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Kinetic studies of the action of cellulase upon sodium carboxymethyl cellulose
ABCHIVI~;A
OF
BIOCHEMISTRY
Kinetic
KAKL-ERIK
AND
BIOPHYSICS
133, 233-237 (1969)
Studies
of the Action
of Cellulase
Sodium
Carboxymethyl
Cellulose
ERIKSSOiY
HERP;T
AKD
HOLCER
upon
HOI,LMARI<
Swedish Forest 1’rodwl.s Research Laboralo~~~~, Slockholm, Sweden Iteceived
March
4, 1969; accepted
.Jlme 3, l!Xi!l
Kinetic studies of the hydrolysis of carboxymethyl cellulose (CM-cellulose) by cellulase have been carried ollt. It has been formd that the hydrolysis of three (or more) adjacent unsubstituted glucosyl residues is the initial and dominating reaction. The total reaction at extended reaction time, measured by reducing end-group determinations, has been found to exceed the concentration of three adjacent, unsubstituted glucosyl residues, showing that ot.her groupings are also involved. The shape of the reaction curve indicates that there are different reaction rates for different groupings. The Michaelis-Menten constant, K, , has been viscometrically determined as a function of the degree of substitution (DS). The K, value was found to increase with DS. The K,, value could be extrapolated to DS = 0; i.e., cellulose in a hypothetical solution. The value obtained was 3 X 1OP g/liter.
This paper presents the results of investigations concerning the action of cellulases upon carboxymethyl cellulose (CMcellulose). In particular, the influence of the degree of substitution (DS)’ and the location of the substituents upon the rate of enzymic hydrolysis was studied. CM-cellulose has been widely used as a substrate for the determination of cellulase activity (1). This use has been extended since the viscometric method, developed by Almin et al. (2,3), made it possible to determine the cellulase activity in absolute terms. Whether a CM-cellulose solution is a suitable substrate for the determination of cellulase activity or not can be questioned as long as the influence of the substituents; i.e., their amount, location, and shape, is not known in detail. Reese et al. (4,s) found that the DS value influences the rate of enzymic hydrolysis so that the higher the DS the lower the rate 1 The complete substitlltion hydroxyls on each anhydroglucose DS of 3.0.
of a11 three unit leads to a
of hydrolysis. Almin and Eriksson (6), however, found that the activity measured in bonds broken per unit time is not influenced by the DS provided this is >0.5. Almin et al. (3) also tried to determine the Michaelis-Alenten constant, K, , for the hydrolysis of a CM-cellulose with a DX of 0.83. Unfortunately, this value is just below the suitable range for K, determinations, since the concentrations of substrate required are so low that the activity determinations by viscosity measurements become inaccurate. It has been suggested by Reese (5) that the enzymic hydrolysis takes place where two adjacent unsubstituted glucosyl residues are present. Klop and Iiooiman (7), using very long reaction times, found that the glycosidic bond between two glucosyl residues could be hydrolyzed if the aglycone group was unsubstituted or B-substituted, but not if substituted at C-2 or C-3. Recently, Wirick (8) suggested that points of enzymic attack consisted of three or more adjacent unsubstituted glucosyl residues. In the present investigation K, has been
233
234
ERIKSSON
AND
determined as a function of DS. It has been shown that hydrolysis primarily occurs when three (or more) adjacent unsubstituted glucosyl residues are present. MATERIALS
AN11 METHODS
Enzymes. The following enzyme preparations were used in the investigations: A commercial enzyme powder, Meicelase, manufactured by Meiji Seika Kaisha Lt)d., Japan, from the fungus Trichoderma viride was used. The powder was treated as follows. ,4 1O7o suspension of the enzyme powder in 0.1 M ammonium acetate buffer (pH 5) was stirred for 24 hr at 4”. The clear enzyme solution was precipitated by (NH4)804 at 90% saturation. The precipitate was dissolved in water and the solution dialyzed in collodion tubes against wat,er, 0.02 M ammonium acetate buffer (pH 5), and 0.02 M pyridinium acetate buffer (pH 5)) successively. The prepared enzyme solution, called enzyme E, had 3.1 cellulase units/ml (cf. Ref. 2, 3). 9 purified cellulase from the fungus SlerelLm sanguinolentum was used. It, was obtained from the Pl fraction of the purificat,ion procedure described in Ref. (9). The solution of the purified cellulase, called enzyme F, had 1.5 cellulase units/ml. C&f-cellulose preparations. Five CM-cellulose preparations were used with the following properties: Designation of the CM-cellulose
Degree of substitution (IX)
CMC 3 CD ti34 CMC 4 CMC 5 Cl) 645
0.85 0.95 1.04 1.25 1.33
Degree of polymerization (DP) (viscosity average)
382 460 517 434 610
The CM-cellulose preparations were manufactured by Uddeholms AB, Sweden. The DP values of CMC 3, CMC 4, CMC 5, and all the DS values were determined by Uddeholms AB, whereas the DP values of Cl> 63-l and CD 645 were determined at the Central Laboratory of the Swedish Cellulose Industry. The methods employed for the DS and DP determinations have been reported in Ref.
