Microb. Ecol.7:331-341(1981)
hIICI~OBI~ILECOLOGV
Activity of Bacteriolytic Enzymes Adsorbed to Clays Guno Haskfi* Department of Microbial Ecology, University of Lund, Helgonav~igen 5, S-223 62 Lund, Sweden
Abstract. Myxococcus virescens is able to produce extracellular bacteriolytic enzymes that are rapidly adsorbed on montmorillonite. These adsorbed enzymes are active and can be assayed by measuring the release of UV-absorbing materials in mixtures containing Micrococcus luteus cells. The activity of the clay-adsorbed enzymes is, however, considerably lower than that of the unadsorbed enzymes. Both unadsorbed and adsorbed enzymes have their maximum activity at approximately the same pH. At lower clay-enzyme concentrations, the activity is proportional to the concentration. If, however, increasing amounts of clay are added to a fixed volume of clay-enzyme suspension, the activity remains almost unchanged until a definite limit is reached, then the activity decreases rapidly. This limit was dependent only on the ratio of the amounts of enzyme and clay and not on the absolute concentration of the enzyme. The montmorillonite-adsorbed bacteriolytic enzymes from M. virescens were not active against gram-negative bacteria, and no activity against purified cell walls from M. luteus could be measured. Montmorillonite-adsorbed egg white lysozyme was not active on M. luteus cells.
Introduction Some myxobacteria can lyse other bacteria and feed on the released nutrients. They produce extracellular bacteriolytic enzymes that break bonds in the cell wail of other bacteria. These enzymes are rapidly adsorbed on soil particles, especially on clay materials [8]. The bacteriolytic enzymes can be desorbed if the pH or ionic strength is increased, but if the myxobacteria are to be able to feed on other living bacteria in nature, the bacteriolytic enzymes must be active in the adsorbed state. This paper deals with experiments concerning the activity of clay-adsorbed bacteriolytic enzymes from
Myxococcus virescens.
Materials and Methods Enzyme-Clay Suspension A cell-free culture solution was obtained from M+ virescens as previously described [5, 8]. It was stored at - 2 5 ~ until used. This solution (250 ml) was mixed with a montmorillonite suspension (17 ml, 300 rag) with the aid of a magnetic stirrer at 23~ [8]. After an hour, the suspension was centrifuged at 48,000 x g for 12 min. *Present address: Swedish Sugar Co. Ltd., Research Laboratories, Box 6, S-23200 Arl~Sv, Sweden.
0095-3628/81/0007-0331 $02.20 9 1981 Springer-Verlag New York Inc.
332
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The clear solution contained no bacteriolytic activity. The enzyme-montmorillonite particles were washed once with 35 ml of distilled water, recentrifuged, and then suspended in 0.05 M phosphate buffer, pH 7.5, to a volume of 25 ml. After centrifugation of the washed clay-enzyme particles, the supematant did not contain any enzyme activity. This standard enzyme-clay suspension was stored at 4~ and used for experiments during 2 weeks. Only a minor decrease in activity could be observed during that time. The cell-free culture solution contained 5.72 mg protein/nil. Of this protein content, the three bacteriolytic enzymes represented only 0.42 #g/ml, and the alanyl-lysyl endopeptidase was the main part (0.34/ag/ml). At the montmorillonite 36% of the proteins and 100% of the bacteriolytic enzymes were adsorbed. The standard enzyme-clay suspension thus contained 12 mg clay/mi, resulting in !.72 mg protein/mg clay and 0.35/,tg bacteriolytic enzymes/rag clay. in experiments on the effects of different clay concentrations, a standard montmorillonite suspension was used. It contained the same amount of clay (12 mg/ml) as the standard enzyme-montraorilonite suspension. The previously [7] purified alanyl-lysyl endopeptidase (enzyme 111)was used in some experiments either as a solution or as a montmorillonite-adsorbed suspension (0.28 /ag enzyme/mg clay). The enzyme d a y suspensions were made in the same buffer and pH and with the same amount of clay and the same enzyme activity as the clay-adsorbed unpurified enzymes described above. " Although great care was taken to standardize all handling with the enzyme suspensions, some smaller differences between different suspensions could not be avoided, and therefore different experiments are not always totally comparable.
Enzyme Assay For determination of the activity of nonadsorbed bacteriolytic enzymes, the method previously described was used [9]. This method is based on turbidity measurements in suspensions of intact cells ofMicrococcus luteus. It cannot, however, be used in montmorillonite suspensions, as the bacterial cells are precipitated when mixed with the clay. Instead, the act~.vity of clay-adsorbed enzymes was assayed by the ability to produce nonadsorbed UV-absorbing material. In the standard assay, a suspension ofM. luteus cells in Tris-HC 1 buffer, pH 8.0, was used. The concentration of cells was the same as in the previously used method (0.36 mg/ml). A suspension of clay-adsorbed enzymes was mixed in centrifuge tubes to a total volume of 10 ml and at a final concentration of 0.05 M Tris-HCI. The tubes were incubated at 37~ in a shaker for 1 h, cooled, and centrifuged at 48,000 • g for 12 min. The absorbancy of the clear solution was determined at 280 nm in a Beckman DB-GT Spectrophotometer (10 mm cell). In the figures, the values are corrected for blanks without any addition of enzymes. This was the standard assay used, unless otherwise stated. The UV-absorbing material produced by the action of the clay-adsorbed enzymes was adsorbed to the montroorillortite to a very little extent. Less than 5% was adsorbed when 2 mg clay was added per ml of an enzyme-digested cell suspension.
