Protein & Peptide Letters, 2009, 16, 1098-1105
1098
Invertase from Hyper Producer Strain of Aspergillus niger: Physiochemical Properties, Thermodynamics and Active Site Residues Heat of Ionization
Habibullah Nadeem, Muhammad Hamid Rashid*, Muhammad Riaz, Bibi Asma,
Muhammad Rizwan Javed and Raheela Perveen
Enzyme Engineering Lab., National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang
Road, Faisalabad, Pakistan
Abstract: Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyperproducer strain of Aspergillus niger (34.1 U ml-1), along with its physiochemical properties, kinetics and thermodynamics
of stability-function. The Invertase showed great potential for industry as being highly efficient (kcat = 24167 s-1 at 65 °C,
pH 5.0) and stable (half life= 12 h at 56°C).
Keywords: Thermostability, Enthalpy, Entropy, Free energy, Ionization energy.
INTRODUCTION
Invertase (-fructofuranosidase: E.C.3.2.1.26) catalyse
the hydrolysis of sucrose into glucose and fructose. It is one
of the most widely used enzymes in the food industry, especially in the preparation of jams and candies [1], where fructose is preferred over sucrose because it is sweeter and does
not crystallize easily.
Using waste biomass to produce biofuel (ethanol) can
reduce the use of fossil fuels, reduce greenhouse gas emissions and reduce pollution and waste management problems
[2]. Biologically alcohols are produced by the action of microbes and enzymes through the fermentation of sugars.
Ethanol can be used in petrol engines as a replacement for
gasoline; it can be mixed with gasoline to any percentage.
Gasoline with ethanol added has higher octane, hence the
engine can typically burn hotter and more efficiently. Molasses, a sub-product in sugar production is rich in sucrose and
has been used as a carbon source in the production of fructofuranosidase by Saccharomyces cerevisiae [3]. Invertases have high potential to be used in biofuel production
because they have a tendency to enhance the glucose content
in molasses, which is considered to be an excellent ethanol
feedstock.
Invertase has been mainly produced from Saccharomyces
cerevisiae and commercial fungal strains of the Aspergillus
genus, such as A. niger, A. oryzae and A. ficcum [4]. Aspergillus niger is a filamentous, non-pathogenic fungus that
belongs to the “Generally Recognized As Safe (GRAS)”
microbial organisms, which are used for the production of
enzymes [5]. Although there are many reports on isolation
and characterization of invertases from various microbes but
on an average less than 1% of the potential microbes have
*Address correspondence to this author at the Principal Scientist, National
Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang
Road, Faisalabad, Pakistan; Fax: +92-041-2651472;
E-mails: hamidcomboh@gmail.com; mhrashid@nibge.org
0929-8665/09 $55.00+.00
been identified [6]. So the need to isolate and identify organisms, which are either hyper-producers and/or sufficiently
robust to withstand conditions of the intended application
and/or are producers of novel enzymes is highly significant.
This current report deals with the purification, kinetic and
thermodynamic properties of a highly efficient and stable fructofuranosidase from a local, hyper-producing strain of A.
niger. We considered that this biocatalyst has great potential
to be used in the food industry and in biofuel production.
MATERIALS AND METHODS
Microorganism
A pure local strain of Aspergillus niger was obtained
from NIBGE, Faisalabad. The culture was maintained on
potato dextrose agar (PDA) plates and slants as described
earlier [7]. All chemicals were purchased from MP Biomedicals, USA.
Inoculum Preparation & Biomass Estimation
For inoculum preparation a loop full of spores from a 5 to
6 days old A. niger slant was transferred into a 250 ml Erlenmeyer flask containing 50 ml of sterile Vogel’s medium
and 8-10 acid washed glass beads were added. The composition of the growth medium was (gl-1): glucose (20.0), trisodium citrate (2.5), KH2PO4 (5.0), NH4NO3 (2.0), (NH4)2SO4
(4.0), MgSO4.7H2O (0.2), peptone (2.0), microelement solution (10 ml) and vitamin solution (5.0 ml). The pH of the
medium was adjusted to 5.0 using 1M HCl/1M NaOH. The
flask was incubated at 30 °C on an orbital shaker at 150 rpm
for 48h to get a homogenous cell suspension.
