Process Biochemistry 35 (1999) 111 – 117

www.elsevier.com/locate/procbio

Modeling of enzymatic protein hydrolysis

M.C. Márquez *, M.A. Vázquez
Chemical Engineering Department, Faculty of Chemical Sciences, Uni6ersity of Salamanca, Plaza de los Caı́dos 1 – 5, 37008 -Salamanca, Spain

Received 17 July 1998; received in revised form 15 February 1999; accepted 28 February 1999

Abstract

Studies on proteolysis of hemoglobin by Alcalase 0.6L are described. Trials were conducted in a batch reactor at different
operating conditions. Degrees of hydrolysis in the proteolysates were determined and kinetics of the reaction was considered in
detail in relation to substrate concentration, enzyme concentration, temperature, degree of hydrolysis and reaction time. From the
results a general kinetic equation for the enzymic hydrolysis of proteins is suggested. The proposed kinetic model allows an easier
determination of kinetic parameters using only a few experiments. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Enzymic hydrolysis; Enzyme; Hemoglobin; Kinetics; Protein; Kinetic parameters

1. Introduction The purpose of the present work was to show that

Biotechnology reactions are usually carried out using
fed-batch reactors since greater flexibility is provided in
terms of product manufacture. Optimization of batch
bioreactors depends on the kinetics of the processes [1]
which can become extremely complicated for enzymic
hydrolysis of proteins. The large number of peptide
bonds which are cleaved, both in parallel and in series,
during the hydrolysis of most polymer molecules sets its
natural limitation to the possibility of estimating the
basic kinetic parameters.
This is a general problem in establishing kinetic
models for the hydrolysis of macromolecular substrates.
If the kinetic model is too simple, its inadequate repre-
sentation of the true mechanism will limit its general
applicability [2–4], while a complicated model cannot
be analyzed statistically with sufficient precision by the
often relatively crude kinetic experiments [5 – 7].
To circumvent these problem, simple empirical rate
equations may be applied to express hydrolysis curves.
The use of empirical rate equations does not mean that
all information in the mechanism is lost; much informa-
tion can be obtained by researching empirical hydroly-
sis curves under different conditions.
* Corresponding author. Tel.: +34-92-3294479; fax: + 34-92-
3294574.
E-mail address: mcm@gugu.usal.es (M.C. Márquez)
hydrolysis curves of proteins catalyzed by enzymes in
solution can be modeled by a simple empirical equation
from which kinetic parameters can be deduced.
Hemoglobin and Alcalase were chosen as substrate
and enzyme, respectively, for this study. Although the
batch hydrolysis of hemoglobin with Alcalase has been
extensively analyzed [8–10], little researches on process
kinetics have been carried out. The only kinetic model
applied [3] was not supported by sufficient experimental
data and therefore remains speculative.
The experimental data obtained in this research were
used to demonstrate the application of empirical equa-
tions for the determination of reaction rates constants
and reaction energies.
2. Materials and methods
The substrate used in this research was bovine
hemoglobin from Sigma.
The enzyme complex used in this work was Alcalase
0.6L (Novo Industri), a non-specific bacterial endo-pep-
tidase from Bacillus licheniformis, with Subtilisin Carls-
berg as its main component (specific activity= 0.6 AU
g − 1; density of 1.26 g ml − 1).
Hydrolysis experiments were performed in a 0.5 l
jacketed reactor with magnetic stirring and pH and

0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 0 4 1 - 2
112 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117

