Enzyme and Microbial Technology 39 (2006) 252–258

Enzymatic inhibition and thermal inactivation in the hydrolysis

of chestnut purée with an amylases mixture
Cristina López, Ana Torrado, Pablo Fuciños, Nelson P. Guerra, Lorenzo Pastrana ∗
Department of Biochemistry, Genetics and Immunology, University of Vigo, 32004 Ourense, Spain
Received 25 May 2005; accepted 24 October 2005

Abstract
Hydrolysis of starch present in 225 g L−1 chestnut purée was performed at 70 ◦ C in a sole step with a thermostable ␣-amylase (Termamyl 120L,
type S) and glucoamylase (AMG 300L) mixture. Applying an optimal ratio for both enzymes at a high enzyme concentration (60 enzymatic
units g−1 raw chestnut, corresponding to 13.5 enzymatic units mL−1 chestnut purée) total polysaccharide conversion to glucose was obtained in
15 min. Complete hydrolysis was not possible when operating at a 10-fold lower enzyme concentration after 48 h incubation. Glucoamylase product
inhibition and thermal deactivation appeared as the main reasons for the incomplete reaction of the enzymes mixture at the lower concentration
assayed. An empirical model was applied to describe substrate and competitive and non competitive product inhibition and operational kinetic
parameters were calculated for the enzymes mixture in chestnut purée. While ␣-amylase was not affected by the thermal treatment in the operational
conditions, glucoamylase showed a strong thermal deactivation that did not follow first order kinetics but fitted to a parallel biexponential model,
which points towards the presence in AMG 300L of two glucoamylase forms with different thermostabilities. An empirical model was developed to
describe thermal deactivation of the enzymes mixture in chestnut purée considering the presence of different glucoamylase forms and a sinergistic
effect between them and the ␣-amylase.
© 2005 Elsevier Inc. All rights reserved.

Keywords: Chestnut starch; One-step hydrolysis; Glucoamylase; ␣-Amylase; Substrate and product inhibition; Thermal deactivation

1. Introduction ondly, the production of an alcoholic beverage from distillation

In some countries, chestnut trees culture plays an impor-
tant environmental and economic role due to their landscape
qualities and the production of wood and chestnuts. The fruit
is generally manufactured in local industries to obtain differ-
ent confectionery high-added-value products like marron glace.
Since these delicatessen products require high quality chestnuts
(in terms of size, shape or suitability for peeling and sweet pro-
cessing), only a small percentage of the annual chestnut crop
is destined to industrial transformation. However, in basis of
their starchy nature, overproduction of chestnuts can be used in
the formulation of alternative foods or for biotechnological pur-
poses. In Galicia (NW of Spain) – one of the most productive
chestnut areas in Europe – two realistic industrial alternatives
for exploitation of overproduction are being tested: firstly, the
development of sweet flour for celiac people without gluten, sec-
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.
E-mail address: pastrana@uvigo.es (L. Pastrana).
of fermented chestnut. In the first case, only partial enzymatic
hydrolysis of starch is required, while in the second full hydrol-
ysis is needed.
Industrial enzymatic hydrolysis of starch is influenced by
variables related to the chemical and physical starch nature
(relative proportion of amylose and amylopectin or granule
crystallinity) and its suspensions (viscosity, resistance to ret-
rogradation or gelatinization degree), but also by those vari-
ables associated with the catalytic process (pH, temperature,
enzyme/substrate ratio or enzymatic deactivation and inhibi-
tion phenomena). These last phenomena were mainly studied
in starch–glucoamylase systems [1–4], but there are also studies
with ␣-amylase [5–8]. In the case of hydrolysis of usual starch
sources like corn, potato, wheat or rice, these effects are well
documented [9–11]. Nevertheless, due to the different nature
of these substrates, enzymatic preparations, enzymatic assay
methods and kinetic models applied, important differences in
the values of the kinetic parameters, as well as in the diagnosis
of the existence of a concrete type of inhibition, are found in the
literature. Thus, the determination and evaluation of enzymatic

