Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.     

N-hydroxysuccinimide carbonates and carbamates are useful reactive reagents for coupling ligands to lysines on proteins

Ligands containing amino or hydroxyl groups were converted to their corresponding activated N-hydroxysuccinimidyl carbamate and carbonate by reaction with disuccinimidyl carbonate (DSC). The latter reagents can be used for the group-specific modification of primary amines as an alternative to the widespread usage of N-hydroxysuccinimide esters. Biotin and 2,4-dinitrophenyl (DNP) derivatives were used as examples to demonstrate the approach. Biotin and DNP were each extended by attaching two different spacer arms, carrying either a hydroxyl group or a primary amine as terminal functions. The latter were then activated via their conversion to N-hydroxysuccinimide carbonates and carbamates, respectively. The usefulness of these reagents for protein modification was investigated. The modified proteins obtained exhibited similar stability and activity characteristics compared to those modified with active N-hydroxysuccinimdyl esters. The activation of hydroxy- or amino-terminating compounds with DSC represents a general method that can be applied to any ligand which contains these functional groups for its covalent coupling to amines.     

A new method for measuring sulfotransferase

Agarose can be activated by adding cyanogen bromide, dissolved in acetonitrile, to beads suspended in a solution of sodium carbonate. The necessity for manual titration and the use of a pH meter are thus eliminated. Activation for 1 min results in coupling capacities comparable to those reported for the titration method. The coupling capacity is influenced by the initial carbonate concentration and by the duration and temperature of the activation reaction. The coupling capacities of the activated gels are very reproducible.     

含环碳酸酯基固定化酶载体的合成及其性能研究

 环碳酸酯基具有于温和条件下与氨基反应形成羟基与氨基甲酸酯的特征 .通过反相悬浮聚合 ,以乙烯撑碳酸酯为反应性单体 ,亲水性N ,N′ 亚甲基双丙烯酰胺为交联剂 ,分别选用N 乙烯基吡咯烷酮和丙烯酸 β 羟乙酯两种亲水性共单体为合成酶载体的成分 ,首次以二甲基甲酰胺和线型高分子聚乙二醇 40 0为复合致孔剂合成了两种性能优越的 ,可快速且可保持酶活力的固载胰蛋白酶的大孔型交联高分子酶载体 .其以σ共价键形成的固定化酶和以纯物理吸附形式吸附的酶同时存在于树脂骨架之中 ,根据相似相容原理 ,固载于载体上的酶分子容易吸附溶液中酶分子使其以物理吸附的形式存在 ,后者在经过 6次使用后近乎定量脱落 ,最终保持 85%活力不变者当为σ共价键结合酶 .纯σ共价键结合于高分子骨架的酶不易脱落 ,利用这一特性可使固定化酶的催化过程简化 ,可避免繁杂而冗长的分离脱落酶的过程 .可望将这种高分子酶载体用于临床医学中属于多肽类的分子量为 2 0 0 0左右的中级分子量毒素的无泄露排除 .     

The co-operative effect of physical and covalent protein adsorption on heterofunctional supports

It has been found that the enzymes penicillin G acylase from Escherichia coli (PGA) and lipase from Bacillus thermocatenulatus (BTL) did not significantly adsorb on highly activated amino-agarose beads at pH 7 (a support where 85–90% of a crude extract of proteins become adsorbed). Moreover, it has been found that these enzymes do not covalently immobilize on highly activated epoxy-agarose beads at pH 7. However, both enzymes slowly immobilize on heterofunctional supports having a high density of amino–epoxy groups. The immobilized enzymes retain a high percentage of activity (more than 90% for PGA and 60% for BTL). On the other hand, the immobilization of a crude extract of proteins on amino–epoxy supports under conditions where only a limited protein ionic exchange was permitted (by using high ionic strength or lowly activated supports), also permitted a similar high immobilization yield of the proteins. Similarly, glutamate dehydrogenase (GDH) and β-galactosidase from Thermus thermophilus can be fully immobilized under conditions where less than 20% of these enzymes can be ionically exchanged in the aminated support. The results suggested that the percentage of proteins that may be physically adsorbed on the support becomes irreversibly immobilized by the covalent reaction between the nucleophilic groups in the protein surface and the very near epoxy groups of the support (in an almost intramolecular reaction). Thus, using these supports, it is possible to immobilize almost all the proteins by anionic exchange, that is, the area with the highest density in anionic groups. In many cases, this region could not correspond to the protein regions usually utilized to immobilize proteins. This way, it is possible to achieve, in a very simple fashion and without modifying the protein, new orientations of some immobilized enzymes and proteins.     