Analytical Methods The procedures for the determination of reducing sugars and free amino groups were principally those described by Ghuysen et al. [3].
Protein Assay Protein content in the solutions was determined by the method of Lowry et al. [ 1 ! ], with bovine serum albumin as standard.
Results
1. Bacteriolytic Activity of Adsorbed Enzymes This experiment was performed to determine the activity of clay-adsorbed bacteriolytic enzymes from Myxobacteria and to investigate differences in activity between adsorbed and nonadsorbed enzymes.
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File. 1. Effect of time on the bactcriolysisofM. tuteuscells by bactcriolyucenzymes from M. vireseens. The cells were degraded by a suspension of unpurified and unadsorbed enzymes (o), of montmoriiionite-adsorbed enzymes (o), and of desorbed and purified enzymes (-).
To four flasks, each containing 50 ml of the M. luteus cell suspension in 0.05 M Tris-HCl buffer, pH 8.0,250/.tl of the following solutions was added: flask I, a cell-free culture solution from M. virescens (180 BEU/ml, cf [5]); flask 2, the same volume of the culture solution adsorbed on 3.0 mg montmorillonite according to the standard method; flask 3, a solution of purified alanyl-lysyl-endopeptidase (enzyme UI) corresponding to that amount (0.085 #g) which can be purified from 250/zl culture solution by adsorption on and desorption from 3.0 mg montmorillonite [8]; and flask 4, a montmorillonite suspension (3.0 mg) used as a blank. Figure 1 shows that the clay-adsorbed enzymes were active on M. luteus cells. However, they were not as active as the corresponding amount of culture solution and not even as active as the enzymes which can be desorbed from the same amount of clay. These differences might be due to several reasons. Since the activity increases when the enzymes are eluted, there must be some kind of inhibition of the activity when the enzymes are adsorbed. The clay-adsorbed bacteriolytic enzymes and the eluted enzymes reached after 6 h the same level of absorbance, which indicates that the desorbed enzymes are the same as those which are active in the clay-enzyme complex. However, the ability of these enzymes to degrade bacterial cell walls is not equal to that of the culture solution. This unpurified solution contains several bacteriolytic and proteolytic enzymes that can work synergetically on the peptidoglycans.
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2. Activity at Different pHs The same enzyme suspensions as in experiment 1 were used. The concentrations were chosen so that the activity at pH 8.8 would be about the same. The enzyme activities were measured with the standard assay but with a universal buffer [19] instead of phosphate buffer. This buffer consists of phosphate, citrate, and borate salts in water and has a nearly constant ionic strength (0.07-0.10M) over a wide pH range. The activity peak of the adsorbed enzymes was about the same as that for the unadsorbed enzymes with respect to pH (Fig. 2). These results do not agree with those concerning pure proteinases [14] and pure chitinases [16]. The activity peak of adsorbed proteinases and chitinases was greater by 1.0 pH unit than that for the unadsorbed enzymes. The clay-adsorbed bacteriolytic enzymes had less activity than the unadsorbed enzymes at low pH. At high pH, there was no difference between the three activity curves, probably because the bacteriolytic enzymes are desorbed from the clay at a pH above 9 [8].
Clay-AdsorbedEnzymes
335
3. Activity at Different Concentrations of Clay Different volumes (0--70 #1) of the standard montmorillonite suspension were mixed with 4 /al of the enzyme-montmorillonite suspension. The activities were assayed according to the standard procedure. Although 4 tzl of the enzyme-clay suspension gave an absorbance proportional to the volume of the enzyme-clay suspension, the addition of increasing amounts of montmorillonite decreased the activities (Fig. 3A). This may have resulted from an increasing adsorption of degradation products on the clay particles, but the addition of more montmorillonite to the centrifuged, clay-free reaction products did not significantly change the absorbance in comparison with the addition of the same volume of water. Consequently, the lower activity with higher amounts of montmorillonite indicates that the activity is inhibited in one way or another. In Fig. 3B, the absorbance is plotted against the ratio of the amounts of enzymes and of clay. When this ratio was decreased from 1.72 mg protein/mg clay, as in the stock enzyme-montmorillonite suspension, the absorbance did not change until the ratio fell below a value of about 0.6 mg/mg. To determine whether this was also valid for different amounts of enzymes, the next experiment was performed.
4. Activity at Different Enzyme/Clay Ratios In three series of tubes 5, 10, and 20/zl of the enzyme-montmorillonite suspensions was used. Different volumes of the standard montmorillonite suspension were added to the tubes to obtain final enzyme/clay ratios between 0.09 and 1.72 mg/mg clay. In a fourth series, M. luteus cells were mixed with enzyme-free clay suspensions. The activity was assayed according to the standard method. The enzyme activity decreased when the clay was added (Fig. 4A). Larger amounts of enzyme needed larger amounts of clay to be inhibited to the same extent, and when the activity was plotted against the enzyme/clay ratio, the curves for different amounts of enzyme coincided well (Fig. 4B). Therefore, the activity is dependent on the ratio of the amounts of enzyme and clay and not only on the absolute amount of enzyme. The absorbances of the blanks were unchanged until the addition of the montmorillonite suspension exceeded 0.25 ml, which corresponded to 8 mg clay/rag microbial cells. At higher concentrations, the absorbance increased rapidly. At lower clay concentrations, M. luteus cells always precipitate together with the clays, but at higher concentrations the cells do not precipitate. It was also impossible to get clear supernatants at the centrifugation rates employed. This might explain the increase in UV absorption. Skujing et al. [ 16] have reported similar artifacts. It seems less probable that the cells have been autolyzed as a result of oxygen transfer (cf [17]), as the clay concentration is less than 1%.
