Immobilized Glucose Isomerase Research Articles

Glucose isomerase catalysed isomerisation reactions of (2 R,3 R)-configured aldofuranoses into the corresponding open-chain 2-ketoses

Immobilised glucose isomerase (EC 5.3.1.5) accepted various (2 R,3 R)-configured aldofuranoses such as d-erythrose, as well as homologous C-5-modified d-ribose derivatives, as substrates. In the case of d-crythrose, quantitative conversion into d- glycero-tetrulose took place. d-Ribofuranoses were converted into the corresponding open-chain 2-ketoses in isolated yields of 65%. Surprisingly, l-crythrose also turned out to be a substrate of this enzyme.

Simultaneous effects of immobilization and substrate protection on the thermodynamics of glucose isomerase activity and inactivation

The influence of enzyme immobilization on the main thermodynamic quantities of glucose isomerization to fructose and vice versa was studied in batch tests by using a commercial immobilized glucose isomerase. The values calculated for the immobilized system showed, when compared with those reported for the native enzyme, higher equilibrium constants at T > 70°C and simultaneous increases in both standard enthalpy and entropy changes of reaction. Activation enthalpy and entropy changes of both forward and reverse enzyme-catalyzed reactions were calculated from the initial glucose isomerase activity evaluated at different temperatures by the Briggs-Haldane model. The activation enthalpy of the forward reaction was about 24% less than that of the native enzyme. Enzyme inactivation tests performed at different temperatures and sugar equilibrium concentrations confirmed the existence of the so-called substrate protection phenomenon which resulted in a 29–36% reduction in the decay constant. A slight increase in the activation free enthalpy of the inactivation reaction was also shown.

Immobilized Glucose Isomerase Use in Mirin Production

ABSTRACTThe conversion of glucose in mirin (an alcoholic seasoning) to fructose using immobilized glucose isomerase (IGI) was studied in order to produce sweetened mirin without chill haze. The initial conversion velocity (Cm/tm) with IGI was affected by temperature, pH, and ethanol concentration. This reaction was first order, for which the temperature coefficient (Q10) over the range 20–55°C was 2.0. The value of Cm/tm was a maximum at pH 8.0, and decreased with an increase in ethanol. Conversions carried out in a continuous column reaction system had Cm/tm values 36 times higher than that in a batch system. Mirin treated with IGI did not form chill haze.

Simulation of a reactor for glucose isomerization to fructose by immobilized glucose isomerase with continuous enzyme renewal

Two different dispositions of laboratory-scaled columns have been tested to simulate the isomerization of glucose to fructose in a mobile bed reactor where exhausted immobilized glucose isomerase is continuously renewed. If the simulation columns working at 65°C are arranged in parallel and connected to a section for final enzyme exploitation at 75°C, a syrup with constant composition can be produced, at relatively constant total throughput, by feeding the individual columns at flow rate decreasing according to the enzyme decay profile and following a programmed disphased mode of operation.

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase in the presence of substrate protection

The activity of immobilized glucose isomerase of Streptomyces murinus has been tested batchwise under different conditions in order to gather the related kinetic parameters necessary to optimize an immobilized enzyme column for the continuous production of high fructose corn syrup (HFCS). To this purpose, the Briggs-Haldane model incorporating an apparent first-order inactivation constant has been used with success. A comparison of the equilibrium constants and of the maximum theoretical conversion yields calculated at different temperatures with those estimated for the native enzyme demonstrates that the immobilization favours the transformation of glucose to fructose only at T > 70 °C, as a possible consequence of a combined effect of catalysis and equilibrium thermodynamics enhancement. Enzyme inactivation has also been tested at different temperatures and sugar concentrations to evaluate the related kinetic parameters under different conditions of substrate protection.

Resistance of biocatalyst fixed beds undergoing particle destruction

The hydrodynamic resistance in a biocatalyst particle fixed bed reactor was studied. The biocatalyst used was immobilized glucose isomerase supported by an organic carrier composed of cross-linked proteins. The bed structural deterioration caused by compression-enhanced particle fragmentation and washing-out was considered. The resistance functions (friction factor vs. Re) for four different cases of polydisperse material with increasing spread of the size distribution, corresponding to different extents of particle destruction, were evaluated. The experiments were repeated with three different values of the bed compression, corresponding to different sample positions in the large-scale isomerization column. The relationships obtained exhibited slopes slightly greater than those predicted theoretically for stable packings. This effect can be explained by the increased irregularity of voids due to bed degradation, accompanied by the formation of hollow spaces and sudden changes in the cross-section of the channels. A semiempirical correlation recommended by Reichelt was found to represent the experimental data well for the practically important region of Re > 0.5.

