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Purification of Myrothecium verrucaria cellulase
Purification
of Myrothecium
verrucariu Cellulase*
D. R. Whitaker From the Division
of Applied
Biology, National Ottawa, Canada
Research Laboratories,
Received November 17, 1952 INTRODUCTION Current knowledge of the mechanism of cellulose breakdown by enzymes has been based on the properties of crude or slightly purified enzyme preparations or of cellulolytic organisms. On such evidence it has been often postulated that cellulose is hydrolyzed by a complex of two or more enzymes working in sequence (1). Among the organisms claimed to have a multienzyme cellulase system is the mold Myrothecium verrucariu (Alb. and Schw.) Ditm. ex Fr. (2, 3). The United States Department of Agriculture (USDA) strain No. 1334.2 of this mold has been used extensively as a test organism for studies on cellulose breakdown by fungi (1). The object of the present work was to purify the cellulolytic enzyme system of M. verrucaria. For reasons given later, this system is designated simply as cellulase. A note on some of the methods used has been published previously (4). METHODS AND RESULTS 1. Production of Enzyme The enzyme was obtained from culture filtrates of Myrothecium verrucuria, strain USDA 1334.2. Procedures for culturing this organism for cellulase production have been described by Greathouse (5) and by Saunders et al. (6). The culture media used in the present work (Table I) were based partly on that of Saunders et al. (6). The cellulose substrate was a cotton linters (Grade 27, Hercules Powder Co.) ground in a Wiley mill to pass a l-mm. mesh sieve, extracted with benzene-alcohol (2: 1 v/v), washed with ethanol, and vacuum dried. All media were autoclaved 1 Issued as National
Research
Council
253
No. 2942.
254
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WHITAKER
at 15 lb. steam pressure: the tubes of cellulose-agar for 20 min., the flasks of shake culture medium for 30 min., and t’he flasks of submerged culture medium for 1 hr. The mold was cultured by the following procedure. Slopes of cellulose-agar in 8 X 1 in. tubes were inoculated with spores of M. verrucaria and incubated at 30°C. for 5 days. Each slope was then flooded with 25 ml. of sterile water and the spores and mycelium scraped into suspension with a sterile knife. The suspension from each slope was added to a l-l. Erlenmeyer shake flask containing 309 ml. of shake culture medium. To increase the agitation during shaking, the shake flasks were baffled (Fig. la), each baffle being about 1% in. long and 34 in. high. The flasks were incubated at 30°C. for 7 days on a rotary shaker which described a horizontal circle of 1 in. diameter at 113 r.p.m. The contents of each flask were TABLE Composition Shake culture
I
of Media
per Liter
medium :
Cotton linters NHaNOa NaNOa KHzPOa KzHPOd NaH2POd*H20 Na2HPOd MgSOd.7H20
.s. 30 0.60 3.80 0.20 0.15 2.09 1.50 0.30
w. 0.054 0.06 0.2 2.0 0.08 0.4 1.2 0.04
Submerged culture medium: Shake culture medium plus 1.0 g. glucose. Cellulose-agar : Shake culture medium plus 15 g. agar. then added to a 12-1. flask (Fig. lb) containing 6 1. of submerged culture medium. These flasks, in batches of six, were incubated at 30°C. for 12 days with vigorous aeration and rotary shaking. The air for the flasks was washed and partially saturated with water by passage through a coarse-porosity sintered-glass disk at the bottom of a 9-l. bottle containing 6 1. of approximately 0.02 N sulfuric acid, and then passed through a spray trap to a manifold connected to the flasks. The conditions of rotary shaking were the same as for the shake flasks. With flasks of this size, these shaking conditions were sufficient to control foaming. At the end of the incubation period, the medium in each flask was examined microscopically for possible bacterial contamination. It was then filtered through several layers of cheesecloth on a Biichner funnel to remove the bulk of the mold mycelium and residual cellulose. The filtrate from six flasks varied in volume from 23 to 31 1. It was chilled to about 5°C. and clarified in a Sharples supercentrifuge (medium jet). A 599-ml. sample was withdrawn and freeze-dried. The remainder of the filtrate was stored overnight at -1°C. pending concentration.
MYROTHECIUM
VERRUCARIA
255
CELLULASE
2. Assay of Enzyme Activity Enzyme activity was assayed from the amount of soluble reducing sugar formed when the enzyme was incubated with sub&rate. Two substrates were used, viz., “swollen linters” (7), ground extracted linters swollen in cold alkali under the conditions found by Jgrgensen (8) to cause no degradation of cellulose; and “precipitated cellulose” (7), a partly degraded and finely dispersed cellulose prepared by dissolving
@ FIG. merged
1. Flasks used culture flasks.
in cellulase
production:
A,
shake
culture
flasks
;
B,
sub-
absorbent cotton in sulfuric acid and immediately filtering into ice water. The assays were made against an enzyme standard which, during trials of purification methods on a batch of culture filtrate, was the freezedried culture filtrate of that batch. Assays were made at very low enzyme concentrations to avoid limitation of reaction rate by substrate concentration. Under such conditions, reducing sugar formation is strongly stimulated by very low concentrations of protein (9). Sufficient albumin was therefore added to the assay medium to eliminate any differences
2x
D.
R.
WHITAKER
in protein stimulation due to differences in the non-cellulase content of the enzyme preparations.
protein
The assay medium had the following composition: (a) acetate buffer of pH 5.6 (25°C.) containing Dowicide G (sodium pentachlorophenate) as antiseptic, 10 ml.; (b) crystalline bovine plasma albumin (Armour), 300 pg.; (c) substrate, 200 mg.; (d) enzyme solution; and (e) glass-distilled water to bring the final volume to 20 ml. The acetate buffer was prepared from 0.1 M sodium acetate and 0.1 M acetic acid. Dowicide G (about 0.5 g./l.) was added and the buffer stored at 2”C., the excess antiseptic being filtered off after a few days. The assay medium was incubated for 17 hr. in a rotary-shaken water bath at a temperature of 35 f 0.05%. The assay flasks were 125-ml. Erlenmeyer flasks with baffles at the bottom, each about 1 in. long and >i in. high, similar to those of the shake culture flasks. The stoppers of the flasks were protected from splashing by glass covers. The shaker described a horizontal circle of 236 in. diameter at 70 r.p.m. After incubation, the assay medium was centrifuged at about 2000 r.p.m. and samples of the supernatant solution (1 ml. for precipitated cellulose, 3 ml. for swollen linters) analyzed for reducing sugar by the micro method of Somogyi. The reducing sugar titers of controls containing enzyme but no substrate were negligible. The titers of controls containing substrate but no enzyme were appreciable, though very low, for precipitated cellulose due to traces of reducing sugar retained in the preparation of this substrate. This titer was subtracted from the enzyme titer. The enzyme solutions were assayed in duplicate at three enzyme concentrations. The enzyme standard was used at concentrations corresponding to 0.05, 0.10, and 0.20 ml. of culture filtrate in 20 ml. of assay medium. Other enzyme preparations were diluted to give approximately the same reducing sugar titers, the required dilution being estimated approximately from their protein concentration as measured by transmittance at 276 mcc. The titers for each concentration were averaged, the percentage deviation of individual titers from their mean rarely exceeding 570,. The concentration of enzyme standard corresponding to the titer of an enzyme preparation was determined graphically from a plot of the concentration and titer of the standard. From the known dilution of the preparation in the assay flasks, its enzyme activity per milliliter could then be expressed as a percentage of that of the standard. Use of three enzyme concentrations generally insured that at least two titers of an enzyme preparation were within the plotted range of the enzyme standard. Separate estimates of enzyme activity from the two titers usually agreed within 5% of their mean. Relative enzyme activities were converted to arbitrary cellulase units by assigning to the enzyme standard an arbitrary unit activity$oward each substrate. The convention followed in triaIs of purification procedures was to define the activity of the enzyme standard in use as one cellulase unit per milligram of protein. Protein nitrogen was determined by the Kjeldahl procedure with trichloroacetic acid at 2.570 concentration as protein precipitant. Protein concentrations were taken as protein N concentrations X 6.25.
