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Chapter4:Purificationandcharacterization
98
CHAPTER 4
PURIFICATION AND CHARACTERIZATION OF
INULINASE
Chapter4:Purificationandcharacterization
99
4.1 INTRODUCTION
Industries uses large amount of sugars and hence, new sources of sugars are always
been sought. Recently, the sugar industries have faced intense competition from High
Fructose Syrup (HFS) as a low-cost alternative sweetener. For producing HFS,
conventional processes are based on the usage of starch as a raw material. However,
inulin, being a reservoir of fructose, proves to be a better raw material compared to
starch for HFS production (Zhang et al., 2010).
Inulinases are classified among the hydrolase that target on the β-2, 1 linkage of inulin
and hydrolyze it to fructose and glucose. They can be divided into exoinulinase and
endo- inulinase depending on their mode of action. Exoinulinase catalyzes the
removal of terminal fructose residues from the non-reducing end of inulin molecule
while the endoinulinase hydrolyze the internal linkages in inulin to yield inulotriose,
inulotetraose and inulopentaose as the main products (Chi et al., 2009). Endoinulinase
lack invertase activity while most of the exoinulinase shows invertase activity coupled
with inulin hydrolytic activity.
Microorganisms are the best sources for commercial production of inulinases because
of their ease of cultivation and high yields of the enzyme. Members of the genus
Aspergillus, Penicillium, Kluyveromyces, Cryptococcus, Pichia, Bacillus, etc. has
been proved to be high inulinase producers. Microbial inulinases (2, 1 β-D fructan
fructanohydrolase, E.C.3.2.1.7) are stable at high temperatures, a characteristic which
is favourable for avoidance of microbial contamination and high solubility of the
substrate (Pessoni et al., 1999). Fungal inulinases are frequently composed of a
mixture of fructanohydrolases with high activity and stability. Inulinases of fungal
origin have mostly been extra-cellular in nature and have generally been exo-acting
(Pandey et al., 1999). These hydrolase are usually inducible and able to hydrolyse
sucrose and raffinose along with inulin.
Owing to the prospect of applying inulinase in nutraceuticals and pharmaceuticals,
profound study has been carried out regarding the synthesis of inulinase by various
microorganisms and the search for high inulinase producers as well as the purification
of these enzymes has received increasing attention. As a part of this, the potential
producers of inulinase have been identified and dependence of its catalytic activity on
temperature, pH, substrate concentration, metal ions, activators, inhibitors, etc. has
been determined. It has been established that fermentation is the most suitable and
attractive method for inulinase production, but purification of inulinase usually
Chapter4:Purificationandcharacterization
100
involves several steps that end up the process expensive and time consuming. The
purification and properties of inulinase have been studied in many fungal species
(Gupta et al., 1997; Ettalibi and Baratti, 1987; Jing et al., 2003) and it has been
observed that most of the reports on purification of extra-cellular inulinases produced
by fungi, yeast and bacteria deals with the conventional method of centrifugation,
ultra filtration, salt/solvent precipitation followed by various column chromatography
techniques (Treichel et al., 2014). For the inulinase of intracellular nature, an added
step of cell wall destruction is needed prior to the conventional purification
procedures.
Purification of enzymes remove other contaminating enzymes from crude
preparations and helps to study their true characteristics which further makes it easier
to decide their most suitable end applications. In this chapter we report partial
purification of inulinase produced by newly isolated fungi, Aspergillus tubingensis
CR16 as well as study of its biochemical properties.
4.2 MATERIAL AND METHODS
4.2.1 Materials
All the chemicals used were of analytical grade. DEAE- Celluose and Sephdex G-150
were obtained from Sigma. Inulin (chicory), ammonium sulphate and potato dextrose
agar (PDA) were from Hi-media.
4.2.2 Enzyme production and extraction
Inulinase was produced under statistically optimized conditions as described in
Chapter 3, section 3.2.4. Enzyme was extracted as per the procedure described in
section.3.2.3.3
Supernatant was considered as the crude enzyme solution and was subjected to further
purification steps.