5. Experiments with a Purified Bacteriolytic Enzyme In the hitherto described experiments, enzyme-clay suspensions with unpurified enzymes were used. To determine if the same results were obtained with purified
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enzymes, experiments were performed with an. alanyl-lysyl-endopeptidase (enzyme HI) purified from M. virescens culture solutions. The activities at different endopeptidase concentrations and at different enzyme/clay ratios agreed well with those obtained with unpurified enzymes (cf Experiments 3 and 4). In Fig. 5 the activities at different concentrations of clay are shown (of Fig. 3B). 6. Activity on Purified Cell Walls
Experiments were performed on the ability of montmorillonite-adsorbed bacteriolytic enzymes to degrade pure cell walls. Instead of intact bacterial cells, purified cell walls of
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M. luteus were used at a concentration of 0.3 mg/ml. Otherwise the experiments were r u n at the same pH, temperature, time, etc. as in the standard assay. After centxifugation of the s u s p e n s i o n s , the a m o u n t s of reducing sugars and free a m i n o groups were d e t e r m i n e d . Different concentrations of the standard clay-enzyme suspensions o f u n p u r i f i e d as well as purified m y x o b a c t e d a l e n z y m e s were used. In none of these cases was it possible to show a n y degradation products.
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7. Activity on Different Gram-Positive and Gram-Negative Bacteria Cell-free culture solutions from M. virescens exhibit bacteriolytic activity against cells o f M . luteus and, to a small extent, also against cells ofBaciUus subtilis and some strains o f B. megaterium. They are, however, inactive against gram-negative bacteria (cf review by Dworkin [2]). Experiments with standard suspensions of clay-adsobed bacteriolytic enzymes
Clay-AdsorbedEnzymes
339
showed that the enzymes were active against the above mentioned gram-positive bacteria but were unable to degrade Escherichia coli, Aerobacter aerogenes, or Pseudomonas fluorescens. Consequently, activity of myxobacteria against gramnegative bacteria in soil seems not to depend on this mechanism, and these bacteria may be degraded only by cell-to-cell contact or by synergetic effects between bacteriolytic enzymes and other extracellular substances [10].
8. Experiments with Lysozyme Yermoljeva and Bouianowskaja, [ 18] reported in 1938 that kaolinite-adsorbed egg white lysozyme is inactive against M. luteus cells. To determine whether the same is valid for montmorillonite-adsorbed lysozyme, some experiments were performed. Buffer, temperature, time, and concentration of clay were the same as in experiment 3 described above. At lysozyme concentrations exceeding 0.1 mg/mg clay, bacterial cells were lysed, but at lower concentrations there was no detectable lysis. The activity at high enzyme concentration may have been due to enzyme molecules which, because of overloading of the clay, were easily desorbed when mixed with the bacterial suspension. If moderate amounts of lysozyme (10 #g/mg clay) were adsorbed on montmorillonite, which was previously loaded with bacteriolytic enzymes from M. virescens, the activity was not increased. Clay-adsorbed lysozyme was also unable to degrade pure cell walls (cfExperiment 6).
Discussion The assay of activity of clay-adsorbed bacteriolytic enzymes on bacterial cells is complex as a result of different adsorption phenomena between clay and bacteriolytic enzymes, clay and nonbacteriolytic proteins, clay and cells, and clay and degradation products. The bacteriolytic enzymes cleave bonds in the peptidoglycan layer of the cell wall, which in turn causes the osmotic rupture of the cytoplasmic membrane. The intracellular proteins are thus almost completely dissolved in the surrounding buffer solution, where they can be assayed as UV-absorbing material. The more direct method of assaying degradation products of the peptidoglycan was impossible to use, due to adsorption or, more likely, to too low concentrations. When M. luteus ceils are mixed with montmorillonite, the bacteria are precipitated together with the clay particles. Otherwise,the montmorillonite did not result in any alteration of the cells per se. McLaren and Estermann [13] and Skujin~ et al. [16] have shown that the sequence of component addition is of great importance for the activity of clay-adsorbed enzymes. This does not seem to be valid for the myxobacterial enzymes, as the addition of an enzyme solution to a suspension of clay-precipitated bacteria gave the same amount of UV-absorbing material as the addition of a bacterial suspension to clay-adsorbed enzymes. The bacteriolytic enzymes from M. virescens do not seem to desorb under the conditions of the standard assay. Neither water nor Universal buffer at a pH below 8 seemed to desorb the enzymes [8]. That it is possible to double the amount of clay
340
G. Haskii
without any decrease in activity (Figs. 3B, 413, and 5B) further proves the great stability of the enzyme-clay complex. The bacteriolytic enzymes from M. virescens were partly inhibited when they were adsorbed on the clay (Fig. 1). This inhibition could be caused by such factors as steric hindrance, diffusional resistance, blocking of the active sites' electrostatic effects, or reversible denaturation. The culture solution contains, in addition to some proteinases, at least three bacteriolytic enzymes, but only one of them--enzyme Ill--could be eluted from the clay [8]. The two glucosidases [6] are probably inactivated when adsorbed. Consequently, this is a further reason for the lower activity of the adsorbed enzymes. The clay-adsorbed bacteriolytic enzymes were active in different buffers--Tris-HCl, phosphate, and the Universal buffer (phosphate-citrate-borate)--but not in Veronal (barbiturate) buffer. Nonadsorbed enzymes are active even in Veronal buffer. The ratio of the amounts of bacteriolytic enzymes and the clay material seems to be important to the activity. Consequently, the enzyme concentration cannot be the only limiting factor for the activity. Surface adsorption of proteins changes the charge characteristics and pH of the clay [4]. Such changes can affect the activity of adsorbed enzymes, and they are probably of significance in systems in which both enzymes and substrate are adsorbed. Culture solutions of M. virescens contain at least three bacteriolytic enzymes [6]. Only one of these, the endopeptidase, is active when it is adsorbed to montmorillonite or kaolinite. Clay-adsorbed lysozyme is, on the other hand, inactive. This is probably the result of the inability of the adsorbed enzyme molecules to come in contact with the bacterial cell wall due to steric hindrance, blocking of the active sites, and/or electrostatic effects. In recent years, immobilized enzymes and their reaction kinetics have received a great deal of attention (cf review by Chibata [1]). Some results concerning the kinetics of soil-adsorbed enzymes have also been published (cf reviews by McLaren [12] and Skujinw [ 15]). Similar investigations concerning bacteriolytic enzymes are also needed. However, before any such investigations can be undertaken, an assay must be devised for the quantitative determination of degradation products. As a substrate for such an assay, synthetic or natural small molecular components of cell wall peptidoglycans are suggested, making it feasible to determine the reasons for the activity of some clayadsorbed bacteriolytic enzymes and the inactivity of others. Acknowledgments. The skillful technical assistance of Mrs. Ingrid Blomqvist is gratefully acknowledged. This investigation has been supported financially by grants from the Swedish Natural Science Council and from M. Bergwalls" Foundation.
