Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

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.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Immobilization of lipase into mesoporous silica particles by physical adsorption

Mesoporous silica particles for immobilization of lipase from Candida rugosa were prepared by precipitation and aggregation of primary particles from highly basic sodium silicate solution but without addition of templates. The average pore size of the material was 15.8 nm, which allowed enzyme adsorption inside the pores and high enzyme loading. Specific surface area of the material was found to be 359 m²g^?1. A loading of 100 mglipasegdrysilica^?1 was obtained at initial enzyme concentration of 1.8 mgmL^?1 by physical adsorption. The FTIR spectrum showed the structural conformation of lipase to be retained after adsorption into the mesoporous silica support. Although the efficiency of the mesoporous biocatalyst was shown to be lower than that of the free enzyme, the immobilized enzyme showed enhanced thermal stability and could be desorbed with Triton X-100, indicating the hydrophobic nature of the adsorption.     

Determination of intrinsic parameters for immobilization reactions of catalase and amyloglucosidase in porous glass supports

Experiments have been carried out for immobilizing enzyme-catalase and amyloglucosidase in controlled-pore glass particles of two different pore sizes. The experimental results have been analyzed, initial-stage analysis for the rate parameters of immobilization reactions and long-time analysis for determining the evolution of the immobilization process. These investigations suggest that the overall process of immobilization is controlled by the restricted diffusion of enzymes into the pores of the support. As a result, immobilized enzyme (IME) can penetrate only up to a certain distance into the support. The penetration depth of IME for the support-enzyme system mentioned have been evaluated from the experimental bulk enzyme concentration data in a batch recirculation reactor.     

Hydrolysis of soluble starch using Bacillus licheniformis alpha-amylase immobilized on superporous CELBEADS

In the present work, indigenously prepared rigid superporous (pore size of approximately 3 microm) cross-linked cellulose matrix (CELBEADS) has been used as a support for the immobilization of Bacillus licheniformis alpha-amylase (BLA). Optimum pH and temperature, and Michaelis-Menten constants were determined for both free and immobilized BLA. Immobilized BLA was observed to produce a different saccharide profile than free BLA at any value of dextrose equivalent. It was observed that pH, temperature, and initial starch concentration has a significant effect on the saccharide profile of starch hydrolysate produced using immobilized BLA in the batch mode, whereas the ratio of concentration of enzyme units to initial starch concentration has no influence on the same. Hence immobilized BLA can be used as an additional tool for production of maltodextrins with different saccharide profiles. Immobilized BLA has better thermostability than free BLA. Immobilized BLA was found to retain full activity even after eight batches of hydrolysis, each of 8h duration at 55 degrees C and 90 mg/mL initial starch concentration. A semiempirical model has been used for the prediction of saccharide composition of starch hydrolysate with respect to time.     

One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.     

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.     

Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are 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.     

Protein‐coated polymer as a matrix for enzyme immobilization: Immobilization of trypsin on bovine serum albumin‐coated allyl glycidyl ether–ethylene glycol dimethacrylate copolymer

Allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE‐25) shows excellent bovine serum albumin (BSA) adsorption (up to 16% (w/w)) at pH 8.0 and the adsorbed BSA is strongly bound. This protein‐coated polymer provides a novel matrix with naturally existing functional groups such as thiol, amino, and carboxylic acid that are available for covalent immobilization of functional enzymes. Employing appropriate strategies, trypsin as a model protein was covalently bound to BSA‐coated matrix both independently, and in a stepwise manner on the same matrix, with less than 5% loss of enzyme activity during immobilization. Glutaraldehyde crosslinking after immobilization provide stable enzyme preparation with activity of 510 units/g recycled up to six times without loss of enzyme activity. AFM studies reveal that the polymer surface has protein peaks and valleys rather than a uniform monolayer distribution of the protein and the immobilized enzyme preparation can best be described as polymer supported cross‐linked enzyme aggregates (CLEAs). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:317–323, 2014     

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.     