4.2.3 Enzyme Assay and Protein Assay
Inulinase and invertase assays were performed as described in Chapter 3, section
3.2.5. Protein assay was performed as per indicated in Chapter 3, section 3.2.6
4.2.4 Purification of Inulinase
4.2.4.1 Ammonium sulphate precipitation
Crude inulinase was subjected to precipitation with ammonium sulphate (40-80%)
under mild stirring conditions at 4°C. The solution was kept overnight for the
saturation purpose. Precipitates were recovered by centrifugation at 3000 rpm for 20
Chapter4:Purificationandcharacterization
101
minutes at 4°C, suspended in specific volume of 0.2M sodium acetate buffer pH-5.0
and dialyzed against the same buffer for the removal of residual ammonium sulphate.
Dialyzed enzyme was concentrated by ultra filtration using 30KDa cut off membrane
(Vivaspin Centrifugal Concentrator, Sigma-Aldrich).
4.2.4.2 Gel permeation chromatography
Concentrated enzyme preparation was applied onto a Sephadex G-150 column (1x10
cm) and was eluted with 0.2M sodium acetate buffer (pH-5.0) at the flow rate of 0.25
ml/minute. Enzyme activity and protein elution profile was monitored.
4.2.4.3 Ion exchange chromatography on DEAE cellulose column
Fractions containing inulinase activity were pooled and applied onto a DEAE
Cellulose column (1x10 cm) pre-equilibrated with 0.2M sodium acetate buffer pH-
5.0.The unadsorbed protein was eluted with the starting buffer while adsorb protein
was eluted from the column with a linear NaCl gradient (0.1 to 1M) prepared in the
same buffer at the flow rate of 0.4 ml/minute. Each fraction was checked for enzyme
activity and protein content.
4.2.5 Native and SDS polyacrylamide gel electrophoresis
Native as well as SDS PAGE was carried out using the Mini Dual Vertical
electrophoretic system (Tarsons). A separation gel with 12% acrylamide cross-linked
with bismethyleneacrylamide with pH-8.8 was used with 5% stacking gel. The
electrophoresis buffer was composed of a Tris-glycine system. A voltage of 200V and
a starting current of 100 mA were applied for the process and 30µl of precleaned
enzyme was separated within 1.5 h. After separation, protein bands were visualized
using silver staining.
4.2.6 Activity staining
Activity staining of native gel was carried out as per the procedure described by
Praznik and Baumgartner (1995) with suitable modifications. The gel was immersed
in 1% inulin solution in 0.2M sodium acetate buffer pH-5.0 for 1h at 50°C and then
was treated with 0.1% triphenyl tetrazolium chloride (TTC) in 0.1M NaOH solution
for 15 minutes in the dark and for 15 minutes at 100°C for the colour development.
4.2.7 Assay of enzyme activities of the band
The native bands corresponding to silver stained bands were cut down and macerated
in tubes containing 0.9 ml of 1% inulin prepared in 0.2M Na-acetate buffer pH-5.0.
The reaction tubes were incubated at 60°C for 3 h and terminated by boiling in water
Chapter4:Purificationandcharacterization
102
bath for 10 minutes. Samples were checked for the release of reducing sugars by DNS
(Miller, 1939).
4.2.8 Study of physicochemical characteristics of purified inulinase and invertase
The purified enzyme was analyzed for the study of its physicochemical properties.
