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Studies on cellulolytic enzymes
-4RCHIVES
OF
BIOCHEMISTRY
.iND
Studies VI. Specificity
130, 28&294
BIOPHYSICS
(1969)
on Cellulolytic
and Mode Cellulase
of Action on Different from Penicillium
GORAN Institute of Biochemistry,
Enzymes Substrates
of a
notatum
PETTERSSON
University
of Uppsala,
Uppsala,
Sweden
Received October 11, 1968; accepted November 26, 1968 Lichenin (a mixed 0-1.4 and p-1.3 glucan) is the only polysaccharide of several tested that is degraded by the enzyme. The Penicillium cellulase has a limited ability to attack crystalline cellulose in contrast to amorphous cellulose, which is readily attacked. Cellobiose is not attacked, while the rate of degradation of the higher members of the series of oligoglucosides increases with increasing D.P. The half-lifes of cellotriose, cellotetraose and cellopentaose were found to be 3700, 50, and 3 min, respectively. The central bonds of cellotetraose and cellopentaose were found to be the preferred points of cleavage. The K, values for the oligosaccharides displayed a pronounced tendency to decrease with chain length. Kinetic data suggested that the specificity region of the cellulase is five glucose units in length.
Culture filtrates from strongly collulolytic microorganisms often lack the ability to degrade native cotton fibers. These findings have been interpreted in terms of the Cl-C, hypothesis according to Reese (1). The C1 factor present during the growth of the microorganism would thus have disappeared due to, for instance, instability or adsorption on solid components in the culture media (mycelia or cellulose). During the last few years, however, culture filtrates capable of solubilizing cotton fibers have been produced from the moulds Trichodermu viride and Trichoderma koningi (Mandels and Reese (2) ; Haliwell (3) ; Li et al. (4) ; Selby and Maitland (5). It has also been possible to isolate different components in the cellulolytic enzyme system (4, 5), (Cl endo and exoglucanases). However, the mechanism of action of the C1 component is still obscure. After all, it seems evident that the Trichoderma species studied have a multienzymatic mechanism for cellulose degradation. attack have
Evidence for an unienzymatic been presented previously by
Whitaker
al. (6) working
et
with
material
from Myrothercium verrucaria. Selby and Maitland (7) working with material from the same organism have presented data which, on the contrary, seem to support a multienzymatic mechanism. Even if most data in the literature today seem to favor a multienzymatic mechanism, it is too early to draw any general conclusions about the mechanism for microbial cellulose degradation. The enzyme preparation used for the purification of the cellulose studied here is like some of the Trichoderma preparations of commercial origin. The cellulase has been purified to the extent of physico-chemical homogeneity (8, 9). Its molecular weight has been determined to be 35,000 and the amino acid composition is known (9). The further characterization has included studies of the protein conformation (lo), the effect of different inhibitors (11) and the role of different amino acid side chains for the enzymic function-particularly the role of the tryptophyl residues (12). The present paper is concerned with the mode of action
STUDIES
ON CELLULOLYTIC
on different types of cellulose substrates and the activity toward other polysaccharides. MATERIALS
AND
METHODS
Enzyme. The cellulase from Penicillium notalum was purified as described previously (8). Substrates. The following polysaccharides were used : Avicel (a microcrystalline cellulose from American Viscose Corporation, Marcus Hook, Pennsylvania, USA), dextran (Dextran 150 obtained from AB Pharmacia, Uppsala, Sweden), starch (Starch hydrolyzed from Connaught Medical Research Laboratories, Toronto, Canada), pullulan,l pustulan,’ laminarin,’ mannan’ and lichenin. The following oligosaccharides were used: Cellobiose (from Kebo AB, Stockholm, Sweden), cellotriose, cellotetraose, cellopentaose, cellohexaose, and a mixture of xylodextrinsl D.P. 5-7. G3, Gs, and Ge3 were isolated from a hydrochloric acid hydrolyzate of Whatman cellulose powder and were separated by chromatography using the charcoal-celite-2.5Y stearic acid procedure according to Miller et al. (13). Most of the cellotetraose used for this work was isolated after acetolysis and deacetylation of cellulose (13) and fractionation of the product by repeated gel filtrations on Sephadex G-25, as described by Flodin and Aspberg (16). Oligosaccharides were reduced with NaBH4 according to Cole and King (17). Following the reduction step the product was desalted on Sephadex G-15. Cellodextrins with D.P. > 7 were obtained after acid hydrolysis of 100 g cellulose for 2 hr at 25”. The hydrolyzate was treated as described above. The fraction used was eluted from the charcoal-celite adsorbent with 25% ethanol (v/v) after that the lower oligosaccharides had been removed with 20% ethanol (v/v). Thinlayer chromatography revealed that no oligosaccharides of lower D.P. than 7 was present. Acid swollen cellulose was prepared according to Walseth (18). Analytical methods. Reducing end-groups were determined with the DNS (dinitro salicylic acid) 1 A gift from Dr Hlkan BjBrndal, Swedish Forest Products Research Laboratories, Paper Technology Department, Stockholm, Sweden. 2 A gift from Dr Bo GBransson at the Department of Microbiology of the Royal Agricultural College, Uppsala, Sweden. 3 Abbreviations: G1, Gz, Ga, G4, Gs, and Gg p-1.4-oligoglucosides of the polymer series of cellulose; G~H, Gsn, G~H, and G~H the corresponding series in which the reducing glucose has been reduced to sorbitol.
