Immobilization Of Inulinase Research Articles

APPLICATION OF SUPER-CROSSLINKED POLYMERS AS CARRIERS OF HETEROGENEOUS BIOCATALYSTS FOR INULINHYDROLYSIS REACTION

n this work the study of adsorption immobilization of inulinase on super-crosslinked macroporous sorbents based on styrene and divinylbenzene: low-base anion exchanger A100, high-base anion exchanger A500R, strong acid cation exchanger C100H was carried out. The influence of the duration of the sorption process, the value of the concentration of hydrogen ions and the concentration of protein in solution on the amount of immobilized enzyme and the activity of the obtained heterogeneous biocatalysts is considered. It was revealed that adsorption reaches its max-imum value on average after 4 h at pH of 4.7-5.0 for the sorbents under consideration. The activity of the obtained heterogeneous biocatalysts is 64.8-83.5% of the activity of free inulinase. Although the activity of inulinase during adsorption on the polymer carriers used in the work decreases, the integral amount of the resulting product will be higher than for the native catalyst.The isotherms of inulinase adsorption on super-crosslinked polymers have been studied. The high stability of the obtained heterogeneous biocatalysts is explained by the high sorption capacity of super-crosslinkedsorbents. It was revealed that immobilized inulinase can be used during 8-11 consecutive cycles of the substrate hydrolysis reaction.Using the BET adsorption theory, the enzyme sorption isotherms were analyzed and the equilibrium sorption parameters werecalculated: the maximum amount of sorbed inulinase, the constants characterizing the sorbate-sorbent and sorbate-sorbate interaction. Among the considered carriers, the highest value of the sorption equilibrium constant (KL=4.25±0.04 l/mmol) corresponds to the inulinase-cation exchanger biocatalyst, the sorption ca-pacity of which is also the largest. The data obtained is of interest for evaluating the effectiveness of the use of sorbents as inulinase carriers in subsequent technological operations of processing of inulin-containing raw materials.

Production, purification and immobilization of inulinases from Aspergillius species

Inulinases are enzymes catalysing hydrolysis of polyfructosans to produce fructose or fructooligosaccharides; these properties have attracted interest of many researchers towards exploring various plant sources (agro food waste) as substrates. According to the literature, various microorganisms, such as fungi, yeast, bacteria, and actinomycetes, can synthesize inulinase. Producer microorganisms can be the micromycetes Aspergillus, Penicillium, Rhizopus and Fusarium, the yeast Kluyveromyces and the bacteria Clostridium thermosuccinogenes and Bacillus subtilis, using inulin, sucrose, fructose, lactose, raffinose, xylose as sources of carbon. The present study mainly envisages inulinase produced by Aspergillius species grown on a fermentation medium based on different carbon sources (inulin and agro-alimentary by-products). The crude extract was purified by fractional precipitation with ammonium sulphate. The dissolved and dialyzed precipitate was further purified by ion exchange chromatography on DEAE-Sephadex. The experiments led to the obtaining of an inulinase with a specific activity of 43.34 U/mg proteins. Inulinase immobilization tests on chitosan beads have led to a stable biocatalyst at temperature and pH variations that could be useful for obtaining fructose syrup.

Development of a sustainable route for the production of high-fructose syrup from the polyfructan inulin.

The authors used mesoporous silica microspheres as a support for the immobilization of inulinase from Aspergillus brasiliensis MTCC 1344 by the process of cross‐linking. Under optimized operating conditions of pH 6.0, particle/enzyme ratio of 2.0:1.0 and glutaraldehyde concentration of 7 mM, a maximum immobilization yield of 90.7% was obtained after a cross‐linking time of 12.25 h. Subsequently, the cross‐linked inulinase was utilized for the hydrolysis of 5% inulin, and a maximum fructose concentration of 31.7 g/L was achieved under the optimum conditions of pH 6.0 and temperature 60°C in 3 h. Furthermore, on performing reusability studies during inulin hydrolysis, it was observed that the immobilized inulinase could be reused up to 10 subsequent cycles of hydrolysis, thus providing a facile and commercially attractive process of high‐fructose syrup production.

