The separation of glutathione and glutamic acid using a simulated moving-bed adsorber system

Glutathione (GSH) and glutamic acid (Glu) were continuously separated using a simulated moving-bed adsorber system. In this system, a specially-designed multi-port rotary valve was used to move the adsorbent particles, and its rotation speed and the liquid flow rate in the adsorber were controlled by a personal computer. Under the optimal conditions for the simulated moving-bed adsorber, both the purity and the yield coefficient of GSH in the raffinate stream at a steady state reached around 99%, and the concentration of GSH and the production of GSH for the amount of adsorbent used were greater by as much as ten times and eighteen times than for the conventional batch operation. The adsorption isotherms of GSH and Glu, which were non-linear and concetration-dependent, wre well expressed using several parameters, and also the courses of GSH and Glu concentrations in transient changes to the steady-state condition could be predicted well by the intermittent moving-bed model.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Inulin hydrolysis by the Debaryomyces phaffii inulinase immobilized on Deae cellulose

Debaryomyces phaffii inulinase was immobilized by adsorption on DEAE-cellulose. The immobilization caused a drop in optimum pH, a slight increase in optimum temperature and an important increase in the thermal stability of the system. The activity yield of immobilized inulinase was 75%.A continuous reactor operation was carried out. The utilization of this system permited the production of fructose syrup from inulin.     

Continuous production of fructose syrup and ethanol from hydrolysed Jerusalem artichoke juice

Summary The results from this study showed that Jerusalem artichoke juice can be used for the production of very enriched fructose syrup by selective conversion of glucose to ethanol in a continuous process using immobilized cells ofSaccharomyces cerevisiae ATCC 36859. The product contained up to 99% of the total carbohydrates as fructose compared to 76% in the feed. Using Jerusalem artichoke juice supplemented with some glucose a product was obtained with 7.5% w/v ethanol which made ethanol recovery economically favourable. It was found that some fructose was consumed in these continuous processes; the glucose/fructose conversion rate ratio was regulated by the glucose concentration in the product stream.     

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.     

Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.     

Biosorption of 2,4-dichlorophenol by immobilized white-rot fungus Phanerochaete chrysosporium from aqueous solutions

The fungus Phanerochaete chrysosporium was immobilized in several polymer matrices: Ca-alginate, Ca-alginate-polyvinyl alcohol (PVA) and pectin, and was then used as a biosorbent for removing 2,4-dichlorophenol (2,4-DCP) in wastewater. Immobilization of P. chrysosporium onto pectin was less efficient than that onto other matrices because of its poor mechanical strength and low adsorption efficiency. Ca-alginate immobilized fungal beads with biocompatibility exhibited good mechanical strength and adsorption efficiency over 60%. Among the different biomass dosages in Ca-alginate immobilized fungal beads, 1.25% (w/v) was the optimum. The adsorption data of 2,4-DCP on the blank Ca-alginate beads, free, and immobilized fungal biomass could be described by the Langmuir and Freundlich isotherms very well. Desorption operation was efficiently completed by using distilled water as eluant, and the desorption efficiency reached 82.16% at an optimum solid/liquid ratio of 14.3. The consecutive adsorption/desorption cycles studies employing the Ca-alginate immobilized fungal beads demonstrated that the immobilized fungal biomass could be reused in five cycles without significant loss of adsorption efficiency and adsorbent weight.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Recovery of Acinetobacter radioresistens lipase by hydrophobic adsorption to n-hexadecane coated on nonwoven fabric

A simple and clean adsorption/desorption process was proposed for recovering Acinetobacter radioresistens lipase from fermentation broth. The adsorbent used was n-hexadecane coated on a hydrophobic nonwoven fabric (NWF). n-Hexadecane has a melting point of 16-18 degrees C, and its affinity for lipase decreases markedly from liquid to solid state. Accordingly, performing the adsorption and desorption above and below, respectively, the melting point would need no extraneous materials for separation. The adsorption isotherms at various temperatures were found to follow the Langmuir model. Simulation of the batch adsorption/desorption process showed that there exists an optimal amount of adsorbent for both concentration factor and enzyme recovery; the process is restrained by equilibrium. The performance of column adsorption/desorption could also be simulated using the adsorption isotherm, and it was shown that the concentration factor was proportional to the amount of adsorbent used. The benefits of this process include easy preparation of adsorbent, low operational cost, no extraneous materials needed, negligible enzyme denaturation, high efficiency, and simple process simulation.     

Modified reactive SMB for production of high concentrated fructose syrup by isomerization of glucose to fructose

This work presents modifications to the Hashimoto's hybrid simulated moving bed reactor (SMBR) system which was used to produce 55% high fructose syrup (HFS55). The purpose of this study is to develop a new SMBR system to overcome the disadvantages of Hashimoto system (3-zone SMB with seven reactors), i.e., low utility of reactors when feed being a 50/50 blend of glucose and fructose. Two different configurations of modified system were presented in this paper: the first configuration is 4-zone SMB with one reactor, while the other one consists of one additional reactor. Both of these configurations aim at improving the concentration and purity of glucose at the inlet of the reactor, which will lead to both high productivity and high purity of fructose in the product. A state-of-the-art optimization technique, viz., non-dominated sorting genetic algorithm (NSGA) is used in finding the optimal design and operating parameters for the modified reactive SMB and Varicol processes. Compared with the Hashimoto's system, high productivity and purity of fructose can be achieved in these new systems using less number of reactors.     