Optimum temperature for inulinase as well as invertase activity was determined by
performing the enzyme assay in the temperature range of 30°C to 70°C. Optimum pH
was analyzed by performing the enzyme assay at different pH ranging from pH – 3.0
to pH – 8.0 with 0.2M citrate buffer for pH 3, 0.2M sodium acetate buffer for pH 4
and 5 and 0.2M sodium phosphate buffer for pH 6, 7 and 8. Effect of metal ions on
inulinase and invertase activity was detected by incubating the enzyme with the salts
of different metals viz. Hg, Fe, K, Na, Ca, Co and Mg in 1mM concentration at 30°C
for 1 h. Enzyme assay was performed after incubation and relative activity was
calculated. Effect of surfactants and additives on inulinase and invertase was checked
by performing the enzyme reaction in the presence of different surfactants namely
Tween 20, Tween 80, Triton X-100, PEG in 1% concentration and SDS and EDTA in
1mM concentration. Thermostability of inulinase and invertase was performed by
exposing purified enzyme to 50°C, 60°C and 70°C for 10 h. Samples were withdrawn
periodically and were analyzed for residual activity. Kinetic parameters were analyzed
at substrate concentration 0.1- 2% inulin and 0.1-1% sucrose, for inulinase and
invertase respectively. Km and Vmax were calculated according to the lineweaver
burk plot.
4.2.9 Enzyme activity on different substrates
Inulinase action was analyzed for its hydrolytic capacity on different substrates. Each
substrate namely inulin (chicory), inulin (dahlia), sucrose and raffinose in 1%
concentration were mixed with purified inulinase and reaction was carried out at 60°C
for 20 minutes. After termination of the reaction under boiling water bath for 10
minutes, the reaction products were analyzed using DNS (Miller 1939).
4.2.10 Qualitative analysis of products of inulin hydrolysis by TLC (Thin Layer
Chromatography)
Products of inulin hydrolysis in the course of time were qualitatively analyzed by thin
layer chromatography (TLC) using the solvent system isopropanol: ethyl acetate:
water in ratio 5:2.5:2.5. Plate was sprayed with the spraying reagent and was
Chapter4:Purificationandcharacterization
103
incubated at 100°C for colour development. Samples were analyzed against glucose
(1 mg/ml), fructose (1 mg/ml) and sucrose (1 mg/ml) as standards.
4.2.11 Transfructosylation
Transfructosylation ability of the partially purified enzyme was analysed. The reaction
mixture consisting of 9.8 ml of 60% sucrose solution prepared in 0.2M sodium acetate
buffer, pH 5.0, and 0.2 ml of enzyme solution was incubated at 60°C for 1 h. The
fructose present in the reaction mixture was estimated by DNS and glucose
concentration was determined by GOD-POD method. Transfructosylation was
determined by the difference between glucose and fructose.
4.3 RESULTS AND DISCUSSION
4.3.1 Purification of Inulinase
Crude enzyme produced under SSF conditions using wheat bran and 10% CSL, was
purified initially by ammonium sulphate fractionation at 40-80% saturation.
Ammonium sulphate saturation resulted in recovery of almost 70% inulinase activity
(Table 4.1) and 64.9% of invertase activity (Table 4.2), with the fold purification of
4.3 and 4.1 respectively. Concentration with ultra filtration increased the purification
of inulinase to 5.6 but does not showed any significant purification of invertase.
Further purification of the proteins thereafter by gel permeation chromatography on
Sephadex G-150 column resulted in the removal of large portion of contaminating
protein and inulinase fraction was eluted as single broad peak as per shown in Fig.4.1.
This step purified the enzyme to nearly double. The passage of Sephadex G-150
pooled fractions (4, 5 and 6) through DEAE Cellulose column resulted in three active
peaks displaying inulinase activity (Fig.4.2). The pooled fractions (9, 10 and 11) after
this step showed that inulinase was purified up to almost 35 fold but the yield was
only 2.4%. (Table4.1). The pooled fractions from gel permeation chromatography as
well as ion exchange chromatography were also analyzed for invertase activity and it
was found that invertase activity was purified up to 25 fold. However, the yield of
invertase was very low (0.17%) after the purification procedure.
There are many reports on purification of inulinase but the fold purification of
inulinase obtained in the present study was higher compared to that reported by
Treichel et al., (2014); Fawzi (2011); Ohta et al., (2002); Belamri et al., (1994).
