Immobilization of proteins on partially hydrolyzed agarose beads

Treatment of agarose beads with mild acid (0.2 M HCl, 55 degrees C, several hours) hydrolyzes some of the glycosidic bonds between D-galactosyl residues and 3,6-anhydro-L-galactosyl residues, and thus produces aldehydo-groups useful for immobilization of amino compounds by reductive amination with NaCNBH3. More than 20 mg (0.3 mumol) of bovine serum albumin could be coupled per gram of partially hydrolyzed agarose beads. Arthrobacter neuraminidase immobilized by this method was useful for desialylation of sialyl glycoconjugates, and was found not to leach from the gel and to be much more thermostable than the free enzyme.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Immobilization of lipase onto micron-size magnetic beads

A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 microm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40-0.55 mmolg(-1) supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization (sigma(s)) of 14.6 emicrog(-1). Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mgg(-1) support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20s.     

Immobilization of acetate kinase and phosphotransacetylase on derivatized glass beads

The enzymes acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) and phosphotransacetylase (acetyl coenzyme A: orthophosphate acetyltransferase, EC 2.3.1.8) were separately immobilized onto controlled pore glass CPG and silica beads (pore size 50 nm). Different coupling techniques were screened and immobilized enzymes were subjected to storage stability tests. The selected method, the CPG γ-aminopropyl glutaraldehyde succinate dihydrazide, was further optimized to improve the activity of the enzyme-loaded glass beads.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Immobilization of proteins on liposome surface

Different methods for the immobilization of proteins (enzymes and immunoglobulins) on liposomes are reviewed. These methods include adsorption, incorporation, covalent binding and binding of preliminarily modified proteins with the liposomal surface. Literature data are compared, optimal immobilization conditions are discussed and requirements for the immobilization processes are formulated. The possibility of using liposome-protein conjugates for drug targeting is especially discussed.     

Immobilization of proteins on magnetic nanoparticles

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation ofN-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticle. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the activation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.     

Immobilization of phospholipid vesicles on alkyl derivatives of agarose gel beads

We have immobilized phospholipid vesicles on hydrophobic derivatives of agarose gel beads. The vesicles were prepared from cholate-solubilized egg yolk phospholipids by gel filtration in 0.2 M NaCl at pH 7.1, which produced small vesicles, or in 0.5 M (NH₄)₂SO₄ at pH 8.0, which yielded large ones. The small vesicles eluted with K_d 0.4–0.6 and the large ones with K_d 0.05 on Sepharose 4B. Butyl, octyl and dodecyl sulfide derivatives of Sepharose 4B were synthesized using 1,4-butanediol diglycidyl ether and alkyl mercaptans (Maisano, F., Belew, M. and Porath, J. (1985) J. Chromatogr. 321, 305–317). The phospholipid vesicles were immobolized on 0.6–1-ml columns of these adsorbents in the salt solution that had been used for the preparation of the liposomes. A ligand concentration of 8 μmol per ml gel was sufficient for immobilization of small as well as large vesicles. The capacity of immobilization per ml gel was at least 20–100 and 1.5–3 μmol of phospholipids for small and large vesicles, respectively. The rate of adsorption of small vesicles was initially 0.3–0.5 μmol of phospholipids per min per ml gel, but decreased later to 0.2–0.3 μmol/min per ml as the gel bead surfaces approached saturation. These rates were determined at a vesicle concentration corresponding to 1.2 mM phospholipids and at room temperature. The butyl adsorbent gave a higher initial adsorption rate but a lower capacity than the dodecyl adsorbent, probably due to differences in the energy thresholds for ligand penetration through the hydrophilic surface layer of the vesicles, and to differences in the binding strength. The maximal concentration of adsorbed small vesicles that we achieved, 100 μ mol of phospholipids per milliliter octyl surfide-Sepharose 4B, would be equivalent to close-packing of the spherical phospholipid vesicles in 40% of the accessible volume of the gel beads.     

Immobilization of cell-associated enzymes by entrapment in polymethacrylamide beads

Immobilization of E. coil, Aspergillus niger and Aspergillus japonicus was accomplished by entrapment in polymethacrylamide beads via a two-phase polymerization using soybean oil as the suspension medium. The immobilized E. coli cells containing penicillin G acylase possessed a higher activity than that reported in the literature. The mmobilized mycelia of Aspergillus sp. containing β-fructofuranosidase were effective for catalyzing the formation of fructooligosaccharides.     

Immobilization of proteins on aldehyde-activated polyacrylamide supports

A method has been developed for the immobilization of proteins on derivatized polyacrylamide gels. Aminoethyl Bio-Gel P-150 was converted to its stable N-2,3-dihydroxypropyl derivative by borohydride reduction of the Schiff base formed with glyceraldehyde. Periodate oxidation of the modified gel provided a reactive aldehyde, which was subsequently coupled to protein by reductive amination with sodium cyanoborohydride. Coupling efficiencies were found to be >90% for concanavalin A and bovine serum albumin, and the gels contained as much as 5 and 20 mg of protein/ml of gel, respectively. Immobilized concanavalin A retained 89% of its binding capacity and was demonstrated to be chemically stable with variations in pH, and changes in concentrations of Triton X-100 and sodium dodecyl sulfate (at concentrations <0.1%). Bovine β-hexosaminidase and β-glucuronidase, higher molecular weight proteins, were also bound with retention of activity, but with less efficiency. This procedure provides an efficient method for the covalent immobilization of proteins.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Immobilization of human thrombomodulin on glass beads and its anticoagulant activity.

Human thrombomodulin (TM) was for the first time immobilized on glass beads by the reaction between the carboxyl group of TM and the amino group of glass beads using water-soluble carbodiimide. Immobilized human TM exhibited both anticoagulant activity and inhibition of platelet aggregation of human blood.     

Immobilization of beta-galactosidase, albumin, and gamma-globulin on epoxy-activated acrylic beads

A comparative study was conducted into the immobilization of beta-galactosidase, albumin, and gamma-globulin on an epoxy-activated polyacrylic matrix (oxirane C, R?hm-Pharma GmbH, Darmstadt). The kinetic parameters of the immobilized beta-galactosidase were investigated with three kinds of miniaturized analytical reactors; namely, stirred batch, continuous stirred-tank, and packed-bed reactors. The optimum binding conditions, saturation activity and Michaelis constant of immobilized beta-galactosidase are given, together with determinations of the binding capacity of the oxirane C matrix for the three proteins investigated. For beta-galactosidase a saturation activity of 1300 U/g oxirane C was reached. The maximum binding, achieved by experiment, was 140 mg/g with 0.69 yield for albumin, 120 mg/g with 0.61 yield for gamma-globulin, and 40 mg/g with 0.42 yield for beta-galactosidase. From these data the inner surface of the matrix as a function of the size of the bound proteins was estimated.     

Immobilization of lipase to chitosan beads using a natural cross-linker

Genipin, a reagent of plant origin was used for the immobilization of lipase by cross-linking to chitosan beads. The catalytic properties and operational and storage stabilities of the immobilized lipase were compared with the soluble lipase. Under optimum conditions, 198 microg protein was bound per g chitosan with a protein-coupling yield of 35%. The hydrolytic activity was 10.8 U/g chitosan and the relative specific activity was 108%. The immobilized lipase showed better thermal and pH stabilities compared to the soluble form. The immobilized enzyme exhibited mass transfer limitations as reflected by a higher apparent K(m) value and a lower energy of activation. The immobilized enzyme retained about 74% of its initial activity after five hydrolytic cycles.