Response surface optimization for the continuous glucose isomerization process

Production of fructose via a continuous glucose isomerization process was optimized using response surface methodology. Glucose isomerization was performed using immobilized glucose isomerase in a flow-through tubular reactor. Process factors eg pH (7.0–7.8), temperature (50–60°C), flow rate (5–17 ml min−1) and glucose content (30–50% w/w) of the feedstock solution were simultaneously tested according to a central composite experimental design. Measured responses such as % isomerization, and fructose yield (gh−1) has an excellent correlation with tested factors. The highest desirability,D, (geometric mean of % isomerization and fructose yield) was obtained when the feedstock (56–60°C) had 34–36% glucose, a pH of 7.4–7.8 and was pumped at 15 ml min−1.

Simulation of glucose isomerase reactor: optimum operating temperature

A design equation for immobilized glucose isomerase (IGI) packed bed reactor is developed assuming enzyme deactivation and substrate protection. The developed equation is used to simulate the performance of the reactor at various temperatures (50–80 °C). Enzyme deactivation is significant at high temperature. Substrate protection showed to have significant effect in reducing enzyme deactivation and increasing the enzyme half-life. Factors affecting the optimum operating temperature are discussed. The optimum operating temperature is greatly influenced by the operating period and to a lesser extent with both initial glucose concentration and glucose conversion.

High fructose syrup production using fructose-selective liquid membranes

Fructose is separated from a fructose–glucose mixture using a polymer-supported liquid membrane containing a boronic acid carrier; addition of immobilized glucose isomerase to a solution of glucose in the source compartment of a liquid membrane transport cell produces a receiving phase mixture that is more than 80% fructose.

Kinetic study of fructose‐glucose isomerization in a recirculation reactor

AbstractWe have studied the equilibrium and kinetics of fructose‐to‐glucose isomerization using a commercial immobilized glucose isomerase, Sweetzyme T® (from NOVO), in a packed‐bed recirculation reactor which enabled us to eliminate the influence of external‐mass transfer and which could function as a differential reactor when a sufficient flow rate was used. The results obtained have also been corrected with regard to the influence of internal transport in the immobilized enzyme particles.Isomerization kinetics is a pseudo first‐order reversible reaction in the ranges studied: 303–333 K, pH 7.5 and 0.5 to 2.0 mol/L hexose concentrations. We compare our results with those found in the literature, and propose equations for the influence of temperature on the equilibrium and on the kinetic constants.

Glucose isomerase (EC 5.3.1.5) as a reagent in carbohydrate synthesis: success and failures with the isomerisation of non-natural derivatives of d-

The properties of glucose isomerase (EC 5.3.1.5) and its synthetic applications are briefly reviewed and recent results with non-natural substrates are discussed. Commercially available immobilized glucose isomerase from Streptomyces murinus, previously discovered to accept d-glucose as well as d-fructose derivatives with modifications at C-3, C-5, and C-6, was also found to be an efficient catalyst for the conversion of 5,6-dimodified derivatives of d-glucose as well as 5-modified d-xylofuranose into the corresponding open-chain 2-ketoses. The equilibrium mixture in all but one case investigated favoured the respective ketose in ratios of 3:1 to 4:1. These results are in agreement with molecular modelling experiments suggesting that the free energies of the furanoid aldoses serving as starting materials are generally slightly higher than the values for the corresponding open-chain ketose. Dimerisation of the open-chain sugar derivatives was not observed.

Immobilization ofStreptomyces atratus glucose isomerase on various supports

The immobilization ofStreptomycs atratus glucose isomerase has been achieved and the periods of half-life of the immobilized glucose isomerase have been determined on various supports, among which the most acceptable are cotton lint and polyurethane. The stability of the immobilized preparations has been demonstrated.

Analysis of substrate protection of an immobilized glucose isomerase reactor

The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme-substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, eta(D), along the length of the packed bed. The axial distribution profile of eta(D) was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long-term operation.

