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[7] Extraction of enzymes from microorganisms (bacteria and yeast)
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EXTRACTION OF ENZYMES FROM MICROORGANISMS
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the method has so far been applied mostly with animal tissues, the procedures described above should be just as suitable for obtaining active enzymes from plant tissues, yeasts, and bacteria. [7] E x t r a c t i o n of E n z y m e s f r o m M i c r o o r g a n i s m s
(Bacteria and Yeast)
By I. C. GUNSALTJS Principle The methods which have proved effective in liberating enzymes from microbial cells have been largely mechanical rupture of the cell wall and membrane, frequently with fragmentation of the latter. In specific instances enzymatic, ~ including autolysis, and chemical 2 treatments have proved useful. The choice of procedure will depend on the particular species or strain of microorganism used, including the ease with which the cell is ruptured, the quantity of enzyme required, and the type of preparation from which the extract is prepared. The choice will also vary with the sensitivity and the localization of the enzyme within the cell. Most methods in use were empirically derived; thus, consulting the original literature in specific cases is useful--for example, a recent review by Hugo z carries more than a hundred references to specific cases. Bacterial and yeast cells for the preparation of enzyme extracts may be obtained as products of the fermentation industries; that is, brewer's yeast as sludge from a spent fermentation tank, baker's yeast in pound cakes or in bulk--usually starch-free--from the baking or food industries. A few bacterial strains are available from pharmaceutical procedures which do not impair enzyme activity. More frequently, however, bacterial cells are grown by the investigator, usually under conditions which support optimum growth or a maximum enzyme yield. 4 In the latter case, the age and enzymatic content of the cells, the means of harvest--usually Sharples continuous centrifuge--and the type of cell preparation from which the extract is prepared can be controlled. For small quantities, batch centrifugation may be employed, in which case aeration during harvest can also be controlled, should this be important to the investiga' M. Penrose and J. H. Quastel, Proc. Roy. Sac. (London) B107, 168 (1930); M. F. Utter, L. O. Krampitz, and C. H. Werkman, Arch. Biochem. 9, 285 (1946). P. V. B. Cowles, Yale J. Biol. and Med. 19, 835 (1947). W. B. Hugo, Baderiol. Revs. 18, 87 (1954). 4 A. J. Wood and I. C. Gunsalus, J. Bacteriol. 44, 333 (1942) ; E. F. Gale, Bacteriol. Revs. 7, 139 (1943).
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GENERAL PREPARATIVE PROCEDURES
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tion. In such cases freshly harvested cell paste with a minimum of autolysis is obtained. In the use of freshly harvested or commercially prepared ceU paste, cakes, or sludges, washing may be employed if the experiments so indicate, using caution with aged cells lest the enzymes be extracted by the washing procedure. The cells may be preserved by storage at reduced temperatures, for example, in the deep-freeze or dry ice chest at - 10 to - 40 °; or they may be reduced immediately to extracts, in which state the enzymes are usually stable at deep-freeze temperatures. If neither of these proves convenient, stable dried cell preparations can usually be obtained by air, vacuum, or solvent drying procedures. In summary: The extraction and preservation of the activity of any given enzyme is an empirical process based on the investigator's own or his colleagues' experience. In the absence of example, the original literature offers a wide variety of methods and of organisms used successfully for various enzymes2 Dried Cell Preparations The ease of release of enzymes from cells depends to a great extent on their previous treatment--drying may serve to preserve enzyme activity or may be used as a means of liberating or extracting enzymes. Dried cell preparations frequently have sufficiently lost the "permeability" properties of living cells to serve as enzyme preparations--that is, they will metabolize phosphate esters and polybasic acids and can be activated by coenzymesL-and the coenzymes and metabolic intermediates are often leeched from the cells during the drying procedure or after resuspending in buffers. In many cases the drying procedure involves enzymatic autolysis, which enhances enzyme liberation. Procedure
Dried cell preparations can be obtained by: 1. Air drying--frequently accompanied by autolysis, 2. Slow vacuum drying--usually from cell pastes, also accompanied by autolysis and yielding glass-like preparations which can be later powdered readily by mortar and pestle; 3. Lyophylization--desiccation from the frozen state, usually of a 10 to 20% by wet weight cell suspension; 4. Dehydrating with water-miscible solvents. 5 I. C. Gunsalus and W. W. Umbreit, J. Bacteriol. 49, 347 (1945); I. C. Gunsalus, W. D. Bellamy, and W. W. Umbreit, J. Biol. Chem. 1§5, 685 (1944).
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EXTRACTION OF ENZYMES FROM MICROORGANISMS
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Air Drying (of Yeast). The finding by Lebedev s that air-dried yeast could be extracted by mild grinding or by stirring with buffer greatly accelerated studies with soluble enzymes. Since Lebedev's finding, many methods have been employed for drying 7 and for extracting 8 air-dried yeast and bacteria. The important factors considered in the early experiments are summarized by Harden2 For more recent data specific manuscripts should be consulted. In general, yeast at the consistency of yeast cake, or ceils solidly packed by centrifugation after washing, are crumbled by hand or by forcing through a 10-mesh sieve or potato dicer onto trays in layers less than an inch deep and allowed to dry 2 to 3 days at temperatures ranging from 25 to 30 °, occasionally as high as 35 to 40 ° . The layer of yeast may be turned at half-day intervals to promote drying; a fan may be employed to accelerate the process. Air-dried yeast has undergone partial autolysis and will usually yield active enzyme extracts if suspended in 3 to 5 vol. of water or suitable buffer and stirred 2 to 3 hours at room temperature, s,9 (Occasionally yeast is pretreated--for example, mild bicarbonate hydrolysis before extraction, s) The cell debris can then be removed by centrifugation, and the enzymes subjected to further purification if desired. If a particular enzyme is not solubilized by the drying procedure, one of the more rigorous methods of extraction may be resorted to. Slow Drying in Vacuum. This procedure has been advantageously applied to enzyme extraction of several types of bacteria.l°-l~ The packed ceils obtained by centrifugation are transferred by a spatula to a beaker or evaporating dish, placed in a desiccator over calcium chloride, P20~ or anhydrous calcium sulfate (Drierite), and the desiccator evacuated with an aspirator or vacuum pump, sealed, and allowed to stand, usually overnight. Occasionally a thick, 20 % wet weight, cell suspension is dried by a similar procedure. The dried preparations are usually of hard, glassy consistency and have undergone some autolysis. The cells may be extracted by suspending directly in buffer but usually more successfully by grinding the dried cell mass to a powder with a mortar and pestle before extraction. If the paste has been deposited in layers more than 0.5 to 1 cm. thick, gentle grindins followed by further drying may be required. More labile enzymes are destroyed by the slow drying procedure; 6 A. yon. Lebedev, Compt. rend. 152, 49 (1911). C. Neuberg and H. Lustig, Arch. Biochem. 1, 191 (1943). 8 B. L. Horeeker and P. Z. Smyrniotis, J. Biol. Chem. 193~ 371 (1951). 9A. Harden, "Alcoholic Fermentation," Chapter 2. Longmans, Green, & Co., London, 1923. 10F. Lipmann, Cold Spring Harbor Symposia Quant. Biol. 7~ 248 (1939). 11E. R. Stadtman and H. A. Barker, J. Biol. Chem. 180, 1085 (1949). 1~B. P. Sleeper, M. Tsuehida, and R. Y. Stanier, J. Baeteriol. 59, 129 (1950).