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.10.012
 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258
inhibition must be experimentally performed for each particu-
lar system. Thermal inactivation of amylases is a well-known
process [12–14], which affects their industrial performance.
Variations of thermolability are also found for glucoamylases
from different origins and forms. Unfortunately, hydrolytic stud-
ies of indigenous starches that constitute local alternative sources
are scarce and, in the particular case of chestnut starch, only
information about the description of the reological properties of
starch pastes is available [15].
In a previous work, we optimized the enzyme/substrate ratio
and the relative proportion of ␣-amylase and glucoamylase in
the enzymatic mixture to hydrolyze chestnut purée in a sin-
gle step [16]. A synergistic effect, similar to that described
by Fujii and Kawamura with potato starch [17], was postu-
lated for these two enzymes to explain total starch conver-
sion to glucose only when the enzymatic mixture included
a ratio of ␣-amylase/glucoamylase activity of 0.351 and the
enzyme/substrate ratio was the highest assayed (60 enzy-
matic units g−1 raw chestnut). Nevertheless, starch hydrol-
ysis was always uncompleted in reaction mixtures with low
enzyme/substrate ratio (6 enzymatic units g−1 raw chestnut).
Although no kinetic experiments were made, enzymatic inhibi-
tion and/or thermal deactivation were postulated as causes for
reaction stopping before total starch hydrolysis to glucose in
these conditions.
In this work, we have studied the existence of inhibition
phenomena and thermal deactivation of the ␣-amylase and
glucoamylase low concentration mixture in chestnut purée
hydrolysis. Simple models were empirically applied to this
complex system in order to describe the technological pro-
cess.
2. Materials and methods
2.1. Materials: substrate and enzymes
The percentages of the main components in the peeled raw chestnut used in
this work were 56.9 water, 36.7 total sugars, 6.5 sucrose, 30.2 starch and 2.24
protein [16]. Chestnut purée was prepared at different concentrations by mixing
chopped chestnuts with 50 mM phosphate–citric buffer (pH 4.75), containing
glucose in adequate concentrations for product inhibition assays. It was gela-
tinized by heating in autoclave for 1 h at 100 ◦ C for simultaneous sterilization
so that the hydrolysate could be used as substrate for fermentation. After hot
homogenization (3 min in an Ultraturrax at 9500 rpm), the suspension (purée)
was immediately used as substrate (or maintained at 70 ◦ C) to avoid retrograda-
tion.
Two thermostable commercial amylases were used: Termamyl 120L type
S ␣-amylase (EC 3.2.1.1) and AMG 300L glucoamylase (EC 3.2.1.3), both
purchased from Novo Nordisk A/S (Bagsvaerd, Denmark).
2.2. Hydrolysis conditions
Experiments were done in sealed 100 mL bottles loaded with 50 mL of
chestnut purée for hydrolysis kinetics and thermal deactivation assays, or in
sealed 15-mL tubes loaded with 5 mL of chestnut purée for inhibition assays.
Due to the viscosity of the more concentrated purées, loading was done by
weight after measuring the density of each purée. The mixture of enzymes
was added as an aqueous solution prepared to provide the total enzymatic
units (considered as the sum of the individual activities of ␣-amylase and glu-
coamylase, measured as described by Murado et al. [18]) indicated in each
assay, and 12 ppm of CaCl2 in the final reaction mixture. The ratio of ␣-
253
Fig. 1. Kinetics of 225 g L−1 chestnut purée hydrolysis at 70 ◦ C with an ␣-
amylase and glucoamylase mixture (ratio: 0.35/0.65) at two enzyme concentra-
tions: () 13.5 EU mL−1 and () 1.35 EU mL−1 chestnut purée.
amylase/glucoamylase applied was always 0.35/0.65 (based on one total enzy-
matic unit) according with the conditions optimized in a previous paper [16].
The volume of the enzymatic solution was 10% of the total volume of reac-
tion.
Hydrolysis was carried out at 70 ◦ C in a thermostatized bath with orbital
agitation (100 rpm). Samples were taken off at fixed times after stopping the
reaction by the addition of 10N NaOH (never more than 1% of the total volume
of reaction, giving pH ∼ 12) excepting for the thermal deactivation assays. In all
cases samples were centrifuged (12,000 × g) and the supernatants were recov-
ered for analytical determinations (total and reducing sugars). In the assays of
thermal deactivation the supernatants were loaded at a PD-10 column and equi-
librated in 50 mM phosphate–citric buffer (pH 4.75) to eliminate the reducing
sugars in the mixture that interfere with the measurement of amylolytic activity,
as described by Murado et al. [18].
Hydrolysis (H) was defined as the ratio between the reducing sugars present
in the supernatant obtained from the mixture of reaction and the total sugars
(excepting cellulose, pentosan and sucrose) in the chestnut purée. According to
this definition and the sugar composition of chestnut, 100% hydrolysis corre-
sponds with total conversion of starch to glucose. The initial rate of reaction in
the inhibition assays was measured as the amount of reducing sugars generated
after 3 min of incubation.
2.3. Product inhibition
Product inhibition assays were performed by adding different concentrations
of glucose to different concentrations of chestnut purée and measuring the initial
hydrolysis rates. The highest purée concentration assayed was lower than in the
kinetic assay showed in Fig. 1 to reduce the effect of substrate inhibition. Glucose
concentrations (between 0 and 9 g L−1 ) were selected considering glucose levels
measured at the beginning of the incubation, corresponding to conditions of
initial hydrolysis rates. The difficulty of detecting low enzymatic activities in the