References 1. Chibata, Ichiro (ed.): Immobilized Enzymes. Research Development, pp. 108--147. Halsted Press, New York (1978) 2. Dworkin, M.: Biology of the myxobacteria. Annu. Rev. Microbiol. 20, 75-106 (1966) 3. Ghuysen, J. M., D. J. Tipper, and J. L. Strominger: Enzymes that degrade bacterial cell walls. Methods Enzymol. 8,685-699 (1966) 4. Hatter, R. D., and G. Stotzky: X-ray diffraction, electron microscopy, electrophoretic mobility, and pH of some stable smectite-protein complexes. Soil Sci. Soc. Am. Proc. 37, 116-123 (1973) 5. Hask~, G.: Extracellular lytic enzymes of Myxococcus virescens. I. Separation of the bacteriolytic enzymes from the butk of proteinases. Physiol. Plant. 25, 86--89 (i971) 6. Hask~, G.: Extracellular lytic enzymes of Myxococcua virescens. H. Purification of three bacteriolyfc
Clay-Adsorbed Enzymes
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
341
enzymes and determination of their molecular weights and isoelectric points. Physiol. Plant. 26, 221-229 (1972) Hask~l, G.: Extracellular lyric enzymes ofMyxococcus virescens. IV. Purification and characterization of a D-alanyl-e-N-lysine endopepridase. Physiol. Plant. 31,252-256 (1974) Hask~,G.: Influence of clay minearls on sorption of bacteriolytic enzymes. Micmb. Ecol. 1, 234-245 (1975) Hask~, G., and S. St~dal: Variants of Myxococcus virescens exhibiting dispersed growth. Growth and production of extracellular enzymes and slime. Physiol. Plant. 24, 136-142 ( 1971 ) Hask~, G., B. Noren, and G. Odham: Effects of fatty acids on the activity of bacteriolytic enzymes. Physiol. Plant. 27, 187-194 (1972) Lowry, H. O., N. J. Rosebrough, A. L. Fan', and R. J. Randall: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) McLaren, A. D.: Kinetics and consecutive reactions of soil enzymes. In R. G. Burns (ed:): Soil Enzymes, pp. 97-116. Academic Press, London (1978) Mcl.amn, A. D., and E. F. Estermann: The adsorption and reactions of enzymes and proteins on kaolinite. III. The isolation of enzyme-substrate complexes. Arch. Biochem. Biophys. 61, 158-173 (1956) McLaren, A. D., and E. F. Estermann: lnfluence of pH on the activity of chymotrypsin at a solid-liquid interface. Arch. Biochem. Biophys. 68, 157-160 (1957) Skujin[, J.: Extracellular enzymes in soil. CRC Crit. Rev. Microbiol. 4,383-421 (1976) Skujin~, J., A. Pukite, and A. D. McLaren: Adsorption and activity of chitinase on kaolinite. Soil Biol. Biochem. 6, 179-182 (1974) Stotzky, G., and L. T. Rem: Influence of clay minerals on microorganisms. IV. Montmorillonite and kaolinite on fungi. Can. J. Micmbiol. 13, 1535-1550 (1967) Yermoljeva, Z. W., and I. Bouianowskaja: Le lysozyme, ses propri6t6s et ses applications. Acta Med. URSS 1,248-257 (1938) Ostling, S., and P. Virtama: A modified preparation of the universal buffer described by Theorell and Stenhagen. Acta Physiol. Scand. 11,289-293 (1946)
hIICI~OBI~ILECOLOGV
Activity of Bacteriolytic Enzymes Adsorbed to Clays Guno Haskfi* Department of Microbial Ecology, University of Lund, Helgonav~igen 5, S-223 62 Lund, Sweden
Abstract. Myxococcus virescens is able to produce extracellular bacteriolytic enzymes that are rapidly adsorbed on montmorillonite. These adsorbed enzymes are active and can be assayed by measuring the release of UV-absorbing materials in mixtures containing Micrococcus luteus cells. The activity of the clay-adsorbed enzymes is, however, considerably lower than that of the unadsorbed enzymes. Both unadsorbed and adsorbed enzymes have their maximum activity at approximately the same pH. At lower clay-enzyme concentrations, the activity is proportional to the concentration. If, however, increasing amounts of clay are added to a fixed volume of clay-enzyme suspension, the activity remains almost unchanged until a definite limit is reached, then the activity decreases rapidly. This limit was dependent only on the ratio of the amounts of enzyme and clay and not on the absolute concentration of the enzyme. The montmorillonite-adsorbed bacteriolytic enzymes from M. virescens were not active against gram-negative bacteria, and no activity against purified cell walls from M. luteus could be measured. Montmorillonite-adsorbed egg white lysozyme was not active on M. luteus cells.