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GENERAL PREPARATIVE PROCEDURES
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thus partial reaction sequences may be studied successfully with such preparations. '~ As with air-dried yeast, the soluble enzymes can usually be extracted from the slow vacuum-dried bacterial or yeast preparations by gentle agitation with buffer or water. The incorporation of reducing substances-that is, cysteine, glutathione, or sodium sulfide~--before drying, especially with metabolically anaerobic bacteria, has been helpful in preventing the loss of otherwise labile enzymes. These additions have been particularly beneficial in preserving those enzymes which appear to possess mercapto groups or to be sulfhydryl activated. H Lyophylization. Drying quick-frozen, thick cell suspensions in vacuum has proved especially beneficial in preserving labile enzymes. In fact, a similar procedure on a smaller scale is in common use to preserve stock cultures in a viable state. ~3 For enzyme work, vacuum-dried cells are usually stored in the presence of air in a sufficiently tight container to prevent rehydration. The cells soon lose their viability and in many cases are more permeable to substrates, including cofactors, than are viable cells. 6 Usually, more drastic means are required to extract enzymes from lyophylized than from air or slow vacuum-dried cells, very probably because autolysis does not occur. Lyophylization of masses of cells can be accomplished most conveniently by suspending 10 to 40 % wet weight of cells in distilled water. With some cells a washing step to remove salts and residual media ingredients is desirable to reduce autorespiration (endogenous substrate oxidation); with large batches of cells a quick freeze before drying is also desirable. For preparations up to 10 to 15 g. of cells, a single 100-mm. Petri dish containing 10 to 40 ml. of suspension can be placed over 4- to 8-mesh Drierite in a 250-ram. desiccator and evacuated to less than 0.1 mm. mercury. Under these conditions the rate of evaporation will be rapid enough to cause the suspension to freeze. If the vacuum is preserved--usually it is well to keep the pump running-the preparation will dry without thawing in a period of 6 to 8 hours (usually well to leave overnight) to yield a porous, friable mass which may be reduced to a powder by working gently with a spatula on a clean paper. The enzyme activities in lyophylized cells may remain nearly constant for several years if stored at - 2 0 ° and protected from the re-entry of water. The moisture content of lyophylized cells is frequently less than 0.1%, usually lower than in air-dried preparations. To prepare large batches of lyophylized cells, shell freezing in plasma bottles or round-bottom distillation flasks, up to 2- to 3-1. volume, is useful. The desiccation is usually accomplished by connecting the flask la L. A. Rogers, J. Bac~eriol. 57, 137 (1949) ; L. Atkin, W. Moses, and P. P. Gray, ibid. §7, 575 (1949).
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EXTRACTION OF ENZYMES FROM MICROORGANISMS
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to a large-diameter, short-path vacuum system containing a mechanically refrigerated, or dry ice-cooled, receiver to remove the water by sublimation. Convenient units for this purpose are available commercially or m a y be fabricated from glass or from metal. 14,15 Acetone (Solvent) Dried Preparations. Large quantities of ceils m a y be conveniently dried by adding an aqueous suspension to a large volume of water-miscible solvent at a sufficiently low temperature to prevent denaturation of the cell proteins. The most common procedure is to add an aqueous, or buffered, cell suspension slowly with vigorous stirring to not less than 10 volumes of acetone previously cooled to - 2 0 ° or to the temperature of dry ice. After a brief stirring the cells are allowed to settle, the supernatant, which frequently contains g u m m y material, is decanted, and the residual solvent is removed on a Buchner filter. The filter cake of ceils is washed on the Buchner with 2 to 5 vol. of - 2 0 ° a c e t o n e - - o r suspended in 2 to 5 vol. of chilled acetone and again collected on a B u c h n e r - and washed once with 2 to 3 vol. of - 2 0 ° peroxide-free ether, sucked dry on the filter, and transferred to a large sheet of wrapping paper and worked gently with a spatula until the solvent has evaporated, leaving a dry powder. If the atmosphere is humid, the dried ceils should not remain in contact with the air for more than a few minutes, and the removal of solvent can be completed, although less satisfactorily, in a desiccator in the presence of paraffin to absorb the solvent. Acetone-dried preparations usually contain no living cells--bacterial endospores being an exception--and retain f e w e r of the permeability properties of the viable cell than do lyophylized preparations.. If freshly prepared cells are acetone-dried, little, if any, autolysis will have occurred. Acetone-dried cells m a y yield active enzyme extracts on stirring with suitable buffer or with water, 18 or more rigorous extraction m a y be required. ~7 Other water-miscible solvents including dioxane ~s have also been employed, although less frequently, to prepare dried cells. Toluene, chloroform, or similar solvents ~9 m a y be used to alter the permeability of ceils, permitting enzyme assays without respect to cell permeability. This type of preparation is seldom subjected to further 14 E. W. Flosdorf, "Freeze-Drying," Reinhold Publishing Co., New York, 1949; Virtis Co., Inc., Yonkers, New York (metal). 15 W. J. Elser, R. A. Thomas, and E. I. Steffen, J. Immunol. 28, 433 (1935); D. H. Campbell and D. Pressman, Science 99, 285 (1944) (glass). 16 E. F. Gale, Advances in Enzymol. 6, 1 (1946); G. W. E. Plaut and H. A. Lardy, J. Biol. Chem. 180, 13 (1949). 17 M. I. Dolin, Thesis, Indiana University, 1950. ~s Procedures similar to J. B. Sumner and A. L. Dounce, J. Biol. Chem. 127, 439 (1939). 19 E. Buchner and R. Rapp, Ber. 80, 2668 (1897); see also ref. 9, p. 36.
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GENERAL PREPARATIVE PROCEDURES
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purification, even to the extent of removing the enzymes from cellular debris. Toluene may, however, be used in conjunction with grinding procedures to enhance enzyme liberation. ~° Butanol, 2~ among other solvents, has been used to solubilize particle-bound enzymes. Methods for the solubilization and analysis of the enzymatic components of the cytochrome-containing bacterial particles have, however, not been devised.
