Rapid and efficient bioconversion of chicory inulin to fructose by immobilized thermostable inulinase from Aspergillus tubingensis CR16
© Trivedi et al. 2015
Received: 1 April 2015
Accepted: 29 June 2015
Published: 15 July 2015
Fructose, a monosaccharide, has gained wide applications in food, pharmaceutical and medical industries because of its favourable properties and health benefits. Biocatalytic production of fructose from inulin employing inulinase is the most promising alternative for fructose production. For commercial production, use of immobilized inulinase is advantageous as it offers reutilization of enzyme and increase in stability. In order to meet the demand of concentrated fructose syrup, inulin hydrolysis at high substrate loading is essential.
Inulinase was immobilized on chitosan particles and employed for fructose production by inulin hydrolysis. Fourier transform infrared spectroscopy (FTIR) analysis confirmed linkage of inulinase with chitosan particles. Immobilized biocatalyst displayed significant increase in thermostability at 60 and 65 °C. Statistical model was proposed with an objective of optimizing enzymatic inulin hydrolytic process. At high substrate loading (17.5 % inulin), using 9.9 U/g immobilized inulinase at 60 °C in 12 h, maximum sugar yield was 171.1 ± 0.3 mg/ml and productivity was 14.25 g/l/h. Immobilized enzyme was reused for ten cycles. Raw inulin from chicory and asparagus was extracted and supplied in 17.5 % for enzymatic hydrolysis as a replacement of pure inulin. More than 70 % chicory inulin and 85 % asparagus inulin were hydrolyzed under optimized parameters at 60 °C. Results of high performance liquid chromatography confirmed the release of fructose after inulin hydrolysis.
The present findings prove potentiality of immobilized thermostable inulinase from Aspergillus tubingensis CR16 for efficient production of fructose syrup. Successful immobilization of inulinase on chitosan increased its stability and provided the benefit of enzyme reutilization. Box-Behnken design gave a significant model for inulin hydrolysis. Extraction of raw inulin from chicory and asparagus and their enzymatic hydrolysis using immobilized inulinase suggested that it can be a remarkable cost-effective process for large-scale fructose production.
KeywordsChitosan particles Inulinase Immobilization Thermostable Inulin hydrolysis Chicory inulin Fructose
From the industrial point of view, fructose is becoming an increasingly important ingredient in food and pharmaceutical products. Fructose is a GRAS sweetener with flavour-enhancing properties. It shows higher solubility and is 1.5 times sweeter than sucrose and thus has preferably replaced sucrose in many foods and beverages . Commercial production of fructose syrup is based on either multienzymatic hydrolysis of starch or less favoured invert sugar production with invertase. Multienzymatic hydrolysis of starch requires action of three different enzymes with different reaction conditions. Starch is hydrolyzed to dextrins by α-amylase, then saccharified to glucose by glucoamylase and finally glucose is isomerised to fructose by glucose isomerase. However, due to the thermodynamical equilibrium, this process yields only 45 % fructose. Another less-favoured approach for obtaining fructose syrup is from the invert syrup obtained from sucrose hydrolysis by the action of invertase. But this process also requires separation of fructose from glucose and thus not preferable at commercial level. Hence, fructose production by inulin hydrolysis is the most advantageous approach as it requires only a single step enzymatic reaction with high fructose yield. Inulin is a well-known carbohydrate polymer made up of linear chain of β-2,1-linked d-fructofuranose molecules which is terminated by a glucose molecule in a sucrose type linkage. Inulin, as a storage polymer, is found in the roots and tubers of many plants of Compositae family, e.g. Jerusalem artichoke, Dahlia, Chicory etc., and because of high content of d-fructose (>75 %), it is recognized as a raw material for the production of high fructose syrup . In addition to this, inulin has better solubility than starch, in water at higher temperature and forms less viscous solution even at higher concentration . Total or partial hydrolysis of inulin, leading to syrups with high fructose content, can be achieved by the action of inulinases . Inulinases are classified as endoinulinase (2,1-β-d-fructan fructanohydrolase—EC.184.108.40.206) which act on internal linkages of inulin to release oligosaccharides as end products and exo inulinase (β-d-fructan fructanohydrolase—EC.220.127.116.11) which act on terminal molecules to release fructose as an end product. Most of the exoinulinases can hydrolyse sucrose along with inulin. The production of fructose syrup from inulin can be achieved through enzymatic hydrolysis by exoinulinase acting either alone or synergistically with endoinulinase.
