Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Determination of total and active immobilized enzyme distribution in porous solid supports

The total and active immobilized enzyme (IME) distributions in porous supports are studied both theoretically and experimentally. In order to determine experimentally the enzyme distribution profiles within a single particle, we construct a diffusion cell containing controlled-pore glass particles such that the cell would mimic a large pellet support. Our purpose is to study the interplay between the diffusion process within the interparticle void space and immobilization process in the controlled-pore glass particles onto the evolution of the (total and active) enzyme distributions. A mathematical model is developed to describe the interaction of various processes within the diffusion cell. The immobilized enzymes are determined for a system of trypsin and controlled-pore glass particles. The total amount of enzymes are determined by the amino acid analysis, and the active fraction is obtained by an active-site titration. The experimentally measured total IME profiles compare very well with that predicted by the model. The determined active enzyme profile is found to be nonuniform one, and it represents about 40% of the total enzyme immobilized in the support particles.     

Diffusion and chemical reaction rates with nonuniform enzyme distribution: an experimental approach

The study of the effects of nonuniform distributions of immobilized beta-galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two beta-galactosidase substrates: lactose and ONPG in a commercial silica-alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized beta-galactosidase of different diameter (1 x 10(-4) to 5 x 10(-3) m) as catalyst, employing a temperature of 40 degrees C for lactose and 25 and 40 degrees C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 x 10(-3) m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution.     

Influence of intraparticle diffusional limitation on the observed kinetics of immobilized enzymes and on catalyst design

Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes. These solutions have also been compared with experimental data for enzymes immobilized to DEAE-cellulose particles. The influence of particle size and enzyme content on catalyst design is discussed.     

Mass transfer effects in solvent-free fat interesterification reactions: influences on catalyst design

The use of solvent-free systems in the oil and fats industry is commonplace. Initial studies on interesterification were carried out in solvent systems because the lipase was immobilized solely by adsorption onto particles of diatomaceous earth. In this study, the mass transfer characteristics associated with the continuous interesterification of olive oil in a solvent-free system have been examined, for lipase immobilized on the three ion-exchange materials: Duolite ES562, Duolite ES568, and Spheroil DEA. The process of immobilization is influenced by the internal structure of the material and this in turn influences the interesterification activity of the catalyst. Individually prepared catalysts for the three support materials have shown that external mass transfer limitations are unlikely even at low flowrates.In the case of Spherosil DEA, with a mean pore diameter of 1480 A, the wide pores would be expected to reduce internal mass transfer limitations; however, it is more likely that the reduction in activity with increased catalyst loading is due to the lipase molecules being immobilized in a tightly packed monolayer. In such a situation, some active sites of the lipase molecules would become inaccessible to substrate molecules leading to an observed reduction in activity. For Duolite ES568, the observed results are very similar to those seen for Spherosil DEA, however, the pore structure of this support material indicate that some internal mass transfer limitations may also be occurring. Yet the contribution of the individual effects cannot be determined. The results observed for the support Duolite ES562 are different than those observed for the other materials and reflect the heterogeneity of Duolite ES562. The large proportion of narrow pores in the support mean that, for the catalysts examined, immobilization is most likely to have occurred in the external pores of the particles, and as such no internal mass transfer limitation is observed.It is clear that for interesterification the material chosen for enzyme immobilization will have an important role in determining the catalyst efficiency. External mass transfer limitations are very minor and observed internal mass transfer limitations may be caused by both internal mass transfer and the manner in which the immobilization process occurs. (c) 1994 John Wiley & Sons, Inc.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

Enzyme loading in a solid support with nonuniform pore size distribution

The concept of pore size distribution is incorporated into the Clark model of enzyme immobilization in the present study. This refined model predicted that in the case of small harmonic pore radius with the same surface area and porosity of the support, more enzyme could be loaded in a support with nonuniform pores than that with uniform pores. In comparing the enzyme loading efficiency of the support with two different pore size distributions, the one with Gaussian distribution had the greater amount of enzyme immobilized than the other one with Rajagopalan's distribution. Furthermore, more enzyme could be loaded in a support with wider Gaussian pore size distribution than that with narrower distribution. The immobilized enzyme profile in the solid support with pore size distribution displayed a stepwise pattern which differed appreciably from the sigmoidal profile predicted for the support with uniform pore size. This stepwise enzyme distribution profile became sigmoidal with decreasing h(T) or increasing k. The new model could be used for designing protocols for an enzyme immobilization process.     

Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology

Utilizing whole cell biocatalyst instead of free or immobilized enzyme is a potential way to reduce the cost of catalyst in lipase-catalyzed biodiesel production. Rhizopus oryzae (R. oryzae) IFO4697 whole cell immobilized within biomass support particles (BSPs) was used for the methanolysis of soybean oil for biodiesel production in this paper. tert-Butanol was demonstrated to be an ideal reaction medium, in which the negative effects caused by substrate methanol could be eliminated effectively. A central composite design was adopted to study the effect of tert-butanol quantity, methanol quantity, water content and dry biomass of the immobilized cell on biodiesel (methyl ester) yield. Each factor was studied in five levels. Using response surface methodology, a quadratic polynomial equation was obtained for methyl ester yield by multiple regression analysis. Biodiesel yield of 72% could be obtained under the optimal conditions and further verification experiments confirmed the validity of the predicted model.     

Parameters in the construction of an immobilized dual enzyme catalyst.

The glucose oxidase and catalase activities immobilized to the gamma-aminopropyltriethoxysilane derivative of nickel-impregnated silica alumina was controlled by several factors. The most important of these was enzyme concentration. In constructing the dual immobilized enzyme catalyst, competition between the two enzymes for available binding sites was observed. The order of addition of the various reactants during immobilization was also important. Higher glucose oxidase activities were immobilized when glutaraldehyde was added concurrently with enzyme, while maximal coupling of catalase occurred if glutaraldehyde was first added to react with the amino derivative of the silica alumina support, excess reagent washed away, and then the catalase added. Bovine serum albumin, which aids in the crosslinking of glucose oxidase, hindered the coupling of the enzyme to the support particles.     

In situ visualization and effect of glycerol in lipase-catalyzed ethanolysis of rapeseed oil

Immobilized lipases can be used in biodiesel production to overcome many disadvantages of the conventional base-catalyzed process. However, the glycerol by-product poses a potential problem for the biocatalytic process as it is known to inhibit immobilized lipases, most likely by clogging of the catalyst particles. In this paper, this negative effect was further investigated and confirmed in ethanolysis of rapeseed oil. A dyeing method was developed for in situ visualization of glycerol in order to study its partitioning and accumulation during the ethanolysis reaction. The method was used to illustrate the interaction of glycerol with immobilized lipases and thus provided an aid for screening supports for lipase immobilization according to their interaction with glycerol. Glycerol was found to have great affinity for silica, less for polystyrene and no affinity for supports made from polymethylmethacrylate and polypropylene. It was also found that the immobilization of enzyme on the support influenced the adsorption of glycerol to the surface of the enzyme carrier.     

Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schro?n, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

几种离子液体的微波法合成及其对脂肪酶催化效果的影响

采用微波法合成9种目标离子液体,对中间体[Bmim]Br的合成条件及其离子液体对全细胞催化剂催化效果的影响进行考察.直接将产脂肪酶真菌粗状假丝酵母(Candida valida) T2细胞固定在聚氨酯颗粒中,制备固定化细胞催化剂,将其应用于合成离子液体介质中催化甲醇与大豆油酯交换反应制备生物柴油.结果表明:微波功率200 W下间隙照射100 s,中间体[Bmim]Br的收率达95.16%,有效地提高了离子液合成产率;在[Bmim]PF6离子液中固定化细胞酶催化转酯化反应30 h,大豆油的转化率达42%,反应效果较其他8种合成离子液体好;固定化细胞颗粒和[Bmim]PF6重复使用4次,其油脂转化率和酶活保持率分别达到29%和69%,表现出较好的催化反应稳定性.     