Inulinase immobilization onto glutaraldehyde activated duolite XAD for the production of fructose from inulin

Abstract In the present practice, central composite rotatable design (CCRD) of response surface methodology (RSM) was used to optimise inulinase immobilization onto duolite XAD by gultaraldehyde (GA) cross-linking. After optimization, at optimal levels of variables (GA concentration 1.59%, enzyme coupling time 12 h, GA treatment time 4.0 h and enzyme load 22.7 IU), maximal inulinase activity yield and immobilization yield attained was 53.2% and 76.45%, respectively. A very narrow difference between predicted and experimental results confirms the authenticity of the present model. Besides, coefficient of determination (R2) value approaching one also authenticates the affluence of the designed polynomial model. Maximum hydrolysis (79.5%) of inulin by immobilized biocatalyst was achieved under agitation (125 rpm) mode of hydrolysis. Fructose production from inulin using inulinase immobilized onto duolite XAD was successfully performed for 10 consecutive batch cycles.

Shallow porous microsphere carriers with core-shell structure based on glass beads cross-linking chitosan for immobilizing inulinase

We develop a new type of shallow porous microsphere carriers with core-shell structure by using glass beads as cores, chitosan as coating layer, glutaraldehyde as cross-linking agent and stearic acid powder as porogen. The characterization of FT-IR, SEM, XRD, TGA-DSC and XPS demonstrates that the modification in each step is feasible and chitosan forms a uniformly coated graft layer on the surface, accounting for 40 % of the total weight. Then, this study focuses on immobilization of inulinase on the shallow porous microsphere carriers. The optimal conditions for immobilization are: pH value is 5.0; cross-linking time is 2.5 h; the molar ratio of free enzyme, glutaraldehyde and carrier amino group is 5.0 × 10−3:0.3:1. Among them, pH value has the most significant influence on the reaction process. The prepared immobilized inulinase preserves 73 % activity of free inulinase, and the suitable ranges of pH and temperature widen. Thermal stability experiments are operated at 70 °C for 3 h, when immobilized inulinase still maintains 69.20 % of initial activities whereas free inulinase only maintains 33.79 %. The immobilized inulinase still maintains 77.93 % of initial activities after 10 reuses, and its calculated half-life is 22 times, demonstrating developmental potential in industrial application.

Immobilization of fungal inulinase on hetero-functionalized carbon nanofibers for the production of fructose from inulin

The present study describes the response surface optimization (RSM) of inulinase immobilization on carbon nanofibers (CNFs), modified using 3-aminopropyl-triethoxysilane (APTES) and glutaraldehyde (GA). Immobilized protein content and inulinase activity were used as indicators of immobilization. Under RSM optimised conditions viz., sonication time 120 min, APTES concentration 3.0%, GA concentration 2.0%, GA activation time 75 min, enzyme load 27.5 IU and enzyme coupling time 2.75 h, inulinase activity yield and immobilization yield was 63.92% and 70.87%, respectively. The results revealed successful linking of inulinase onto APTES-GA modified CNFs. Besides, the good agreement between predicted and experimental values authenticates the significance of the model. The developed immobilized biocatalyst showed high operational stability (30 cycles) for fructose production from inulin in a batch system.

Inulinase immobilization on polyethylene glycol/polypyrrole multiwall carbon nanotubes producing a catalyst with enhanced thermal and operational stability.

This paper describes the development of a simple method for mixed non-covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes (f-MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N-H functional groups from PPy and -COO- from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345μg protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV-Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half-life (t 1/2) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.

Immobilization of Inulinase on Aminated Multiwalled Carbon Nanotubes by Glutaraldehyde Cross-Linking for the Production of Fructose

In the present work, an attempt was made to enhance immobilization yield and operational stability of inulinase (Exo-II) from Penicillium oxalicum onto amino (+NH2) terminated multiwalled carbon nanotubes (MWCNTs) through glutaraldehyde (GA) cross-linking. A statistical study using response surface methodology (RSM) was employed to optimise inulinase immobilization onto 3-aminopropyl-triethoxysilane (APTES) and GA modified surfaces of MWCNTs. Under optimized conditions (APTES concentration 4%; sonication time 4 h; GA concentration 2%; GA activation time 75 min; enzyme load 27.5 IU and enzyme coupling time 2.75 h), inulinase activity and immobilization yield obtained were 73.9% and 84.9%, respectively. An increase in inulinase activity (1.22-fold) and immobilization yield (1.14-fold) was observed after modification of aminated MWCNTs by GA cross-linking. Developed immobilized biocatalyst shown excellent operational stability for 43 cycles for fructose production in a batch system. Increased immobilization yield and operational stability of the developed biocatalyst onto APTES-GA functionalized MWCNTs justifies it a good candidate for fructose production from inulin.

