World J Microbiol Biotechnol (2014) 30:1063–1073
DOI 10.1007/s11274-013-1525-8
ORIGINAL PAPER
Immobilized Sclerotinia sclerotiorum invertase to produce invert
sugar syrup from industrial beet molasses by product
Refka Mouelhi • Ferid Abidi • Said Galai •
M. Nejib Marzouki
Received: 8 May 2013 / Accepted: 12 October 2013 / Published online: 19 October 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract The fungus Sclerotinia sclerotiorum produces
invertase activity during cultivation on many agroindustrial
residues. The molasses induced invertase was purified by
DEAE-cellulose chromatography. The molecular mass of
the purified enzyme was estimated at 48 kDa. Optimal
temperature was determined at 60 °C and thermal stability
up to 65 °C. The enzyme was stable between pH 2.0 and
8.0; optimum pH was about 5.5. Apparent Km and Vmax for
sucrose were estimated to be respectively 5.8 mM and
0.11 lmol/min. The invertase was activated by b-mercap-
toethanol. Free enzyme exhibited 80 % of its original
activity after two month’s storage at 4 °C and 50 % after
1 week at 25 °C. In order to investigate an industrial
application, the enzyme was immobilized on alginate and
examined for invert sugar production by molasses hydro-
lysis in a continuous bioreactor. The yield of immobilized
invertase was about 78 % and the activity yield was 59 %.
Interestingly the immobilized enzyme hydrolyzed beet
molasses consuming nearly all sucrose. It retained all of its
initial activity after being used for 4 cycles and about 65 %
at the sixth cycle. Regarding productivity; 20 g/l of
molasses by-product gave the best invert sugar production
46.21 g/day/100 g substrate related to optimal sucrose
conversion of 41.6 %.
Ferid Abidi and Galai Said have contributed equally to this work.
R. Mouelhi (&)  F. Abidi  S. Galai  M. N. Marzouki
Laboratory of Protein Engineering and Bioactive Molecules
(LIP-MB), National Institute of Applied Sciences and
Technology, University of Carthage, B. P. 676,
1080 Tunis Cedex, Tunisia
e-mail: mouelhirefka@yahoo.fr
M. N. Marzouki
e-mail: mnmarzouki@yahoo.fr; mn.marzouki@insat.rnu.tn
Keywords Invertase  Purification  Inverts sugar
syrup  Immobilization  Sclerotinia sclerotiorum 
Bioreactor
Introduction
The conversion of industrial waste, in particular sugar
industry by-product, represents an important option for
both the exploitation of energy and the reduction of pol-
lution. Several studies have already shown the ability of
invertase for the valorization of molasses (Shaheen et al.
2011; Ye et al. 2012). Therefore invertase also named (b-
fructofuranosidase, EC 3.2.1.26) which catalyses the
hydrolysis of sucrose into an equimolar mixture of glucose
and fructose known as invert sugar offer an attractive
alternative for molasses exploitation. The enzymatic
activity of invertases has been characterized mainly in
plants (Alberto et al. 2004). Among microorganisms,
Saccharomyces cerevisae (Herwig et al. 2001), Candida
utilis (Belcarz et al. 2002), and Aspergillus niger (Gomez
et al. 2000) have been widely studied. Several invertases
have also been characterized such as those from Asper-
gillus niveus (Guimaraes et al. 2009).Invertase was also
produced from different yeast strains such as C. utilis
(Belcarz et al. 2002) and Rhodotorula glutinis (Rubio et al.
2002). Extensive studies have been done by using synthetic
medium for preparation of invertase while little attention
has been paid to its production from unconventional
inexpensive sources (Shaheen et al. 2011; Guimaraes et al.
2009). Invertase is used for the preparation of invert sugar,
which is largely applied as feed stocks in fermentation
processes and sweetener in food and pharmaceutical
industries.
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Various techniques for improving the production of
invert sugar, including continuous culturing and immobi-
lization cells have been evaluated (Kurup et al. 2005).
Many methods namely adsorption, covalent bonding,
cross-linking, entrapment, and encapsulation are widely
used for immobilization. Among the different immobili-
zation techniques, entrapment in calcium alginate gel has
been one of the most used methods for whole enzyme
entrapment due to its simplicity and non-toxic character
(Haroldo and Hélia 2010). Alginate has been used for the
immobilization of microorganisms but it requires an addi-
tional process for the enzymes immobilization. In this aim
the present study deals with the production, purification
and biochemical characterization of invertase by the fila-
mentous fungus Sclerotinia sclerotiorum using molasses as
substrate. Thus the purpose of this work was to investigate
the continuous process of invert sugar production in bio-
reactor. As well as kinetic and rheological study of
molasses hydrolysis by immobilized invertase was carried
out.
Materials and method
Materials and chemical
Filamentous fungi S. sclerotiorum strain used in this study
was purchased from the collection of the Laboratory of
Plant Protection, National Institute of the Agronomic
Research of Tunisia, and maintained on potato-dextrose-
agar (PDA) slopes. All other chemicals were obtained from
readily available commercial sources.
Growth conditions and screening of invertase
producing fungi
Invertase production was initially achieved by fungi culture
on complete medium potato dextrose broth (PDB). Mycelia
plugs (4 mm diameter) from a 3-dayold PDA culture, were
transferred to 20 ml of PDB and the culture was grown for
3 days at 25 °C, with shaking at 150 rpm, then used to
inoculate liquid basal medium used firstly for screening
hyper-productive fungi of extracellular invertase. The
culture medium for fungal growth and invertase production
used at this stage was composed of 2 g/l (w/v) yeast
extract, 1.0 g/l (w/v) KCl, 0.5 g/l (w/v) Mg(SO4), 6.0 g/l
(w/v) KH2PO4, 14.3 g/l (w/v) K2HPO4, 4.3 g/l (w/v)
NaNO3, 1.3 g/l (w/v) (NH4)2SO4, along with 1 % (w/v)
sucrose as an inducer. To 1 l of this medium we added
