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Characterization of Crude Thermostable Exoinulinase Produced by Saccharomyces sp

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Studies were carried out to investigate the properties of extracellular inulinase produced by Saccharomyces sp. in solid state fermentation of wheat bran. The pattern of inulin hydrolysis by the crude inulinase enzyme as revealed by thin layer chromatography analysis of the products of hydrolysis showed the inulinase to be exo-acting. Temperature and pH studies revealed that the inulinase preparation had an optimum activity of 125.4 units per gram of dry substrate (U/gds) at 50ºC while an optimum inulinase activity of 128.3 U/gds was observed at pH 4.5. The crude inulinase preparation retained 82% of its activity at 50ºC after heating for two hours. Effect of various thermal stabilizers on the activity of the crude inulinase showed that glycerol had the best thermal stabilizing effect on the activity of the crude inulinase preparation. Potassium and calcium ions were found to enhance the activity of the enzyme while mercury and silver ions completely inhibited the inulinase activity. The crude inulinase preparation had a V max and K m values of 416 and 1.47 mM, respectively.
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AU J.T. 16(2): 81-88 (Oct. 2012)
Research Paper
81
Characterization of Crude Thermostable Exoinulinase
Produced by Saccharomyces sp.
Emmanuel Oluwaseun Garuba and Anthony Abiodun Onilude
Department of Microbiology University of Ibadan
Ibadan, Oyo state, Nigeria
E-mail: <oluwaseungaruba@live.com; aa.onilude@ui.edu.ng>
Abstract
Studies were carried out to investigate the properties of extracellular inulinase
produced by Saccharomyces sp. in solid state fermentation of wheat bran. The pattern
of inulin hydrolysis by the crude inulinase enzyme as revealed by thin layer
chromatography analysis of the products of hydrolysis showed the inulinase to be exo-
acting. Temperature and pH studies revealed that the inulinase preparation had an
optimum activity of 125.4 units per gram of dry substrate (U/gds) at 50ºC while an
optimum inulinase activity of 128.3 U/gds was observed at pH 4.5. The crude inulinase
preparation retained 82% of its activity at 50ºC after heating for two hours. Effect of
various thermal stabilizers on the activity of the crude inulinase showed that glycerol
had the best thermal stabilizing effect on the activity of the crude inulinase preparation.
Potassium and calcium ions were found to enhance the activity of the enzyme while
mercury and silver ions completely inhibited the inulinase activity. The crude inulinase
preparation had a V
max
and K
m
values of 416 and 1.47 mM, respectively.
Keywords: Fermentation, wheat bran, temperature, pH, thermal stability.
glycerol.
Introduction
Inulin is a polydispersed fructan
consisting m  -(21)-D-fructosyl-
fructose links terminated by a sucrose residue
(De Leenheer 1996). It is reported to be the
next abundant polysaccharides after starch
(Kango and Jain 2011), found in many plants
of compositae and gramineae and also
accumulated in the underground roots and
tubers of several plants including Vernonia
herbacea, Jerusalem artichoke (Helianthus
tuberosus), chicory (Cichorium intibus,
Cichorium endivia), and dahlia (Dahlia
pinnata) where it acts as storage
polysaccharide (Gupta and Kaur 1997). Inulin
from various sources are reported to vary in
there degree of polymerization (Chi et al.
2011). Furthermore, the degree of
polymerization is reported to be a function of
the harvesting period, and storage time after
harvesting (Chi et al. 2011). Inulinasesare
-2,1
linkage of inulin, resulting in the formation of
fructose, glucose and inulooligosaccharides
depending on the pattern of action of inulinase
(Onilude et al. 2012). The inulinases have
become the cynosure of researchers primarily
due to its application in the production of high
fructose syrup (Garca-Aguirre et al. 2009;
Mutanda et al. 2009; Ricca et al. 2009) and
fructooligosaccharides (Yun et al. 2000;
Mutanda et al. 2008; Risso et al. 2012) via the
hydrolysis of inulin. Inulinases have also been
reported to be useful in the production of
ethanol via the fermentation of the fructose
produced during the hydrolysis of inulin
(Nakamura et al. 1996; Zhang et al. 2010a and
b), acetone and butanol, pullulan exopoly-
saccharide, gluconic acid and sorbitol, and
alsosingle-cell oil and single-cell protein
production (Chi et al. 2011). Although
inulinases from various organisms have been
reported (Uzunova et al. 2001; Zherebtsov et
al. 2002; Sharma et al. 2006; Singh et al. 2007;
Yuan and Bai 2008; Mazutti et al. 2010), only
a few of these organisms have been able to
AU J.T. 16(2): 81-88 (Oct. 2012)
Research Paper
82
produce sufficient inulinases which fulfil the
desired characteristics of high temperature
optimum and thermal stability (for the
successful application of inulinases in the
various industries)hence, the need to screen for
as well isolate new inulinase-producing
microorganism that can meet the various
inulinase demand of the various inulinase
utilizing industries. Onilude et al. (2012)
reported inulinase production by a newly
isolated Saccharomyces sp. in solid state
fermentation using wheat bran as substrate.
