Substrate and water adsorption phenomena in a gas/solid enzymatic reactor

The adsorption of water and substrates to dry deposited alcohol dehydrogenase from Lactobacillus brevis (LBADH) is studied in a continuous enzymatic gas/solid reactor. This work is aiming at obtaining a deeper and more thorough understanding of the enzyme microenvironment in the gas/solid system of acetophenone reduction with concomitant oxidation of 2-propanol. Extensive water adsorption studies showed that the effect of sucrose in the enzyme preparation on the water adsorption isotherm is significant for water activities exceeding 0.5 and reaches a factor 2 with respect to bead mass at water activity of 0.9. Significant hysteresis during water desorption is identified, resulting in up to 0.6 mg_water/mg_protein, for lyophilized enzyme preparation and up to 10 mg_water/mg_protein for deposited enzyme preparation. The adsorption of the substrates is quantified here for the first time. Whereas the adsorption of the main substrate, acetophenone, may reach a significant level of up to 6 mg/mg_protein, at an acetophenone activity of 0.32, the secondary substrate, 2-propanol is not adsorbed at a detectable degree. The presence of water leads to a decrease of the adsorbed amount of acetophenone, by approximately 25%, at a water activity of 0.54.     

Evaluating adsorbed-phase activity coefficient models using a 2D-lattice model

Despite the wide use of the real adsorbed solution theory to predict multicomponent adsorption equilibrium, the models used for the adsorbed phase activity coefficients are usually borrowed from the gas–liquid phase equilibria. In this work, the accuracy of the Wilson and NRTL models for evaluating adsorbed phase activity coefficients is tested using a 2D-lattice model. An accurate model for adsorbed-phase activity coefficients should have no problem in fitting adsorption data obtained using this simple lattice model. The results, however, show that the commonly used Wilson and NRTL models cannot describe the adsorbed phase activity coefficients for slightly non-ideal to strong non-ideal mixtures. Therefore, until new models for adsorbed phase activity coefficients are developed, we should use existing models for liquids with care. In the second part of this work, the use of Monte Carlo simulations on a segregated 2D-lattice model, for predicting adsorption of mixtures is investigated. The segregated model assumes that the competition for adsorption occurs at isolated adsorption sites, and that the molecules from each adsorption site interact with the bulk phase independently. Two binary mixtures in two adsorbent materials were used as case studies for testing the predictions of the segregated 2D-lattice model: the binary system CO₂–N₂ in the hypothetical pure silica zeolite PCOD8200029, with isolated adsorption sites and normal preference for adsorption, and the binary system CO₂–C₃H₈ in pure silica mordenite (MOR), with isolated adsorption sites and inverse site preference. The segregated 2D-lattice model provides accurate predictions for the system CO₂–N₂ in PCOD8200029 but fails in predicting the adsorption behaviour of CO₂–C₃H₈ in pure silica MOR. The predictions of the segregated ideal adsorbed solution theory model are superior to those of the 2D-lattice model.     

Adsorption and recovery of cellulases during hydrolysis of newspaper

The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C_xA; were adsorbed preferentially to those showing C₁A; afterwards ths situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzyme that had remained in solution after typical hydrolysis showed a potential saving of up to 40%.     

Congo Red attached monosize poly(HEMA-co-MMA) microspheres for use in reversible enzyme immobilisation

Monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) (poly(HEMA-co-MMA)), microspheres were prepared by dispersion polymerisation of HEMA and MMA in an ethanol–water medium in the presence of an initiator (α,α′-azobisisobutyronitrile, AIBN). An affinity dye, i.e. Congo Red (CR) was attached covalently and then Fe³⁺ ions were incorporated. The poly(HEMA-co-MMA)-CR attached and poly(HEMA-co-MMA)-CR-Fe³⁺ incorporated microspheres were used in the immobilisation of glucose oxidase (GOD) via adsorption. The adsorption capacities of these microspheres were determined by varying the concentration of GOD in the adsorption medium. GOD adsorption capacities of the Fe³⁺ incorporated microspheres (165 mg g⁻¹) was greater than that of the dye-attached microspheres (126 mg g⁻¹). The non-specific adsorption of the GOD on the poly(HEMA-co-MMA) microspheres was negligible. The K_m values for both immobilised poly(HEMA-co-MMA)-CR-GOD (7.2) and poly(HEMA-co-MMA)-CR-Fe³⁺-GOD (6.8) were higher than that of the free enzyme (6.6 mM). Optimum reaction pH was 5.0 for free and 7.0 for both immobilised preparations. Optimum reaction temperature of the adsorbed enzymes was 10 °C higher than that of the free enzyme and was significantly broader. After 10 successive uses the retained activity of the adsorbed enzyme was 93%. It was observed that enzyme could be repeatedly adsorbed and desorbed on the CR attached poly(HEMA-co-MMA) microspheres without significant loss in adsorption capacity or enzyme activity.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Conversion of cellobiose to glucose using immobilized β-glucosidase reactors