Mechanical Rupture of Cells Mechanical rupture of the rigid cell wall is employed as a means of liberating particularly the soluble enzymes, which presumably occur in the cell cytoplasm. Such procedures also fragment the more friable cell membrane but do not usually remove the particle-bound enzymes from their cellular components. These more rigorous methods are used for cells which do not yield extracts after autolysis or drying and are not subject to known specific enzyme treatment. ~,22Most mechanical metbods of cell rupture combine techniques, such as the addition of abrasives, freezing the cell mass--with the ice crystals serving as abrasive--or the combination of crushing and shearing forces. The mechanical rupture of microbial cells to yield active enzyme preparations will be discussed under the following headings: 1. Mechanical pressure; hydraulic or fly press. 2. Pressure release; rapid release of compressed gas. 3. Sonic or ultrasonic waves; tuned magnetostriction or piezoelectric oscillator. 4. Mechanical shaking with abrasives; tuning fork or blendorhomogenizer. 5. Grinding; mechanical or manual. Among the abrasives most commonly used with these procedures are fine quartz sand, powdered Pyrex glass, micro-size ballotini or reflector beads, Carborundum, jeweler's rouge, powdered dry ice, and alumina. The most effective particle size is 500-mesh or finer; uniform particle size affords more effective cell fragmentation. In the use of all the abrasives tested, except alumina, mechanical forces seem to predominate. With alumina a chemical process seems also to be involved. 23,24 The use of abrasives, particularly alumina, will be discussed in greater detail below. j. Berger, M. J. Johnson, and W. H. Peterson, Enzymologia 4, 31-35 (1937). 31 R. K. Morton, Nature 166, 1092 (1950). 2~L. H. Stickland, Biochem. J. 23, 1187 (1929). 23.TH. McIlwain, J. Gen. Microbiol. 2, 288 (1948). 24O. Hayaishi~and R. Y. Stanier, J. Baeteriol. 62, 691 (1951). s0
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EXTRACTION OF ENZYMES FROM MICROORGANISMS
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Mechanical Pressure. Two means of preparing microbial "iuices" or cell extracts employing hydraulic pressure have been employed. Buchner and Hahn's (1903) 2~ preparation of yeast zymase, by applying hydraulic pressure to a mixture of yeast and kieselguhr, constituted an important step forward in establishing on a firm basis the enzyme concept. An excellent review of these early studies, including grinding with quartz sand and preparation of extracts from dried cells and from fresh yeast by hydraulic pressure, appears in Chapter 2 of Harden's monograph, "Alcoholic Fermentation. ''9 Buchner and Hahn mixed kieselguhr with 3 to 4 parts of yeast paste, subjected the mass to hydraulic pressure, and were able to collect 30 to 50% of the cell volume as an extract-zymase. Such concentrated extracts were used in early experiments, before diffusible cofactor requirements were recognized as one of the prime causes for loss of activity on dilution. The hydraulic press, although useful, has not been employed extensively with bacteria and seems not to be readily applicable to larger quantities of cells. More recently, Hughes 2Bhas introduced the use of a fly press to apply instantaneously pressures up to 10 to 15 tons per square inch. The cells may be mixed with an abrasive or cooled to - 2 0 to - 3 0 °, in which case the ice crystals serve as abrasive. Rupture of a very high percentage of the cells has been reported3.~6--up to 99% of the ceils in Escherichia coli pastes. Bacterial endospores have yielded enzyme preparations by repeated treatment in the Hughes press, interspersed with alternate freezing and thawing. 27 Pressure Release. The rupture of microorganisms by the sudden release of pressure after compressing a water-soluble gas has recently been reinvestigated by Fraser ~8for the release of bacteriaphage and of enzymes from E. coll. Fraser's procedure can be applied to small volumes, 5 to 10 ml., and to dilute cell suspensions, 108 cells per milliliter. A single decompression passage using nitrous oxide at pressures up to 900 psi ruptured.more than 75 % of the cells in an E. coli suspension. The method can be applied to heavier cell suspensions, but the percentage of cells ruptured decreases. Some organisms are refractory even to several decompression passages. This procedure has been especially useful in releasing particulate cellular components, including bacteriaphage. Sonic Waves. Treatment of cell suspensions at sonic frequencies in the Raytheon 50-watt 9-kc. or 200-watt 10-kc. magnetostriction oscillator, ~ E. Buchner and H. Haehn, " D i e Zymase Garung," p. 58. R. Oldenburg, Munich, 1903; see also ref. 9, p. 23. ~e D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951). ~7 M. R. Pollock, J. Gen. Microbiol. 8, 186 (1953). ~*D. Fraser, Nature 167, 33 (1951).
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GENERAL P R E P A R A T ~ E PROCEDURES
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either alone or with finely powdered abrasives, has simplified and accelerated studies with bacterial extracts. The two Raytheon instruments are similar except for power output and size of the sample cup. The suspension is treated in a cylindrical stainless steel, water-cooled cup, to which the power is applied through a bottom diaphragm activated by a laminated nickel rod held in an oscillating magnetic field. The instrument is " t u n e d " for maximum energy input, but to date no means of measuring the power actually delivered to the liquid in the treatment cup has been devised--thus each treatment is more or less empiricaI. The treatment cup of the 9-kc. machine has a total volume of 60 ml., in which 25 to 35 ml. of cell suspension can be treated satisfactorily; the 10-kc. cup volume is 150 ml., in which 60 to 100 ml. can be treated. The best disintegration has been obtained with cell suspensions containing 50 to 100 rag. dry weight per milliliter; thus the 10-kc. machine will allow treatment of 2 to 10 g. of dry cells. Gram-negative rods (pseudomonads or coli-aerogenes types) are readily ruptured--a 10- to 15-minute treatment in the ]0-kc. machine will break more than 90% of the cells--thus allowing treatment of up to 25 to 50 g. of cells per hour. The Gram-positive organisms of the Bacillus and Clostridium genera are also readily ruptured, but the lactic acid bacteria, the staphylococci and the corynebacteria, are more refractory, frequently requiring periods of 30 to 60 minutes in the 10-kc. machine, and as long as 3 hours in the 9-kc. machine. In some cases even this extended treatment releases less than half of the enzyme content in soluble form, the remainder being recoverable in the cell residue. 17 Yeast cells and bacterial endospores are more refractory to rupture in the Raytheon equipment, only limited success having been obtained to date. The use of a quartz (piezoelectric) crystal employing supersonic frequencies up to 600 kc. previously employed ~9 has proved less effective than the magnetostriction method because of the small volumes which may be employed, the critical nature of the tuning, the relatively dilute suspensions which may be successfully treated, and the extended period of treatment--up to 4 hours--required. Mechanical Shaking with Abrasives. Another method applied to cell suspensions is rapid shaking--300 to 3000 oscillations a minute---with regular small particles (50 to 500 ~). Important variables are the size and uniformity of the particles, the speed of shaking, and the nature and density of the cell suspension. 3° Mechanical shaking with a Mickle 3~ or 39 p. K. Stumpf, D. E. Green, and F. W. Smith, J. Bacteriol. 51, 487 (1946). ~0 G. Furness, J. Gen. Microblol. 7, 335 (1952), P. D. Cooper, J. Gen. Micro~iol. 9, 199
(1953). al H. Mickle, J. Roy. Microscop. Soc. 65, 10 (1948).
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Microid 3° mechanical shaker, using ballotini beads, grade 9 to 16 (average diameter 300 to 100 u), at cell densities up to 2 to 5 mg. dry weight per milliliter, has been used to liberate enzymes and to prepare cell walls for examination2 °.32 The Mickle disintegrator (H. Mickle, Hampton, Middlesex) carries two 15-ml. cups, each mounted on the end of an electrically driven tuning fork, and the Microid shaker (Griffin & Tatlock, London) carries four 3 X 14-cm. (about 100-ml. volume) cups at speeds up to about 2500 oscillations per minute, or two cups at speeds to 3000 oscillations per minute. There are few data available on enzyme extraction by either of these procedures, but those available indicate promise. Lamanna and Mallette 3~ have recently reported the rupture of dense cell suspensions, particularly yeast, by disintegration through mechanical agitation in the presence of grade 10 glow beads (60 to 80 mesh, 200-mu average diameter), obtainable from Minnesota Mining & Manufacturing Company, Minneapolis. For this purpose they employed a Waring blendor or a Virtis homogenizer. As typical examples, 99 % of the cells in a pound of yeast were ruptured in the presence of 800 g. of beads in a total volume of approximately 1 1. by 90-minute treatment in the Waring blendor. To avoid surface denaturation by foaming, a paraffin block carved to fit the top of the Waring cup was pressed tightly against the liquid surface. With the Virtis homogenizer, which operates at higher speeds, 99% of the cells of a 1.5-ml. E. coli suspension, containing 4 X 10J° organisms per milliliter and 2 g. of beads, were ruptured after 5 minutes' treatment. Heat generated by either of these instruments can be dissipated by water cooling--the micro cup of the Waring blendor has been ice-cooled by filling with crushed ice an 8- to 10-inch cone soldered to its base. Lamanna and Mallette give no data on enzyme extraction--presumably the enzymes would be liberated and not destroyed by these procedures. Grinding. Cell suspensions have been milled mechanically by feeding continuously through a Booth-Green mill, ~4which employs a motor-driven cone and close-fitting roller bearing which turns a machined race. The heat generated is dissipated by circulating the cell suspension with a small metering pump through a heat exchanger submerged in ice water. This procedure, useful in early experiments, has now been largely replaced by more convenient methods which may be applied to larger quantities of cells. As a typica~ example of the Booth-Green procedure, 2 hours were required to extract E. coli or Bacillus subtilis enzymes and as much as 4 hours for refractory organisms such as Sarcina lutea. ~2 M. R. J. Sa|ton and R. W. Home, Biochim. et Biophys. Acta 7, 177 (1951). ~ C. L a m a n n a and M. F. Mallette, J. Bacteriol. 67, 503 (1954). 3~ B. H. Booth and D. E. Green, Biochem. J. 32, 855 (1938).