1 ml of oligo-elements solution containing (g/l): CoCl2
(2.0); CuCl2 (2.0); MnSO4–H2O (1.6); ZnSO4–H2O (1.4);
CaCl2 (2.0); FeSO4–H2O (5.0) (Smaali et al. 2003). The
initial pH of the medium was adjusted to 5.5 by adding HCl
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10 % (w/v). The medium was sterilized by autoclaving at
120 °C for 45 min. The culture was grown in 500 ml
erlenmeyer flasks containing 100 ml media. Biomass yield
was determined after 12 days incubation at 25 °C under
shaking at 150 rpm. Invertase production was measured at
various intervals. The cell-free supernatant obtained by
filtration and centrifugation was used for the determination
of extracellular invertase activities.
Induction effect of various inducers
The metabolite induction of the enzymes synthesis was
investigated. The fungus was cultivated at 25 °C with
agitation at 150 rpm in erlenmeyer flasks containing
100 ml liquid medium adjusted at pH 5.5 supplemented
with 1 % (w/v) of different invertase inducer substrates
such us molasses, hay, barley bran, straw, wheat bran and
cores of date. The culture liquid obtained after mycelium
removal, as described below was used for invertase activ-
ities determination. Biomass was then measured in myce-
lium fraction.
Preparation of crude enzyme extract
Culture media and cells were harvested after 12 days by
vacuum filtration through Whatman No. 1 filter paper on
Buchner funnel, and centrifuged at 6,000 rpm and 4 °C for
15 min. The supernatant obtained was used as crude
enzyme preparation. Protein concentration was determined
by Bradford method using Bio-Rad kit (Bradford 1976).
Bovine serum albumin (Sigma) was used as protein
standard.
Fungal biomass measurements
Culture media were filtered through pre-weighed Whatman
No. 1 filter paper. The filtrate was centrifuged as described
above to recover mycelium. The mycelium was dried at
80 °C for 24 h and stored in the desiccators’ chamber
before being weighed.
Enzyme assay
Invertase activity was determined by incubating 10 ll of
crude enzyme solution or partially purified ezyme with
490 ll of 0.3 M sucrose in 50 mM acetate buffer (pH 5.5)
at 60 °C for 20 min. One ml of dinitrosalicylic acid reagent
was added and heated for 15 min in a boiling water bath to
stop enzymatic reaction and quantify product. Finally the
absorbance was assayed at 540 nm in spectrophotometer
(Miller 1959). One unit of invertase (IU) is defined as the
amount of enzyme which liberates 1 lmol of glucose/min/ml
under the assay condition.
World J Microbiol Biotechnol (2014) 30:1063–1073
Kinetic analysis
Using soluble sucrose as substrate, kinetic parameters of
purified invertase were investigated in 50 mM sodium
acetate buffer, pH 5.5, at 60 °C. The purified enzyme was
added to 1 ml of reaction mixture containing 0.03 M of
sucrose. The initial kinetic parameters were then deter-
mined and, Michaelis constants (Km) and (Vmax), were
calculated using a Lineweaver–Burk plot.
Enzyme purification: anion exchange chromatography
The extract was loaded onto a DEAE-cellulose chromato-
graphic column (25 cm 9 2.6 cm) equilibrated with
sodium acetate buffer, 25 mM, pH 5.5. DEAE-cellulose
Fast Flow resin used was purchased from Pharmacia
(Uppsala, Sweden). The enzyme was eluted with a linear
salt concentration gradient (NaCl, 0–1 M) in the same
buffer and 3.0 ml fractions were collected at a flow rate of
20 ml/h. The A280 and invertase activity were monitored.
Fractions showing invertase activities were pooled before
electrophoresis analysis and conserved at -20 °C for fur-
ther studies.
PAGE analysis and zymography
After exchange chromatography on DEAE-cellulose active
fractions were applied onto SDS-PAGE and analyzed by
zymography as described below.SDS-PAGE was carried
out as described by Laemmli et al. (1970) in a discontin-
uous system made of 5 % stacking gel and 10 % running
gel by using a Mini-Protein II gel system (Bio-Rad). Pro-
teins bands were stained by silver nitrate method (Blum
et al. 1987).The first step in purifying proteins from poly-
acrylamide gels is to locate the active band of interest in
the gel by zymography. After renaturation of enzyme in gel
with isopropanol and acetic acid; invertase was visualized
by incubating gel in sucrose (1 %) at 37 °C for 30.
Immediately gel was put into the 2, 3, 5 Triphenyl Tetra-
zolium Chloride (TTC) solution and heat for 3 or 4 min
until red colored band corresponding to invertase activity
appeared and then washed with 7.5 % acetic acid. After gel
electrophoresis; we used a clean scalpel to cut off a strip,
invertase is extracted from the gel after the grinding step,
suspension and centrifugation. An aliquot of the superna-
tant may be tested for the presence of purified invertase by
subjecting it to SDS-PAGE.
The SDS low-molecular-weight standard mixture
(Pharmacia) was used in order to determine the apparent
molecular weight of the purified invertase. Molecular mass
markers (25–225 kDa; Sigma-Aldrich, France) were
included and invertase activity was detected in the gels as
previously described.
1065
Influence of temperature on enzyme activity
and stability
Invertase activity was determined at various temperatures
between 50 and 75 °C in 50 mM sodium acetate buffer, for
30 min at pH 5.5, using soluble molasses as substrate and
the dinitrosalicylic acid method as described previously.
The stability was investigated by measuring the residual
activity at 60 °C after invertase incubation for 30 min at
various temperatures between 55 and 65 °C in the presence
or in the absence of substrate.
Influence of pH on enzyme activity and stability
Optimum pH of the invertase was determined at 60 °C with
the standard assay at different pH values using the fol-
lowing buffers: 50 mM citrate–phosphate buffers (pH
2.5–7), 50 mM Tris–HCl buffers (pH 7.5–8.5), and 50 mM
glycine-NaOH buffers (pH 9.5–10.5). pH stability was
studied by measuring residual activity at pH 5.5, 60 °C,
after enzyme incubation at various pH values between 4
