Agar-based magnetic affinity support for protein adsorption

Magnetic colloidal particles were prepared by a coprecipitation method. The particles were composed of nanometer-sized superparamagnetic Fe(3)O(4) particles stabilized by lauric acid. Then, magnetic agar gel beads were produced by a water-in-oil emulsification method using a mixture of agar solution and the magnetic colloidal particles as the aqueous phase. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare an agar-based magnetic affinity support (MAS) for protein adsorption. The support showed good magnetic responsiveness in a magnetic field. Bovine serum albumin (BSA) was used as a model protein to test adsorption equilibrium and kinetic behavior of the MAS. The adsorption equilibrium of BSA to the MAS was described by the Langmuir-type isotherm. Adsorption capacity of the MAS for BSA was up to 25 mg/mL at a CB coupling density of 1.6 micromol/mL. The effect of ionic strength on BSA adsorption was complex, exhibiting a maximum capacity at an ionic strength of 0.06 mol/L. The adsorption of BSA to the MAS was also influenced by pH. Uptake rate of BSA to the MAS was analyzed using a pore diffusion model. The pore diffusion coefficient was estimated to be 1.75 x 10(-11) m(2)/s. Finally, recycled use of the MAS demonstrated the stability of the MAS in protein adsorption and magnetic responsiveness.     

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.     

Analysis of lysozyme in cheese by immunocapture mass spectrometry

The enzyme lysozyme is used as a preservative to prevent late blowing of ripened cheese, caused by Clostridium tyrobutyricum. Since the enzyme is extracted from hen egg white, lysozyme has to be declared on food product labels as a potential allergen. Here, a method is reported that combines immunocapture purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis for the detection of lysozyme in cheese samples. Cheese extracts were treated with magnetic particles coated with a monoclonal antibody directed against lysozyme. After immunocapture purification, lysozyme was detected by MALDI-TOF-MS. The limit of detection of the assay was about 5 mg/kg lysozyme in cheese. The method reliably distinguished between cheese samples which had been produced with and without lysozyme. Thus, the novel assay allows the reliable, sensitive, and specific detection of lysozyme in a food matrix. The assay could be easily adapted to other target peptides and proteins in complex food matrices and, therefore, has a broad application potential, e.g. for the analysis of allergens.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.     

Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).     

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Characterization of protein capacity of nanocation exchanger particles as filling material for functional magnetic beads for bioseparation purposes

In this study we describe the synthesis and characterization of nanocation exchanger particles (NCEX) as the functional filling material for magnetic beads. Polystyrene NCEX particles were synthesized from styrene via a mini-emulsion polymerization. The coupling of cation exchanger groups was done with chlorosulfuric acid after the polymerization reaction. The NCEX particles have an average diameter of 160-260 nm. Their ion exchange capacity amounts up to 4.58 mval/g. In an adsorption experiment it was possible to adsorb 192 mg lysozyme/g NCEX. Depending on the equilibrium concentration of lysozyme in the bulk solution 70-85% of the attached protein was desorbed. NCEX particles were used to produce magnetic beads with cation exchanger properties. Therefore an innovative production process for the synthesis of magnetic beads from different single components was used. The produced magnetic beads contained 40 wt % NCEX material and showed an ion exchanger capacity of 2 mequiv/g. It was possible to adsorb 75 mg lysozyme/g magnetic beads with a maximum recovery rate of 95%.     

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.     

Yeast cell debris and protein partitioning in the poly(ethylene glycol)-poly(vinyl alcohol) biphasic system

Yeast cells, cell debris and protein partitioning have been investigated in the poly(ethylene glycol) (PEG) 8000/poly(vinyl alcohol) (PVA) 10,000 system. Cells and cell debris partition into the lower (PVA) phase over the pH range 4.8-7.5, and with up to 0.37 M KCl at pH 5.9. Protein partitioning is more pH-dependent in the PEG/PVA system than in the PEG/dextran system, and a significant fraction of the total protein is found at the interface at lower pH values. Significant, rapid purification of overproduced pyruvate kinase in a PEG/PVA system containing Blue Sepharose CL-6B particles is demonstrated.     

