STUDIES OF AN ENZYME PRODUCED BY BACILLUS FULMINANS

STUDIES OF AN ENZYME PRODUCED BY BACILLUS FULMINANS THAT INACTIVATES BLOOD GROUP 0 SUBSTANCE1 INGEBORG NAYLOR AND HAROLD BAER Department of Microbiology, Tulane University School of Medicine, New Orleans, Louisiana Iseki and Tsunoda (1952) isolated an organism, identified as Bacillus fulminans, an extract of which was capable of specifically inactivating blood group 0 substance. A recent report by Yosizawa (1957) showed that the enzyme liberated fucose as a result of its action. The present study provides an improved procedure for the isolation and purification of this enzyme, a description of some of its properties, and information relating to its mode of action. MATERIALS AND METHODS B. fulminans, according to the 6th edition of Bergey's Manual of Determinative Bacteriology (Breed et al., 1948) (this organism is not referred to in Vol. I of the 7th edition), is a variety of Bacillus cereus and, using the scheme devised by Smith et al. (1952), the organism which we obtained from Dr. Iseki proved to be a strain of B. cereus; two strains of B. cereus from our stock collection did not, however, produce the blood group 0 inactivating enzyme. Our preparations of blood group 0 substances were isolated from saliva, human ovarian cyst fluid, porcine stomach linings, and from commercial porcine gastric mucin by the phenol extraction method of Annison and Morgan (1952); the isolation procedures and properties of the substances are described in a previous publication by Baer et al. (1954). When saliva was to be tested, it was collected over a period of not more than ½ hr, then, unless used immediately, frozen and maintained at -20 C. For use, the saliva was rapidly thawed and centrifuged at 2 to 6 C to effect clarification. Blood group 0 activity was determined by the test of inhibition of hemagglutination, employing either high titered normal chicken serum or an extract of Ulex europeus seeds as sources of group 1 Supported by research grant 2964 of the National Institute of Allergy and Infectious Diseases, Department of Health, Education, and Welfare. Received for publication December 5, 1958 O red blood cell agglutinins. A mixture of 0.1 ml of a saline solution of blood group 0 substance and 0.2 ml of anti-o reagent were incubated at 37 C for ½ hr and then 0.1 ml of 4 per cent saline suspension of human group 0 cells added. Following a further ½f hr of incubation, the tubes were centrifuged for 1 to 2 min at about 1000 rpm and, after gentle resuspension of the cells, the presence of agglutination was ascertained, usually at 30 X magnification. Blood group A and B activity was determined similarly using commercial typing serum as a source of antibody. Nitrogen was determined by the modified Kjeldahl procedure of Hiller et al. (1949). Fucose was determined by the method of Dische and Shettles (1951). Proteolytic activity was determined by placing a drop of about 0.01 ml of solution on the surface of a 0.4 per cent gelatin agar plate, incubating at 37 C overnight, and then flooding the plate with a solution containing HgCl2, 15 g; concentrated HCl, 20 ml; and water, 100 ml. This procedure was adapted from Smith et al. (1952). Paper chromatography was done on Whatman no. 1 paper by the ascending method, the solvent being butanol-ethanol-water (4: 1: 1). Ammoniacal silver nitrate and aniline phthalate sprays were employed to detect reducing substances, and ninhydrin was used to reveal the presence of amino acids and amino sugars. To isolate the enzyme, the bacteria were grown for about 24 hr on heart infusion agar and the growth removed with cotton swabs; care was taken not to prolong the time of incubation because excessive spore formation reduced the yield. Although Iseki and Tsunoda (1952) did not supply details for the isolation, the following procedure was found to give an active preparation. The growth from 15 petri dishes was suspended in 30 ml of 0.066 M phosphate buffer, ph 7.0, and incubated at 37 C for 24 hr. Following centrifugation to remove debris, 13.7 g of ammonium sulfate was added and, after 24 hr at 4 C, the precipitate 771

