Journal of Chemical Technology and Biotechnology
J Chem Technol Biotechnol 77:15±20 (online: 2001)
DOI: 10.1002/jctb.520
Hydrolysis of carboxymethylcellulose with
mixtures of cellulase and b-1,4-glucosidase
V Bravo,* MP Páez, M Aoulad El-Hadj, A Reyes and AI Garcı́a
Departamento de Ingenierı´a Quı´mica, Facultad de Ciencias, Universidad de Granada, c/Fuentenueva, Granada E-18071, Spain
Abstract: We studied the hydrolysis of carboxymethylcellulose at 50 °C and pH 4.9 with a commercial
preparation of cellulase (Celluclast) supplemented with a commercial b-1,4-glucosidase product
(Novozym). The initial concentration of carboxymethylcellulose was varied between 2 and 12.5 kg m 3
for assays with Celluclast and two Novozym/Celluclast ratios (0.5 and 1). We determined the conversion to glucose and the overall conversion in terms of glucose equivalent. Conversion to glucose
turned out to be directly proportional to the product of experimental time multiplied by the concentration of Celluclast and the constant of proportionality increased concomitantly with the concentration of Novozym. Overall conversion remained unaffected by the concentration of Novozym.
# 2001 Society of Chemical Industry
Keywords: carboxymethylcellulose; cellulase; b-1,4-glucosidase; enzymatic hydrolysis; Celluclast; Novozym
intensity of the treatment with Celluclast
(min 1 (kg m 3) 1)
Intensity of the treatment with Celluclast
(min kg m 3)
NOTATION
C
Cf ,
e0
e03
G
Gf ,
k, K
ka
KICP
KM
P
Pf ,
S
t
tR
tS
xC
xp
XC
XCC
XCN
Xp
Cellobiose concentration (mol m 3)
Cellobiose concentration for complete
hydrolysis (mol m 3)
Concentration of Celluclast (kg m 3)
Concentration of Novozym (kg m 3)
Glucose concentration (mol m 3)
Glucose concentration for complete
hydrolysis (mol m 3)
Kinetic parameters
Rate constant (mol m 3 min 1 (kg m 3) 1)
Constant for competitive inhibition by the
product
Michaelis constant (mol m 3)
Total potential glucose for the hydrolysis of
CMC (mol m 3)
Total potential glucose for the complete
hydrolysis of CMC (mol m 3)
substrate concentration (kg m 3)
Corrected reaction time (min)
Reaction time (min)
Stop time (min)
Conversion to glucose
Overall conversion expressed in terms of
glucose equivalent
Derivative of conversion to glucose versus
intensity of the treatment with Celluclast
(min 1 (kg m 3) 1)
Ordinate at the origin in eqn (5) (min 1 (kg
Celluclast m 3) 1)
Slope in eqn (5) (min 1 (kg Novozym
m 3) 1)
Derivative of overall conversion versus
y
a1
a2
a3
Mass fraction of endoglucanase in
Celluclast
Mass fraction of exoglucanase in Celluclast
Mass fraction of b-1,4-glucosidase in
Celluclast
Subscripts
0
Initial value
en
Endoglucanase
ex
Exoglucanase
INTRODUCTION
One fundamental step in processes designed to take
advantage of residual lignocellulose biomass is to
hydrolyse the pretreated residues using cellulases.
Given their relatively high overall hydrolytic yield the
most widely used cellulases are those from Trichoderma
viride and Trichoderma reesei.
The enzymatic hydrolysis of cellulose is a heterogeneous catalytic process involving insoluble cellulose
and a soluble cellulase catalyst, the accepted mechanism of this reaction being as follows:1±3
(i) Endoglucanase, E1, forms intermediate compounds with cellulose chains and hydrolyses them
at random, giving rise to less polymerised chains
and soluble reducing sugars with a degree of
* Correspondence to: V Bravo, Departamento de Ingenierı́a Quı́mica, Facultad de Ciencias, Universidad de Granada, c/Fuentenueva,
Granada E-18071, Spain
E-mail: [email protected]
(Received 12 April 2001; revised version received 27 July 2001; accepted 2 August 2001)
# 2001 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2002/$30.00
15
V Bravo et al
polymerisation of less than six. Cellobiose and
glucose are usually present in this soluble fraction.