MYROTHECIUM
VERRUCARIA
CELLULASE
257
3. Concentration of the Enzyme The protein concentration of the culture filtrates varied from about 0.15 to 0.25 mg. protein/ml. As this concentration was too low for direct application of the purification methods tested, the culture filtrates were first concentrated. The method used in the work reported here was a sequence of vacuum evaporation and concentration by slow freezing.2 The culture filtrate was first concentrated by two passages through a Bartholomew evaporator (10). The evaporator was operated to give a concentration of at least 2:l on each passage at a feed rate of from 3 to 4 1. culture filtrate/hr. The heat input was a mixture of air and steam at 90°C. The vacuum was from an oil pump protected by large Dry Ice traps. At the high feed rate and evaporation temperatures of 2%32”C., there was no measurable loss in enzyme activity during concentration. At the low feed rate and evaporation temperatures of 3539”C., there were losses in enzyme activity up to about 5%, presumably from occasional local overheating along the column. The evaporated filtrates were concentrated by slow freezing in a cold room at -12°C. to bring the final concentration to about 1O:l. The filtrates were frozen in a 6- or 8-1. glass jar insulated with s in. of hair felt on the sides and with 2 in. of hair felt on the top and bottom. An inner packing of glass wool extended from the lid to within about 36 in. of the surface of the filtrate. Under these conditions, ice formation was from the walls inward, leaving a concentrated solution in a core at the center. After 3-4 days in the cold room, the concentrate was decanted. The ice was finely chipped, spun in a basket centrifuge at 1X., washed with a small volume of cold water, and recentrifuged. The solutions removed by centrifuging were added to the concentrate. This slow-freezing procedure caused no loss in enzyme activity, and the recovery was usually about 95%.
4. PuriJication
of the Enzyme
(a) Initial Precipitation and Extraction. After concentration, the enzyme was precipitated by ammonium sulfate and extracted with water. The protein concentration at this stage was still rather low and, to precipitate the enzyme completely, it was found necessary to saturate with ammonium sulfate under conditions giving further concentration. It was found desirable to first remove, at approximately 30yo ammonium sulfate saturation, a precipitate containing certain colloids which were difficult to remove at later stages of purification. This precipitate also contained traces of residual cellulose and protein denatured during evaporation. When precipitated at pH 6.5-7.0 it contained very little cellulase, but at lower pH’s its cellulase content became very appreciable. 2 The method since adopted is concentration at 18°C. in a Mojonnier Temperature Evaporator (Model LTFL, Mojonnier Bros. Co., Chicago).
Low-
258
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The detailed procedure was as follows. To each 2 1. of concentrate (pH ML. 6.5) wa.s added 100 ml. of 1 M phosphate buffer of pH 6.8. Nine hundred milliliters of ammonium sulfate solution (saturated at. 2°C.) was t,hen added by capillary inflow with stirring. After standing overnight at, 2”C., the solution was centrifuged at high speed (Sorva.11 centrifuge, 10,000 r.p.m.). Tho supernatant solution was added to a 4-l. beaker in which was suspended a cellophane dialysis sac packed with 1 kg. of finely ground ammonium sulfate. The ends of the sac were connected to overflow tubes at the top of the beaker to drain off the solution rising in the sac. Ammonium sulfate was replenished as it dissolved. After 5 days at 2”C., saturation was complete and the volume of the concentrate had been reduced by approximately two-thirds. The saturated concentrate was centrifuged as before. The supernatant solution was discarded and the sediment recentrifuged to minimum volume. The sediment was shaken for 6 hr. at 1°C. with 200 ml. of distilled water, and the suspension was centrifuged as before. The clear supernatant solution was decanted, and the sediment was shaken overnight with 50 ml. of distilled water and again centrifuged. The final sediment was discarded. The supernatant solutions were combined and designated “Initial Extract.”
The Initial Extract contained nearly all the cellulase activity of the original concentrate. Its cellulase activity per unit protein concentration was approximately the same as that of the culture filtrate before concentration. At pH 6.8 and 5.0, the extract gave only one peak in the ultracentriftige. The components present in the electrophoretic pattern at pH 6.8 are shown in Fig. 2. The components were designated as: component (a), the component migrating very slowly at pH 6.8; component (b), the component of intermediate mobility; and component (c), the minor, fasting-moving components. This pattern is from an extract obtained under conditions of ammonium sulfate precipitation giving less complete precipitation of cellulase than those described above. With complete precipitation, the relative area of component (b) increased and, at the protein concentrations permitted by the dark color of the extract, component (c) was less clearly resolved into its separate components. The Initial Extracts were the starting products for the fractionation methods examined. For fractionations at low ionic strength, the extracts were dialyzed at 1°C. to remove ammonium sulfate. The dialysis membranes were of gold-beater’s skin tied over glass tubes of 8 cm. diameter. (Cellophane membranes could not be used at low salt concentrations as they were rapidly attacked by the enzyme.) As minute holes were occasionally present in some samples of these membranes, the membranes were tested for leaks by starch dialysis, membranes not retaining starch being rejected.
MYROTHECIUM
VERRUCARIA
CELLULASE
259
(b) Fractionation Methods. The following methods were tested: (1) Fractionation by Ammonium Sulfate. Trials of ammonium sulfate fractionation were made at 1°C. between pH 4.0 and 7.0. The best fraction&ions obtained were at an initial pH of 5.6 and an initial protein concentration of 1% (W/V). Under these conditions, the precipitate removed at 50yo ammonium sulfate saturation was enriched in components (a) and (c) of the Initial Extract; the precipitate removed at 100~o saturation was enriched in component (b) and in enzyme activity toward swollen linters, precipitated cellulose, and cellobiose (4). Refractionations of the latter precipitate between closer saturation levels were not very effective as successive fractions showed relatively slight changes in the ratio of their enzyme activity to their protein content. (8) Adsorption on Calcium Phosphate Gel. The procedure followed was based on the methods of Swingle and Tiselius (11). After dialysis against 0.001 M KH.zPO,-N~~HPO, (pH 6.8), the protein in Initial Extracts was adsorbed on columns of Car(POa)l gel-Celite Analytical Filter Aid (1:5) and washed with 1%
IL2
YIN.
195 MIN.
2. Electrophoretic components of Initial Extract. Descending boundary on left, ascending boundary on right. Migration toward anode. Protein concn., 0.61%; buffer, phosphate, pH 6.8 (25”C.), and ionic strength 0.2; current, 0.020 amp.; temp., -0.l”C. FIG.
saline. The adsorbed protein was eluted at 1°C. with 0.05 M KHzPO,-N&HP04 (pH 6.8). It moved down the column behind two bands of pigment: a pink band moving very rapidly in front and a yellowish brown band moving slowly just in advance of the protein. The protein was collected in small fractions. The transmittance of these fractions at 276 w indicated that the protein was eluted with a very sharp concentration front followed by a long continuously decreasing tail. The ratio of cellulase activity to protein content was greatest for the first protein fraction and decreased with succeeding fractions. The highest ratio obtained was approximately twice that of the starting product. The enrichment in component (b) of the eleetrophoretic pattern was approximately the same. (3) Fractionation with Ethanol. Conditions for ethanol fractionation were determined on Initial Extracts dialyzed against 0.002 M phosphate buffer of pH 6.8. Ionic strength was varied by addition of sodium acetate, and pH by addition of acetic acid. Ethanol was added by capillary inflow with continuous shaking, at 0°C. for ethanol concentrations below 10% (V/Y) or at -7°C. for higher concentrations. After standing overnight, the precipitates were removed on a high-speed centrifuge and dissolved in dilute acetate buffer of pH 5.6.
260
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WHITAKER
The effects of pH and ionic strength on ethanol fractionation were marked. At constant ionic strength, decreasing the pH from 6.8 to 4.5 decreased the ethanol concentration required to commence and to complete protein precipitation. At constant pH, decreasing the ionic strength had a similar effect. The best fractionations obtained were at pH 5.0 at low ionic strength. The results of a fractionation at pH 5.0 and ionic strength 0.02 are given in Table II. In this fractionation, the precipitate forming at each ethanol concentration was centrifuged down, washed with a few milliliters of acetate buffer of corresponding ethanol concentration, and recentrifuged. The supernatant solutions were combined and ethanol was added to give the next ethanol concentration. The cellulase assays were with swollen linters as substrate. The units of cellulase activity are arbitrary. The enrichment in cellulase activity of the fractions precipitating between 10 and 25?& ethanol concentration was accompanied by the disappearance of component (c) from their electrophoretic patterns at pH 6.8. Similar results were TABLE Ethanol
Fractionation
Fraction
Starting
of Initial
Extracts
Ethanol concentration at which precipitated, .a/o
II at pH 6.0 and Ionic
Temp.
1 2 3 4 Final supernatant
Cellulase units
0.02 Cellulase to protein ratio
2920
2920
1.00
-1 -7 -7 -12
235 75 1715 509 254
142 51 2020 613 -
0.61 0.68 1.18 1.21 -
Totals
2790
2830
product (vol. 90 ml.) % 5 10 15 25
Protein m.