Kochhar et al., (1999) has attempted ethanol precipitation of inulinase but obtained
only 8% activity yield of inulinase.
Chapter4:Purificationandcharacterization
104
Table 4.1: Purification of Inulinase
Steps Inulinase
(U/ml) Total units
Protein (mg/ml)
Total protein
(mg)
Specific Activity (U/mg)
Fold purification
% yield
I/S
Crude 22.6 5650 19.5 4875 1.15 1 100 0.20Ammonium sulphate precipitation (40-80%)
262.7 3916.3 52.2 840.4 5.0 4.3 69 0.21
Ultra filtration (30KDa)
580.0 2900 88.0 440.0 6.5 5.6 51 0.24
Sephadex G-150
168.05 840.25 13.9 69.5 12.0 10.4 14.8 0.25
DEAE Cellulose
28.1 140.5 0.7 3.5 40.1 34.8 2.4 0.28
Table 4.2: Purification of Invertase
Steps Invertase
(U/ml) Total units
Protein (mg/ml)
Total protein
(mg)
Specific Activity (U/mg)
Fold purification
% yield
Crude 113.0 28,250 19.5 4875 5.7 1 100
Ammonium sulphate precipitation (40-80%)
1224.1 18,362 52.2 840.4 23.4 4.1 64.9
Ultra filtration (30KDa)
2416.3 12081.
5 88.0 440.0 27.4 4.8 42.7
Sephadex G-150
672.2 3361 13.9 69.5 48.3 8.4 11.8
DEAE Cellulose
100.3 501.5 0.7 3.5 143.2 25.1 0.17
Chapter4:Purificationandcharacterization
105
Figure 4.1: Elution profile of inulinase in gel permeation chromatography using
Sephadex G-150
Figure 4.2: Elution profile of inulinase in ion exchange chromatography using
DEAE cellulose
4.3.2 Native and SDS PAGE of purified fractions of inulinase
The native page is an effective method to separate enzymes with identical properties.
The ionic strength of buffer and pH are the main factors in PAGE (Jing et al., 2003).
The enzyme preparations after the purification procedure showed the presence of four
0
5
10
15
20
25
0
20
40
60
80
100
120
140
160
180
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
mg/
ml
U/m
l
Fractions
Inulinase (U/ml) Protein mg/ml
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
mg/
ml
U/m
l
Fractions
Inulinase (U/ml)
Chapter4:Purificationandcharacterization
106
bands on native PAGE (Fig. 4.3). Hence we were able to achieve partial purification
of inulinase. SDS PAGE results (Fig. 4.4) also showed the presence of many bands
and hence the enzyme cannot be considered as completely purified inulinase. To
check whether the bands present were of inulinase enzyme or some other
contaminating protein, unstained portion of the native gel containing the bands
corresponding to the silver stained bands were cut down, macerated and subjected to
inulinase assay. Results of the reaction shown in Table 4.3 indicated that all the bands
present on native gel displayed inulinase activities to different extent. Hence it can be
considered that there may be the presence of multiple forms of enzyme in Aspergillus
tubingensis CR16 displaying inulinase activity. It has been reported that fructose can
also be obtained by the synergistic actions of exo-inulinase and endo-inulinase;
however it is difficult to determine whether they coexist at the same time. Like other
glycosidases, such as endoglucanse and exoglucanse, exo-inulinase and endo-
inulinase are also very similar in properties hence difficult to distinguish and separate
the two enzymes using conventional methods (Jing et al., 2003). There are reports on
the coexistence of more than one inulinase with endo action, exo action as well as
invertase in Aspergillus ficuum (Ettalibi and Baratti, 1987; Jing et al., 2003).
