Enzyme Glucose Isomerase Research Articles

KINETIC STUDY COMPARISON OF IMMOBILIZED GLUCOSE ISOMERASE (GENSWEET AND SWEETZYME IT) IN STIRRED TANK REACTOR AND PACKED BED REACTOR

D- Glucose/xylose isomerase catalysis the reversible isomerization of aldoses to ketoses such as D-glucose and D-xylose to D-fructose and D-xylose respectively. High fructose corn syrup (HFCS), a low calorie sugar substitute for cane sugar, utilizes Glucose isomerase enzyme for conversion of glucose to fructose. The conversion of glucose to fructose favors more at high temperature, providing an incentive to utilize thermostable and thermoactive glucose isomerase in High fructose corn syrup (HFCS) production. Present studies emphasize on enzymatic conversion and optimization using Sweetzyme IT extra & Gensweet, commercially available glucose isomerases. The experiments were carried out for enzymatic conversion of glucose to fructose using Gensweet and Sweetzyme in Packed bed reactor (PBR) and Stirred tank reactor (STR). Maximum conversion was seen in Stirred tank reactor (STR) using both of these enzymes, approx 10 % more Fructose conversion comparing it to packed bed reactor (PBR). Also, Stirred tank reactor (STR) reaction conditions such as pH, buffers, cofactor (MgSO4) requirement were optimized to achieve optimum enzyme activity. Analysis of enzymatic conversion samples was done using HPLC-RID (using Zorbax Column). The importance of the divalent cation MgCl2 for optimal enzyme activity was investigated. The enzyme performed best at pH 7.5 and 60°C, using 10mM MgSO4 as a cofactor. Utilizing Gensweet in Stirred tank reactor (STR), the maximum fructose transformation was 44 %. The most activity was detected with Sodium phosphate buffers, and EPPS buffers at pH 7 and 8, accordingly, whereas the least activity was reported with TRIS HCl buffer.

Fructose syrup production from Onggok with isomerization process by Mg/Al hydrotalcite catalyst and glucose isomerase enzyme

Onggok, solid waste of tapioca still contains high carbohydrate. This component can be converted to liquid sugar (glucose and fructose). Hydrolysis process convert carbohydrate become glucose syrup while isomerization process would change those glucose become fructose syrup. This study aims to produce fructose from Onggok with isomerization by Mg/Al hydrotalcite catalyst and compare the productivity 500ml Onggok substrate (10.32 percent DS) was hydrolyzed become glucose in liquefaction and saccharification process. Liquefaction took placed in 95 degree C, pH 6.5, for 1 hour, by adding 0.067 percent (v/v) α-amylase while the saccharification was start by adding 0.067 percent (v/v) glucoamylase at 60 degree C, pH 5, for 1 hour. Isomerization with Mg/Al hydrotalcite took placed in 50 ml hydrolysate sugar, 1 percent (b/v) catalyst, and 100 degree C while process with glucose isomerase was at 100 ml substrate, 60 degree C, pH 8.2, and same catalyst ratio (1 percent). Isomerization process for each catalyst would be hold in 3, 5, and 7 hours. The best result of isomerization productivity was 39.29 g/L. h for process with Mg/Al hydrotalcite catalyst, while isomerization yield was 6.18 percent for process with glucose isomerization enzyme.

Safety evaluation of the food enzyme glucose isomerase from Streptomycesmurinus (strain NZYM-GA).

The food enzyme is a glucose isomerase (d‐xylose aldose‐ketose‐isomerase; EC 5.3.1.5) produced with a non‐genetically modified Streptomyces murinus strain NZYM‐GA by Novozymes A/S. The glucose isomerase is intended only to be used in an immobilised form in glucose isomerisation for the production of high fructose syrups. Residual amounts of total organic solids are removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The maximum entropy production and maximum Shannon information entropy in enzyme kinetics

We demonstrate that the maximum entropy production principle (MEPP) serves as a physical selection principle for the description of the most probable non-equilibrium steady states in simple enzymatic reactions. A theoretical approach is developed, which enables maximization of the density of entropy production with respect to the enzyme rate constants for the enzyme reaction in a steady state. Mass and Gibbs free energy conservations are considered as optimization constraints. In such a way computed optimal enzyme rate constants in a steady state yield also the most uniform probability distribution of the enzyme states. This accounts for the maximal Shannon information entropy. By means of the stability analysis it is also demonstrated that maximal density of entropy production in that enzyme reaction requires flexible enzyme structure, which enables rapid transitions between different enzyme states. These results are supported by an example, in which density of entropy production and Shannon information entropy are numerically maximized for the enzyme Glucose Isomerase.