presence of reducing sugars prevented to assay concentrations of glucose higher
than 9 g L−1 and made it necessary to increase slightly the total concentration
of the enzymatic mixture from 1.35 to 4.95 EU mL−1 chestnut purée.
2.4. Thermal deactivation
Thermal deactivation of the ␣-amylase and glucoamylase mixture at low
enzyme concentration (1.35 EU mL−1 ) was studied in the operational conditions
in presence and absence of substrate (buffered chestnut purée and only buffer),
measuring at different times the residual amylolytic activity. To avoid inhibitory
effects in the determination of the amylolytic activity, aliquots were passed
through PD-10 columns, equilibrated in 50 mM phosphate-citric pH 4.75 buffer,
to eliminate glucose from the reaction medium before the analysis.
254 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258
2.5. Analytical methods
Quantification of total (TS) and reducing sugars (RS), and identification of
the soluble sugars (mono-, di- and oligosaccharides) were done as described in a
previous paper [16]. Amylolytic activity (AA) was measured in enzymatic units
(EU), defined as the amount of reducing sugars (expressed in g L−1 ) generated in
10 min of incubation at 40 ◦ C of a 24 g L−1 buffered soluble starch solution (pH
5.0) with a volume of enzyme corresponding to 20% of the volume of the sub-
strate solution, as described by Murado et al. [18]. All analytical determinations
were made in duplicate.
2.6. Modelling and statistical methods
All experiments were done in duplicate. Adjustment of experimental data to
models described in the text was done by non-linear least squares fitting using the
Solver utility from Microsoft® Excel 2002. Performance of models was tested
by comparing the regression coefficient (r2 ) between predicted and observed
data.
3. Results and discussion
3.1. Hydrolysis kinetics of chestnut purée
Fig. 1 shows the hydrolysis kinetics of 225 g L−1 chestnut
purée performed in a single step with an ␣-amylase and glu-
coamylase mixture and two enzyme/substrate ratios (60 and
6 EU g−1 raw chestnut), corresponding respectively to total
enzyme concentrations of 13.5 and 1.35 EU mL−1 chestnut
purée. Total hydrolysis was obtained after approximately 15 min
of incubation only in the series with the higher enzyme con-
centration, while at the lower enzyme/substrate ratio total starch
conversion was not possible even after 48 h incubation. Anyhow,
hydrolysis after the two first hours was ∼90% the maximum
value reached in this case.
Glucose was the main product generated in both cases
throughout all the process (data not shown), reaching concentra-
tions around 66 and 57 g L−1 after 48 h incubation at high and
low enzyme/substrate ratio, respectively. Maltose concentration
in the mixture (produced by ␣-amylase) was maximal in the first
minutes of hydrolysis, decreasing until the end of the incuba-
tion time more quickly when the high enzyme/substrate ratio
was applied. The highest maltose level corresponded to the low
enzyme/substrate assay but it did not reach more than 10 g L−1 .
No oligosaccharides higher than maltose were detected.
Fig. 2. Initial rates of chestnut purée hydrolysis with an amylases mixture for different purée concentrations at two enzyme concentrations: (A) 1.35 and (B)
13.5 EU mL−1 chestnut purée. Symbols represent the experimental points, and solid lines represent the fittings to Michaelis–Menten’s models: with substrate
inhibition (model 1; A) and without substrate inhibition (B).
In basis of the hydrolytic behaviour of both amylases, the
inability of the low enzyme concentration to reach total hydrol-
ysis of a 225 g L−1 chestnut purée could be explained if product
(glucose) inhibition and/or thermal inactivation occurred, affect-
ing more the series with low enzyme/substrate ratio. Substrate
inhibition must be also taken into account.
3.2. Substrate inhibition
Since substrate inhibition reduces the hydrolysis rate mainly
at the beginning of the reaction, the existence of this kind of
inhibition was studied as an additive effect to product inhibition.
Substrate inhibition assays were performed at the two enzyme
concentrations (1.35 and 13.5 EU mL−1 chestnut purée) by mea-
suring the initial reaction rates with different chestnut concen-
trations. 300 g L−1 was the highest value assayed due to the
pastes high viscosity. Despite of the complexity of this system,
which is composed by two enzymes where one of them produces
substrates for the other, a typical profile of substrate inhibition
for a single enzyme and substrate system was obtained for the
lower enzyme concentration assayed (1.35 EU mL−1 ; Fig. 2A).
Therefore, experimental points were acceptably fitted to Hal-
dane’s model (1) to obtain operational empirical parameters
(vm = 17.4 g L−1 min−1 ; Km  = 194.5 g L−1 ; K  = 0.010 L g−1 ;
2
s
r = 0.954):
vm S
v=  + S + K S 2
(1)
Km s
where v and vm are, respectively, initial and maximal initial
reaction rates, S is the substrate concentration, and Km and K 
s
represent operational Michaelis–Menten’s and inhibition con-
stants for the enzymes mixture in chestnut purée.
Although this inhibition model is based on an uncompetitive
mechanism due to the formation of an inactive enzyme–substrate
complex involving two molecules of substrate [19], the high vis-
cosity of the concentrated chestnut purées at the beginning of
the process, when the initial reaction rate was measured, could
cause diffusional restrictions contributing to the substrate inhi-
bition effect, as described for systems with glucoamylase and
starch that also fit to Haldane’s model [1–3]. Considering the
liquefying action attributed to ␣-amylases, glucoamylases must
 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258 255

