Original paper
KINETIC AND THERMODYNAMIC PARAMETERS OF
THE THERMOSTABLE XYLANASE PRODUCTION BY
Rhizomucor pusillus
Armando Robledo1, Cristóbal N. Aguilar1, Ruth E. Belmares-Cerda1, Juan C. Contreras-Esquivel1, Mario
.
A. Cruz-Hernández2 and Julio C. Montañéz-Sáenz3
1
Food Research Department. School of Chemistry. Universidad Autónoma de Coahuila. Unidad Saltillo.
Blvd. Venustiano Carranza PO BOX 252. ZIP 2500. Coahuila, México.
2
Food Science & Technology Department. Universidad Autónoma Agraria Antonio Narro. Unidad Saltillo.
Blvd Antonio Narro 1923. Col. Buenavista. ZIP 25315. Coahuila, México.
3
Chemical Engineering Department. School of Chemistry. Universidad Autónoma de Coahuila. Unidad
Saltillo. Blvd. Venustiano Carranza e Ing. José Cárdenas s/n. ZIP 25280. Coahuila, México.
Corresponding author at: Department of Chemical Engineering, School of Chemistry, Universidad
Autónoma de Coahuila, Saltillo, 25280, México. Tel.: +52 844 4161238; fax: +52 844 4159534.
E-mail: julio.montanez@uadec.edu.mx
1
ABSTRACT
A thermostable extracellular xylanase from Rhizomucor pusillus was produced using a 120 h solid-state
culture and exhibited maximal enzyme activity at pH 6.0 and 70 °C. Thermal inactivation of the pure
enzyme followed first-order kinetics. The activation and inactivation energies were 14.73 and 356.95
kJ/mol, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the xylanase was
highly thermostable. This is the first report on the thermodynamic parameters of xylanase produced by Rh.
pusillus.
Keywords: Xylanase, solid state fermentation, thermodynamics, Rhizomucor pusillus.
2
thermodynamic and kinetic parameters of the
extracellular xylanase produced by Rhizomucor
pusillus strain in solid substrate fermentation
(SSF).
1.
INTRODUCTION
Xylanases are group of enzymes mainly
consisting of endoxylanase (EC 3.2.1.8) which
primarily cleaves β-1,4 linked xylan backbone and
β-xylosidase (EC 3.2.1.37) which converts
xylooligomers to monomeric xylose sub-unit
(Garai and Kumar 2013). These hydrolytic
enzymes are produced by a variety of
microorganisms including bacteria, yeast and
filamentous fungi (Lakshmi et al. 2009; Garai and
Kumar 2013). Xylanases have a wide industrial
application in the conversion of lignocellulose to
sugars such as xylitol a food sweetener and
furfural which is used in the plastic industry
(Ncube et al. 2012). Other potential applications
include the beer and juice clarification; improving
digestibility of animal feed; achieve the complete
saccharification of lignocellulosic biomass for
ethanol production as an alternative fuel (Michelin
et al. 2012); improving nutritional value in bread
making (Kapilan and Arasaratnam 2011) among
others.
2.
MATERIALS AND METHODS
2.1. Microorganism and inoculum
Rh. pusillus SOC-4A isolated from corn
cob silage (Robledo et al. 2014) was from the
Food Research Department collection (access
code HM999962.1) of the Universidad Autónoma
de Coahuila, México. The strain was growth on
potato dextrose agar (PDA) and incubated at 50 ºC
for 7-day-old. Stock cultures were maintained on
potato dextrose agar at 4 ºC and routinely cultured.
2.2. Xylanase production
The xylanase production was carried out
in tray bioreactors, each containing 3 g of corn cob
(CC) used as substrate and support, and 11 mL of
minimum Czapek-Dox culture medium (g/L):
7.65, NaNO3; 3.04, KH2PO4; 1.52, MgSO4▪7H2O;
1.52, KCl. Milled CC was sieved to obtain an
homogeneous particle size of 1.0 mm. To be used
as carrier, the materials were pre-treated by
boiling for 10 min, washed three times with
distilled water, and subsequently dried at 60 ºC for
48 h to constant weight (Mussatto et al. 2009).
The pH was adjusted to 6.0 before sterilization.
After sterilization at 121°C for 15 min, each tray
was inoculated with fresh fungal spores
(1x107/gram of dry support, referred as: gds) and
the plates were incubated at 55 °C for 7 days.
Samples were obtained each 24 h and xylanase
activity was measured.