and 8 at 25 °C for 24 h.
Effects of metal ions, detergents, and reducing agents
on invertase activity
For determining the influence of metal ions, the invertase
activity was assayed in the standard conditions in the
presence of 2 mM of various metal ions (Co2?, Mn2?,
Fe2?, Cu2?, Zn2? and Mg2); or 100 mM CaCl2; or 4 mM
of the following reagents (SDS, ethylenediaminetetraacetic
acid (EDTA), b-mercaptoethanol (b-ME)) and 5 % (w/v)
Glycerol or sorbitol or 100 mM-1 M NaCl. The activity
assayed without metal ions was considered to be the ref-
erence value (100 %).
The enzyme solution was preincubated at 60 °C for
30 min in the presence of various chemical reagents and
then the residual invertase activity was measured under the
standard assay conditions. Invertase activity assayed
without denaturing agents was considered as control.
Storage stability
The stability of invertase during storage 25 and 4 °C was
investigated. Enzyme preparation was mixed with acetate
buffer 50 mM (pH 5.5) and stored at room temperature and
4 °C. Then aliquots were withdrawn at desired time
intervals to test the remaining activities.
Molasses characteristics
The composition of molasses varies with the variety of
sugar beet, the agro climatic conditions of the region, sugar
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Table 1 Characteristics of beet molasses used in the fermentation invertase. The effluent from the column was collected in
experiments the same flask which served as the product reservoir. A
Parameter value Value flow breaker was installed between the column and the
pump, which prevented the contamination of the feed tank.
Brix (degree) 72
The samples for analyses were taken from the outlet
Density (g/l) 1,290 compartment of column and the total reduced sugar con-
Total sugar, g/kg molasses 836 centrations were monitored during the course of continuous
Glucose (%) 7.59 ± 1.93 hydrolysis. About 70 % of the column was filled with the
Fructose (%) 5.05 ± 0.08 alginate capsules. The extra space was anticipated for bead
Sucrose (%) 50.39 ± 0.20 expansion by the fresh media. A packed-bed bioreactor was
used in continuous mode and closed cycle for hydrolyze of
molasses at 45 °C and pH 5.5. The working volume of the
manufacturing process, handling and storage (Shaheen et al.
reactor prior to packing was 179, 54 ml and the column
2011). Table 1 summarizes the chemical composition of
volume after packing was 25 ml. The hydraulic retention
sugar beet molasses provided by Sugar industry of Tunisia
time for the feed in the reactor column was 154, 91 h. The
(Beja, Tunisia).It contained 50.39 ± 0.20 % sucrose,
concentration of 20 g/l of molasses were used (the corre-
5.05 ± 0.08 % fructose and 7.59 ± 1.93 % glucose.
sponding sucrose concentration were measured to be about
10 g/l).
Enzyme immobilization
Thin layer chromatography (TLC)
The invertase was entrapped in calcium alginate capsules
by dropping 10 ml of aqueous solution of sodium alginate
The hydrolysis products of molasses by immobilized
(2.5 %) containing 20 U invertase (185 U/mg) into 100 ml
invertase in a bioreactor were analyzed after 4 days at
of 0.1 M CaCl2 solution (buffered at pH 5.5). The alginate
45 °C by TLC. Samples were subjected to TLC separation
drops solidified upon contact with CaCl2, forming capsules
that was realized with a silica gel plate (Kiesel gel 60 F254;
(diameter of 5 mm) and thus entrapping invertase. The
E. Merck AG, Darmstadt, Germany). After developing the
spheres were allowed to harden for 30 min and then
products with a solvent system of n-butanol/acetic acid/
washed with acetate buffer to remove excess calcium ions
water (4:1:1, v/v/v), spots were visualized by spraying with
and enzyme. The capsules were stored at 4 °C in acetate
10 % sulfuric acid in ethanol and heating at 120 °C during
buffer until use. Immobilization yield is defined as the ratio
10 min. Sucrose(S), glucose (G) and fructose (F)(Sigma)
of the activity of immobilized enzyme to the activity of the
were used as standards.
free enzyme used. Immobilization was estimated as
follows:
Repeated batch hydrolysis
Immobilization yield (%) ¼ ðTotal enzyme activity 
non - immobilized activityÞ= Total enzyme activity  100:
In repeated batch hydrolysis reactions were performed in
Sucrose 10 % in acetate buffer (50 mM, pH 5.5) was
bioreactor at 45 °C with molasses solution (2 % w/v,
reacted with immobilized invertase (100 capsules having
100 ml) and 15.6 UI of invertase immobilized onto alginate
15.6 IU) or free invertase at 45 °C for 20 min. The reaction
capsules. After each cycle the immobilized enzyme were
mixture was agitated at 450 rev/min with a magnetic stir-
recovered by a simple filtration, washed with acetate buffer
rer. The 10 ll reaction mixture, was added to 990 ll DNS
and used for the next batch run. After reacting at pH 5.5 for
and boiled for 15 min to inactivate the enzyme. The
several hours, the hydrolysate was harvested for assay. The
amount of glucose or fructose formed from sucrose was
immobilized invertase were used for the next batch
determined by TLC. Activity was estimated as follows:
hydrolysis with the same initial molasses (corresponding to
Activity yield ð%Þ ¼ Immobilized activity= 10 g/l sucrose). The experiment was carried out until there
ðTotal enzyme activity  non - immobilized activityÞ was a noticeable reduction in the enzyme activity.
 100:
Rheological measurements and flow characterization
Experimental bioreactor setup
Rheological measurements were carried out using a
The immobilized invertase reactor was a tubular column, Rheotest (VEB-MLW, Werk, Germany) Searle type rhe-
(volume = 20 ml). A peristaltic pump (Heighdolf 5101, ometer, equipped with a coaxial cylinder sensor system
Germany) was used to transfer the molasses solution from (radii ratio of 1.06). The instrument can be operated at 44
a conical flask to the column containing immobilized different speeds, which are changed stepwise with a