The biomass content of the inoculum was estimated on
the basis of absorptiometry (cells turbidity) by measurement
of the light transmitted at 610 nm [8]. The standard curve for
biomass estimation was prepared as follows: 50 ml culture
was harvested and centrifuged at 15,300g at 4 °C for 15
© 2009 Bentham Science Publishers Ltd.
Invertase from Hyper Producer Strain of Aspergillus niger
min. The supernatant was separated and the pellet was resuspended in 0.89% (w/v) saline solution and centrifuged (23 times) as above. Then the pellet was freeze dried and
ground to fine powder with pestle and mortar. Appropriate
stock solutions were made, which were used to prepare the
final stock (1 mg ml-1). Various dilutions of the final stock
(1-10 folds) were made and the optical density (OD) of each
dilution was noted at 610 nm. Distilled water was used as a
blank and calibration curve was plotted between OD and mg
of cells.
Invertase Production
Invertase or sucrase was produced under submerged
growth conditions in 250 ml Erlenmeyer flasks containing
Vogel’s medium with sucrose (2% w/v) as sole carbon
source. Flasks were autoclaved for 15 min at 121 °C, 1.1
kgfcm-2 and inoculated (92 mg cells per 50 ml growth medium). The flasks were incubated at 30±1 °C for 60 hours.
Isolation of Crude Invertase
After 60 hours of cultivation, the crude enzyme was extracted from the growth medium by filtration through Whatman filter paper. The extract was centrifuged at 25,900 g
for 15 min at 4 °C to remove the suspended particles.
Invertase Assay
Activity of the enzyme was determined as described [9].
Briefly, an appropriate amount of invertase was reacted with
1% (w/v) sucrose solution in 50 mM Na-acetate buffer (pH
5.0) at 50 °C for 15 min. The reaction was quenched by placing the tubes in boiling water for 5 min and then cooled immediately in ice. The released glucose was measured using
glucose measuring kit (Fluitest® GLU, Made in Germany).
One unit was defined as the amount of invertase that releases 1 mol of glucose min-1 from sucrose at 50 °C, pH
5.0.
Protein Assay
Protein was estimated as described by Bradford [10] using bovine serum albumin as a standard.
Purification of Invertase
Crude invertase was subjected to a four-step purification
procedure. After optimization of invertase precipitation solid
ammonium sulfate was added bit by bit to the crude enzyme
to make 30% saturation at 0 °C. The invertase solution was
left overnight at 4 °C and then centrifuged at 25,900g for
20 min. The pellet of precipitated proteins was discarded and
more ammonium sulfate was added to the supernatant to
make 70% saturation at 0 °C. The solution was kept overnight at 4 °C and centrifuged as described above. This time,
the supernatant was discarded, whereas the pellet containing
invertase was dissolved in 50 mM Na-acetate buffer (pH 5.0)
and dialyzed over night against distilled water to remove
salts.
The invertase after ammonium sulfate precipitation was
loaded on a Hiload anion exchange (Q-Sepharose) column of
Fast Protein Liquid Chromatography (FPLC) system, using a
Protein & Peptide Letters, 2009, Vol. 16, No. 9
1099
superloop of 50 ml at a flow rate of 2 ml min-1. A linear gradient of NaCl (0-1M) in 20 mM Tris-HCl, pH 7.5 was used
as elution buffer. The fractions containing invertase were
pooled. Solid ammonium sulfate was added to the dialyzed
sample from the Hiload column to a final concentration of 2
M. The enzyme solution was filtered through 0.22 m filter
paper and was applied to a hydrophobic interaction (Phenyl
Superose) column using a superloop of 50 ml at a flow rate
of 1.0 ml min-1. The elution was carried out with a linear
gradient of ammonium sulphate (2-0 M) in 50 mM sodium
phosphate buffer pH 7. Active fractions were collected. The
dialyzed sample after HIC was loaded on a Mono Q column,
using a superloop of 50 ml at a flow rate of 1.0 ml min-1. A
linear gradient of NaCl (0-1 M) in 20 mM Tris-HCl, pH 7.5
was used as an elution buffer. The fractions containing invertase were pooled. The pooled fractions after each purification
step were dialyzed extensively against distilled water at 4 °C
and total enzyme activity and proteins were estimated in the
pools.