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GENERAL PREPARATIVE PROCEDURES
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The majority of the grinding procedures now in use are applied to (1) cell pastes, (2) frozen cell pastes, or (3) dried cells. With both hand and mechanical grinding, abrasives are usually added. As with the hydraulic press, the most effective abrasives are of small, uniform particle size and appear to exert a mechanical effect in the rupture of the cells. Freezing procedures, and the addition of dry ice, appear also to involve abrasive action of the ice, or the dry ice crystals; a single exception, the use of powdered alumina, introduced by McIlwain, 23 appears also to exert a chemical action. ~4 Cell pastes mixed with powdered Pyrex or, more recently, with other abrasives, have been ground by forcing the mixture between a motordriven, ice-filled cone and a close-fitting funnel. 35 In early studies this grinding method was used to prepare concentrated extracts which were of considerable value in establishing pathways in microbial systems. More recently, alumina grinding and the use of sonic oscillators have somewhat replaced this method because of the rather laborious nature of the method, the difficulty in collecting the ground celIs without loss, and the necessity for recycling if a high percentage of the ceils is to be ruptured. It is possible that the use of uniform-size, small glass beads or other fine-mesh abrasives, including alumina, would enhance the effectiveness and thereby the usefulness of this procedure. McIlwain suggests that one criterion for effective grinding is uniform particle size and particles of a size approximating that of the cells to be ruptured. With the introduction of alumina, it was found that cell pastes to which one or two weights of alumina, 500 mesh or finer, had been added could be ruptured by hand grinding for 2 to 5 minutes with a chilled mortar and pestle, 23 in many cases liberating more than 80% of an enzymatic activity measurable in the cell suspension or dried cells. An empirical but convenient method of determining when the paste has been ground sufficiently is to observe a change to a darker, more viscous (tacky), somewhat moister preparation. With further experience, the grade and exact size of alumina particles seem not to be highly important, so long as the alumina used does not contain excess alkalinity. McIlwain suggests Griffin and Tatlock's "microid polishing alumina" which has been washed and dried at 100°. More recently in this country, "levigated alumina," obtainable from Buehler, Ltd., Metallurgical Apparatus, Evanston, Illinois, ~4 or Alcoa A-301, - 3 2 5 mesh, or A-303 alumina, obtainable from Aluminum Company of America, Chemicals Division, Pittsburgh, Pennsylvania, have been used with equal success. After grinding cell pastes, or dried cells, to which buffer or water has 35 G. Kalnitsky, M. J. Utter, and C. H. Werkraan, J. Bacteriol. 49, 595 (1945).
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been added along with alumina, the thick paste can be diluted gradually to 50 to 100 mg. dry weight of cells per milliliter, and the alumina, unruptured cells, and cell walls removed by centrifugation at 10,000 to 12,000 r.p.m, in an SS-1 ServaU centrifuge or other centrifuge giving approximately the same gravitational field--that is, 15,000 to 20,000 X g. Differential centrifugation has been applied to separate unbroken cells and cell walls from the fragmented amber fraction, presumably the cell membrane, which contains the hydrogen transport and some of the substrate dehydrogenase enzymes26 Centrifugation of ground cells to separate debris is best accomplished by dilution of the extracts to 50 to 100 mg. dry cell weight per milliliter to reduce the density, and particularly the viscosity, sufficiently to allow effective separation. The fractionation of bacterial extracts proceeds best after removal of the nucleic acids--otherwise sharp separations are not possible. 36,37 The problems encountered in fractionation of microbial extracts will be dealt with elsewhere. Dried cells can also be ruptured by alumina grinding with a mortar and pestle, followed by an addition of a suitable buffer and continued grinding. By this procedure active extracts of both easily rupture cells, i.e., E. coli, ~7 and more difficultly ruptured cells, such as Streptococcus faecalis, 17 have been prepared. From 100 mg. dry weight of E. coil, 15 to 20 mg. of protein is extractable; from S. faecalis, 8 to 10 mg. These extracts are subject to fractionation after removal of the nucleic acids. Both organisms, as dry powders, can be similarly ruptured by grinding with powdered dry ice with analogous yields of protein, but so far these extracts have resisted purification. Other Procedures. For special cases a vacuum ball mill 8s has yielded active cell extracts, particularly of those enzymes subject to air oxidation. Similar procedures have been employed to obtain labile antigens, frequently by submerging the ball mill in a dry ice-solvent mixture to render the cells more brittle. Enzymatic methods have been employed in very few cases, owing to the limited variety of cells which can be ruptured with specific enzymes, as for example, lysozyme. 1 It may well prove that cell wall hydrolyzing enzymes, or even proteolytic enzymes, will come into wider use as these methods are explored. ~ Chemical lysis has so far been relegated to agents such as glycine, 86 I. C. Gunsalus, R. Y. Stanier, and C. F. Gunsalus, J. Bacteriol. 66, 535, 548 (1953). a7 S. Korkes, A. del Campillo, I. C. Gunsalus, and S. Ochoa, J. Biol. Chem. 195~ 721 (1951). 38 W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques," p. 129. Burgess Press, Minneapolis, 1949.
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GENERAL PREPARATIVE PROCEDURES
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which exerts generalized action on proteins, often with denaturation. Recently, polyelectrolytes (i.e., a dimethyl aminoethyl acryllate polymer) active in low concentration have been introduced by Puck 39 for the liberation of bacteriaphage and promise also to be useful in the liberation of enzymes. These methods have not, however, been explored sufficiently to allow general application. s0 T. T. Puck, Cold Spring Harbor Symposia Quant. Biol. 18, 153 (1953).
[8] Extraction of Soluble Enzymes from H/gher Plants By ALVIN NASON Preparation
Principle. The procedures outlined below are intended to extract soluble protein containing maximum enzymatic activity from plant cells under conditions least favorable for protein denaturation. The use of a buffered extracting solution, generally ranging on the alkaline side, is usually desirable. The addition of sulfhydryl compounds, other reducing substances, or metal-binding agents may enhance the extraction, activity, and stability of some enzymes, depending on their properties. 1 Highspeed centrifugation is necessary to free the extract of chloroplast fragments, grana, and mitochondria. Reagents 0.1 M potassium phosphate buffer, pH 7.5, with or without 10-3 M cysteine or Versene (ethylenediaminetetraacetic acid). Chilled acetone ( - 15°). Procedures. The variety of procedures which have been used for obtaining soluble cell-free enzyme extracts from higher plants can be classified into the following three main groups. In most cases either fresh or frozen tissue may be used as starting material, depending on the activity obtained in the final preparation. In the case of seed materials, the seed may be soaked overnight by covering with tap water and then extracting essentially by the methods below; or the dried seeds may first be ground or milled to a powder and then extracted essentially by method 2. METHOD 1. Extracts of pulpy tissues such as storage organs (e.g., the carrot or potato) can be prepared by first passing the tissue through a meat grinder. This is followed by pressing out the juice by hand or mechanical press through muslin or three to four layers of cheesecloth. An active extract of starch phosphorylase has been prepared from potato 1See M. Gibbs, Vol. I [62].