Evaluation of enzyme distribution in spherical supports by computed fluorescence tomography

The overall catalytic properties of technical biocatalysts are strongly dependent on the spatial distribution of the enzyme immobilized within the porous support. Microfluorometry of support particles grafted with fluorochrome-tagged enzymes offered a convenient means to study these profiles without destruction of the support matrix. Equatorial point scanning as well as a much more sensitive slit scanning of a variety of fluorescent enzyme-support conjugates were studied and compared. The principal problem with this technique, the evaluation of local enzyme densities from a measured integral signal, could be solved mathematically. The resulting relationships resemble those obtained for a simple symmetrical case in computed X-ray tomography which led us to term this technique computed fluorescence tomography. Measurements and data evaluation are demonstrated by means of examples. The general conclusion reached is that inhomogeneous enzyme distributions form more often than hitherto realized.     

Fundamental studies of glucose oxidase immobilization on controlled pore glass

Immobilization experiments have been performed with glucose oxidase as enzyme and controlled-pore glass of different pore sizes as support for chemical coupling. The experimental results have been analyzed for comparison with the theoretical model predictions. Analysis of the initial stage of the process gives the fundamental characteristic of the immobilization reaction. These investigations allow us to study the influence of the degree of diffusional restriction on the evolution of the immobilization process and spatial distribution of immobilized enzyme. Nonuniformly distributed concentrations have been achieved within the porous matrix, and suggestions have been made in designing such profiles by choosing appropriate experimental parameters.     

Determination of intraparticle immobilized enzyme distribution in porous support by confocal scanning microscopy

Summary A method for determining the distribution of immobilized protein within a porous support has been developed. The method is based on confocal microscopy of polyacrylamide gel beads coupled to fluorescein isothiocyanate labelled enzyme. It is applied to immobilized soybean lipoxygenase. This technique allows the quantitative and qualitative analysis of protein distribution profile in intact polymer beads without splitting or cutting the carrier.     

固定化脂肪酶催化制备L-薄荷醇

用大孔树脂NKA固定高选择性的脂肪酶,催化有机相中转酯化反应,从而拆分八异构体消旋薄荷醇来制备L-薄荷醇。研究pH、载体与酶比例对固定化酶制备的影响及固定化酶的反应稳定性;考察温度、转酯化过程醇酯比例、及底物醇异构组成变化对拆分效果的影响。结果表明:固定化酶的最适pH为8,载体与酶的比例为5∶1时,所得固定化酶的反应稳定性比游离酶的反应稳定性提高了约50%;转酯化反应的最优温度为40℃,醇酯比例为1.5∶1时最佳,改进八异构体消旋薄荷醇组分比例后,非对映体选择率dep达到了95.1%。     

Thermo-sensitive amphiphilic block copolymer poly (styrene-<Emphasis Type="Italic">b</Emphasis>-N-isopropylacrylamide) with switchable catalytic activity immobilizing pectinase

Pectinase was immobilized onto thermo-sensitive amphiphilic block copolymers poly(styrene-b-Nisopropylacrylamide) PS-b-poly(N-isopropylacrylamide) (PNIPAM) by covalent attachment. Biochemical studies have found that the stability of the PS-b-PNIPAM support is not impeded by the bound proteins despite that up to 242.5 mg of enzyme is immobilized per gram of carrier particles. The immobilized enzyme retained nearly 65% of its initial activity over 30 days, and the optimum temperature and pH also increased to the range of 60 ∼ 70°C and 4.0 ∼ 6.0, respectively. The immobilized enzyme also exhibited great operational stability, and more than 60% residual activity was observed in the immobilized enzyme after 10 batch reactions. Moreover, the lower critical solution temperature of the PS-b-PNIPAM support could be switched on or off by a small change in solution temperature. Thus, the immobilized pectinase could be recovered and showed durable activity during the recycle process.     

Operational stability of immobilized d-glucose isomerase in a continuous feed stirred tank reactor

d-Glucose isomerization has been studied using immobilized cells of Streptomyces phaeochromogenes in a continuous feed stirred tank reactor (CSTR) where the external film diffusion resistance was negligible. Experiments conducted with various sizes of enzyme particles indicated that a strong internal diffusion resistance improved the apparent stability of these particles. The performance equations of the CSTR were constructed by associating the material balances for the inside porous support matrix with the bulk liquid phase, and enzyme deactivation was also taken into consideration. An iterative method together with the orthogonal collocation method is proposed for the evaluation of effectiveness factor and the substrate concentration profile within the enzyme particles. The numerical results offer an alternative analytical proof for the observation that under strong internal diffusion control the apparent operational stability of immobilized enzyme is improved.