In the last decade, several industrial
process have been developed in order to use the
agro-industrial and agricultural residues in solid
substrate
fermentation
(SSF),
minimizing
pollution and generate an industrial interest due its
high availability and low acquisition costs. SSF is
characterized by growing microorganism on solid
support in absence of free water. In some cases
fungi grow well on solid substrates and produce
large amount of enzymes and metabolites
compared to liquid state fermentation (Garai and
Kumar 2013). Sometimes enzyme produced under
SSF offers better thermo stability than that of
produced by submerged fermentation (Saqib et al.
2010). These process are achieved in bioreactors
where transport phenomena and modeling
microbial growth kinetics needs to be understood
to determine the overall performance of a SSFbioreactor (Mitchell et al. 2012). Mathematical
models represent a convenient, concise, and
powerful way of describing these phenomena and
their interactions and provide a sound foundation
for
process
development,
control,
and
optimization.
2.3. Xylanolitic extract (XE)
After incubation, the fermented material
was diluted with 37 mL of solvent, composed by
NaCl solution (0.9 %) with Tween 80 (0.1 %), in 3
g of cultured material homogenized during 5 min
at 133 rpm (Maciel et al. 2009). Solids were
filtered under vacuum through Whatman n° 01
filter paper, and further filtered through a 0.22 µm
Millipore membrane filter paper, using the clear
supernatant as raw extract (enzyme source). From
the raw extract, total sugar consumption (Dubois
et al. 1956) and biomass content (Robledo et al.
2008) were estimated.
Is well know, that some thermophilic
fungi produce xylan-degrading enzymes and at the
same time, have attracted growing attention
because of their exceptional potential as sources of
thermostable xylanases. These fungi have been
isolated from soil and other vegetal materials non
related to the corn cob. In this paper, we report the
3
equal, such that the biomass profile is symmetrical
by rotation around the transition point. Substrate
consumption can be modeled using a two-term
expression proposed by Pirt (1975) as follows:
2.4. Xylanase activity assay
Xylanase activity was determined by
mixing 0.07 mL of beechwood xylan 1 % (w/v)
prepared in 50 mM acetate buffer pH 6.2 with
0.03 mL of the enzyme source. The enzymesubstrate mixture was incubated at 55 °C for 5
min. The released reducing sugars were
determined by the use of 3,5-dinitrosalicylic acid
(DNS) method (Miller 1959) with xylose as
standard (Bailey M.J. 1992). One unit of xylanase
is defined as the amount of enzyme that liberates 1
µmol of xylose equivalent per minute, under assay
conditions. Each experiment was carried out in
triplicate and results were taken as the mean of
three.
−
𝑑𝑡
= 𝜇𝑚𝑎𝑥 ∙ 𝑋 ∙ (1 −
𝑋𝑚𝑎𝑥
)
1
𝑌𝑋⁄𝑆
∙
𝑑𝑋
𝑑𝑡
+𝑚∙𝑋
(3)
𝑋−𝑋0
𝑌𝑋⁄𝑆
−
𝑚∙𝑋𝑚𝑎𝑥
𝜇𝑚𝑎𝑥
𝑋𝑚𝑎𝑥 −𝑋0
∙ ln [
𝑋𝑚𝑎𝑥 −𝑋
]
(4)
Kinetics of product formation can be
modeled using the Luedeking and Piret (1959)
equation as follows:
𝑑𝑃
𝑑𝑡
= 𝑌𝑃⁄𝑋 ∙
𝑑𝑋
𝑑𝑡
+𝑘∙𝑋
(5)
Where P is the product concentration YP/X
the product yield in terms of biomass (units of
product per unit of biomass) and k the secondary
coefficient of product formation or destruction.
Here the coefficient k can be negative, zero, or
positive, since product formation or destruction is
not necessarily related to growth. Again it is
possible to solve Eq. (6) as a function of biomass:
𝑃(𝑡) = 𝑃0 + 𝑌𝑃⁄𝑋 ∙ (𝑋 − 𝑋0 ) +
𝑘∙𝑋𝑚𝑎𝑥
𝜇𝑚𝑎𝑥
∙ ln [
𝑋𝑚𝑎𝑥−𝑋0
𝑋𝑚𝑎𝑥 −𝑋
]
(6)
Regarding the effect of temperature, most
attention has been given on the maximal growth
rate (µmax) and inactivation rate constant (k).
Simple empirical equations are normally fitted by
least squares regression to growth rate data
collected between the minimum and maximum
growth temperatures. Several expressions have
been used to describe the variation in specific
growth rate with temperature (Mitchell et al.