Cellobiose is an intermediate in the enzymatic hydrolysis of cellulose to glucose and acts as an inhibitor for the cellulase enzymes. The conversion of cellobiose to glucose was studied with β-glucosidase adsorbed on Amberlite DP-1, a cation-exchange resin. The best overall pH for adsorption and reactor operation was near 5.0. The K_m values increased with increasing enzyme loading due to competitive inhibition. The maximum practical enzyme loading was about 28 units/g resin. The immobilized enzyme was operated continously in both packed bed and fluidized bed reactors, giving half-lives between 200 and 375 h.     

Improved biochemical characteristics of crosslinked β-glucosidase on nanoporous silica foams

Large mesoporous cellular foam (LMCF) materials were synthesized using the microemulsion templating route. For the enzyme stabilization, β-glucosidase was immobilized onto mesocellular silica foams (MCFs) in a simple and effective way, a process achieved using enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of crosslinked enzyme aggregates (CLEAs) of nanometer scale. The structural and chemical properties of these prepared materials were characterized by TG, CPMAS NMR and nitrogen adsorption measurements. The crosslinked immobilizates retained activity over wider ranges of temperature and pH than those of the free enzyme. Kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The resulting CLEA was proved to be active and recyclable up to 10 cycles without much loss in activity. This demonstrates its prospects for commercial applications. The immobilizate exhibited enhanced storage stability characteristics than the native enzyme. In contrast to adsorbed GL and covalently bound glucosidase, the resulting crosslinked enzyme aggregates (CLEAs) showed an impressive stability with high enzyme loadings.     

Adsorption of major endoglucanase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated A _max and K _ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.     

Inactivation of liver aldolase in fasting rabbits: Evidence for the accumulation of two different immunoreactive forms

During prolonged starvation the activity of aldolase in crude rabbit liver extracts decreases to less than one-half the value observed in extracts of livers from fed animals. The specific activity of the enzyme purified by adsorption on phosphocellulose and elution with substrate is also approximately one-half that of the purified native enzyme. Both the level of enzyme activity and the specific activity are restored to normal within 36 h of refeeding. After removal of active aldolase from the liver extracts by adsorption on phosphocellulose an additional immunoreactive protein can be isolated by adsorption on antialdolase-Sepharose and elution with 4 m MgCl₂. This protein is devoid of catalytic activity and in livers of fasted rabbits accounts for nearly 40% of the total immunoreactive material. It has also been detected in extracts prepared from livers of fed rabbits, where it accounts for 10–20% of the total protein adsorbed by antialdolase-Sepharose. The low-activity enzyme isolated from livers of fasted rabbits cannot be reactivated by sulfhydryl compounds; it shows similar sensitivity to heat and denaturing agents as the enzyme isolated from livers of fed rabbits. The activity ratios with fructose 1,6-bisphosphate, fructose 1-phosphate, and triose phosphate are similar to those observed for the native liver enzyme.     

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

Reactivity of phosphorene with a 3d element trioxide (CrO<Subscript>3</Subscript>) considering van der Waals molecular interactions: a DFT-D2 study

First-principle calculations are performed to investigate the interaction between clean black phosphorene and the CrO₃ molecule which is known to be a powerful oxidizer and a suspected carcinogen. Van der Waals forces are included in all calculations through empirical corrections. Energetics studies are first done to determine the structural stability. Then charge density, Löwdin population analysis and electronic states are evaluated. Results show that the CrO₃ molecule, with an acceptor electron character, is chemisorbed on the phosphorene surface inducing minimal geometrical distortions, however, after adsorption, a partial charge gradient is produced between the P atoms located at the phosphorene upper and lower planes. Furthermore, variations on the CrO₃ concentration causes different interaction strengths. At high concentrations of adsorbed CrO₃ molecules, the interaction with the surface becomes stronger due to an increased steric effect between neighboring molecules. Nevertheless, this effect along with the geometrical distortions produced on the phosphorene structure, due to the large number of molecules adsorbed, leads to a decrement on the adsorption energy. It is expected that the reported results may render phosphorene as a promising material for application as a gas sensor.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.     

Activity losses among T4 lysozyme charge variants after adsorption to colloidal silica.