Fig. 3. Initial rates of chestnut purée hydrolysis at different concentrations of glucose as inhibitor and different purée concentrations with: (A) the amylases mixture
(total amylolytic activity: 4.95 EU mL−1 ), (B) ␣-amylase (amylolytic activity: 1.73 EU mL−1 ) and (C) glucoamylase (amylolytic activity: 3.22 EU mL−1 ). Symbols
represent the experimental points for the different concentrations of glucose: (A) () 0 g L−1 , () 3 g L−1 , (
) 5 g L−1 and (♦) 8 g L−1 ; (B) () 0 g L−1 , () 2 g L−1 ,
(
) 4 g L−1 , (♦) 6 g L−1 and () 8 g L−1 ; (C) () 0 g L−1 , () 2 g L−1 , (
) 6 g L−1 and (♦) 9 g L−1 . Solid lines represent the fittings to Michaelis–Menten’s model
with: mixed competitive and non competitive product and substrate inhibition (model 4; A and C) and only substrate inhibition (model 1; B).

be more affected than ␣-amylases by substrate inhibition when plot was not clear enough to decide the type of product inhibition.
this is, at least, partially due to diffusional restrictions. Considering the enzymes mixture as a sole enzyme to simplify
When the higher enzyme concentration ratio was applied the system, all the experimental results were fitted to competitive
(13.5 EU mL−1 ; Fig. 2B), the inhibitory profile disappeared (2) and non competitive (3) product inhibition models, which
and a good fit to Michaelis–Menten’s model without inhibi- were modified to include an additional term for substrate inhi-
tion was obtained (vm = 50.8 g L−1 min−1 ; Km  = 194.5 g L−1 ; bition.
2
r = 0.991). This behaviour supports the hypothesis of mass vm S
transfer limitations as a cause of the substrate inhibition pro- v=
 (2)
file observed for the low enzyme concentration. In effect, it is