Introduction Some myxobacteria can lyse other bacteria and feed on the released nutrients. They produce extracellular bacteriolytic enzymes that break bonds in the cell wail of other bacteria. These enzymes are rapidly adsorbed on soil particles, especially on clay materials [8]. The bacteriolytic enzymes can be desorbed if the pH or ionic strength is increased, but if the myxobacteria are to be able to feed on other living bacteria in nature, the bacteriolytic enzymes must be active in the adsorbed state. This paper deals with experiments concerning the activity of clay-adsorbed bacteriolytic enzymes from
Myxococcus virescens.
Materials and Methods Enzyme-Clay Suspension A cell-free culture solution was obtained from M+ virescens as previously described [5, 8]. It was stored at - 2 5 ~ until used. This solution (250 ml) was mixed with a montmorillonite suspension (17 ml, 300 rag) with the aid of a magnetic stirrer at 23~ [8]. After an hour, the suspension was centrifuged at 48,000 x g for 12 min. *Present address: Swedish Sugar Co. Ltd., Research Laboratories, Box 6, S-23200 Arl~Sv, Sweden.
0095-3628/81/0007-0331 $02.20 9 1981 Springer-Verlag New York Inc.
332
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The clear solution contained no bacteriolytic activity. The enzyme-montmorillonite particles were washed once with 35 ml of distilled water, recentrifuged, and then suspended in 0.05 M phosphate buffer, pH 7.5, to a volume of 25 ml. After centrifugation of the washed clay-enzyme particles, the supematant did not contain any enzyme activity. This standard enzyme-clay suspension was stored at 4~ and used for experiments during 2 weeks. Only a minor decrease in activity could be observed during that time. The cell-free culture solution contained 5.72 mg protein/nil. Of this protein content, the three bacteriolytic enzymes represented only 0.42 #g/ml, and the alanyl-lysyl endopeptidase was the main part (0.34/ag/ml). At the montmorillonite 36% of the proteins and 100% of the bacteriolytic enzymes were adsorbed. The standard enzyme-clay suspension thus contained 12 mg clay/mi, resulting in !.72 mg protein/mg clay and 0.35/,tg bacteriolytic enzymes/rag clay. in experiments on the effects of different clay concentrations, a standard montmorillonite suspension was used. It contained the same amount of clay (12 mg/ml) as the standard enzyme-montraorilonite suspension. The previously [7] purified alanyl-lysyl endopeptidase (enzyme 111)was used in some experiments either as a solution or as a montmorillonite-adsorbed suspension (0.28 /ag enzyme/mg clay). The enzyme d a y suspensions were made in the same buffer and pH and with the same amount of clay and the same enzyme activity as the clay-adsorbed unpurified enzymes described above. " Although great care was taken to standardize all handling with the enzyme suspensions, some smaller differences between different suspensions could not be avoided, and therefore different experiments are not always totally comparable.
Enzyme Assay For determination of the activity of nonadsorbed bacteriolytic enzymes, the method previously described was used [9]. This method is based on turbidity measurements in suspensions of intact cells ofMicrococcus luteus. It cannot, however, be used in montmorillonite suspensions, as the bacterial cells are precipitated when mixed with the clay. Instead, the act~.vity of clay-adsorbed enzymes was assayed by the ability to produce nonadsorbed UV-absorbing material. In the standard assay, a suspension ofM. luteus cells in Tris-HC 1 buffer, pH 8.0, was used. The concentration of cells was the same as in the previously used method (0.36 mg/ml). A suspension of clay-adsorbed enzymes was mixed in centrifuge tubes to a total volume of 10 ml and at a final concentration of 0.05 M Tris-HCI. The tubes were incubated at 37~ in a shaker for 1 h, cooled, and centrifuged at 48,000 • g for 12 min. The absorbancy of the clear solution was determined at 280 nm in a Beckman DB-GT Spectrophotometer (10 mm cell). In the figures, the values are corrected for blanks without any addition of enzymes. This was the standard assay used, unless otherwise stated. The UV-absorbing material produced by the action of the clay-adsorbed enzymes was adsorbed to the montroorillortite to a very little extent. Less than 5% was adsorbed when 2 mg clay was added per ml of an enzyme-digested cell suspension.
Analytical Methods The procedures for the determination of reducing sugars and free amino groups were principally those described by Ghuysen et al. [3].
Protein Assay Protein content in the solutions was determined by the method of Lowry et al. [ 1 ! ], with bovine serum albumin as standard.
Results
1. Bacteriolytic Activity of Adsorbed Enzymes This experiment was performed to determine the activity of clay-adsorbed bacteriolytic enzymes from Myxobacteria and to investigate differences in activity between adsorbed and nonadsorbed enzymes.
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File. 1. Effect of time on the bactcriolysisofM. tuteuscells by bactcriolyucenzymes from M. vireseens. The cells were degraded by a suspension of unpurified and unadsorbed enzymes (o), of montmoriiionite-adsorbed enzymes (o), and of desorbed and purified enzymes (-).