This paper reports the characteristic of the
crude inulinase produced by this
Saccharomyces sp. in solid state fermentation
of wheat bran.
Materials and Methods
Microorganism
The inulinase-producing Saccharomyces
sp. used in this study was obtained from the
Culture Collection Centre of The Department
of Microbiology University of Ibadan. The
organism was previously isolated from
spontaneously fermented sugar-cane (Onilude
et al. 2012). It was sub-cultured on yeast
extract-peptone-sucrose (YPS) agar medium
containing (g/l) yeast extract 2.5, peptone 5.0,
sucrose 15.0 and agar 20.0 incubated at 30°C
for 48 hours.
Inoculum Preparation
Inoculum was prepared by transferring 1-
ml cell suspension of 24 hold culture of the
organism into a liquid medium (100 ml)
containing (g/l) sucrose (20.0), yeast extract
(5.0), K
2
HPO
4
(5.0), NH
4
Cl (1.5), KCl 1.15),
and MgSO
4
7H2O (0.65), in an Erlenmeyer
flask as described by Mazutti et al. (2006).
Flasks were incubated at 35°C and 150 rpm for
24 hours and 2 ml of this was used as the
inoculum.
Solid State Fermentation
Solid state fermentation was carried out
as described by Onilude et al. (2012) using
wheat bran as substrate. The wheat bran was
inoculated with a 2-ml suspension of the
inoculum above. The moisture content of the
wheat bran was adjusted to 65% (w/v) while
the pH was adjusted to 5.5. Incubation was
done at 35ºC for 72 hours after which the
inulinase produced was extracted as described
by Mazutti et al. (2006).
Assay of Inulinase
Inulinase activity was determined
according to Burkert et al. (2006). Crude
enzyme extract (0.1 ml) was incubated at 50°C
for 15 min with 0.9 ml of 0.1 M sodium acetate
buffer (pH 5.5) containing 2% inulin.
Thereafter, the enzyme was inactivated by
keeping the reaction mixture at 90°C for 10
min. The reaction mixture was then assayed for
glucose as a reducing sugar using the DNSA
method (Miller 1959). Absorbance of the
reaction mixture was measured using a Jenway
Spectrophotometer at 540 nm. One unit of
inulinase activity was defined as the amount of
     
fructose per minute under standard assay
conditions.
Pattern of Inulin Hydrolysis
Pattern of hydrolysis of inulin (whether
exo- or endo-acting) by the crude inulinase
extract was determined by spotting the reaction
product (after incubating inulin and the crude
inulinase for 2 hours at 50
o
C) on a pre-coated
TLC plate (Merck Germany). The plate was
developed with a solvent system of chloroform:
acetic acid: water (30:35:5 v/v/v) at room
temperature and sugars were visualized by
heating the plate at 120
o
C for 10 min after
      -naphtol
(containing 10% phosphoric acid) acid as
described by Azhari et al. (1989).
Determination of Optimum pH and pH
Stability of the Crude Inulinase
The effect of pH on the activity of the
crude enzyme was determined by incubating
0.1 ml of the crude enzyme with 0.9 ml of
inulin substrate preparation at pHs between
3.0-7.0 using 0.1 M sodium acetate for pH 3.0-
5.5, citrate phosphate buffer for pH 6.0-6.5,
sodium phosphate buffer for pH 6.5-7.0 and
thereafter measuring the reducing sugar
liberated as stated above (Chen et al. 2009).