Km 1 + KI + S + Ks S 2
iC
reasonable to suppose that the higher amount of ␣-amylase at the
vm S
higher enzymes mixture concentration was enough to decrease v=
 (3)
medium viscosity and reduce diffusional restrictions.  + S + K S 2 ) 1 + I
(Km s 
KiNC
In any case, it is expected that ␣-amylase activity, which
decreases sharply the starch paste viscosity, will reduce quickly where I is the inhibitor (glucose) concentration, and KiC  and

substrate inhibition at the lower enzyme concentration along the 

incubation.
3.3. Product inhibition
To verify the existence of glucose inhibition on hydrolysis
at the low enzyme/substrate ratio, inhibition assays were per-
formed with the amylases mixture as described in Section 2.3.
Results shown in Fig. 3A indicate the existence of glucose
inhibition at these low concentrations, but the Lineweaver–Burk
KiNC are operational competitive and non competitive inhibition
constants, respectively.
Acceptable values of the parameters and regression coeffi-
cients were obtained in both cases, the non competitive inhibition
constant being slightly higher than the competitive one (Table 1).
The difficulty for assigning inhibition by glucose to com-
petitive or non competitive mechanisms could be attributed to
applying one enzyme inhibition models to a mixture composed
by two amylases working simultaneously, with different reaction
mechanisms. To clarify the glucose inhibitory effect on the amy-

Table 1
Operational kinetic constants for the enzymes mixture at low concentration and for each enzyme assayed separately as described in the text, calculated according to
models 1–4 based on Michaelis–Menten’s model considering substrate and two types of product inhibition
Type of inhibition Model vm (g L−1 min−1 ) Km (g L−1 ) KiC (g L−1 ) KiNC (g L−1 ) Ks (L g−1 ) r2

α-Amylase
Substrate 1 10.0 210.0 – – 0.0022 0.976
Glucoamylase
Substrate + product (C) 2 13.0 148.5 11.4 – 0.0048 0.986
Substrate + product (NC) 3 13.2 168.7 – 27.2 0.0039 0.979
Substrate + product (Mm) 4 12.9 148.4 12.3 454.8 0.0047 0.991
Amylases mixture
Substrate + product (C) 2 17.6 182.4 9.0 – 0.0044 0.987
Substrate + product (NC) 3 17.5 198.1 – 17.1 0.0031 0.980
Substrate + product (Mm) 4 18.5 194.5 9.7 319.9 0.0048 0.986

C: competitive; NC: non competitive; Mm: mixed model (competitive and non competitive).
256 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258
lases mixture, new product inhibition experiments were done.
Each enzyme was separately assayed in the same concentration
as in the mixture (␣-amylase: 1.73 EU mL−1 ; glucoamylase:
3.22 EU mL−1 ; Fig. 3B and C). Inhibition was only observed
for glucoamylase in this range of glucose concentrations. The
adjustments to the modified competitive (2) and non competi-
tive (3) inhibition models gave acceptable regression coefficients
(Table 1), but the two types of inhibition could not be distin-
guished.
This apparently confusing situation could be explained con-
sidering the glucoamylase reaction mechanism, based on the
presence of seven subsites in the active centre, which are the
joining points for the glucose monomers of the polysaccha-
ride, and the catalytic site between subsites 1 and 2. There
are different theories that postulate the initial union of one
substrate monomer to subsites 1 or 2 and the posterior reor-
ganisation of the enzyme–substrate complex to occupy all the
subsites [20]. The existence of different points for the interaction
substrate–enzyme which have different effect on the viability of
the complex could justify competitive inhibition when glucose
joints to the first union subsite (being 1 or 2 depending on the
theory), and non competitive inhibition when glucose binds to
one of the other subsites, making difficult the correct formation
of the enzyme–substrate complex. According to this supposi-
tion, Matsumura et al. [21] have described the implication of
two subsites in glucoamylase inhibition by glucose. In addition,
the existence of mixed inhibition mechanisms (competitive and
non competitive) has been yet described for other enzymes as a
human kynureninase [22].
Taking it into account, the results for glucoamylase were
again fitted to a mixed model (4) including simultaneously
competitive and non competitive product inhibition as well as
substrate inhibition, and the adjustment was better than for mod-
els 2 and 3 (Table 1).
vm S
v=

