Preparation of Zn2+‐chelated poly(HEMA‐MAH) cryogel for affinity purification of chicken egg lysozyme

Supermacroporous poly(2‐hydroxyethyl methacrylate) [poly(HEMA)]‐based monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with N‐methacryloyl‐L ‐histidine methyl ester (MAH) as functional comonomer and N,N′‐methylene‐bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for affinity purification of lysozyme from chicken egg white. The monolithic cryogel containing a continuous polymeric matrix having interconnected pores of 10–50 μm size was loaded with Zn²⁺ ions to form the metal chelate with poly(HEMA‐MAH) cryogel. Poly(HEMA‐MAH) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA‐MAH) monolithic cryogel was 5.62 g H₂O/g cryogel. Poly(HEMA‐MAH) cryogel containing 45.8 μmol MAH/g was used in the adsorption/desorption of lysozyme from aqueous solutions. The nonspecific adsorption of lysozyme was very low (7.5 mg/g). The maximum amount of lysozyme adsorption from aqueous solution in phosphate buffer was 209 mg/g at pH 7.0. It was observed that lysozyme could be repeatedly adsorbed and desorbed with the poly(HEMA‐MAH) cyogel without significant loss of adsorption capacity. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

利用聚丙烯酰胺基阳离子交换型晶胶介质从鸡蛋清中分离溶菌酶(英文)

An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer (pH 7.8) containing dif-ferent concentrations of NaCl is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purity lysozyme from chicken egg white.     

内嵌SiO_2纳米颗粒的超大孔复合晶胶的制备(英文)

Supermacroporous composite cryogels embedded with SiO2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide(AAm) and N,N-methylene-bis-acrylamide(MBAAm) containing SiO2 nanoparticles(mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08) under the freezing-temperature variation condition in glass columns.The properties of these composite cryogels were measured.The height equivalent to theoretical plate(HETP) of the cryogel beds at different liquid flow rates was determined by residence time distribution(RTD) using tracer pulse-response method.The composite cryogel matrix embedded with the mass fraction of SiO2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization.Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid(AMPSA) was carried out to evaluate the protein breakthrough and elution characteristics.The chromatography can be carried out at relatively high superficial velocity,i.e.,15 cm·min-1,indicating the satisfactory mechanical strength due to the embedded nanoparticles.     

Adsorption behaviours of lysozyme onto poly-hydroxyethyl methacrylate cryogels containing methacryloyl antipyrine-Ce(III)

In this study, Ce³⁺-based cryogel with methacryloyl antipyrine (MAAP) and 2-hydroxyethyl methacrylic acid (HEMA) [p(HEMA-MAAP-Ce³⁺] was prepared. MAAP-Ce³⁺ complex was characterized by UV–near infrared and energy-dispersive X-ray spectroscopy. Cryogel was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and swelling test. Pore size of the cryogel was found to be about 30–50?µm. The effects of flow rate, pH, temperature, and initial enzyme concentration have been investigated. Maximum adsorption capacity was found to be 57.84?mg?g^?1 cryogel at pH 6.0. After seven times of adsorption–desorption cycles of same cryogel, it was observed that there is negligible decrease in the adsorption capacity.     

A purification method for specific serine proteases using one-step affinity chromatography

A simple affinity chromatography procedure for specific isolation of serine proteases is described. The procedure was tested using enzymes from five microbial and one plant source. Feather keratin, covalently bound to controlled-pore glass, was the support and magnesium chloride was used in the elution buffers instead of zinc chloride. This enabled one-step isolation of serine proteases present in the biological materials used. The small (15 cm × 1 cm) controlled-pore keratin-glass column allowed high flow rates and protected the proteases from autodigestion during the chromatography process. The serine proteases were eluted from the column with good recovery (40–84·6%) and a purification efficiency between 5 and 7. The purified proteases were homogeneous by polyacrylamide gel electrophoresis.     

冰胶印迹聚合物固定化脂肪酶的制备及催化性能

利用冰冻凝胶(cryogel,简称冰胶)印迹聚合物实现了脂肪酶的固定化.在脂肪酶存在的条件下,以过硫酸铵/亚硫酸氢钠为引发剂,由丙烯酰胺、N,N-亚甲基双丙烯酰胺、丙烯酸、烯丙胺共聚而得到印迹聚合物固定化酶.通过催化三油酸甘油酯与甲醇的酯交换反应,发现冰胶固定化脂肪酶、常规凝胶固定化脂肪酶、游离脂肪酶具有相似的催化性能.冰胶固定化酶与相应的凝胶固定化酶显示出类似的稳定性,而传质方面则优于常规凝胶固定化酶,因此冰胶印迹聚合物固定化有望成为一种具有吸引力的酶固定化方法.     

