Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model

The substrate specificity of immobilized D-glucose isomerase (EC 5.3. 1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with alpha-, beta-, and equilibrated D-glucose solutions indicates that beta anomer as well as alpha anomer is a substrate of the glucose isomerase at pH 7.5 and 60 degrees C. For substrate concentration of 0.028 mol l(-1) (1% w/v), the initial conversion rate of alpha-D-glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than beta-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of alpha- and beta-D-glucose with different rate constants.     

A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system.     

Mechanism of thermoinactivation of immobilized glucose isomerase

Irreversible thermoinactivation of immobilized glucose isomerase from Streptomyces olivochromogenes has been mechanistically investigated at the pH-optimum of enzymatic activity (pH 8.0). Ligands (high fructose corn syrup and the competitive inhibitor xylitol) greatly stabilize the immobilized enzyme at high temperatures. At 90 degrees C in the presence of 2M xylitol, irreversible inactivation of immobilized glucose isomerase is caused by deamidation of its asparagine/glutamine residues. On the basis of the data obtained, it appears that the time-dependent decay of glucose isomerase activity in industrial bioreactors is brought about by oxidation of the enzyme's cysteine residue and/or heat-induced deleterious reactions with high fructose corn syrup or its impurities.     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

Properties of glucose isomerase from Streptomyces robeus S-606

Optimal conditions of the glucose isomerase fixation in a cell are determined by thermal treatment of Str. robeus S-606 biomass. Under these conditions the maximal enzyme activation (by 50-55 percent) is simultaneously observed. Basic properties of glucose isomerase fixed inside the cell are studied in comparison with the enzymic cell-free extract of this enzyme. The pH-optimum for preparations coincides and is observed at pH 7.5; the temperature optimum for the soluble enzyme is 70 degrees C, and for the intracellular enzyme it is higher by 5 degrees C. Thermostability of the intracellular enzyme is also higher than that of the soluble one. The Michaelis constants are calculated for the glucose isomerase preparations in a form of producer cells and enzymic extract: they equal to 0.375 M and 0.285 M, respectively. A comparison of properties permits considering intracellular glucose isomerase as an immobilized enzymic preparation.     

Experimental operation of immobilized multienzyme systems in optimal and suboptimal configurations

Two mixed immobilized enzyme systems, glucoamylase–glucose isomerase and glucose isomerase–glucose oxidase–catalase, were operated to verify theoretical predictions that optimal bifunctional catalyst configutations could exist superior to those where the catalysts were mixed uniformly or arranged sequentially in a tubular reactor. The experimental results for all three configurations conformed to the theoretical values sufficiently closely to support of optimal catalyst profiles.     

Studies on Sugar-isomerizing Enzymes

The effect of a borate on the isomerization reaction between glucose and fructose which is catalyzed by a glucose isomerase was investigated. The yield of fructose was dependent on both the ratio of sugar to the borate and pH. A maximum of 88 to 90% of glucose was converted into fructose when the isomerization reaction was carried out at around pH 7.5 and in the presence of an appropriate amount of the borate which forms a complex between one molecule of sugar and one molecle of boric acid.     

Immobilized enzyme catalysis with reaction-generated pH change

Many enzyme-catalyzed reactions involve the liberation or consumption of hydrogen ions. In this paper a mathematical model is employed to investigate how such reactions behave when the enzyme is immobilized. Shifted pH optima, disappearance of an optimum pH, insensitivity to bulk pH, and very large effectiveness factors are some of the phenomena which appear as a result of pH coupling between the reaction and the enzyme's activity. Several of the qualitative features revealed by the model are consistent with earlier experimental observations. In addition, preliminary guidelines for optimal choice of enzyme support are suggested.     