ENZYMES
method (19) or by the spectrophotometric method according to Somogyi (20). The orcinol method (14) was used for determination of the oligosaccharides. Standard curves were made for each member in the series Gl-G5. The enzymic degradation of the oligosaccharides was followed by determining the change in optical rotation at 365 nm as a function of time at 40”. A recording PerkinElmer 141 Polarimeter equipped with a lo-cm microcell was used. Analytical separation methods. Thin-layer chromatography was done on 20 X 20.cm plates coated with a 0.25-mm layer of Kieselguhr G buffered with 0.02 M sodium acetate. Prior to use, the plates were kept at 100” for at least 24 hr. The samples were applied 2.5 cm from the bottom edge of the plate and the chromatogram was developed in 65yo isopropanol (v/v)-ethylacetate 1:1 (21) using a sandwich chamber. When the solvent front was about 2 cm from the top of the plate, the experiment was interrupted and the plate was dried at room temperature. The plate was then sprayed with the anisaldehyde-sulfuric acid reagent (22) and heated to 105” for 10 min. A good separation of the cellodextrins with D.P. from 1 to 6 was achieved (see Fig. 1). For quantitative analysis of the cellodextrins the microcolumn method was used as described by Miller (15). A constant flow rate was used during the experiment. Degradation of noncellulose polysaccharides. 0.50/, Solutions of the following polysaccharides : Dextran, starch, pullulan, pustulan, laminarin, mannan, and lichenin were prepared in 0.05 M sodium acetate buffer pH 5.0. Two-milliliter samples of each polysaccharide solution were incubated at 40” with 0.1 ml cellulase solution (AZ80 = 0.28). After 15. and 60.min incubation, the samples were analyzed for reducing end-groups with the DNS method as described in (19). The test for xylanase activity was carried out in t,he same way except that due to the small amounts of xylodextrins available only 0.5-ml samples of substrate were used. Degradation of cellulose and physically modifed cellulose. Ten millileters of a 1% suspension of microcrystalline cellulose (Avicel) were prepared in 0.05 M sodium acetate buffer pH 5.0. The suspension contained 0.047, merthiolate (w/v) and 0.047, (w/v) sodium azide as preservatives. A solution of 200 ~1 cellulase (A280 = 18) in the same buffer were added and the suspension was placed on a thermostated rotary shaker at 37”. Samples were withdrawn and tested for reducing sugars with the Somogyi spectrophotometric method (20) each day during a period of 16 days. The experiment was then interrupted by heating the suspension
288
PETTERSSON
to 100” for 20 min. Remaining cellulose was removed by centrifugation and the concentration of saccharides in the supernate was determined with the orcinol method. The composition of oligosaccharides was determined by the microcolumn technique as described previously. In another experiment, the time course of hydrolysis of acid swollen cellulose (see under Materials) was determined. The experiment was performed as described above except that the activity tests had to be done with time intervals as short as 15 min during the first 2 hr, due to the high initial rate of hydrolysis. The mode of action against cellodextrins and modified cellodextrins. The enzymic hydrolysis of the ,8-1.4-oligo-glucosides was followed by determining the change in composition with time by means of the microcolumn method of Miller (15). Oligosaccharide of 12 mg in 4 ml 0.05 M sodium acetate buffer pH 5.0 were incubated with 200 ~1 enzyme (A280 = 2.1) in the same buffer at 40”. At various time intervals, 400 ~1 of the reaction mixture were removed, mixed with 1 ml of hot ethanol, and evaporated to dryness. The sample was dissolved in 200 ~1 of distilled water and subjected to column chromatography. The proper time interval for each oligosaccharide was determined beforehand by TLC. The Michaelis parameters K, and V for the enzyme was determined from Lineweaver-Burk plots for G4, Gs, G8, and a mixture of cellodextrins with D.P. >7. The initial velocities for the enaymic hydrolysis of Gq, Gg, and Gg were determined by measuring the change in optical rotation with time. The enzymic hydrolysis of the cellodextrin mixture (D.P. >7) was followed by measuring the increase in reducing end-groups. Samples were removed from the solution immediately after adding the enzyme and then after different time intervals and tested for reducing sugars with the Somogyi spectrophotometric method (20). The initial linear part of the curve obtained by plotting the reducing values vs. time was used for the calculation of v, K, and V were also determined for the reduced cellodextrins G~H and Gjn using the reducing end-group method. RESULTS
The activity towards noncellulose polysaccharides. To study the specificity of the enzyme the following polysaccharides were tested as substrates : Dextran (a-1.6-glucan), starch (a-1.4-glucan), pullulan (mixed a-1.4and a-1.6-glucan), laminarin @-1.3-glucan), pustulan (p-1.6-glucan), mannan (p-1.4linked mannose), Iichenin (mixed p-1.3 and
P-1 A-glucan), and a xylodextrin mixture (P-1.4 linked xylose). Only lichenin was degraded of the polysaccharides tested. The degradation products were identified by thin layer chromatography. Assuming that the hydrolysis products have the same Rp values as the corresponding p-1.4 oligosaccharides run as standard, it seems that oligosaccharides with D.P. from 3-6 are enriched during the enzymic hydrolysis (Fig. 1). The hydrolysis of cellulose. A degradation experiment of crystalline cellulose (Avicel) is illustrated in Fig. 2. The hydrolysis is very slow during the first 6 days, after which there is a rapid increase until the plateau value is reached after 12 days. Analysis of the supernate revealed that it contained glucose, cellobiose, and cellotriose in the molar ratio 1: 1: 0.15. The amount of oligosaccharide corresponded to approximately 2% of the weight of the cellulose at the start of the experiment. Acid swollen cellulose is much more rapidly hydrolyzed, as is shown by Fig. 3. It is especially the initial phase of the hydrolysis that is rapid. By the end of 4 days, 12 % of the cellulose was solubilized. Glucose, cellobiose, and cellotriose were recovered in the molar ratio 1:2.2:0.5. Enx ymic hydrolysis of the p-1.4-oligoglucosides. To determine the rate and the method of degradation of the p-1.4-oligoglucosides the sugars were incubated with the enzyme and samples were taken out during the reaction and subjected to microcolumn chromatography (Figs. 4, 5, and 6). Cellobiose was not hydrolyzed, while the rate of hydrolysis of the higher oligoglucosides increased with D.P. The half-lives obtained at constant enzyme concentration for Ga, Gq, and Gj were thus found to be 3700, 50, and 3 min, respectively. G8 is hydrolyzed in a very straightforward way producing Gz and G1 in equimolar quantities (Fig. 7). The hydrolysis products of Gq are Gs, Ge, and G1 (Fig. 8). Gz seems to be formed in higher amounts than Gs and G1. This is especially striking during the later part of the reaction. Gz and G1 are formed in approximate1 y equimolar amounts during the early stage of the reaction. Hydrolysis of Gj gives all kinds of products (Fig. 9). G, (not shown in Fig. 9) is
STUDIES
ON CELLULOLYTIC
289
ENZYMES
FIG. 1. Thin layer chromatography of lichenin after hydrolysis with the cellulase from The chromatogram was developed for 30 min. with 65% isopropanol:ethylacetate 1:l. The samples contained (from the left to the right): B-1.4-oligoglucosides with DP l-6 (glucose moves closest to the front). Cellobiose, Hydrolyzates of lichenin samples taken after 2, 4, 6, and 24 hr of hydrolysis. Cellobiose P-1.4-oligoglucosides with DP 1-G.
P. n&&m.
also formed but due to the low orcinol values obtained an accurate determination of the amounts was impossible. However, results from several experiments indicate that G1 is formed in approximately the same amounts as Gq during the initial part of the reaction. From Fig. 9 it is apparent that oligosaccharides are formed in the following relative amounts Gz > Ga > Gd. Furthermore, it is evident that Gt and Ga are formed in approximately the same amounts during the initial stage of the reaction, whereas during later stages more G2 than G, is formed. The
kinetic
parameters
Km and
b
there
kf is the velocity constant first order with respect to substrate when [S] << [Km1derived for the cleavage of short chain polymers by Hanson (23) were determined for the p-1.4oligoglucosides cellotetraose, cellopentaose, and cellohexaose and for reduced cellotetraose and cellopentaose. The polarimetric technique used for the determination of the rate of hydrolysis of the oligosaccharides is classical in enzymology. Modern instrumentation has made
E o.4 f o,3 9 2b 0.2
3 0.1 7F” 2
4
6
8
Days
IO
12
14
J 16
FIG. 2. Formation of reducing sugars from crystalline cellulose (Avicel) determined as glucase, upon degradation with the cellulase from P. n&turn. Substrate: 10 ml of a 1% suspension of Avicel in 0.05 M sodium acetate buffer pH 5.0. Enzyme: 200 pl L42eo = 18. Te.perat,Llre: 370.
it possible to increase the precision of the method by measurements at shorter wavelengths where the optical rotation is much higher than at the sodium D line. The fact that the method also allows the continuous registration of the enzymic hydrolysis makes it particularly valuable. Mutarotation of substrates or products of the reaction may of course be a complication even if it is possible (Whitaker (24)) that mutarotation
290
PETTERSSON
F‘ iO.4 $0.3 E
c 0.2 B bO1 : T 10
20
Frocfion
FIG. 3. Formation of reducing sugars from acid swollen cellulose determined as glucose upon degradation with the cellulase from P. notatum. The experimental conditions were the same as in Fig. 2.