Sub-Unit Molecular Mass
The purity of the purified invertase and its sub-unit molecular mass was determined by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as
described [11]. Protein markers of Fermentas (10-200 kDa)
were used as standard. The gel containing different molecular weight markers was stained with 0.1% Coomassie brilliant blue R-250 solution.
The subunit molecular mass of invertase was also confirmed by using 10% SDS denaturing-renaturing PAGE
(SDS-DR-PAGE). The sample was prepared by incubating
purified invertase at 50 °C for 45 min with SDS sample
buffer in the presence of -mercaptoethanol (2% v/v). PAGE
was run at constant voltage (70 V) and the temperature was
controlled (10 °C) by using a refrigerated circulating water
bath. The gel was re-natured to remove the SDS by treating
with 20% (v/v) isopropanol in 50 mM sodium acetate buffer,
pH 5.0 (two washes of 45 min each). Then the gel was immersed in 50 mM sodium acetate buffer pH 5.0 to remove
isopropanol (two changes of buffer each of 45 min). Invertase activity staining was done as follows: The re-natured gel
was over-laid on agarose gel (0.8% w/v) prepared in Naacetate buffer containing sucrose (1.0% w/v) as a substrate.
The gels were incubated at 50 °C for 90 min with continuous
sprinkling of buffer to avoid drying of the gels and after incubation the agarose gel was cut as a replica copy of the renatured SDS-gel, and stained by immersing in the glucose
detection kit solution. A pink-colored activity band appeared
after 15 min of staining.
Optimum Temperature, Activation Energy & Temperature Quotient (Q10)
The optimum temperature and activation energy (Ea) of
invertase were determined by incubating an appropriate
amount of the enzyme in 1% sucrose solution at various
temperatures ranging from 25-70 °C in 50 mM Na-acetate
buffer for 15 min at pH 5.0. The activation energy was calculated by using an Arrhenius plot as described earlier [7]. The
effect of the temperature on the rate of the reaction was expressed in terms of Q10, which is the factor by which the rate
1100 Protein & Peptide Letters, 2009, Vol. 16, No. 9
Nadeem et al.
increases or decreases, due to an increase or decrease in temperature by 10 °C. The Q10 was calculated by rearranging the
equation given by Dixon and Webb [12].
Q10 = Antilog (E10/RT2)
(1)
Where,
(Kd) were determined as described [15, 16]. The energy of
activation for irreversible thermal denaturation Ea(d) was
determined from an Arrhenius plot.
Thermodynamics of irreversible thermal stability of invertase was determined by rearranging the Eyring’s absolute
rate equation derived from the transition state theory [14].
E = Ea = Activation Energy of sucrose hydrolysis
Kd = (kbT/h).e (-H*/RT).e (S*/R)
*
Optimum pH
The effect of pH on sucrose hydrolysis was determined
by assaying the invertase at various pHs ranging from 2-11,
and pH optima were determined at 50, 55, 60 and 65 °C. The
buffers used were as follows: Glutamic acid/HCl (pH 2-2.9),
Na-acetate/acetic acid (pH 3.2-5.3), MES/KOH (pH 5.6-6.5),
MOPS/KOH (pH 6.8-7.4), HEPES/KOH (pH 7.7-8.3), Glycine/NaOH (pH 8.6-9.8) and CAPSO/NaOH (pH 10.1-11.0).
Heat of Ionization (HI) of Active Site Residues
The pKa values of acidic and basic limbs of invertase
active site residues were determined at different temperatures
(50, 55, 60 and 65 °C) as described by Dixon and Webb. The
effect of temperature on the pH optima was determined as
mentioned above. HI was determined by plotting pKa values of active site residues determined at different temperatures against 1/T [12].