Inulin hydrolysis using inulinase proves particularly more effective for industrial purposes if an immobilized form of enzyme is used, as it offers several technological advantages . Chitosan, a partially deacetylated polymer of n-acetyl glucosamine, consists of β (1,4)-linked d-glucosamine residues and is relatively reactive natural biopolymer. Over the past two decades, various attempts for enzyme immobilization on chitosan particles have been reported. However, preparation processes are found to be tedious and time consuming or contain agents that are harmful to humans, which limit their applications in food and pharmaceutical industries . Thus, there is a need of a simple, yet effective method of immobilization which can be applied for fructose production . In order to meet the demand of concentrated fructose syrup, inulin hydrolysis at high substrate loading is essential. In addition to that, lower complexities as well as concomitant cost reduction are foreseen for the operation of process. This objective can be achieved through the replacement of pure inulin by raw inulin for enzymatic hydrolysis for fructose production. Chicory and asparagus are among the well-known inulin rich crops. In chicory, inulin is stored as a reserve carbohydrate in the fleshy tap root and constitutes about 70–80 % of root dry weight . Asparagus is a perennial herb, which stores up to 15 % inulin in its roots. Raw inulin extraction and hydrolysis from dahlia and asparagus have been studied well, but the reports which describe inulin extraction from chicory and its hydrolysis are very few. In addition to this, studies reported in the literature do not take into account high inulin concentration for inulin hydrolysis which can directly render concentrated fructose syrup.
In the present communication, we report immobilization of indigenously produced inulinase of Aspergillus tubingensis CR16 on chitosan particles and its utilization for the bioconversion of inulin to fructose. Properties of free and immobilized inulinase have been compared. Systematic evaluation of key operational parameters for inulin hydrolysis has been carried out, and an optimized process has been developed using immobilized inulinase. Additionally, raw inulin was extracted from chicory and asparagus and was subjected to enzymatic hydrolysis along with pure chicory inulin. It is worth denoting that we are describing here, inulin bioconversion in a batch reactor, at the highest reported inulin concentration at industrially preferred temperature (60 °C) using immobilized inulinase.
Inulin (chicory), glutaraldehyde solution and potato dextrose agar (PDA) were from HiMedia, chitosan from shrimp shells (deacetylation ≥75 %) and tripolyphosphate (TPP) were from Aldrich (Sigma). Wheat bran was bought from local vendors of Anand. Corn steep liquor (CSL) was procured from Anil Starch Ltd., Ahmedabad, India. Dry chicory roots were provided by Pioneer Chicory Factory, Anand, India. Asparagus roots were procured from the Directorate of Medicinal Plants and Herbs, Anand. After milling and sieving of dry chicory roots and asparagus roots, the resultant powder was directly used for extraction.
All the other chemicals used were of analytical grade.
Inulinase was produced using wheat bran and corn steep liquor as a substrate under solid state fermentation by A. tubingensis CR16 as describe in our earlier work . Crude inulinase was partially purified by ammonium sulphate precipitation (40–80 %) followed by dialysis against 0.2 M sodium acetate buffer (pH 5.0) and was used for immobilization.
Immobilization of inulinase on chitosan particles
Chitosan particles were synthesized by ionic gelation of chitosan with TPP anions as described by Dounighi et al.  with suitable modifications. Size determination and zeta potential of particles were determined by Zetatrac (Microtrac 10.6.1). Surface characteristics of chitosan particles were observed using scanning electron microscopy.
In order to confirm the cross-linking of inulinase on chitosan particles as well as analysing their chemical functionalization, fabricated chitosan particles (with and without inulinase) were subjected to IR analysis using ABB-MB3000 IR analyzer, (ABB India Ltd.)
Inulinase was assayed by measuring the concentration of reducing sugars by 3,5-dinitrosalicylic acid (DNS) . Reaction mixture, consisting of 0.1 ml of appropriately diluted enzyme and 0.9 ml of 1 % inulin in 0.2 M sodium acetate buffer (pH 5), was incubated at 60 °C for 20 min and was terminated by further incubation in boiling water bath for 10 min. One unit of inulinase activity was defined as the amount of enzyme necessary to release 1 μmol of fructose per minute under the above conditions. Total sugars were analysed as reducing sugars obtained after complete acid hydrolysis of inulin (H2SO4, pH 2.0, 100 °C, 1 h).