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World J Microbiol Biotechnol (2014) 30:1063–1073 1067

a Invertase Activity [ U/ml ] 9 the enzyme activity till the 12th day of incubation. Similar
Sucrose 1%
8 cores of date 1% behavior was reported for extracellular invertase produc-
molasses 1%
7
straw 1%
tion from A. niger (Sirisansaneeyakul et al. 2000) and from
6 barley bran 1%
S. cerevisiae (Mona and Mohamed 2009). Contrasting with
wheat bran 1%
5 hay 1%
data reported for invertase production by A.flavus (Uma
4
3
et al. 2010). High levels of b-d-fructofuranosidase were
2 produced by S. sclerotiorum when sucrose was used as
1 carbon source, compared to those produced by Saccharo-
0 myces cerevisiae (Shafiq et al. 2003).The important
0 2 4 6 8 10 12 14
Time [days] enzyme production from S. sclerotiorum is suitable for
b 100 10 industrial processes and represents an advantage in regards
Wet weight of the other microorganisms already proposed for such

Invertase Activity [U/ml]

80 dry weight 8 purpose.
Weight [ g/l ]

Activity
As for other previously studied fungi species the pro-
60 6
duction of S. sclerotiorum invertase was found to be
40 4
influenced by the concentration of inducer substrate in the
culture medium. Medium containing 1 % sucrose pre-
20 2 sented the highest invertase activity 8.35 U/ml. 0.5 % of
sucrose also permitted synthesis of the enzyme 6.8 U/ml. A
0 0 high concentration of sucrose in the culture medium
repressed production of the enzyme, whereas the use of 2
and 4 % sucrose as inducer allowed depression of invertase
Inducers production 5 and 4 U/ml respectively.
The cultivation of the fungi strain S. sclerotiorum on
Fig. 1 Kinetics production of invertase by the fungus S. sclerotiorum different Tunisian agro industrial wastes without any
with various inducers (a). Determination of the wet and the dry
additive nutriments was investigated for the production of
weight of mycelium and the invertase activity (b)
extracellular invertase after 12 days of growth (Fig. 1a).
The data showed a wide variation in the yield of extra-
selector switch. The speed of the rotating cylinder varied cellular enzyme through the tested carbon sources at 1 %
from 0.028 to 243 rpm. A thermostatic bath containing (1.2–7.3 U/ml). The highest yield of invertase was produced
ethyl alcohol was used to control the working temperature by using 1 % molasses residue as a medium (7.3 U/ml),
within the range 45 °C. Shear stress values at the surface of followed by wheat bran and core of date which showed
the internal cylinder were obtained by multiplying torque lower amounts of invertase (5.1, and 3.4 U/ml respec-
readings by the rheometer constant. The widely known tively). Little yield of extracellular invertase was produced
empirical expression for the stress tensor was used for by using barley bran, while negligible amounts of invertase
describing the flow behavior of sugar syrup. were detected by using hay and straw (0.8 U/ml). In
another case we monitored the biomass and the invertase
activity over time. However the secretion of invertase
Results and discussion presents a different profile according to the inducer. The
kinetic of production showed that the fungus behave on the
Production of invertase onto induced medium different way towards inducers (Fig. 1b).
Molasses is a mixture of sucrose and reducing sugars
Production of invertase by S. sclerotiorum was followed by (RSs) such as glucose and fructose, it has also many
estimating the enzyme activities at different time intervals unidentified components. In order to investigate their
(Fig. 1a). The highest level of invertase activity was obtained abilities to induce invertase, we used it at the same con-
in medium supplemented by 1 % sucrose (8.35 U/ml), under centration on sucrose. 1 % molasses containing 0.5 %
orbital agitation (100 rpm), at 25 °C, for 12 days. The sucrose produced 7.3 U/ml enzymes however sucrose
produced invertase activity was considerably higher than solution at 0.5 % produced lower activity 6.8 U/ml.
A. niger (Gomez et al. 2000), and Saccahromycopsis fib- Therefore the enzyme reaction is activated to some extent
uligera (Gogoi et al. 1998), but significantly lower than by another sugar in molasses such as fructose and glucose.
most from yeast. It can be observed that extracellular From this study, it can be concluded that the molasses can
invertase reached a maximum activity after 12 days culture be more effectively used as a substrate for the production
(1,950 U/g dry weight mycelia), followed by a decline in of enzyme. For this reason, molasses was chosen as the