(6). The CM-cellulose solutions were prepared as described previously (2). Capillary visconaeter. The viscometer used was viscometer 1 described in Ref. (3). Ten milliliters of the CM-cellulose solution in sodium-acetate buffer, pH 5.0, 0.05 M was incubated with 10 or 20 ~1 of a solution of enzyme E diluted 10 times. The
HOLLMARK viscometer runs and the calculation of the enzymic activity, 8, were carried out according to the methods described in Ref. (2,3). Determination OJ” reducing end groups. These determinations were made on CMC 3 (DS 0.85) with a concentration of 1 g/lit,er. The enzyme used was enzyme F. The ratio of enzyme to CM-celll~lose solution was 1:lOO ml/ml. The reaction temperature was held at 25”. The amount of reducing end groups was determined according to Somogyi (10) RESULTS
The Michaelis-Menten constant, K, , was determined for the T. viride enzymic hydrolysis of five CM-cellulose preparations of different DS in order to investigate the influence of DS upon K, . The viscometric method described by Almin and Eriksson (2) offers a convenient method for the determination of reaction velocities with this type of substrate. If the reaction velocity is determined for a series of substrate concentrations, it can be used to determine the Michaelis-Menten constant by the method of Lineweaver and Burk (11). The K, values obtained with the five CM-cellulose preparations as substrates are given in Table I, where K, is expressed in grams per liter as well as in equivalents of p-1,4-glucosidic bonds per liter. It can be seen that K, increases with increasing DS. This is to be expected, since it can be assumed that the effective substrate concentration is only the concentration of hydrolyzable p-1,4-glucosidic bonds. The maximum reaction velocity, (V), which can also be determined from the Lineweaver and Burk diagram (11)) does not varv significantly with DS (see Table I). This is”in accordance with our earlier findings (6). In addition to the viscosity measurements, an experiment was carried out with S. sanyuinolentunz enzyme in which the course of the reaction was followed by measuring the increase in reducing end groups. The extent of the reaction versus t,ime is shown in Fig. 1. The reaction rate shows an initial rapid period which begins to slow down above the point marked SO”which is the cube of so , the fraction of unsubstituted glycosyl residues. The calculation of so from DS
.
ACTION /
Designation of the CJI-cellulose
OF CELLULAW I i
235
K,,so3 (equiv @-l,?-glucosidic bonds/liter)
Km in/liter)
of
x
10-a
X
10-S
22.0 X 10m6
2.4 2.5 4.7
x
10-s
22.4
10-C
3.7
x
10-S
10-C
1.7 x
0.85
0.11
0.48
x
lo-”
0.0385
18.5
x
10-F
G34
0.95
0.20
0.84
x
10-a
0.0227
19.1
x
10-C
CMC
1
1.04
0.10
1 .63
x
low
0.0133
CMC
5
1.25
1.67 2.50
fi.38 x $1,313 x
lo-:: 10-Z
0.00351 0.00195
cn1c CD
cm
3
615
1 .x3
a The calcrkrtion
of su from IIS is given
Extent (mole
FIG.
uring
1. Enzyrnic the
increase
ill I:ef.