(ii) Exoglucanase, E2, binds to the non-reducing ends
of cellulose chains and their derivative oligosaccharides, splitting off cellobiose.
(iii) Finally, b-1,4-glucosidase, E3, hydrolyses cellobiose into glucose in a homogeneous phase.
If, as is generally the case, the cellulases lack b-1,4glucosidase activity the ®nal product of hydrolysis will
be mainly cellobiose.4 Thus in order to avoid inhibition by high concentrations of cellobiose it is necessary
to supplement the cellulases with b-glucosidases,
obtained either from other microorganisms or from
vegetable ®bres. These can be added to the culture
medium either freely or in immobilised form.5,6 For
example, b-1,4-glucosidase can be obtained very
cheaply from the yeast Aspergillus, which is of considerable commercial interest.
To discover more about the mechanisms of the
enzymatic reactions that take place during the hydrolysis of cellulose, whilst at the same time avoiding
the complexities of such a heterogeneous system, it is
possible in principle to use soluble substrates of known
characteristics, such as cellobiose and carboxymethylcellulose, which allow homogeneous conditions to be
used. Thus in the past7,8 we have studied the hydrolysis of cellobiose with Novozym, a commercial
preparation containing highly active b-1,4-glucosidases derived from Aspergillus niger. We also considered it is interesting to assess the effect of
supplementing cellulases with b-1,4-glucosidases derived from other sources. The prime object of the
research described here was to investigate the possible
bene®ts to CMC hydrolysis of supplementing Celluclast (a commercially produced liquid cellulase derived from T reesei but very low in b-1,4-glucosidase
activity) with Novozym.
EXPERIMENTAL
Carboxymethylcellulose of low viscosity was bought
from Sigma (C-5678) and Celluclast and Novozym
were supplied by Novo Nordisk. Both enzymes were
used under the experimental conditions recommended
by the manufacturers, 50 °C and pH 4.9, which were
obtained by preparing the solutions with the appropriate proportions of sodium-acetate buffer. Checks
were made to ensure that no signi®cant change in pH
occurred during the course of the experiments.
Hydrolysis was carried out in temperature-controlled test tubes by mixing 3 cm3 of CMC solution
with 0.3 cm3 of the enzyme solution. To study the
hydrolysis of CMC with Celluclast alone and with
mixtures of Celluclast and Novozym we carried out
experiments with different initial concentrations of
CMC (2, 5, 7.5, 10 and 12.5 kg m 3) in which the
ratio between the concentrations of Novozym and
Celluclast was also varied (0, 0.5 and 1).
16
The GOD±Perid glucose±oxidase method9 was
used to analyse the glucose concentration (G) resulting
from the hydrolysis of CMC and the 3,5-dinitrosalicylic acid technique (DNS)10 was applied to determine the concentration of reducing sugars produced.
Con®rmation by HPLC that glucose and cellobiose
are to all intents and purposes the main products of the
reaction means that the quantity of cellobiose (C)
present can be arrived at by simple subtraction.
To ensure that neither the carboxymethylcellulose
nor the enzymes themselves interfered in any way with
the experimental results their solutions were analysed
before mixing. It was found that Novozym contained a
small quantity of glucose (0.136 g glucose g 1 enzyme)
and reducing sugars (0.182 g glucose g 1 enzyme),
both of which were taken into account in our calculations. Initial tests with cellobiose were also made7,11
to con®rm that there was no appreciable deactivation
of either Celluclast or Novozym during the hydrolytic
process at 50 °C and pH 4.9.
The reaction was stopped by dipping the test tubes
into boiling water for 5 min, thus denaturing the
enzyme, as described elsewhere.12,13 Other changes in
both temperature and pH were assayed to stop the
reaction and no signi®cant differences were found in
the subsequent glucose analyses, for which reason we
decided to use the boiling water method as being both
simple and ef®cient. When the conversion values are
represented versus time for the different initial concentrations of cellobiose it can be seen7,11 that the
experimental points ®t straight lines that do not
however pass through the origin but the ordinates of
which at the origin tend towards the conversion values
of the experiments made at zero reaction time. Thus
we determined that there was a delay in the stopping of
the reaction, tS, which would explain the conversion
results detected in the experiments at zero reaction
time. That is to say, there is a stopping time,
calculated7 to be 5.75 min, which when added to the
reaction time, tR, allows us to arrive at a corrected
reaction time, t, which determines an initial conversion
of zero.