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.     

Multi‐cycle recovery of lactoferrin and lactoperoxidase from crude whey using fimbriated high‐capacity magnetic cation exchangers and a novel “rotor–stator” high‐gradient magnetic separator

Cerium (IV) initiated “graft‐from” polymerization reactions were employed to convert M‐PVA magnetic particles into polyacrylic acid‐fimbriated magnetic cation exchange supports displaying ultra‐high binding capacity for basic target proteins. The modifications, which were performed at 25 mg and 2.5 g scales, delivered maximum binding capacities (Q_max) for hen egg white lysozyme in excess of 320 mg g^?1, combined with sub‐micromolar dissociation constants (0.45–0.69 µm) and “tightness of binding” values greater than 49 L g^?1. Two batches of polyacrylic acid‐fimbriated magnetic cation exchangers were combined to form a 5 g pooled batch exhibiting Q_max values for lysozyme, lactoferrin, and lactoperoxidase of 404, 585, and 685 mg g^?1, respectively. These magnetic cation exchangers were subsequently employed together with a newly designed “rotor–stator” type HGMF rig, in five sequential cycles of recovery of lactoferrin and lactoperoxidase from 2 L batches of a crude sweet bovine whey feedstock. Lactoferrin purification performance was observed to remain relatively constant from one HGMF cycle to the next over the five operating cycles, with yields between 40% and 49% combined with purification and concentration factors of 37‐ to 46‐fold and 1.3‐ to 1.6‐fold, respectively. The far superior multi‐cycle HGMF performance seen here compared to that observed in our earlier studies can be directly attributed to the combined use of improved high capacity adsorbents and superior particle resuspension afforded by the new “rotor–stator” HGMS design. Biotechnol. Bioeng. 2013; 110: 1714–1725. © 2013 Wiley Periodicals, Inc.     

Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing

In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving (i) chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K(d) approximately 0.08 microM) was exhibited by Cu(2+)-charged type II support materials. Equilibrium adsorption of L1 to these nonporous supports was achieved very rapidly (<300 s), and approximately 90% of the tightly bound L1 could be desorbed in just one elution step by including >100 mM imidazole in the equilibration buffer. The influence of feedstock complexity on L1 adsorption to the Cu(2+)-charged type II magnetic chelators was studied using various dilutions of four crude chemical E. coli cell extracts containing denatured L1 protein. Undiminished L1 adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L(-1)) and spermine-treated chemical cell extract (feedstock B). Efficient recovery of L1 from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu(2+)-chelator particles following batch adsorption of L1. Over 70% of the initial L1 present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of approximately 10.     

Effect of alcohols on aqueous lysozyme-lysozyme interactions from static light-scattering measurements

Alcohols have been widely used as protein denaturants, precipitants and crystallization reagents. We have studied the effect of alcohols on aqueous hen-egg lysozyme self-interactions by measuring the osmotic second virial coefficient (B22) using static light scattering. Addition of alcohols increases B22, indicating stronger protein-protein repulsion or weaker attraction. For the monohydric alcohols used in this study (methanol, ethanol, 1-propanol, n-butanol, iso-butanol and trifluoroethanol), B22 for lysozyme reaches a common plateau at approximately 5% (v/v) alcohol, while glycerol increases B22 more than monohydric alcohols. For a 0.05 M NaCl hen-egg lysozyme solution at pH 7, B22 increases from 2.4 x 10(-4) to 4.7 x 10(-4) ml mol/g2 upon addition of monohydric alcohols and to 5.8 x 10(-4) ml mol/g2 upon addition of glycerol. We describe the alcohol effect using a simple model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions. In this model, the increased lysozyme repulsive forces in the presence of monohydric alcohols are interpreted in terms of adsorption of alcohol molecules on hydrophobic sites on the protein surface. This adsorption reduces attractive hydrophobic protein-protein interactions. A thicker lysozyme hydration layer in aqueous glycerol solution can explain the glycerol-increased lysozyme-lysozyme repulsion.     

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well-known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5-80 m(2)/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.     

Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification

Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.     

Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.