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.     

Mathematical modeling of the integrated process of mercury bioremediation in the industrial bioreactor

The mathematical model of the integrated process of mercury contaminated wastewater bioremediation in a fixed-bed industrial bioreactor is presented. An activated carbon packing in the bioreactor plays the role of an adsorbent for ionic mercury and at the same time of a carrier material for immobilization of mercury-reducing bacteria. The model includes three basic stages of the bioremediation process: mass transfer in the liquid phase, adsorption of mercury onto activated carbon and ionic mercury bioreduction to Hg(0) by immobilized microorganisms. Model calculations were verified using experimental data obtained during the process of industrial wastewater bioremediation in the bioreactor of 1 m³ volume. It was found that the presented model reflects the properties of the real system quite well. Numerical simulation of the bioremediation process confirmed the experimentally observed positive effect of the integration of ionic mercury adsorption and bioreduction in one apparatus.     

Electrospun gold nanofiber electrodes for biosensors

A new form of high surface area bioelectrode, based on nanofibers of electrospun gold with immobilized fructose dehydrogenase, was developed. The gold fibers were prepared by electroless deposition of gold nanoparticles on an electrospun poly(acrylonitrile)-HAuCl(4) fiber. The material was characterized using electron microscopy, XRD and BET, as well as cyclic voltammetry and biochemical assay of the immobilized enzyme. The electrochemical surface area of the gold microfibers was 0.32 ± 0.04 m(2)/g. Fructose dehydrogenase was covalently coupled to the gold surface through glutaraldehyde crosslinks to a cystamine monolayer. The enzyme exhibited mediated electron transfer directly to the gold electrode and catalytic currents characteristic of fructose oxidation in the presence of a ferrocene methanol mediator were observed. The limit of detection of fructose was 11.7 μM and the K(M) of the immobilized enzyme was 5mM. The microfiber electrode was stable over 20 cycles with a 3.05% standard deviation. The response time of the sensor was less than 2.2s and reached half maximum value within 3.6s. The sensor was proven to be accurate and precise in both serum and popular beverages sweetened with high fructose corn syrup. The addition of glucose isomerase enabled the sensor to perform with glucose, thus expanding the available analyte selection for the sensor.     

Dynamic simulation of the integrated bioreactor

The article reports numerical simulation for the system combining selective adsorption of fructose and an enzyme reaction to produce higher fructose syrup. A procedure for the dynamic simulation of the process in the frame of equations for mass transfer in fixed bed, is obtained by making a solute material balance. A fixed bed model incorporating axial dispersion and a linear driving force for mass transfer was successfully used to estimate optimum operating conditions (switch times and fluid flow rates)     

Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance

A strategy for the optimization of an expanded bed adsorption process has been developed by studying a model system involving the adsorption of lysozyme onto the adsorbent STREAMLINE SP. The hydrodynamic and adsorption properties of this ion exchange adsorbent in a variety of viscosities of feedstocks have been compared by analyzing bed expansion characteristics, liquid phase dispersion characteristics, equilibrium adsorption isotherms, and mass transfer characteristics. Additionally, the influences of the degree of bed expansion on adsorption performance have been investigated by frontal analysis. In these experiments, viscous feedstocks were simulated by the inclusion of glycerol in the adsorption buffers. Breakthrough curves for lysozyme were characterized and compared in terms of overall purification processing time and productivity. On the basis of these results, the relative productivities of different operating modes with the same process liquid were found to be almost the same. However, the processing time for each purification cycle decreased with increasing velocity of process liquid. It is demonstrated that an adsorption process carried out at a constant degree of bed expansion (twice its settled bed height, corresponding to bed voidage of 0.7) is more efficient, when characterized by the apparent dynamic binding capacity, than operation at a constant liquid velocity of 300 cm/h. These results have significant implications on the design and operation of the expanded bed adsorption procedures. The advantages and problems encountered in the use of expanded bed techniques for the direct extraction of proteins from unclarified feedstocks are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite^TM IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.     

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.     

High fructose formation from sugarcane syrup and molasses usingZymomonas mobilis mutants

High fructose recovery yields were obtained using sugarcane syrup and C-molasses (equal to blackstrap molasses) and a fructokinase negative mutant ofZymomonas mobilis. The fructose recovery was 95.7% with sugarcane syrup and 99.4% with 300 g/L C-molasses or mixtures of both. High fructose corn syrup of a 48/52 mixture of glucose and fructose gave only a 65–70% fructose recovery due to high sorbitol formation.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

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.