Energy conservation and maximal entropy production in enzyme reactions

A procedure for maximization of the density of entropy production in a single stationary two-step enzyme reaction is developed. Under the constraints of mass conservation, fixed equilibrium constant of a reaction and fixed products of forward and backward enzyme rate constants the existence of maximum in the density of entropy production is demonstrated. In the state with maximal density of entropy production the optimal enzyme rate constants, the stationary concentrations of the substrate and the product, the stationary product yield as well as the stationary reaction flux are calculated. The test, whether these calculated values of the reaction parameters are consistent with their corresponding measured values, is performed for the enzyme Glucose Isomerase. It is found that calculated and measured rate constants agree within an order of magnitude, whereas the calculated reaction flux and the product yield differ from their corresponding measured values for less than 20 % and 5 %, respectively. This indicates that the enzyme Glucose Isomerase, considered in a non-equilibrium stationary state, as found in experiments using the continuous stirred tank reactors, possibly operates close to the state with the maximum in the density of entropy production.

Tandem Catalytic Conversion of Glucose to 5-Hydroxymethylfurfural with an Immobilized Enzyme and a Solid Acid

Conversion of cellulosic biomass to renewable chemicals such as 5-hydroxymethylfurfural (HMF) is of high current interest. Herein, we report a rare example of one-pot synthesis of HMF from glucose by tandem catalysis. The system is composed of a thermophilic glucose isomerase enzyme for glucose isomerization to fructose and a solid acid catalyst for fructose dehydration to HMF. A base (−NH2) functionalized mesoporous silica (aminopropyl-FMS) with large pore size was deployed successfully to immobilize and protect the thermophilic glucose isomerase in organic solvents at high temperature. The combination of this catalyst with a Bronsted acid (−SO3H) functionalized mesoporous silica (propylsulfonic acid-FMS) allowed us to conduct a one-pot transformation of glucose to HMF directly in a monophasic solvent system composed of tetrahydrofuran (THF) and H2O (4:1 v/v) with 61% yield of fructose and 30% yield of HMF at temperatures >363 K in 24 h.

Application of a novel type impinging streams reactor in glucose conversion to fructose using glucose isomerase enzyme

BACKGROUND The isomerization of glucose to fructose is one of the most widely used processes in the food industry to produce dietetic foods and drinks. Since this reaction is affected by external mass transfer resistances, application of an impinging jets technique in reducing external resistances may be justified. RESULTS Conversion of glucose to fructose was studied in a novel type of impinging streams reactor using glucose isomerase enzyme. Response surface methodology was applied to determine the effects of certain pertinent parameters of the process including axial velocity (A), feed concentration (B), nozzle flow rates (C) and enzyme loading (D) on the performance of the apparatus. A quadratic polynomial multiple regression model was selected and analysis of variance revealed a high coefficient of determination (R2) value equal to 0.972. The optimum process conditions for fructose production were established as, A = 0.1 m s-1, B = 1.5 kg m-3, C = 3.3e-5 m3 s-1 and D = 0.0035 kg. CONCLUSION It was found that an impinging streams reactor had higher performance capability in comparison with conventional reactors in solid–liquid enzyme reactions. In such a reactor 30% conversion of glucose was obtained within 9 s while the same conversion in packed-bed reactors requires 5 min. © 2013 Society of Chemical Industry

Optimization of glucose isomerase production by Streptomyces albaduncus

The effect of environmental factors on glucose isomerase productivity of Saudi Arabia isolated Streptomyces albaduncus was studied. During growth of the tested microorganism in the basal medium at 30°C, the glucose isomerase production reached the maximum level (6530 U/L) after 96 h incubation period. Irrespective of the carbon source, replacement of xylose with different carbohydrate did not increase the enzyme productivity. On the other hand, the highest enzyme productivity was obtained when corn steep liquor (2.0%, w/v) and yeast extract (1%) were used as nitrogen sources. Also, the optimum initial pH for maximum growth and enzyme productivity was 7. Further improvement in the glucose isomerase production was obtained after the addition of 0.01% (w/v) DL-isoleucine to the culture medium. Accordingly, optimization of the environmental condition associated with increase in the glucose isomerase enzyme productivity from S. albaduncus up to 15328 U/L which is approximately 2.3 more times as compared to that obtained under the initial condition. Finally, a promising production of glucose isomerase (14100 U/L) at large scale level was achieved by cultivation of the tested strain in the optimum environmental conditions using laboratory fermentor of 14 L. Key words: Glucose isomerase, fermentation, environmental condition, Streptomyces albaduncus.