To four flasks, each containing 50 ml of the M. luteus cell suspension in 0.05 M Tris-HCl buffer, pH 8.0,250/.tl of the following solutions was added: flask I, a cell-free culture solution from M. virescens (180 BEU/ml, cf [5]); flask 2, the same volume of the culture solution adsorbed on 3.0 mg montmorillonite according to the standard method; flask 3, a solution of purified alanyl-lysyl-endopeptidase (enzyme UI) corresponding to that amount (0.085 #g) which can be purified from 250/zl culture solution by adsorption on and desorption from 3.0 mg montmorillonite [8]; and flask 4, a montmorillonite suspension (3.0 mg) used as a blank. Figure 1 shows that the clay-adsorbed enzymes were active on M. luteus cells. However, they were not as active as the corresponding amount of culture solution and not even as active as the enzymes which can be desorbed from the same amount of clay. These differences might be due to several reasons. Since the activity increases when the enzymes are eluted, there must be some kind of inhibition of the activity when the enzymes are adsorbed. The clay-adsorbed bacteriolytic enzymes and the eluted enzymes reached after 6 h the same level of absorbance, which indicates that the desorbed enzymes are the same as those which are active in the clay-enzyme complex. However, the ability of these enzymes to degrade bacterial cell walls is not equal to that of the culture solution. This unpurified solution contains several bacteriolytic and proteolytic enzymes that can work synergetically on the peptidoglycans.
334
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"
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Fig. 2. Activity of bacteriolytic enzymes in a Universal buffer at different pHs. The culture solution was unpurified and unadsorbe,d (o), adsorbed on montmorillonite (o), and desorbed from montmorillonite and purified (=). The maximum activity for each enzyme system was taken as reference (100%).
2. Activity at Different pHs The same enzyme suspensions as in experiment 1 were used. The concentrations were chosen so that the activity at pH 8.8 would be about the same. The enzyme activities were measured with the standard assay but with a universal buffer [19] instead of phosphate buffer. This buffer consists of phosphate, citrate, and borate salts in water and has a nearly constant ionic strength (0.07-0.10M) over a wide pH range. The activity peak of the adsorbed enzymes was about the same as that for the unadsorbed enzymes with respect to pH (Fig. 2). These results do not agree with those concerning pure proteinases [14] and pure chitinases [16]. The activity peak of adsorbed proteinases and chitinases was greater by 1.0 pH unit than that for the unadsorbed enzymes. The clay-adsorbed bacteriolytic enzymes had less activity than the unadsorbed enzymes at low pH. At high pH, there was no difference between the three activity curves, probably because the bacteriolytic enzymes are desorbed from the clay at a pH above 9 [8].
Clay-AdsorbedEnzymes
335
3. Activity at Different Concentrations of Clay Different volumes (0--70 #1) of the standard montmorillonite suspension were mixed with 4 /al of the enzyme-montmorillonite suspension. The activities were assayed according to the standard procedure. Although 4 tzl of the enzyme-clay suspension gave an absorbance proportional to the volume of the enzyme-clay suspension, the addition of increasing amounts of montmorillonite decreased the activities (Fig. 3A). This may have resulted from an increasing adsorption of degradation products on the clay particles, but the addition of more montmorillonite to the centrifuged, clay-free reaction products did not significantly change the absorbance in comparison with the addition of the same volume of water. Consequently, the lower activity with higher amounts of montmorillonite indicates that the activity is inhibited in one way or another. In Fig. 3B, the absorbance is plotted against the ratio of the amounts of enzymes and of clay. When this ratio was decreased from 1.72 mg protein/mg clay, as in the stock enzyme-montmorillonite suspension, the absorbance did not change until the ratio fell below a value of about 0.6 mg/mg. To determine whether this was also valid for different amounts of enzymes, the next experiment was performed.
4. Activity at Different Enzyme/Clay Ratios In three series of tubes 5, 10, and 20/zl of the enzyme-montmorillonite suspensions was used. Different volumes of the standard montmorillonite suspension were added to the tubes to obtain final enzyme/clay ratios between 0.09 and 1.72 mg/mg clay. In a fourth series, M. luteus cells were mixed with enzyme-free clay suspensions. The activity was assayed according to the standard method. The enzyme activity decreased when the clay was added (Fig. 4A). Larger amounts of enzyme needed larger amounts of clay to be inhibited to the same extent, and when the activity was plotted against the enzyme/clay ratio, the curves for different amounts of enzyme coincided well (Fig. 4B). Therefore, the activity is dependent on the ratio of the amounts of enzyme and clay and not only on the absolute amount of enzyme. The absorbances of the blanks were unchanged until the addition of the montmorillonite suspension exceeded 0.25 ml, which corresponded to 8 mg clay/rag microbial cells. At higher concentrations, the absorbance increased rapidly. At lower clay concentrations, M. luteus cells always precipitate together with the clays, but at higher concentrations the cells do not precipitate. It was also impossible to get clear supernatants at the centrifugation rates employed. This might explain the increase in UV absorption. Skujing et al. [ 16] have reported similar artifacts. It seems less probable that the cells have been autolyzed as a result of oxygen transfer (cf [17]), as the clay concentration is less than 1%.