The pH stability of the enzyme was also
studied by incubating the crude enzyme at
AU J.T. 16(2): 81-88 (Oct. 2012)
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83
different pHs followed by incubation with
inulin substrate and assaying for reducing sugar
liberated afterwards.
Determination of Optimum Temperature
and Thermostability of the Crude Inulinase
The optimum temperature for the activity
of the crude inulinase was determined by
incubating 0.1 ml of the enzyme with 0.9 ml of
the substrate mixture at different temperature
ranges (40, 45, 50, 55, 60, 65 and 70°C) for 15
min at pH 4.5 and the liberated reducing sugar
measured as described above. Thermal stability
of the enzyme was also studied by incubating
the crude enzyme at the various temperatures
(40, 50, 60 and 70ºC) for 2 hours and assaying
for the residual inulinase activity afterwards.
Effect of Thermal Stabilizers on the Crude
Enzyme Activity
The effect of various thermal stabilizers
on the activity of the enzyme was done as
described by Gill et al. (2006) by incubating
the enzyme with 10% w/v of stabilizers
(ethylene glycol, sorbitol, glycerol, dextran) at
70ºC for 3 hours. Then the residual activity was
estimated by incubating 0.1 ml of the recovered
enzyme in each experiment with 0.9 ml of 2%
inulin substrate in 0.1 M sodium acetate buffer
at pH 5.5 and the amount of released reducing
sugars was estimated. In all experiments, each
set up was replicated thrice.
Effect of Various Metal Ions and Other
Chemicals on the Activity of the Inulinase
The effect of various metal ions on
inulinase activity was investigated by
incubating 0.1 ml of the crude enzyme solution
with 0.9 ml of the inulin containing 2 mM of
K
+
, Fe
2+
, Mg
2+
, Ca
2+
, Mn
2+
, Al
3+
, Cu
2+
, Zn
2+
and Ag
+
in 0.5 M sodium phosphate buffer (pH
7.0) at 50 °C for 30 min, then assaying for
residual enzyme activity.
Also, the effects of 2 mM EDTA and 10
mM urea on inulinase activity were determined
by incubating 0.1 ml of the crude enzyme
solution with 0.9 ml of inulin containing EDTA
and urea at 50 °C for 30 min, then assaying for
the residual enzyme activity as previously
stated.
Kinetic Parameters of the Crude Inulinase
For the investigation of the kinetic
parameters of the crude inulinase preparation,
0.4 U/ml of the crude inulinase preparation was
incubated with the inulin concentration ranging
from 0.2×10
-4
to 4.0×10
-4
M as described by
Zang et al. (2004) and the inulinase activity
measured for each of the substrate
concentrations as described earlier. The K
m
and
V
max
values were determined by the method of
Lineweaver-Burk.
Results and Discussion
The results of the pattern of inulin
hydrolysis by the crude inulinase preparation
from Saccharomyces sp. is shown in Fig. 1.
Exo-acting inulinase releases fructose as the
main product of hydrolysis while endo-acting
inulinases release mixture of fructose and other
inulooligosaccharides (Kango and Jain 2011).
The absence of any inulooligosaccharides in
the reaction mixture suggests that the inulinase
preparation from this Saccharomyces sp. is
exo-acting. Similar inulinase preparations that
are exo-acting have been isolated and purified
from various inulinase-producing organisms
such as: Kluyveromyces marxianus strain CBS
6556 (Rouwenhorst et al. 1998),
Chrysosporium pannorum AHU 9700 (Xiao et
al. 1989); Aspergillus fumigatus (Gill et al.
2006); Penicillium sp. TN-88 (Moriyama et al.
2002); and Aspergillus awomari (Arand et al.
2002). The exo-acting nature of this inulinase
makes it of potential importance in the food
industries where it can be used in the
production of ultra high fructose syrup.
The results of the effect of pH on activity
of the crude inulinase preparation presented in
Fig. 2 showed that the optimum pH was 4.5
with an activity of 128.3 U/gds while the
lowest activity of 35.3 U/gds was observed at
pH 7.0. Similar values were found in the
literature. Kluyveromycessp. Y-85 maximum
inulinase activity was observed at pH 4.5
(Wenling et al. 1999) while with K. marxianus
DMS 70106, Pessoa and Vitolo (1999)
described a high inulinase activity between pHs
3.2 and 5.0.