Maximum reaction velocity (V) (equiv of 8-1, l-glucosidic bonds/liter.sec)
x
18.2 x
10-8
(12)
of hydrolysis fraction)
hydrolysis in
reducing
of
CMC
end
3 incltbated
with
enzyme
F.
The
reaction
was
followed
by
meas-
groups.
is given by Spurlin (12). The fraction can be calculated for any CAI-cellulose preparation of known LX, provided that the distribution of substituents is random, and the relative reactivities of the three hydroxyl groups are those proposed by Croon and Purves (13). The validity of our arguments depends upon the acceptance of these assumptions. The cube of this fraction, so3,denotes the ratio between the number of possible sites for enzymic attack along the C&-cellulose chain (assuming that the enzyme requires three adjacent unsubstituted
glucosyl residues), and the number of sites in an unsubstituted chain. DISCUSSION
If the K, values obtained (Table I) are multiplied by the factor so3, a product is obtained which is independent of DX. An interpretation of this is that during the incubation time used for the viscosity experiments the dominating reaction is the hydrolysis of p-1,4-glucosidic bonds included in or adjacent to a so3grouping. However, since the final extent of reaction, as seen in Fig. 1,
236
ERIKRSON
AND
is greater than the value of so3, it is evident that other groupings also are hydrolyzed, although less rapidly. Since the product K,so3 was found to be independent of DS is should be possible to calculate the value of K, for DX = 0 and so3 = 1. It would then have been advantageous for the extrapolation to employ values of DS closer to DX = 0 than those used. Rowever, this would involve the experimental difficulties mentioned in the introduction. At DS = 0 the substrate is cellulose. If no complications arise at lower degrees of substitution, it can be assumed that the calculated value of K,s,+ will also be representative for cellulose. This will give us the Michaelis-hlenten constant for the hypothetical state of cellulose in an aqueous solution.
K, , cell. soln = 2 X 1OP equiv of p-1 ,4-glucosidic bonds/liter The knowledge of this value may be useful for the determination of the accessibility of cellulose to enzymic hydrolysis. This will, however, require a reliable mathematical approach for the expression of cellulose concentrations in the heterogeneous reaction mixture. At this stage no approach of that kind is known to us. The information supplied by the reaction curve in Fig. 1 can be treated as follows. The common Michaelis-type reaction path is kz ES-EfI’
E+S+ * 1
the rate equation of which is
Vt = K, In a
a-x
+ x
(1)
where V = kz[E]o; K, = ‘-lk-
k2 ;
a = [S]o = (x) tern, K, is the MichaelisMenten constant, a is the initial substrate concentration, x the product concentration at time t, and [E]o the initial enzyme concentration.
HOLLMARK
The reaction curve in Fig. 1 can be checked against Eq. (1) by plotting x/t versus l/t.ln (a/a - x). By doing this, it was found that the reaction curve cannot be described by Eq. (1). The shape of the reaction curve can be explained, however, if it is assumed that the experimentally found reaction curve is the sum of a number of reaction curves with different sets of parameters. Equation (1) can then be written
Vi t = K,i ln a+, 2
The experimentally
z
+ xi
(la)
found value of t is then
xYezp= f: xi i=1
(2)
CONCLUSION
From our data it seems reasonable to conclude that the enzymic hydrolysis of CM-cellulose is composed of a series of reactions, each involving a definite pattern of substituents which influence the rate of hydrolysis. ;\Iaking the assumption that the number of adjacent unsubstituted glucosyl residues is the only factor influencing the rate of hydrolysis at a certain point on the CM-cellulose chain. it can be said that hydrolysis occurs easily when three adjacent unsubstituted glucosyl residues are present, and very slowly when only two adjacent unsubstituted residues are present. It is not known whether a group of four unsubstituted residues is attacked even faster than a group of three, because the concentration of such groups is so small that it is not possible to detect their contribution. Disregarding assumptions of the above kind it may be concluded that a series of simultaneous hydrolysis reactions occur, each reaction contributing to the total reaction. Apparently, this is due to a reactivity distribution in the high molecular weight substrate. Similar behavior has often been observed when high molecular weight substrates are used. ACKNOWLEDGMENTS The authors are indebted to Prof. Biirje Steenberg and Prof. Torbjiirn Norin for discussions and valuable help during the preparation of this maw
ACTION uscript. The English Fredricks.