5. Experiments with a Purified Bacteriolytic Enzyme In the hitherto described experiments, enzyme-clay suspensions with unpurified enzymes were used. To determine if the same results were obtained with purified
336
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Fig. 3. Activity at different concentrations of clay. Four microliters of the enzyme-montmorillonite suspension was mixed with 0-70p I of the pure clay suspension. The activity is plotted (A) vs the total amount of clay (mg/ml) and (B) the amount of proteins/weight of clay (mg/mg). The activities are not corrected for blanks not containing any enzymes.
enzymes, experiments were performed with an. alanyl-lysyl-endopeptidase (enzyme HI) purified from M. virescens culture solutions. The activities at different endopeptidase concentrations and at different enzyme/clay ratios agreed well with those obtained with unpurified enzymes (cf Experiments 3 and 4). In Fig. 5 the activities at different concentrations of clay are shown (of Fig. 3B). 6. Activity on Purified Cell Walls
Experiments were performed on the ability of montmorillonite-adsorbed bacteriolytic enzymes to degrade pure cell walls. Instead of intact bacterial cells, purified cell walls of
Clay-Adsorbed Enzymes
337 ,|
/
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Fig. 4. Activity at different enzyme/clay ratios. To three series of tubes containing 5 (e), 10 (o), and 20 (m)p1, respectively, of enzyme-clay suspension, 0-500tt I of the pure montmorilionitesuspension was added. Blanks were run without enzymes (-). The activities in B are corrected for blanks. For other legends see Fig. 3.
M. luteus were used at a concentration of 0.3 mg/ml. Otherwise the experiments were r u n at the same pH, temperature, time, etc. as in the standard assay. After centxifugation of the s u s p e n s i o n s , the a m o u n t s of reducing sugars and free a m i n o groups were d e t e r m i n e d . Different concentrations of the standard clay-enzyme suspensions o f u n p u r i f i e d as well as purified m y x o b a c t e d a l e n z y m e s were used. In none of these cases was it possible to show a n y degradation products.
338
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Fig. 5. Activity of purified endopeptidase at different concentrations of clay. Four microliters of the enzyme-montmorillonite suspension (0.28,u g enzyme/mg clay) was mixed with 0-70gl of the pure clay suspension. The activities are corrected for blanks without enzymes.
7. Activity on Different Gram-Positive and Gram-Negative Bacteria Cell-free culture solutions from M. virescens exhibit bacteriolytic activity against cells o f M . luteus and, to a small extent, also against cells ofBaciUus subtilis and some strains o f B. megaterium. They are, however, inactive against gram-negative bacteria (cf review by Dworkin [2]). Experiments with standard suspensions of clay-adsobed bacteriolytic enzymes
Clay-AdsorbedEnzymes
339
showed that the enzymes were active against the above mentioned gram-positive bacteria but were unable to degrade Escherichia coli, Aerobacter aerogenes, or Pseudomonas fluorescens. Consequently, activity of myxobacteria against gramnegative bacteria in soil seems not to depend on this mechanism, and these bacteria may be degraded only by cell-to-cell contact or by synergetic effects between bacteriolytic enzymes and other extracellular substances [10].
8. Experiments with Lysozyme Yermoljeva and Bouianowskaja, [ 18] reported in 1938 that kaolinite-adsorbed egg white lysozyme is inactive against M. luteus cells. To determine whether the same is valid for montmorillonite-adsorbed lysozyme, some experiments were performed. Buffer, temperature, time, and concentration of clay were the same as in experiment 3 described above. At lysozyme concentrations exceeding 0.1 mg/mg clay, bacterial cells were lysed, but at lower concentrations there was no detectable lysis. The activity at high enzyme concentration may have been due to enzyme molecules which, because of overloading of the clay, were easily desorbed when mixed with the bacterial suspension. If moderate amounts of lysozyme (10 #g/mg clay) were adsorbed on montmorillonite, which was previously loaded with bacteriolytic enzymes from M. virescens, the activity was not increased. Clay-adsorbed lysozyme was also unable to degrade pure cell walls (cfExperiment 6).
Discussion The assay of activity of clay-adsorbed bacteriolytic enzymes on bacterial cells is complex as a result of different adsorption phenomena between clay and bacteriolytic enzymes, clay and nonbacteriolytic proteins, clay and cells, and clay and degradation products. The bacteriolytic enzymes cleave bonds in the peptidoglycan layer of the cell wall, which in turn causes the osmotic rupture of the cytoplasmic membrane. The intracellular proteins are thus almost completely dissolved in the surrounding buffer solution, where they can be assayed as UV-absorbing material. The more direct method of assaying degradation products of the peptidoglycan was impossible to use, due to adsorption or, more likely, to too low concentrations. When M. luteus ceils are mixed with montmorillonite, the bacteria are precipitated together with the clay particles. Otherwise,the montmorillonite did not result in any alteration of the cells per se. McLaren and Estermann [13] and Skujin~ et al. [16] have shown that the sequence of component addition is of great importance for the activity of clay-adsorbed enzymes. This does not seem to be valid for the myxobacterial enzymes, as the addition of an enzyme solution to a suspension of clay-precipitated bacteria gave the same amount of UV-absorbing material as the addition of a bacterial suspension to clay-adsorbed enzymes. The bacteriolytic enzymes from M. virescens do not seem to desorb under the conditions of the standard assay. Neither water nor Universal buffer at a pH below 8 seemed to desorb the enzymes [8]. That it is possible to double the amount of clay