temperature control. Samples were prepared by sus- Table 1

pK and a values for different temperatures
pending a given amount of hemoglobin (0.5 – 3.0 g) in
400 ml of hot water (35, 45, 55°C). Solutions were then T (°C) pK a
adjusted to pH 7.0 with NaOH 0.5 N. Alcalase was
added (1.89× 10 − 2 − 9.45 ×10 − 2 AU l − 1) and stirred 35 7.45 0.25
(300 rpm). All experiments were carried out in duplicate 45 7.20 0.38
55 7.00 0.49
(figures show the average values of the experiments);
the average standard deviation calculated for these
experiments was 0.03. Temperature and pH were cho-
where pK is the average pK value of the a-amino
sen following the indications of maxima activity and
groups liberated during the hydrolysis. pK and a values
stability for Alcalase provided by Novo [11].
used are showed in the Table 1 [8].
Kinetics was followed by base (1 N NaOH) con-
sumption, during hydrolysis, since the addition of base
in order to maintain constant pH is proportional to the
degree of hydrolysis [8]. 3. Results and discussion
Proteinases attack peptide bonds in proteins as
follows: 3.1. Kinetic model
1. Opening of the peptide bond:
Enzyme Experimental curves obtained (Figs. 1–3) can be
CHR%CONHCHR%% + H2O “ CHR%COOH fitted by the following kinetic equation:
+NH2CHR%% d(DH)
= a exp[−b · (DH)] (4)
2. Proton exchange: dt
CHR%COOHNH2CHR% where a and b have different values for the different
+ experiments.
“ %CHR%COO + NH3CHR%%
−
In agreement with previous studies, this form for the
3. Titration of amino group: kinetic equation can be explained if the hydrolysis is
+ modeled as a zero order reaction with a simultaneous
NH3CHR%% +OH − X NH2CHR%% +H2O
second order inactivation of the enzyme [12].
Under neutral or alkaline hydrolysis conditions, the Then if the hydrolysis reaction is
dissociation of the amino groups becomes significant k1 k2
E +S ? ES“ E+ P
and the continuous base consumption necessary to k -1

maintain constant pH during the reaction is then a then the reaction rate will be determined by the irre-
convenient mean of controlling the process. versible stage
The degree of hydrolysis, DH, is defined as the ratio
of the number of peptide bonds cleaved (per weight d(DH)
r= s0 = k2ES (5)
unit), h, to the total number of peptide bonds (per dt
weight unit), ht:
If enzymic inactivation reaction is
h
DH= 100 (1)
ht
The total number of peptide bonds per weight unit,
ht, in a protein can be calculated from its amino acid
composition. For hemoglobin a value of 8.3 eqv kg − 1
has been used [8].
The relationship between DH and base consumption
is given by the following equation:
BNB 1 1
DH = 100 (2)
Mp a ht
B is the base consumption, NB is the normality of the
base, Mp is the mass of protein (N × 6.25) and a is the
average degree of dissociation of a-NH2 groups:
10pH − pK
a= (3) Fig. 1. Hydrolysis curves for different initial substrate concentrations
1 +10pH − pK (e0 =5.67 ×10 − 2 AU l − 1; T =55°C and pH 7.0).
 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117 113

where KM is the Michaelis–Menten constant

k − 1 + k2
KM = (10)
k1
Substitution of Eq. (9) into Eq. (8) and simplification
S= s0 yield the expression for the free enzyme
concentration
KMe
E= (11)
KM + s0
If KM  s0, Eq. (11) can be reduced to
KMe
E= (12)
s0
Insertion of this Eq. (12) in Eq. (7) gives
Fig. 2. Hydrolysis curves for different initial enzyme concentrations
(s0 =2.50 g l − 1; T =55°C and pH 7.0). d(DH) k 1
− = 2 (13)
k3 de k3KM e
E +ES “ Ea + Ei +P
Integration of Eq. (13) between the limits of the
then the kinetic equation for this process is given by

initial and final reaction conditions provides:

−
de
= k3EES (6) e =e0 −
k3KM
(DH)
n (14)
dt k2

The relationship between the two mechanisms con- Combination of Eq. (14) with Eqs. (5), (9) and (12)


sidered, Eqs. (5) and (6), provides the ratio: shows

− s0
d(DH) k
= 2 (7)
r= k2e0 exp −
k3KM
(DH)
n (15)
k2
de k3E
and then
Since all enzyme present is either free or complexed,
the total enzyme concentration at a given moment, k2e0
a= (16)
would be in the form: s0
e =E+ ES (8) k3KM
b= (17)
k2
At steady state, the mass balance for the ES complex
leads to
4. Model validation and kinetic parameter calculation
ES
ES= (9)
KM From the mechanism and the kinetic equation the
proposed kinetic parameters for enzymic hydrolysis of
hemoglobin can be calculated.