772 NAYLOR AND BAER [VOL. 77 that formed was dissolved in 10 ml of phosphate buffer, ph 7.0, then dialyzed in the cold against distilled water to eliminate all ammonium sulfate. The addition of an equal volume of acetone resulted in a precipitate which was separated by centrifugation and dissolved in 10 ml of phosphate buffer, ph 7.0. This solution, which inactivated blood group 0 substance, was employed as a crude enzyme preparation to study some of its properties and as the starting material for further purification procedures. Three different batches of this type had total nondialyzable solids varying from 11 to 26 mg with the nitrogen content ranging from 7 to 17 per cent. These values were based on the use of about 1 g of dry bacterial solids as starting material. The enzyme was tested for activity at varying temperatures and at varying ph. To determine the effect of ph, enzyme solutions were dialyzed in the cold against 0.066 M phosphate buffers in the ph range 2.0 to 8.0, dialysis being carried out for several days with daily change of buffer. The effect of various inhibitors was also determined including some well-known enzyme inhibitors and certain sugars which are either components of blood group substances or closely related sugars. Since efforts to produce the enzyme by growth of the organisms in heart infusion broth failed to yield active preparations, various sugars were added to this base medium to determine whether the enzyme was adaptive. RESULTS Upon testing the enzyme for activity at varying temperatures, it was found to be slowly active at 6 C and still active after heating to 70 C for 10 min or 75 C for 5 min; heating to 85 C for 10 min almost completely destroyed enzymatic activity. Studies with inhibitors showed that sodium azide, 1 mg per ml and Versene, 12.5 mg per ml, could completely inactivate the enzyme, whereas potassium fluoride at 1 mg per ml and phenol at 2 mg per ml had no effect. In attempting to adjust the ph of enzyme solutions by dialysis against buffers of varying ph, it was found that no change in the enzyme occurred until the ph was lowered to below 5.0 when a precipitate formed. The precipitates were removed by centrifugation, the sediment dissolved in buffer, ph 7.0, and the supernatant adjusted to this ph with 1 N NaOH. All solutions were then analyzed for their nitrogen content and TABLE 1 Purification of crude enzyme from Bacillus fulminans by dialysis against 0.066 M phosphate buffers of varying ph ph 2.0 2.5 2.9 3.6 3.9 4.6 5.0 5.6 6.0 6.6 N Mg 168 176 188 188 176 132 60 28 16 12 Precipitate Activity* 4t + I~~~~~~ Supernatant N l9b 195 165 180 150 165 235 300 320 355 370 Activity* + + + * Activity is measured as the ability to partially or completely inactivate 25,g of porcine blood group substance in 15 min at 37 C. t This material cotuld not inactivate 25,ug, cotuld partially inactivate 12.5,ug, and completely inactivate 6,g. their ability to inactivate hog blood group 0 substance. The results appear in table 1. It will be seen that a precipitate appeared rather abruptly as the ph was lowered but that continued reduction in ph failed to induce any significant increase in the quantity of precipitate and only about ' of the nitrogen content was precipitable by this procedure. Although the activity was retained in the precipitate at ph 3.9, it disappeared sharply below ph 3.6 and then could not be found in either the precipitate or supernatant. It was at first assumed that a portion of the enzyme essential for activity had been lost by dialysis but the adjustment of an enzyme solution to ph 3.0 to produce a precipitate and then readjustment to 7.0 resulted in a solution devoid of enzymatic activity. This was true even if the solution readjusted to ph 7.0 was permitted to stand overnight. Precipitation of the crude enzyme by dialysis at ph 3.9 was therefore employed as a purification procedure, with some success, as established by the following means. With the crude enzyme solution, an amount containing 80 to 100 /ug of N was required to inactivate 50,ug of blood group substance in 15 min at 37 C although purified material containing only 5 to 10,ug of N had

1950] BACTERIAL ENZYME AND BLOOD GROUP 0 SUBSTANCE 773 equal activity. Secondly, the crude enzyme possessed some blood group A activity, presumably arising from the meat extract of the medium, whereas the purified material did not demonstrate this activity. The crude material also had a strong tendency to hemolyze erythrocytes whereas the purified material was relatively inert in this respect. Finally, the crude enzyme showed strong proteolytic activity as evidenced by its action on gelatin whereas the purified enzyme had only a trace of this activity. Inspection of table 1 shows that at ph 4.6 there was a precipitate of material that was inactive, the activity remaining in the supernatant, whereas at ph 3.9, the precipitate possessed the activity with none in the supernatant. By dissolving the precipitate obtained at ph 3.9 in phosphate buffer, ph 7.0, then adjusting the ph to 4.6, an inert material was, in fact, precipitated; further dialysis of the supernatant against ph 3.9 buffer then precipitated the active material. By this procedure an additional increase in purity of about twofold was obtained as established by determination of ratios of nitrogen to activity. Because organisms grown in heart infusion broth failed to yield an active enzyme preparation and since the only apparent difference between the broth and the agar medium was the agar, it was hypothesized that the difference