Synthesis and characterization of an acrylamide-based water-soluble affinity polymer for trypsin purification

An acrylic polymer capable of specifically binding the enzyme trypsin has been synthesized by the copolymerization of acrylamide and N-acryloyl-m-aminobenzamidine. The object of the study was to discover suitable conditions for the synthesis of an affinity polymer which could be used in an ultrafiltration system for the biospecific affinity isolation of trypsin from mixtures containing other proteins. The polymer was purified by washing in an ultrafiltration system using a membrane with nominal molecular cutoff of 300 000. The molecular weight of the polymer product was varied by varying the concentration of initiator. The products were studied by nuclear magnetic resonance spectroscopy, by infrared spectroscopy, by viscometry and by their ability to inhibit trypsin in the absence and presence of ammonium sulphate at concentrations commonly found in crude protein solutions. No inhibition towards chymotrypsin activity was detected.     

Harnessing metal ion affinity for the purification of plasmid DNA

The recent surge in plasmid DNA (pDNA) vaccine research has generated demand for an efficient large-scale pDNA purification process. In this study, three process intensification strategies harnessing the interactions of pDNA, RNA and endotoxin with immobilised and/or free transition metal ions were investigated for the purification of plasmid pcDNA3.1D, containing dengue fever antigenic gene (NS3), from the alkaline cell lysate. In the first process scheme, alkaline cell lysate was applied to Cu²⁺-iminodiacetic acid (IDA) for simultaneous removal of RNA and endotoxin. Subsequent addition of CuCl₂ to the supernatant afforded selective precipitation of pDNA, resulting in substantially purified pDNA at an overall recovery yield of 92% with significant reduction in RNA (96%) and endotoxin (>99%). In the second process scheme, pDNA, RNA and endotoxin were first isolated from other impurities in alkaline cell lysate by CuCl₂ precipitation. Addition of EDTA to the precipitated pellets selectively solubilised pDNA and RNA while endotoxin remained insoluble. Subsequent application of solubilised pDNA and RNA to Cu²⁺-IDA resulted in highly purified pDNA with almost complete removal of RNA and any residual endotoxin (∼100% pDNA recovery, ∼100% removal of RNA and endotoxin). In the third process scheme, RNA and endotoxin were first removed from alkaline cell lysate by ZnCl₂ precipitation. pDNA in the supernatant was then recovered by CuCl₂ precipitation (64% pDNA recovery, 86% RNA and >99% endotoxin removal). In essence, the combination of metal ion (CuCl₂) precipitation, followed by immobilised metal affinity chromatography (IMAC) employing Cu²⁺-IDA, provides a great potential to achieve significant intensification of pDNA purification process with improved efficiency and throughput.     

Preparation and characterization of carbon cryogel microspheres

Carbon cryogel microspheres (CC microspheres) were successfully synthesized by an inverse emulsion polymerization of resorcinol with formaldehyde, followed by freeze drying and pyrolysis in an inert atmosphere. CC microspheres were characterized by scanning electron microscopy, elemental analysis and various gas adsorption measurements. By changing both the temperature for preparing the emulsion and pyrolysis temperature, it was possible to prepare both mesoporous microspheres and microspheres covered with ultramicroporous surfaces which pore sizes were smaller than the minimum molecular dimensions of ethane or carbon dioxide. Hydrophobicity of the obtained CC microspheres increased with the increase in pyrolysis temperature. The possibility of using the obtained mesoporous CC microspheres as column packing materials for high-performance liquid chromatography was also shown.     

Performance of pseudo-specific cryogel in lysozyme purification from chicken egg white

The application of cryogels for biomolecule purification has expanded due to their adsorption efficiency and operational advantages. In this study, polyacrylamide cryogels functionalized with l -phenylalanine (cryogel-Phe) via the glutaraldehyde method were designed for lysozyme adsorption. Cryogel functionalization was confirmed by Fourier-transform infrared spectroscopy and Kjeldahl analysis, indicating the immobilization of 458.65 mg_{phenylalanine} g_cryogel⁻¹. Cryogel-Phe showed high porosity (0.95) and a Young's modulus of 526.71 kPa. Thermogravimetric analysis indicated that thermal degradation occurred above 200°C. Differential scanning calorimetry and X-ray diffraction confirmed that the cryogel material was amorphous. In addition, the column presented a hydraulic permeability of 4.15 × 10⁻¹³ m², axial dispersion ranging from 10⁻⁷ to 10⁻⁶ m² s⁻¹, and a height equivalent to a theoretical plate ranging from 0.10 to 0.21 cm. The highest adsorption of lysozyme (67.65 mg g⁻¹) was obtained using sodium thiocyanate saline solution (0.025 mol L⁻¹, pH 5.0). The ability of the cryogel-Phe column to capture and purify lysozyme was confirmed by high enzymatic activity (1294.17 U ml⁻¹), purity (87.92%), purification factor (11.49), and sulphate-polyacrylamide electrophoresis gel (SDS-PAGE) electrophoresis gel.     