HI = -slope 2.303R
(2)
Kinetics & Thermodynamics of Sucrose Hydrolysis
The kinetic constants (Vmax, Km, kcat and kcat/Km) were
determined by incubating fixed amounts of invertase with
varied concentrations of sucrose as a substrate ranging from
0.025 to 1.5% (w/v) at 65 °C, pH 5.0 as described previously
[13].
The thermodynamic parameters for substrate hydrolysis
were calculated by rearranging the Eyring’s absolute rate
equation derived from the transition state theory [14].
kcat = (kbT/h)e(-H*/RT).e(S*/R)
*
H = Ea – RT
(4)
*
G (Free energy of activation) = -RT ln (kcat h/kb.T)
*
*
(3)
*
S = (H - G )/T
(5)
(6)
The free energy of substrate binding and transition state
formation was calculated using the following derivations:
G*E-S (free energy of substrate binding) = -RT ln Ka
(7)
Where, Ka = 1/Km
G*E-T (free energy for transition state formation) =
-RT ln(kcat /Km)
(8)
Thermodynamics of Irreversible Thermal Stability of
Invertase
The enzyme solution in 10 mM Tris-HCl buffer (pH 7)
was incubated at various temperatures (56, 59, 62, 65 °C) in
the absence of substrate. Aliquots were withdrawn at different time intervals, cooled on ice for 30 min and assayed for
-fructofuranosidase (invertase) activity at 50 °C. The data
were fitted to a first order plot and inactivation rate constants
*
(9)
*
H , G , and S of irreversible thermal stability were
calculated by applying Eq. # 4, 5 and 6 with the modifications that in Eq # 4 Ea(d) was used instead of Ea and in Eq #
5 Kd was used in place of kcat.
RESULTS AND DISCUSSION
Production & Purification of Invertase
The local isolate of Aspergillus niger proved to be a hyper producer strain and maximum extra-cellular invertase
production (34.1 U ml-1) under submerged growth conditions
occurred after 60 h of incubation on sucrose (2 % w/v) as
sole carbon source. Bhatti et al. [17] reported 9.9 U ml-1 invertase from Fusarium solani after 96 h of growth on 2 %
molasses. On the other hand, Aspergillus ochraceus produced 12.5 U ml-1 invertase [18], while 2.4 U ml-1 were produced by Aspergillus niger IMI303386 on 2 % sucrose [19].
The invertase of A. niger was purified up to homogeneity
by a four step purification procedure Table (1). The onset
and complete invertase precipitation occurred at 30% and
70% ammonium sulfate saturation at 0 °C, respectively and
invertase was 2.27 fold purified. The invertase was eluted at
440 mM NaCl on FPLC Hiload anion exchange chromatography Fig. (1Ai). Afterwards, the enzyme was purified by
hydrophobic interaction FPLC chromatography using a linear ammonium sulfate gradient (2-0 M) was used. A single
broad peak of invertase was obtained at 850 mM ammonium
sulfate Fig. (1Aii). Further purification was done by FPLC
Mono Q anion exchange chromatography using a linear gradient of 0-1 M NaCl and invertase was eluted at 783.5 mM
NaCl Fig. (1Aiii). Crude invertase was 101 fold purified
with a recovery of 22% and the specific activity of purified
invertase was 28,465 U mg-1 protein (Table 1). The purity of
the enzyme was checked on 10% SDS-PAGE, which was
stained with Coomassie blue and a single band was obtained
Fig. (1B).
Quang et al. [19] purified -fructofuranosidase from Aspergillus niger IMI 303386 up to 50 fold by using a threestep purification procedure and recovery of the enzyme was
42%. A single protein band of invertase was found on 10%
SDS-PAGE and the subunit molecular mass of the enzyme
was 116 kDa, which was also confirmed by doing SDS-DRPAGE and the same result was found Fig. (1B). Sub-unit
molecular mass of the invertase was close to other reported
values i.e.120-130 kDa from Aspergillus niger IMI303386
[19] and 71-111 kDa from Aspergillus niger AS0023 [20].