4.1. Reaction rate constant determination

Before reaction rate constant determination, the infl-

Fig. 3. Hydrolysis curves for different temperatures (s0 = 2.50 g l − 1;
e0 =5.67 ×10 − 2 AU l − 1 and pH 7.0).
uence of initial substrate and enzyme concentration on
a and b parameters was checked.
For analysis of the influence of substrate concentra-
tion (Fig. 1), the initial enzyme concentration was
5.67× 10 − 2 AU l − 1, temperature 55°C and pH 7.0.
Values of a and b for different concentrations of
hemoglobin were calculated from nonlinear regression
analysis. Parameter b did not exhibit any trend when
the initial substrate concentration, s0, changed and its
values lie within a very small range. Thus, b can be
considered independent of these variable and constant
114 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117

Table 2
Kinetic parameters obtained from hydrolysis curves for different
initial substrate concentrationsa

s0 (g l−1) a (b= 0.12) (min−1)

1.25 1.70
2.50 0.63
5.00 0.45
7.50 0.27

a
e0 = 5.67×10−2 AU l−1; T= 55°C and pH 7.0.

in the operating conditions with an average value of

0.12. However, a values calculated from this average
value (Table 2) showed a clear dependence on the initial
substrate concentration.
Analysis of influence of initial enzyme concentration
was carried out for an initial substrate concentration of
2.5 g l − 1, temperature 55°C and pH 7.0 (Fig. 2).
The value of the kinetic parameter b did not present
either any dependence on initial enzyme concentration,
e0, and its average value can be also considered 0.12. In
contrast, a values calculated from this average value of
0.12 (Table 3) increased when the initial enzyme con-
centration increased. This fact agrees with the increase
of overall rate reaction observed in Fig. 2 for high e0.
A plot of values of parameter a calculated from the
average value of b, b =0.12, versus e0/s0 (Fig. 4) leads
to a straight line through (0,0). The linear regression
analysis on the figure shows a correlation coefficient of
0.9937 and yields the expression:
e0
a= 39.19
s0
which agrees with Eq. (16) deduced from the proposed
mechanism. Then the validity of the above hypothesis
for kinetic model is verified and the reaction rate con-
stant value, k2, is determined: 39.19 g AU − 1 min − 1.
4.2. Enzymic inacti6ation constant
Previous considerations and combination of Eqs. (6),
(9) and (12) lead to
de e2 e2
− =k3KM =kd
dt s0 s0
Fig. 4. Variation of kinetic parameter a for different e0/s0 values.
where kd depends on various equilibrium and reaction
rate parameters (k3 and KM = (k − 1 + k2)/k1). In accor-
dance with the previously proposed second order inacti-
vation of the enzyme, kd is considered an enzymic
inactivation constant which can be determined from a
and b parameters:
ke kK e e
ab= 2 0 3 M = k3KM 0 = kd 0 (20)
s0 k2 s0 s0
As would be expected, from a plot of ab as a function
of e0/s0, a straight line was obtained (correlation coeffi-
cient= 0.9937), as depicted in Fig. 5. From the slope of
this line the enzymic inactivation constant was calcu-
lated: 4.66 g AU − 1 min − 1.
4.3. Hydrolysis acti6ation energy and enzymic
(18)
inacti6ation energy
For the determination of hydrolysis activation and
enzymic inactivation energies, the variation of tempera-
ture was analyzed when initial substrate concentration,
initial enzyme concentration and pH were fixed at 2.50
g l − 1, 5.67×10 − 2 AU l − 1 and 7.0, respectively (Fig.
3).
(19)

Table 3
Kinetic parameters obtained from hydrolysis curves for different
initial enzyme concentrationsa

e0×102 (AU l−1) a (b=0.12) (min−1)

3.78 0.46
5.67 0.63
7.56 1.11
9.45 1.59

a
s0 = 2.50 g l−1; T =55°C and pH 7.0. Fig. 5. Variation of kinetic parameters ab for different e0/s0 values.
 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117 115

Table 4
Kinetic parameters obtained from hydrolysis curves for different
temperaturesa

T (°C) b a (min−1)

35 0.22 0.56
45 0.18 0.59
55 0.12 0.63

a
s0 = 2.50 g l−1; e0 = 5.67×10−2 AU l−1 and pH 7.0.

