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. (c) 1993 John Wiley & Sons, Inc.     

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.     

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.     

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.     

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.     

Generalized linearization of kinetics of glucose isomerization to fructose by immobilized glucose isomerase

The kinetic parameters of both glucose isomerization to fructose and immobilized glucose isomerase (GI) inactivation calculated under different conditions are compared and discussed. Utilizing these figures, the possibility of generalizing a linear model, previously proposed for the kinetics of glucose isomerization by immobilized glucose isomerase, is investigated, so as to apply them to whole ranges of temperature and concentrations of actual interest in industrial processes. The proposed model is a satisfactory approximation of the more involved Briggs-Haldane approach and substantially simplifies the problem of optimizing an industrial fixed-bed column for high-fructose corn syrup (HFCS) production.     

Xylose(glucose) isomerase reactivity of immobilized Arthrobacter sp

The study of the xylose/glucose isomerase-containing Arthrobacter sp. B-5 cells immobilized in cobalt hydroxide gel showed that immobilization increases the substrate affinity of the enzyme, its thermo- and pH-optima of action and stability, and makes unnecessary the addition of stabilizing cobalt ions to the isomerization medium.     

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.A reactor model was established analogous to heterogeneous catalytic reactor model taking into account the effect of fluid mass transfer and reversible kinetics. The investigated catalyst system consists of immobilized Streptomyces bambergiensis cells containing the enzyme glucose isomerase, which catalyzes the isomerization of glucose to fructose.List of Symbols A ₀, A ₁, A ₂ parameters in axial dispersion reactor model - c _go, c_g, c_gemol m^–3 glucose concentration at time t=0, at any time and at equilibrium conditions - c _gsmol m^–3 glucose concentration at particle surface - C dimensionless glucose concentration - d _pm particle diameter - d _rm diameter of reactor tube - Da Damkohler number - D _eff m² s^–1 effective glucose diffusion coefficient in Ca-alginate gel beads - k _fm s^–1 film transfer coefficient - K _e equilibrium constant - K _mg, K_mfmol m^–3 Michaelis-Menten constant for glucose and fructose, respectively - K _mmol m^–3 modified Michaelis-Menten constant - K dimensionless parameter - K ^* dimensionless parameter - L m length of reactor tube - Pe Peclet number - Pe _p particle Peclet number - Q m³ s^–1 volumetric flow rate - (-r _g) mol m^–3 s^–1 reaction rate - Re _p Reynolds particle number - Sc Schmidt number - Sh Sherwood number - t s time - v ₀ m s^–1 linear superficial fluid velocity - V _mg, V_mfmol g^–1 s^–1 maximal reaction rate for glucose and fructose, respectively - V _mmol m^–3 s^–1 modified maximal reaction rate for glucose - V _mg ^x mol m^–2 s^–1 maximal reaction rate for glucose - X _g, X_ge glucose conversion and glucose conversion at equilibrium conditions - X normalized conversion - Y dimensionless glucose concentration - void fraction of fixed bed - effectiveness factor of biocatalyst - Pa s kinematic viscosity of substrate - ₁ s first absolute weighted moment - ₂ s² second central weighted moment - _gkg m^–3 substrate density - _pkg m^–3 particle density - ² dimensionless variance of RTD curve - s residence time     

Evaluation of diffusional resistances in the process of glucose isomerization to fructose by immobilized glucose isomerase

A kinetic model presented in a previous work is employed to carry out a systematic study dealing with the relative importance of intraparticle and interparticle diffusional resistances in the process of glucose isomerization to fructose by immobilized glucose isomerase. An analytical generalized expression of the effectiveness factor is obtained, which promises to be particularly useful for design purposes. Finally, the role of each of the main parameters influencing the catalyst effectiveness factor is put in evidence and discussed within the whole range of possible operative conditions.     

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.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

The performance of immobilized glucose isomerase supported by shrimp chitin in various types of reactors

Five different types of reactors were employed for glucose isomerization using shrimp shell as the support on which to immobilize the glucose isomerase. The Michaelis-Menten constants and effective diffusivity of glucose in the immobilized enzyme bed were experimentally determined and used in a theoretical analysis of the radial-flow reactor. The fractional conversions of the radial-flow, fluidizedbed, and packed-bed reactors with the same -residence time were found experimentally to be almost the same. This result reveals that the use of radial-flow and fluidized-bed reactors for this immobilized enzyme system is highly feasible.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Immobilization of glucose isomerase

The immobilization of glucose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column. Of the covalent bonding methods, the colloidal silica-glutaraldehyde method showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Ludox HS-30 bound glucose isomerase column showed a half-life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s, fructose 45%). Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Amberlite IRA-904 resine-bound glucose isomerase showed a half-life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d.s., fructose 45%). Based on the ease of the immobilization process, the possibility of carrier reuse and the extensive use already achieved by ion exchange resins in the sugar industry, IRA-904 resin was selected as the candidate for commercialization.     

Effects of external diffusion and design geometry on the performance of immobilized glucose isomerase reactor system

Using immobilized glucose isomerase, the effects of superficial velocity of the reaction solution flowing through a packed-bed reactor on the apparent kinetic constants of reversible reaction system were studied. The results showed that the apparent kinetic constants, both V_m″ and K_m″, of the forward reaction varied significantly as the superficial velocity is changed, whereas those of the reverse reaction varied only slightly. Using the kinetic data determined experimentally, computer simulation of the enzyme reactor performance was carried out, and the importance of the external mass transfer in the proximity of immobilized-enzyme particles was recognized. The reactor performance, expressed in terms of productivity, was examined as a function of the reactor height-to-diameter ratio, H/D. The productivity of the reactor system goes through a maximum value at a H/D ratio of about 1.6. and decreases as the H/D ratio increases. Theoretical analysis of the reaction kinetics of immobilized-enzyme system that has reversible reaction kinetics is also presented. The experimental results showed good agreement with the results found from the theoretical analysis and the computer simulation studies. Based on the principles of the methods and the results presented in this paper, it is anticipated that one can predict the optimal design and operating conditions for the glucose isomerase reactor system and that application of the results could be extended to other enzyme systems with reversible reaction kinetics.     

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.     

The continuous production of glucose isomerase

Summary It is shown that the enzyme glucose isomerase may be produced effectively by suitable continuous culture techniques using species of Arthrobacter and Mycobacterium. Carbon-limited growth conditions gave better carbon conversion efficiencies and higher specific enzyme activities than batch or nitrogen-limited conditions.This work was completed whilst the author was a member of the staff of I.C.I. Agricultural Division, Billingham, Teesside. Its contents are the subject of British Patent 1 492 258.