Figure 4.3: Native PAGE gel of purified fractions (Lane 1: Sephadex G-150,
Lane 2: DEAE cellulose chromatography fraction 9, Lane 3: DEAE cellulose
chromatography fraction 10, Lane 4: pooled fraction)
Chapter4:Purificationandcharacterization
107
Figure 4.4: SDS PAGE gel of pooled fractions of DEAE Cellulose
chromatography (Lane 1: Markers, Lane 3: fraction 9, Lane 4: fraction 10, Lane
5: fraction 11, Lane 6: pooled fraction 9, 10 and 11)
Table 4.3: Inulinase activity of bands obtained on native gel
Band No. Inulinase U/ml (60°C) 1 0.69 2 9.2 3 3.4 4 5.4
4.3.3 Activity staining of inulinase
Activity staining of the purified inulinase was done on the preparative gel by exposing
the gel to 1% TTC and incubating it in dark for 20 minutes. Fig.4.5 shows that from
the all the four separated bands observed on native gel, only single band showed zone
of hydrolysis with TTC. In the presence of reducing sugars, TTC gets reduced to
triphenylformazon which is a red coloured water insoluble compound which can be
visualized as red coloured band on processed gel. Baumgartner and Praznik (1994)
have got similar results in their study of purification of crude inulinase from
Novozyme, in which they have reported a range of different bands in the protein
Chapter4:Purificationandcharacterization
108
staining of the gel after electrophoresis. But from multiple bands, only two of the
bands gave positive results during the activity staining. This may be due to the lesser
concentration of enzyme present in the band, which may not be enough for the desired
reaction.
Figure 4.5: Activity staining of inulinase
4.3.4 Characterization of inulinase and invertase
The purified enzyme preparation obtained from Aspergillus tubingensis CR16 was
also found to possess invertase activity coupled with inulinase activity. The naming of
a β-fructosidase as an inulinase or invertase is based on its relative hydrolytic capacity
for inulin and sucrose (I/S). The inulin and sucrose hydrolytic activities in the purified
preparations could either be due to two different enzymes or one enzyme showing
broad specificity or one enzyme having two different active sites (Gupta et al., 1997).
The partially purified enzyme preparation was found to have I/S ratio of 0.28. Hence
the physicochemical properties of both the enzyme activities were analyzed.
4.3.4.1 Effect of temperature on inulinase and invertase activity
Inulinase and invertase activity of partially purified enzyme was studied at different
temperatures ranging from 40°C to 70°C. The results (Fig.4.6) showed that optimum
Chapter4:Purificationandcharacterization
109
temperature for inulin hydrolytic activity as well as sucrose hydrolytic activity was
60°C. However the enzyme showed significant invertase activity even at 50°C.
Higher temperature optimum of inulinase is an extremely important factor for the
application of these enzymes for commercial production of fructose or
fructooligosaccharide from inulin, since high temperature (60°C or higher) ensure
proper solubility of inulin and prevent microbial contamination (Vandamme &
Derycke, 1983; Singh & Gill, 2006) Most of the fungal inulinases have been reported
to have temperature optima in the range of 45°C to 55°C, however there are studies
which report the temperature optima of inulinase produced by A. awamori and A.
ficuum to be 60°C (Ohta et al., 2004).
Figure 4.6: Temperature optima of partially purified inulinase produced by
Aspergillus tubingensis CR16
4.3.4.2 Effect of pH on inulinase activity
The influence of pH on partially purified inulinase was studied at different pH range
from 4 to 8. The result (Fig.4.7) shows that nonetheless the maximum activity
appeared at pH 5.0, the enzyme was also appreciably active in wide pH range from 4
to 6 for the inulin hydrolysis. Invertase activity also showed pH optima of pH 5.0
However, it was not considerably active at pH higher than that. Enzymes obtained
from different sources normally have variable pH optima, possibly due to different
amino acid compositions, which in turn affect their ionization in a solution. Hence the
0
20
40
60
80
100
120
20 30 40 50 60 70 80
% R
elat
ive
Act
ivit
y
Temperature (°C)
Inulinase activity Invertase activity
Chapter4:Purificationandcharacterization
110
enzyme active on a broad pH range is always preferable for applications in food
industries (Sarup et al., 2006)
For industrial application enzyme with larger activity in acidic pH range, as the one
here described, are suitable since they make bacterial contamination difficult (Saber
and Naggar, 2009).
