Stability of native and cross-linked crystalline glucose isomerase.

Stabilities of native and cross-linked crystalline forms of Streptomyces rubiginosus glucose isomerase were compared in buffer and in 45% glucose/fructose solutions. The cross-linked crystalline form of the enzyme was more stable in the presence of substrate while in a buffer solution the native enzyme was more stable. Inactivation of native enzyme in buffer did not obey first-order kinetics but proceeded with a rapid first phase followed by a stable phase. This stabilization is interpreted to be a result of a conformational change in the protein structure. Inactivation of the native enzyme in buffer was directly related to protein precipitation. In the presence of high substrate concentration, the inactivation was related to browning reactions between the enzyme and the reactive sugar, resulting in soluble sugar-protein complexes.     

Cross-linked glucose isomerase crystals as a liquid chromatographic separation material

Cross-linked crystals of glucose isomerase (CLGI) were characterized as a liquid chromatographic separation material. The experiments were done with crystals having an average diameter of 83 μm. Porosity (epsilon(p)) and pore size distribution of the CLGI crystals were measured with size exclusion chromatography using D(2)O and polyethylene glycols as probes. CLGI material was capable of separating <1000 g/mol polyethylene glycols. Fifty two percent (epsilon(p) = 0.47) of the total crystal volume was in pores. Pore size measurement showed that CLGI crystals were microporous material, having an average apparent pore diameter of 29 +/- 0.08 ?. CLGI material separated n-alcohols C(1) to C(8) based on the hydrophobic interaction between the protein material and the carbon chain of the alcohols. Height equivalent to a theoretical plate (HETP, in millimeters) ranged from 1.6 to 0.89 for the C(1) to C(7) n-alcohol series. Despite the large crystal size, CLGI as a chirally active phase effectively separated D- and L-arabitol (R(s) = 0.58) and showed potential for chiral separation of amino acids.     

Production,purification and immobilization of glucose isomerase from Streptomyces olivochromogenes PTCC 1547

Production of glucose isomerase from Streptomyces olivochromogenes PTCC 1457 was followed by its purification and immobilization. Different immobilization methods including the use of a hydrophobic support were investigated.     

Production of glucose isomerase by different strains ofStreptomyces

Summary The glucose isomerase activity ofStreptomyces haeochromogenes strains 1 and 2 varies considerably with the assay conditions (pH, glucose concentration,etc.). Nine other species of streptomyces were tested under conditions optimal forS.phaeochromogenes 2. The highest enzyme activity was found inS.nigrificans 3014.     

Simultaneous catalysis and product separation by cross-linked enzyme crystals

Crystalline cross-linked xylose isomerase (CLXI, EC 5.3.1.5) and xylanase (CLX, EC 3.2.1.8) were studied in a packed-bed reactor for simultaneous catalytic reaction and separation of substrates from reaction products. Streptomyces rubiginosus xylose isomerase catalyzed a slow isomerization of L-arabinose to L-ribulose and an epimerization to L-ribose. In equilibrium the reaction mixture contained 52.5% arabinose, 22.5% ribulose, and 25% ribose. In a packed-bed column filled with CLXI, a simultaneous reaction and separation resulted in fractions where arabinose concentration varied between 100-0%, ribulose between 0-55%, and ribose between 0-100%. Trichoderma reesei xylanase II hydrolyzed and transferred xylotetraose mainly to xylotriose and xylobiose. In a packed-bed column filled with CLX, xylotetraose rapidly reacted to xylobiose and xylose by a mechanism that is not yet fully understood.     

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.     

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.     

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     

Metabolism of D-arabinose by Aerobacter aerogenes: purification of the isomerase

In Aerobacter aerogenes, the mutational event permitting the utilization of d-arabinose as a source of carbon and energy is a regulatory mutation resulting in the constitutive synthesis of certain enzymes of the l-fucose catabolic pathway. l-Fucose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. This enzyme was purified to homogeneity as indicated by a single band in disc-gel electrophoretic columns and single peaks with column chromatography and ultracentrifugation from the wild-type PRL-R3 strain, induced with l-fucose and two constitutive mutants, 502 and 510. The ratios of the activities of this isomerase on d-arabinose and l-fucose remained constant throughout all purifications. The apparent K(m) of the isomerase from the wild-type strain induced with l-fucose and from the constitutive mutant strains was 5.0 x 10(-2)m for l-fucose and 1.5 x 10(-1)m for d-arabinose. A strain 531 possessing an apparent alteration in the isomerase was isolated from the strain 502. This altered isomerase exhibited a lowered K(m) for d-arabinose.     

Effect of minerals on glucose isomerase production by Streptomyces kanamyceticus

Summary A study has been made of the mineral requirements of Streptomyces kanamyceticus KCC S-0433 for production of glucose isomerase. The optimal concentrations of MgSO₄ and K₂HPO₄ for enzyme production are 0.07% and 0.05%, respectively. The elements Fe, Mn and Zn are required at levels of 10, 3 and 3 mg/l, respectively. Cu, Co and Ca have inhibitory effects on the production of the enzyme.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Media optimization studies for glucose isomerase production by arthrobacter species

Media optimization studies are carried out with the objective of maximising glucose isomerase production by Arthrobacter sp. The recommended media consists of 1.0% (w/v) xylose, 1.0% peptone, 0.5% yeast extract, 0.025% MgSO₄·7H₂O, 0.6% (NH₄)₂HPO₄ and 0.2% KH₂PO₄. Activity of the enzyme produced in this media is 11.2 units/ml. Growth cycle for batch cultivation is studied and the lag period is 2 hours, followed by exponential phase extending upto 32 hours.     

Substrate protection of immobilized glucose isomerase

Using commercial immobilized glucose isomerase (SWETASE(R), Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner. The data analysis was carried out based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrates was also considered. The protection factor was proposed to elucidate the dependence of the degree of substrate protection. The existence of the protection of glucose isomerase by the substrates has been verified experimentally. Also, the enzyme-substrate complex deactivates with a decay constant which is one-half that of the free enzyme. Theoretical analysis of enzyme deactivation with substrate protection offers an effective understanding which is essential for enzyme replacement and process optimization.     

Synthesis of sorbent from cross-linked starch for affinity purification of glucose(mannose)-specific lectins]

Cross-linked starch gel for the affinity chromatography of D-glucose (D-mannose)-specific lectins is suggested. In order to optimize hydrodynamic properties of gel 30% starch has been hydrolysed by HCI at 70 degrees C during 60 min and then cross-linked by epichlorohydrin under alkaline conditions. Every 100 g of starch require 18 ml of epichlorohydrin and 36 ml of 8 N KOH. The gel obtained has been successfully used for the purification of lectins from Pisum sativum L., Lens culinaris L., Vicia sativa L., and Vicia faba L. seeds. These lectins, purified on starch gel do not differ from sephadex-purified samples.     

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.     

Co-immobilization of cellulase and glucose isomerase by molecular deposition technique

Cellulase was immobilized and co-immobilized with glucose isomerase within p-trimethylamine polystyrene beads using a molecular deposition technique. The co-immobilized enzyme system directly converted insoluble cellulose to glucose and fructose in 60:40 molar ratio. The co-immobilized enzyme still retained 50% of its initial activity at 50°C after 4 recycles of 5 hours for each time.