Immobilized Inulinase on Grafted Alginate Beads Prepared by the One-Step and the Two-Steps Methods (original) (raw)
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IMMOBILIZATION OF GLUCOSE ISOMERASE IN SURFACE-MODIFIED ALGINATE GEL BEADS
Journal of Food Biochemistry, 2008
In this study, glucose isomerase enzyme was entrapped into modified and nonmodified calcium alginate gel beads. Various characteristics of free and immobilized enzymes such as the optimum pH, temperature and dependence of activity on storage and operational stability were evaluated. The optimum pH and temperature of free and immobilized glucose isomerase were found to be the same values as 7.5 and 60C, respectively. For free and immobilized enzymes, kinetic parameters were calculated as 1.79 ¥ 10 -2 and 8.27 ¥ 10 -3 mol/L for K m , and 2.39 ¥ 10 -3 and 6.03 ¥ 10 -3 mol/L min for V max , respectively. After 42 days of storage at 4C, free enzyme retained 56% of its initial activity, while for the immobilized enzyme, this value was observed as 86%. The immobilized samples were used repeatedly 22 times by retaining more than 85% of their initial activity.
Enzyme and Microbial Technology, 1985
fructan fructanohydrolase, EC3.2.1.7} activity have been immobilized in open pore gelatin pellets with retention of >90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6. O. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mo1-1, respectively. K m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.
From Inulin to Fructose Syrups Using Sol–Gel Immobilized Inulinase
Applied Biochemistry and Biotechnology
The present work aims to provide the basic characterization of sol–gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature– and pH–activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis–Menten kinetics suggest that immobilization in sol–gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol–gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol–gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol–gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.
Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology
Biocatalysis and Biotransformation, 2012
This work focuses on the immobilization of a crude inulinase extract obtained by solid-state fermentation using spraydrying technology. Maltodextrin and arabic gum were used as immobilizing agents. The effects of inlet air temperature, maltodextrin/arabic gum ratio and mass fraction of crude enzyme extract on the activity of immobilized inulinase were assessed using a central composite rotatable design (CCRD) (2 3 ). The optimum operational conditions for the immobilization of inulinase by spray-drying was obtained at an inlet air temperature of 200 ° C, mass fraction of crude enzyme extract of 0.5 wt% and using only arabic gum as immobilizing agent. The immobilized enzyme had good thermostability, comparable with other inulinases obtained from different microorganisms. The method used gave good enzyme activity after immobilization and could be applied to other enzymes which have good thermal stability.
Enzyme immobilization and its applications in food processing: A review
International Journal of Chemical Studies
The "immobilized enzymes" are the enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities which can be used repeatedly and continuously. Enzyme immobilization provides an excellent base for increasing availability of enzyme to the substrate with greater turnover over a considerable period of time. Immobilized enzymes are preferred over their free counterpart due to their prolonged availability that reduces redundant downstream and purification processes. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. The choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. The industrial applications of immobilised enzymes are progressively increasing. Immobilized enzymes find use in a number of biotechnological products with applications in diagnostics, bio affinity chromatography, and biosensors. Immobilised enzymes find wide applications in the food industry. With these immobilised enzymes, it is possible to obtain different types of sugar syrups, lactose free milk, clarified and debittered juices and wines. Immobilised enzymes can be employed for the production of different active packaging material like oxygen scavenging, anti-microbial films. However, commercialization of immobilized enzymes is still at a slower pace because of their costs and storage problems. Research should be focused to overcome the current limitations related to immobilization techniques, so as to expand the horizon for all-round application. In future, immobilized enzymes are going to play a vital role in various industries including pharmaceuticals, chemicals, food and fuel.
In recent times, inulinase has emerged as one the most prominent and industrially upcoming enzymes applied to meet the ever increasing demand of D-fructose and fructooligosaccharides (FOS) as sweetener and prebiotics in the food and pharmaceutical industry, respectively. This review deals with types of inulinase and the attempts made to modify it for better thermal stability and shelf life. The ease of immobilization of inulinase has led us to the path of experimenting with different methods of enzyme immobilization since 1979. Several modes of immobilization ranging from simple cross-linking of enzymes onto a polymer support to nanoparticles have been applied over the years. The approach and concept of this review provide a yet unexplored focus on pioneering advances for the development of white biotechnology, for instance production of immobilized inulinase-based reusable biocatalysts and bioreactors designed for their use and for the continuous production of fructose and FOS.
Immobilization of commercial inulinase on alginate–chitosan beads
Sustainable Chemical Processes, 2014
The commercial inulinase obtained from Aspergillus niger was effectively immobilized on alginate-chitosan beads which were hardened with glutaraldehyde. The immobilization conditions were studied using Plackett & Burmann experimental design and central composite rotational design (CCRD). The effects of chitosan, glutaraldehyde, sodium alginate and calcium chloride concentrations in order to obtain a better immobilization yield were optimized. In the Plackett & Burman experimental design, the sodium alginate and calcium chloride had a significant effect (p < 0.1), but only the calcium chloride showed a positive effect, indicating that as higher the concentration, better is the immobilization yield. In the central composite rotational design (CCRD), the best results were obtained in the central point, using sodium alginate (1% w/v) and calcium chloride (4% w/v) as conditions for inulinase immobilization. By the CCRD, the optimal immobilization strategy was: chitosan (0.1% w/v), glutaraldehyde (0.1% v/v), sodium alginate (1% w/v) and calcium chloride (4% w/v). In this condition, the enzyme loading capacity was 668 U/g gel beads and the effect of temperature on the immobilized enzyme activity was also evaluated, showing better activity at 50°C. The immobilized enzyme maintained 76% of its activity in six days at room temperature.
A novel alginate–CMC gel beads for efficient covalent inulinase immobilization
Colloid and Polymer Science, 2017
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.