Fructose production from inulin using fungal inulinase immobilized on 3-aminopropyl-triethoxysilane functionalized multiwalled carbon nanotubes

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. Km and Vmax of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose.

Amobilisasi Inulinase Aspergillus clavatus Gmn 11.3 Galur Lokal Indonesia dengan Matrik Karbon Aktif

Two types of inulinase are produced by Aspergillus clavatus Gmn 11.3 within the 3rd and 5th days of fermentation. The optimum condition of two types of immobilized inulinase is achieved using 20 grams of activated carbon, 200 meshes with protein adsorption of 96.71% and 96.19% respectively. Following immobilization of inulinase, incubation was carried out for 30 hours to hydrolyze inulin. After incubation, the proteins retained on the matrix are 66.96% of the 3 days fermentation enzymes and 37.36% for 5 days of fermentation enzyme.

Production, characterization, and immobilization of inulinase produced by Pseudozyma sp. (CCMB 306)

ABSTRACTThe present paper focuses on the production and characterization of inulinase produced by Pseudozyma sp. (CCMB 306) and on the subsequent immobilization of this enzyme on solid supports (eggshell, celite, Sepabeads®, silica). A primary aim of the present study was to investigate the effects of glucose and yeast extract concentrations (g/L) on the enzyme produced by Pseudozyma sp. according to response surface methodology (RSM). RSM was also used to study the optimum pH and temperature. The results for the optimization of inulinase production showed that the highest enzyme activity was observed for glucose and yeast extract concentrations of 10 and 1 g/L, respectively. The inulinase activity peaked at 50°C and pH 7.0. The results for enzyme immobilization on solid supports showed that celite, in an incubation medium containing acetone, was the best support for inulinases produced by Pseudozyma sp.

A novel alginate–CMC gel beads for efficient covalent inulinase immobilization

Covalent immobilization of inulinase, produced by the marine-derived fungus Aspergillus terreus, on novel gel beads was made by the combination of alginate and carboxymethyl cellulose. Optimization of the loading time and loading units was done by response surface methodology. The bound enzyme displayed a change in optimum operating pH from 5.0 to 5.5 while the optimum operating temperature increased from 50 to 55 °C. K m value has been increased (from 3.6 to 7.1 mg/ml) in comparison with the free enzyme. However, the V max was lowered (from 145 to 77.5 U/g carrier) after immobilization. The immobilized inulinase showed enhancement in thermal stability against high temperature. There was an observed increase in half-lives and D values which revealed the improvement in the enzyme thermal stability. Thermodynamically, after immobilization, a remarkable increase in enthalpy and free energy was observed due to the enhancement of enzyme stability. Immobilized inulinase showed retention of 60% of its original activity after 10 successive cycles. Stability and reusability of immobilized inulinase on alginate–CMC enable the enzyme to be more convenient for industrial application.

The molecular mechanism of adsorption immobilization of inulinase on polymer matrices

The conditions and mechanisms of the immobilization of inulinase on polymeric carriers were studied using the VION KN-1 and KU-2 cation-exchangers, VION AN-1 and AV-17-2P anion-exchangers, and the ampholyte KOPAN-90. The calculated data showed a significant role of van der Waals interactions and hydrogen bonding in the formation of virtually all inulinase complexes with the immobilization matrices. The AV-17-2P anion-exchanger was the only one of the studied polymer matrices that was unable to form hydrogen bonds with inulinase. The mechanisms of the interaction between inulinase and various ampholytes and cation and anion exchange resins differ from each other. The strongest differences are observed in mechanisms of the sorption of inulinase on VION KN-1 and chitosan matrices. Approximately 87% of the identical amino-acid residues are involved in the interaction of the enzyme with the KU-2 and AV-17-2P resins and the VION AN-1 and KOPAN-90 fibers.

Immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system

An extracellular inulinase was partially purified by ethanol precipitation and gel exclusion chromatography from a cell free extract of Kluyveromyces marxianus. Partially purified inulinase exhibited 420 IU/mg specific activity and it was immobilized on chitosan beads. Activity yield of immobilized inulinase was optimized with glutaraldehyde concentration (1–5%), glutaraldehyde treatment time (30–240min), enzyme coupling-time (2–16h) and enzyme loading (5–30 IU) as functions. Under the optimized conditions maximum yield 65.5% of immobilized inulinase was obtained. Maximum hydrolysis of inulin 84.5% and 78.2% was observed at 125rpm after 4h by immobilized and free enzyme, respectively. A retention-time of 4h and 5h was found optimal for the hydrolysis of inulin under agitation (125rpm) by free and immobilized enzyme, respectively. The recycling of the developed immobilized biocatalyst was carried out after 5h of inulin hydrolysis in a batch system. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. This is the first report on the immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system.

The interaction of inulinase molecules with a chitosan matrix: UV-induced changes in the functional properties of immobilized inulinase

We have developed a technique for the immobilization of inulinase on chitosans with different molecular weights. The acid-soluble mid-molecular-weight (200 kDa) and high-molecular-weight (350 kDa) chitosans are shown to be promising matrices for inulinase adsorption. We assumed that the formation of an inulinase-chitosan matrix complex occurs mainly due to hydrophobic interactions; electrostatic interactions also play an essential role. The enzyme complex with high-molecular-weight chitosan is more stable against the action of UV light and temperature. This allows us to recommend chitosan as a catalyst in industry for the production of fructose from inulin-containing vegetable raw mateials.

Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica: Inulin hydrolysis in a continuous mode operation

Abstract Inulinase hydrolyzes inulin and produce high fructose syrup in a single-step reaction. However, finding the best working conditions and the highest inulinase stability, especially in continuous mode operations are of critical value. Native (endo-inulinase (EC 3.2.1.7)) and chemically modified inulinases (endo-inulinase stabilized with pyridoxal 5′-phosphate/ascorbic acid in a semi-rational chemical modification approach) were immobilized on octadecyl substituted nanoporous silica (with the average pore size of 66 nm) by using dioxane to extend the hydrophobic interactions between inulinase refolding intermediates and the hydrophobic alkyl moieties of the support. Native and modified inulinases revealed their maximum surface hydrophobicity at 40% and 50% dioxane in water, respectively. Immobilized inulinases (native and modified) showed 7.89 and 3.73 fold increase in their half lives compared to the free native sample., higher reusability compared to free inulinases (native and modified) and also could retain their activities after 10 hydrolysis cycles in a continuous mode of experimental conditions.

Preparation of Zinc Tungstate (ZnWO4) Particles by Solvo-hydrothermal Technique and their Application as Support for Inulinase Immobilization

ZnWO4 particles were synthesized as a single-phase by a simple and easy solvo-hydrothermal technique using water-ethylene glycol mixture as solvent, without using surfactant. Physical properties of produced particles were analyzed by X-ray diffraction (XRD), infrared spectroscopy (FTIR), surface area (BET), particles size distribution and atomic force microscopy (AFM). This material was used as support for inulinase immobilization by physical adsorption and the influence of temperature (30 and 50 oC) was evaluated. Material with mesoporous characteristic and with a surface area of 35.5 m2.g-1 was obtained. According to the findings, ZnWO4 present a satisfactory inulinase adsorption, and the better result was 605 U.g-1 support at 30 oC. Therefore, ZnWO4 particles prepared by one-step solvo/hydrothermal route provide a new potential support for inulinase immobilization.

In silico design of high-affinity ligands for the immobilization of inulinase

Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase – an enzyme that cleaves inulin and fructose-containing polymers to fructose – has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking.The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors» to the surface.

Production of Copper and Cobalt Aluminate Spinels and Their Application As Supports for Inulinase Immobilization

Copper and cobalt aluminates were obtained through the use of chitosan as template. In this synthesis route, chitosan is eliminated by heating, and a porous material is produced. These oxides were used as supports for inulinase immobilization by adsorption process. Physical properties of produced particles were analyzed by X-ray diffraction (XRD) and nitrogen adsorption-desorption isotherms. Both oxides presented particles containing mesoporous characteristics and high surface area, which is desirable for applications in enzyme immobilization processes. The results revealed that the copper and cobalt aluminates exhibit high inulinase immobilization efficiencies, which makes them promising supports for enzyme immobilization.

Rapid and efficient bioconversion of chicory inulin to fructose by immobilized thermostable inulinase from Aspergillus tubingensis CR16

Abstract Background 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. Results 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. Conclusions 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.