Figure 4.7: pH optima of partially purified inulinase produced by Aspergillus
tubingensis CR16
4.3.4.3 Effect of metal ions on inulinase and invertase activity
Metal ions may act as co-enzymes or they may be present as a part of catalytic site of
the enzyme or may affect enzyme activity. Most of the metal ions serves as either
enzyme co factors, or prosthetic groups and can participate with the enzyme to
accelerate the rate of reaction through several mechanisms. (Sarup et al., 2006). Thus
the effect of various metal ions at 1mM concentrations was checked on inulinase
activity. Among the cations studied, Hg+2 completely inhibited inulinase and invertase
activity (Fig 4.8). The inhibition of enzyme activity by mercury ions may indicate the
importance of thiol containing amino acid in the enzyme activity (Sheng et al., 2008).
Fe+2 and Co+2 ions also inhibited inulinase activity completely while, invertase was
found to be significantly active in the presence of those two ions compared to
inulinase. Inulinase activity was found to be more negatively influenced in the
0
20
40
60
80
100
120
2 3 4 5 6 7 8 9
%R
elat
ive
acti
vity
pH
Inulinase activity Invertase activity
Chapter4:Purificationandcharacterization
111
presence of all the other metal ions compared to invertase activity. None of the cations
showed positive influence on inulinase or invertase activity compared to control.
Figure 4.8: Effect of metal ions on inulinase and invertase activity
4.3.4.4 Effect of additives on inulinase and invertase activity
Effect of various surfactants and metal chelating agents were checked on both
inulinase and invertase activity. The presence of Tween 80 acted positively for
inulinase activity. There was a considerable increase about 60% in inulinase activity
as compared to control. While Tween 80 did not affect invertase activity but instead
the presence of PEG increased invertase activity about 20% compared to control
(Fig.4.9). It may be possible that the enzyme substrate interaction gets improved by
the presence of Tween 80 and helps in increased mobilization of enzyme among the
substrate reaction sites (Kim et al., 2006). The presence of macromolecules in the
reaction mixture can modulate the activity of the enzyme in a complex fashion. The
presence of PEG on the reaction media seems to induce a decrease in the stability of
enzyme substrate complex, favouring the transition towards the product formation.
This phenomenon might be originated by conformational changes on the enzyme due
to its interaction with PEG molecules. Calderon et al., (2013) has reported that the
presence of PEG increased the hydrolysis of p-nitrophenyltrimethyl acetate
hydrolysis. Additionally, the amphipathicity of the surfactant may play a role in
‐20
0
20
40
60
80
100
120
Control HgCl2 FeCl KCl NaCl CaCl2 CoCl2 MgCl2
%R
elat
ive
acti
vity
Metal ions
Inulinase activity Invertase acitivity
Chapter4:Purificationandcharacterization
112
exposing the active sites available for enzyme substrate interaction (Evans and
Abdullah. 2012).
Figure 4.9: Effect of additives on inulinase and invertase activity
4.3.4.5 Thermostability of Inulinase
Application of enzyme in industrial processes often shows thermal inactivation of the
enzyme. The thermal stability of partially purified inulinase was studied in the
temperature range of 50°C to 70°C. The partially purified inulinase was found to be
thermostable with the retention of about 60% of its inulin as well as sucrose
hydrolytic activity even after 8 h (Fig. 4.10), with a half life of 7.9 hrs at 50°C. At
60°C, the enzyme was found less stable and its invertase activity got completely
inactivated after 2 h of exposure. Inulinase activity was still retained up to 12.6% of
its activity (Fig. 4.11). At 70ºC inulinase and invertase both were found to be
denatured within 30 minutes of exposure. The results obtained in the present study
were better compared to those obtained by Ohta et al (2002), who has reported
thermostability studies of inulinase from Rhizopus sp TN-96 from 20-80⁰C and the
complete inactivation of inulinase was observed at 60⁰C in 30 minutes. P. K. Gill et al
(2006) has reported comparision of thermostability of inulinase from A. fumigatus and
Novozyme (commercially available inulinase) which showed Novozyme retained
only 5.2% activity after 2 hrs at 60⁰C in the presence of inulin.