EXTRACTION OF ENZYMES FROM MICROORGANISMS
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the method has so far been applied mostly with animal tissues, the procedures described above should be just as suitable for obtaining active enzymes from plant tissues, yeasts, and bacteria. [7] E x t r a c t i o n of E n z y m e s f r o m M i c r o o r g a n i s m s
(Bacteria and Yeast)
By I. C. GUNSALTJS Principle The methods which have proved effective in liberating enzymes from microbial cells have been largely mechanical rupture of the cell wall and membrane, frequently with fragmentation of the latter. In specific instances enzymatic, ~ including autolysis, and chemical 2 treatments have proved useful. The choice of procedure will depend on the particular species or strain of microorganism used, including the ease with which the cell is ruptured, the quantity of enzyme required, and the type of preparation from which the extract is prepared. The choice will also vary with the sensitivity and the localization of the enzyme within the cell. Most methods in use were empirically derived; thus, consulting the original literature in specific cases is useful--for example, a recent review by Hugo z carries more than a hundred references to specific cases. Bacterial and yeast cells for the preparation of enzyme extracts may be obtained as products of the fermentation industries; that is, brewer's yeast as sludge from a spent fermentation tank, baker's yeast in pound cakes or in bulk--usually starch-free--from the baking or food industries. A few bacterial strains are available from pharmaceutical procedures which do not impair enzyme activity. More frequently, however, bacterial cells are grown by the investigator, usually under conditions which support optimum growth or a maximum enzyme yield. 4 In the latter case, the age and enzymatic content of the cells, the means of harvest--usually Sharples continuous centrifuge--and the type of cell preparation from which the extract is prepared can be controlled. For small quantities, batch centrifugation may be employed, in which case aeration during harvest can also be controlled, should this be important to the investiga' M. Penrose and J. H. Quastel, Proc. Roy. Sac. (London) B107, 168 (1930); M. F. Utter, L. O. Krampitz, and C. H. Werkman, Arch. Biochem. 9, 285 (1946). P. V. B. Cowles, Yale J. Biol. and Med. 19, 835 (1947). W. B. Hugo, Baderiol. Revs. 18, 87 (1954). 4 A. J. Wood and I. C. Gunsalus, J. Bacteriol. 44, 333 (1942) ; E. F. Gale, Bacteriol. Revs. 7, 139 (1943).
52
GENERAL PREPARATIVE PROCEDURES
[7]
tion. In such cases freshly harvested cell paste with a minimum of autolysis is obtained. In the use of freshly harvested or commercially prepared ceU paste, cakes, or sludges, washing may be employed if the experiments so indicate, using caution with aged cells lest the enzymes be extracted by the washing procedure. The cells may be preserved by storage at reduced temperatures, for example, in the deep-freeze or dry ice chest at - 10 to - 40 °; or they may be reduced immediately to extracts, in which state the enzymes are usually stable at deep-freeze temperatures. If neither of these proves convenient, stable dried cell preparations can usually be obtained by air, vacuum, or solvent drying procedures. In summary: The extraction and preservation of the activity of any given enzyme is an empirical process based on the investigator's own or his colleagues' experience. In the absence of example, the original literature offers a wide variety of methods and of organisms used successfully for various enzymes2 Dried Cell Preparations The ease of release of enzymes from cells depends to a great extent on their previous treatment--drying may serve to preserve enzyme activity or may be used as a means of liberating or extracting enzymes. Dried cell preparations frequently have sufficiently lost the "permeability" properties of living cells to serve as enzyme preparations--that is, they will metabolize phosphate esters and polybasic acids and can be activated by coenzymesL-and the coenzymes and metabolic intermediates are often leeched from the cells during the drying procedure or after resuspending in buffers. In many cases the drying procedure involves enzymatic autolysis, which enhances enzyme liberation. Procedure
Dried cell preparations can be obtained by: 1. Air drying--frequently accompanied by autolysis, 2. Slow vacuum drying--usually from cell pastes, also accompanied by autolysis and yielding glass-like preparations which can be later powdered readily by mortar and pestle; 3. Lyophylization--desiccation from the frozen state, usually of a 10 to 20% by wet weight cell suspension; 4. Dehydrating with water-miscible solvents. 5 I. C. Gunsalus and W. W. Umbreit, J. Bacteriol. 49, 347 (1945); I. C. Gunsalus, W. D. Bellamy, and W. W. Umbreit, J. Biol. Chem. 1§5, 685 (1944).
[7]
EXTRACTION OF ENZYMES FROM MICROORGANISMS
53
Air Drying (of Yeast). The finding by Lebedev s that air-dried yeast could be extracted by mild grinding or by stirring with buffer greatly accelerated studies with soluble enzymes. Since Lebedev's finding, many methods have been employed for drying 7 and for extracting 8 air-dried yeast and bacteria. The important factors considered in the early experiments are summarized by Harden2 For more recent data specific manuscripts should be consulted. In general, yeast at the consistency of yeast cake, or ceils solidly packed by centrifugation after washing, are crumbled by hand or by forcing through a 10-mesh sieve or potato dicer onto trays in layers less than an inch deep and allowed to dry 2 to 3 days at temperatures ranging from 25 to 30 °, occasionally as high as 35 to 40 ° . The layer of yeast may be turned at half-day intervals to promote drying; a fan may be employed to accelerate the process. Air-dried yeast has undergone partial autolysis and will usually yield active enzyme extracts if suspended in 3 to 5 vol. of water or suitable buffer and stirred 2 to 3 hours at room temperature, s,9 (Occasionally yeast is pretreated--for example, mild bicarbonate hydrolysis before extraction, s) The cell debris can then be removed by centrifugation, and the enzymes subjected to further purification if desired. If a particular enzyme is not solubilized by the drying procedure, one of the more rigorous methods of extraction may be resorted to. Slow Drying in Vacuum. This procedure has been advantageously applied to enzyme extraction of several types of bacteria.l°-l~ The packed ceils obtained by centrifugation are transferred by a spatula to a beaker or evaporating dish, placed in a desiccator over calcium chloride, P20~ or anhydrous calcium sulfate (Drierite), and the desiccator evacuated with an aspirator or vacuum pump, sealed, and allowed to stand, usually overnight. Occasionally a thick, 20 % wet weight, cell suspension is dried by a similar procedure. The dried preparations are usually of hard, glassy consistency and have undergone some autolysis. The cells may be extracted by suspending directly in buffer but usually more successfully by grinding the dried cell mass to a powder with a mortar and pestle before extraction. If the paste has been deposited in layers more than 0.5 to 1 cm. thick, gentle grindins followed by further drying may be required. More labile enzymes are destroyed by the slow drying procedure; 6 A. yon. Lebedev, Compt. rend. 152, 49 (1911). C. Neuberg and H. Lustig, Arch. Biochem. 1, 191 (1943). 8 B. L. Horeeker and P. Z. Smyrniotis, J. Biol. Chem. 193~ 371 (1951). 9A. Harden, "Alcoholic Fermentation," Chapter 2. Longmans, Green, & Co., London, 1923. 10F. Lipmann, Cold Spring Harbor Symposia Quant. Biol. 7~ 248 (1939). 11E. R. Stadtman and H. A. Barker, J. Biol. Chem. 180, 1085 (1949). 1~B. P. Sleeper, M. Tsuehida, and R. Y. Stanier, J. Baeteriol. 59, 129 (1950).