30 40 number
FIG. 5. Column chromatography of cellotetraose (GJ after enzymic hydrolysis with the cellulase from P. not&m. Sample was taken 60 min after start of the hydrolysis. For further experimental details see the text.
F‘ c 0.8
0
Lx 0.6 PI ii
0.4
B 0 0.2 :
Frocfion Frocfion
number
FIG. 4. Column chromatography
of cellotriose (GJ after enzymic hydrolysis with the cellulase from P. not&urn. Sample was taken 48 hr after start of the hydrolysis. The concentration of oligosaccharide was determined with the orcinol method. For further experimental details see the text.
is not rate limiting at temperatures as high as 40”. The effect of mutarotation and other technical aspects of the polarimetric method for assaying enzymes will be discussed elsewhere. The results of a degradation experiment are reproduced in Fig. 10. The substrate is Gh at a concentration of 5 mg/ml. The curve is composed of an initial lag phase, a nearly linear part, and a later part where the velocity decreases with time. The velocity used for the determination of K, was derived from the slope of the part of the curve immediately after the lag phase. A Lineweaver-Burk plot from a degradation experiment with Gd is shown in Fig. 11.
number
FIG. 6. Column chromatography of cellopentaose (G,) after hydrolysis with the cellulase of P. notatum. Sample was taken 6 min after start of the hydrolysis. For further experimental details see the text.
Km and Icf values for the p-1.4 oligosaccharides are summarized in Table I. S represents the velocity constant, first order with respect to substrate, when S << Km (23). The polarimetric technique was used for Gs, Cd, Gg, and Ge and the reducing end group method for GbH, GsH, and for the cellodextrin mixture with D.P. > 7. A Lineweaver-Burk plot of data from the degradation of G4H is shown in Fig. 12. Gz and Gsa are not attacked by the enzyme and Ga is degraded very slowly. K, and i& were not determined for Ga but the enzyme activity was dependent upon substrate concentration even in the range 100-120 mg/ml. Km must thus be greater than 50 g/liter. It is evident that Km decreases with the D.P. of the substrate while kf increases. It is also evident the reduced
STUDIES
ON CELLULOLYTIC
291
E?JZYRIES
50
100
150
MiflUf.C?S I
20
I
40
/
60 Hours
I
80
I
IOC
FIG. 7. Time course of the hydrolysis of cellotriose with the cellulase from P. not&urn. The distribut.ion of oligosaccharides was determined at different time intervals with the microcolumn method (15). Experimental conditions: 12 mg of oligosaccharide in 4 ml 0.05 M sodium acetate buffer pH 5.0 were incubated with 200 ~1 enzyme (A280 = 2.1) at 40”.
FIG. 10. The change in optical rotation with time upon hydrolysis of cellotetraose with the cellulase from P. not&urn. Substrate: Ga 5 mg/ml in 0.05 M sodium acetate buffer pH 5.0. Enzyme: 100 ~1 (ABO = 2.37) added to 1 ml substrate solution. Temperature : 40”. lo-cm cell. Wavelength of registration 365 nm.
I
I 0.2
I 0.4
I 0.6
I 0.8
~lml/mgj
Minu
fes
FIG. 8. Time course of the hydrolysis tetraose with the cellulase from P. Conditions as in Fig. 7.
of cellonot&urn.
FIG. 11. Double reciprocal plot of rate of hydrolysis of cellotetraose catalyzed by the cellulase from P. not&urn. Initial velocities determined from the change in optical rotation at 365 nm with time (see Fig. 10). pH 5.0, 0.05 M sodium acetate buffer. Temperature: 40”.
members of the oligosaccharide series give higher K, values than the corresponding unreduced oligosaccharides. The differences are particularly pronounced between Gq and GIH and less pronounced between Gg and G 5H. DISCUSSION
d
5
10
15
20
Minutes
FIG. 9. Time course of the hydrolysis pentaose with the cellulase from I’. Conditions as in Fig. 7.
of cellonotatum.
Several CX- and P-linked glucans were tested together with a mannan and a xylodextrin mixture (D.P. 5-7) as substrates for the enzyme. Only lichenin, which is a mixed glucan containing p-l.4 and p-1.3 bonds, was found to be degraded. The products of the reaction were oligoglucosides with D.P. from 3-6. Perlin (25) has shown
292
PETTERSSON TABLE
I
THE EMPIRICAL CONSTANTS K,,, AND k/ FOR THE HYDROLYSIS OF p-1.4 OLIGOGLUCOSIDES WITH THE Ponicillium CELLULASE Kf = K:
mole. 1-1 degree.1. mole-lmin-1 Cellotriose Cellotetraose Cellopentaose Cellohexaose Cellodextrinc (D.P.>7) Reduced cellotetraose Reduced cellopentaose
>900.10-4 250.10-4 10.10-4 6.7.10-” 3.7.10-4
a a a
910.W4
b
13.10-4
0.72= 7.7” 6.3” -
;
b
-
(1Determined from polarimetric measurements. b Determined with the reducing end-group method (see the text). c The average D.P. was considered to be 9.
QI 0 I 8 30 k
I
; 20 \ t? 2 IO $1-k
O.IO
020
0.30
FIG. 12. Double reciprocal plot of rate of hydrolysis of reduced cellotetraose catalyzed by the cellulase from P. notatum. Initial velocities determined from the increase in reducing groups upon hydrolysis of the substrate. Experimental conditions as in Fig. 11.