Poly(acrylamide‐allyl glycidyl ether) cryogel as a novel stationary phase in dye‐affinity chromatography

Poly(acrylamide‐allyl glycidyl ether) [poly(AAm‐AGE)] cryogel was prepared by bulk polymerization which proceeds in an aqueous solution of monomers frozen inside a glass column (cryo‐polymerization). After thawing, the monolithic cryogel contains a continuous polymeric matrix having interconnected pores of 10–100 μm size. Cibacron Blue F3GA was immobilized by covalent binding onto poly(AAm‐AGE) cryogel via epoxy groups. Poly(AAm‐AGE) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(AAm‐AGE) monolithic cryogel was 6.84 g H₂O/g cryogel. Poly(AAm‐AGE) cryogel containing 68.9 μmol Cibacron Blue F3GA/g was used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.2 mg/g). The maximum amount of HSA adsorption from aqueous solution in acetate buffer was 27 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 74.2 mg/g). Desorption of HSA with a purity of 92% from Cibacron Blue F3GA attached poly(AAm‐AGE) cryogel was achieved using 0.1M Tris/HCl buffer containing 0.5M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with poly(AAm‐AGE) cryogel without significant loss in the adsorption capacity. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Nonporous monosize polymeric sorbents: Dye and metal chelate affinity separation of lysozyme

Lysozyme adsorption onto dye‐attached nonporous monosize poly(2‐hydroxyethyl‐methacrylate‐methylmethacrylate) [poly(HEMA‐MMA)] microspheres was investigated. Poly(HEMA‐MMA) microspheres were prepared by dispersion polymerization. The monochloro‐triazine dye, Cibacron Blue F3GA, was immobilized covalently as dye–ligand. These dye‐affinity microspheres were used in the lysozyme adsorption–desorption studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye‐attached and metal‐chelated microspheres were studied in a batch reactor. Effect of Cu(II) chelation on lysozyme adsorption was also studied. The nonspecific adsorption of lysozyme on the poly(HEMA‐MMA) microspheres was 3.6 mg/g. Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 247.8 mg/g. Lysozyme adsorption capacity of the Cu(II) incorporated microspheres (318.9 mg/g) was greater than that of the Cibacron Blue F3GA‐attached microspheres. Significant amount of the adsorbed lysozyme (up to 97%) was desorbed in 1 h in the desorption medium containing 1.0M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. In order to examine the effects of separation conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We conclude that dye‐ and metal‐chelate affinity chromatography with poly(HEMA‐MMA) microspheres can be applied for lysozyme separation without causing any significant changes and denaturation. Repeated adsorption/desorption processes showed that these novel dye‐attached monosize microspheres are suitable for lysozyme adsorption. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 115–124, 2000     

Comparison of affinity membrane adsorbers for the purification of recombinant human erythropoietin (rhEPO)

In this work affinity membrane adsorbers were investigated for the chromatographic purification of recombinant human erythropoietin (rhEPO) produced in mammalian cells. Cibacron Blue (CB), IDA‐Cu⁺², wheat germ agglutinin (WGA), concanavalin A (ConA) and an anti‐EPO monoclonal antibody (MAb) were tested as affinity ligands, attached to microporous Sartobind^® membranes. In experiments carried out with cell culture supernatant, the best results were obtained with Sartobind–CB, Sartobind–WGA and Sartobind–MAb membranes. The thermodynamic parameters were determined by adsorption isotherms of rhEPO onto the membranes. Sartobind–ConA presented the lowest affinity for rhEPO, as evidenced by a lower association constant. For Sartobind–CB, Sartobind–IDA‐Cu⁺² and Sartobind–MAb K_A was in the order of 10⁵ L mol^?1, whereas for Sartobind–WGA it was 10⁶ L mol^?1. Sartobind–CB eluates were also investigated by RP‐HPLC. The purity level achieved in this one‐step purification strategy was 55%, indicating that the Sartobind–CB membrane is a promising affinity membrane for rhEPO purification. Copyright © 2007 Society of Chemical Industry     

Preparation of polyacrylamide-based supermacroporous monolithic cryogel beds under freezing-temperature variation conditions