text was revised
OF CELLULA,CE
by Dr. Pet,er
REFERENCES Svensk Kern. Tidskr. 79, 660 (1967). ALMIN, K. E., AND ERIKSSON, K.-E., Biochim. Biophys. Acta 139, 238 (1967). A4~~~~, K. E., ERIKSSON, K.-E., AND JANSSON, C., Biochim. Biophys. Acta 139, 228 (1967). REESX, E. T., Sru, R. G. H., AND LEVINSON, H. S.. .J. Bacterial. 69. 485 (1950). REESI:,‘E. T., Ind. Eng.‘Chem. 49,‘89 (1957). ALMIN, K. E., AND EHIKSSON, K.-E., Arch. Biochem. Biophys. 124, 129 (1968).
1. ERIKSSOK.
2. 3. 4. 5. 6.
K.-E.,
“37
7. KI,OP, W., AND KO~IMAN, P., Biochim. Riophys. Ach 99, 102 (1965). 8. WIRICI~, M. G., J. Polymer Sci. 6, 1965 (1968). 9. ERIKSSON, K.-E., AND PIWIXRSSDN, B., .I&. Biochem. Niophys. 134, 142 (1968). 10. SoMocuI, M., J. Riol. Chem. 196, 19 (1952). 11. LINI:WI;AWX, II., AND BURK, l)., J. Am. Chem. Sot. 56, 658 (1934). 12. OTT, E;., SI’UHMN, 11. &I., AND ~ItAk’PLIN, RI. W., in “Cellulose arid Cellulose Deriwt 1ves “, Part. 2, p. 676, Iuterscieuce, New York (1951). 13. CROON, I., AND PURVKS, C. B., Saensk Papperstidn. 62, 876 (1959).
OF
BIOCHEMISTRY
Kinetic
KAKL-ERIK
AND
BIOPHYSICS
133, 233-237 (1969)
Studies
of the Action
of Cellulase
Sodium
Carboxymethyl
Cellulose
ERIKSSOiY
HERP;T
AKD
HOLCER
upon
HOI,LMARI<
Swedish Forest 1’rodwl.s Research Laboralo~~~~, Slockholm, Sweden Iteceived
March
4, 1969; accepted
.Jlme 3, l!Xi!l
Kinetic studies of the hydrolysis of carboxymethyl cellulose (CM-cellulose) by cellulase have been carried ollt. It has been formd that the hydrolysis of three (or more) adjacent unsubstituted glucosyl residues is the initial and dominating reaction. The total reaction at extended reaction time, measured by reducing end-group determinations, has been found to exceed the concentration of three adjacent, unsubstituted glucosyl residues, showing that ot.her groupings are also involved. The shape of the reaction curve indicates that there are different reaction rates for different groupings. The Michaelis-Menten constant, K, , has been viscometrically determined as a function of the degree of substitution (DS). The K, value was found to increase with DS. The K,, value could be extrapolated to DS = 0; i.e., cellulose in a hypothetical solution. The value obtained was 3 X 1OP g/liter.
This paper presents the results of investigations concerning the action of cellulases upon carboxymethyl cellulose (CMcellulose). In particular, the influence of the degree of substitution (DS)’ and the location of the substituents upon the rate of enzymic hydrolysis was studied. CM-cellulose has been widely used as a substrate for the determination of cellulase activity (1). This use has been extended since the viscometric method, developed by Almin et al. (2,3), made it possible to determine the cellulase activity in absolute terms. Whether a CM-cellulose solution is a suitable substrate for the determination of cellulase activity or not can be questioned as long as the influence of the substituents; i.e., their amount, location, and shape, is not known in detail. Reese et al. (4,s) found that the DS value influences the rate of enzymic hydrolysis so that the higher the DS the lower the rate 1 The complete substitlltion hydroxyls on each anhydroglucose DS of 3.0.
