Immobilized glucose oxidase–catalase and their deactivation in a differential-bed loop reactor

Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.     

Performance of the continuous glucose isomerase reactor system for the production of fructose syrup

Glucose isomerase in the form of heat-treated whole-cell enzyme prepared from Streptomyces phaeochromogenus follows the reversible single-substrate reaction kinetics in isomerization of glucose to fructose. Based on the Kinetic constants determined and the mathematical model of the reactor system developed, the preformance of a plug-flow-type continuous-enzyme reactor system was studied experimentally and also simulated with the aid of a computer for the ultimate objective of optimization of the glucose isomerase reactor system. The enzyme decay function for both the enzyme storage and during the use in the continuous reactor, was found to follow the first-order decay kinetics. When the enzyme decay function is taken into consideration, the ideal homogeneous enzyme reactor kinetics provided a satisfactory working model without further complicatin of the mathematical model, and the results of computer simulation were found to be in good agreement with the experimental results. Under a given set of constraints the performance of the continuous glucose isomerase reactor system can be predicted by using the computer simulation method described in this paper. The important parameters studied for the optimization of reactor operation were enzyme loading, mean space time of the reactor, substrate feed concentration, enzyme decay constants, and the fractional conversion, in addition to the kinetic constants. All these parameters have significant effect on the productivity. Some unique properties of the glucose isomerization reaction and its effects on the performance of the continuous glucose isomerase reactor system have been studied and discussed. The reaction kinetics of glucose isomerase and the effects of both the enzyme loading and the changes in reaction rate within a continuous reactor on the productivity are all found to be of particular importance to this enzyme reactor system.     

Analysis of the coupled transport, reaction, and deactivation phenomena in the immobilized glucose oxidase and catalase system.

In a previous paper, the overall or macrokinetics of the immobilized glucose oxidase--catalase system has been presented. In this paper a detailed analysis of the interaction of diffusion and reaction in this system will be presented. The mathematical treatment includes two consecutive reactions with two-substrate kinetics. Furthermore, the deactivation of both enzymes due to the intermediate product peroxide is taken into account. The predicted results suggest that the efficiency of the glucose oxidase reaction depends on the concentration ranges of the two substrates. Furthermore, the external mass-transfer rate may cause a shift from glucose limitation to oxygen limitation. The efficiency of the coupled system is always higher than that predicted for the uncoupled reaction path. The calculations show that the economics of the coupled system depend a great deal on the deactivation of the enzymes.     

Immobilized glucoamylase on porous glass

A study was made to determine the controlling mass transfer resistance in the overall reaction rate for conversion of maltose to glucose, catalyzed by glucoamylase immobilized onto porous glass. For normal operation of a packed column and air-stirred batch reactor, the rate controlling step was found to be the internal resistance of simultaneous pore diffusion and chemical reaction. Experimental effectiveness factors were determined and are compared with those derived from a theoretical diffusion model based on Michaelis-Menten kinetics. Also given are temperature and pH relationships for the free and immobilized glucoamylase.     

Application of porous teflon tubing method to automatic fed-batch culture of microorganisms. I. Mass transfer through porous teflon tubing

For the purpose of a rational design for an automatic feedback control system incorporating a porous Teflon tubing sensor in semibatch culture, steady-state mass-transfer characteristics of tubing sensors have been investigated theoretically and experimentally, and also dynamic responses have been studied experimentally. A distributed mathematical model for steady-state diffusion has been solved numerically and its solution has been shown as useful for the sensor design. The overall mass-transfer resistance of radial diffusion has been shown to be the sum of external liquid-film mass-transfer resistance and membrane diffusion resistance. The steady-state experiments using ethanol dissolved in water revealed that its transfer into the tubing was controlled by the molecular diffusion within the tubing-wall membrane. Oxygen transfer from external water into the tubing was shown experimentally to be controlled by the liquid-film resistance outside the tubing. In general, the radial mass transfer of a substance having a small Henry's constant is controlled by the liquid-film resistance. The response of the tubing sensor-detector-recorder system for the stepwise addition of ethanol into the external water could not be represented by a simple combined system of the first-order delay with lag time. The responses depend on the characteristics of the tubing as well as flow rate of the carrier gas, etc., but they were quite excellent in all cases (e.g., 90% in 20 s).     