Generation of a pH gradient in an immobilized enzyme system

Several examples of two-step sequential reactions exist where, because of the poor equilibrium conversion by the first reaction, it is desirable to conduct the two reactions simultaneously. In such a scheme, the product of the first reaction is continuously removed by the second reaction, thus not allowing the first reaction to approach chemical equilibrium. Therefore, the first reaction is allowed to proceed in the desired direction at an appreciable rate. However, in many biochemical applications where enzyme catalysts are involved, the enzyme's activities are strong functions of pH. Where the pH optima of the first and second reaction differ by three to four units, the above reaction scheme would be difficult to implement. In these cases, the two reactions can be separated by a thin permeable membrane across which the desired pH gradient is maintained. In this article, it was shown, both by theory and experiment, that a thin, flat membrane of immobilized urease can accomplish this goal when one face of the membrane is exposed to the acidic bulk solution (pH(b) = 4.5) containing a small quantity of urea (0.01 M). In this particular case, the ammonia that was produced in the membrane consumed the incoming hydrogen ions and thus maintained the desired pH gradient. Experimental results indicate that with sufficient urease loading, the face of the membrane opposite to the bulk solution could be maintained at a pH that would allow many enzymes to realize their maximum activities ( approximately 7.5). It was also found that this pH gradient could be maintained even in the presence of a buffer, which greatly enhances the transport of protons into the membrane. (c) 1993 John Wiley & Sons, Inc.     

Preparation and Some Properties of Chitosan Bound Enzymes

An immobilization method using chitosan prepared from chitin as an insoluble carrier was investigated. Glucose isomerase, urease, glucamylase, trypsin and glucose oxidase were attached to chitosan by the aid of water soluble carbodiimide. Their activity yields were as follows; glucose isomerase 32%, urease 44%, glucamylase 8%, trypsin 10%, glucose oxidase 37%.

Immobilized glucose isomerase showed no significant changes in optimal temperature and heat stability. But pH optimum of reaction and pH stability range were somewhat lowered. The inhibitory effects of bivalent metal ions were considerably reduced by immobilization and similar tendency was observed for buffer reagents such as Tris or veronal. Immobilized glucose isomerase was inhibited by 8 m urea or 6 m guanidine hydrochloride in nearly the same way as free enzyme. With SDS, cysteine or mercaptoethanol free glucose isomerase was scarcely affected by these reagents, while immobilized enzyme considerably suffered to a loss of its activity.     

Sugar cane bagasse as a possible source of fermentable carbohydrates. II. Optimization of the xylose isomerase reaction for isomerization of xylose as well as sugar cane bagasse hydrolyzate to xylulose in laboratory-scale units

Both the forward and backward reactions of xylose isomerase (Sweetzyme Q) with xylose and glucose as substrates have been studied in terms of kinetics and thermodynamics. The relationship between the two reactions can thus be determined. Much attention has been given to the reaction with xylose as substrate. The optimal conditions of the xylose reaction in terms of pH, buffer, metal ions, substrate concentration, temperature, and ionic strength have been determined. These findings did not differ much from those reported for the glucose reaction. Equilibrium constants for the aldose to ketose conversion were more favorable in the case of glucose. The results obtained with continuous isomerization of xylose in columns packed with either Sweetzyme Q or Taka-Sweet were very similar to those obtained from batch isomerization processes. Particle size had a definite effect on reaction rate, which indicates that diffusion limitations do occur with the immobilized enzyme particles. Heat stability of Sweetzyme Q was good with t(1/2) of 118, 248, and 1200 h at 70, 55, and 40 degrees C, respectively. A novel method for the separation of xylose-xylulose mixtures with water as eluant on a specially prepared Dowex 1 x 8 column was developed. This technique has the capability of producing pure xylulose for industrial or research applications. A writ for a patent regarding this technique is at present prepared.     