Now, both a and b changed with the temperature

(Table 4).
Due to dependence of a on reaction rate constant
k2, the change in a caused by a change in tempera-
ture follows the integrated Arrhenius relationship:
DEA
ln a = − +AA
RT
where DEA is the hydrolysis activation energy, AA is
the frequency factor and R is the gas constant.
From the slope of the regression line in the plot of
ln a versus 1/T, Fig. 6 (correlation coefficient=
0.9965), the value of the activation energy can be
determined: 4938.18 J mol − 1.
The change in kd caused by a change in tempera-
ture can be determined from a and b parameters
which also followed the integrated Arrhenius relation-
ship:
DED
ln ab= − + AD
RT
so that from the plot of (ln ab) versus 1/T, Fig. 7
(correlation coefficient=0.9712), the Alcalase inacti-
vation energy, DED, for experimental conditions can
be determined: 25353.54 J mol − 1.
These results differ from those determined by
Adler-Nissen [8] for the enzyme Alcalase. He sug-
gested average values of 50000 J mol − 1 and 270000 J
Fig. 7. Arrhenius plot for enzymatic inactivation energy determina-
tion.
(21)
mol − 1 for DEA and DED, respectively. This disagree-
ment could be due to some fundamental differences
in the Adler-Nissen experimental procedure: casein
and soya isolate were used as substrates, the enzyme
was saturated with substrate throughout the hydroly-
sis reaction (s0 = 8%), a higher pH was used (pH ]8)
and an inactivation reaction of first order was sup-
posed.
On the one hand, when casein and soya protein
isolate are used as substrate with Alcalase the same
behavior was found and similar hydrolysis curves
were obtained if the substrate was casein or soya iso-
late. However, when hemoglobin was used as sub-
(22)
strate there was a surprisingly large difference
between the hydrolysis curves. In accordance with
Adler-Nissen, this result could serve as a sufficient
warning against extrapolating from activity data [8].
When kinetic investigations were carried out at al-
kaline pH and high substrate concentrations, results
were not immediately applicable to processes carried
out at neutral pH, where the substrate was insoluble
and at low substrate concentration (s0 5 4%), where
substrate saturation was not ensured [8].
Finally, when an inactivation reaction of first order
with respect to e0 is supposed, the results are neces-
sarily different from these for an inactivation reaction
of second order with respect to e0. This second order
reaction is acknowledged by Adler-Nissen for operat-
ing temperatures below 50–55°C [8], which agrees
with the present results.

