Studies on Bacterial Protease. Part XVIII. Proteolytic Specificity of Neutral Protease

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1 [Agr. Biol. Chem., Vol. 31, No. 6, p. 718 `723, 1967] Studies on Bacterial Protease Part XVIII. Proteolytic Specificity of Neutral Protease of Bacillus subtilis var. amylosacchariticus By Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKUMOTO Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka Received February 14, 1967 Substrate specificity of the crystalline neutral protease of B. amylosacchariticus was investigated using the B-chain of oxidized beef insulin as the substrate, and the results were compared with those of proteases obtained from other strains of Bacillus subtilis. The neutral protease split the B-chain at eleven sites of the peptide linkages, indicating the narrow specificity as compared with subtilopeptidase A. The results also indicated that the peptide bonds susceptible to the action of the neutral protease were mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. The enzyme showed potent activities in casein digestion at near neutrality and in milk clotting at ph 5.6, whereas it was completely inert on esters and keratin, and only slightly active toward elastin. INTRODUCTION Our previous papers have reported the purification method" of the neutral protease of Bacillus subtilis var. amylosacchariticus Fukumoto, a strain of saccharifying a-amylase-producing Bacillus subtilis2), and characterized the enzyme as a zinc-metalloprotein.3) It was also shown that physical and chemical properties of the enzyme" were distinct from those of B. subtilis neutral proteases so far reported.5-8) The Abbreviation: DNP, Dinitrophenylated; DNPOH, Dinitrophenol; DNPNH2, Dinitroaniline; DNFB, Dinitrofluorobenzene; 1) D. Tsuru, T. Yamamoto and J. Fukumoto, This journal, 30, 651 (1966). 2) J. Fukumoto, T. Yamamoto and K. Ichikawa, Proc. Japan Acad., 27, 352 (1951). 3) D. Tsuru, H. Kira, T. Yamamoto and J. Fukumoto, This journal, 30, 856 (1966). 4) D. Tsuru, H. Kira, T. Yamamoto and J. Fukumoto, ibid., 30, 1164 (1966). 5) J. D. McConn, D. Tsuru and K. T. Yasunobu, J. Biol. Chem., 239, 3706 (1964). 6) D. Tsuru, J. D. McConn and K. T. Yasunobu, Biochem. Biophys. Res. Comm., 15, 367 (1964); J. Biol. Chem., 240, 2415 (1965) authors thus proposed to name the former enzyme as B. subtilis neutral protease II and the latter as the neutral protease I3,4). In a previous paper", the peptidolytic activity of these two types of neutral proteases was compared on various synthetic peptides, and hippuryl-l-leucineamide was found to be the best substrate among the compounds tested for both enzymes. The paper also showed that these enzymes were completely inert toward several esters of amino acid and low fatty acid which were good substrates for an alkaline protease produced by B. amylosacchariticus9) The present paper describes the specificity of B. subtilis neutral protease II on the B- chain of oxidized beef insulin. The pro- 7) J. Fukumoto and H. Negoro, Proc. Japan Acad., 27, 441 (1951). 8) J. Fukumoto, T. Yamamoto and K. Ichikawa, J. Agr. Chem. Soc. Japan, 32, 230, 233 (1958). 9) D. Tsuru, H. Kira, T. Yamamoto and J. Fukumoto, This journal, 30, 1261 (1966).