Enzyme structure and function depend to some extent on enzyme net charge and charge location. Altering the charge of even a single residue may affect the interaction between enzyme and substrate such that all catalytic activity is lost. In this study we investigated the effect of net charge and charge location on the enzymatic activity of synthetic mutants of bacteriophage T4 lysozyme in the presence of colloidal silica. Enzymatic activity decreased upon adsorption, and these changes were variant-specific. Results were interpreted with reference to differences in adsorbed enzyme structure and orientation, and electrostatic effects. By exploring the effects of enzyme charge on adsorption, it may be possible to gain a better understanding of how enzyme structure influences adsorption and function at an interface.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Immobilization of oxyreductases on inorganic supports based on alumina. Immobilization of lactate dehydrogenase on alumina by adsorption

Lactate dehydrogenase (LDH) was adsorbed on low-(γ, η) and high -(θ, α) temperature forms of alumina. θ-Al₂O₃ exhibited the greatest adsorption ability. The maximum adsorption value was 30 mg LDH/g of a carrier. The conditions for irreversible adsorption have been determined. An adsorption isotherm on θ-Al₂O₃ for pH 6.0 has been obtained; the LDH_ads surface area and the carrier surface portion accessible to the enzyme molecules have been calculated. The reaction kinetic parameter were determined by taking into account the reaction proceeding in the intradiffusional region. The specific catalytic activity (A_spec) of LDH_ads at small surface coverage of θ-Al₂O₃ is five times less than A_spec of the native enzyme and K_M^imm with respect to NADH exceeds K_M^nat by two orders or magnitude. The is evidence for a strong LDH–Al₂O₃ interaction and a considerable deformation of the enzyme globule. A_spec and K_M decrease as the amount of the enzyme attached to the carrier increases. Due to adsorption. LDH becomes thermostable and durable. The LDH_ads samples conserve 20–40% of their activity at room temperature during a year.     

Immobilized xanthine oxidase: Kinetics, (in)stability,and stabilization by coimmobilization with superoxide dismutase and catalase

Milk xanthine oxidase was immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to n-octylamine-substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (K_i′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the K_i for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (K_m′) for adsorbed xanthine oxidase was also higher than the K_m for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O₂^? and H₂O₂ side-products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde-crosslinked gelatin matrix, considerably enhanced the operational half-life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).     

Theoretical Study of the TiO2 and MgO Surface Acidity and the Adsorption of Acids and Bases

We present ab-initio periodic Hartree–Fock calculations (crystal program) of small molecules on TiO₂ and MgO. The adsorption of the molecules may be molecular or dissociative. This depends on their acid and basic properties in the gas phase. For the molecular adsorption, the molecules are adsorbed as bases on Ti(+IV) sites, the adsorption energies correlate with the proton affinities. The dissociations on the surface correlate with the gas phase cleavages: thus, the dissociation of MeOH leads to a preferential basic cleavage (the fragment HO– is adsorbed on a Ti⁺⁴ ion and the fragment Me⁺ is adsorbed on a O²– ion of the oxide). The opposite result is obtained with MeSH. Another important factor is the adsorbate–adsorbate interaction: favorable cases are a sequence of H-bonds for the hydroxyl groups resulting from the water dissociation and the mode of adsorption for the ammonium ions. Lateral interactions also force the adsorbed CO₂ molecules to bend over the surface so that their mutual orientation resembles the geometry of the CO₂ dimer. With respect to water adsorption, MgO appears to be a basic oxide. As experimentally observed, NH₃ adsorbs preferentially on TiO₂ and CO₂ on MgO. However, this difference of reactivity should not be expressed in terms of acid vs. basic behaviour but in terms of hard and soft acidity. The MgO surface is a 'soft' acidic surface that reacts preferentially with the soft base, CO₂.     

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K _m value. Starch-binding assays showed that the K _d and B _max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold.     

In situ structure and activity studies of an enzyme adsorbed on spectroscopically undetectable particles

The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased beta-structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surfaces.     

Adsorption of thermomonospora fusca E(5) cellulase on silanized silica

The adsorption kinetics and dodeceyltrimethylammonium-bromide-mediated elution of Thermomonospora fusca E(5) cellulase were recorded in situ, at hydrophobic, silanized silica. Experiments were performed at different solution concentrations, ranging from 0.001 to 0.70 mg/mL. Plateau values of adsorbed mass generally increased with increasing solution concentration, with the adsorbed layer being only partially eluted by buffer. Treatment with surfactant removed more of the adsorbed enzyme in each case, with the remaining adsorbed mass varying little among experiments. Adsorption of E(5) into this nonremovable state was suggested to occur early in the adsorption process and continue until some critical surface concentration was reached. Beyond this critical value of adsorbed mass, adsorption progressed with the protein adopting more loosely bound states. Adsorption kinetic data were interpreted with reference to an adsorption mechanism allowing for irreversible adsorption into two dissimilar states. These states were distinguished by differences in occupied interfacial area, and binding strength, presumably a result of differences in structure. Comparison of the data to the kinetic model based on this mechanism showed that the fraction of adsorbed molecules present in the more tightly bound state decreased as adsorption occurred from solutions of increasing concentration. However, the absolute values of more tightly bound molecules were less dependent on adsorption conditions.