RESULTS AND DISCUSSION
To express the initial concentration of substrate in
terms of the total potential glucose14 we ®rst carried
out the complete hydrolysis of CMC with Celluclast
alone15 and determined that the ratios between the
concentrations of both glucose and cellobiose, Gf , and
Cf , and CMC, S0, remained practically constant for
hydrolysis times 100 h at about Gf /S0 = 0.56 mol
glucose kg 1 CMC and Cf /S0 = 0.52 mol cellobiose
kg 1 CMC. Thus the value of the total product of
complete hydrolysis, expressed in terms of glucose
equivalent, Pf /S0 = 1.6 mol glucose equivalent kg 1
CMC, also remains constant.
Using the total product of complete hydrolysis, Pf ,
the maximum obtainable over long periods, we can
de®ne a conversion to glucose, xC = G/Pf , for the
J Chem Technol Biotechnol 77:15±20 (online: 2001)
Hydrolysis of carboxymethylcellulose
hydrolysis of CMC, the maximum value of which can
be expressed in terms of the ratio Gf /Pf , with a value of
0.35, and an overall conversion, ie cellobiose plus
glucose, expressed in terms of glucose equivalent,
xP = P/Pf , the maximum value of which, for complete
hydrolysis, will be 1.
Furthermore, we found15 no signi®cant variations in
either xP or xC versus time when we made experiments
in which we kept the product of the enzyme
concentration multiplied by the corrected reaction
time, e0(tR ‡ tS) constant. This product, given that
there was no enzyme deactivation, is a suitable variable
for establishing the intensity of the treatment with
Celluclast, including the effect of the stopping time.
Thus the possible synergism that might exist between
the endoglucanases and exoglucanases which has been
suggested by some authors16±18 does not, in fact, need
to be taken into account.
Kinetic model for the hydrolysis of CMC
The three enzymes included in the Celluclast enzyme
complex play different roles in the hydrolysis of CMC,
which may be expressed by the pathways shown in
Scheme 1.19
CMC is ®rst hydrolysed by the glucanases (E1 and
E2), thus releasing shorter CMC chains (CMC'),
which are also hydrolysable. Thus the total substrate,
S, susceptible to hydrolysis by the glucanases at any
moment during the process can be expressed by the
difference between the glucose equivalent obtained in
complete hydrolysis and that obtained up to the
moment in question, and according to overall conversion this can be given as:
S ˆ Pf
P ˆ Pf …1
xp †
…1†
It should be emphasised that by expressing the
cellobiose as glucose equivalent it is possible to
establish the substrate available for the glucanases
independently of b-1,4-glucosidase (E3) activity.
Endoglucanase (E1) activity produces mainly glucose whilst exoglucanase (E2) generates cellobiose,
which in turn exerts competitive inhibition upon the
exoglucanases themselves.
Finally the b-1,4-glucosidases (E3) hydrolyse the
cellobiose into glucose.
The concentration of each of the enzymes can be
related to Celluclast concentration, e0, by introducing
the fractions of Celluclast corresponding to the endoglucanases, exoglucanases and b-1,4-glucosidases (a1,
a2 and a3).
If the intermediate compounds involved in the
reaction (E1S, E2S, E2C and E3C) are in a steady
state and we take into account the intensity of the
treatment with Celluclast and the conversion to
glucose and the overall conversion, then:
dxC
ka;en …1 xp †
ˆ
‡
KM;en ‡ Pf …1 xp †
dy
ka …xp xC †
Pf
KM ‡ …xp xC †
2
dxp
ka;en …1 xp †
ˆ
KM;en ‡ Pf …1 xp †
dy
2ka;ex …1 xp †
‡
Pf
KM;ex ‡ Pf …1 xp † ‡ KICP;ex …xp
2
xC †
…2†
…3†
which represents a kinetic model for the activity of
Celluclast upon CMC already assayed19 in the pH
range from 3.9 to 5.9. At 50 °C and pH 4.9 we
obtained the following values for the kinetic parameters:
Glucose production from cellobiose by the b-1,4glucosidases: ka = 0.0084 mol m 3 (min kg m 3) 1 and
KM = 1.162 mol m 3.