2 Studies on Bacterial Protease. Part XVIII 719 teolytic activity on casein, keratin, and milkclotting activity of the enzyme were also examined, comparing with those of other several proteases of various origins. MATERIALS AND METHODS Enzymes B. subtilis neutral proteases I5) and II1) were purified as described previously. The homo geneity was confirmed by their specific activity, rechromatography on DEAE-sephadex A-50 and by some physicochemical methods. A twice crystallized pepsin preparation was the products of Washington Biochemical Co., and partially purified rennin was purchased from Tokyo Kasei Co., Ltd., and used after a further purification by the column chromato graphy on DEAE-sephadex A-50 at ph 5.4*. A purified preparation of aminopeptidase of Bacillus subtilis var. amyloliquefaciens Fukumoto was kindly supplied by Mr. N. Minamiura10). Rhizopus chinensis acid protease purified in a crystalline form by us11) was also used in the experiment. Enzyme activity assay Proteolytic activity of various proteases was assayed at their own optimal ph values by the method described previously1,11) using casein as the substrate. Milk-clotting activity was measured as follows: Skim milk, 9g, was suspended in 100 ml of 2x 10-2 M lactic acid-4x 10-s M calcium chloride solution, ph 5.6, and stirred vigorously for 10 minutes at 30 C using a homogenizer. To 10 ml of the skim milk solution was added one ml of enzyme and the reaction mixture was incubated at 30 C. If the clotting occurred at thirty minutes after incubation, the enzyme was defined to be 10 units as the milk-clotting activity. Keratinase and elastase activities were kindly measured at Shionogi Research Laboratory according to the methods of Sacchar et al.12) and Nickerson et al.15), respectively. The B-chain peptide of oxidized insulin was prepared from beef insulin according to the method described by Hayashi14) and used after a further purification by the column chromatography of 10) N. Minamiura, J. Fukumoto, and T. Yama moto, This journal, 30, 186 (1966). 11) J. Fukumoto, D. Tsuru and T. Yamamoto, ibid., 31, 710 (1967). * Unpublished. 12) L. S. Saccha.r et al., Proc. Soc. Exp. Biol. Med., 90, 323 (1955). 13) W.J. Nickerson, J.J. Noval and R. S. Robinson, Biochim. Biophys. Acta, 77, 73 (1963). 14) H. Hayashi, Seikagaku, 32, 411 (1960). Sephadex G-50 equilibrated with 10-3M sodium acetate. Pauly reagent was prepared by the method of Smith15) and applied on paper chromatograms to detect peptides containing histidine or tyrosine. Identification of DNP-amino acids was performed by two-dimensional paper chromatography using the solvent systems of toluene-pyridine-ethylene chloro hydrin-ammonia and 1.5 M phosphate buffer, ph 6.016). For the determination of amino acid, paper chromato graphy was also carried out with the solvent systems of butanol/acetic acid/water (4:1:2) and phenol/water (3:1) on Toyo Roshi No. 50 filter paper and, after color development by the ninhydrin reaction, the amounts of each amino acid were quantitatively estimated with an automatic recording densitometer, Toyo Densitol. RESULTS AND DISCUSSION I. Proteolytic Activity on Casein, Elastin, and Keratin, and Milk-Clotting Activity of Neutral Protease. Proteolytic activity of neutral protease II TABLE I. VARIOUS PROTEOLYTIC ACTIVITIES OF SEVERAL PROTEASES * Prepared from the culture broth of B. amylosac chariticus. ** Partially purified preparation. *** Highly purified preparation. (PU): Proteolytic activity toward casein at their own optimal ph values. /: Not checked 15) E. Smith, "Chromatographic Techniques," William Heinemann Medical Books Ltd., London, (1958), p ) H. Fraenkel-Conrat, J. I. Harris and A. L. L evy, "Methods of Biochemical Analysis," Academic Press Inc., New York, 2, (1955), p. 359.

720 Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKUMOTO on casein, elastin and keratin, and the milkclotting activity were examined, comparing with those of several proteases obtained