of a11 three unit leads to a
of hydrolysis. Almin and Eriksson (6), however, found that the activity measured in bonds broken per unit time is not influenced by the DS provided this is >0.5. Almin et al. (3) also tried to determine the Michaelis-Alenten constant, K, , for the hydrolysis of a CM-cellulose with a DX of 0.83. Unfortunately, this value is just below the suitable range for K, determinations, since the concentrations of substrate required are so low that the activity determinations by viscosity measurements become inaccurate. It has been suggested by Reese (5) that the enzymic hydrolysis takes place where two adjacent unsubstituted glucosyl residues are present. Klop and Iiooiman (7), using very long reaction times, found that the glycosidic bond between two glucosyl residues could be hydrolyzed if the aglycone group was unsubstituted or B-substituted, but not if substituted at C-2 or C-3. Recently, Wirick (8) suggested that points of enzymic attack consisted of three or more adjacent unsubstituted glucosyl residues. In the present investigation K, has been
233
234
ERIKSSON
AND
determined as a function of DS. It has been shown that hydrolysis primarily occurs when three (or more) adjacent unsubstituted glucosyl residues are present. MATERIALS
AN11 METHODS
Enzymes. The following enzyme preparations were used in the investigations: A commercial enzyme powder, Meicelase, manufactured by Meiji Seika Kaisha Lt)d., Japan, from the fungus Trichoderma viride was used. The powder was treated as follows. ,4 1O7o suspension of the enzyme powder in 0.1 M ammonium acetate buffer (pH 5) was stirred for 24 hr at 4”. The clear enzyme solution was precipitated by (NH4)804 at 90% saturation. The precipitate was dissolved in water and the solution dialyzed in collodion tubes against wat,er, 0.02 M ammonium acetate buffer (pH 5), and 0.02 M pyridinium acetate buffer (pH 5)) successively. The prepared enzyme solution, called enzyme E, had 3.1 cellulase units/ml (cf. Ref. 2, 3). 9 purified cellulase from the fungus SlerelLm sanguinolentum was used. It, was obtained from the Pl fraction of the purificat,ion procedure described in Ref. (9). The solution of the purified cellulase, called enzyme F, had 1.5 cellulase units/ml. C&f-cellulose preparations. Five CM-cellulose preparations were used with the following properties: Designation of the CM-cellulose
Degree of substitution (IX)
CMC 3 CD ti34 CMC 4 CMC 5 Cl) 645
0.85 0.95 1.04 1.25 1.33
Degree of polymerization (DP) (viscosity average)
382 460 517 434 610
The CM-cellulose preparations were manufactured by Uddeholms AB, Sweden. The DP values of CMC 3, CMC 4, CMC 5, and all the DS values were determined by Uddeholms AB, whereas the DP values of Cl> 63-l and CD 645 were determined at the Central Laboratory of the Swedish Cellulose Industry. The methods employed for the DS and DP determinations have been reported in Ref.