0
20
40
60
80
100
120
140
160
180
control Triton X‐100
Tween 80 Tween 20 SDS PEG EDTA
% R
elat
ive
acti
vity
Additives
Inulinase activity Invertase activity
Chapter4:Purificationandcharacterization
113
Industrial inulin hydrolysis is carried at 60ºC, to prevent microbial contamination and
also to permit the use of higher inulin concentration due to increased solubility. Thus
thermostable inulinolytic enzyme would be expected to play an important role in food
and chemical industries. Higher thermostability of the industrially important enzymes
brings down the cost of production because lower amount of enzyme is lost during the
process (Vandamme and Derycke, 1983; Cruz et al, 1998).
Figure 4.10: Thermostability of inulinase and invertase at 50°C
Figure 4.11: Thermostability of inulinase and invertase at 60°C
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8
% R
esid
ual
act
ivit
y
Time (h)
Inulinase activity Invertase activity
0
20
40
60
80
100
0 1 2 3 4 5 6 7 8
% R
esid
ual a
ctiv
ity
Time (h)
Inulinase activity Invertase activity
Chapter4:Purificationandcharacterization
114
4.3.4.6 Effect of substrate concentration and study of enzyme kinetics
Km and Vmax are the two parameters which define the kinetic behaviour of an
enzyme as a function of substrate concentration [S]. The studies of kinetic parameters
indicate that the apparent Km for inulin (chicory inulin) and sucrose was 3.33 mg/ml
(Fig. 4.12) and 1.11 mg/ml (Fig. 4.13) respectively. Vmax for inulinase was 34.48
mg/ml/min and for invertase, Vmax was 108.69 mg/ml/min. The results showed that
the enzyme shows more affinity towards sucrose compared to inulin and also showed
higher reaction velocity towards sucrose. If an enzyme has a small value of Km, it
achieves its maximum catalytic efficiency at low substrate concentration. Hence the
smaller the value of Km, the more efficient is the enzyme. Another important kinetic
parameter, Vmax is reached when all the enzyme sites are saturated with substrate.
Higher the value of Vmax, more efficient is the enzyme. However, Km and Vmax of
enzyme depends on the particular substrate as well as the reaction conditions.
Inulinases have shown great divergence in Km and Vmax values. It is possible that
the great multiplicity of forms of this enzyme explain these differences (Cruz et al.,
1998).
Figure 4.12: Lineweaver Burk plot of inulinase
y = 0.1064x + 0.0298R² = 0.9754
‐0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
‐1 ‐0.5 0 0.5 1 1.5 2 2.5 3
1/[V
]
1/[S]
Chapter4:Purificationandcharacterization
115
Figure 4.13: Lineweaver Burk plot of invertase
4.3.4.7 Substrate specificity of purified inulinase
The ability to hydrolyze inulin from two different sources as well as raffinose and
sucrose was checked by using respective substrates in 1% concentration. It was
evident from the results (Fig. 4.14) that purified enzyme showed maximum activity on
sucrose due to its high invertase activity. However it was also able to significantly
hydrolyze raffinose along with inulin. Raffinose is a trisaccharide composed of
galactose, glucose and fructose. Soy sources and cottonseed meal are good sources of
raffinose. Humans and other monogastric animals cannot produce the enzyme which
can hydrolyze raffinose and hence they passes into a lower gut where they are
fermented by gas producing bacteria in turn causing intestinal disturbances (Khane et
al., 1994). Hence it is desirable to remove raffinose from soy products. Although,
inulin hydrolysis is the major application of inulinase, it can also be utilized for
raffinose hydrolytic processes. Ability of enzyme to show hydrolytic activity on
various substrates provides wider prospects for the application of enzyme at
commercial level. The inulinase preparation from Kluyveromyces marxianus YS-1
was found to be active on 2% inulin, sucrose and raffinose (Sarup et al., 2006).