Use of iterative OASIS with low resolution SAD laboratory X-ray source (Cr Kα) data sets

Anomalous scattering methods are widely used for phasing macromolecular structures. OASIS program works on a direct methods procedure and is used to break the phase ambiguity intrinsic in a single wavelength anomalous diffraction (SAD) experiment. An iterative phasing/model-building procedure for processing SAD data includes the initial direct method phasing of SAD data, density modification, automatic model building and direct methods phasing of SAD data with feed back from partial structure information. This dual-space procedure has been tested on two experimental Cr Kα SAD data sets, 2.3 A for S-SAD and 2.4 A for I-SAD, of an enzyme glucose isomerase with an approximate molecular weight of 44 kDa (388 residues). HYSS in PHENIX was used to locate the anomalous scatterers for both the data sets (11 sulfurs for 2.3 A data and 9 iodines for 2.4 A data). Phasing and density modification were carried out using OASIS, DM and model building was then carried out using ARP/wARP. Two iterative cycles were necessary for the successful structure solution and refinement of the 2.3 A data set which built 384 residues out of 388. Regular ARP/wARP failed for the 2.4 A resolution data and hence the modified version of REFMAC5 was used in ARP/wARP. A successful model could be built after three iterative cycles of OASIS using this modified version which built 382 residues out of 388 residues. Minimal manual model building was required at this stage and the structure determination was completed using regular REFMAC5. All the computations mentioned here were carried out using Pentium IV PC.

IMMOBILIZATION OF GLUCOSE ISOMERASE IN SURFACE-MODIFIED ALGINATE GEL BEADS

ABSTRACT 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 Km, and 2.39 × 10−3 and 6.03 × 10−3 mol/L min for Vmax, 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. PRACTICAL APPLICATIONS Glucose isomerase (GI) serves as an interesting model for studying structure–function relationships by advanced biochemical and genetic engineering techniques. Besides its academic importance, it has received increased attention by industries for its use in producing high-fructose corn syrup (HFCS) and for its potential application in the production of ethanol from hemicelluloses. The use of GI is expensive because it is an intracellular enzyme, and large quantities are needed to compensate for the high Km for glucose. Therefore, it is important to immobilize GI for its industrial applications. Recently, several methods for immobilizing GI have been developed. However, only a few are economical and yield enzyme preparations with properties that are suitable for commercial production of HFCS and ethanol.

Development and verification of a model to describe an immobilized glucose isomerase packed bed bioreactor

In this paper, the performance of immobilized packed bed glucose isomerase enzyme was mathematically modeled. A modified Michaelis–Menten type relation was used to describe the enzyme kinetics. Mass transfer inside the biocatalyst particle and through the bed column was analyzed simultaneously. Using measured data, physicochemical properties including diffusivity, viscosity and density of sugar solutions were correlated with its concentrations and were used to provide precision in solving the set of model equations. Model equations were solved using the Runge–Kutta and Gauss–Seidel algorithms and finite difference numerical method in MATLAB environment. Model output was used to demonstrate the effect of parameters such as velocity and bulk substrate concentration on the concentration profile within the biocatalyst particle, effectiveness factor and bulk substrate concentration along the bed. Model predictions were further validated against experimental data collected from a lab scale isomerization bioreactor. Measurements of the overall bioreactor conversion at various substrate concentrations were shown to lie within 5% of the values as put forth by the model. Using experimental data, a criteria was proposed to maximize the isomerization conversion from the immobilized bioreactor.

Glucose isomerization in simulated moving bed reactor by Glucose isomerase

Studies were carried out on the production of high-fructose syrup by Simulated Moving Bed (SMB) technology. A mathematical model and numerical methodology were used to predict the behavior and performance of the simulated moving bed reactors and to verify some important aspects for application of this technology in the isomerization process. The developed algorithm used the strategy that considered equivalences between simulated moving bed reactors and true moving bed reactors. The kinetic parameters of the enzymatic reaction were obtained experimentally using discontinuous reactors by the Lineweaver-Burk technique. Mass transfer effects in the reaction conversion using the immobilized enzyme glucose isomerase were investigated. In the SMB reactive system, the operational variable flow rate of feed stream was evaluated to determine its influence on system performance. Results showed that there were some flow rate values at which greater purities could be obtained.

Analysis of the high-fructose syrup production using reactive SMB technology

This paper deals with the production of high-fructose syrup (HFS) from glucose isomerization using reactive simulated moving bed technology. The reversible reaction is catalyzed by the immobilized enzyme glucose isomerase. In a simulated moving bed reactor (SMBR), reaction and separation processes can be coupled to achieve complete reactant conversion. The isomerization kinetics is experimentally determined at 328 K by the Lineweaver–Burk technique. Basic adsorption data for the sugar isomers (glucose and fructose) were obtained with cationic exchange resin as adsorbent. A mathematical model based on the analogy with true moving bed reactor and its numerical solution based on finite volume method were used for the prediction of the behaviour and performance of a SMBR. A new SMBR configuration for glucose isomerization is proposed and the effect of process parameters on its performance is addressed.