5. Model application

The validity of the kinetic model and the kinetic

parameters obtained is shown in Fig. 8, where the
adequacy between the calculated values of hydrolysis
degree versus time (solid curves) and the experimental
Fig. 6. Arrhenius plot for hydrolysis activation energy determination. data is observed at different operating conditions.
116 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117
6. Conclusions
The objective of this paper has been to represent the
rate of enzyme-catalyzed hydrolysis of proteins mathe-
matically. Without suitable rate expressions, reactors
cannot be designed nor can experiments employing
enzymes in solution be modeled.
The proposed kinetic relationship is a simple expo-
nential equation Eq. (4)which agrees with published
data in previous studies on kinetics of enzymic hydroly-
sis for other animal and vegetable proteins. Thus, the
same equations was found when Alcalase was used for
hydrolyzing casein [12], chickpea protein [13] or lactal-
bumin [14]. The operating conditions in these reports
were different from those used in this paper: studies
were carried out at pH 8, 50°C and substrate satura-
tion. This suggests that the same kinetic equation is
applicable for different substrates and operating
conditions.
Bacterial peptidases such as Alcalase follow the pro-
posed equation together with other bacterial proteases
and enzyme preparations from animal sources. Studies
of hydrolysis of whey protein with MKC Protease (a
bacterial protease obtained from Bacillus subtilis) [14],
PEM (a mixture of pancreatic enzymes including
porcine trypsin, bovine trypsin and bovine chy-
motrypsin) [14] or a mixture of both preparations [15]
also could use the Eq. (4) to fit data obtained at pH
8 – 10 and temperature 50 – 70°C. This suggests that the
same kinetic equation is applicable for different en-
zyme –substrate systems and again for different operat-
ing conditions.
Although the proposed kinetic equation is the same
for all cases, the reaction mechanism can be different
when some substrate or product inhibitions take place.
Thus, chickpea hydrolysis [13] and whey protein hy-
drolysis [14,15] were modeled as substrate-inhibited re-
Fig. 8. Kinetic model application (, s0 = 2.50 g l − 1; e0 =1.89 ×
10 − 2 AU l − 1; T =55°C and pH= 7.0.
, s0 = 1.25 g l − 1; e0 = AU−1 min−1)
5.67× 10 − 2 AU l − 1; T= 50°C and pH 7. , s0 = 7.50 g l − 1;
e0 =2.20 ×10 − 2 AU l − 1; T= 55°C and pH 7).
actions. The only different in these cases of inhibitor
presence is that a kinetic parameter in Eq. (4) depends
on an apparent reaction rate constant which includes
not only the reaction rate constant but also an inhibi-
tion rate constant. This seems to indicate that the
proposed kinetic Eq. (4) could be of general
application.
Consequently, it is worth noting that the developed
reaction model is capable of reproducing the kinetic
behavior of a system over a wide range of operating
conditions, thus providing a useful tool for the optimal
reactor design. Moreover, this provides a suitable
model which can be applied not only to hemoglobin
hydrolysis reaction investigated in this work but also to
the description of other enzyme–protein systems that fit
the same mathematical equation. Finally, it allows the
calculation of the necessary kinetic parameters from a
few experiments: Kinetic constants can be determined
by varying s0 and e0 (by plotting a or ab against e0/s0,
a straight line with slope k2 or kd, respectively, is
obtained; although constants can be determined di-
rectly from one only experiments, obtained values could
be less reliable) and energies DEA, DED, can be deter-
mined by varying temperature (a plot of ln a or ln ab
against 1/T leads a straight line with slope DEA or DED,
respectively).
Appendix A. Nomenclature
AA, AD frequency factors, dimensionless
a kinetic parameter (min−1)
B base consumption (l)
b kinetic parameter, dimensionless
DH hydrolysis degree (%)
E free enzyme
Ea active form of enzyme
Ei inactive form of enzyme
e enzyme concentration (AU l−1)
e0 initial enzyme concentration (AU l−1)
ES enzyme–substrate complex
h hydrolysis equivalents (eqv kg−1)
ht total number of peptide bonds in a protein
(eqv kg−1)
KM Michaelis–Menten constant (g l−1)
k1 reaction rate constant for proteolysis (l
AU−1 min−1)
k−1 reaction rate constant for proteolysis (g
AU−1 min−1)
k2 reaction rate constant for proteolysis (g
AU−1 min−1)
k3 reaction rate constant for inactivation (l
AU−1 min−1)
kd reaction rate constant for inactivation (g
Mp mass of protein (kg)
P product
 M.C. Márquez, M.A. Vázquez / Process Biochemistry 35 (1999) 111–117
R gas constant, 8.314 (J mol−1 K−1)
r reaction rate (g l−1 min−1)
S free substrate
s substrate concentration (g l−1)
s0 initial substrate concentration (g l−1)
T temperature (°C or K)
a degree of dissociation of the a-amino group
DEA hydrolysis activation energy (J mol−1)
DED enzyme inactivation energy (J mol−1)
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