Three bands of 95, 65 and 37 kDa molecular mass were reported for -fructofuranosidase of A. japonicus MU-2 [21].
The invertase of Aspergillus niger expressed in E. coli displayed a molecular mass of 75 kDa estimated by SDS-PAGE
[22]. A single wide band of -fructofuranosidase from Candida utilis with molecular mass of 150 kDa is also reported
[23].
Invertase from Hyper Producer Strain of Aspergillus niger
Table 1.
Protein & Peptide Letters, 2009, Vol. 16, No. 9
1101
Purification of Invertase from Aspergillus niger Grown on Sucrose Under Submerged Conditions
Total Protein
(mg)
Total Units (U)
Specific Activity
(U mg-1)
Purification
Factor
%
Recovery
Crude enzyme
291.6
82188
282
1.00
100
(NH4)2 SO4 precipitation
98.10
62900
641
2.27
77
Hiload anion exchange column chromatography
12.60
46013
3652
12.95
56
Hydrophobic interaction column chromatography
4.53
31038
6852
24.30
38
Mono Q anion exchange column chromatography
0.641
18246
28465
101
22
Step
All quoted values were after dialysis against distilled water.
Figure 1. A) Fast Protein Liquid Chromatography of invertase; i, Hiload anion exchange chromatography on a Q-Sepharose column using a
0-1 M NaCl gradient ii, Hydrophobic interaction chromatography on a Phenyl Superose column using a 2-0 M (NH4)2SO4 gradient iii, MonoQ anion exchange chromatography using a 0-1 M NaCl gradient.
B) a, 10 % SDS-PAGE of purified invertase of Aspergillus niger. Lane-1: Fermentas protein markers #SM0661 (10-200 kDa); Lane-2: purified invertase. b, activity staining of A. niger invertase applied on 10% SDS-DR-PAGE.
1102 Protein & Peptide Letters, 2009, Vol. 16, No. 9
Nadeem et al.
Effect of pH
Hydrolysis of sucrose by purified invertase at various
pHs ranging from 2–11 was determined at different temperatures (5065 °C), and at 65 °C (temp optimum) the enzyme
exhibited optimal activity in the pH range 2.9-5.6, while
maximum activity was at pH 4.1. Our results agreed with
that reported for -fructofuranosidase from A. niger AS0023.
We found a drift in pH optimum towards the acidic side
when the temperature was lowered below the optimum temperature. The pH optimum at 55 & 60 °C was 3.5, while it
was 2.9 at 50 °C. -fructofuranosidase from A. niger showed
optimal activity at pH 5.5 [24]. Activity of the enzyme was
inhibited rapidly below and above the optimum pH range.
Heat of Ionization (HI) of Active Site Residues
Here we report for the first time about the heat of ionization of the invertase active site residues. The pKa1 and pKa2
of the active site residues controlling Vmax were determined
as described by Dixon and Webb and at 65 °C their values
were 2.6 and 7.0, respectively Fig. (2). The pKa values of
both acidic and basic limbs showed an increasing trend with
the increase in temp. At 50, 55, 60 and 65 °C, pKa1 values
were 1.55, 1.95, 2.35 and 2.6, while pKa2 values were 6.3,
6.4, 6.9 and 7.0, respectively. The heat of ionization for the
acidic limb (HI(AL)) and the basic limb (HI(BL)) of A. niger
invertase was determined as described by Dixon and Webb and
Figure 3. Dixon plot for the determination of Heat of ionization of
A. niger invertase active site residues. Data presented are average
values ± SD of n = 3 experiments.
was equal to 148.58 and 108.68 kJ mol-1, respectively Fig. (3).
Figure 4. Arrhenius plot to determine the effect of temperature on
activity and activation energy for sucrose hydrolysis by invertase of
A. niger. Data presented are average values ± SD of n = 3 experiments.
Figure 2. Dixon plot for the determination of pKa of ionizable
groups of A. niger invertase active site residues. Data presented are
average values ± SD of n = 3 experiments.