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a 0,5 9 b
0,0- 1M Nacl 8
0,4 7

Abs at 280nm
6

Activity [U/ml ]
0,3
Abs at 280 nm 5
Activity [U/ml]
4
0,2
3

0,1 2
1
0 0
1
5
9
13
17
21
25
29
33
37
41
45
49
53
57
61
65
69
73
Fractions

Fig. 2 DEAE-cellulose chromatography profile of invertase (a). chromatography; Lane 3 Zymogram of invertase activity of pooled
Silver staining SDS—gel electrophoresis of the purified invertase anion –exchange chromatography fractions; lane 4 SDS PAGE of
from S. sclerotiorum (b). M: Standard protein markers; lane 1 crude extracted protein from the zymogram gel
culture filtrate; lane 2 active fraction after anion—exchange

Table 2 Purification of extracellular S. sclerotiorum invertase

Purification steps Protein(mg) Total activity (units) Activity U/ml Specific activity (U/mg) Purification fold Yield (%)

Crude extract 29.75 195 6.5 6.55 1 100

DEAE-cellulose 0.57 94.54 7.87 165.85 25.32 48.5

most promising substrate for invertase production and was
used throughout the course of this work. So, the present
study still continues using molasses residue for production
of higher yield of invertase from S. sclerotiorum. This
value (1,950 U/g dry biomass) was better than that
obtained with Sclerotium rolfsii invertase (Kotwal and
Shankar 1997) and certainly higher than that obtained by
Mamma et al. (2008), who found that the highest invertase
activity produced by A. niger cultivated on agro industrial
residue was 72.5 U/g dry weight mycelia. Thus it is con-
cluded that higher level of invertase can be produced
practically from molasses as a cheap source of sucrose.
Purification of b-fructofuranosidase
The crude extract containing 29.7 mg of protein and 195 U
of activity was applied to a DEAE-cellulose to carry out
anion exchange chromatography. The extracellular invert-
ase was eluted from the column with a linear gradient of
0–1 M NaCl. The proteins were detected by measuring
absorbance at 280 nm and the associated enzyme activity
was measured by DNS methods as described on materials
and methods. After that the active fractions eluted at 0.4 M
NaCl were collected (Fig. 2a). The pool has a specific
activity of 165.8 U/mg proteins. About 25.32 fold of
purification and 48.5 % recovery of enzyme activity were
gained after this step (Table 2). The purification fold
obtained was much better than those obtained by Rubio
et al. (2002), Mona and Mohamed (2009) and Uma et al.
(2010).
The pool of DEAE purification step was analyzed by
SDS-PAGE. A large part of the contaminating proteins was
removed after this step as shown by SDS polyacrylamide
gel electrophoresis (Fig. 2b, lane 2). After identification of
the enzyme of interest by zymography using TTC as
staining solution (Fig. 2b, lane 3), the active protein band
was cut and analyzed on renaturated SDS-PAGE (Fig. 2-
b).Thus Lane 4 showed a single polypeptide band with a
mobility corresponding to a molecular mass about 48 kD.
This molecular weight was lower than that of extracellular
b-fructofuranosidase from X. dendrorhous reported previ-
ously (Linde et al. 2009).However, one from S. occiden-
talis had an approximate molecular weight of 85 kDa
(Álvaro-Benito et al. 2007) and another from R. glutinis
had around 47 kDa (Rubio et al. 2002). It was thus sug-
gested that S.sclerotiorum invertase purified in this work
had comparable molecular weight with others cited inver-
tases (Rubio et al. 2002).
Effect of temperature on activity and stability
DEAE purified S.sclerotiorum b-fructofuranosidase
exhibited optimal activity at 60 °C. The activity of the
enzyme was retained at 55 °C for 2 h and about 80 % of
the residual activity was retained at 60 and 65 °C for 1 h.
Considering the results obtained, this optimal temperature