AU J.T. 16(2): 81-88 (Oct. 2012)
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84
Fig. 1. TLC analysis showing the pattern of
hydrolysis of inulin by the crude inulinase by
Saccharomyces sp. (Lane 1); sugar standards
(Lane 2).
Fig. 2. Effect of different pH values on the
activity of inulinase produced by
Saccharomyces sp. Data are presented as
mean ± SEM, n = 3.
Kushi et al. (2000) and Ettalibi and
Baratti (2001) obtained maximum inulinase
activity at pH 4.7 for K. marxianus var.
bulgaricus and Aspergillus ficcum,
respectively. Loss of inulinase activity beyond
the optimum pH could be as a result of the
changes in the state of acidic or basic amino
acids in the protein. Changes in pH may also
change the shape or charge properties of the
substrate so that either the substrate cannot
bind to the active site or it cannot undergo
catalysis.
At 50
o
C, the crude inulinase preparation
was stable over a wide range of pH (4.0-7.0)
with the enzyme activity drastically reduced
outside this range and the enzyme almost lost
its activity beyond 3.0 and 9.0 (data not
shown). The results of optimum temperature
determination and thermal stability of the crude
inulinase are presented in Figs. 3 and 4. The
data showed that the crude inulinase
preparation from Saccharomyces sp. had an
optimum temperature of 50
o
C. Optimum
activity at 50
o
C has been reported for inulinase
preparations from various organisms by
Workman and Day (1983) and Cruz-Guerrero
et al. (1995). High inulinase activity at
relatively elevated temperatures is an
interesting factor from the industrial point of
view considering the limited solubility of inulin
at room temperatures (Ettalibi and Baratti
2001; Gill et al. 2006). The crude enzyme
retained 82% (106.49 U/gds) of its activity
after 2 hours of heat treatment (at 50
o
C), 69%
(88.53 U/gds) (at 60
o
C) and 30% (38.49 U/gds)
at (70
o
C) while there was complete inactivation
of inulinase activity at 80
o
C.A good thermal
stability at 50
o
C but not at 60
o
C for the strain
K. marxianus CDBB-L-278 was also described
by Cruz-Guerrero et al. (1995).
Fig. 3. Effect of different temperature values on
the activity of inulinase produced by
Saccharomyces sp. Data are presented as
mean ± SEM, n = 2.
Fig. 4. Thermal stability of inulinase produced
by Saccharomyces sp. Data are presented as
means ± SEM, n = 3.
Fructose
Sucrose
Rafffinose
Origin
1 2
AU J.T. 16(2): 81-88 (Oct. 2012)
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85
Similarly, Gill et al. (2006) reported 76%
residual of activity of inulinase by an inulinase
isoform of Aspergillus fumigatus. Higher
temperature optimum and pH stability by the
inulinase from Saccharomyces sp. could also
be as a result of the fact that the enzyme was
prepared in solid state fermentation (Pandey et
al. 2000). Enzymes with higher optimal
temperature of activity and stability at high
temperatures are attractive for biotechnological
application in various industrial sectors.
The results of the effects of various
thermal stabilizers on the thermal stability of
the crude inulinase (represented in Fig. 5)
showed that glycerol had the best stabilizing
effect with an activity of 106.01 U/gds as
compared to 81.12 U/gds (control experiment).
Similar results have been previously reported
by Öngen-Baysal et al. (1994) and Taylor et al.
(1995). Glycerol having the best stabilizing
effect could be as a result of preferential
exclusion of the polyols with proteins, which
increases with an increasing polyol size (Liu et
al. 2010), resulting in an indirect interactions
that prevent the protein from thermal unfolding
(Taravati et al. 2007).
The effect of various metal ions at 2 mM
concentration on the inulinase activity of the
extracellular inulinase from Saccharomyces sp.
is presented in Table 1. A total loss of activity
was observed in the presence of Hg
+
and Ag
+
while in the presence of K
+
and Ca
2+
, the
inulinase activity increased from 100±0.08
U/gds to 116.8±0.41 U/gds and 104.6±4.20
U/gds, respectively. A reduced activity was
also observed in the presence of Fe
2+
(Table 1).