54
GENERAL PREPARATIVE PROCEDURES
[7]
thus partial reaction sequences may be studied successfully with such preparations. '~ As with air-dried yeast, the soluble enzymes can usually be extracted from the slow vacuum-dried bacterial or yeast preparations by gentle agitation with buffer or water. The incorporation of reducing substances-that is, cysteine, glutathione, or sodium sulfide~--before drying, especially with metabolically anaerobic bacteria, has been helpful in preventing the loss of otherwise labile enzymes. These additions have been particularly beneficial in preserving those enzymes which appear to possess mercapto groups or to be sulfhydryl activated. H Lyophylization. Drying quick-frozen, thick cell suspensions in vacuum has proved especially beneficial in preserving labile enzymes. In fact, a similar procedure on a smaller scale is in common use to preserve stock cultures in a viable state. ~3 For enzyme work, vacuum-dried cells are usually stored in the presence of air in a sufficiently tight container to prevent rehydration. The cells soon lose their viability and in many cases are more permeable to substrates, including cofactors, than are viable cells. 6 Usually, more drastic means are required to extract enzymes from lyophylized than from air or slow vacuum-dried cells, very probably because autolysis does not occur. Lyophylization of masses of cells can be accomplished most conveniently by suspending 10 to 40 % wet weight of cells in distilled water. With some cells a washing step to remove salts and residual media ingredients is desirable to reduce autorespiration (endogenous substrate oxidation); with large batches of cells a quick freeze before drying is also desirable. For preparations up to 10 to 15 g. of cells, a single 100-mm. Petri dish containing 10 to 40 ml. of suspension can be placed over 4- to 8-mesh Drierite in a 250-ram. desiccator and evacuated to less than 0.1 mm. mercury. Under these conditions the rate of evaporation will be rapid enough to cause the suspension to freeze. If the vacuum is preserved--usually it is well to keep the pump running-the preparation will dry without thawing in a period of 6 to 8 hours (usually well to leave overnight) to yield a porous, friable mass which may be reduced to a powder by working gently with a spatula on a clean paper. The enzyme activities in lyophylized cells may remain nearly constant for several years if stored at - 2 0 ° and protected from the re-entry of water. The moisture content of lyophylized cells is frequently less than 0.1%, usually lower than in air-dried preparations. To prepare large batches of lyophylized cells, shell freezing in plasma bottles or round-bottom distillation flasks, up to 2- to 3-1. volume, is useful. The desiccation is usually accomplished by connecting the flask la L. A. Rogers, J. Bac~eriol. 57, 137 (1949) ; L. Atkin, W. Moses, and P. P. Gray, ibid. §7, 575 (1949).
[7]
EXTRACTION OF ENZYMES FROM MICROORGANISMS
55
to a large-diameter, short-path vacuum system containing a mechanically refrigerated, or dry ice-cooled, receiver to remove the water by sublimation. Convenient units for this purpose are available commercially or m a y be fabricated from glass or from metal. 14,15 Acetone (Solvent) Dried Preparations. Large quantities of ceils m a y be conveniently dried by adding an aqueous suspension to a large volume of water-miscible solvent at a sufficiently low temperature to prevent denaturation of the cell proteins. The most common procedure is to add an aqueous, or buffered, cell suspension slowly with vigorous stirring to not less than 10 volumes of acetone previously cooled to - 2 0 ° or to the temperature of dry ice. After a brief stirring the cells are allowed to settle, the supernatant, which frequently contains g u m m y material, is decanted, and the residual solvent is removed on a Buchner filter. The filter cake of ceils is washed on the Buchner with 2 to 5 vol. of - 2 0 ° a c e t o n e - - o r suspended in 2 to 5 vol. of chilled acetone and again collected on a B u c h n e r - and washed once with 2 to 3 vol. of - 2 0 ° peroxide-free ether, sucked dry on the filter, and transferred to a large sheet of wrapping paper and worked gently with a spatula until the solvent has evaporated, leaving a dry powder. If the atmosphere is humid, the dried ceils should not remain in contact with the air for more than a few minutes, and the removal of solvent can be completed, although less satisfactorily, in a desiccator in the presence of paraffin to absorb the solvent. Acetone-dried preparations usually contain no living cells--bacterial endospores being an exception--and retain f e w e r of the permeability properties of the viable cell than do lyophylized preparations.. If freshly prepared cells are acetone-dried, little, if any, autolysis will have occurred. Acetone-dried cells m a y yield active enzyme extracts on stirring with suitable buffer or with water, 18 or more rigorous extraction m a y be required. ~7 Other water-miscible solvents including dioxane ~s have also been employed, although less frequently, to prepare dried cells. Toluene, chloroform, or similar solvents ~9 m a y be used to alter the permeability of ceils, permitting enzyme assays without respect to cell permeability. This type of preparation is seldom subjected to further 14 E. W. Flosdorf, "Freeze-Drying," Reinhold Publishing Co., New York, 1949; Virtis Co., Inc., Yonkers, New York (metal). 15 W. J. Elser, R. A. Thomas, and E. I. Steffen, J. Immunol. 28, 433 (1935); D. H. Campbell and D. Pressman, Science 99, 285 (1944) (glass). 16 E. F. Gale, Advances in Enzymol. 6, 1 (1946); G. W. E. Plaut and H. A. Lardy, J. Biol. Chem. 180, 13 (1949). 17 M. I. Dolin, Thesis, Indiana University, 1950. ~s Procedures similar to J. B. Sumner and A. L. Dounce, J. Biol. Chem. 127, 439 (1939). 19 E. Buchner and R. Rapp, Ber. 80, 2668 (1897); see also ref. 9, p. 36.
56
GENERAL PREPARATIVE PROCEDURES
[7]
purification, even to the extent of removing the enzymes from cellular debris. Toluene may, however, be used in conjunction with grinding procedures to enhance enzyme liberation. ~° Butanol, 2~ among other solvents, has been used to solubilize particle-bound enzymes. Methods for the solubilization and analysis of the enzymatic components of the cytochrome-containing bacterial particles have, however, not been devised.