3 720 Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKUMOTO on casein, elastin and keratin, and the milkclotting activity were examined, comparing with those of several proteases obtained from various sources. The results are summarized in Table I. The neutral protease II was found to be much more active than several other proteases in casein digestion activity at their optimal ph values. This was also the case in milk-clotting activity at ph 5.6. However, the enzyme showed only a slight activity on elastin and was completely inert on keratin, in sharp contrast with B. subtilis alkaline proteases which showed high for the second run. The papers were dried at room temperature before staining with ninhydrin. One of the the sheet prepared as the marker of peptide maps was sprayed with 0.2 % solution of ninhydrin in watersaturated butanol and heated at 100 C for five minutes to bring the full color development of ninhydrin reaction. Peptide map of the digestion products is diagrammatically re presented in Fig. 1. After disappearance of proteolytic activity on both substrates. Ac cording to the experimental results of Mori hara17), a purified preparation of keratinase obtained from Streptomyces fradiae revealed activities of 80 units on keratin and 100 units on elastin per mg of the enzyme under the same condition. On the other hand, Strep. fradiae elastase was reported to show 700 units of elastase activity per mg protein. II. Specificity of Neutral Protease II on the B-chain of Oxidized Insulin. Digestion of the B-chain of oxidized insulin by the enzyme and the fractionation of peptides were carried out as follows: About 10 mg of the B-chain was dissolved in 0.2 ml of 10-2m Tris-HC1 buffer solution, ph 7.2, which contained 250ƒÊg of the enzyme and incubated for twenty-four hours at 37 C under toluene. The digestion was stopped by placing the tube in a boiling water-bath for ten minutes and the mixture was evaporated to dryness under vacuo. Dried digests were dissolved in a minimum quantity of water, spotted on four sheets of Toyo Roshi No. 50 filter paper and subjected to the twodimensional paper chromatography by the descending method to separate the resulted peptides. The solvent systems used were butanol/acetic acid/water (4:1:2 by vol.) for the first run and butanol/pyridine/water (1:1:1) 17) K. Morihara, T. Oka and H. Tsuzuki, The 18th Symposia on Enz. Chem. Japan, 238 (1966). FIG. 1. Peptide Map of Digestion Products of Oxidized Insulin B-chain by Neutral Protease II. text. The experimental details are described in the The black and dotted spots indicate peptides containing histidine and tyrosine, respectively. ninhydrin reaction, the paper chromatogram was stained with the Pauly reagent to detect the peptides containing histidine or tyrosine as shown by shadowed spots in Fig. 1. About 15 peptides were separated by the paper chromatography and 8 of them gave a positive reaction with the Pauly reagent. The other sheets which were prepared for elution of peptides and subsequent amino acid analysis were sprayed with 0.02 % ninhydrin solution in acetone, heated for five minutes at 50 C and allowed to stand overnight in the dark. Comparing with the peptide map of strongly stained sheets (Fig. 1), areas around the faintly stained spots on the papers were cut off from the sheets and

4 Studies on Bacterial Protease. Part XVIII 721 dipped into acetone solution to remove excess ninhydrin. The paper strips were then eluted with 1.5 ml of water and the eluates were brought to dryness in a vacuum desiccator. The eluates of identical spots from 4 to 8 sheets were pooled and used for the analyses of amino acid composition and amino-terminal amino acid. An aliquot of each peptide was hydrolyzed by 6 N hydrochloric acid in a sealed evacuated tube for twenty-four hours at 110 C, and, after removal of hydrochloric acid in vacuo, the hydrolyzate was subjected to paper chromatography as described above. The quantitative analysis of each amino acid was carried out using a Toyo Densitol after staining with ninhydrin. A part of the peptides, on the other hand, was digested by the aminopeptidase of B. subtilis to identify the amino-terminal amino acid. About one mg of each peptide was dissolved in 0.05 ml of 5 x 10-3 M Tris HCl-2 x 10-3M MnCl2, ph 7.5, containing an appro priate quantity of purified B. subtilis' amino peptidase free from protease activity, followed by the incubation at 30 C. An aliquot of the reaction mixture was periodically removed and the liberated amino acids were identified by paper chromatography. In order to identify again the amino -terminal amino acid by the Sanger's DNP method, another part of the pooled peptides was dissolved in 0.1 ml of 1 % trimethylamine solution and mixed with 0.2 ml of 5 % 2, 4- TABLE II. AMINO ACID COMPOSITION AND AMINO-TERMINAL AMINO ACID OF PEPTIDES SEPARATED BY PAPER CHROMATOGRAPHY. * P: Determined by the aminopeptidase method. D: Determined by the DNP-method. ** Supposed to be derived from contaminated peptides or amino acids, because of the low contents. Peptide No. used here is that shown in Fig. 1 and peptide No. 4 was found to be contaminated with another peptide, No. 4'. /: Not detected. -: Pauly reaction negative.