that lichenin gives cellobiose as a product when degraded with cellulase, but not laminaribose. In our experiment only minor amounts of cellobiose could be detected (see Fig. 1). The major products formed were oligosaccharides with D.P. 5 or 6. It is thus possible that our Penidium cellulase has a more restricted mode of action than the cellulase used by Perlin. The Penicillium enzyme showed a very limited ability to degrade crystalline cellulose (see Fig. 2). Only about 2% of the
cellulose was solubilized after 12 days. On the contrary, acid swollen cellulose is rapidly degraded. Fig. 3 shows that most of the degradation occurs during the first hour of the reaction. By the end of 4 days, 12 % of the cellulose was solubilized. More extensive hydrolysis was observed when freshly prepared cellulose was used. The cellulose utilized for the actual experiment was prepared 1 month prior to use. Nevertheless, it is obvious that there are parts of the swollen cellulose that are not hydrolyzed by the enzyme. The enzyme does not attack cellobiose, while higher members of the oligoglucoside series are attacked at increasing rate. The half-lives obtained at a constant enzyme concentration were thus found to be 3700, 50, and 3 min for cellobiose, cellotetraose, and cellopentaose, respectively. All possible products are formed upon degradation of cellotriose, cellotetraose, and cellopentaose. The relative amounts of oligosaccharides indicate, however, that the central linkages are the preferred points of cleavage (Figs. 8 and 9). The rate of hydrolysis of the terminal bonds increases like the overall rate of hydrolysis in the series cellotriose to cellopentaose. The major products of hydrolysis of cellopentaose are cellobiose and cellotriose (Fig. 9). These sugars are formed in the same amounts during the initial part of the reaction while later more cellobiose is formed than cellotriose, probably due to hydrolysis of the cellotetraose initially formed. The method of degradation seems to resemble closely what has been found for the cellulase from Myrothecium verrucuriu (26), except that the Myrothecium enzyme was found to hydrolyze cellobiose. The empirical constants K, and k, for the hydrolysis of the /3-1.4-oligoglucosides have been determined. The initial velocities were determined by a polarimetric method which, to the author’s knowledge, has not been used for this purpose before. The method has many advantages. It is, for instance, the only method that offers the possibility of continuous registration of the enzymic hydrolysis of oligosaccharides. It is also far less tedious than other possible methods. Nevertheless, it must be emphasized that
STUDIES
ON CELLULOLYTIC
much work remains to be done before all the potentialities of the technique can be realized. The effect of mutarotation of the hydrolysis products is, for instance, not known. This means that the numerical values of the constants in Table I might be of limited significance. The figures are nevertheless considered good enough to demonstrate a trend in the order of magnitude. The figures indicate that K, decreases with the D.P. of the substrate. The decrease is very drastic going from cellotetraose to cellopentaose but less pronounced thereafter. The decrease in K, with increasing chain length of the oligoglucosides might reflect an increasing tendency to form enzyme substrate complexes. It is nevertheless surprising that the addition of a p-sorbityl residue to cellotetraose has about the same effect as the addition of a glucoyl residue. The same trend in K, values for the oligoglucosides has been observed earlier by Whitaker (6) and by Li et al. (4) working with other cellulases. Table I shows that the k, value increases about ten-fold going from cellotetraose to cellopentaose, while there is a slight decrease (possibility insignificant) going from cellopentaose to cellohexaose. According to Hanson (23) this trend in the values of kf means that the specificity region of the enzyme is five glucose units in length. It should be observed that there is a similar very drastic change in the K, values going from cellotetraose to cellopentaose. Applying the same argument Whitaker (26) and Hanstein and Whitaker (27) showed that the specificity region of the Myrothecium cellulase was at least five glucose units in length. The present data indicates that the Penicillium cellulase functions as an endoglucanase and should be designated as a /3-1.4-glucan 4-glucanohydrolase (EC 3.2.1.4). The typical preference for the cleavage of nonterminal bonds in the short chain oligosaccharides does not rule out the possibility that longer chains are cleaved in an approximately random way. Although in most cases the properties of the substrate, (substitution or accessibility of linkages) rather than the intrinsic properties of the enzyme, determine the method of degradation.
ENZYMES
293
The degradation pattern obtained on oligoglucosides resembles closely that of other cellulolytic enzymes (26-28). The only exception seems to be the exoglucanase from Trichoderma viride (4) which prefers terminal linkages. The Penicillium enzyme attacks crystalline cellulose very slowly, while swollen cellulose is readily attacked. The enzyme was found to degrade cellotriose very slowly. Thereafter, the rate of degradation increased rapidly with the D.P. of the oligosaccharide chain up to a limit of five glucose residues. In crystalline cellulose the polysaccharide chains are very closely packed and it is improbable that longer segments of polysaccharide chains are accessible to the enzyme. It is thus possible to explain the slow rate of hydrolysis of native cellulose, in view of the kinetic experiments with the oligoglucosides. It is of interest to note that after degradation of crystalline cellulose, the molar ratio of glucose to cellobiose was approximately 1: 1, which is the ratio expected upon degradation of cellotriose. The swollen cellulose gave glucose and cellobiose approximately in the molar ratio 1:2, which is the expected ratio upon degradation of longer oligosaccharides. It thus seems that these results further support the hypothesis that only short segments of polysaccharide chains in crystalline cellulose are accessible to the enzyme. ACKNOWLEDGMENTS I thank my teacher Professor J. his kind interest in this work and Mrs. Fredriksson for valuable technical The work has been supported in part from the Hiertn-Retzius foundation.
Porath for Ulla-Britt assistance. by a grant
REFERENCES 1. REI~:SE, E. T., SIU, 11. G. H., AND LEVINSSON, H. S., J. Hacteriol. 69,485 (1950). 2. MANDELS, M. AND Rnkss, E. T., Develop. Ind. Microbial. 6, 5 (1964). 3. HALLIWELL, G., Rio&em. J. 100, 315 (1966). 4. LI, L. H., FLORA, R. M., AND KIXG, K. W., Arch. Biochem. Biophys. 111, 439 (1965). 5. SELBY, K. .\ND MMTLAND, C. C.,Biochem. J. 104, 716 (1967). 6. WHITAKER, I). ii., HANSON, K. R., ago D.4TTs P. K., Can. J. Biochem. Physiol. 41, 671 (1963).
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PETTERSSON
7. SELBY, K. AND MAITLAND, C. C., Biochem. J. 94, 578 (1965). 8. PETTERSSON, G., Arch. Biochem. Biophys. 133, 307 (1968). 9. PETTERSSON, G. AND EAKF,R, D. L., Arch. Biothem. Biophys. 124,497 (1968). 10. PETTERSSON, G. AND ANDERSSON, L., Arch. Biochem. Biophys. 124, 497 (1968). 11. ERIKSSON, K.-E. AND PETTERSSON, G., Arch. Biochem. Biophys. 134, 160 (1968). 12. PETTERSSON, G., Arch. Biochem. Biophys. 126,776 (1968). 13. MILLER, G. L., DEAN, J., AND BLUM, R., Arch. Biochem. Biophys. 91, 21 (1960). 14. VASSEUR, E., Acta Chem. Stand. 2, 693 (1948). 15. MILLER, G. L., Anal. Biochem. 2, 133 (1960). 16. FLODIN, P. AND ASPBERG, K., Biol. Struct. Function 1,345 (1961). 17. COLE, F. E. AND KING, K. W., Biochim. Biophys. Acta 81, 122 (1964). 18. WALSETH, C. S., Tappi 36, 228 (1952).
19. MILLER,
G. L., BLUM, R., GLENNON, W. E., AND BURTON, A. L., Anal. Biochem. 2, 127 (1960).
20. SOMOGYI, M., J. Biol. Chem. 196,19 (1952). 21. JANSSON, J. C., Personal communications. 22. STAHL, E., Diinnsicht-Chromatographie Ein
Laboratoriumshandbuch. 2. Auflage. p. 817. Springer, Berlin (1967). 23. HANSSON, K. R., Biochemistry 1,723 (1962). 24. WHITAKER, D. R., Arch. Biochem. Biophys. 63, 436 (1954). 25. PERLIN, A. S., in
“Advances in Enzymic Hydrolysis of Cellulose and Related Material.” E. T. Reese, (ed.) p. 188. Pergamon Press, New York (1963). 26. WHITAKER, D. R., Arch. Biochem. Biophys. 63,439 (1954). 27. HANSTEIN, E. G. AND WHITAKER, D. R., Can J. Biochem. Physiol. 41, 707 (1963). 28. CLARKE, A. E. AND STONE, B. A., Biochem. J
96,802 (1965).