Characterization of immobilized and free inulinase
Influence of immobilization on physico-chemical properties of inulinase was evaluated. Effect of temperature and pH on inulinase activity was studied in the range of 50–70 °C and pH 3–8 (0.2 M citrate buffer for pH 3, 0.2 M sodium acetate buffer for pH 4, 5 and 6, 0.2 M sodium phosphate buffer for pH 7 and 8), respectively, for free and immobilized inulinase. Temperature stability of immobilized and free inulinase was analysed by incubating enzymes at 60 and 65 °C for 4 h. Samples were withdrawn at different time intervals and were analysed for residual inulinase activity. Half-life of immobilized and free inulinase was calculated.
Study of enzyme kinetics
Effect of substrate concentration was evaluated in the range of 0.2 to 2 % inulin concentration at 60 °C. Km and Vmax for free and immobilized inulinase were determined using Lineweaver-Burk plot.
Optimization of inulin hydrolysis using three factor Box-Behnken design
BBD design for optimization of inulin hydrolysis as per coded and decoded values of parameters
Enzyme concentration (U/g)
Substrate concentration (%)
Predicted ln sugar released (μg/ml)
Experimental ln sugar released (μg/ml)
Systems were analysed for the release of reducing sugars at specific time interval as planned in BBD. Control reactions were also carried out to discount the presence of sugar out of enzymatic hydrolysis. Statistical analysis was done using software MINITAB 16. Graphs were generated to highlight the roles played by various factors and to emphasize the roles played by physical constraints and biochemical aspects in the final sugar yield released from inulin hydrolysis.
Runs were carried out under optimized conditions, and reducing sugars were estimated by DNS. The extent of inulin hydrolysis (%) was calculated as ratio of reducing sugars released from enzymatic inulin hydrolysis to that from acid hydrolysis multiplied by 100.
High performance liquid chromatography (HPLC) of inulin hydrolysate was performed against standard glucose, fructose and sucrose solutions using HPLC (Shimadzu, Japan) equipped with Bio-Rad Aminex-87C column with mobile phase 5 mM H2SO4 and 0.6 ml/min flow rate at column temperature 65 °C using RID detector.
Extraction of inulin and raw inulin hydrolysis
Dried chicory roots and asparagus roots were ground to powdered form. Extraction of inulin from chicory root powder and asparagus root powder was carried out under boiling conditions. Extracts were filtered through muslin cloth. Filtrate was analysed for reducing sugars and total sugars, and inulin content was determined. Raw inulin concentration in both the extracts was adjusted to 17.5 %, and hydrolysis was carried out using optimized parameters.
Reusability of immobilized inulinase
Immobilized inulinase was reused for hydrolysis of 17.5 % inulin using 10 U/g of enzyme loading for ten successive cycles of 8 h at 60 °C and pH 5.0. Biocatalyst was recovered at 6000 rpm, at 4 °C for 20 min. Hydrolysates were analysed for the amount of reducing sugar released from inulin hydrolysis. The amount of sugar released in the first cycle was considered to be 100 %, and the sugar yield of the successive cycles was presented as percentage relative sugar yield.
Results and discussion
Fabrication of chitosan particles
Immobilization of inulinase
Immobilization of inulinase was performed on activated chitosan particles. Chitosan particles were activated using 1.25 % glutaraldehyde. Free amino groups present in the chitosan molecule react with free aldehyde group of glutaraldehyde to form a cross-linked complex. Furthermore, the amino groups present on the enzyme molecule can complex with the free aldehyde groups of glutaraldehyde enabling covalent cross-linking of the enzyme.
Partially purified inulinase (200 U) was added to chitosan particles (1 g) and was incubated for 18 h. Immobilization yield of 66.4 ± 0.9 % and activity yield of 36.4 ± 0.4 % were achieved after 18 h of incubation. The amount of enzyme units attached per gram of support (132 U/g) was much higher as compared to other such studies carried out by Gill et al. , Ettalibi et al.  and Yun et al. . Gill et al.  reported immobilization of 30 U/g of inulinase on chitin with an immobilization yield of 78 % after incubation time of 24 h. Ettalibi et al.  have reported inulinase loading of 50 U/g of porous glass beads as a support and achieved 77 % of immobilization yield after 24 h of incubation. Similarly, Yun et al.  achieved immobilization of 72 U/g of inulinase on chitosan as a support material, but they were able to immobilize 217 U/g of inulinase on polystyrene carrier UF93 as a support material.