5 722 Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKUMOTO dinitrofluorobenzene solution in ethanol. The mixture was incubated at 30 C for four hours in the dark. To the reaction mixture was added 0.1ml of 1 % trimethylamine solution and the unreacted DNFB was removed by extraction three times with 2 ml portions of ether. The aqueous solution was evaporated to dryness in vacuo and the residue was hydrolyzed with 0.2 ml of 6 N hydrochloric acid for eight hours at 105 C in a sealed evacuated tube. The hydrolyzate was diluted to 1.5 ml with deionized water, extracted four FIG. 2. Paper Chromatogram of DNP-Amino Acids Obtained from DNP-Peptides Mixture. A: tyrosine; B: DNPNH2; C: phenylalanine; D: DNPOH; E. glycine; F: leucine; G: valine; H: alanine; I: serine; J: threonine; K: glutamic acid; L: aspartic acid; times with 2.5ml of ether and three times with 2ml portions of ethyl acetate. Etherand ethyl acetate extracts were, respectively, washed twice with 2ml of deionized water and evaporated to dryness in vacuo. After sublimation for removal of dinitrophenol, the DNP-amino acid fractions were applied on Toyo Roshi No. 51 filter paper (40 x 40cm) and chromatographed two-dimensionally using the solvent system described above14). Dinitrophenylation was also carried out directly on the digestion products before separation to each peptide. As shown in Fig. 2, the DNP-amino acids obtained from the hydrolyzate of DNP-peptides mixture were valine, leucine, phenylalanine, tyrosine, alanine, glycine serine, and aspartic acid in a decreasing order, together with trace amounts of glutamic acid and threonine. A summary of the analytical results and the probable structure of peptides obtained from the B-chain are listed in Table II and Fig. 3. The substrate specificity of the neutral protease I was also investigated by the same method as described above and almost similar peptide map was obtained except that the other three spots appeared, accompanying by the disapparence of peptides Nos. 5, 7 and 8, as shown in Fig. 4. From the analyses of amino acid composition and FIG. 3. Possible Amino Acid Sequence of Peptides Separated from Digestion Products of Oxidized Insulin B-Chain by Neutral Protease II.

6 Studies on Bacterial Protease. Part XVIII 723 FIG. 4. Peptide Map of Digestion Products of Oxidized Insulin B-Chain by Neutral Protease I. Spots A, B and C indicate peptides which were not found in digests by neutral protease II. amino-terminal amino acid (Table II), the possible amino acid sequence of each peptide was deduced*. In Fig. 5, peptide bonds of oxidized insulin B-chain susceptible to the proteolytic action of the neutral proteases I and II are indicated by arrows. The result obtained with another B. subtilis protease, subtilopeptidase A, is also shown there.", 19) The solid arrows designate the major sites of the enzyme attack while the broken arrows show bonds that were also cleaved but were found sometimes intact. The neutral proteases I and II of B. subtilis were thus found to show minor differences in their proteolytic specificity on the B-chain of oxidized insulin, while to be quite different from subtilopeptidase A. The former two enzyme revealed narrower specificity than the latter. The results presented here also indicate that the peptide bonds susceptible to the action of the neutral protease are mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. Acknowledgements Our thanks are due to Dr. K. Morihara for his courtesy in the assay of keratinase and elastase activity, and to Dr. K. Tomoda for his kind supply of the two types of proteases used in the experiment. FIG. 5. Amino Acid Sequence of B-chain of Oxidized Beef Insulin and the Possible Site of Hydrolysis by B. subtilis Proteases. * The possible amino acid sequence of peptides A, B and C in Fig. 4 was deduced to be AspN-G1uN- His-Leu-Cys-Gly-Ser-His, Ala-Leu-Tyr and Leu-Val- Cys-Gly-Glu-Agr, respectively, from their amino acid analyses and amino-terminal amino acid determina tion. 18) H. Tuppy, Monatsch., 34, 996 (1953). 19) R. L. Hill, Adv. Prot. Chem., 20, 37 (1965).

By Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKIIMOTO

By Daisuke TSURU, Heizo KIRA, Takehiko YAMAMOTO and Juichiro FUKIIMOTO [Agr. Biol. Chem., Vol. 30, No. 12, p. 1261 `1268, 1966] Studies on Bacterial Protease Part XVI. Purification, Crystallization and Some Enzymatic Properties of Alkaline Protease of Bacillus subtilis var.