OF
BIOCHEMISTRY
.iND
Studies VI. Specificity
130, 28&294
BIOPHYSICS
(1969)
on Cellulolytic
and Mode Cellulase
of Action on Different from Penicillium
GORAN Institute of Biochemistry,
Enzymes Substrates
of a
notatum
PETTERSSON
University
of Uppsala,
Uppsala,
Sweden
Received October 11, 1968; accepted November 26, 1968 Lichenin (a mixed 0-1.4 and p-1.3 glucan) is the only polysaccharide of several tested that is degraded by the enzyme. The Penicillium cellulase has a limited ability to attack crystalline cellulose in contrast to amorphous cellulose, which is readily attacked. Cellobiose is not attacked, while the rate of degradation of the higher members of the series of oligoglucosides increases with increasing D.P. The half-lifes of cellotriose, cellotetraose and cellopentaose were found to be 3700, 50, and 3 min, respectively. The central bonds of cellotetraose and cellopentaose were found to be the preferred points of cleavage. The K, values for the oligosaccharides displayed a pronounced tendency to decrease with chain length. Kinetic data suggested that the specificity region of the cellulase is five glucose units in length.
Culture filtrates from strongly collulolytic microorganisms often lack the ability to degrade native cotton fibers. These findings have been interpreted in terms of the Cl-C, hypothesis according to Reese (1). The C1 factor present during the growth of the microorganism would thus have disappeared due to, for instance, instability or adsorption on solid components in the culture media (mycelia or cellulose). During the last few years, however, culture filtrates capable of solubilizing cotton fibers have been produced from the moulds Trichodermu viride and Trichoderma koningi (Mandels and Reese (2) ; Haliwell (3) ; Li et al. (4) ; Selby and Maitland (5). It has also been possible to isolate different components in the cellulolytic enzyme system (4, 5), (Cl endo and exoglucanases). However, the mechanism of action of the C1 component is still obscure. After all, it seems evident that the Trichoderma species studied have a multienzymatic mechanism for cellulose degradation. attack have
Evidence for an unienzymatic been presented previously by
Whitaker
al. (6) working
et
with
material
from Myrothercium verrucaria. Selby and Maitland (7) working with material from the same organism have presented data which, on the contrary, seem to support a multienzymatic mechanism. Even if most data in the literature today seem to favor a multienzymatic mechanism, it is too early to draw any general conclusions about the mechanism for microbial cellulose degradation. The enzyme preparation used for the purification of the cellulose studied here is like some of the Trichoderma preparations of commercial origin. The cellulase has been purified to the extent of physico-chemical homogeneity (8, 9). Its molecular weight has been determined to be 35,000 and the amino acid composition is known (9). The further characterization has included studies of the protein conformation (lo), the effect of different inhibitors (11) and the role of different amino acid side chains for the enzymic function-particularly the role of the tryptophyl residues (12). The present paper is concerned with the mode of action
STUDIES
ON CELLULOLYTIC
on different types of cellulose substrates and the activity toward other polysaccharides. MATERIALS
AND
METHODS
Enzyme. The cellulase from Penicillium notalum was purified as described previously (8). Substrates. The following polysaccharides were used : Avicel (a microcrystalline cellulose from American Viscose Corporation, Marcus Hook, Pennsylvania, USA), dextran (Dextran 150 obtained from AB Pharmacia, Uppsala, Sweden), starch (Starch hydrolyzed from Connaught Medical Research Laboratories, Toronto, Canada), pullulan,l pustulan,’ laminarin,’ mannan’ and lichenin. The following oligosaccharides were used: Cellobiose (from Kebo AB, Stockholm, Sweden), cellotriose, cellotetraose, cellopentaose, cellohexaose, and a mixture of xylodextrinsl D.P. 5-7. G3, Gs, and Ge3 were isolated from a hydrochloric acid hydrolyzate of Whatman cellulose powder and were separated by chromatography using the charcoal-celite-2.5Y stearic acid procedure according to Miller et al. (13). Most of the cellotetraose used for this work was isolated after acetolysis and deacetylation of cellulose (13) and fractionation of the product by repeated gel filtrations on Sephadex G-25, as described by Flodin and Aspberg (16). Oligosaccharides were reduced with NaBH4 according to Cole and King (17). Following the reduction step the product was desalted on Sephadex G-15. Cellodextrins with D.P. > 7 were obtained after acid hydrolysis of 100 g cellulose for 2 hr at 25”. The hydrolyzate was treated as described above. The fraction used was eluted from the charcoal-celite adsorbent with 25% ethanol (v/v) after that the lower oligosaccharides had been removed with 20% ethanol (v/v). Thinlayer chromatography revealed that no oligosaccharides of lower D.P. than 7 was present. Acid swollen cellulose was prepared according to Walseth (18). Analytical methods. Reducing end-groups were determined with the DNS (dinitro salicylic acid) 1 A gift from Dr Hlkan BjBrndal, Swedish Forest Products Research Laboratories, Paper Technology Department, Stockholm, Sweden. 2 A gift from Dr Bo GBransson at the Department of Microbiology of the Royal Agricultural College, Uppsala, Sweden. 3 Abbreviations: G1, Gz, Ga, G4, Gs, and Gg p-1.4-oligoglucosides of the polymer series of cellulose; G~H, Gsn, G~H, and G~H the corresponding series in which the reducing glucose has been reduced to sorbitol.