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well.     

Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.     

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.     

Activating hydroxyl groups of polymeric carriers using 4-fluorobenzenesulfonyl chloride.

4-Fluorobenzenesulfonyl chloride (fosyl chloride), due to the strong electron-withdrawing property of its fluoride atom, is found to be an excellent activating agent for the covalent attachment of biologicals to a variety of solid supports (e.g. functionalized polystyrene microspheres, Sepharose beads, or cellulose rods and hollow fibers). This reagent reacts rapidly with primary or secondary hydroxyl groups, at ambient temperature and pressure, to form 4-fluorobenzenesulfonate leaving groups. The activated solid support can be used immediately or preserved for several months without loss of activity by freeze-drying or by storage at 4 degrees C in aqueous solution at pH 5. Enzymes, antibodies, avidin, and other biologicals can be covalently attached to the activated solid phase with excellent retention of biological function. Potential therapeutic applications of the fosyl chloride chemistry for bioselective separation of human lymphocyte subsets from whole blood and tumor cells from bone marrow are presented.     

Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions

Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.     

Method for lipase-catalyzed carbonate synthesis via one- and two-step alkoxycarbonylation reactions

Lipase-catalyzed alkoxycarbonylation methods offer potential advantages over the currently practiced industrial scale chemical synthesis of carbonates. We report a method for synthesis of organic carbonates via lipase-catalyzed alkoxycarbonylation between diphenyl carbonate and various alcohols in hexane. This method utilizes precursors that are readily available and does not involve extensive purification of the intermediate. In a two-step process, the two phenyl groups of diphenyl carbonate were substituted by two alcohol nucleophiles. The approach was demonstrated for two-step synthesis of 14 different disubstituted carbonate products. The rates of reaction for the two steps were much slower if the order of nucleophile addition was reversed. Under optimal conditions, complete conversion of diphenyl carbonate occurred within 8-15 h at 50 degrees C, which is a significant improvement from 50-90 h at 24 degrees C. A kinetic model for the alkoxycarbonylation reaction was derived based on the Michaelis-Menten equation, which simplified to first-order kinetics at low and equimolar concentration of substrates.     

Immobilization of cells via activated cell walls

Summary Cell walls of Saccharomyces cerevisiae and S. uvarum were activated by periodate oxidation of vicinal diol groups in cell wall polysaccharides. The aldehyde groups thus generated allow the yeast cells to be covalently bound to modified bead cellulose or macroporous glycidyl methacrylate supports, or to enzymes such as glucose oxidase and catalase.     

Enzymatic synthesis of carbonate monomers and polycarbonates

Diphenyl carbonate is an attractive monomer for copolymerization with Bisphenol-A to produce the strong, high melting polycarbonate, Bisphenol-A Polycarbonate. Diphenyl carbonate is an ideal candidate for this polymerization as the phenols constitute good leaving groups during polymerization. Industrially, diphenyl carbonate is produced via the phosgenation of a phenolic sodium salt. Using phosgene creates additional safety hazards as well as concerns in treating or disposing of the reaction by-products. The enzymatic synthesis of diphenyl carbonate via alcoholysis of dimethyl carbonate by phenol is presented. While the process is environmentally benign and eliminates the considerable safety issues related to the use of phosgene, phenol is a poor nucleophile and conversion to diphenyl carbonate is limited. Enzyme catalyzed condensation polymerization of carbonate monomers and diols is a more feasible and direct enzymatic route to polycarbonate. We describe an AA-BB condensation polymerization to make polycarbonates using enzymes at ambient conditions. Molecular weights of up to 8, 500 MW are achieved. Unlike the industrial polymerization, this process is performed without the use of acid catalysts, significant energy input, or high temperature or pressure.     