Effect of Temperature
The optimum temperature of invertase from A. niger for
hydrolysis of sucrose was found to be 65 °C and its activity
was lost rapidly at 70 °C. -fructofuranosidase with exactly
the same optimum temperature (60–65 °C) was reported in
Aspergillus japonicus [21]. However, -fructofuranosidase
purified from Candida utilis [23] showed optimum activity
at 70 °C and that of Aspergillus niger AS0023 at 55 °C [20],
whereas maximum activity at 50 °C was also reported from
Aspergillus niger IMI303386 [19].
The activation energy (Ea) of invertase for sucrose hydrolysis determined from an Arrhenius plot was 33.76 kJ
mol-1 Fig. (4). The Ea for sucrose hydrolysis by invertase
from Aspergillus niger was 8.32 fold higher than required by
-fructofuranosidase from Fusarium solani, 5.45 fold higher
than -fructofuranosidase isolated from baker’s yeast, while
1.24 fold higher than that from Rhodotorula glutinis
[17,25,26]. This indicates that larger amount of energy was
required to form the activated complex by -fructofuranosidase obtained from A. niger as compared to other reported -fructofuranosidases. The temperature quotient for
the enzyme was 1.01.
Kinetics & Thermodynamics of Substrate Hydrolysis
The Michaelis Menten constant (Km) and Vmax of invertase from Aspergillus niger for sucrose hydrolysis at 65 °C
were determined from a Lineweaver Burk plot and were
equal to 0.117 M sucrose and 12500 U mg-1 protein, respectively Fig. (5). The Km of A. niger invertase for sucrose was
smaller than that of A. japonicus (0.227 M) [21], while was
higher than 0.020 M for Schwanniomyces occidentalis -
Invertase from Hyper Producer Strain of Aspergillus niger
fructofuranosidase [1] and 0.060 M for A. niger fructofuranosidase [27]. This indicated that the invertase
produced by A. niger has higher affinity for sucrose than that
of A. japonicus, while has lower affinity than that of S. occidentalis. The Kcat of A. niger invertase was 24,167 s-1 and
specificity constant (Kcat / Km) was 206553. Extremely high
turn over confirmed that the A. niger invertase was very efficient in sucrose hydrolysis and has great potential for industrial application.
Protein & Peptide Letters, 2009, Vol. 16, No. 9
1103
elevated temperatures in the presence of substrates [29].
Pseudo first order plots Fig. (6) were applied to determine
the extent of thermal inactivation and results regarding thermal inactivation of the invertase are presented in Table (2).
The thermal inactivation of enzyme is accompanied by the
disruption of non covalent linkages, including hydrophobic
interactions, with a concomitant increase in the enthalpy of
activation i.e., H* [7]. The opening up of enzyme structure
is accompanied by an increase in the disorder (randomness)
or entropy of activation (S*) [30]. The same trend is observed by us in case of A. niger -fructofuranosidase between the temperature range of 56-65 °C Table (2). The enzyme was very stable at 56 °C and exhibited half life of 11.9
hours. At higher temp half life decreased sharply i.e.,105 min
at 59 °C, 13 min at 62 °C and just 10 min at 65 °C.
Figure 5. Lineweaver-Burk plot for the determination of kinetic
constants (Vmax, Km) for sucrose hydrolysis at 65°C, pH 5 by invertase of A. niger. Data presented are average values ± SD of n = 3
experiments. The turn over (Kcat) was calculated using the relation:
Vmax/[e]. Invertase concentration [e] in the reaction mixture was
8.621 10-3 H mole.