2012). Thermal inactivation of the enzyme was
determined by incubating the raw extract at
particular temperatures. Aliquots were withdrawn
at different times, cooled on ice-water bath for 2 h
and assayed for enzyme activity under the
standard conditions. This procedure was repeated
at five different temperatures ranging from 70 to
(1)
The integrated form of this equation, used
for performing model fitting and parameter
estimation in this study, is presented in equation
(2)
𝑋(𝑡) = 𝑋𝑚𝑎𝑥 ⁄1 − ((𝑋𝑚𝑎𝑥 − 𝑋0 )⁄𝑋0 ) ∙ 𝑒 −𝜇𝑚𝑎𝑥∙𝑡
=
𝑆(𝑡) = 𝑆0 −
Based on the data collected during
fermentative process with Rh. pusillus SOC-4A, a
series of mathematical models (Equations (1)–(6))
were developed to describe the correlation
between substrate consumption and biomass and
xylanase production. Several kinetic equations
with varying complexity, such as linear,
exponential, logistic and power-law logistic
models, have been suggested by researchers to
describe fungal biomass growth during cultivation
involving solids (Mitchell et al. 2012; Shi et al.
2012). Among these, the logistic equation
describes a limitation on growth, and for this
reason has quite commonly been incorporated into
models of growth in SSF (Mitchell et al. 2012).
The model describes growth in microbial
population as a function of maximum biomass
density (Xmax), specific growth rate (µmax), and
time (t) as described in equation (1).
𝑋
𝑑𝑡
Where S is the substrate concentration
(measured as total sugars in g per gds), YX/S the
biomass yield coefficient (g X/g S) and m the
maintenance coefficient (g S/g X·h). Solution of
equation (3) can be obtained as a function of X as
follows:
2.5. Thermodynamic and kinetic parameters
calculation
𝑑𝑥
𝑑𝑆
(2)
This kinetic model describes early
acceleration followed by deceleration, with the
rates of acceleration and deceleration being nearly
4
90 °C. The activation energy was estimated by the
Arrhenius equation expressed in logarithmic
terms.
ln𝑘 = ln𝐴 + (
−𝐸𝑎
𝑅
1
1
𝑇
𝑇𝑟𝑒𝑓
)∙( −
)
Natural materials used in SSF as both
substrate and support, have several carbon sources
that can be employed by the microorganism for
growth. Figure 1 depicts the biomass estimation
behavior in the solid state media containing corncob as a support-substrate.
(7)
60
Biomass (mg/gds)
Where k is the first order rate constant for
inactivation, A is the pre-exponential factor, Ea is
the activation energy, R is the universal gas
constant and T and Tref are the experimental and
reference temperatures. The slope (-Ea/R) and the
intercept (lnA) were obtained by linear regression.
The thermodynamic data were calculated by
rearranging the Eyring absolute rate equation to
study the overall thermodynamic parameters in the
temperature range of 70–90 °C.
50
40
30
20
10
0
0
24
48
72
96 120 144
Time (h)
𝑘=
𝑇∙𝑘𝐵
ℎ
∙e
∆𝑆∗
𝑅
−∆𝐻∗
𝑅∙𝑇
∙e
Figure 1. Biomass estimation
production by Rh. pusillus in SSF
(8)
Equation (8) can be linearised as:
𝑘
𝑘
ln ( ) = ln ( 𝐵) +
𝑇
∆𝑆 ∗
ℎ
𝑅
−
∆𝐻 ∗
𝑅
∙
1
𝑇
xylanase
Microorganism
showed
a
characteristically lag phase between the first 24 h,
with a diauxic phase (48 – 72 h) and an
exponential growth until the maximum values of
33 mg/gds. The characteristic diauxic phase is
present in processes where various carbon sources
are used, like in a natural complex matrix, where
the microorganism adaptation and assimilation of
the easily metabolizable sugars is generated. Saha
(2003) reported for corn-cob values of 45 %
cellulose, 35 % hemicellulose and 15 % lignin,
and microorganism will tend to employ the greater
energy efficiency provider with less requirements
to metabolize (Parés and Juárez 1997; RodríguezFernández et al. 2011). As shown in Figure 1,
Logistic equation (Eq. 1) predicted the fungal
biomass with quite precision (R2 > 0.95) during
the fermentative process. The low value of µmax
(0.10 /h) in comparison with reported by other
authors (Hernández-Rodríguez et al. 2009) implies
that the cell growth is related with the enzyme
production, in order to degrade the substrate
present in the media.
(9)
where k, T, kB, h, ΔS*, ΔH* and R are
inactivation rate constant, absolute temperature,
Boltzmann constant, Planck’s constant, entropy of
activation, enthalpy of activation and gas constant,
respectively.