Workman and Day (1983) reported that various
metal ions affect the activity of enzymes by
either inhibiting or stimulating enzyme activity.
The complete inhibitory effect of Hg
2+
observed in this study has also been reported
earlier (Ettalibi and Baratti 1987; Kochhar et
al. 1997; Sharma et al. 2006).
The inhibitory effect observed could be
due to the presence some thio (-SH) group in
the active site of the enzyme which is
necessary for the catalytic activity of the
inulinase. In this work, EDTA had no
significant effect on enzyme activity. This
suggests that the inulinase preparation is not
dependent on divalent ion as they affect the
catalytic activity of the enzyme as it relates to
divalent cofactors (Guimarães et al. 2007). The
effect of metal ions on enzyme activity may be
relevant when considering the use of substrate
with high salt content (Bhatti et al. 2006). The
kinetic parameters, as investigated by a Line-
weaver Burk plot, revealed that the crude
inulinase had a K
m
and K
cat
of 2.86 mM and
1.71×10
2
s
-1
, respectively. High K
m
and K
cat
values by the inulinase preparation imply a
high affinity for inulin substrate by the enzyme
(Gill et al. 2006).
Fig. 5. Effect of different thermal stabilizers on
inulinase activity produced by Saccharomyces
sp. Data are presented as mean ± SEM, n = 3.
Table 1. Effect of various metal ions on
inulinase activity by Saccharomyces sp.
Metal
ions
Concentration
(mM)
Inulinase
activity (U/gds)
Control
-
119±0.08
Mg
2+
2
84.2±0.29
Cu
2+
2
44.2±0.49
Mn
2+
2
48.5±6.12
Ag
+
2
n.d.
K
+
2
126.8±0.41
Ca
2+
2
134.6±4.20
Fe
2+
2
n.d.
Zn
2+
2
86.3±2.83
Na
+
2
135.2±1.42
Hg
2+
2
n.d.
Urea
10
58.0±0.49
EDTA
2
122.5±0.09
Note: Data are presented as mean ± S.D; n.d. = not
detected; and n = 3.
AU J.T. 16(2): 81-88 (Oct. 2012)
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86
Conclusion
In conclusion, this work presents the
characteristics of crude inulinase preparation
from Saccharomyces sp. The pattern of inulin
hydrolysis by this enzyme, the relatively high
thermal stability and high activity at slightly
acidic pH makes it of potential importance in
the production of fructose from inulin.
However, there is a need to further investigate
the kinetics of inulin hydrolysis by this crude
inulinase preparation in order to develop an
appropriate model the application of the
enzyme in fructose production.
References
Arand, M.; Golubev, A.M.; Brandao Neto,
J.R.; Polikarpov, I.; Wattiez, R.; Korneeva,
O.S.; Eneyskaya, E.V.; Kulminskaya, A.A.;
Shabalin, K.A.; Shishliannikov, S.M.;
Chepurnaya, O.V.; and Neustroev, K.N.
2002. Purification, characterization, gene
cloning and preliminary X-ray data of the
exo-inulinase from Aspergillus awamori.
Biochemical Journal 362(1): 131-5.
Azhari, R.; Szlak, A.M.; Ilan, E.; Sideman, S.;
and Lotan, N. 1989. Purification and
characterization of endo- and exo-inulinase,
Biotechnology and Applied Biochemistry
11(1):105-17.
Bhatti, H.N.; Asgher, M.; Abbas, A.; Nawaz,
R.; and Sheikh, M.A. 2006. Studies on
kinetics and thermostability of a novel acid
invertase from Fusarium solani. Journal of
Agricultural and Food Chemistry 54(13):
4,617-23.
Burkert, J.F.M.; Kalil, S.J.; Filho, F.M.; and
Rodrigues, M.I. 2006. Parameters
optimization for enzymatic assays using
experimental design. Brazilian Journal of
Chemical Engineering 23(2): 163-70.
Chen, H.-Q.; Chen, X.-M.; Li, Y.; Wang, J.;
Jin, Z.-Y.; Xu, X.-M.; Zhao, J.-W.; Chen,
T.-X.; and Xie, Z.-J. 2009. Purification and
characterization of exo- and endo-inulinase
from Aspergillus ficuum JNSP5-06. Food
Chemistry 115(4): 1,206-12.