Mechanical Rupture of Cells Mechanical rupture of the rigid cell wall is employed as a means of liberating particularly the soluble enzymes, which presumably occur in the cell cytoplasm. Such procedures also fragment the more friable cell membrane but do not usually remove the particle-bound enzymes from their cellular components. These more rigorous methods are used for cells which do not yield extracts after autolysis or drying and are not subject to known specific enzyme treatment. ~,22Most mechanical metbods of cell rupture combine techniques, such as the addition of abrasives, freezing the cell mass--with the ice crystals serving as abrasive--or the combination of crushing and shearing forces. The mechanical rupture of microbial cells to yield active enzyme preparations will be discussed under the following headings: 1. Mechanical pressure; hydraulic or fly press. 2. Pressure release; rapid release of compressed gas. 3. Sonic or ultrasonic waves; tuned magnetostriction or piezoelectric oscillator. 4. Mechanical shaking with abrasives; tuning fork or blendorhomogenizer. 5. Grinding; mechanical or manual. Among the abrasives most commonly used with these procedures are fine quartz sand, powdered Pyrex glass, micro-size ballotini or reflector beads, Carborundum, jeweler's rouge, powdered dry ice, and alumina. The most effective particle size is 500-mesh or finer; uniform particle size affords more effective cell fragmentation. In the use of all the abrasives tested, except alumina, mechanical forces seem to predominate. With alumina a chemical process seems also to be involved. 23,24 The use of abrasives, particularly alumina, will be discussed in greater detail below. j. Berger, M. J. Johnson, and W. H. Peterson, Enzymologia 4, 31-35 (1937). 31 R. K. Morton, Nature 166, 1092 (1950). 2~L. H. Stickland, Biochem. J. 23, 1187 (1929). 23.TH. McIlwain, J. Gen. Microbiol. 2, 288 (1948). 24O. Hayaishi~and R. Y. Stanier, J. Baeteriol. 62, 691 (1951). s0
[7]
EXTRACTION OF ENZYMES FROM MICROORGANISMS
57
Mechanical Pressure. Two means of preparing microbial "iuices" or cell extracts employing hydraulic pressure have been employed. Buchner and Hahn's (1903) 2~ preparation of yeast zymase, by applying hydraulic pressure to a mixture of yeast and kieselguhr, constituted an important step forward in establishing on a firm basis the enzyme concept. An excellent review of these early studies, including grinding with quartz sand and preparation of extracts from dried cells and from fresh yeast by hydraulic pressure, appears in Chapter 2 of Harden's monograph, "Alcoholic Fermentation. ''9 Buchner and Hahn mixed kieselguhr with 3 to 4 parts of yeast paste, subjected the mass to hydraulic pressure, and were able to collect 30 to 50% of the cell volume as an extract-zymase. Such concentrated extracts were used in early experiments, before diffusible cofactor requirements were recognized as one of the prime causes for loss of activity on dilution. The hydraulic press, although useful, has not been employed extensively with bacteria and seems not to be readily applicable to larger quantities of cells. More recently, Hughes 2Bhas introduced the use of a fly press to apply instantaneously pressures up to 10 to 15 tons per square inch. The cells may be mixed with an abrasive or cooled to - 2 0 to - 3 0 °, in which case the ice crystals serve as abrasive. Rupture of a very high percentage of the cells has been reported3.~6--up to 99% of the ceils in Escherichia coli pastes. Bacterial endospores have yielded enzyme preparations by repeated treatment in the Hughes press, interspersed with alternate freezing and thawing. 27 Pressure Release. The rupture of microorganisms by the sudden release of pressure after compressing a water-soluble gas has recently been reinvestigated by Fraser ~8for the release of bacteriaphage and of enzymes from E. coll. Fraser's procedure can be applied to small volumes, 5 to 10 ml., and to dilute cell suspensions, 108 cells per milliliter. A single decompression passage using nitrous oxide at pressures up to 900 psi ruptured.more than 75 % of the cells in an E. coli suspension. The method can be applied to heavier cell suspensions, but the percentage of cells ruptured decreases. Some organisms are refractory even to several decompression passages. This procedure has been especially useful in releasing particulate cellular components, including bacteriaphage. Sonic Waves. Treatment of cell suspensions at sonic frequencies in the Raytheon 50-watt 9-kc. or 200-watt 10-kc. magnetostriction oscillator, ~ E. Buchner and H. Haehn, " D i e Zymase Garung," p. 58. R. Oldenburg, Munich, 1903; see also ref. 9, p. 23. ~e D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951). ~7 M. R. Pollock, J. Gen. Microbiol. 8, 186 (1953). ~*D. Fraser, Nature 167, 33 (1951).
58
GENERAL P R E P A R A T ~ E PROCEDURES
[7]
either alone or with finely powdered abrasives, has simplified and accelerated studies with bacterial extracts. The two Raytheon instruments are similar except for power output and size of the sample cup. The suspension is treated in a cylindrical stainless steel, water-cooled cup, to which the power is applied through a bottom diaphragm activated by a laminated nickel rod held in an oscillating magnetic field. The instrument is " t u n e d " for maximum energy input, but to date no means of measuring the power actually delivered to the liquid in the treatment cup has been devised--thus each treatment is more or less empiricaI. The treatment cup of the 9-kc. machine has a total volume of 60 ml., in which 25 to 35 ml. of cell suspension can be treated satisfactorily; the 10-kc. cup volume is 150 ml., in which 60 to 100 ml. can be treated. The best disintegration has been obtained with cell suspensions containing 50 to 100 rag. dry weight per milliliter; thus the 10-kc. machine will allow treatment of 2 to 10 g. of dry cells. Gram-negative rods (pseudomonads or coli-aerogenes types) are readily ruptured--a 10- to 15-minute treatment in the ]0-kc. machine will break more than 90% of the cells--thus allowing treatment of up to 25 to 50 g. of cells per hour. The Gram-positive organisms of the Bacillus and Clostridium genera are also readily ruptured, but the lactic acid bacteria, the staphylococci and the corynebacteria, are more refractory, frequently requiring periods of 30 to 60 minutes in the 10-kc. machine, and as long as 3 hours in the 9-kc. machine. In some cases even this extended treatment releases less than half of the enzyme content in soluble form, the remainder being recoverable in the cell residue. 17 Yeast cells and bacterial endospores are more refractory to rupture in the Raytheon equipment, only limited success having been obtained to date. The use of a quartz (piezoelectric) crystal employing supersonic frequencies up to 600 kc. previously employed ~9 has proved less effective than the magnetostriction method because of the small volumes which may be employed, the critical nature of the tuning, the relatively dilute suspensions which may be successfully treated, and the extended period of treatment--up to 4 hours--required. Mechanical Shaking with Abrasives. Another method applied to cell suspensions is rapid shaking--300 to 3000 oscillations a minute---with regular small particles (50 to 500 ~). Important variables are the size and uniformity of the particles, the speed of shaking, and the nature and density of the cell suspension. 3° Mechanical shaking with a Mickle 3~ or 39 p. K. Stumpf, D. E. Green, and F. W. Smith, J. Bacteriol. 51, 487 (1946). ~0 G. Furness, J. Gen. Microblol. 7, 335 (1952), P. D. Cooper, J. Gen. Micro~iol. 9, 199
(1953). al H. Mickle, J. Roy. Microscop. Soc. 65, 10 (1948).
[7]
EXTRACTION OF ENZYMES FROM MICROORGANISMS
59
Microid 3° mechanical shaker, using ballotini beads, grade 9 to 16 (average diameter 300 to 100 u), at cell densities up to 2 to 5 mg. dry weight per milliliter, has been used to liberate enzymes and to prepare cell walls for examination2 °.32 The Mickle disintegrator (H. Mickle, Hampton, Middlesex) carries two 15-ml. cups, each mounted on the end of an electrically driven tuning fork, and the Microid shaker (Griffin & Tatlock, London) carries four 3 X 14-cm. (about 100-ml. volume) cups at speeds up to about 2500 oscillations per minute, or two cups at speeds to 3000 oscillations per minute. There are few data available on enzyme extraction by either of these procedures, but those available indicate promise. Lamanna and Mallette 3~ have recently reported the rupture of dense cell suspensions, particularly yeast, by disintegration through mechanical agitation in the presence of grade 10 glow beads (60 to 80 mesh, 200-mu average diameter), obtainable from Minnesota Mining & Manufacturing Company, Minneapolis. For this purpose they employed a Waring blendor or a Virtis homogenizer. As typical examples, 99 % of the cells in a pound of yeast were ruptured in the presence of 800 g. of beads in a total volume of approximately 1 1. by 90-minute treatment in the Waring blendor. To avoid surface denaturation by foaming, a paraffin block carved to fit the top of the Waring cup was pressed tightly against the liquid surface. With the Virtis homogenizer, which operates at higher speeds, 99% of the cells of a 1.5-ml. E. coli suspension, containing 4 X 10J° organisms per milliliter and 2 g. of beads, were ruptured after 5 minutes' treatment. Heat generated by either of these instruments can be dissipated by water cooling--the micro cup of the Waring blendor has been ice-cooled by filling with crushed ice an 8- to 10-inch cone soldered to its base. Lamanna and Mallette give no data on enzyme extraction--presumably the enzymes would be liberated and not destroyed by these procedures. Grinding. Cell suspensions have been milled mechanically by feeding continuously through a Booth-Green mill, ~4which employs a motor-driven cone and close-fitting roller bearing which turns a machined race. The heat generated is dissipated by circulating the cell suspension with a small metering pump through a heat exchanger submerged in ice water. This procedure, useful in early experiments, has now been largely replaced by more convenient methods which may be applied to larger quantities of cells. As a typica~ example of the Booth-Green procedure, 2 hours were required to extract E. coli or Bacillus subtilis enzymes and as much as 4 hours for refractory organisms such as Sarcina lutea. ~2 M. R. J. Sa|ton and R. W. Home, Biochim. et Biophys. Acta 7, 177 (1951). ~ C. L a m a n n a and M. F. Mallette, J. Bacteriol. 67, 503 (1954). 3~ B. H. Booth and D. E. Green, Biochem. J. 32, 855 (1938).