High throughput technique in structural bioinformatics

High Throughput techniques are necessary for solvin g the three dimensional structures of macromolecule s because the macromolecular sequences available exceed far i n number than the available three-dimensional struc ture. Experimental phasing using anomalous data is essential to solve non-homologous protein structures. Su lfur atom is commonly present in protein structures in the form of cysteine and methionine residues. With recent pr ogress in data collection, Sulfur ‐ SAD phasing methods are extensively used for phasing macromolecular structures. S -SAD phasing has been tested on experimental Cr K α data set, 2.4 A, of an enzyme glucose isomerase of molecular weight 44 kDa. The combination of PHENIX and ARP/wARP built 379 residues out of 388 residues using 11 sulfurs located initially from the SAD data. Truncate the d ata to 3 A resolution for testing the power of S-SA D. The above combination built 283 residues out of 388 residues using 11 sulfurs. Successive model could be built u sing OASIS which built 375 residues out of 388 residues. Minim al manual model building was required at this stage and the structure determination was completed using REFMAC5.

Equilibrium Kinetics of D-Glucose to D-Fructose Isomerization Catalyzed by Glucose Isomerase enzyme from Streptomyces phaeochromogenus

The time course of the glucose isomerization was investigated. At equilibrium the fructose content in the reaction mixture was 52% of the initial glucose concentration. The equilibrium constants K1 and K-1 for the isomerization reaction were determined at 60 degrees C. Two graphical plots were developed to reduce the problems of determining the individual constant. These plot methods for chemical reaction kinetics were applied with equal validity to the kinetics of glucose isomerization process.

Production of ethanol from lignocellulosic materials

In the bioconversion of lignocellulosic materials to ethanol, the cost of the raw material and the cost of the enzymes have been identified as the major costs for the product. In addition, many lignocellulosic materials contain considerable amounts of five-carbon sugars which are not easily fermented to ethanol. The possibility of improving the cost-intensive steps was investigated. The steam pre-treatment of a fast-growing species of Salix (Q082) was optimized with respect to treatment time and temperature, to give the best glucose yield after enzymatic hydrolysis. It was found that the semi-continuous production of cellulolytic enzymes with Trichoderem reesei Rutgers C30 in an aqueous two-phase system (an extractive fermentation system) increased the amount of recoverable enzyme activity compared with a batch process. It was also found that the enzyme consumption in the hydrolysis of cellulolytic materials, in filter paper units (FPU) g −1 reducing sugars (RS), could be considerably reduced by recirculating the cellulolytic enzymes in an aqueous two-phase system combined with an ultra-filtration unit. For the fermentation of pentoses, it was found that the combination of the commercial enzyme glucose isomerase and regular bakers' yeast, Saccharomyces cerevisiae, was superior to xylose fermenting yeasts. Yields and productivities comparable to those obtained in hexose fermentation could be achieved.

Safety evaluation of Streptomyces murinus glucose isomerase

A glucose isomerase enzyme, obtained from Streptomyces murinus, was produced by a fermentation process and subjected to a series of tests to investigate its safety in use and manufacture. It was not mutagenic (Ames test, using liquid culture) nor did it provoke chromosomal damage (rat bone marrow cytogenetics test). It did not contain (nor did the organism produce) antimicrobial activity or macrolidpolyene antibiotics. It had no teratogenic activity when administered to pregnant rats at 100 000 ppm in the diet. It was without effect upon rats when administered at this dietary concentration for 4 weeks. Dietary administration at 5000, 15 000 or 50 000 ppm to rats for 13 weeks resulted in nephrocalcinosis in females at all dosages (probably a physiological response to the altered calcium:phosphate ratio in the admixed diet) and status spongiosus in the brains of males receiving 50 000 ppm. As the finding of nephrocalcinosis in rats is generally agreed to be of no toxicological importance with regard to the use in man, the dietary concentration of 15 000 ppm was considered to be highest no-effect level. This level corresponds to an intake of some 1000 mg/kg/day, which represents approximately 8000 times the human intake based on a conservative estimation.

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.

Immobilization of cells containing glucose isomerase using a multifunctional crosslinking reagent

This paper describes a simple method for the immobilization of cells containing glucose isomerase enzyme activity, by using an epoxypolyamine crosslinking reagent. One kg of immobilized preparation could convert 5,700 kg of glucose to glucose-fructose syrups; the conversion was 42%.

ChemInform Abstract: GLUCOSE‐ISOMERASE‐CATALYZED D‐GLUCOSE ‐ D‐FRUCTOSE INTERCONVERSION: MECHANISM AND REACTIVE SPECIES

The isomerization of (1-13C)-D-glucose to an equilibrium mixture of (1-13C)-D-glucose and (1-13C)-D-fructose by the enzyme glucose isomerase (E.C. 5.3.1.5) has been followed using 13C NMR spectroscopy. It is concluded that the α-D-glucopyranose and the α-D-fructofuranose form are the reactive species for the enzyme.