Immobilization of glucose isomerase on Indion 48-R

The glucose isomerase from Streptomyces sp. NCIM 2730 was produced using wheat bran or bagasse hydrolyzate as an inducer and immobilized on an inexpensive anion exchange resin Indion 48-R. The adsorption capacity and efficiency of the resin were found to be 25 U g −1 and 60%, respectively. The properties of soluble and immobilized glucose isomerase were compared. The stability of the enzyme at higher temperatures and acidic pH is increased due to immobilization. Concentrated glucose syrup (25%) was efficiently converted to fructose by the immobilized enzyme. After the seventh reuse, the conversion capacity of immobilized glucose isomerase was reduced to 61%, with a 35% loss in enzyme activity.

Immobilization of glucose isomerase onto granular chicken bone.

Glucose isomerase was immobilized onto granular chicken bone (BIOBONE) by adsorption. The amount of activity bound relative to an equal amount of free enzyme was 32 +/- 1%, with the estimated specific activity decreasing from 11.1 +/- 0.7 to 3.9 +/- 0.5 U/mg protein with immobilization. Compared with the free enzyme, immobilized glucose isomerase showed a threefold increase in the Km for fructose and a fivefold decrease in Vmax. High operating temperatures were possible (greater than 55 degrees C), but continuous use and long-term storage studies showed gradual losses of activity. Both the binding and the activity of the bone-immobilized enzyme were highly resistant to treatments with detergent, ethanol, and KCl. Studies to determine mass transfer limitation effects on immobilized glucose isomerase showed that these were insignificant for this system.

Reactor design for the enzymatic isomerization of glucose to fructose

A comprehensive methodology is presented for the design of reactors using immobilized enzymes as catalysts. The design is based on material balances and rate equations for enzyme action and decay and considers the effect of mass transfer limitations on the expression of enzyme activity. The enzymatic isomerization of glucose into fructose with a commercial immobilized glucose isomerase was selected as a case study. Results obtained are consistent with data obtained from existing high-fructose syrup plants. The methodology may be extended to other cases, provided sound expressions for enzyme action and decay are available and a simple flow pattern within the reactor might be assumed.

Development of reactor model for glucose isomerization catalyzed by whole-cell immobilized glucose isomerase

Based on the kinetic constants determined and the mathematical model of the reactor system developed, the performance of axial flow packed bed continuous enzyme reactor system was studied experimentally and also simulated with the aid of a computer for ultimate objective of optimization of the glucose isomerase reactor system.

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory‐scale reactor

In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.

One-step conversion of cellulose to fructose using coimmobilized cellulase, β-glucosidase, and glucose isomerase

Glucose isomerase was immobilized by itself and coimmobilized with cellulase and β-glucosidase using a polyurethane foam (Hypol® FHP 2002). Approximately 50% of the enzyme added was immobilized. The immobilized enzyme was active at pH values as low as 6.8. When immobilized alone, the Km for Mg2+ increased by 5.5fold and the Km for fructose increased 62%. The half-life of the immobilized glucose isomerase was approximately 160 h of continuous hydrolysis, with a substantial (about 35–40%) amount of activity remaining even after 1000 h. When all three enzymes were immobilized together, the system was found capable of functioning at pH 7.0 to produce fructose from both soluble and insoluble cellulose substrates. At this pH, the glucose:fructose ratio was 70:30. The advantageous properties of the foam as a support for enzyme immobilization and the efficiency of the one-step conversion process outlined combine to make this system appear valuable for use in high fructose syrup production.

An automated glucose isomerase reactor system with online flow injection analyzers for monitoring of pH, glucose and fructose concentrations

The literature on glucose isomerization with immobilized glucose isomerase has been reviewed with respect to thermodynamics and kinetics of the reaction and the industrial process layout. A laboratory scale fixed-bed reactor for isomerization of glucose with immobilized glucose isomerase has been constructed. A system for sample withdrawal and three flow injection analyzers—measuring glucose, fructose and pH—has been developed, automated and used to monitor the process. The equipment is controlled by a personal computer with interface expansion boards. The isomerization process has been monitored in steady-state operation as well as in pH transients. Using a commercial immobilized glucose isomerase (Sweetzyme Q from NOVO Industri A/S), the isomerization process has been studied with respect to equilibrium conversion, composition of the feed syrup, film diffusion resistance, enzyme deactivation and buffering effect of the immobilized enzyme. The experience from computerization of the equipment and the use of flow injection analyzers is summed up and the potential of such analyzers for monitoring biotechnological processes is discussed.