∆G = ∆H − T∆S (10)
2.6. Data analysis
All the SSF experiments were conducted
by triplicate and the average values are reported.
Data were analyzed using an analyses of variance
(ANOVA) procedure, comparing treatment means
using the Tukey’s range procedure (p ≤ 0.05) in
Minitab® 16.1.0. In order to achieve the best fit of
the experimental data, model fitting and parameter
estimation was performed by minimizing the sum
square error between experimental and modelpredicted values, using the nonlinear least-squares
method provided by Polymath® software 6.0.204.
3.
during
Under non-optimized conditions xylanase
production (Figure 2) was initiated at 24 h and
reached the highest yield of enzyme activity at 96
h with 18 U/gds, no showing statistical difference
compared to 48 and 72 h. Complexity of the
substrates allow the synthesis of several enzymes
from the microorganism, capable to hydrolyze the
polymeric matrix. Further fermentation to 96 h did
RESULTS AND DISCUSSION
3.1. Kinetics analysis of xylanase production
5
not increase enzyme activity values. Azad et al.
(2013) reported the same fermentation time to
obtain the highest enzyme activity using Rh.
pusillus BPJ-2 on wheat bran, were they reached
0.085 U/mL at 4th fermentation day.
important to distinguish whether productivity is
based on a very productive strain (high specific
production rate qP) or simply because biomass is
produced in large quantities.
The enzyme activity is related to the
concentration of total sugars in the medium, which
are present as two major carbon sources, cellulose
and hemicellulose. Figure 3 shows a decrease of
total sugar content from 24 h, showing a
correlation with the growth lag phase of 0-24 h
(Figure 1), and to the estimation of enzymatic
activity (Figure 2) to remain without variation
from 48 h until the end of the evaluation of
fermentation.
25
Product (U/gds)
20
15
10
5
0
24
48 72 96
Time (h)
120 144
Total sugars content
(mg/gds)
0
Figure 2. Time course of xylanase production by Rh.
pusillus on SSF.
Luedeking and Piret (1959) model was
used to analyze the behavior of the enzyme
production and yield to growth relation. We know
that it is possible to predict the production and
stability of the enzyme in the system with constant
k value. If k is < 0, the system presented a convex
fermentation curve, which indicated that the
secondary specific rate of enzyme instability can
be related to the rate of enzyme production
associated to the vegetative growth of the
microorganism. From the production point of
view, a positive value of k helps to identify
fermentation conditions in which the excreted
enzyme activity will be stable in the fermentation
broth and may be a significant factor for better
enzyme productivity. In this case, the xylanase
production appears to be associated to the fungal
growth due that the value of product yield (YP/X =
2.66 U/mg X) was greater than k (-0.1010)
suggesting that the enzyme production is
necessary, first for the degradation of the substrate
and consequently achieve the microorganism
growth. Similar behavior were reported by Shi et
al. (2012) when they obtain negative k values for
the
cellulase
production
Phanerochaete
chrysosporium observing that the enzyme
production resembled multiple phases due to the
heterogeneous and recalcitrant nature of substrate.
Zhao et al. (2010) observed that a high YP/X value
indicates a higher contribution of the growth of
individual cells to the specific metabolite
production. On a different point of view, it is
120
100
80
60
40
20
0
0
24
48 72 96 120 144
Time (h)
Figure 3. Total sugar consumption during xylanase
production by Rh. pusillus in SSF
This behavior suggests the presence of
enzymes that degrade the complex matrix, and
release monomeric sugars that may be used for
metabolic functions of the organism. Azad et al.
(2013) reported a strain of Rh. pusillus with low
cellulolytic activity on CMC-agar while the
xylanolytic activity was much greater using
Xylan-agar media. Somkuti (1974) suggested that
the strains of Mucor (Rhizomucor) may exhibit
cellulolytic activity as long as it is induced.
Table 1 gives the list of the kinetic
parameters obtained in the production of xylanase
by Rh. pusillus in SSF. Major maintenance
coefficient values correspond to the application of
energy to carry out endogenous processes, where
the growth stops and the biomass remains
constant. The biomass needs to consume energy
and substrate to maintain its viability and to
realize its basic metabolic activities like
respiration, secondary metabolisms., turnover of
proteins and active transport (Pirt 1975;
Rodríguez-Fernández et al. 2011).
6
Table 1. Principal kinetic parameters for the xylanase
production by Rh. pusillus on corn cob.