 
 (4)
 I I
Km 1+ 
KiC
+ S + Ks S 2 1+ 
KiNC
Applying this mixed model, the non competitive inhibition con-
stant was clearly higher than for the competitive inhibition,
Fig. 4. Amylolytic activity (AA), expressed as percentages referred to the initial value (1.35 EU mL−1 ), of an ␣-amylase and glucoamylase mixture (respective
ratio: 0.35/0.65) along the incubation at 70 ◦ C in: (A) 50 mM citric–phosphate buffer pH 4.75 and (B) 225 g L−1 buffered chestnut purée. Symbols represent the
experimental points; dashed and solid lines represent, respectively, the fittings to a first order model and model 6.
which means the small contribution of this kind of inhibition at
these low glucose concentrations. This behaviour is in agreement
with the preceding hypothesis considering the higher number of
non joining substrate subsites (from 3 to 7).
When comparing the calculated operational Michaelis–
Menten’s constant Km  for glucoamylase with other Michaelis–
Menten’s constants reported in the literature for this amylase,
it must be considered that this parameter is referred to chest-
nut purée concentration as an operational substrate. Km  was
then calculated with respect to starch taking into account that
starch content in chestnut is ∼30%. The new Km  value thus
−1
calculated (∼45 g L ) was higher than Km values for the same
enzyme with soluble starch (0.7354 g L−1 at 23 ◦ C [3]) and other
glucoamylases (0.8 g L−1 at 70 ◦ C for Thermomyces lanugi-
nosus glucoamylase [23]; 0.40 g L−1 at 60 ◦ C for Thermomucor
indicae-seudaticae glucoamylase [24]). Nevertheless, these val-
ues increases as the substrates are less adequate for this enzyme
(2.4 g L−1 in raw starch at 60 ◦ C for Thermomucor indicae-
seudaticae glucoamylase [24]; 6.5 g L−1 in maltose at 70 ◦ C
for Thermomyces lanuginosus glucoamylase [23]; 18 g L−1 in
molasses at 67 ◦ C for Rhizopus glucoamylase [25]), which
suggests that complex substrates as chestnut purée starch or
molasses are less suitability than starch to be hydrolyzed by
this amylase.
Mixed model 4 was applied to calculate the operational
kinetic parameters for the enzymes mixture in this system and
a good fitting was obtained (Table 1). Although this equa-
tion is referred to initial reaction times, it was also used to
estimate hydrolysis rates along all the incubation period for
the kinetic assay (Fig. 1) at low enzyme concentration. Glu-
cose concentrations at every sampling time were taken as
inhibitor concentrations in the model. After 48 h reaction, the
estimated hydrolysis rate was around 40% the rate if no glu-
cose was present. This percentage could be even lower if sub-
strate consumption along the process is considered. Therefore,
that low value points out to glucose inhibition as an impor-
tant reason for the decrease of the amylases mixture ability
to hydrolyze the starch present in 225 g L−1 chestnut purée
at the lower enzymes concentration. Anyhow, this effect is
not strong enough to explain the low hydrolysis percentage
 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258
Table 2
Parameters and regression coefficients corresponding to the fitting of the experimental values to thermal deactivation models 5 and 6 for the glucoamylase alone and
the enzymes mixture incubated at low concentration at 70 ◦ C in chestnut purée
Enzyme Model AA␣ (%) a ka (min−1 )
Glucoamylase 5 – 78.4 0.029
Enzymes mixture 6 26.1 57.5 0.020
obtained in these conditions at the end of the incubation time
(Fig. 1).
3.4. Thermal deactivation
Thermal deactivation of the ␣-amylase and glucoamylase
mixture at low enzyme concentration was studied in presence
and absence of substrate (buffered chestnut purée and only
buffer). Results (Fig. 4) showed a strong decrease of the enzy-
matic activity in both cases, being higher when the amylases
mixture was incubated in buffer, as it was expected considering
the favourable effect of the substrate on enzymatic thermal sta-
bility. Anywise, the loss of activity in chestnut purée was higher
than 60% after 3 h of incubation. Therefore, thermal deactiva-
tion appears to be an important cause of the incomplete reaction
at the lower enzyme concentration assayed.
As expected when more than one enzyme is present, results
corresponding to both assays of enzymatic thermal stability in
chestnut purée and buffer did not fit to a first order deactivation