Glucose production from CMC by endoglucanases:
ka,en = 0.066 mol m 3 (min kg m 3) 1 and KM,en =
0.382 mol m 3.
For the hydrolysis of CMC with mixtures of
Celluclast and Novozym the kinetic model needs to
be modi®ed only in so far as to take into account the
simultaneous activity of both b-1,4-glucosidases on
the cellobiose, that is to say, adding a term to the
equation for xc, whilst the higher transformation of
cellobiose into glucose should not modify the total
product, P, expressed in terms of glucose equivalent
and thus eqn (3) continues to be applicable for xp.
Conversion to glucose
From the glucose concentration we calculated the
values of xC, which are set out in Figs 1±5, versus the
intensity of the treatment with Celluclast, y, for each of
the initial concentrations of CMC assayed. The results
obtained from experiments carried out with mixtures
of various concentrations of Celluclast and Novozym,
expressed as e03/e0 are also included in these ®gures. It
can be seen that for all the initial CMC concentrations
Scheme 1
J Chem Technol Biotechnol 77:15±20 (online: 2001)
17
V Bravo et al
Figure 1. Variation in conversion to glucose versus intensity of the
treatment with Celluclast at S0 = 2 kg m 3 and for different ratios of enzyme
concentrations. The solid line derives from eqn (8) and the dotted line
corresponds to the directly proportional relationship between xC and y,
eqn (4).
Figure 3. Variation in conversion to glucose versus intensity of the
treatment with Celluclast at S0 = 7.5 kg m 3 and for different ratios of
enzyme concentrations. The solid line derives from eqn (8) and the dotted
line corresponds to the directly proportional relationship between xC and y,
eqn (4).
and all the e03/e0 ratios the values of xC versus y adjust
satisfactorily to straight lines that pass through the
origin (dotted lines), indicating a directly proportional
relationship between conversion to glucose and the
intensity of the treatment with Celluclast, and thus:
dxC xC
ˆ
ˆ XC
dy
y
…4†
To determine the effect upon XC of adding
Novozym to the solution we adjusted the values of
xC/y versus those of the ratio e03/e0 by linear regression
and concluded that for all the initial concentrations of
CMC assayed there was a linear relationship between
either variables.
XC ˆ XCC ‡ XCN
e03
e0
…5†
Figure 2. Variation in conversion to glucose versus intensity of the
treatment with Celluclast at S0 = 5 kg m 3 and for different ratios of enzyme
concentrations. The solid line derives from eqn (8) and the dotted line
corresponds to the directly proportional relationship between xC and y,
eqn (4).
18
Figure 4. Variation in conversion to glucose versus intensity of the
treatment with Celluclast at S0 = 10kg m 3 and for different ratios of enzyme
concentrations. The solid line derives from eqn (8) and the dotted line
corresponds to the directly proportional relationship between xC and y,
eqn (4).
Figure 5. Variation in conversion to glucose versus intensity of the
treatment with Celluclast at S0 = 12.5 kg m 3 and for different ratios of
enzyme concentrations. The solid line derives from eqn (8) and the dotted
line corresponds to the directly proportional relationship between xC and y,
eqn (4).
J Chem Technol Biotechnol 77:15±20 (online: 2001)
Hydrolysis of carboxymethylcellulose
Table 1. Values for the total product of complete hydrolysis, Pf, and for the parameters in eqn (5)
S0 (kg m 3)
Pf (mol m 3)
12.5
10
7.5
5
2
20
16
12
8
3.2
so that the ordinate at the origin
XCC ˆ
XCC (min
xC
XCN e03 t
ˆ
y
1
(kg Celluclast m 3) 1)
XCN (min
0.0039
0.0052
0.0067
0.0094
0.0172
xC
e0 t
e03ˆ0
…6†
represents glucose production without Novozym,
e03 = 0, and the slope
xC XCC e0 t
xC
ˆ
XCN ˆ
…7†
e03 t
e03 t e0ˆ0
1
(kg Novozym m 3) 1)
0.0105
0.0114
0.0158
0.0194
0.0306
ka, KM, ka,en and KM,en indicated above together with
those of XCN from Table 1 the solid lines in Figs 1±5
were obtained, which reproduced the experimental
values satisfactorily.