Chi, Z.-M.; Zhang, T.; Cao, T.-S.; Liu, X.-Y.;
Cui, W.; and Zhao, C.-H. 2011.
Biotechnological potential of inulin for
bioprocesses. Bioresource Technology
102(6): 4,295-303.
Cruz-Guerrero, A.; Garcia-Peña, I.; Barzana,
E.; Garcia-Garibay, M.; and Gomes-Ruiz, L.
1995. Kluyveromyces marxianus CDBB-L-
278: a wild inulinase hyperproducing strain.
Journal of Fermentation and Bioengineering
80(2): 159-63.
De Leenheer, L. 1996. Chapter 4. Production
and use of inulin: industrial reality with a
promising future. In: van Bekkum, H.;
Röper, H.; and Voragen, A.G.J. (eds.).
Carbohydrates as Organic Raw Materials.
Vol. 3. VCH Publishers Inc., New York,
NY, USA, pp. 67-92.
Ettalibi, M.; and Baratti, J.C. 1987.
Purification, properties and comparison of
invertase, exoinulinases and endoinulinases
of Aspergillus ficuum. Applied
Microbiology and Biotechnology 26(1): 13-
20.
Ettalibi, M.; and Baratti, J.C. 2001. Sucrose
hydrolysis by thermostable immobilized
inulinases from Aspergillus ficuum. Enzyme
and Microbial Technology 28(7-8): 596-601.
Garca-Aguirre, M.; Senz-lvaro, V.A.;
Rodrguez-Soto, M.A.; Vicente-Magueyal,
F.J.; Botello-lvarez, E.; Jimenez-Islas, H.;
Crdenas-Manrquez, M.; Rico-Martnez,
R.; and Navarrete-Bolaos, J.L. 2009.
Strategy for biotechnological process design
applied to the enzymatic hydrolysis of agave
fructo-oligosaccharides to obtain fructose-
rich syrups. Journal of Agricultural and
Food Chemistry 57(21): 10,205-10.
Gill, P.K.; Manhas, R.K.; and Singh, P. 2006.
Purification and properties of a heat-stable
exoinulinase isoform from Aspergillus
fumigatus. Bioresource Technology 97(7):
894-902.
Guimarães, L.H.S.; Terenzi, H.F.; Polizeli, M.
de L.T. de M.; and Jorge, J.A. 2007.
Production and characterization of a
thermostable extracellular -D-
fructofuranosidase produced by Aspergillus
ochraceus with agroindustrial residues as
carbon sources. Enzyme and Microbial
Technology 42(1): 52-7
Gupta, A.K.; and Kaur, N. 1997. Fructan
storing plants - a potential source of high
AU J.T. 16(2): 81-88 (Oct. 2012)
Research Paper
87
fructose syrups. Journal of Scientific and
Industrial Research 56(8): 447-52.
Kango, N.; and Jain, S.C. 2011. Production and
properties of microbial inulinases: recent
advances. Food Biotechnology 25(3): 165-
212.
Kochhar, A.; Kaur, N.; and Gupta, A.K. 1997.
Inulinase from Aspergillus versicolor: a
potent enzyme for producing fructose from
inulin. Journal of Scientific and Indusrial
Research 56(12): 721-6.
Kushi, R.T.; Monti, R.; and Contiero, J. 2000.
Production, purification and characterization
of an extracellular inulinase from
Kluyveromyces marxianus var. bulgaricus.
Journal of Industrial Microbiology and
Biotechnology 25(2): 63-9.
Liu, F.-F.; Ji, L.; Zhang, L.; Dong, X.-Y.; and
Sun, Y. 2010. Molecular basis for polyol-
induced protein stability revealed by
molecular dynamics simulations. Journal of
Chemical Physics 132(22): 225,103-11.
Mazutti, M.; Bender, J.P.; Treichel, H.; and Di
Luccio, M. 2006. Optimization of inulinase
production by solid-state fermentation using
sugarcane bagasse as substrate. Enzyme and
Microbial Technology 39(1): 56-9.
