Chapter 4: Purification and characterization CHAPTER 4 PURIFICATION AND CHARACTERIZATION OF INULINASE

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1 CHAPTER 4 PURIFICATION AND CHARACTERIZATION OF INULINASE 98

2 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 ) 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 99

3 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 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 Enzyme production and extraction Inulinase was produced under statistically optimized conditions as described in Chapter 3, section Enzyme was extracted as per the procedure described in section Supernatant was considered as the crude enzyme solution and was subjected to further purification steps Enzyme Assay and Protein Assay Inulinase and invertase assays were performed as described in Chapter 3, section Protein assay was performed as per indicated in Chapter 3, section Purification of Inulinase 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

4 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) 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 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 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 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 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 101

5 bath for 10 minutes. Samples were checked for the release of reducing sugars by DNS (Miller, 1939) 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 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) 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 102

6 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 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 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. 103

7 Steps Table 4.1: Purification of Inulinase Inulinase (U/ml) Total units Protein (mg/ml) Total protein (mg) Specific Activity (U/mg) Fold purification % yield Crude Ammonium sulphate precipitation (40-80%) Ultra filtration (30KDa) Sephadex G DEAE Cellulose I/S Table 4.2: Purification of Invertase Steps Crude Ammonium sulphate precipitation (40-80%) Ultra filtration (30KDa) Sephadex G-150 DEAE Cellulose Invertase (U/ml) Total units Protein (mg/ml) Total protein (mg) Specific Activity (U/mg) Fold purification % yield , ,

8 Inulinase (U/ml) Protein mg/ml 20 U/ml mg/ml Fractions 0 Figure 4.1: Elution profile of inulinase in gel permeation chromatography using Sephadex G-150 Inulinase (U/ml) U/ml Fractions mg/ml Figure 4.2: Elution profile of inulinase in ion exchange chromatography using DEAE cellulose 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 105

9 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 endoinulinase 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) 106

10 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) 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 107

11 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 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 Hence the physicochemical properties of both the enzyme activities were analyzed 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 108

12 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). 120 Inulinase activity Invertase activity % Relative Activity Temperature ( C) Figure 4.6: Temperature optima of partially purified inulinase produced by Aspergillus tubingensis CR 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 109

13 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). Inulinase activity Invertase activity %Relative activity ph Figure 4.7: ph optima of partially purified inulinase produced by Aspergillus tubingensis CR 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 110

14 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 Inulinase activity Invertase acitivity %Relative activity Control HgCl2 FeCl KCl NaCl CaCl2 CoCl2 MgCl2 Metal ions Figure 4.8: Effect of metal ions on inulinase and invertase activity 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 111

15 exposing the active sites available for enzyme substrate interaction (Evans and Abdullah. 2012). % Relative activity control Triton X 100 Inulinase activity Tween 80 Tween 20 SDS PEG EDTA Additives Invertase activity Figure 4.9: Effect of additives on inulinase and invertase activity 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. 112

16 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). 120 Inulinase activity Invertase activity % Residual activity Time (h) Figure 4.10: Thermostability of inulinase and invertase at 50 C % Residual activity Inulinase activity Invertase activity Time (h) Figure 4.11: Thermostability of inulinase and invertase at 60 C 113

17 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 mg/ml/min and for invertase, Vmax was 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) y = x R² = /[V] /[S] Figure 4.12: Lineweaver Burk plot of inulinase 114

18 0.07 y = x R² = /[V] /[S] Figure 4.13: Lineweaver Burk plot of invertase 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). 115

19 35 Enzyme activity (U/ml) Inulin (chicory) Inulin (Dahlia) Sucrose Raffinose 1% Substrate Figure 4.14: Substrate specificity of purified inulinase 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). 116

20 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) 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 117

21 et al., (2005) has reported FOS production using FTase (15U/ml) obtained from Aspergillus oryzae CFR 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. REFERENCES Arand M., Golubev A. M., Neto J. R. N., Polikarpov I., Wattiez R. and Korneeva O. S. (2002). Purification, characterization, gene cloning and preliminary X-ray data of the exoinulinase from Aspergillus awamori. Biochemical Journal, 362: Baumgartner S. and Praznik W., (1994). Purification of exo- and endoinulinase from crude inulinase extract for the analysis of fructans. International Journal of Biological Macromolecules, 17(5): Belamri M., Sassi A. H., Savart M., Elarak T. A. and Cottin P., (1994). Purification and properties of an extracellular inulinase like β-fructosidase from Bacillus stearothermophilus. Letters in Applied Microbiology. 19: Calderson C. and Lissi E. (2013). Polyethylene glycol effect on the transient and steady state phases of p-nitrophenyltrimethyl acetate hydrolysis catalyzed by α- chymotrpsin. Journal of Chile Chemical Soceity, 58(4): Chi Z., Chi Z., Zhang T., Lin G., and Yue L., (2009). Inulinase expressing microorganisms and applications of inulinases. Applied Microbiology and Biotechnology, 82:

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