340
G. Haskii
without any decrease in activity (Figs. 3B, 413, and 5B) further proves the great stability of the enzyme-clay complex. The bacteriolytic enzymes from M. virescens were partly inhibited when they were adsorbed on the clay (Fig. 1). This inhibition could be caused by such factors as steric hindrance, diffusional resistance, blocking of the active sites' electrostatic effects, or reversible denaturation. The culture solution contains, in addition to some proteinases, at least three bacteriolytic enzymes, but only one of them--enzyme Ill--could be eluted from the clay [8]. The two glucosidases [6] are probably inactivated when adsorbed. Consequently, this is a further reason for the lower activity of the adsorbed enzymes. The clay-adsorbed bacteriolytic enzymes were active in different buffers--Tris-HCl, phosphate, and the Universal buffer (phosphate-citrate-borate)--but not in Veronal (barbiturate) buffer. Nonadsorbed enzymes are active even in Veronal buffer. The ratio of the amounts of bacteriolytic enzymes and the clay material seems to be important to the activity. Consequently, the enzyme concentration cannot be the only limiting factor for the activity. Surface adsorption of proteins changes the charge characteristics and pH of the clay [4]. Such changes can affect the activity of adsorbed enzymes, and they are probably of significance in systems in which both enzymes and substrate are adsorbed. Culture solutions of M. virescens contain at least three bacteriolytic enzymes [6]. Only one of these, the endopeptidase, is active when it is adsorbed to montmorillonite or kaolinite. Clay-adsorbed lysozyme is, on the other hand, inactive. This is probably the result of the inability of the adsorbed enzyme molecules to come in contact with the bacterial cell wall due to steric hindrance, blocking of the active sites, and/or electrostatic effects. In recent years, immobilized enzymes and their reaction kinetics have received a great deal of attention (cf review by Chibata [1]). Some results concerning the kinetics of soil-adsorbed enzymes have also been published (cf reviews by McLaren [12] and Skujinw [ 15]). Similar investigations concerning bacteriolytic enzymes are also needed. However, before any such investigations can be undertaken, an assay must be devised for the quantitative determination of degradation products. As a substrate for such an assay, synthetic or natural small molecular components of cell wall peptidoglycans are suggested, making it feasible to determine the reasons for the activity of some clayadsorbed bacteriolytic enzymes and the inactivity of others. Acknowledgments. The skillful technical assistance of Mrs. Ingrid Blomqvist is gratefully acknowledged. This investigation has been supported financially by grants from the Swedish Natural Science Council and from M. Bergwalls" Foundation.
References 1. Chibata, Ichiro (ed.): Immobilized Enzymes. Research Development, pp. 108--147. Halsted Press, New York (1978) 2. Dworkin, M.: Biology of the myxobacteria. Annu. Rev. Microbiol. 20, 75-106 (1966) 3. Ghuysen, J. M., D. J. Tipper, and J. L. Strominger: Enzymes that degrade bacterial cell walls. Methods Enzymol. 8,685-699 (1966) 4. Hatter, R. D., and G. Stotzky: X-ray diffraction, electron microscopy, electrophoretic mobility, and pH of some stable smectite-protein complexes. Soil Sci. Soc. Am. Proc. 37, 116-123 (1973) 5. Hask~, G.: Extracellular lytic enzymes of Myxococcus virescens. I. Separation of the bacteriolytic enzymes from the butk of proteinases. Physiol. Plant. 25, 86--89 (i971) 6. Hask~, G.: Extracellular lytic enzymes of Myxococcua virescens. H. Purification of three bacteriolyfc
Clay-Adsorbed Enzymes
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
341
enzymes and determination of their molecular weights and isoelectric points. Physiol. Plant. 26, 221-229 (1972) Hask~l, G.: Extracellular lyric enzymes ofMyxococcus virescens. IV. Purification and characterization of a D-alanyl-e-N-lysine endopepridase. Physiol. Plant. 31,252-256 (1974) Hask~,G.: Influence of clay minearls on sorption of bacteriolytic enzymes. Micmb. Ecol. 1, 234-245 (1975) Hask~, G., and S. St~dal: Variants of Myxococcus virescens exhibiting dispersed growth. Growth and production of extracellular enzymes and slime. Physiol. Plant. 24, 136-142 ( 1971 ) Hask~, G., B. Noren, and G. Odham: Effects of fatty acids on the activity of bacteriolytic enzymes. Physiol. Plant. 27, 187-194 (1972) Lowry, H. O., N. J. Rosebrough, A. L. Fan', and R. J. Randall: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) McLaren, A. D.: Kinetics and consecutive reactions of soil enzymes. In R. G. Burns (ed:): Soil Enzymes, pp. 97-116. Academic Press, London (1978) Mcl.amn, A. D., and E. F. Estermann: The adsorption and reactions of enzymes and proteins on kaolinite. III. The isolation of enzyme-substrate complexes. Arch. Biochem. Biophys. 61, 158-173 (1956) McLaren, A. D., and E. F. Estermann: lnfluence of pH on the activity of chymotrypsin at a solid-liquid interface. Arch. Biochem. Biophys. 68, 157-160 (1957) Skujin[, J.: Extracellular enzymes in soil. CRC Crit. Rev. Microbiol. 4,383-421 (1976) Skujin~, J., A. Pukite, and A. D. McLaren: Adsorption and activity of chitinase on kaolinite. Soil Biol. Biochem. 6, 179-182 (1974) Stotzky, G., and L. T. Rem: Influence of clay minerals on microorganisms. IV. Montmorillonite and kaolinite on fungi. Can. J. Micmbiol. 13, 1535-1550 (1967) Yermoljeva, Z. W., and I. Bouianowskaja: Le lysozyme, ses propri6t6s et ses applications. Acta Med. URSS 1,248-257 (1938) Ostling, S., and P. Virtama: A modified preparation of the universal buffer described by Theorell and Stenhagen. Acta Physiol. Scand. 11,289-293 (1946)