(6). The CM-cellulose solutions were prepared as described previously (2). Capillary visconaeter. The viscometer used was viscometer 1 described in Ref. (3). Ten milliliters of the CM-cellulose solution in sodium-acetate buffer, pH 5.0, 0.05 M was incubated with 10 or 20 ~1 of a solution of enzyme E diluted 10 times. The
HOLLMARK viscometer runs and the calculation of the enzymic activity, 8, were carried out according to the methods described in Ref. (2,3). Determination OJ” reducing end groups. These determinations were made on CMC 3 (DS 0.85) with a concentration of 1 g/lit,er. The enzyme used was enzyme F. The ratio of enzyme to CM-celll~lose solution was 1:lOO ml/ml. The reaction temperature was held at 25”. The amount of reducing end groups was determined according to Somogyi (10) RESULTS
The Michaelis-Menten constant, K, , was determined for the T. viride enzymic hydrolysis of five CM-cellulose preparations of different DS in order to investigate the influence of DS upon K, . The viscometric method described by Almin and Eriksson (2) offers a convenient method for the determination of reaction velocities with this type of substrate. If the reaction velocity is determined for a series of substrate concentrations, it can be used to determine the Michaelis-Menten constant by the method of Lineweaver and Burk (11). The K, values obtained with the five CM-cellulose preparations as substrates are given in Table I, where K, is expressed in grams per liter as well as in equivalents of p-1,4-glucosidic bonds per liter. It can be seen that K, increases with increasing DS. This is to be expected, since it can be assumed that the effective substrate concentration is only the concentration of hydrolyzable p-1,4-glucosidic bonds. The maximum reaction velocity, (V), which can also be determined from the Lineweaver and Burk diagram (11)) does not varv significantly with DS (see Table I). This is”in accordance with our earlier findings (6). In addition to the viscosity measurements, an experiment was carried out with S. sanyuinolentunz enzyme in which the course of the reaction was followed by measuring the increase in reducing end groups. The extent of the reaction versus t,ime is shown in Fig. 1. The reaction rate shows an initial rapid period which begins to slow down above the point marked SO”which is the cube of so , the fraction of unsubstituted glycosyl residues. The calculation of so from DS
.
ACTION /
Designation of the CJI-cellulose
OF CELLULAW I i
235
K,,so3 (equiv @-l,?-glucosidic bonds/liter)
Km in/liter)
of
x
10-a
X
10-S
22.0 X 10m6
2.4 2.5 4.7
x
10-s
22.4
10-C
3.7
x
10-S
10-C
1.7 x
0.85
0.11
0.48
x
lo-”
0.0385
18.5
x
10-F
G34
0.95
0.20
0.84
x
10-a
0.0227
19.1
x
10-C
CMC
1
1.04
0.10
1 .63
x
low
0.0133
CMC
5
1.25
1.67 2.50
fi.38 x $1,313 x
lo-:: 10-Z
0.00351 0.00195
cn1c CD
cm
3
615
1 .x3
a The calcrkrtion
of su from IIS is given
Extent (mole
FIG.
uring
1. Enzyrnic the
increase
ill I:ef.
Maximum reaction velocity (V) (equiv of 8-1, l-glucosidic bonds/liter.sec)
x
18.2 x
10-8
(12)
of hydrolysis fraction)
hydrolysis in
reducing
of
CMC
end
3 incltbated
with
enzyme
F.
The
reaction
was
followed
by
meas-
groups.
is given by Spurlin (12). The fraction can be calculated for any CAI-cellulose preparation of known LX, provided that the distribution of substituents is random, and the relative reactivities of the three hydroxyl groups are those proposed by Croon and Purves (13). The validity of our arguments depends upon the acceptance of these assumptions. The cube of this fraction, so3,denotes the ratio between the number of possible sites for enzymic attack along the C&-cellulose chain (assuming that the enzyme requires three adjacent unsubstituted
glucosyl residues), and the number of sites in an unsubstituted chain. DISCUSSION
If the K, values obtained (Table I) are multiplied by the factor so3, a product is obtained which is independent of DX. An interpretation of this is that during the incubation time used for the viscosity experiments the dominating reaction is the hydrolysis of p-1,4-glucosidic bonds included in or adjacent to a so3grouping. However, since the final extent of reaction, as seen in Fig. 1,
236
ERIKRSON
AND
is greater than the value of so3, it is evident that other groupings also are hydrolyzed, although less rapidly. Since the product K,so3 was found to be independent of DS is should be possible to calculate the value of K, for DX = 0 and so3 = 1. It would then have been advantageous for the extrapolation to employ values of DS closer to DX = 0 than those used. Rowever, this would involve the experimental difficulties mentioned in the introduction. At DS = 0 the substrate is cellulose. If no complications arise at lower degrees of substitution, it can be assumed that the calculated value of K,s,+ will also be representative for cellulose. This will give us the Michaelis-hlenten constant for the hypothetical state of cellulose in an aqueous solution.
