.~~ Thermolysin is quite different from the other neutral proteases having very high thermostability and amino acid composition,15 although its specificity of bond cleavage on both synthetic peptides and B-chain of insulin is ~i r n i l a r .~.~~ A preliminary account of the distinctions between the B. subtilis and B. thermoproteolyticus enzymes has been published.'? This paper gives a detailed account of the isolation of B. subtilis neutral protease by a new method,...

Measurements of the rate of hydrolysis of a series of esters of methyl-phosphonic acid in water have established the rate order Me > Et > Pri > neopentyl in alkaline solution, and Me > Et = neopentyl < Pri in acid solution. Substitution at the phosphorus atom has little effect on the reactivity in acid but reduces the rates considerably in alkaline solution, indicating that the latter reaction normally occurs at the phosphorus atom. The acid hydrolysis of dineopentyl methylphosphonate leads to the formation 2-methylbut-2-ene. This ester therefore reacts by preliminary ionisation of the C-0 bond and the relative reactivities in acid solution suggest that the ethyl and isopropyl esters react similarly.

snmmaryThe alkaline proteases of B. subtilis NRRL B3411, B. pumilis, and B. lichenijormis have been isolated by fractionation followed by ion exchange chromatography and their homogeneity demonstrated. General enzyme properties of the B. subtilis NRRL B3411 alkaline protease have been studied and attempts made to differentiate a group of alkaline protesses. It is clear that the alkaline proteases known as Subtilisins or Subtilopeptidases are not exclusive to B. subtilis but are common to many BaciUi and therefore the generic name Bacillopeptidases has been proposed. It is clear too that on the basis of the effect of pH on activity, amino acid composition, esterase activity, and immunological crossreactions the Bacillopeptidsses can be divided into two groups or types: ( a ) Bacillopeptidase A (Subtilisin A or Subtilopeptidase A) which includes Subtiliiin Carlsberg, B. lichaifonnis, and B. pumilis alkaline proteases; (b) Bacillopeptidase B (Subtiliiin B or Subtilopeptidase B) which includes B subtilis NRRL B3411, Subtilisin Novo, Subtilisin BPN' (Nagarse), alkaline protease Daiwa Kasei, and (probably) B. subtilis oar. amylosacchariticus. At present, no further differentiation is possible and whether or not the enzymes within group A or B are identical remains an open question.Methods for examination of crude enzyme mixtures or fermentation beers are described and from the examination of a number of crude enzymes and fermentation beers it appears that organisms producing Bacillopeptidase A do not produce neutral protease or amylase, while organisms producing Bacillopeptidase B produce a neutral protease and amylase as well. 213

SummaryThe amylase of Bacillus sublilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 607, recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 f 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at 70°C in the presence of 0.01M Ca++ and 0.1 R;I NaCl over a broad pH range from 5.5-9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying a-amylase as determined by analysis of hydrolysis products and immunological studies.

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