y = 0.0101x + 0.0092R² = 0.9744
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
‐1 0 1 2 3 4 5 6
1/[V
]
1/[S]
Chapter4:Purificationandcharacterization
116
Figure 4.14: Substrate specificity of purified inulinase
4.3.4.8 Qualitative analysis of the products of inulin hydrolysis by TLC (Thin
Layer Chromatography)
To determine exo- or endoacting nature of inulinase, TLC analysis of the reaction
products of inulin treated with inulinase was done (Fig.4.15). Fructose was the only
sugar detected on TLC plate, supported the view that inulinase was an end group
cleaving enzyme. Thus inulinase produced by Aspergillus tubingensis CR16 was an
exoinulinase. Inulinases belong to the group of fructanohydrolases and can be
classified as endoinulinase (2,1-β-D-fructan fructanohydrolase) which hydrolyze
internal β-2,1 fructofuranosidic linkages to yield inulotriose, -tetraose and pentaose as
the main products. In contrast, exoinulinase (β-D-fructan fructohydrolase) splits
terminal fructose units. Fructose is an important ingredient in food and
pharmaceutical industry (Gill et al., 2006). Fructose is considered as a safe alternative
to sucrose because it has beneficial effects in diabetic patients, increases iron
absorption in children, high solubility, low viscosity, higher sweetening capacity and
thus can be used as a low calorie sweetener (Pandey et al., 1999).
0
5
10
15
20
25
30
35
Inulin (chicory) Inulin (Dahlia) Sucrose Raffinose
Enz
yme
acti
vity
(U
/ml)
1% Substrate
Chapter4:Purificationandcharacterization
117
Figure 4.15: Qualitative Analysis of the products of inulin hydrolysis by TLC
(Thin Layer Chormatography) (F: Fructose; G: Glucose; EC: Enzyme control:
SC: Substrate control)
4.3.4.9 Transfructosylation
Since the production and application of Fructooligosaccharides (FOS) have gained
commercial importance because of their favourable functional properties, there is a
need to search for newer and potential processes for their production. The synthesis of
FOS is studied using enzymes with high transfructosylation activity, where the best
enzymes are from fungi such as Aspergillus niger, Aspergillus japonicus,
Aureobasidium pullulans and Fusarium oxysporum (Santos and Maugeri, 2007).
Hence, partially purified enzyme was analyzed for its transfructosylation ability at
high sucrose concentration. The analysis revealed that partially purified inulinase
from Aspergillus tubingensis CR16 did not display any transfructosylation capacity.
Over the years, a number of transfructosylating enzymes as well as exo-inulinases
from Aspergillus sp. have been described (Arand et al., 2002; Moriyama et al., 2003).
Goosen et al., (2008) have described exoinulinase of Aspergillus niger N402 with
significant transfructosylation activity at increasing sucrose concentration. Sangeetha
Chapter4:Purificationandcharacterization
118
et al., (2005) has reported FOS production using FTase (15U/ml) obtained from
Aspergillus oryzae CFR 202.
4.4 CONCLUSION
Inulinase produced by Aspergillus tubingensis CR16 was partially purified (35 fold)
by ammonium sulphate precipitation followed by gel permeation chromatography and
ion exchange chromatography. The purified enzyme preparations displayed the
possibility of the presence of multiple inulinases which also showed sucrose
hydrolytic activity along with inulinase activity. The enzyme was also able to act on
raffinose along with sucrose and inulin. Purified inulinase showed high temperature
optima and low pH optima, the properties which are preferred during industrial
processes involved in inulin hydrolysis.
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