Strength
“C.
obtained in fractionations at pH 5.0 and ionic strength 0.002 except that the precipitation of the fraction with the low cellulase to protein ratio was complete at 5% ethanol concentration. (4) Fractionation with Polymethacrylic Acid (PMA). The procedure followed in trials of precipitation by PMA was based on the methods of Morawetz and Hughes (12). PMA was prepared from methacrylic acid (technical grade, Matheson Co., East Rutherford, N. J.). It was dissolved in water by shaking overnight, brought to the desired pH with sodium hydroxide, and diluted to the required concentrations with acetate buffer of the same pH and various ionic strengths. The enzyme solutions were ethanol-fractionated Initial Extracts. The protein concentration was 1% and the pH and ionic strength were varied by addition of acetic acid and sodium acetate. The enzyme solutions were mixed with an equal volume of PMA in buffer of the same pH and ionic strength and left overnight at 1°C. The protein-PMA precipitates were centrifuged down and dissolved in 0.05 M acetate buffer of pH 6.5. PMA was then removed by precipitation with barium chloride. The conditions chosen for precipitation by PMA were determined by the
MYROTHECIUM
VERRUCARIA
CELLULASE
261
following considerations. It was found desirable to precipitate with PMA at low ionic strengths. Protein (and cellulase) precipitation began at about pH 4.7. In buffer of ionic strength 0.02, cellulase precipitation was complete at pH 4.5 with a PMA to protein ratio (w/w) of 0.5 and complete at pH 4.2 with a ratio of 0.25. However at such low ionic strengths, precipitation of protein in the enzyme solutions commenced, without addition of PMA, at about pH 4.4 and increased with decreasing pH. The cellulase to protein ratio of this precipitate varied from cu. 0.7-0.9. To obtain high cellulase to protein ratios from the PMA precipitate it was necessary to first remove the precipitate forming before PMA addition. Ratios of up to twice that of the starting product were then obtained. However, when the ionic strength was very low, the precipitate removed contained a considerable amount of cellulase. Conditions were therefore chosen to minimize the loss of cellulase in this precipitate without sacrificing enrichment in enzyme activity in the PMA precipitate. The best conditions found were a pH of 4.35, ionic strength of 0.01, and a PMA to protein ratio of 0.25 (aide infra).
(c) Purijication Procedure. A protocol of the purification procedure adopted is given in Table III. The concentrate used as starting product was obtained from approximately 20 1. of a culture filtrate with a protein content of 0.215 mg. protein/ml. and, by definition, a cellulase activity toward each substrate of one cellulase unit/mg. protein. Throughout this purification procedure, the fractions were freeze-dried at each stage. Aliquots were taken for enzyme assay and protein analysis, and part of each main fraction was retained for future reference. 5. Properties of PuriJied Cellulase (a) Physical Properties. The purified cellulase from the above fractionation was electrophoretically homogeneous at pH 6.82, 5.03, and 4.33 (Figs. 3a, b, and c). These pH’s are respectively above, near, and below the isoelectric point. The product was also homogeneous at pH 5.03 in the ultracentrifuge (Fig. 3d). (b) Activity Toward Other Substrates. Additional substrates tested were: (a) unswollen linters, the ground cotton linters used in the assay medium; (6) cellobiose (Kerfoots reagent); and (c) carboxymethylcellulose (Type 5OT, Hercules Powder Co.), a water-soluble substituted cellulose of a low degree of polymerization (D.P.) (mean D.P. ca. 150) and low degree of substitution (cu. 0.5). The product was dialyzed free of salt, precipitated with ethanol, and vacuum-dried. It was dissolved in the assay medium by shaking for several hr. at 35°C. before addition of enzyme. The activity of the purified cellulase toward 1% concentrations of these substrates was assayed by the procedure previously described. Reducing sugar formation from the cellulose substrates was determined by the micro-Somogyi method,
TABLE Protocol
III
of Purijication
Procedure Precipitated cellulose
cellulase unit,
A. Precipitation with (NH4)zSOa Concentrated culture filtrate 1990 ml., pH 6.6, 1°C. 100 ml. 1 M phosphate, pH 6.8 900 ml. satd. (NH,)zSO, Centrifuged. Sediment Supernatant saturated with (NH,)&WI by dialysis. Centrifuged. Supernatant discarded. Sediment Extracted twice with water (200 ml., 50 ml.). Centrifuged after each extraction; supernatants combined. Initial czlracl Sediment
4240
4060
4060
304
36
37
3650 215
3690 56
3780 59
Total B. Fractionation with ethanol 6.8 g. Initial extract protein (dialyzed against 0.01 M phosphate, pH 6.8) Vol. 96 ml. pH to 4.95 Ethanol to 5% (v/u) at 1°C. Centrifuged, sediment washed and recentrifuged. Sediment Combined supernatants Ethnkl to 25% (V/V) at -7°C. Sediment A Supernatant
4170
3780
3880
2830
2900
401
387
1762 309
2210 151
2110 169
Total 6. Fractionation with PMA 1.6% g. Sediment A protein Dissolved in 162 ml. of 0.01 M sodium acetate at 1°C. Glacial acetic added by capillary inflow to pH 4.35. Centrifuged, sediment washed, and recentrifuged. Sediment Supernatants 1132 ml. of 0.25% PMA in 0.01 M sodium 4.35. Centrifuged, acetate at pH washed, and recentrifuged. Supernatant Sediment Dissolved in 80 ml. of 0.05 M sodium acetate at pH 6.5; 6 ml. 10% BaCla added; centrifuged and sediment (Ba PMA) discarded. Supernatant (Purijied cellulase)
2730
2760
2670
1620
2030
1940
100
81
78
510
73
60
724
1620
1640
1430
1770
1800
Total 262
MYROTHECIUM
VERRUCARIA
263
CELLULASE
and the extent of cellobiose hydrolysis by Goebel’s modification (13) of the Willstltter-Schudel method. The same enzyme standard was used as in Table III, its activity toward each substrate being likewise defined as one unit/mg. protein. The concentrations of the standard used in these assays corresponded to 0.025, 0.05, and 0.1 ml. of culture filtrate/20 ml. for carboxymethylcellulose; 0.1, 0.2 and 0.4 ml. for unswollen linters; and 3.7, 7.5, and 11.2 ml. for cellobiose.
A
s
A
Apn6.82
+4
++
pH5.03
ptl5.03
---I
C
A +-
P pH4.33
Fxo. 3. Electrophoretic and ultracentrifuge patterns of purified cellulase. A, B, and C, electrophoretic patterns; current, 0.020 amp.; temp., -O.l’C., ionic strength, 0.2; pH measured at 1°C. A, ascending boundary; time, 221 min.; buffer, phosphate. B, descending boundary; time, 187 min.; buffer, acetate. C, descending boundary; time, 225 min.; buffer, acetate. D, ultracentrifuge pattern (Spinco Model E ultracentrifuge); speed, 60,000 r.p.m.; time, 32 min.; buffer, acetate; ionic strength, 0.2.
The assays gave the following cellulase to protein ratios for these substrates: unswollen linters, 2.12; carboxymethylcellulose, 2.05; and cellobiose, 2.22. These ratios are thus approximately the same as those calculated for swollen linters, 2.24, and precipitated cellulose, 2.26, from the data in Table III. Reducing sugar titer-enzyme concentration curves of purified cellulase are given in Fig. 4 for the cellulose substrates. Comparable data
264
D.
R. WHITAKER
for cellobiose are: 1570 pg. protein N/20 ml., 7.55y0 hydrolysis; 3140 pg., 13.1y0 hydrolysis; and 4730 pg., 18.6% hydrolysis. (c) Reducing Sugar End Products. The identity of the soluble end products formed from swollen linters and precipitated cellulose under assay conditions was investigated as follows. Purified cellulase was incubated with substrate in baffled 2809ml. Fernbach flasks containing 409 ml. of assay medium. For each substrate, the assay medium was at pH 5.6 in one flask and at pH 6.8 in a second Aask (buffer, 296 ml. of 0.05
FIG. 4. Reducing sugar formation from cellulose substrates by purified cellulase at low enzyme concentrations. Titers (micro-Somogyi) : carboxymethylcellulose and precipitated cellulose/ml. of assay medium; swollen linters/3 ml. of assay medium; unswollen linters/5 ml. of assay medium.