Optimization of the activity in porous media of proton-generating immobilized enzymatic reactions by weak acid facilitation

In addition to the role of maintaining the pH, buffers can also facilitate the transport of H(+) ions in acid-generating systems. The role of this facilitation in proton transport in porous pellets on acid-generating immobilized enzymic reactions is examined. The activity in these systems can be maximized by a proper control of facilitation, which involves the determination of the appropriate variables out of (1) the concentration of the weak acid, (2) the pH of the medium, (3) the bulk substrate concentration, and (4) the type of weak acid. Since the intrinsic activity (IA) of the immobilized enzyme is such that it exhibits an optimum with respect to the pH, a partial (optimal) removal of diffusional limitation by facilitation maximizes the activity when the bulk pH is larger than this optimum pH. A complete removal of diffusional limitations, however, maximizes the activity when the bulk pH is less than or equal to the above optimum pH. The control of the diffusional resistance can be achieved by controlling the extent of facilitation, hence by adjusting the parameters mentioned above. Computations have been carried out to examine the effect of each of these parameters on the activity of the immobilized enzyme. It is found that when the bulk pH is less than or equal to the optimum pH of the intrinsic activity of the immobilized enzyme, there exists a lower limit on the amount of weak acid required to maximize the activity. However, an optimum amount of weak acid is required to maximize the activity when the bulk pH is higher than that optimum pH. For a given activity the amount of weak acid is minimal if the pK of the weak acid is close to the bulk pH. The effect of coupling between the proton and substrate transport on activity control is also examined and the effect of geometry on activity is evaluated for spherical, cylindrical, and flat-plate configurations.     

树状黄杆菌变株U-616葡萄糖异构酶的研究

以树状黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍａｒａｂｏｒｅｓｃｅｎｓ）ＮＲＲＬ１１０２２为出发菌株,用紫外线对其进行诱变,经筛选得到一株葡萄糖异构酶的高产菌株Ｕ－６１６,其酶活力提高３１％。经保存三年和多次传代复测,其产酶能力保持稳定。其生长和产酶需较高的溶氧水平,最适产酶温度为３０℃,最适产酶ｐＨ为７．０－７．５,铁离子对其生长和产酶无明显的影响。所产葡萄糖异构酶的最适温度为６０－８０℃,最适ｐＨ为７．５－８．５,Ｃｏ２＋和Ｍｇ２＋对酶有激活作用,对金属离子耐受性较强,对Ｃａ２＋不敏感,热稳定性较好。树状黄杆菌变株Ｕ－６１６是一株产胞内葡萄糖异构酶的优良菌株。     

Studies on microbial glucose isomerase: 4. Characteristics of immobilized whole-cell glucose isomerase from Streptomyces spp.

Whole-cell glucose isomerase from a Streptomyces spp. was immobilized by entrapment in gelatin matrices crosslinked with glutaraldehyde. The resultant immobilized enzyme preparation had up to 40% recovery yield of the activity and showed relatively long stabilities during storage and the isomerizing reaction. The storage half-life of the preparation was 19 months at 5°C and the half-life of the enzyme during operation was 260 days in the presence of 1 mM Co²⁺ and 80 days in the absence of the metal ion. Optimum pH and temperature were 7.5 and 70–75°C, respectively. The K_m values for glucose and fructose were 0.29 and 0.46 m, respectively, with a maximum theoretical conversion yield of 56%. The simulation results based on the reversible one-substrate enzyme kinetic model agreed well with the experimental data obtained from a batch reactor. The continuous operation of packed bed reactors demonstrated that some effects of the external film diffusion resistance were apparent at low flow rates of the substrate feed solution, whereas the internal pore diffusion resistance was negligible up to the pellet size used in this work.     

Peptide synthesis with immobilized carboxypeptidase Y

Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 mum microparticulate amino-silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino-silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed-bed reactors for peptide synthesis. Packed-bed reactors (40 x 4.6 mm, 60 x 4.6 mm) were used to catalyze the synthesis of 170 mg N-benzoyl-L-arginyl-L-methioninamide, 380 mg N-benzoyl-L-arginyl-L-methionyl-L-leucinamide, and 200 mg N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L-methionyl-L-leucyl-L-phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N-benzoyl-L-arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.