In the present studies, activity yield was lesser compared to immobilization yield. It is possible that because of immobilization, some active sites may remain unexposed at high enzyme loading. Similar studies have been carried out by Valerio et al. , and they have suggested that when higher enzyme concentration is immobilized, the binding sites of the carrier get saturated with enzyme which further leads to diffusion limitation phenomena.
FTIR analysis of inulinase immobilized on chitosan particles
Effect of temperature on immobilized and free inulinase activity
Effect of pH on immobilized and free inulinase activity
Determination of kinetic parameters
Study of kinetic parameters of free and immobilized inulinase was studied in the range of 0.2 to 2 % inulin. Michaelis-Menten constant calculated for immobilized and free inulinase, from Lineweaver-Burk plot, was 8.3 and 7.4 mg/ml, respectively, showing that immobilization decreased the affinity of inulinase for inulin. This might be because of the change in the charged groups of active sites due to the formation of additional bond between enzymes and support . Vmax for immobilized and free inulinase remained almost unaffected by immobilization (52.6 and 52.0 μmol/ml/min, respectively) revealing that immobilization did not interfere with the reaction velocity.
Optimization of inulin hydrolysis
Regression coefficients of ln sugar released
Standard error coefficient
Enzyme concentration (U/g)
Substrate concentration (%)
Enzyme concentration (U/g) × Enzyme concentration (U/g)
Substrate concentration (%) × Substrate concentration (%)
Time (h) × Time (h)
Enzyme concentration (U/g) × Substrate concentration (%)
Enzyme concentration (U/g) × Time (h)
Substrate concentration (%) × Time (h)
Analysis of variance for ln sugar released
Degree of freedom
Sequential sum of squares
Adjusted sum of squares
Adjusted mean squares
Validation of the experimental model
In order to determine the accuracy of the model and to validate the model, experiments were repeated in triplicates using the predicted optimized parameters. The yield of reducing sugars was 171.1 ± 0.3 mg/ml at 17.5 % inulin using 9.9 U/g of inulinase after 12 h reaction time at 60 °C and was in close accordance with the predicted value (ln 12.00). When compared with the reducing sugars released from the acid hydrolysis of inulin, it was found that more than 95 % of inulin hydrolysis was achieved through enzymatic inulin hydrolysis, using optimized parameters.
There are many studies on inulin hydrolysis but to the best of our knowledge, this is the first report of inulin hydrolysis at high substrate concentration with high sugar yield using inulinase immobilized on chitosan particles. The results obtained in the present work were better as compared to the previous reports. Matunda et al.  have shown 70.9 % fructose yield by hydrolysis of 15 % inulin using free inulinase from Aspergillus ficuum after 48 h at 50 °C, while Sirisansaneeyakul et al.  reported hydrolysis of 10 g/l inulin with fructose yield of 3.7 % after 20 h at 40 °C using mixed inulinases from Aspergillus niger and Candida guilliermondii.
Raw inulin extraction and its enzymatic hydrolysis
Reusability of immobilized biocatalyst for inulin hydrolysis
The present study describes the successful application of immobilized inulinase from A. tubingensis CR16 for the production of fructose syrup by inulin hydrolysis. Inulinase was immobilized on chitosan particles with 66.4 ± 0.9 % immobilization yield and 36.4 ± 0.4 % activity yield. Immobilization increased thermal stability of inulinase. Statistical optimization of inulin hydrolysis depicted the parameters which could give the maximum possible fructose yield. Also, it is worth mentioning that even at very high inulin concentration (17.5 %) more than 95 % inulin hydrolysis was achieved from pure inulin. Along with that, high concentration of raw inulin from chicory roots and asparagus roots were hydrolysed at 60 °C after 12 h in a single step. Immobilized inulinase was found to be reusable for ten cycles. Thus, the above findings prove the potential of immobilized inulinase for inulin hydrolysis for large-scale fructose production at industrial level.
ST acknowledges the staff of Shree P.M.Patel College of Paramedical Science and Technology for their kind cooperation.
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