Mazutti, M.A.; Skrowonski, A.; Boni, G.;
Zabot, G.L.; Silva, M.F.; de Oliveira, D.; Di
Luccio, M.; Filho, F.M.; Rodriguez, M.I.;
and Treichel, H. 2010. Partial
characterization of inulinases obtained by
submerged and solid-state fermentation
using agroindustrial residues as substrates: a
comparative study. Applied Biochemistry
and Biotechnology 160(3): 682-93.
Miller, G.L. 1959. Use of dinitrosalicylic acid
reagent for determination of reducing sugar.
Analytical Chemistry 31(3): 426-8.
Moriyama, S.; Akimoto, H.; Suetsugu, N.;
Kawasaki, S.; Nakamura, T.; and Ohta, K.
2002. Purification and properties of an
extracellular exoinulinase from Penicillium
sp. strain TN-88 and sequence analysis of the
encoding gene. Bioscience, Biotechnology,
and Biochemistry 66(9): 1,887-96.
Mutanda, T.; Wilhelmi, B.S.; and Whiteley,
C.G. 2008. Response surface methodology:
synthesis of inulooligosaccharides with an
endoinulinase from Aspergillus niger.
Enzyme and Microbial Technology 43(4-5):
362-8.
Mutanda, T.; Wilhelmi, B.; and Whiteley, C.G.
2009. Controlled production of fructose by
an exoinulinase from Aspergillus ficuum.
Applied Biochemistry and Biotechnology
159(1): 65-77.
Nakamura, T.; Ogata, Y.; Hamada, S.; and
Ohta, K. 1996. Ethanol production from
Jerusalem artichoke tubers by Aspergillus
niger and Saccharomyces cerevisiae.
Journal of Fermentation and Bioengineering
81(6): 564-6.
Öngen-Baysal, G.; Suha Sukan, .; and
Vassilev, N. 1994. Production and
properties of inulinase from Aspergillus
niger. Biotechnology Letters 16(3): 275-80.
Onilude, A.A; Fadahunsi, I.F.; and Garuba
E.O. 2012. Inulinase Production by
Saccharomyces sp. in solid state
fermentation using wheat bran as substrate.
Annals of Microbiology 62(2): 843-8.
Pandey, A.; Soccol, C. R.; Nigam, P.; and
Soccol, V.T. 2000. Biotechnological
potential of agro-industrial residues. I:
sugarcane bagasse. Bioresource Technology
74(1): 69-80.
Pessoa, A., Jr.; and Vitolo, M. 1999. Inulinase
from Kluyveromyces marxianus: culture
medium composition and enzyme
extraction. Brazilian Journal of Chemical
Engineering 16(3): 1-14.
Ricca, E.; Calabrò, V.; Curcio, S.; and Iorio, G.
2009. Fructose production by chicory inulin
enzymatic hydrolysis: a kinetic study and
reaction mechanism. Process Biochemistry
44(4): 466-70.
Risso, F.V.A.; Mazutti, M.A.; Treichel, H.;
Costa, F.; Maugeri, F.; and Rodrigues, M.I.
2012. Comparison between systems for
synthesis of fructooligosaccharides from
sucrose using free inulinase from
Kluyveromyces marxianus NRRL Y-7571.
Food and Bioprocess Technology 5(1): 331-7.
Rouwenhorst, R.J.; Visser, L.E.; van der Baan,
A.A.; Scheffers, W.A.; and van Dijken, J.P.
1998. Production, distribution, and kinetic
properties of inulinase in continuous
cultures of Kluyveromyces marxianus CBS
6556. Applied and Environmental
Microbiology 54(5): 1,131-7.
AU J.T. 16(2): 81-88 (Oct. 2012)
Research Paper
88
Sharma, A.D.; Kainth, S.; and Gill, P.K. 2006.
Inulinase production using garlic (Allium
sativum) powder as a potential substrate in
Streptomyces sp. Journal of Food
Engineering 77(3): 486-91.
Singh, R.S.; Sooch, B.S.; and Puri, M. 2007.
Optimization of medium and process
parameters for the production of inulinase
from a newly isolated Kluyveromyces
marxianus YS-1. Bioresource Technology
98(13): 2,518-25.
Taylor L.S.; York, P.; Williams, A.C.;
Edwards, H.G.; Mehta, V.; Jackson, G.S.;
Badcoe, I.G.; and Clarke, A.R. 1995.