K, , cell. soln = 2 X 1OP equiv of p-1 ,4-glucosidic bonds/liter The knowledge of this value may be useful for the determination of the accessibility of cellulose to enzymic hydrolysis. This will, however, require a reliable mathematical approach for the expression of cellulose concentrations in the heterogeneous reaction mixture. At this stage no approach of that kind is known to us. The information supplied by the reaction curve in Fig. 1 can be treated as follows. The common Michaelis-type reaction path is kz ES-EfI’
E+S+ * 1
the rate equation of which is
Vt = K, In a
a-x
+ x
(1)
where V = kz[E]o; K, = ‘-lk-
k2 ;
a = [S]o = (x) tern, K, is the MichaelisMenten constant, a is the initial substrate concentration, x the product concentration at time t, and [E]o the initial enzyme concentration.
HOLLMARK
The reaction curve in Fig. 1 can be checked against Eq. (1) by plotting x/t versus l/t.ln (a/a - x). By doing this, it was found that the reaction curve cannot be described by Eq. (1). The shape of the reaction curve can be explained, however, if it is assumed that the experimentally found reaction curve is the sum of a number of reaction curves with different sets of parameters. Equation (1) can then be written
Vi t = K,i ln a+, 2
The experimentally
z
+ xi
(la)
found value of t is then
xYezp= f: xi i=1
(2)
CONCLUSION
From our data it seems reasonable to conclude that the enzymic hydrolysis of CM-cellulose is composed of a series of reactions, each involving a definite pattern of substituents which influence the rate of hydrolysis. ;\Iaking the assumption that the number of adjacent unsubstituted glucosyl residues is the only factor influencing the rate of hydrolysis at a certain point on the CM-cellulose chain. it can be said that hydrolysis occurs easily when three adjacent unsubstituted glucosyl residues are present, and very slowly when only two adjacent unsubstituted residues are present. It is not known whether a group of four unsubstituted residues is attacked even faster than a group of three, because the concentration of such groups is so small that it is not possible to detect their contribution. Disregarding assumptions of the above kind it may be concluded that a series of simultaneous hydrolysis reactions occur, each reaction contributing to the total reaction. Apparently, this is due to a reactivity distribution in the high molecular weight substrate. Similar behavior has often been observed when high molecular weight substrates are used. ACKNOWLEDGMENTS The authors are indebted to Prof. Biirje Steenberg and Prof. Torbjiirn Norin for discussions and valuable help during the preparation of this maw
ACTION uscript. The English Fredricks.
text was revised
OF CELLULA,CE
by Dr. Pet,er
REFERENCES Svensk Kern. Tidskr. 79, 660 (1967). ALMIN, K. E., AND ERIKSSON, K.-E., Biochim. Biophys. Acta 139, 238 (1967). A4~~~~, K. E., ERIKSSON, K.-E., AND JANSSON, C., Biochim. Biophys. Acta 139, 228 (1967). REESX, E. T., Sru, R. G. H., AND LEVINSON, H. S.. .J. Bacterial. 69. 485 (1950). REESI:,‘E. T., Ind. Eng.‘Chem. 49,‘89 (1957). ALMIN, K. E., AND EHIKSSON, K.-E., Arch. Biochem. Biophys. 124, 129 (1968).
1. ERIKSSOK.
2. 3. 4. 5. 6.
K.-E.,
“37
7. KI,OP, W., AND KO~IMAN, P., Biochim. Riophys. Ach 99, 102 (1965). 8. WIRICI~, M. G., J. Polymer Sci. 6, 1965 (1968). 9. ERIKSSON, K.-E., AND PIWIXRSSDN, B., .I&. Biochem. Niophys. 134, 142 (1968). 10. SoMocuI, M., J. Riol. Chem. 196, 19 (1952). 11. LINI:WI;AWX, II., AND BURK, l)., J. Am. Chem. Sot. 56, 658 (1934). 12. OTT, E;., SI’UHMN, 11. &I., AND ~ItAk’PLIN, RI. W., in “Cellulose arid Cellulose Deriwt 1ves “, Part. 2, p. 676, Iuterscieuce, New York (1951). 13. CROON, I., AND PURVKS, C. B., Saensk Papperstidn. 62, 876 (1959).