Chapter 4- Purification and Characterizationshodhganga.inflibnet.ac.in/bitstream/10603/70210/11/11_chapter4.pdfinulinase and invertase activity was detected by incubating the enzyme

Chapter4:Purificationandcharacterization

98

CHAPTER 4

PURIFICATION AND CHARACTERIZATION OF

INULINASE


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

endoinulinase 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


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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


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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


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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


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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.


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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


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

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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Fractions

Inulinase (U/ml) Protein mg/ml

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Inulinase (U/ml)


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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)


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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


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


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

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100

120

20 30 40 50 60 70 80

% R

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Act

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Temperature (°C)

Inulinase activity Invertase activity


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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

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120

2 3 4 5 6 7 8 9

%R

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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

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40

60

80

100

120

Control HgCl2 FeCl KCl NaCl CaCl2 CoCl2 MgCl2

%R

elat

ive

acti

vity

Metal ions

Inulinase activity Invertase acitivity


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.

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control Triton X‐100

Tween 80 Tween 20 SDS PEG EDTA

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Additives



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

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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]


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]


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


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


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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Documents

Chapter 4- Purification and Characterizationshodhganga.inflibnet.ac.in/bitstream/10603/70210/11/11_chapter4.pdfinulinase and invertase activity was detected by incubating the enzyme