ENZYMES
method (19) or by the spectrophotometric method according to Somogyi (20). The orcinol method (14) was used for determination of the oligosaccharides. Standard curves were made for each member in the series Gl-G5. The enzymic degradation of the oligosaccharides was followed by determining the change in optical rotation at 365 nm as a function of time at 40”. A recording PerkinElmer 141 Polarimeter equipped with a lo-cm microcell was used. Analytical separation methods. Thin-layer chromatography was done on 20 X 20.cm plates coated with a 0.25-mm layer of Kieselguhr G buffered with 0.02 M sodium acetate. Prior to use, the plates were kept at 100” for at least 24 hr. The samples were applied 2.5 cm from the bottom edge of the plate and the chromatogram was developed in 65yo isopropanol (v/v)-ethylacetate 1:1 (21) using a sandwich chamber. When the solvent front was about 2 cm from the top of the plate, the experiment was interrupted and the plate was dried at room temperature. The plate was then sprayed with the anisaldehyde-sulfuric acid reagent (22) and heated to 105” for 10 min. A good separation of the cellodextrins with D.P. from 1 to 6 was achieved (see Fig. 1). For quantitative analysis of the cellodextrins the microcolumn method was used as described by Miller (15). A constant flow rate was used during the experiment. Degradation of noncellulose polysaccharides. 0.50/, Solutions of the following polysaccharides : Dextran, starch, pullulan, pustulan, laminarin, mannan, and lichenin were prepared in 0.05 M sodium acetate buffer pH 5.0. Two-milliliter samples of each polysaccharide solution were incubated at 40” with 0.1 ml cellulase solution (AZ80 = 0.28). After 15. and 60.min incubation, the samples were analyzed for reducing end-groups with the DNS method as described in (19). The test for xylanase activity was carried out in t,he same way except that due to the small amounts of xylodextrins available only 0.5-ml samples of substrate were used. Degradation of cellulose and physically modifed cellulose. Ten millileters of a 1% suspension of microcrystalline cellulose (Avicel) were prepared in 0.05 M sodium acetate buffer pH 5.0. The suspension contained 0.047, merthiolate (w/v) and 0.047, (w/v) sodium azide as preservatives. A solution of 200 ~1 cellulase (A280 = 18) in the same buffer were added and the suspension was placed on a thermostated rotary shaker at 37”. Samples were withdrawn and tested for reducing sugars with the Somogyi spectrophotometric method (20) each day during a period of 16 days. The experiment was then interrupted by heating the suspension
288
PETTERSSON
to 100” for 20 min. Remaining cellulose was removed by centrifugation and the concentration of saccharides in the supernate was determined with the orcinol method. The composition of oligosaccharides was determined by the microcolumn technique as described previously. In another experiment, the time course of hydrolysis of acid swollen cellulose (see under Materials) was determined. The experiment was performed as described above except that the activity tests had to be done with time intervals as short as 15 min during the first 2 hr, due to the high initial rate of hydrolysis. The mode of action against cellodextrins and modified cellodextrins. The enzymic hydrolysis of the ,8-1.4-oligo-glucosides was followed by determining the change in composition with time by means of the microcolumn method of Miller (15). Oligosaccharide of 12 mg in 4 ml 0.05 M sodium acetate buffer pH 5.0 were incubated with 200 ~1 enzyme (A280 = 2.1) in the same buffer at 40”. At various time intervals, 400 ~1 of the reaction mixture were removed, mixed with 1 ml of hot ethanol, and evaporated to dryness. The sample was dissolved in 200 ~1 of distilled water and subjected to column chromatography. The proper time interval for each oligosaccharide was determined beforehand by TLC. The Michaelis parameters K, and V for the enzyme was determined from Lineweaver-Burk plots for G4, Gs, G8, and a mixture of cellodextrins with D.P. >7. The initial velocities for the enaymic hydrolysis of Gq, Gg, and Gg were determined by measuring the change in optical rotation with time. The enzymic hydrolysis of the cellodextrin mixture (D.P. >7) was followed by measuring the increase in reducing end-groups. Samples were removed from the solution immediately after adding the enzyme and then after different time intervals and tested for reducing sugars with the Somogyi spectrophotometric method (20). The initial linear part of the curve obtained by plotting the reducing values vs. time was used for the calculation of v, K, and V were also determined for the reduced cellodextrins G~H and Gjn using the reducing end-group method. RESULTS
The activity towards noncellulose polysaccharides. To study the specificity of the enzyme the following polysaccharides were tested as substrates : Dextran (a-1.6-glucan), starch (a-1.4-glucan), pullulan (mixed a-1.4and a-1.6-glucan), laminarin @-1.3-glucan), pustulan (p-1.6-glucan), mannan (p-1.4linked mannose), Iichenin (mixed p-1.3 and
P-1 A-glucan), and a xylodextrin mixture (P-1.4 linked xylose). Only lichenin was degraded of the polysaccharides tested. The degradation products were identified by thin layer chromatography. Assuming that the hydrolysis products have the same Rp values as the corresponding p-1.4 oligosaccharides run as standard, it seems that oligosaccharides with D.P. from 3-6 are enriched during the enzymic hydrolysis (Fig. 1). The hydrolysis of cellulose. A degradation experiment of crystalline cellulose (Avicel) is illustrated in Fig. 2. The hydrolysis is very slow during the first 6 days, after which there is a rapid increase until the plateau value is reached after 12 days. Analysis of the supernate revealed that it contained glucose, cellobiose, and cellotriose in the molar ratio 1: 1: 0.15. The amount of oligosaccharide corresponded to approximately 2% of the weight of the cellulose at the start of the experiment. Acid swollen cellulose is much more rapidly hydrolyzed, as is shown by Fig. 3. It is especially the initial phase of the hydrolysis that is rapid. By the end of 4 days, 12 % of the cellulose was solubilized. Glucose, cellobiose, and cellotriose were recovered in the molar ratio 1:2.2:0.5. Enx ymic hydrolysis of the p-1.4-oligoglucosides. To determine the rate and the method of degradation of the p-1.4-oligoglucosides the sugars were incubated with the enzyme and samples were taken out during the reaction and subjected to microcolumn chromatography (Figs. 4, 5, and 6). Cellobiose was not hydrolyzed, while the rate of hydrolysis of the higher oligoglucosides increased with D.P. The half-lives obtained at constant enzyme concentration for Ga, Gq, and Gj were thus found to be 3700, 50, and 3 min, respectively. G8 is hydrolyzed in a very straightforward way producing Gz and G1 in equimolar quantities (Fig. 7). The hydrolysis products of Gq are Gs, Ge, and G1 (Fig. 8). Gz seems to be formed in higher amounts than Gs and G1. This is especially striking during the later part of the reaction. Gz and G1 are formed in approximate1 y equimolar amounts during the early stage of the reaction. Hydrolysis of Gj gives all kinds of products (Fig. 9). G, (not shown in Fig. 9) is
STUDIES
ON CELLULOLYTIC
289
ENZYMES
FIG. 1. Thin layer chromatography of lichenin after hydrolysis with the cellulase from The chromatogram was developed for 30 min. with 65% isopropanol:ethylacetate 1:l. The samples contained (from the left to the right): B-1.4-oligoglucosides with DP l-6 (glucose moves closest to the front). Cellobiose, Hydrolyzates of lichenin samples taken after 2, 4, 6, and 24 hr of hydrolysis. Cellobiose P-1.4-oligoglucosides with DP 1-G.
P. n&&m.
also formed but due to the low orcinol values obtained an accurate determination of the amounts was impossible. However, results from several experiments indicate that G1 is formed in approximately the same amounts as Gq during the initial part of the reaction. From Fig. 9 it is apparent that oligosaccharides are formed in the following relative amounts Gz > Ga > Gd. Furthermore, it is evident that Gt and Ga are formed in approximately the same amounts during the initial stage of the reaction, whereas during later stages more G2 than G, is formed. The
kinetic
parameters
Km and
b
there
kf is the velocity constant first order with respect to substrate when [S] << [Km1derived for the cleavage of short chain polymers by Hanson (23) were determined for the p-1.4oligoglucosides cellotetraose, cellopentaose, and cellohexaose and for reduced cellotetraose and cellopentaose. The polarimetric technique used for the determination of the rate of hydrolysis of the oligosaccharides is classical in enzymology. Modern instrumentation has made
E o.4 f o,3 9 2b 0.2
3 0.1 7F” 2
4
6
8
Days
IO
12
14
J 16
FIG. 2. Formation of reducing sugars from crystalline cellulose (Avicel) determined as glucase, upon degradation with the cellulase from P. n&turn. Substrate: 10 ml of a 1% suspension of Avicel in 0.05 M sodium acetate buffer pH 5.0. Enzyme: 200 pl L42eo = 18. Te.perat,Llre: 370.
it possible to increase the precision of the method by measurements at shorter wavelengths where the optical rotation is much higher than at the sodium D line. The fact that the method also allows the continuous registration of the enzymic hydrolysis makes it particularly valuable. Mutarotation of substrates or products of the reaction may of course be a complication even if it is possible (Whitaker (24)) that mutarotation
290
PETTERSSON
F‘ iO.4 $0.3 E
c 0.2 B bO1 : T 10
20
Frocfion
FIG. 3. Formation of reducing sugars from acid swollen cellulose determined as glucose upon degradation with the cellulase from P. notatum. The experimental conditions were the same as in Fig. 2.