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was higher than that determined for A. niger invertase
(55 °C) (L’Hocine et al. 2000). The enzyme was also more
thermostable than those from R.glutinis (Rubio et al. 2002)
and Azotobacter chroococcum (De la Vega et al. 1991).
Therefore S. sclerotiorum invertase was highly stable at a
wide range of temperature. Invertase with higher activity
and good thermal stability at high temperatures was
attractive for biotechnological application in various
industrial sectors.
Effects of pH on invertase activity and stability
Invertase activity assayed in standard conditions over a
broad pH range from pH 2.5 to pH 8.5 showed an optimum
at pH 5.5 and more than 50 % of the maximum activity
was measured between pH 4.0 and 6.5. This value was the
same as the optimum pH of the extracellular enzyme from
S. occidentalis (pH 5.5) (Álvaro-Benito et al. 2007).It was
slightly lower than that found for X. dendrorhous (pH
5.0–6.5) (Linde et al. 2009) and higher to that observed for
R. glutinis (Rubio et al. 2002). Thus optimal pH from 2.6 to
6.5 has been reported for invertases from different yeast
and fungi (Chaudhuri and Maheshwari 1996; Hernalsteens
and Maugeri 2008).
Based on the results obtained, the invertase from
S.sclerotiorum is completely stable in pH range 4.0–7.0 at
25 °C.The enzyme lost only 30–15 % of its activity at pH
below 4.0 or above 7.0 respectively.These results partially
agree with that reported by L’Hocine et al. (2000).Hence it
can be concluded that invertase from S.sclerotiorum has at
least an important potential for industrial use as its privi-
leged stability.
Effect of metal ions and reagents on activity of enzyme
All the metals are used at 2 mM,Co2? and Mn2? caused a
10 % fold increase however a slight stimulation of
invertase activity was obtained by Fe2?, Ca2?, Cu2? and
Zn2?. These results suggest that S. sclerotiorum b-fructo-
furanosidase is not modulated by divalent metal ions.
Similar behavior was observed for invertases from A. niger
(L’Hocine et al. 2000) and S. cerevisiae (Mona and Mo-
hamed 2009). In contrast, these same ions inhibited the
enzymatic activity in other fungi, such as Thermomyces
lanuginosus (Chaudhuri and Maheshwari 1996).
The invertase activity increased with Mg2?. In short,
invertases from R. glutinis and F. solani were also acti-
vated by Mg2? (Mamma et al. 2008).
EDTA showed no significant effect on enzyme activity
but B-mercaptoethanol increased the enzyme activity about
60 % whereas SDS reduced its activity. The presence of
sorbitol and glycerol had no significant effect on sucrose
hydrolysis and this suggested that the enzyme retains most
1069
of its catalytic activity even at higher concentrations of
these additives. These two reagents can be used as stabi-
lizers of the enzyme. In order to investigate the effects of
salt stress on enzyme activity, NaCl was tested in varied
concentrations (100 mM–1 M). NaCl had no effect on
enzyme activity (data not shown). This result suggested
that invertase could tolerate perfectly the salt stress.
Storage stability
Biotechnological applications of invertase depend on its
stability during extract preservation. One of the most
important parameters to be considered in free enzyme is
storage stability, so we analyzed the stability of the enzyme
at various times. Results show that invertase remains
totally active at 4 °C after one month and retained 80 % of
its activity at 4 °C after two month storage. The enzyme
remain active (50 % of residual activity) after one week at
25 °C. Consequently invertase is very stable at 4 °C and
can be considered stable at room temperature.
Determination of kinetic parameters
The Michaelis–Menten constant (Km) value of the pure
enzyme was found to be 5.8 mM while its Vmax was
0.11 lmol/min. The Km value seemed to be lower than
those prepared from R. glutinis (Rubio et al. 2002) and S.
cerevisiae (Mona and Mohamed 2009). Values showed that
invertase from S. sclerotiorum have higher affinity to
sucrose than those from other microorganisms like Can-
dida sp (Hernalsteens and Maugeri 2008) and A. niger
(L’Hocine et al. 2000).
Immobilization
Immobilization is a strategy not only for protecting enzyme
but also for bioreactor conception. The reusability of the
immobilized enzyme is another very important factor
interested in its application for the continuous process. For
invertase immobilization, many methods have been
described such as gel entrapment and co-immobilization of
invertase within a gel fiber (Nakane et al. 2000), covalent
attachment (Tümtürk et al. 2000) and ionic adsorption
(Torres et al. 2002). Different kinds of natural and syn-
thetic polymers have also been carried out for enzyme
immobilization (Müfrettin 2011). A variety of supports
such as chitosan (Gomez et al. 2006) and agarose-guar gum
biopolymer membrane (Bagal and Karve 2006) are used.
Recent investigations of nanomaterials for the adsorption,
encapsulation and cross-linking of enzymes have been
achieved (Sotiropoulou and Chaniotakis 2003; Wang et al.
2009). For the implementation in an industrial process,
scientists have studied sucrose conversion by immobilized
123
1070
invertase in many types of bioreactors such as a multiple
air-lift loop designed by Bakker et al. (1994). Furthermore,
different enzymes such as b-glycosidases, b-xylosidase and
b-glucosidase produced by the fungus S. sclerotiorum were
immobilized using different supports and evaluated for use
in continuous reactors (Gargouri et al. 2006).
In this context, the present research was based on the
entrapment of S. sclerotiorum invertase in calcium alginate
capsules. Whereas in the following section, the effects of
some parameters such as alginate and substrate concen-
tration were analyzed and the reuse of the immobilized
invertase was also studied for continuous process in bio-
reactor that we designed.