Sucrose reduces the efficiency of protein
denaturation by a chaotropic agent.
Biochimica et Biophysica Acta 1,253(1):
39-46.
Taravati, A.; Shokrzadeh, M.; Ebadi, A.G.;
Valipour, P.; Hassan, A.T.M.; and Farrakhi
F. 2007. Various effects of sugar and
polyols on the protein structure and
function: role as osmolyte on protein
stability. World Applied Sciences Journal
2(4): 353-62.
Uzunova, K.; Vassileva, A.; Kambourova, M.;
Ivanova, V.; Spasova, D.; Mandeva, R.;
Derekova, A.; and Tonkova, A. 2001.
Production and properties of a bacterial
thermostable exo-inulinase. Zeitschrift für
Naturforschung C 56(11-12): 1,022-28.
Wenling, W.; Huiying, W.W.L.; and Shiyuan,
W. 1999. Continuous preparation of fructose
syrups from Jerusalem artichoke tuber using
immobilized intracellular inulinase from
Kluyveromyces sp. Y-85. Process
Biochemistry 34(6-7): 643-6.
Workman, W.E.; and Day, D.F. 1983.
Purification and properties of the beta-
fructofuranosidase from Kluyveromyces
fragilis. Federation of European
Biochemical Societies (FEBS) Letters
160(1-2): 16-20.
Yuan, W.; and Bai, F. 2008. Optimization of
medium and process parameters for the
production of inulinase from Kluyveromyces
marxianus Y1. Journal of Biotechnology
136S: S317-8.
Yun, J.W.; Park, J.P.; Song, C.H.; Lee, C.Y.;
Kim, J.H.; and Song, S.K. 2000. Continuous
production of inulo-oligosaccharides from
chicory juice by immobilized endoinulinase.
Bioprocess Engineering 22(3): 189-94.
Zhang, T.; Chi, Z.; Chi, Z.; Parrou, J.L.; and
Gong, F. 2010a. Expression of the inulinase
gene from the marine-derived Pichia
guilliermondii in Saccharomyces sp. W0
and ethanol production from inulin.
Microbial Biotechnology 3(5): 576-82.
Zhang, T.; Chi, Z.; Zhao, C.-H.; Chi, Z.-M.;
and Gong, F. 2010b. Bioethanol production
from hydrolysates of inulin and the tuber
meal of Jerusalem artichoke by
Saccharomyces sp. W0. Bioresource
Technology 101(21): 8,166-70.
Zherebtsov, N.A.; Shelamova, S.A.; and
Abramova, I.N. 2002. Biosynthesis of
inulinases by Bacillus bacteria. Prikladnaya
Biokhimiya i Mikrobiologiya 38(6): 634-8
(in Russian).
... This was done as described by Garuba and Onilude (2012). Crude extracellular inulinase produced the fungal isolates were incubated at 65°C in the absence of inulin and thereafter the residual activity measured as described above. ...
Article
Full-text available
In this study, attempts were made to isolate and characterized inulinase-producing thermophilic fungi from waste dump site located at Awota-Apete, Ibadan, SouthWestern Nigeria. The thermostability of the inulinase produced was also investigated by incubating inulinase produced at 65 °C for three hours and thereafter measuring the residual inulinase activity. Eight different fungi capable of growing on inulin agar at 50 °C were isolated. They were identified as Aspergillus flavus, A. tamarii, A. parasiticus, A. niger, A. oryzae, Rhizopus sp., Penicillium citrinum and Neurospora sp. using cultural, microscopic and the sequence of the Internal Transcribed Spacer regions 1 and 2 (ITS1/ITS2) of the fungal genome. Thermostability studies on the produced inulinase revealed that inulinase from A. tamarii-INU4 retained 13.7U/ml of its activity after incubation at 65°C for 3 hr. This was followed by extracellular inulinase produced by A. flavus-INU2 with 5.4U/ml residual activity after incubation at 65°C for 3hrs. Extracellular inulinase by Penicilium citrinum had the lowest residual activity of 0.2U/ml after incubation at the same condition. Conclusively, this paper reports potential sources of thermostable inulinase-a property which is necessary for the complete hydrolysis of inulin by inulinases in the production of fructose and fructo-oligosaccharides.
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