Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE-agarose, Q1A-Sepabeads, and Sepabeads EC-EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI-Sepabeads than in Q1A-Sepabeads or DEAE-supports, requiring very high ionic strength to remove glucoamylase from the PEI-supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).     

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.     

Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes

The equilibrium and kinetics of adsorption of reactive dye RR222 and Cu²⁺, and the activity of immobilization of acid phosphatase, on highly swollen chitosan beads were examined at 30°C. The chitosan was prepared from shrimp shell wastes and was cross-linked with different dosages of glutaraldehyde or glyoxal (100–80,000 mg/l). It was shown that the amounts of solute adsorption and the immobilization capacity of acid phosphatase on cross-linked chitosan beads were substantially affected by their degree of cross-linking. The cross-linking rate of chitosan with glutaraldehyde could be described by a pseudo-second-order equation and the cross-linking equilibrium by the Freundlich equation. This provided an experimental method to control the degree of cross-linking of chitosan beads. Finally, the activity and lifetime of the immobilized enzyme were measured to evaluate the application potential.     

Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.     

Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.     

The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: Stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports

Glutamate dehydrogenase (GDH) from Thermus thermophilus is a homotrimeric enzyme that tends to dissociate at acidic pH values. GDH is readily adsorbed on highly activated anionic exchangers (HAAE), but hardly adsorbed on lowly activated supports (LAAE) or on highly activated epoxy supports. When using amino-epoxy supports, GDH immobilized on HAAE-epoxy and more slowly on LAAE-epoxy supports. Both immobilized biocatalysts were incubated at pH 10 for different times to increase the multipoint covalent attachment. LAAE-epoxy-GDH was stable at pH 4 and 25 °C, the enzyme stability did not depend on the enzyme concentration and did not release any subunit to the supernatant, in opposition to the results obtained using HAAE-epoxy supports. The general application of this strategy to stabilize multimeric enzymes was verified by immobilizing a crude protein extract. It seems that proteins adsorb on LAAE by the larger region of their surface (that is the one that involves the highest number of enzyme subunits), since it is the only area large enough to permit a multipoint ionic exchange on this LAAE. On the contrary, using HAAE, some proteins may become adsorbed by clusters that were rich in anionic groups and located in a corner of the multimer, involving only some of the subunits in the enzyme immobilization. That way, a careful design of the design of the support permits to take full advantage of the immobilization on heterofunctional supports.     

Reversible immobilization of soybean beta-amylase on phenylboronate-agarose

Soybean beta-amylase (EC 3.2.1.2) wap immobilized on phenylboronate-agarose by strong interactive binding. The insoluble derivative was active and more stable to temperature changes than the free enzyme. The absence of enzyme leakage even in the presence of substrate was demonstrated. Changes in pH over a wide range (4.0-8.0) did not affect the stability of the complex. The support could be recovered by sorbitol elution, which demonstrated the reversibility of the binding. Since the enzyme was not retained on phenylagarose under similar conditions, we rejected hydrophobic interactions as a cause of the strong binding of the enzyme to phenylboronate-agarose. We suggest that the bonding of the enzyme to the phenylboronate ligand occurs by a charge transfer mechanism between the trigonal boronate and the side chain nitrogenated groups. It was concluded that phenylboronate-agarose has good properties as a support, which recommends its use for the preparation of immobilized enzymes.     

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.     

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.     

Reversible covalent immobilization of ligands and proteins on polyacrylamide gels

Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.     

Reversible immobilization of eurasian otters with a combination of ketamine and medetomidine

The efficacy and safety of the combination of medetomidine and ketamine was examined in order to establish an adequate chemical immobilization protocol in the Eurasian otter (Lutra lutra) for use during translocation projects in Spain. Thirty-eight Eurasian otters ranging in body mass from 3 to 8.7 kg (mean 5.3 kg) were successfully anesthetized on 82 occasions. The dosage of ketamine was 5.1+/-0.8 (3.4-6.6) mg/kg (mean +/- SD; range) combined with medetomidine at a dosage of 51+/-8 Rg/kg (34-66 microg/kg). In most cases anaesthetic effect occurred within 3 min and the mean induction time was 5.5+/-3.2 min. The mean pulse rate was 95 beats/min. The mean respiratory rate was 32 respirations/min while the relative oxyhemoglobin saturation was 93%. According to these results, this anesthetic protocol is considered safe and can be recommended in wild caught Eurasian otters for immobilization during translocation projects. It is safe, rapid and can be reversed when needed with atipamezole. However caution is required as heart depression resulting in bradychardia may occur.     