Effect of sodium alginate concentration
Through the concentration of Sodium Alginate solution it
was possible to modify the structure of gel and the acces-
sibility of the substrate to the active site Hence various
concentrations of sodium alginate (0.5–3 %) were used to
test the alginate spheres formulation and the invertase
immobilization yield. The entrapped activity was found
maximal 78 % at 2.5 % (w/v) of sodium alginate. However
lower cohesion of enzyme from capsules occurred at 0.5
and 1 % (w/v) of sodium alginate. Furthermore at 3 % (w/v)
sodium alginate the entrapped activity of the enzyme was
found very low because the high viscosity of gel which
decreased the pores size and thus hindered the penetration
of substrate into the alginate spheres. Different researchers
also reported that sodium alginate concentration range
from 2 to 3 % was suitable for the immobilization of
keratinase and protease (Farag and Hassan 2004; Elibol
and Moreira 2003). The main attraction of using alginate as
the immobilization support is that the gel formation process
requires only simple equipment and that the reagents are
relatively inexpensive and nontoxic.
Effect of calcium chloride concentration on invertase
Concentration of calcium chloride (10–100 mM, pH 5.5)
was also varied in order to obtain stable capsules able to
locked maximum enzyme. Thus it was found that the
partially purified enzyme retained highest activity
with10 mM CaCl2. As calcium chloride concentration
increased beyond 0.1 M the activity decreased. These
results are similar to that reported by Aziz Tanriseven and
Doğan (2001).
Effect of substrate concentration on activity
of immobilized invertase
Partially purified invertase was entrapped in calcium algi-
nate using molasses as a substrate and the activity yield of
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World J Microbiol Biotechnol (2014) 30:1063–1073
immobilized invertase was determined. Then the hydroly-
sis of molasses ranging from 1 to 5 % was studied.
It was found that 2 % molasses allowed the highest
activity yield (64 %). 1 % of molasses also permitted
synthesis with 59 % of activity yield. A high concentra-
tion of molasses repressed activity of the immobilized
invertase, as well as the use of 3, 4 and 5 % allowed
depression of activity (38.15, 30 and 14.89 % respec-
tively). The increase in the molasses concentration with
high viscosity and the existence of undesired might have
caused less access of substrate to the active site of
entrapped enzyme and thus decreased the activity of
immobilized invertase.
Summarizing Alginate/enzyme spheres have been suc-
cessfully formed by using 2.5 % (w/v) of sodium alginate
and 10 mM CaCl2. Thus at these conditions the immobi-
lization yield was 78 % and the activity yield was 64 %.
Interestingly the activity yield is better than that obtained
by immobilized invertase from Aspergillus awamori (Issam
et al. 2012).Therefore optimized conditions were selected
for further experiments.
Bioreactor and analysis of end products
The reactor that we designed is a 20 ml column through
with a solution of 2 % molasses corresponding to 1 %
sucrose in circulation at pH 5.5 and 45 °C using 7.87 U of
enzyme immobilized on alginate spheres at flow rates 1 ml/h.
By taking samples at various times, under these conditions,
RSs are produced and assayed by DNS. Results show that
the concentration of RSs increased with time. After
10 days recirculation of the molasses solution, 45 g/l of
fructose and glucose are produced without disruption of the
alginate-invertase capsules and without any microbial
contamination during this period. The maximum conver-
sion yield of sucrose to invert sugar was 41.6 %.The final
production was higher than that obtained from invertase
immobilized onto polyethylenimine beads for sucrose
hydrolysis (Arica and Bayramoglu 2006). The calculated
productivity of this bioconversion was 46.2 g/d/100 g.
After that production of invert sugar by immobilized
invertase in a bioreactor was analyzed by TLC. Hence we
note the vanishing of sucrose and a gradual appearance of
fructose and glucose in 7 days of hydrolysis. Sucrose was
detected in very small concentration on the 10th day (data
not shown). Besides the good stability of immobilized
invertase (remained fully active after 12-day incubation)
and the higher production of inverted sugars from molas-
ses, S. sclerotiorum invertase has certainly important
properties that distinguish this enzyme from all other
invertase described.
In that case entrapped invertase is qualified in molasses
hydrolysis and fructose syrup production used for the
World J Microbiol Biotechnol (2014) 30:1063–1073
120
100
80
% RS
60
40
20
0
0 1 2 3 4 5 6 7
Cycles
Fig. 3 Reusability of alginate beads (repeated batch) of molasses
cleavage by immobilized invertase in bioreactor for the synthesis of
RS. Samples are taken every day
fermentation process as described by Atiyeh and Duvnjak
(2002). Immobilized derivatives were used in different
types of reactors and in different continuous bioprocesses
(Girelli and Mattei 2005). Similar vertical packed bed
bioreactor using covalently immobilized invertase was
developed by Pabyton et al. (2011). Interestingly, we
completed molasses hydrolysis process by simple entrap-
ment invertase in a packed column. This reactor can be a
new alternative to produce inverted sugar syrup that avoids
chemical or structural enzyme modification during process.
The hydrolysis of all sucrose by the solution recirculation
and the non-accumulation of the substrate residues on their
surfaces may decrease the possibility of microbial con-
tamination. Yet, it could increase the half-life of the
bioreactor.
Reusability of immobilized invertase
To estimate better this production and in order to evaluate
the operational stability of the catalyst, we reuse the algi-
nate-invertase reactor under the same conditions after an