Parameter (Units)
kJ/mol, and thermal inactivation of xylanase
formation was 356.95 (Figure 4). Both values
differ from those reported by literature for
mesophilic organism (138.9 – 177 kJ/mol,
activation and inactivation respectively) (Bokhari
et al. 2010). Lower activation energy for product
formation may be considered as parameter for
thermostable enzymes.
Value
-1
Specific growth rate [µmax] (h )
0.1040
Biomass yield [YX/S] (mg X/mg S)
0.1172
Product yield [YP/X] (U/mg X)
2.6648
Specific production rate [qP] (U/mg
0.2773
X.h)
Specific substrate uptake rate [qS]
0.8875
(mg X/mg S.h)
Maintenance coefficient [ms] (g
-0.3200
S/g X.h)
Product coefficient [k]
-0.1010
0.00
Ln (k)
-1.00
-2.00
-3.00
-4.00
-5.00
The obtained value of YP/X suggests that
xylanase production is growth-associated. While
the negative value of k indicates the apparent
degradation of the xylanase; which means that the
degradation of the xylanase can be considered
first-order with respect to the biomass
concentration
-6.00
2.70 2.75 2.80 2.85 2.90 2.95
1/T x 1000 (1/K)
Figure 4. Arrhenius plot for activation energy
determination.
3.2. Thermodynamics of xylanase production
Values of thermodynamic parameters
were calculated from figures 5 and 6 and are
showed in table 2. The xylanase had 354 kJ/mol
ΔH (inactivation) and 712 J/mol ΔS (inactivation)
values. These values are far from those reported in
literature (111.13 kJ/mol for enthalpy and 502.17
J/mol for entropy) (Bokhari et al. 2010) when
analyze a xylanase from Thermomyces
lanuginosus.
Enzymes used in biotechnology processes
generally suffer denaturation at temperatures over
55 °C, resulting in poor efficiency of hydrolysis
and requiring the use of large amounts of enzyme.
The advantage of using thermostable enzymes,
resides in carrying out the hydrolysis at high
temperatures. Therefore, it is important to
understand the mechanisms of enzyme
inactivation and denaturation. This can be
explained either by the degradation being
controlled by a reversible reaction step, or by the
metabolites resulting from the thermal degradation
exhibiting some light absorbance at that
wavelength. Thermal inactivation occurs in two
steps, as shown below:
Ln (Residual activity)
N↔U→I
1.0
(11)
0.0
-1.0
-2.0
-3.0
-4.0
-5.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Time (min)
Where N is the native enzyme, U is the
unfolded enzyme which could be reversibly
refolded upon cooling, and I is the inactivated
enzyme formed after prolonged exposure to heat
and therefore cannot be recovered on cooling
(Iqbal et al. 2003).
Figure 5. Temperature stability of the xylanase from
Rh. pusillus
Activation energy (Ea) for thermal
activation of xylanase formation was 14.73
7
According to the findings of this study,
the xylanase activity showed thermodynamic
evidence to be a thermostable enzyme, able to
keep activity around temperatures of 65 – 85 °C.
The investigation of the thermal inactivation at a
temperature ranging from 70 to 80 °C showed a
first-order kinetics model. The xylanase
production by Rhizomucor pusillus required less
thermal energy for activation of production
process. The lower values obtained for activation
energy and change in enthalpy of denaturation
suggest that the enzyme is highly stable towards
thermal denaturation. The activity/stability
correlation has recently become a subject of
debate. This work presented evidence that the
enzymes can be active and thermostable at the
same time.
Ln (k/T)
0.00
2.70 2.75 2.80 2.85 2.90 2.95
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00
1/T x 1000 (1/K)
Figure 6. Arrhenius plot between ln(kd/T) and 1/T to
calculate overall ΔH (enthalpy of deactivation) and ΔS
(entropy of deactivation)
Table 2. Kinetic and thermodynamic parameters for
irreversible thermal inactivation of xylanase from Rh.
pusillus.
T
Kd
(°K) (1/min)
t1/2
(min)
ΔH
(kJ/mol)
ΔG
(kJ/mol)
ΔS
(J/mol)
343
0.009
75.33
11.9
110
-285
348
0.030
22.95
11.8
111
-285
353
0.320
2.17
11.8
113
-286
358
0.354
1.96
11.7
114
-287
363
0.278
2.49
11.7
116
-287
5.
ACKNOWLEDGEMENTS
The author ROBLEDO wish to thank
CONACYT (Consejo Nacional de Ciencia y
Tecnología) for their financial support.
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hydrophobic interactions, with a concomitant
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4.
CONCLUSIONS
8
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