M KHaPOrNazHPOc containing a trace of Dowicide G). The enzyme concentration corresponded to 8.9 pg. protein N/20 ml. After 17 hr. incubation at 35”C., the contents of each flask were centrifuged and the supernatant solution freeze-dried. The freeze-dried products were each dissolved in 15 ml. of water and cleared with Zn(OH)z by the method of Somogyi (14). They were then freed of salt in ionexchange columns, first in a column of Duolite (Chemical Process Co., San Francisco) to remove anions and then in a column of Nalcite HCR (Alchem. Ltd., Burlington, Ont., Canada) to remove cations. Washing of the columns with distilled water was continued to about 95% recovery of reducing sugar titer (final volumgr 259 ml .)
MYRO!PHECIUM
VERRUCARIA
265
CELLULASE
The resulting solutions were examined by three procedures : 1. Small samples were evaporated to thin sirups and developed on paper chromatograms. The developing solvent was the organic phase of a mixture of pyridine, ethyl acetate, and water (1:2:2 v/v). After drying, the papers were sprayed with aniline phthalate and heated to 110°C. The resulting chromatograms showed only two components. These corresponded in position with controls of glucose and cellobiose. The two spots were of approximately equal intensity and area for all solutions. Similar evidence of glucose and cellobiose formation by cellulolytic enzymes in culture filtrates of various fungi has been reported by Levinson et al. (3). 2. Two hundred milliliters of each solution was refluxed with phenylhydraaine acetate and sodium acetate. The phenylosazones precipitated were filtered off, washed with water, and converted to phenylosotriazoles by the method of Hann and Hudson (15). The phenylosotriazoles were treated with charcoal and recrystallized from water. Their melting points (corr.) were all in the range 192193°C. rising to 193-194°C. on mixture with authentic phenyl glucosotriazole of m.p. 194-195°C. 3. The solutions were analyzed for reducing sugar by the micro-Somogyi method and for total carbohydrate by the anthrone method as modified by Seifter et al. (16) with glucose as standard. Converting the Somogyi titers to glucose gave anthrone glucose to Somogyi glucose ratios as follows: Swollen linters Precipitated cellulose The ratios are thus of the order to be expected concentrations of glucose and cellobiose.
pH 5.6
pH 6.8
1.39 1.44
1.48 1.62
from
approximately
equimolar
These results therefore suggest that glucose and cellobiose are the main soluble end products of cellulase action under the above assay conditions. (d) Origin of Glucose. In the preceding assays of enzyme activity, the enzvme concentrations required for assay with 1% cellobiose were approximatelg 75X those required for assay with swollen linters and precipitated cellulose. This comparatively low activity toward cellobiose is of significance with regard to the origin of glucose as an end product of cellulose breakdown by this enzyme. It was noted previously (4) that the hydrolysis of cellobiose by partially purified Myrothecium cellulase appeared to be inhibited by increasing concentrations of cellobiose. This observation was confirmed in that no measurable hydrolysis of 5% cellobiose could be detected at enzyme concentrations giving nearly 209$ hydrolysis of 1% cellobiose. The apparently low activity of this cellulase toward cellobiose might therefore be due to assay at
266
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too high a cellobiose concentration. However, in assays at very low cellobiose concentrations-0.01, 0.02, and 0.1% cellobiose-the enzyme concentrations used in precipitated cellulose and swollen linters assay gave no measurable increase in micro-Somogyi titer. As such enzyme concentrations give glucose as a major product from these cellulose substrates, it is concluded that the formation of glucose by this cellulase is not dependent on formation via cellobiose. DLYXJSSION
The above results are considered to suggest a unienzymatic mechanism of cellulose hydrolysis by this cellulase. The following is considered to be the main evidence: (a) The purified enzyme was homogeneous electrophoretically and in the ultracentrifuge. (b) Its enrichment in enzyme activity, relative to the culture filtrate from which it was obtained, was approximately the same for five substrates ranging in degree of polymerization from untreated cellulose to cellobiose. (c) Although enzyme activity was measured from the formation of soluble sugars (which included glucose) at low enzyme concentrations, all fractionation methods examined gave balance sheets in which the recovery of enzyme activity toward two cellulose substrates was in reasonably good agreement with the recovery of total protein. Such balances are considered contrary to the expectations of a multienzyme mechanism, for end-product formation would then be expected to depend not only on total recovery but on the relative concentration of enzyme components in each fraction. This evidence is not considered at variance with results which have been interpreted to indicate a multienzyme mechanism for this mold. Reese and his co-workers (2) have interpreted their finding that certain non-cellulolytic fungi can hydrolyze carboxymethylcellulose as indicating that cellulolytic fungi hydrolyze comparable anhydroglucose chains by an enzyme different from that causing the initial breakdown of cellulose. However the properties of a cellulase must include not only p-1,4polyanhydroglucosidase activity but properties permitting such activity under conditions of adsorption on an insoluble, cellulose surface. The findings of Reese et al. can be equally interpreted as merely indicating that these non-cellulolytic fungi produce an enzyme with the former but not the latter property. The detailed physical and chemical mechanisms involved in cellulase action are undoubtedly complex and cannot yet be discussed in detail.
MYROTHECIUM VERRUCARIA CELLULASE
267
The importance of the fine structure of cellulose in determining accessibility to enzyme attack is indicated by the recent data of Walseth (17). The effects of proteins and dyes adsorbed by cellulose on the action of Myrothetium cellulase (7, 9) suggest the importance of the surface potential of cellulose in cellulase action. The independent formation of cellobiose and glucose from cellulose by low concentrations of this cellulase is suggestive of a random breakdown of the cellulose chain in regions of equal accessibility. More positive evidence favoring this mechanism has been summarized by Greathouse (5). ACKNOWLEDGMENTS The writer wishes to express his thanks to Mr. Donald H. Hambrook for technical assistance throughout this work; to Dr. W. H. Cook and Dr. J. R. Colvin for valuable discussions and cooperation and assistance in the use of the ultracentrifuge and electrophoresis equipment; to Mr. A. E. Castagne for the qualitative chromatographic analyses; and to the Hercules Powder Co. for a generous supply of cotton linters.
SUMMARY
The cellulase in culture filtrates of Myrothecium verrucaria was purified by a sequence of concentration, precipitation by ammonium sulfate, fractionation with ethanol, and precipitation with polymethacrylic acid. The enrichments in enzyme activity toward the following substrates were approximately the same: untreated cotton linters, swollen cotton linters, a cellulose degraded by acid hydrolysis, a substituted soluble cellulose, and cellobiose. The purified enzyme was electrophoretically homogeneous at 3 pH’s and homogeneous in the ultracentrifuge. It is suggested that its mechanism of cellulose breakdown is unienzymatic, and evidence supporting this mechanism is discussed. REFERENCES 1. SIU, R. G. H., Microbial 2. 3. 4. 5. 6.
Decomposition
of Cellulose. Reinhold, New York, 1951. REESE, E. T., SIU, R. G. H., AND LEVINSON, H. S., J. Bact. 69,485 (1950). LEVINSON, H. S., MANDELS, G. R., AND REESE, E. T., Arch. Biochem. and Biophys. 31,351 (1951). WHITAKER, D. R., Nature 166, 1070 (1951). GREATHOUSE, G. A., Textile Research J. 23, 227 (1950). SAUNDERS, P. R., SIU, R. G. H., AND GENEST, R. N., J. Biol. Chem. 174, 697
(1948). 7. BASU, S. N., AND WHITAKER, D. R., Arch. Biochem. and Biophys. 8. J$RGENSEN, L., Studies on the Partial Hydrolysis of Cellulose, Hos Emil Moesture, Oslo, 1950.
42, 12 (1953). p. 140. Trykt
268 9. 10. 11. 12. 13. 14. 15. 16.
D.
R. WHITAKER
WHITAKER, D. R., Science 116, 90 (1952). BARTHOLOMEW, W. H., Anal. Chem. 21, 527 (1949). SWINGLE, S. M., AND TISELIUS, A., B&hem. J. (London) MORAWETZ, H., AND HUGHES, W. H., J. Phys. Chem. 66, GOEBEL, N. F., J. Bid. Chem. 73, 801 (1927). SOMOGYI, M., J. Biol. Chem. 160, 69 (1945). HANN, R. M., AND HUDSON, C. S., J. Am. Chem. Sot. 66, SEIFTER, S., DAYTON, S., NOVIC, B., AND MUNTWYLER, 26, 191 (1950). 17. WALSETH, C., Tappi 36, 233 (1952).
46, 171 (1951). 64 (1952).