30 40 number
FIG. 5. Column chromatography of cellotetraose (GJ after enzymic hydrolysis with the cellulase from P. not&m. Sample was taken 60 min after start of the hydrolysis. For further experimental details see the text.
F‘ c 0.8
0
Lx 0.6 PI ii
0.4
B 0 0.2 :
Frocfion Frocfion
number
FIG. 4. Column chromatography
of cellotriose (GJ after enzymic hydrolysis with the cellulase from P. not&urn. Sample was taken 48 hr after start of the hydrolysis. The concentration of oligosaccharide was determined with the orcinol method. For further experimental details see the text.
is not rate limiting at temperatures as high as 40”. The effect of mutarotation and other technical aspects of the polarimetric method for assaying enzymes will be discussed elsewhere. The results of a degradation experiment are reproduced in Fig. 10. The substrate is Gh at a concentration of 5 mg/ml. The curve is composed of an initial lag phase, a nearly linear part, and a later part where the velocity decreases with time. The velocity used for the determination of K, was derived from the slope of the part of the curve immediately after the lag phase. A Lineweaver-Burk plot from a degradation experiment with Gd is shown in Fig. 11.
number
FIG. 6. Column chromatography of cellopentaose (G,) after hydrolysis with the cellulase of P. notatum. Sample was taken 6 min after start of the hydrolysis. For further experimental details see the text.
Km and Icf values for the p-1.4 oligosaccharides are summarized in Table I. S represents the velocity constant, first order with respect to substrate, when S << Km (23). The polarimetric technique was used for Gs, Cd, Gg, and Ge and the reducing end group method for GbH, GsH, and for the cellodextrin mixture with D.P. > 7. A Lineweaver-Burk plot of data from the degradation of G4H is shown in Fig. 12. Gz and Gsa are not attacked by the enzyme and Ga is degraded very slowly. K, and i& were not determined for Ga but the enzyme activity was dependent upon substrate concentration even in the range 100-120 mg/ml. Km must thus be greater than 50 g/liter. It is evident that Km decreases with the D.P. of the substrate while kf increases. It is also evident the reduced
STUDIES
ON CELLULOLYTIC
291
E?JZYRIES
50
100
150
MiflUf.C?S I
20
I
40
/
60 Hours
I
80
I
IOC
FIG. 7. Time course of the hydrolysis of cellotriose with the cellulase from P. not&urn. The distribut.ion of oligosaccharides was determined at different time intervals with the microcolumn method (15). Experimental conditions: 12 mg of oligosaccharide in 4 ml 0.05 M sodium acetate buffer pH 5.0 were incubated with 200 ~1 enzyme (A280 = 2.1) at 40”.
FIG. 10. The change in optical rotation with time upon hydrolysis of cellotetraose with the cellulase from P. not&urn. Substrate: Ga 5 mg/ml in 0.05 M sodium acetate buffer pH 5.0. Enzyme: 100 ~1 (ABO = 2.37) added to 1 ml substrate solution. Temperature : 40”. lo-cm cell. Wavelength of registration 365 nm.
I
I 0.2
I 0.4
I 0.6
I 0.8
~lml/mgj
Minu
fes
FIG. 8. Time course of the hydrolysis tetraose with the cellulase from P. Conditions as in Fig. 7.
of cellonot&urn.
FIG. 11. Double reciprocal plot of rate of hydrolysis of cellotetraose catalyzed by the cellulase from P. not&urn. Initial velocities determined from the change in optical rotation at 365 nm with time (see Fig. 10). pH 5.0, 0.05 M sodium acetate buffer. Temperature: 40”.
members of the oligosaccharide series give higher K, values than the corresponding unreduced oligosaccharides. The differences are particularly pronounced between Gq and GIH and less pronounced between Gg and G 5H. DISCUSSION
d
5
10
15
20
Minutes
FIG. 9. Time course of the hydrolysis pentaose with the cellulase from I’. Conditions as in Fig. 7.
of cellonotatum.
Several CX- and P-linked glucans were tested together with a mannan and a xylodextrin mixture (D.P. 5-7) as substrates for the enzyme. Only lichenin, which is a mixed glucan containing p-l.4 and p-1.3 bonds, was found to be degraded. The products of the reaction were oligoglucosides with D.P. from 3-6. Perlin (25) has shown
292
PETTERSSON TABLE
I
THE EMPIRICAL CONSTANTS K,,, AND k/ FOR THE HYDROLYSIS OF p-1.4 OLIGOGLUCOSIDES WITH THE Ponicillium CELLULASE Kf = K:
mole. 1-1 degree.1. mole-lmin-1 Cellotriose Cellotetraose Cellopentaose Cellohexaose Cellodextrinc (D.P.>7) Reduced cellotetraose Reduced cellopentaose
>900.10-4 250.10-4 10.10-4 6.7.10-” 3.7.10-4
a a a
910.W4
b
13.10-4
0.72= 7.7” 6.3” -
;
b
-
(1Determined from polarimetric measurements. b Determined with the reducing end-group method (see the text). c The average D.P. was considered to be 9.
QI 0 I 8 30 k
I
; 20 \ t? 2 IO $1-k
O.IO
020
0.30
FIG. 12. Double reciprocal plot of rate of hydrolysis of reduced cellotetraose catalyzed by the cellulase from P. notatum. Initial velocities determined from the increase in reducing groups upon hydrolysis of the substrate. Experimental conditions as in Fig. 11.