Characterization of efficient proteolysis by trypsin loaded macroporous silica

Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.     

Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure–activity changes

The purpose of the present investigation was to study the pH dependence of both the immobilization process and the enzyme activity of a feruloyl esterase (FoFaeC from Fusarium oxysporum) immobilized in mesoporous silica. This was done by interpreting experimental results with theoretical molecular modeling of the enzyme structure. Modeling of the 3D structure of the enzyme together with calculations of the electrostatic surface potential showed that changes in the electrostatic potential of the protein surface were correlated with the pH dependence of the immobilization process. High immobilization yields were associated with an increase in pH. The transesterification activity of both immobilized and free enzyme was studied at different values of pH and the optimal pH of the immobilized enzyme was found to be one unit lower than that for the free enzyme. The surface charge distribution around the binding pocket was identified as being a crucial factor for the accessibility of the active site of the immobilized enzyme, indicating that the orientation of the enzyme inside the pores is pH dependent. Interestingly, it was observed that the immobilization pH affects the specific activity, irrespective of the changes in reaction pH. This was identified as a pH memory effect for the immobilized enzyme. On the other hand, a change in product selectivity of the immobilized enzyme was also observed when the transesterification reaction was run in MOPS buffer instead of citrate phosphate buffer. Molecular docking studies revealed that the MOPS buffer molecule can bind to the enzyme binding pocket, and can therefore be assumed to modulate the product selectivity of the immobilized enzyme toward transesterification.     

Effects of pore characters of mesoporous resorcinol–formaldehyde carbon gels on enzyme immobilization

This paper demonstrates, for the first time, the use of resorcinol–formaldehyde carbon gels (RFCs) as enzyme carriers. The immobilization behavior of Bacillus licheniformis serine protease in RFCs of different pore characters was investigated. RFCs derived with (RF1) and without (RF2) cationic surfactant (trimethylstearylammonium chloride; C18) resulted in predominantly microporous, and mesoporous characters, respectively. It was found that support pore size and volume were key parameters in determining immobilized enzyme loading, specific activity, and stability. RF2, with higher mesopore volume (V_mes: RF1 = 0.21 cm³/g; RF2 = 0.81 cm³/g) and mesopore size radius (RF1 = 1.7–3.8 nm; RF2 = 7.01 nm), accommodated approximately fourfold more enzyme than RF1. Serine protease loading in RF2 could reach as high as 21.05 unit/g support. In addition, RF2 was found to be a better support in terms of serine protease operation and storage stability. Suitable mesopore size likely helped preventing immobilized enzyme from structural denaturation due to external forces and heat. However, immobilized enzyme in RF1 gave 12.8-fold higher specific activity than in RF2, and 2.1-fold higher than soluble enzyme. Enzyme leaching was found to be problematic in both supports, nonetheless, higher desorption was observed in RF2. Enhancement of interaction between serine protease and RFCs as well as pore size adjustment will be necessary for repeated use of the enzyme and further process development.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Stable and efficient immobilization technique of aldolase under consecutive microwave irradiation at low temperature

To establish a stable and efficient immobilization technique under microwave irradiation, a focused microwave reaction system was used, where the temperature was set appropriately in the microwave system and cooling module to produce consecutive microwave irradiation. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA) was rapidly and efficiently immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation. When the output power in the microwave system was set to 30 W, after 3 min, 88.4% of the enzyme protein was coupled to the wall of the support pores and the specific activity of the immobilized enzyme was 2.24 U mg⁻¹, 149.2% higher than that of the free enzyme and 157.0% higher than that of the non-microwave-assisted immobilized enzyme. In catalysis, microwave-assisted immobilized DERA tolerated a wider range of both pH and temperature than other DERA preparations. The thermal and storage stabilities were also significantly improved. This focused; microwave-assisted immobilization technique has proven to be simple, stable and highly efficient. This technique could also be applied to other enzyme immobilizations.