735 (1944). E., Arch. B&hem.
of Myrothecium
verrucariu Cellulase*
D. R. Whitaker From the Division
of Applied
Biology, National Ottawa, Canada
Research Laboratories,
Received November 17, 1952 INTRODUCTION Current knowledge of the mechanism of cellulose breakdown by enzymes has been based on the properties of crude or slightly purified enzyme preparations or of cellulolytic organisms. On such evidence it has been often postulated that cellulose is hydrolyzed by a complex of two or more enzymes working in sequence (1). Among the organisms claimed to have a multienzyme cellulase system is the mold Myrothecium verrucariu (Alb. and Schw.) Ditm. ex Fr. (2, 3). The United States Department of Agriculture (USDA) strain No. 1334.2 of this mold has been used extensively as a test organism for studies on cellulose breakdown by fungi (1). The object of the present work was to purify the cellulolytic enzyme system of M. verrucaria. For reasons given later, this system is designated simply as cellulase. A note on some of the methods used has been published previously (4). METHODS AND RESULTS 1. Production of Enzyme The enzyme was obtained from culture filtrates of Myrothecium verrucuria, strain USDA 1334.2. Procedures for culturing this organism for cellulase production have been described by Greathouse (5) and by Saunders et al. (6). The culture media used in the present work (Table I) were based partly on that of Saunders et al. (6). The cellulose substrate was a cotton linters (Grade 27, Hercules Powder Co.) ground in a Wiley mill to pass a l-mm. mesh sieve, extracted with benzene-alcohol (2: 1 v/v), washed with ethanol, and vacuum dried. All media were autoclaved 1 Issued as National
Research
Council
253
No. 2942.
254
D.
R.
WHITAKER
at 15 lb. steam pressure: the tubes of cellulose-agar for 20 min., the flasks of shake culture medium for 30 min., and t’he flasks of submerged culture medium for 1 hr. The mold was cultured by the following procedure. Slopes of cellulose-agar in 8 X 1 in. tubes were inoculated with spores of M. verrucaria and incubated at 30°C. for 5 days. Each slope was then flooded with 25 ml. of sterile water and the spores and mycelium scraped into suspension with a sterile knife. The suspension from each slope was added to a l-l. Erlenmeyer shake flask containing 309 ml. of shake culture medium. To increase the agitation during shaking, the shake flasks were baffled (Fig. la), each baffle being about 1% in. long and 34 in. high. The flasks were incubated at 30°C. for 7 days on a rotary shaker which described a horizontal circle of 1 in. diameter at 113 r.p.m. The contents of each flask were TABLE Composition Shake culture
I
of Media
per Liter
medium :
Cotton linters NHaNOa NaNOa KHzPOa KzHPOd NaH2POd*H20 Na2HPOd MgSOd.7H20
.s. 30 0.60 3.80 0.20 0.15 2.09 1.50 0.30
w. 0.054 0.06 0.2 2.0 0.08 0.4 1.2 0.04
Submerged culture medium: Shake culture medium plus 1.0 g. glucose. Cellulose-agar : Shake culture medium plus 15 g. agar. then added to a 12-1. flask (Fig. lb) containing 6 1. of submerged culture medium. These flasks, in batches of six, were incubated at 30°C. for 12 days with vigorous aeration and rotary shaking. The air for the flasks was washed and partially saturated with water by passage through a coarse-porosity sintered-glass disk at the bottom of a 9-l. bottle containing 6 1. of approximately 0.02 N sulfuric acid, and then passed through a spray trap to a manifold connected to the flasks. The conditions of rotary shaking were the same as for the shake flasks. With flasks of this size, these shaking conditions were sufficient to control foaming. At the end of the incubation period, the medium in each flask was examined microscopically for possible bacterial contamination. It was then filtered through several layers of cheesecloth on a Biichner funnel to remove the bulk of the mold mycelium and residual cellulose. The filtrate from six flasks varied in volume from 23 to 31 1. It was chilled to about 5°C. and clarified in a Sharples supercentrifuge (medium jet). A 599-ml. sample was withdrawn and freeze-dried. The remainder of the filtrate was stored overnight at -1°C. pending concentration.
MYROTHECIUM
VERRUCARIA
255
CELLULASE
2. Assay of Enzyme Activity Enzyme activity was assayed from the amount of soluble reducing sugar formed when the enzyme was incubated with sub&rate. Two substrates were used, viz., “swollen linters” (7), ground extracted linters swollen in cold alkali under the conditions found by Jgrgensen (8) to cause no degradation of cellulose; and “precipitated cellulose” (7), a partly degraded and finely dispersed cellulose prepared by dissolving
@ FIG. merged
1. Flasks used culture flasks.
in cellulase
production:
A,
shake
culture
flasks
;
B,
sub-
absorbent cotton in sulfuric acid and immediately filtering into ice water. The assays were made against an enzyme standard which, during trials of purification methods on a batch of culture filtrate, was the freezedried culture filtrate of that batch. Assays were made at very low enzyme concentrations to avoid limitation of reaction rate by substrate concentration. Under such conditions, reducing sugar formation is strongly stimulated by very low concentrations of protein (9). Sufficient albumin was therefore added to the assay medium to eliminate any differences
2x
D.
R.
WHITAKER
in protein stimulation due to differences in the non-cellulase content of the enzyme preparations.
protein
The assay medium had the following composition: (a) acetate buffer of pH 5.6 (25°C.) containing Dowicide G (sodium pentachlorophenate) as antiseptic, 10 ml.; (b) crystalline bovine plasma albumin (Armour), 300 pg.; (c) substrate, 200 mg.; (d) enzyme solution; and (e) glass-distilled water to bring the final volume to 20 ml. The acetate buffer was prepared from 0.1 M sodium acetate and 0.1 M acetic acid. Dowicide G (about 0.5 g./l.) was added and the buffer stored at 2”C., the excess antiseptic being filtered off after a few days. The assay medium was incubated for 17 hr. in a rotary-shaken water bath at a temperature of 35 f 0.05%. The assay flasks were 125-ml. Erlenmeyer flasks with baffles at the bottom, each about 1 in. long and >i in. high, similar to those of the shake culture flasks. The stoppers of the flasks were protected from splashing by glass covers. The shaker described a horizontal circle of 236 in. diameter at 70 r.p.m. After incubation, the assay medium was centrifuged at about 2000 r.p.m. and samples of the supernatant solution (1 ml. for precipitated cellulose, 3 ml. for swollen linters) analyzed for reducing sugar by the micro method of Somogyi. The reducing sugar titers of controls containing enzyme but no substrate were negligible. The titers of controls containing substrate but no enzyme were appreciable, though very low, for precipitated cellulose due to traces of reducing sugar retained in the preparation of this substrate. This titer was subtracted from the enzyme titer. The enzyme solutions were assayed in duplicate at three enzyme concentrations. The enzyme standard was used at concentrations corresponding to 0.05, 0.10, and 0.20 ml. of culture filtrate in 20 ml. of assay medium. Other enzyme preparations were diluted to give approximately the same reducing sugar titers, the required dilution being estimated approximately from their protein concentration as measured by transmittance at 276 mcc. The titers for each concentration were averaged, the percentage deviation of individual titers from their mean rarely exceeding 570,. The concentration of enzyme standard corresponding to the titer of an enzyme preparation was determined graphically from a plot of the concentration and titer of the standard. From the known dilution of the preparation in the assay flasks, its enzyme activity per milliliter could then be expressed as a percentage of that of the standard. Use of three enzyme concentrations generally insured that at least two titers of an enzyme preparation were within the plotted range of the enzyme standard. Separate estimates of enzyme activity from the two titers usually agreed within 5% of their mean. Relative enzyme activities were converted to arbitrary cellulase units by assigning to the enzyme standard an arbitrary unit activity$oward each substrate. The convention followed in triaIs of purification procedures was to define the activity of the enzyme standard in use as one cellulase unit per milligram of protein. Protein nitrogen was determined by the Kjeldahl procedure with trichloroacetic acid at 2.570 concentration as protein precipitant. Protein concentrations were taken as protein N concentrations X 6.25.