Glucose reactions with acid and base catalysts in hot compressed water at 473 K

The effects of the homogeneous catalysts (H(2)SO(4) and NaOH) and heterogeneous catalysts (TiO(2) and ZrO(2)) on glucose reactions were examined in hot compressed water (473 K) by a batch-type reactor. From the homogeneous catalyst studies, we confirmed that the acid catalyst promoted dehydration, while isomerization of glucose to fructose was catalyzed by alkali. Anatase TiO(2) was found to act as an acid catalyst to promote formation of 5-hydroxymethylfuraldehyde (HMF). Zirconia (ZrO(2)) was a base catalyst to promote the isomerization of glucose. The effects of the additives were also confirmed through fructose reactions.     

Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol

Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is very similar to those of the corresponding regions of maleylacetoacetate isomerases, enzymes that catalyze the glutathione-dependent isomerization of a cis double bond in maleylacetoacetate to the trans configuration during the catabolism of phenylalanine and tyrosine. Furthermore, tetrachlorohydroquinone dehalogenase catalyzes the isomerization of maleylacetone (an analogue of maleylacetoacetate) at a rate nearly comparable to that of a bona fide bacterial maleylacetoacetate isomerase. Since maleylacetoacetate isomerase is involved in a common and presumably ancient pathway for catabolism of tyrosine, while tetrachlorohydroquinone dehalogenase catalyzes a more specialized reaction, it is likely that tetrachlorohydroquinone dehalogenase arose from a maleylacetoacetate isomerase. The substrates and overall transformations involved in the dehalogenation and isomerization reactions are strikingly different. This enzyme provides a remarkable example of Nature's ability to recruit an enzyme with a useful structural scaffold and elaborate upon its basic catalytic capabilities to generate a catalyst for a newly needed reaction.     

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.     

Contribution of Glucose Phosphorylation to Glucose Metabolism in Brevibacterium fuscum

When the ‘dihydroxyacetone-fermentation’ was carried out in a steady state by the cells of Br. fuscum, it was suggested that the consumption rate of glucose in the medium might be regulated at the initial stages of glucose degradation such as; (a) glucose isomerization, (b) glucose dehydrogenation, and (c) glucose phosphorylation. Of these three enzymatic reactions, the isomerization and the dehydrogenation were proved to be unable to occur or negligible in vivo. So, in consideration of the pool sizes of Mg^{+ +}, Pi, H⁺, glucose, G6P*, ATP, ADP, etc., the intracellular glucokinase** activity was calculated. Results indicate that glucokinase reaction may be the limiting factor for direct glucose metabolism in Br. fuscum.     

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.     

Batch kinetic studies of glucose isomerization to fructose by Arthrobacter species

Glucose can be isomerized to fructose by the catalytic action of the enzyme, glucose isomerase. This enzyme is synthesized by a variety of micro-organisms, predominantly by bacteria. Arthrobacter species cells are grown in a medium standardized specifically to synthesize the enzyme and are then used to isomerize glucose under conditions of no further cell growth. Effect of metal ions on the isomerization is studied and it is found that magnesium promoted the reaction, sodium had no effect and calcium and manganese inhibited the reaction. Rate of reaction per unit of catalyst is found to be constant. Michaelis-Menten model modified for the reversibility of the reaction is suitable to describe the isomerization kinetics and the kinetic parameters are determined and reported.List of Symbols k ₁ rate constant (Glucose to intermediate complex) - k _–1 rate constant (Intermediate complex to glucose) - k ₂ rate constant (Intermediate complex to fructose) - k _–2 rate constant (Fructose to intermediate complex) - v _mf maximum reaction velocity of the forward (GF) reaction - v _mb maximum reaction velocity of the reverse (FG) reaction - K _f Michaelis-Menten constant for the forward (GF) reaction - K _b Michaelis-Menten constant for the reverse (FG) reaction - K _eq equilibrium constant - r _G rate of glucose consumption     

Succinic acid fermentation in a stationary-basket bioreactor with a packed bed of immobilized Actinobacillus succinogenes: 1. Influence of internal diffusion on substrate mass transfer and consumption rate