Overall conversion
which is a quadratic equation with regard to xC and
allows us to determine the value of xC for each pair of
experimental values of xp and y. By using the values of
The overall conversion, meaning the total product
deriving from the hydrolysis of CMC, ie cellobiose
plus glucose, expressed in terms of glucose equivalent,
was calculated from the concentrations of glucose and
cellobiose and the value of Pf , for each series of
experiments. The values for xp versus intensity of the
treatment with Celluclast, y, for two of the series of
experiments made with different initial concentrations
of CMC, S0, are shown as an example in Figs 6 and 7.
It can be seen in these ®gures that, in the same way as
occurs with the other series of experiments at different
S0 values, the values for the various e03/e0 ratios can be
®tted to the same curve and also that overall conversion increases rapidly at ®rst concomitantly with an
increase in intensity of the treatment with Celluclast,
but then gradually slows down, which makes it dif®cult
to apply the initial-rate method.
If xC in eqn (3) of the kinetic model is replaced with
[(XCC ‡ XCNe03/e0) y], in accordance with eqns (4) and
(5) this allows a numerical integration without regard
to eqn (2) to obtain the variation of xp versus y. For this
Runge±Kutta's fourth-order method was used and
taking the kinetic parameter values previously established with Celluclast19 we applied an optimisation
using the simplex method, obtaining the values:
Figure 6. Variation in overall conversion versus intensity of the treatment
with Celluclast at S0 = 2 kg m 3 and for different ratios of enzyme
concentrations. The solid line derives from numerical integration of eqn (3).
Figure 7. Variation in overall conversion versus intensity of the treatment
with Celluclast at S0 = 12.5 kg m 3 and for different ratios of enzyme
concentrations. The solid line derives from numerical integration of eqn (3).
represents production due to the activity of Novozym
and the relationship between increased conversion to
glucose and intensity of the treatment with mixtures of
Novozym and Celluclast. The values obtained for XCC
and XCN for every initial concentration of CMC, S0,
and the corresponding value for the total product of
complete hydrolysis, Pf , are given in Table 1.
According to eqn (2) of the kinetic model and the
directly proportional relationship between xC and y
found in eqn (4).
dxC xC
ˆ
dy
y
2
6 ka;en …1 xp †
‡
ˆ4
KM;en ‡Pf …1 xp †
3
ka …xp xC †
e03 7
‡ XCN 5
Pf
e0
KM ‡ …xp xC †
2
…8†
J Chem Technol Biotechnol 77:15±20 (online: 2001)
19
V Bravo et al
ka,ex = 563 (min kg m 3) 1, KM,ex = 176 mol m 3 and
KICP,ex = 6590, which allowed us to obtain by numerical integration the solid lines shown in Figs 6 and 7,
which ®tted the experimental results satisfactorily.
CONCLUSIONS
The kinetics of the hydrolysis of carboxymethylcellulose with the cellulases contained in Celluclast supplemented with the b-1,4-glucosidases provided by
Novozym can be ®tted to a kinetic model which takes
into account variations in the conversion to glucose
and overall conversion in terms of glucose equivalent
versus the intensity of treatment with Celluclast.
The ratio between the conversion to glucose and the
intensity of the treatment with Celluclast remains
constant throughout the interval assayed and this ratio
increases linearly with the relationship between Novozym and Celluclast concentrations. This increase
results in cellobiose being converted to glucose much
more rapidly and thus reduces the inhibition which
cellobiose exerts over the glucanases.
The fact that the overall conversion is expressed in
terms of glucose equivalent means that its value is not
affected by the increase in the ratio between the concentrations of Novozym and Celluclast, and therefore
the addition of Novozym only modi®es our proposed
kinetic model in as far as it affects the conversion to
glucose.
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