MYROTHECIUM
VERRUCARIA
CELLULASE
257
3. Concentration of the Enzyme The protein concentration of the culture filtrates varied from about 0.15 to 0.25 mg. protein/ml. As this concentration was too low for direct application of the purification methods tested, the culture filtrates were first concentrated. The method used in the work reported here was a sequence of vacuum evaporation and concentration by slow freezing.2 The culture filtrate was first concentrated by two passages through a Bartholomew evaporator (10). The evaporator was operated to give a concentration of at least 2:l on each passage at a feed rate of from 3 to 4 1. culture filtrate/hr. The heat input was a mixture of air and steam at 90°C. The vacuum was from an oil pump protected by large Dry Ice traps. At the high feed rate and evaporation temperatures of 2%32”C., there was no measurable loss in enzyme activity during concentration. At the low feed rate and evaporation temperatures of 3539”C., there were losses in enzyme activity up to about 5%, presumably from occasional local overheating along the column. The evaporated filtrates were concentrated by slow freezing in a cold room at -12°C. to bring the final concentration to about 1O:l. The filtrates were frozen in a 6- or 8-1. glass jar insulated with s in. of hair felt on the sides and with 2 in. of hair felt on the top and bottom. An inner packing of glass wool extended from the lid to within about 36 in. of the surface of the filtrate. Under these conditions, ice formation was from the walls inward, leaving a concentrated solution in a core at the center. After 3-4 days in the cold room, the concentrate was decanted. The ice was finely chipped, spun in a basket centrifuge at 1X., washed with a small volume of cold water, and recentrifuged. The solutions removed by centrifuging were added to the concentrate. This slow-freezing procedure caused no loss in enzyme activity, and the recovery was usually about 95%.
4. PuriJication
of the Enzyme
(a) Initial Precipitation and Extraction. After concentration, the enzyme was precipitated by ammonium sulfate and extracted with water. The protein concentration at this stage was still rather low and, to precipitate the enzyme completely, it was found necessary to saturate with ammonium sulfate under conditions giving further concentration. It was found desirable to first remove, at approximately 30yo ammonium sulfate saturation, a precipitate containing certain colloids which were difficult to remove at later stages of purification. This precipitate also contained traces of residual cellulose and protein denatured during evaporation. When precipitated at pH 6.5-7.0 it contained very little cellulase, but at lower pH’s its cellulase content became very appreciable. 2 The method since adopted is concentration at 18°C. in a Mojonnier Temperature Evaporator (Model LTFL, Mojonnier Bros. Co., Chicago).
Low-
258
D.
R. WHITAKER
The detailed procedure was as follows. To each 2 1. of concentrate (pH ML. 6.5) wa.s added 100 ml. of 1 M phosphate buffer of pH 6.8. Nine hundred milliliters of ammonium sulfate solution (saturated at. 2°C.) was t,hen added by capillary inflow with stirring. After standing overnight at, 2”C., the solution was centrifuged at high speed (Sorva.11 centrifuge, 10,000 r.p.m.). Tho supernatant solution was added to a 4-l. beaker in which was suspended a cellophane dialysis sac packed with 1 kg. of finely ground ammonium sulfate. The ends of the sac were connected to overflow tubes at the top of the beaker to drain off the solution rising in the sac. Ammonium sulfate was replenished as it dissolved. After 5 days at 2”C., saturation was complete and the volume of the concentrate had been reduced by approximately two-thirds. The saturated concentrate was centrifuged as before. The supernatant solution was discarded and the sediment recentrifuged to minimum volume. The sediment was shaken for 6 hr. at 1°C. with 200 ml. of distilled water, and the suspension was centrifuged as before. The clear supernatant solution was decanted, and the sediment was shaken overnight with 50 ml. of distilled water and again centrifuged. The final sediment was discarded. The supernatant solutions were combined and designated “Initial Extract.”
The Initial Extract contained nearly all the cellulase activity of the original concentrate. Its cellulase activity per unit protein concentration was approximately the same as that of the culture filtrate before concentration. At pH 6.8 and 5.0, the extract gave only one peak in the ultracentriftige. The components present in the electrophoretic pattern at pH 6.8 are shown in Fig. 2. The components were designated as: component (a), the component migrating very slowly at pH 6.8; component (b), the component of intermediate mobility; and component (c), the minor, fasting-moving components. This pattern is from an extract obtained under conditions of ammonium sulfate precipitation giving less complete precipitation of cellulase than those described above. With complete precipitation, the relative area of component (b) increased and, at the protein concentrations permitted by the dark color of the extract, component (c) was less clearly resolved into its separate components. The Initial Extracts were the starting products for the fractionation methods examined. For fractionations at low ionic strength, the extracts were dialyzed at 1°C. to remove ammonium sulfate. The dialysis membranes were of gold-beater’s skin tied over glass tubes of 8 cm. diameter. (Cellophane membranes could not be used at low salt concentrations as they were rapidly attacked by the enzyme.) As minute holes were occasionally present in some samples of these membranes, the membranes were tested for leaks by starch dialysis, membranes not retaining starch being rejected.
MYROTHECIUM
VERRUCARIA
CELLULASE
259
(b) Fractionation Methods. The following methods were tested: (1) Fractionation by Ammonium Sulfate. Trials of ammonium sulfate fractionation were made at 1°C. between pH 4.0 and 7.0. The best fraction&ions obtained were at an initial pH of 5.6 and an initial protein concentration of 1% (W/V). Under these conditions, the precipitate removed at 50yo ammonium sulfate saturation was enriched in components (a) and (c) of the Initial Extract; the precipitate removed at 100~o saturation was enriched in component (b) and in enzyme activity toward swollen linters, precipitated cellulose, and cellobiose (4). Refractionations of the latter precipitate between closer saturation levels were not very effective as successive fractions showed relatively slight changes in the ratio of their enzyme activity to their protein content. (8) Adsorption on Calcium Phosphate Gel. The procedure followed was based on the methods of Swingle and Tiselius (11). After dialysis against 0.001 M KH.zPO,-N~~HPO, (pH 6.8), the protein in Initial Extracts was adsorbed on columns of Car(POa)l gel-Celite Analytical Filter Aid (1:5) and washed with 1%
IL2
YIN.
195 MIN.
2. Electrophoretic components of Initial Extract. Descending boundary on left, ascending boundary on right. Migration toward anode. Protein concn., 0.61%; buffer, phosphate, pH 6.8 (25”C.), and ionic strength 0.2; current, 0.020 amp.; temp., -0.l”C. FIG.
saline. The adsorbed protein was eluted at 1°C. with 0.05 M KHzPO,-N&HP04 (pH 6.8). It moved down the column behind two bands of pigment: a pink band moving very rapidly in front and a yellowish brown band moving slowly just in advance of the protein. The protein was collected in small fractions. The transmittance of these fractions at 276 w indicated that the protein was eluted with a very sharp concentration front followed by a long continuously decreasing tail. The ratio of cellulase activity to protein content was greatest for the first protein fraction and decreased with succeeding fractions. The highest ratio obtained was approximately twice that of the starting product. The enrichment in component (b) of the eleetrophoretic pattern was approximately the same. (3) Fractionation with Ethanol. Conditions for ethanol fractionation were determined on Initial Extracts dialyzed against 0.002 M phosphate buffer of pH 6.8. Ionic strength was varied by addition of sodium acetate, and pH by addition of acetic acid. Ethanol was added by capillary inflow with continuous shaking, at 0°C. for ethanol concentrations below 10% (V/Y) or at -7°C. for higher concentrations. After standing overnight, the precipitates were removed on a high-speed centrifuge and dissolved in dilute acetate buffer of pH 5.6.
260
D.
R.
WHITAKER
The effects of pH and ionic strength on ethanol fractionation were marked. At constant ionic strength, decreasing the pH from 6.8 to 4.5 decreased the ethanol concentration required to commence and to complete protein precipitation. At constant pH, decreasing the ionic strength had a similar effect. The best fractionations obtained were at pH 5.0 at low ionic strength. The results of a fractionation at pH 5.0 and ionic strength 0.02 are given in Table II. In this fractionation, the precipitate forming at each ethanol concentration was centrifuged down, washed with a few milliliters of acetate buffer of corresponding ethanol concentration, and recentrifuged. The supernatant solutions were combined and ethanol was added to give the next ethanol concentration. The cellulase assays were with swollen linters as substrate. The units of cellulase activity are arbitrary. The enrichment in cellulase activity of the fractions precipitating between 10 and 25?& ethanol concentration was accompanied by the disappearance of component (c) from their electrophoretic patterns at pH 6.8. Similar results were TABLE Ethanol
Fractionation
Fraction
Starting
of Initial
Extracts
Ethanol concentration at which precipitated, .a/o
II at pH 6.0 and Ionic
Temp.
1 2 3 4 Final supernatant
Cellulase units
0.02 Cellulase to protein ratio
2920
2920
1.00
-1 -7 -7 -12
235 75 1715 509 254
142 51 2020 613 -
0.61 0.68 1.18 1.21 -
Totals
2790
2830
product (vol. 90 ml.) % 5 10 15 25
Protein m.