A comparison of the kinetic properties of native bovine muscle carbonic anhydrase and an activated derivative with modified thiol groups

Steady-state and equilibrium kinetic properties of native bovine carbonic anhydrase III (carbonate hydrolyase, EC 4.2.1.1) and a derivative modified with methyl methanethiosulfonate were investigated. The modified enzyme has a markedly increased CO2 hydration activity compared to the native form with a 3-times higher value of kcat and a 6-10-times higher value of kcat/Km. Qualitatively, the activated enzyme shows the same kinetic behavior as native isoenzyme III. This is reflected in similar pH dependences of the kinetic parameters for CO2 hydration, similar solvent hydrogen isotope effects on these parameters, similar deviations from Michaelis-Menten kinetics for the HCO3- dehydration reaction, and similar behavior of the kinetics of CO2/HCO3- exchange at chemical equilibrium as measured by a 13C-NMR magnetization transfer technique. It is concluded that the conversion of -SH groups to -S-S-CH3 moieties does not change the catalytic mechanism, but leads to an increased rate of CO2/HCO3- interconversion as well as to an increased rate of proton transfer between the active site and the reaction medium.     

Protein immobilization to alumina supports: I. characterization of alumina-organophosphate ligand interactions and use in the attachment of papain

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. (c) 1992 John Wiley & Sons, Inc.     

The thio-Mitsunobu reaction: a useful tool for the preparation of 2,5-anhydro-2-thio- and 3,5-anhydro-3-thiopentofuranosides

The unprotected methyl L-arabinofuranosides, D-ribofuranosides and D-xylofuranosides are transformed into the corresponding S-acetyl-5-thio derivatives by the thio-Mitsunobu reaction. Mesylation and subsequent reaction with sodium hydrogen carbonate led, depending on the configuration of the intermediate, to 2,5-anhydro-2-thio- or 3,5-anhydro-3-thiopentofuranosides. Due to inversion at C-3 or C-2 during the intramolecular nucleophilic displacement the products exhibit L-lyxo-, D-arabino- or D-lyxo-configuration. Analogously, the methyl 2,3-anhydro-D-ribofuranosides yielded 5-thio-S-acetates with intact 2,3-oxirane groups, which were cyclised with sodium hydrogen carbonate by epoxide ring opening and concomitant ring closure to form exclusively 3,5-anhydro-3-thio-D-xylofuranosides. A related 3,5-anhydro-3-seleno-D-lyxofuranoside was obtained by reaction of a 3,5-di-O-mesyl-D-arabinofuranoside with sodium hydrogen selenide. Several X-ray diffraction analyses proved the structures of the products.     

Coupling of ligands to insoluble matrices via aldehyde groups

Summary The linkages formed between glutaraldehyde-activated solid supports and ligands containing amino groups are more stable than bonds formed after oxidation of glucose residues of the support to aldehyde groups. These results show that different types of chemical bonds are formed by the two kinds of activated support. If glutaral dehyde is used for the activation the reduction step is not necessary.Dedicated to Professor Dr. Georg Manecke on the occasion of his 70th birthday     

The phosphorothioite method. Synthesis of deoxyoligonucleotides on polymer supports by using nucleoside phosphorothioites

The development of a new method, which is called "the phosphorothioite method", for synthesizing deoxyoligonucleotides on polymer supports is described. Nucleoside phosphorothioites were easily prepared and readily activated by iodine or silver acetate. In the case of the use of iodine, the present method did not require the oxidation step, because it could be simultaneously performed during condensation and thereby the reaction time could be shorten. This method was applied successfully to the polymer support synthesis.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.