extensive washing with 50 mM acetate buffer pH 5.5.
Hence the activity of entrapped enzyme was assayed for
seven cycles with molasses as a substrate. There was no
significant activity decrease during 4 batch reactions. The
enzyme showed 65 % activity during the fourth and the
sixth reuse. Only in seventh cycle the immobilized
invertase lost 80 % of activity (Fig. 3). This decrease in
activity may be due to the leakage of enzyme during
washing of capsules at the end of each cycle.
The hydrolysis products of molasses by immobilized
invertase in a bioreactor were analyzed by TLC.Fig. 4
shows the evolution of the reaction, sucrose was detected in
very small quantities and completely hydrolyzed at the 4th
day with appearance of fructose-glucose simultaneously
accordingly the results show that the reactor was efficient
1071
G S F M 1 2 3 4
Fig. 4 TLC analysis of molasses cleavage by immobilized invertase
in bioreactor. After 4 days samples were subjected to TLC separation.
Lanes 1–4: samples at various incubation times; G, S, F, M sugar
standards glucose, sucrose, fructose and molasses respectively
on invert sugar production and very suitable for continuous
process. Therefore the alginate-invertase reactor looks
promising as a new alternative to produce inverted sugar
syrup from molasses in a simple bioprocess. Although
experiments have clearly shown advantages due to its
reuse, simple set up and low cost regarding material and
energy use.
Rheological parameters of invert sugar syrup
The knowledge of the rheological properties of invert sugar
is necessary for the design and the optimization of every
process involving fluid flow and heat transfer. Therefore,
the rheological behavior of syrup was experimentally
determined at 45 °C, using a rotational rheometer equipped
with coaxial cylinders. Rheograms of syrup were obtained
in the range of shear rates from 0.7 to 120 s-1.Figure 5a
shows that the viscosity decreases with shear rate. At high
shear rate all compounds were sheared. Furthermore the
final viscosity is similar to that of water which confirms
that all the syrup compounds were sheared. Thus results
indicated that the invert sugar syrup produced from sucrose
behave as pseudoplastic fluids, being well represented by
the Power Law model in the tested ranges (Marques et al.
2006). This decrease of consistency could be explained by
the effect of high temperature and the appearance of the
RSs instead of sucrose. Similar behavior was observed for
the yield stress in the rheological characterization of
aqueous solutions of sucrose (Marques et al. 2006).
Afterward we applied the rheometer to demonstrate the
effect of molasses on the rheological properties of the
mixture sucrose, glucose and fructose. The effect of
molasses on the rheological behavior was showed on rhe-
ogram (Fig. 5b). A significant increase in the viscosity of
syrup was observed. However the viscosity remains high at
the end (0,25 Pa.s) for the reason that the molasses com-
pounds affected the mechanical properties of the syrup.
123
1072
Fig. 5 Rheogram of invert a3
sugar produced by immobilized
invertase in bioreactor using
Viscosity Pa s
sucrose (a). Rheogram of invert 2
sugar produced by immobilized
invertase in bioreactor using
molasses (b)
1
0 0
0 500
Shear rate s-1
Similar rheogram at the same conditions was obtained from
many liquid foods, such as guava puree (Vitali and Rao
1982), sucrose-CMC model solution (Berto et al. 2003).
Finally it can be concluded that even the effect of molasses
on the flow properties of invert sugar syrup, the immobi-
lized invertase keep the sucrose hydrolysis near to 90 %.
Captivatingly, this study is the first step in the invert sugars
production from molasses, offering understanding proper-
ties of the immobilized enzyme for industrial process.
Conclusion
In conclusion, we have shown the ability of S. sclerotiorum
to grow and to produce invertase on a variety of carbon
sources, including beet molasses, utilization of these sub-
strates leads to an ecologically process and economically
advantage. In fact, the immobilized invertase constitutes a
performed method for sucrose hydrolysis and invert sugar
syrup production. Invertase production by S. sclerotiorum
under optimized cultural condition was studied and the
enzyme was isolated, characterized and purified. The pro-
cedure presented here is reproducible, simple, has a low
cost and can easily be adapted. The behavior of invertase
activity at different temperature, pH and substrate con-
centration was analyzed and specially exhibit good stabil-
ity. The immobilized invertase system using alginate was
found efficient. Finally we tried to apply this enzyme in the
development of an enzyme reactor for the synthesis of
invert sugar. The improvement of the bioreactor was fol-
lowed by a rheological study. The results showed inter-
estingly that the product is comparable to other glucose-
fructose syrups and supports its use in food processing and
feed stock in fermentation process. In all, this study proved
that the invertase bioreactor could serve in a promising
process to valorize molasses as industrial waste. Since
further investigations are needed for optimization of the
bioreactor operational conditions. Research is continuing in
our laboratory to investigate valorization of others indus-
trial and agricultural by-product using invertase and others
hydrolytic enzymes. Thus we project to develop invertase
123
World J Microbiol Biotechnol (2014) 30:1063–1073
b 0,4
0,3
Viscosity Pa s
0,2
0,1
1000 1500 0 500 1000 1500
Shear rate s-1
efficient immobilization system for oligosaccharides syn-
thesis in a continuous reactor.
Acknowledgments This work was supported by the financial pro-
ject of LIP-MB Laboratory, INSAT, Carthage University, Ministry of
Higher Education and Scientific Research of Tunisia.
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