This paper is dedicated to the study on external and internal mass transfers of glucose for succinic fermentation under substrate and product inhibitions using a bioreactor with a stationary basket bed of immobilized Actinobacillus succinogenes cells. By means of the substrate mass balance for a single particle of biocatalysts, considering the Jerusalimsky kinetic model including both inhibitory effects, specific mathematical expressions have been developed for describing the profiles of the substrate concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on glucose consumption rate. The results indicated that very low values of internal mass flow could be reached in the particles center. The corresponding region was considered biologically inactive, with its extent varying from 0.24% to 44% from the overall volume of each biocatalyst. By immobilization of bacterial cells and use of a basket bed, the rate of glucose consumption is reduced up to 200 times compared with the succinic fermentation system containing free cells.     

Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support.

Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.     

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.     

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.     

Parameter evaluation and performance studies in a fluidized-bed immobilized enzyme reactor

Dispersion and mass-transfer characteristics and fluidization parameters influencing the performance of a small pilot-plant immobilized enzyme reactor are evaluated. The suitability of a dispersed plug-flow model to predict the conversions obtained in the enzymatic reaction (starch → glucose) catalyzed by amyloglucosidase immobilized to solid and porous carriers is assessed. The performance of a fluidized-bed reactor is compared on the basis of a normalized residence time with that of a fixed bed and found to be superior.     

Whole-cell hollow-fiber reactor: Effectiveness factors

Analytical expressions, which allow the generation of effectiveness factor graphs for a reactor system employing immobilized whole cells a biocatalyst, are presented. In particular hollow-fiber devices (such as dialysis or ultrafiltration units) are considered. Such devices are analogs to a shell-and-tube heat exchanger. Whole cells are entrapped on the shell side: a nutrient solution is circulated through the tubes, substrate diffuses from the tube side, across the fiber, and into the cell mass on the shell side, where it irreversibly reacts to form product. The product back-diffuses into the circulating nutrient solution. The overall substrate mass-transfer process is hypothesized to be either diffusion limited in the hollow-fiber tube wall and/or the shell-side cell suspension and/or reaction limited at the enzyme sites within the whole cells. The first- and zero-order limits of the Michaelis-Menten rate law are used in generating effectiveness factor expressions. The effectiveness factor is a function of reaction order, Thiele modulus, diffusion coefficient ratio (defined as the effective substrate diffusivity in the hollow-fiber membrane wall divided by the effective substrate diffusivity in the cell suspension), partition coefficient, volume of the cell suspension, and hollow-fiber width. Equations for the effectiveness factor are also detailed when the hollow-fiber mass-transfer resistance is far greater than the cell suspension mass-transfer resistance. An effectiveness factor chart is presented specifically for the commercially available C-DAK 4 dialyzer (Cordis Dow Co., Miami, Florida). In general terms the effectiveness factor expressions are applicable for characterizing diffusion and reaction within a catalytically active cylindrical annulus, Whose inner surface offers a diffusional resistance and whose outer surface is impermeable to reactants. Some generalization of the Thiele modulus is undertaken which serves to draw the asymptotes on the effectiveness factor charts together. Comment is made on the variation of the slope of the effectiveness factor graph and its relation to the change in the observed reaction activation energy. Possible application of the model to the catalytic tube wall reactor is discussed.     

Induction of Ears from Maize Seeds in Vitro and Plant Regeneration from Ovaries of Unfertilized Ears

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

A new colorimetric method for determining the isomerization activity of sucrose isomerase

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

Ion exchange resins as catalyst for the isomerization of alpha-pinene to camphene

Camphene is an industrial intermediate compound for commercial chemicals such as isoborneol, isobornyl acetate and camphor. Industrially, the conventional process for camphene production consists of the isomerization of alpha-pinene using acidic TiO2 as catalyst. The use of this catalyst presents problems such as considerable time for preparation, reproducibility and recovery of catalyst from products after the alpha-pinene isomerization. For the first time, a commercial exchange resin was used as catalyst for this reaction. Based on the concentration of product as a function of the reaction time, the path of the alpha-pinene transformation to camphene and byproducts is proposed. Temperature and alpha-pinene/catalyst ratio were studied in order to optimize the yield to camphene production. The obtained results were comparable with those reported for acidic TiO2.