that lichenin gives cellobiose as a product when degraded with cellulase, but not laminaribose. In our experiment only minor amounts of cellobiose could be detected (see Fig. 1). The major products formed were oligosaccharides with D.P. 5 or 6. It is thus possible that our Penidium cellulase has a more restricted mode of action than the cellulase used by Perlin. The Penicillium enzyme showed a very limited ability to degrade crystalline cellulose (see Fig. 2). Only about 2% of the
cellulose was solubilized after 12 days. On the contrary, acid swollen cellulose is rapidly degraded. Fig. 3 shows that most of the degradation occurs during the first hour of the reaction. By the end of 4 days, 12 % of the cellulose was solubilized. More extensive hydrolysis was observed when freshly prepared cellulose was used. The cellulose utilized for the actual experiment was prepared 1 month prior to use. Nevertheless, it is obvious that there are parts of the swollen cellulose that are not hydrolyzed by the enzyme. The enzyme does not attack cellobiose, while higher members of the oligoglucoside series are attacked at increasing rate. The half-lives obtained at a constant enzyme concentration were thus found to be 3700, 50, and 3 min for cellobiose, cellotetraose, and cellopentaose, respectively. All possible products are formed upon degradation of cellotriose, cellotetraose, and cellopentaose. The relative amounts of oligosaccharides indicate, however, that the central linkages are the preferred points of cleavage (Figs. 8 and 9). The rate of hydrolysis of the terminal bonds increases like the overall rate of hydrolysis in the series cellotriose to cellopentaose. The major products of hydrolysis of cellopentaose are cellobiose and cellotriose (Fig. 9). These sugars are formed in the same amounts during the initial part of the reaction while later more cellobiose is formed than cellotriose, probably due to hydrolysis of the cellotetraose initially formed. The method of degradation seems to resemble closely what has been found for the cellulase from Myrothecium verrucuriu (26), except that the Myrothecium enzyme was found to hydrolyze cellobiose. The empirical constants K, and k, for the hydrolysis of the /3-1.4-oligoglucosides have been determined. The initial velocities were determined by a polarimetric method which, to the author’s knowledge, has not been used for this purpose before. The method has many advantages. It is, for instance, the only method that offers the possibility of continuous registration of the enzymic hydrolysis of oligosaccharides. It is also far less tedious than other possible methods. Nevertheless, it must be emphasized that
STUDIES
ON CELLULOLYTIC
much work remains to be done before all the potentialities of the technique can be realized. The effect of mutarotation of the hydrolysis products is, for instance, not known. This means that the numerical values of the constants in Table I might be of limited significance. The figures are nevertheless considered good enough to demonstrate a trend in the order of magnitude. The figures indicate that K, decreases with the D.P. of the substrate. The decrease is very drastic going from cellotetraose to cellopentaose but less pronounced thereafter. The decrease in K, with increasing chain length of the oligoglucosides might reflect an increasing tendency to form enzyme substrate complexes. It is nevertheless surprising that the addition of a p-sorbityl residue to cellotetraose has about the same effect as the addition of a glucoyl residue. The same trend in K, values for the oligoglucosides has been observed earlier by Whitaker (6) and by Li et al. (4) working with other cellulases. Table I shows that the k, value increases about ten-fold going from cellotetraose to cellopentaose, while there is a slight decrease (possibility insignificant) going from cellopentaose to cellohexaose. According to Hanson (23) this trend in the values of kf means that the specificity region of the enzyme is five glucose units in length. It should be observed that there is a similar very drastic change in the K, values going from cellotetraose to cellopentaose. Applying the same argument Whitaker (26) and Hanstein and Whitaker (27) showed that the specificity region of the Myrothecium cellulase was at least five glucose units in length. The present data indicates that the Penicillium cellulase functions as an endoglucanase and should be designated as a /3-1.4-glucan 4-glucanohydrolase (EC 3.2.1.4). The typical preference for the cleavage of nonterminal bonds in the short chain oligosaccharides does not rule out the possibility that longer chains are cleaved in an approximately random way. Although in most cases the properties of the substrate, (substitution or accessibility of linkages) rather than the intrinsic properties of the enzyme, determine the method of degradation.
ENZYMES
293
The degradation pattern obtained on oligoglucosides resembles closely that of other cellulolytic enzymes (26-28). The only exception seems to be the exoglucanase from Trichoderma viride (4) which prefers terminal linkages. The Penicillium enzyme attacks crystalline cellulose very slowly, while swollen cellulose is readily attacked. The enzyme was found to degrade cellotriose very slowly. Thereafter, the rate of degradation increased rapidly with the D.P. of the oligosaccharide chain up to a limit of five glucose residues. In crystalline cellulose the polysaccharide chains are very closely packed and it is improbable that longer segments of polysaccharide chains are accessible to the enzyme. It is thus possible to explain the slow rate of hydrolysis of native cellulose, in view of the kinetic experiments with the oligoglucosides. It is of interest to note that after degradation of crystalline cellulose, the molar ratio of glucose to cellobiose was approximately 1: 1, which is the ratio expected upon degradation of cellotriose. The swollen cellulose gave glucose and cellobiose approximately in the molar ratio 1:2, which is the expected ratio upon degradation of longer oligosaccharides. It thus seems that these results further support the hypothesis that only short segments of polysaccharide chains in crystalline cellulose are accessible to the enzyme. ACKNOWLEDGMENTS I thank my teacher Professor J. his kind interest in this work and Mrs. Fredriksson for valuable technical The work has been supported in part from the Hiertn-Retzius foundation.
Porath for Ulla-Britt assistance. by a grant
REFERENCES 1. REI~:SE, E. T., SIU, 11. G. H., AND LEVINSSON, H. S., J. Hacteriol. 69,485 (1950). 2. MANDELS, M. AND Rnkss, E. T., Develop. Ind. Microbial. 6, 5 (1964). 3. HALLIWELL, G., Rio&em. J. 100, 315 (1966). 4. LI, L. H., FLORA, R. M., AND KIXG, K. W., Arch. Biochem. Biophys. 111, 439 (1965). 5. SELBY, K. .\ND MMTLAND, C. C.,Biochem. J. 104, 716 (1967). 6. WHITAKER, I). ii., HANSON, K. R., ago D.4TTs P. K., Can. J. Biochem. Physiol. 41, 671 (1963).
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7. SELBY, K. AND MAITLAND, C. C., Biochem. J. 94, 578 (1965). 8. PETTERSSON, G., Arch. Biochem. Biophys. 133, 307 (1968). 9. PETTERSSON, G. AND EAKF,R, D. L., Arch. Biothem. Biophys. 124,497 (1968). 10. PETTERSSON, G. AND ANDERSSON, L., Arch. Biochem. Biophys. 124, 497 (1968). 11. ERIKSSON, K.-E. AND PETTERSSON, G., Arch. Biochem. Biophys. 134, 160 (1968). 12. PETTERSSON, G., Arch. Biochem. Biophys. 126,776 (1968). 13. MILLER, G. L., DEAN, J., AND BLUM, R., Arch. Biochem. Biophys. 91, 21 (1960). 14. VASSEUR, E., Acta Chem. Stand. 2, 693 (1948). 15. MILLER, G. L., Anal. Biochem. 2, 133 (1960). 16. FLODIN, P. AND ASPBERG, K., Biol. Struct. Function 1,345 (1961). 17. COLE, F. E. AND KING, K. W., Biochim. Biophys. Acta 81, 122 (1964). 18. WALSETH, C. S., Tappi 36, 228 (1952).
19. MILLER,
G. L., BLUM, R., GLENNON, W. E., AND BURTON, A. L., Anal. Biochem. 2, 127 (1960).
20. SOMOGYI, M., J. Biol. Chem. 196,19 (1952). 21. JANSSON, J. C., Personal communications. 22. STAHL, E., Diinnsicht-Chromatographie Ein
Laboratoriumshandbuch. 2. Auflage. p. 817. Springer, Berlin (1967). 23. HANSSON, K. R., Biochemistry 1,723 (1962). 24. WHITAKER, D. R., Arch. Biochem. Biophys. 63, 436 (1954). 25. PERLIN, A. S., in
“Advances in Enzymic Hydrolysis of Cellulose and Related Material.” E. T. Reese, (ed.) p. 188. Pergamon Press, New York (1963). 26. WHITAKER, D. R., Arch. Biochem. Biophys. 63,439 (1954). 27. HANSTEIN, E. G. AND WHITAKER, D. R., Can J. Biochem. Physiol. 41, 707 (1963). 28. CLARKE, A. E. AND STONE, B. A., Biochem. J
96,802 (1965).