Strength
“C.
obtained in fractionations at pH 5.0 and ionic strength 0.002 except that the precipitation of the fraction with the low cellulase to protein ratio was complete at 5% ethanol concentration. (4) Fractionation with Polymethacrylic Acid (PMA). The procedure followed in trials of precipitation by PMA was based on the methods of Morawetz and Hughes (12). PMA was prepared from methacrylic acid (technical grade, Matheson Co., East Rutherford, N. J.). It was dissolved in water by shaking overnight, brought to the desired pH with sodium hydroxide, and diluted to the required concentrations with acetate buffer of the same pH and various ionic strengths. The enzyme solutions were ethanol-fractionated Initial Extracts. The protein concentration was 1% and the pH and ionic strength were varied by addition of acetic acid and sodium acetate. The enzyme solutions were mixed with an equal volume of PMA in buffer of the same pH and ionic strength and left overnight at 1°C. The protein-PMA precipitates were centrifuged down and dissolved in 0.05 M acetate buffer of pH 6.5. PMA was then removed by precipitation with barium chloride. The conditions chosen for precipitation by PMA were determined by the
MYROTHECIUM
VERRUCARIA
CELLULASE
261
following considerations. It was found desirable to precipitate with PMA at low ionic strengths. Protein (and cellulase) precipitation began at about pH 4.7. In buffer of ionic strength 0.02, cellulase precipitation was complete at pH 4.5 with a PMA to protein ratio (w/w) of 0.5 and complete at pH 4.2 with a ratio of 0.25. However at such low ionic strengths, precipitation of protein in the enzyme solutions commenced, without addition of PMA, at about pH 4.4 and increased with decreasing pH. The cellulase to protein ratio of this precipitate varied from cu. 0.7-0.9. To obtain high cellulase to protein ratios from the PMA precipitate it was necessary to first remove the precipitate forming before PMA addition. Ratios of up to twice that of the starting product were then obtained. However, when the ionic strength was very low, the precipitate removed contained a considerable amount of cellulase. Conditions were therefore chosen to minimize the loss of cellulase in this precipitate without sacrificing enrichment in enzyme activity in the PMA precipitate. The best conditions found were a pH of 4.35, ionic strength of 0.01, and a PMA to protein ratio of 0.25 (aide infra).
(c) Purijication Procedure. A protocol of the purification procedure adopted is given in Table III. The concentrate used as starting product was obtained from approximately 20 1. of a culture filtrate with a protein content of 0.215 mg. protein/ml. and, by definition, a cellulase activity toward each substrate of one cellulase unit/mg. protein. Throughout this purification procedure, the fractions were freeze-dried at each stage. Aliquots were taken for enzyme assay and protein analysis, and part of each main fraction was retained for future reference. 5. Properties of PuriJied Cellulase (a) Physical Properties. The purified cellulase from the above fractionation was electrophoretically homogeneous at pH 6.82, 5.03, and 4.33 (Figs. 3a, b, and c). These pH’s are respectively above, near, and below the isoelectric point. The product was also homogeneous at pH 5.03 in the ultracentrifuge (Fig. 3d). (b) Activity Toward Other Substrates. Additional substrates tested were: (a) unswollen linters, the ground cotton linters used in the assay medium; (6) cellobiose (Kerfoots reagent); and (c) carboxymethylcellulose (Type 5OT, Hercules Powder Co.), a water-soluble substituted cellulose of a low degree of polymerization (D.P.) (mean D.P. ca. 150) and low degree of substitution (cu. 0.5). The product was dialyzed free of salt, precipitated with ethanol, and vacuum-dried. It was dissolved in the assay medium by shaking for several hr. at 35°C. before addition of enzyme. The activity of the purified cellulase toward 1% concentrations of these substrates was assayed by the procedure previously described. Reducing sugar formation from the cellulose substrates was determined by the micro-Somogyi method,
TABLE Protocol
III
of Purijication
Procedure Precipitated cellulose
cellulase unit,
A. Precipitation with (NH4)zSOa Concentrated culture filtrate 1990 ml., pH 6.6, 1°C. 100 ml. 1 M phosphate, pH 6.8 900 ml. satd. (NH,)zSO, Centrifuged. Sediment Supernatant saturated with (NH,)&WI by dialysis. Centrifuged. Supernatant discarded. Sediment Extracted twice with water (200 ml., 50 ml.). Centrifuged after each extraction; supernatants combined. Initial czlracl Sediment
4240
4060
4060
304
36
37
3650 215
3690 56
3780 59
Total B. Fractionation with ethanol 6.8 g. Initial extract protein (dialyzed against 0.01 M phosphate, pH 6.8) Vol. 96 ml. pH to 4.95 Ethanol to 5% (v/u) at 1°C. Centrifuged, sediment washed and recentrifuged. Sediment Combined supernatants Ethnkl to 25% (V/V) at -7°C. Sediment A Supernatant
4170
3780
3880
2830
2900
401
387
1762 309
2210 151
2110 169
Total 6. Fractionation with PMA 1.6% g. Sediment A protein Dissolved in 162 ml. of 0.01 M sodium acetate at 1°C. Glacial acetic added by capillary inflow to pH 4.35. Centrifuged, sediment washed, and recentrifuged. Sediment Supernatants 1132 ml. of 0.25% PMA in 0.01 M sodium 4.35. Centrifuged, acetate at pH washed, and recentrifuged. Supernatant Sediment Dissolved in 80 ml. of 0.05 M sodium acetate at pH 6.5; 6 ml. 10% BaCla added; centrifuged and sediment (Ba PMA) discarded. Supernatant (Purijied cellulase)
2730
2760
2670
1620
2030
1940
100
81
78
510
73
60
724
1620
1640
1430
1770
1800
Total 262
MYROTHECIUM
VERRUCARIA
263
CELLULASE
and the extent of cellobiose hydrolysis by Goebel’s modification (13) of the Willstltter-Schudel method. The same enzyme standard was used as in Table III, its activity toward each substrate being likewise defined as one unit/mg. protein. The concentrations of the standard used in these assays corresponded to 0.025, 0.05, and 0.1 ml. of culture filtrate/20 ml. for carboxymethylcellulose; 0.1, 0.2 and 0.4 ml. for unswollen linters; and 3.7, 7.5, and 11.2 ml. for cellobiose.
A
s
A
Apn6.82
+4
++
pH5.03
ptl5.03
---I
C
A +-
P pH4.33
Fxo. 3. Electrophoretic and ultracentrifuge patterns of purified cellulase. A, B, and C, electrophoretic patterns; current, 0.020 amp.; temp., -O.l’C., ionic strength, 0.2; pH measured at 1°C. A, ascending boundary; time, 221 min.; buffer, phosphate. B, descending boundary; time, 187 min.; buffer, acetate. C, descending boundary; time, 225 min.; buffer, acetate. D, ultracentrifuge pattern (Spinco Model E ultracentrifuge); speed, 60,000 r.p.m.; time, 32 min.; buffer, acetate; ionic strength, 0.2.
The assays gave the following cellulase to protein ratios for these substrates: unswollen linters, 2.12; carboxymethylcellulose, 2.05; and cellobiose, 2.22. These ratios are thus approximately the same as those calculated for swollen linters, 2.24, and precipitated cellulose, 2.26, from the data in Table III. Reducing sugar titer-enzyme concentration curves of purified cellulase are given in Fig. 4 for the cellulose substrates. Comparable data
264
D.
R. WHITAKER
for cellobiose are: 1570 pg. protein N/20 ml., 7.55y0 hydrolysis; 3140 pg., 13.1y0 hydrolysis; and 4730 pg., 18.6% hydrolysis. (c) Reducing Sugar End Products. The identity of the soluble end products formed from swollen linters and precipitated cellulose under assay conditions was investigated as follows. Purified cellulase was incubated with substrate in baffled 2809ml. Fernbach flasks containing 409 ml. of assay medium. For each substrate, the assay medium was at pH 5.6 in one flask and at pH 6.8 in a second Aask (buffer, 296 ml. of 0.05
FIG. 4. Reducing sugar formation from cellulose substrates by purified cellulase at low enzyme concentrations. Titers (micro-Somogyi) : carboxymethylcellulose and precipitated cellulose/ml. of assay medium; swollen linters/3 ml. of assay medium; unswollen linters/5 ml. of assay medium.