might be attributable to the fact that agar and the blood group substance are both galactosecontaining polysaceharides, which further suggested that the enzyme was adaptive. Consequently, various sugars were added to the broth and galactose, fucose, and melibiose were found to provoke enzyme formation by the bacteria, whereas glucose, glucosamine, and lactose did not. Organisms grown in 1 per cent galactose heart infusion broth provided an excellent yield of enzyme. Almost all of the work involving the isolation and purification of the enzyme employed porcine substance from gastric mucin as the test substrate; this substance possesses both blood group O and A activity and since only the 0 activity is destroyed it serves as both a test and control. Table 2 provides a list of additional antigens tested. Not only the mucin OA substance, which is a mixture of 0 and A substances, but also substances 2 and 7 from individual porcine stomach linings lost only the 0 activity, the A activity remaining unaffected. Blood group 0 TABLE 2 Effect of an enzyme from Bacillus fulminans on various blood group substances Ability of Enzyme Treated SrlgclMaterial to Substance Serological Inhibit Anti-A Anti-O Hog (from gastric muicin). OA + Hog 2... OA + - Hog 7 G...OA +_ Cyst 4 phenol sol... Cyst 9 phenol sol... A + Saliva (U. R.)... Saliva (I. N.)... A + substance derived from human ovarian cyst fluid lost its activity, whereas A substance of similar origin suffered no change; the same result was obtained when whole saliva from group 0 and A secretors was the substrate. The enzyme could also eliminate the agglutinability of group 0 cells when these were tested with either chicken anti-o or Ulex extract; the agglutinability of erythrocytes of group A, B, or AB by anti-a and anti-b sera was unaltered subsequent to the exposure of these cells to the enzyme. Since blood group A, B, and 0 substances have, qualitatively, the same composition, it was important to determine the portion of the blood group substance attacked by the enzyme. Samples of blood group substance were reacted with the enzyme until the serological reactivity of the 0 substances had completely disappeared; as controls, blood group 0 substance was mixed with enzyme inactivated by boiling for 5 min, and blood group A substance was treated with active enzyme. The materials were dialyzed against distilled water, concentrated by vacuum distillation, and analyzed by paper chromatography. Only when group 0 substance was reacted with the enzyme was any spot detected and this corresponded to fucose. Analysis of the enzymetreated blood group 0 substance showed it to contain only about 80 per cent of the original fucose. Another procedure employed to elucidate the mechanism of action was to attempt to inhibit the action of the enzyme by various sugars in a manner analogous to the enzyme inhibition car-

774 NAYLOR AND BAER [VO L. 77 TABLE 3 Inhibition of an enzyme from Bacilluts fulmltinans by various sugars SugarsTestedCInciitr lowest Maximal Conc Noninhibitory Coc Sugars Sugars Tested InhiLowest Tested for L-Fucose... 0.025 D-Galatose.0.1 I)-GalactosamineHC1 0.1 Lactose...... 0.5 L-Arabinose... 0.5 Melibiose... 0.5 D-GTlucose 0....5 D-Xylose... 0.5 D-Mannitol... 0.5 L-Rhamnose... 0.5 D-Glucosamine HCI... 0.5 N-Acetylglucosamine... 0.5 ried out by Watkins and imorgan (1955). The sugars employed, their concentrations and the results are given in table 3. Enzyme-sugar mixtures were allowed to remain at room temperature (approximately 26 C) for 3 hr, then porcine blood group substance was added and the mixture permitted to remain at room temperature overnight. The ability of the mixture to inhibit chicken anti-o was then determined. Ulex extract was not suitable for this purpose since its ability to agglutinate group 0 cells was inhibited by fucose. None of the sugars tested inihibited the chicken anti-o. DISCUSSION The procedure outlined by Iseki and Tsunoda (1952) yielded us a preparation from B. fulminans that inactivated blood group 0 activity but not A or B. An additional 20-fold purification could be obtained, based on the ratio of nitrogen to activity, merely by dialysis first against phosphate buffer at ph 4.6 then at ph 3.9 since the enzyme seemed to precipitate rather sharply at the latter ph. At a lower ph, both the precipitate and supernatant were inactive; one possible mechanism for this inactivation might be splitting of the enzyme into protein and coenzyme or metal. Inactivation by Versene would seem to point to a metal-containing enzyme. However, raising the ph to redissolve the inactive precipitate obtained at ph 2.5 failed to effect reactivation and the addition of a number of metal ions M M such as Ca, Mfg, Mn, Cu, and Fe failed to restore activity. Consequently, the nature of the inactivation at low ph is not yet settled. Reaction with