kinetics (Fig. 4). Before proposing an empirical model for the
thermal deactivation of the mixture, each enzyme was assayed
separately to study their kinetics in chestnut purée. Fig. 5 shows
that glucoamylase was the only responsible for the thermal deac-
tivation of the mixture, while the ␣-amylase kept 100% of the
initial activity at the end of the incubation in these conditions.
Neither glucoamylase deactivation followed a first order
kinetics. These behaviour could be explained by the presence of
two different forms of this enzyme in the commercial prepara-
Fig. 5. Amylolytic activity (AA), expressed as percentages referred to the initial
values (0.47 EU mL−1 for the ␣-amylase; 0.88 EU mL−1 for the glucoamylase),
for the ␣-amylase () and the glucoamylase () incubated alone at 70 ◦ C in
225 g L−1 buffered chestnut purée. Symbols represent the experimental points;
dashed and solid lines represent, respectively, the fittings to a first order model
and model 5.
257
b kb (min−1 ) S1 S2 r2
21.6 0.000 – – 0.995
1.7 0.000 0.010 0.029 0.998
tion, as described by other authors [26,27], with different thermal
stabilities. Taking it into account, a parallel biexponential model
based on that proposed by Aymard and Belarbi [28] for the math-
ematical formulation of enzymes mixtures deactivation kinetics
was applied to the experimental data:
AAgt = ae−ka t + be−kb t , (5)
where AAgt represents total glucoamylase activity at time t
expressed as percentage referred to the initial amylolytic activ-
ity, and a and b, and ka and kb are, respectively, pre-exponential
parameters and first order constants for each glucoamylase form
in the commercial enzyme.
As Table 2 shows, fitting provided a good regression coeffi-
cient and suggested the presence of one thermolabile majority
and other thermostable minority enzyme.
A complete model also based on Aymard and Belarbi’s one
was finally proposed for the ␣-amylase and glucoamylase mix-
ture considering the thermal behaviour of each enzyme. This
model was modified to include a synergistic effect between these
two enzymes, described by Fujii et al. [17] as a consequence of
the production of optimal substrates for the glucoamylase by the
action of ␣-amylase on starch.
AAt = AA␣ + ae−ka t + be−kb t + S1 AA␣ (ae−ka t )
+S2 AA␣ (be−kb t ), (6)
where AAt represents the total amylolytic activity for the mix-
ture at time t expressed as percentage referred to the total initial
activity, AA␣ is the ␣-amylase activity expressed as percentage,
and S1 and S2 are the synergism terms between the ␣-amylase
and each glucoamylase form.
As Table 2 shows, good fittings were obtained. When model 6
was applied to the enzymes mixture incubated in buffer without
substrate, no fitting of the amylolytic activity experimental val-
ues was obtained. This different behaviour of the same enzymes
in different media could be related to other components of chest-
nut different from starch (proteins mainly), which could interact
with the enzymes affecting their stability and kinetics. This
hypothesis, as well as the presence of two glucoamylase forms
and their thermal stability will be studied in a next paper.
4. Conclusions
Complete hydrolysis of chestnut purée is achieved in a sole
step if the concentration of an optimal mixture of a thermostable
␣-amylase and glucoamylase is high enough to counteract
enzymes inhibition and thermal deactivation in the operational
conditions. The enzymes mixture was affected by substrate and
product inhibition, the latter described by a simultaneous glu-
258 C. López et al. / Enzyme and Microbial Technology 39 (2006) 252–258
cose competitive and non competitive model according to the
glucoamylase mechanism of reaction. The biphasic exponential
thermal decay kinetics of the glucoamylase suggested the pres-
ence in AMG 300L of two glucoamylase forms with different
thermostability. The empirical model developed to describe the
enzymes mixture thermal deactivation kinetics in chestnut purée
support the presence of these two glucoamylase forms and the
existence of a synergistic effect between the ␣-amylase and each
glucoamylase form.
Acknowledgements
We thank Novo Nordisk A/S and Marron Glace (Ltd.) for the
supply of enzymes and chestnuts, respectively. FEDER Project
IFD97–0020–C02–02 provided the financial support for this
work.
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