Continuous supermacroporous monolithic cryogel is a novel sponge-like chromatographic adsorbent for bioseparation in downstream process and it is always prepared under a constant freezing-temperature condition. In this work, polyacrylamide-based supermacroporous monolithic cryogel beds in different inner diameter glass columns (I.D. 10, 16 and 26 mm) have been prepared by radical cryogenic copolymerization process under the condition of freezing-temperature variation. The matrix microstructure morphologies of these cryogels were analyzed by scanning electron microscopy (SEM) and the axial liquid dispersion characteristics in these cryogel beds were also analyzed by measuring the residence time distributions (RTDs). The formation of cryogel bed under the present situation has been considered as a coinstantaneous process of solvent crystal growth and polymeric monomer copolymerization. Effects of freezing-temperature variation route, cooling rate and redox initiator concentration on the cryogel microstructure and the liquid dispersion characteristics were investigated experimentally to reveal the cooperative mechanisms of these two processes on the formation of cryogel bed. The results showed that the cryogel pore-structures and the axial liquid dispersion coefficients in the cryogel beds depended strongly on the cooling condition, which controls the solvent crystallization process, and the redox initiator concentration, which influences the monomer polymerization.     

功能性载溶菌酶微球的制备与性能

为了制备理化性能优异的溶菌酶微球用于壳聚糖材料的体内降解调控,实验从多参数多指标出发,较为系统全面地考察了载溶菌酶微球的制备与性能。微球产率为5.7%～97.4%,粒径可控制在30～100μm,载药量和包封率理想。微球缓释效果明显,16 d的释放可控制在20%～70%的较大区间内,释放规律符合一级动力学模型。酶活基本保持完好,大部分在90%以上。     

Preparation of highly magnetic chitosan particles and their use for affinity purification of enzymes

A novel protocol for preparation of highly magnetic chitosan particles, with a coercive force as high as 3500 Oe, has been developed. The surface of the particles was functionalized with aldehyde groups to facilitate the attachment of affinity ligands. The optimum conditions for the preparation of highly magnetic chitosan particles and immobilization of trypsin on magnetic particles were obtained; these particles were then used for affinity purification of aprotinin, and the conditions of affinity purification are discussed in detail. The proposed method was successfully applied to the affinity purification of aprotinin. Copyright © 2003 Society of Chemical Industry     

Dye‐affinity hollow‐fibres and their lysozyme adsorption–desorption characteristics

Dye‐affinity adsorption is increasingly used for protein separation. Hollow‐fibres have advantages as adsorbents in comparison to conventional bead supports because they are not compressible and can eliminate internal diffusion limitations. The aim of this study was to explore in detail the performance of polyamide hollow‐fibres to which Reactive Green HE‐4BD was attached for adsorption of lysozyme. The hollow‐fibre was characterized by scanning electron microscopy. These dye‐carrying hollow‐fibres (26.3 µmol g^?1) were used in the lysozyme adsorption–elution studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye‐attached hollow‐fibres was studied in a batch system. The non‐specific adsorption of lysozyme on the polyamide hollow‐fibres was 1.8 mg g^?1. Reactive Green HE‐4BD attachment significantly increased the lysozyme adsorption up to 41.1 mg g^?1. Langmuir adsorption model was found to be applicable in interpreting lead adsorption by Reactive Green HE‐4BD attached hollow fibres. Significant amount of the adsorbed lysozyme (up to 95%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0. In order to determine the effects of adsorption conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We concluded that polyamide dye‐affinity hollow‐fibres can be applied for lysozyme adsorption without causing any significant conformational changes. Repeated adsorption–elution processes showed that these dye‐attached hollow‐fibres are suitable for lysozyme adsorption. © 2001 Society of Chemical Industry     

Biopolymer based ion imprinting cryogel traps for the removal of Tl(I)

The aim of this study is to prepare supermacroporous cryogel with embedded ion imprinted polymer (IIP) particles which can be used for the selective removal of Tl(I) from aqueous solution. In the first step, Tl(I) has complexed with chitosan and crosslinked with ephychlorohydrin. In the second step, the supermacroporous poly(hydroxyethyl methacrylate) (PHEMA) cryogel with embedded IIP particles [PHEMA/MIP composite cryogel] which have been produced by free radical polymerization initiated by N,N,N’,N’-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in ice bath. The template, Tl(I), has been removed from the polymer using 0.1 M EDTA solution. The cryogel is characterized by swelling test, scanning electron microscopy, and Fourier transform infrared spectroscopy. The effective parameters on Tl(I) adsorption process have evaluated by investigating the dependency of the adsorption capacity on flow-rate, Tl(I) concentration and contact time. The maximum adsorption capacity of Tl(I) ions has been found to be 1.55 µgg^?1. The selectivity of the IIP biopolymer cryogel has investigated by competitive adsorption of Cd(II), Cu(II), K(I), and Fe(II). The relative selectivity coefficients of IIP biopolymer cryogel are 3.98 and 62.5 for Cd(II) and Cu(II), respectively. The IIP biopolymer cryogel could be used many times without any significant decrease in the adsorption capacity. As a conclusion it can be said that the IIP biopolymer cryogel could be used for specific separation of Tl(I) ions from nuclear, industrial, and environmental waste.