We could not find any report on the thermodynamic
characterization of microbial invertases. Recently we have
reported thermodynamic properties of sugarcane soluble acid
invertase (SAI) for sucrose hydrolysis [28]. The Gibbs free
energy (G*) of A. niger invertase at 65 °C was very low
(54.77 kJ mol-1) as compared to the sugarcane SAI at 55 °C
(71.2 kJ mol-1). As enzymes speedup the reactions by lowering down the free energy, therefore the A. niger invertase
was very efficient due to its lower G*, which explained that
its transition state (ES*) was spontaneously converted into
the products. Similarly, the change in enthalpy (H*) for
sucrose hydrolysis indicated that the energy required by A.
niger invertase was lower (30.95 kJ mol-1) than that of sugarcane SAI (52.6 kJ mol-1), which explained that the transition state (ES*) of A. niger invertase was more actively
formed. The entropy (S*) of activation of sucrose hydrolysis by A. niger invertase was -70.46 J mol-1 K-1, while S* of
the SAI was -57 J mol-1 K-1, which explained that the higher
activity of A. niger invertase was due to the lowering down
of free energy but was not entropically driven.
Figure 6. Pseudo first order plots for irreversible thermal inactivation of A. niger invertase. The enzyme solution was incubated at
various temperatures (56 to 65 °C) in 10 mM Tris-HCl buffer (pH
7). Data presented are average values ± SD of n = 3 experiments.
Thermal denaturation of enzymes can be considered to
occur in two steps [31] as shown below:
N U*I
Where, N is the native enzyme, U* is the unfolded inactive
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 upon cooling.
Free energy for activation of substrate binding (G*E-S)
and for the formation of transition complex (G*E-T) were 1.16 and -6.61 kJ mol-1, respectively for A. niger invertase,
whereas, 10.8 kJ mol-1 and 2.6 kJ mol-1, respectively were
for the SAI. The lower free energy values for A. niger invertase again provided the evidence for its higher activity.
The activation energy for irreversible thermal denaturation ‘Ea(d)’ of the invertase was determined by applying Arrhenius plot Fig. (7) and Gibbs free energy (G*) for activation of thermal unfolding of enzyme was 99.84 kJ mol-1 .
With an increase in temperature, a slight decrease in free
energy was observed. The enthalpy of activation of thermal
unfolding (H*) of the enzyme at 56 °C was 482.9 kJ mol-1 .
Its value remained almost same upto 65 °C. The entropy of
activation (S*) for unfolding of transition state of the fructofuranosidase was 1.164 kJ mol-1 K-1 at 56 °C, which
was slightly increased at 62 °C Table (2). But the increase or
decrease in entropy was not significant.
Irreversible Thermostability of Invertase
CONCLUSION
Thermostability is the ability of enzyme molecule to resist against thermal unfolding in the absence of substrate,
while thermophilicity is the capability of enzymes to work at
Due to very high catalytic activity of A. niger invertase
(-fructofuranosidase), we considered it as a strong candidate for application in food industry as well as biofuel pro-
1104 Protein & Peptide Letters, 2009, Vol. 16, No. 9
Table 2.
Nadeem et al.
Kinetics and Thermodynamics of Irreversible Thermal Stability of Invertase from Aspergillus niger
Temp
(K)
Kd
(min-1)
t
(min)
H*
(kJ mol-1)
G*
(kJ mol-1)
S*
(kJ mol -1K-1)
329
0.00096
719
482.98
99.84
1.165
332
0.00658
105
482.96
95.47
1.167
335
0.05390
13
482.94
90.50
1.171
338
0.06820
10
482.91
90.68
1.16
Where, Kd = first order rate constant for inactivation, t = half life = 0.693/Kd. Thermodynamic parameters were calculated using equations 4, 5 and 6.
duction from molasses. Especially, it has high potential for
sucrose processing industry to increase sweetness level of
sugar.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Figure 7. Arrhenius plot for determination of activation energy
‘Ea(d)’ for irreversible thermal denaturation of A. niger invertase.
Data presented are average values ± SD of n = 3 experiments.
[15]
[16]
ACKNOWLEDGEMENTS
The work presented is a part of Ph.D. studies of Mr.
Habibullah Nadeem. The project was partly funded by
Higher Education Commission (HEC), Pakistan under the
Indigenous Scholarship Scheme and Pakistan Atomic Energy
Commission. Technical assistance of Mr. Ghulam Ali
Waseer is appreciated.
[17]
[18]
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