various blood group-containing substances clearly demonstrated the specificity of the enzyme. Only group 0 activity was eliminated, whether of porcine or human origin, whether purified or contained in whole saliva or as part of an erythrocyte. The great selectivity appears to be due to attack on a fucose linkage since this was the only recognizable substance split off during the action of the enzyme on purified blood group 0 substance. This would also appear to be the only definite piece of evidence pointing to the inactivation as an enzymatic process rather than a selective inhibition of activity. It was confirmed by analysis of the inactivated, nondialyzable portion of porcine or human blood group 0 substances, indicating that only about 80 per cent of the fucose was still present. This, in general, agrees with the findings of Yosizawa (1957) who found that about 50 per cent of the fucose was removed from blood group O substance by the enzyme. Thus, blood group O substance must possess fucose in at least two types of linkage, part of which is enzyme-sensitive and part enzyme-resistant. Two types of experiments undoubtedly relate to the nature of the fucose linkage, i. e., enzyme inhibition by sugars and the stimulation of adaptive enzyme formation by sugars. Enzyme inhibition experiments showed that fucose, and possibly galactose and galactosamine are involved in activity; glucosamine (loes not seem involved. The activity of arabinose and the inactivity of xylose and glucose would seem to confirm the importance of the galactose-type of configuration. Stimulation of enzyme production by fucose and galactose (galactosamine was not tested because the quantity required made the test prohibitively expensive) and the failure of glucosamine and glucose in this respect lend added significance to the enzyme inhibition results. Yosizawa (1957) also tested the inhibitory effect of sugars on the enzyme but due to the low concentrations that he employed he found only fucose to be inhibitory. The disaccharides lactose and melibiose both inhibited enzyme action, whereas only melibiose stimulated the adaptive enzyme formation. Since glucose neither inhibited enzyme action nor provoked enzyme formation it may be assumed that this portion of the disaccharide was inert and,

1959] BACTERIAL ENZYME AND BLOOD GROUP 0 SUBSTANCE 775 therefore, the important difference between the disaccharides lies in the fact that one is an a-dgalactopyranoside and the other a f3-d-galactopyranoside; the fact that the a-d form inhibits the enzyme and stimulated its formation may indicate that it is the linkage important for the specificity of the blood group 0 substance. These results parallel to some extent those of Watkins and Morgan (1955) employing an enzyme from Trichomonas foetus since this 0 inactivating enzyme was also inhibited by fucose and galactosamine; however, T. foetus enzyme was essentially unaffected by lactose and melibiose. SUMMARY The blood group 0 inactivating enzyme produced by Bacillus fulminans has been partially purified and some of its properties ascertained. It is an adaptive enzyme, produced when the organism is grown in the presence of galactose, fucose, and melibiose. The action of the enzyme is inhibited by fucose, galactose, galactosamine, arabinose, melibiose, and lactose. Part of the fucose of 0 substance is liberated when the latter reacts with the enzyme. REFERENCES ANNISON, E. F. AND MORGAN, W. T. J. 1952 Studies in immunochemistry. II. The isolation and properties of the human blood group H substance. Biochem. J., 52, 247-258. BAER, H., BRINGAZE, J. K., AND McNAMEE, M. 1954 The immunochemistry of blood group 0. I. The production of precipitating and agglutinating antibodies in chickens to various human and hog blood group substances. J. Immunol., 74, 67-80. BREED, R. S., MURRAY, E. G. D., AND HITCHENS, P. A. 1948 Bergey's manual of determinative bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore. DISCHE, Z. AND SHETTLES, L. B. 1951 A new spectrophotometric test for the detection of methyl pentose. J. Biol. Chem., 192, 579-582. HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D. 1949 A study of conditions for Kjeldahl determination of nitrogen in proteins. J. Biol. Chem., 176, 1401-1419. ISEKI, S. AND TSUNODA, S. 1952 On a bacterial enzyme which specifically decomposes 0 substance. Proc. Japan Acad., 28, 370-373. SMITH, N. R., GORDON, R. E., AND CLARK, F. E. 1952 Aerobic spore forming bacteria. U. S. Dept. Agr., Monograph No. 16. WATKINS, M. W. AND MORGAN, W. T. J. 1955 Inhibition by simple sugars of enzymes which decompose the blood-group substances. Nature, 175, 676-677. YOSIZAWA, Z. 1957 Biochemical studies on carbohydrates. CXCV. Digestion of group 0 and AO mucopolysaccharides from pig stomach mucus by the 0 enzyme from B. fulminans. T6hoku J. Exptl. Med., 65, 175-185.