60
GENERAL PREPARATIVE PROCEDURES
[7]
The majority of the grinding procedures now in use are applied to (1) cell pastes, (2) frozen cell pastes, or (3) dried cells. With both hand and mechanical grinding, abrasives are usually added. As with the hydraulic press, the most effective abrasives are of small, uniform particle size and appear to exert a mechanical effect in the rupture of the cells. Freezing procedures, and the addition of dry ice, appear also to involve abrasive action of the ice, or the dry ice crystals; a single exception, the use of powdered alumina, introduced by McIlwain, 23 appears also to exert a chemical action. ~4 Cell pastes mixed with powdered Pyrex or, more recently, with other abrasives, have been ground by forcing the mixture between a motordriven, ice-filled cone and a close-fitting funnel. 35 In early studies this grinding method was used to prepare concentrated extracts which were of considerable value in establishing pathways in microbial systems. More recently, alumina grinding and the use of sonic oscillators have somewhat replaced this method because of the rather laborious nature of the method, the difficulty in collecting the ground celIs without loss, and the necessity for recycling if a high percentage of the ceils is to be ruptured. It is possible that the use of uniform-size, small glass beads or other fine-mesh abrasives, including alumina, would enhance the effectiveness and thereby the usefulness of this procedure. McIlwain suggests that one criterion for effective grinding is uniform particle size and particles of a size approximating that of the cells to be ruptured. With the introduction of alumina, it was found that cell pastes to which one or two weights of alumina, 500 mesh or finer, had been added could be ruptured by hand grinding for 2 to 5 minutes with a chilled mortar and pestle, 23 in many cases liberating more than 80% of an enzymatic activity measurable in the cell suspension or dried cells. An empirical but convenient method of determining when the paste has been ground sufficiently is to observe a change to a darker, more viscous (tacky), somewhat moister preparation. With further experience, the grade and exact size of alumina particles seem not to be highly important, so long as the alumina used does not contain excess alkalinity. McIlwain suggests Griffin and Tatlock's "microid polishing alumina" which has been washed and dried at 100°. More recently in this country, "levigated alumina," obtainable from Buehler, Ltd., Metallurgical Apparatus, Evanston, Illinois, ~4 or Alcoa A-301, - 3 2 5 mesh, or A-303 alumina, obtainable from Aluminum Company of America, Chemicals Division, Pittsburgh, Pennsylvania, have been used with equal success. After grinding cell pastes, or dried cells, to which buffer or water has 35 G. Kalnitsky, M. J. Utter, and C. H. Werkraan, J. Bacteriol. 49, 595 (1945).
[7]
EXTRACTION OF ENZYMES FROM MICROORGANISMS
61
been added along with alumina, the thick paste can be diluted gradually to 50 to 100 mg. dry weight of cells per milliliter, and the alumina, unruptured cells, and cell walls removed by centrifugation at 10,000 to 12,000 r.p.m, in an SS-1 ServaU centrifuge or other centrifuge giving approximately the same gravitational field--that is, 15,000 to 20,000 X g. Differential centrifugation has been applied to separate unbroken cells and cell walls from the fragmented amber fraction, presumably the cell membrane, which contains the hydrogen transport and some of the substrate dehydrogenase enzymes26 Centrifugation of ground cells to separate debris is best accomplished by dilution of the extracts to 50 to 100 mg. dry cell weight per milliliter to reduce the density, and particularly the viscosity, sufficiently to allow effective separation. The fractionation of bacterial extracts proceeds best after removal of the nucleic acids--otherwise sharp separations are not possible. 36,37 The problems encountered in fractionation of microbial extracts will be dealt with elsewhere. Dried cells can also be ruptured by alumina grinding with a mortar and pestle, followed by an addition of a suitable buffer and continued grinding. By this procedure active extracts of both easily rupture cells, i.e., E. coli, ~7 and more difficultly ruptured cells, such as Streptococcus faecalis, 17 have been prepared. From 100 mg. dry weight of E. coil, 15 to 20 mg. of protein is extractable; from S. faecalis, 8 to 10 mg. These extracts are subject to fractionation after removal of the nucleic acids. Both organisms, as dry powders, can be similarly ruptured by grinding with powdered dry ice with analogous yields of protein, but so far these extracts have resisted purification. Other Procedures. For special cases a vacuum ball mill 8s has yielded active cell extracts, particularly of those enzymes subject to air oxidation. Similar procedures have been employed to obtain labile antigens, frequently by submerging the ball mill in a dry ice-solvent mixture to render the cells more brittle. Enzymatic methods have been employed in very few cases, owing to the limited variety of cells which can be ruptured with specific enzymes, as for example, lysozyme. 1 It may well prove that cell wall hydrolyzing enzymes, or even proteolytic enzymes, will come into wider use as these methods are explored. ~ Chemical lysis has so far been relegated to agents such as glycine, 86 I. C. Gunsalus, R. Y. Stanier, and C. F. Gunsalus, J. Bacteriol. 66, 535, 548 (1953). a7 S. Korkes, A. del Campillo, I. C. Gunsalus, and S. Ochoa, J. Biol. Chem. 195~ 721 (1951). 38 W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques," p. 129. Burgess Press, Minneapolis, 1949.
62
GENERAL PREPARATIVE PROCEDURES
~]
which exerts generalized action on proteins, often with denaturation. Recently, polyelectrolytes (i.e., a dimethyl aminoethyl acryllate polymer) active in low concentration have been introduced by Puck 39 for the liberation of bacteriaphage and promise also to be useful in the liberation of enzymes. These methods have not, however, been explored sufficiently to allow general application. s0 T. T. Puck, Cold Spring Harbor Symposia Quant. Biol. 18, 153 (1953).
[8] Extraction of Soluble Enzymes from H/gher Plants By ALVIN NASON Preparation
Principle. The procedures outlined below are intended to extract soluble protein containing maximum enzymatic activity from plant cells under conditions least favorable for protein denaturation. The use of a buffered extracting solution, generally ranging on the alkaline side, is usually desirable. The addition of sulfhydryl compounds, other reducing substances, or metal-binding agents may enhance the extraction, activity, and stability of some enzymes, depending on their properties. 1 Highspeed centrifugation is necessary to free the extract of chloroplast fragments, grana, and mitochondria. Reagents 0.1 M potassium phosphate buffer, pH 7.5, with or without 10-3 M cysteine or Versene (ethylenediaminetetraacetic acid). Chilled acetone ( - 15°). Procedures. The variety of procedures which have been used for obtaining soluble cell-free enzyme extracts from higher plants can be classified into the following three main groups. In most cases either fresh or frozen tissue may be used as starting material, depending on the activity obtained in the final preparation. In the case of seed materials, the seed may be soaked overnight by covering with tap water and then extracting essentially by the methods below; or the dried seeds may first be ground or milled to a powder and then extracted essentially by method 2. METHOD 1. Extracts of pulpy tissues such as storage organs (e.g., the carrot or potato) can be prepared by first passing the tissue through a meat grinder. This is followed by pressing out the juice by hand or mechanical press through muslin or three to four layers of cheesecloth. An active extract of starch phosphorylase has been prepared from potato 1See M. Gibbs, Vol. I [62].