Amyloid fibrils as a nanoscaffold for enzyme immobilization

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross‐linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD‐functionalized amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.     

Reversible immobilization of invertase on Cu-chelated polyvinylimidazole-grafted iron oxide nanoparticles

Polyvinylimidazole (PVI)-grafted iron oxide nanoparticles (PVIgMNP) were prepared by grafting of telomere of PVI on the iron oxide nanoparticles. Different metal ions (Cu²⁺, Zn²⁺, Cr²⁺, Ni²⁺) ions were chelated on polyvinylimidazole-grafted iron oxide nanoparticles, and then the metal-chelated magnetic particles were used in the adsorption of invertase. The maximum invertase immobilization capacity of the PVIgMNP–Cu²⁺ beads was observed to be 142.856 mg/g (invertase/PVIgMNP) at pH 5.0. The values of the maximum reaction rate (V _max) and Michaelis–Menten constant (Km) were determined for the free and immobilized enzymes. The enzyme adsorption–desorption studies, pH effect on the adsorption efficiency, affinity of different metal ions, the kinetic parameters and storage stability of free and immobilized enzymes were evaluated.     

Reversible,lectin-mediated immobilization of plant protoplasts on agarose beads

A technique is described for the reversible, lectin-mediated immobilization of plant protoplasts on agarose beads. Cyanogen-bromide-activated agarose beads were coated with protein (gelatine or bovine serum albumin) and lectins were subsequently linked to the protein layer using glutaraldehyde. The technique has possible applications in protoplast fusion-product isolation, cellrecognition studies, and membrane isolation.Abbreviations BSA bovine serum albumin - Con A concanavalin A - FDA fluorescein diacetate - PNA peanut agglutinin - WGA wheat-germ agglutinin     

Curdlan as a support matrix for immobilization of enzyme

Curdlan, a high molecular weight extracellular β(1→3) glucan produced by pure culture fermentation by Agrobacterium radiobacter NCIM 2443 contains large number of free hydroxyl groups. The reaction of hydroxyl containing supports with epichlorohydrin results in activated epoxy groups that can covalently link with available amino, hydroxyl, or sulfhydryl groups of enzymes, thereby immobilizing it. The present work reports on preparation of epoxy-activated matrix for immobilization of a model enzyme, porcine pancreatic lipase. The binding capacity of the matrix prepared by extraction of epoxy-activated curdlan by isopropyl alcohol was found to be 58.7% with about 0.6% loss of the enzyme activity during immobilization. Further, the specific activity of the enzyme increased marginally from 9.37 to 10.2. The corresponding value was 10.15 for a commercial sample of curdlan, epoxy-activated as for laboratory-isolated curdlan. Sepharose, the most widely used support matrix for the immobilization of enzymes was used for comparison in this study.     

Enhancement of product selectivity via enzyme immobilization in sequential degradation reactions of polymeric substrates

The balance equations pertaining to the modelling of a slap-shaped bead containing immobilized enzyme uniformly distributed which catalyzes the sequential reactions of degradation of a polymeric substrate were written and analytically solved in dimensionless form. The effect of the Thiele modulus on the selectivity of consumption of each multimeric product was studied for a simple case. Whereas plain diffusional regime leads to lower selectivities than plain kinetic regime, improvements in selectivity of species A _i relative to species A_i+1 may be obtained at the expense of higher Thiele moduli within a limited range when the diffusivity of A _i is larger than that of A _i +1, or when the pseudo first order kinetic constant describing the rate of consumption of A _i is lower than that of A_i+1.List of Symbols A _i polymeric substrate containing i monomeric subunits - C _i mol·m^–3 normalized counterpart of C _i - C _i mol·m^–3 concentration of substrate A _i - C _i,0 mol·m^–3 initial concentration of substrate A _i - C _i,0 normalized counterpart of C _i,0 - D _ap,i m²·s^–1 apparent diffusivity of substrate A _i - k _i s^–1 pseudo-first order rate constant - K _m,i mol·m^–3 Michaelis-Menten constant associated with substrate A _i - L m half-thickness of the catalyst slab - N number of monomeric subunits of the largest substrate molecule - Th Thiele modulus - V _i mol·m^–3·s^–1 rate of rection of substrate A _i - V_max,i mol·m^–3·s^–1 maximum rate of reaction under saturating conditions of substrate A _i - x m longitudinal coordinate - S _i,i+1 selectivity of enzyme with respect to substrates with consecutive numbers of monomeric subunits Greek Symbols _i ratio of maximum rates of reaction - _i ratio of apparent diffusivities