M KHaPOrNazHPOc containing a trace of Dowicide G). The enzyme concentration corresponded to 8.9 pg. protein N/20 ml. After 17 hr. incubation at 35”C., the contents of each flask were centrifuged and the supernatant solution freeze-dried. The freeze-dried products were each dissolved in 15 ml. of water and cleared with Zn(OH)z by the method of Somogyi (14). They were then freed of salt in ionexchange columns, first in a column of Duolite (Chemical Process Co., San Francisco) to remove anions and then in a column of Nalcite HCR (Alchem. Ltd., Burlington, Ont., Canada) to remove cations. Washing of the columns with distilled water was continued to about 95% recovery of reducing sugar titer (final volumgr 259 ml .)
MYRO!PHECIUM
VERRUCARIA
265
CELLULASE
The resulting solutions were examined by three procedures : 1. Small samples were evaporated to thin sirups and developed on paper chromatograms. The developing solvent was the organic phase of a mixture of pyridine, ethyl acetate, and water (1:2:2 v/v). After drying, the papers were sprayed with aniline phthalate and heated to 110°C. The resulting chromatograms showed only two components. These corresponded in position with controls of glucose and cellobiose. The two spots were of approximately equal intensity and area for all solutions. Similar evidence of glucose and cellobiose formation by cellulolytic enzymes in culture filtrates of various fungi has been reported by Levinson et al. (3). 2. Two hundred milliliters of each solution was refluxed with phenylhydraaine acetate and sodium acetate. The phenylosazones precipitated were filtered off, washed with water, and converted to phenylosotriazoles by the method of Hann and Hudson (15). The phenylosotriazoles were treated with charcoal and recrystallized from water. Their melting points (corr.) were all in the range 192193°C. rising to 193-194°C. on mixture with authentic phenyl glucosotriazole of m.p. 194-195°C. 3. The solutions were analyzed for reducing sugar by the micro-Somogyi method and for total carbohydrate by the anthrone method as modified by Seifter et al. (16) with glucose as standard. Converting the Somogyi titers to glucose gave anthrone glucose to Somogyi glucose ratios as follows: Swollen linters Precipitated cellulose The ratios are thus of the order to be expected concentrations of glucose and cellobiose.
pH 5.6
pH 6.8
1.39 1.44
1.48 1.62
from
approximately
equimolar
These results therefore suggest that glucose and cellobiose are the main soluble end products of cellulase action under the above assay conditions. (d) Origin of Glucose. In the preceding assays of enzyme activity, the enzvme concentrations required for assay with 1% cellobiose were approximatelg 75X those required for assay with swollen linters and precipitated cellulose. This comparatively low activity toward cellobiose is of significance with regard to the origin of glucose as an end product of cellulose breakdown by this enzyme. It was noted previously (4) that the hydrolysis of cellobiose by partially purified Myrothecium cellulase appeared to be inhibited by increasing concentrations of cellobiose. This observation was confirmed in that no measurable hydrolysis of 5% cellobiose could be detected at enzyme concentrations giving nearly 209$ hydrolysis of 1% cellobiose. The apparently low activity of this cellulase toward cellobiose might therefore be due to assay at
266
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WHITAKER
too high a cellobiose concentration. However, in assays at very low cellobiose concentrations-0.01, 0.02, and 0.1% cellobiose-the enzyme concentrations used in precipitated cellulose and swollen linters assay gave no measurable increase in micro-Somogyi titer. As such enzyme concentrations give glucose as a major product from these cellulose substrates, it is concluded that the formation of glucose by this cellulase is not dependent on formation via cellobiose. DLYXJSSION
The above results are considered to suggest a unienzymatic mechanism of cellulose hydrolysis by this cellulase. The following is considered to be the main evidence: (a) The purified enzyme was homogeneous electrophoretically and in the ultracentrifuge. (b) Its enrichment in enzyme activity, relative to the culture filtrate from which it was obtained, was approximately the same for five substrates ranging in degree of polymerization from untreated cellulose to cellobiose. (c) Although enzyme activity was measured from the formation of soluble sugars (which included glucose) at low enzyme concentrations, all fractionation methods examined gave balance sheets in which the recovery of enzyme activity toward two cellulose substrates was in reasonably good agreement with the recovery of total protein. Such balances are considered contrary to the expectations of a multienzyme mechanism, for end-product formation would then be expected to depend not only on total recovery but on the relative concentration of enzyme components in each fraction. This evidence is not considered at variance with results which have been interpreted to indicate a multienzyme mechanism for this mold. Reese and his co-workers (2) have interpreted their finding that certain non-cellulolytic fungi can hydrolyze carboxymethylcellulose as indicating that cellulolytic fungi hydrolyze comparable anhydroglucose chains by an enzyme different from that causing the initial breakdown of cellulose. However the properties of a cellulase must include not only p-1,4polyanhydroglucosidase activity but properties permitting such activity under conditions of adsorption on an insoluble, cellulose surface. The findings of Reese et al. can be equally interpreted as merely indicating that these non-cellulolytic fungi produce an enzyme with the former but not the latter property. The detailed physical and chemical mechanisms involved in cellulase action are undoubtedly complex and cannot yet be discussed in detail.
MYROTHECIUM VERRUCARIA CELLULASE
267
The importance of the fine structure of cellulose in determining accessibility to enzyme attack is indicated by the recent data of Walseth (17). The effects of proteins and dyes adsorbed by cellulose on the action of Myrothetium cellulase (7, 9) suggest the importance of the surface potential of cellulose in cellulase action. The independent formation of cellobiose and glucose from cellulose by low concentrations of this cellulase is suggestive of a random breakdown of the cellulose chain in regions of equal accessibility. More positive evidence favoring this mechanism has been summarized by Greathouse (5). ACKNOWLEDGMENTS The writer wishes to express his thanks to Mr. Donald H. Hambrook for technical assistance throughout this work; to Dr. W. H. Cook and Dr. J. R. Colvin for valuable discussions and cooperation and assistance in the use of the ultracentrifuge and electrophoresis equipment; to Mr. A. E. Castagne for the qualitative chromatographic analyses; and to the Hercules Powder Co. for a generous supply of cotton linters.
SUMMARY
The cellulase in culture filtrates of Myrothecium verrucaria was purified by a sequence of concentration, precipitation by ammonium sulfate, fractionation with ethanol, and precipitation with polymethacrylic acid. The enrichments in enzyme activity toward the following substrates were approximately the same: untreated cotton linters, swollen cotton linters, a cellulose degraded by acid hydrolysis, a substituted soluble cellulose, and cellobiose. The purified enzyme was electrophoretically homogeneous at 3 pH’s and homogeneous in the ultracentrifuge. It is suggested that its mechanism of cellulose breakdown is unienzymatic, and evidence supporting this mechanism is discussed. REFERENCES 1. SIU, R. G. H., Microbial 2. 3. 4. 5. 6.
Decomposition
of Cellulose. Reinhold, New York, 1951. REESE, E. T., SIU, R. G. H., AND LEVINSON, H. S., J. Bact. 69,485 (1950). LEVINSON, H. S., MANDELS, G. R., AND REESE, E. T., Arch. Biochem. and Biophys. 31,351 (1951). WHITAKER, D. R., Nature 166, 1070 (1951). GREATHOUSE, G. A., Textile Research J. 23, 227 (1950). SAUNDERS, P. R., SIU, R. G. H., AND GENEST, R. N., J. Biol. Chem. 174, 697
(1948). 7. BASU, S. N., AND WHITAKER, D. R., Arch. Biochem. and Biophys. 8. J$RGENSEN, L., Studies on the Partial Hydrolysis of Cellulose, Hos Emil Moesture, Oslo, 1950.
42, 12 (1953). p. 140. Trykt
268 9. 10. 11. 12. 13. 14. 15. 16.
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WHITAKER, D. R., Science 116, 90 (1952). BARTHOLOMEW, W. H., Anal. Chem. 21, 527 (1949). SWINGLE, S. M., AND TISELIUS, A., B&hem. J. (London) MORAWETZ, H., AND HUGHES, W. H., J. Phys. Chem. 66, GOEBEL, N. F., J. Bid. Chem. 73, 801 (1927). SOMOGYI, M., J. Biol. Chem. 160, 69 (1945). HANN, R. M., AND HUDSON, C. S., J. Am. Chem. Sot. 66, SEIFTER, S., DAYTON, S., NOVIC, B., AND MUNTWYLER, 26, 191 (1950). 17. WALSETH, C., Tappi 36, 233 (1952).
46, 171 (1951). 64 (1952).
735 (1944). E., Arch. B&hem.