Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands

In reaction to the comment by Dr. Y.-S. Ho on our publication "Affinity dye-ligand poly(hydroxyethylmethacrylate/chitosan composite membrane for adsorption lysozyme and kinetic properties, Biochemical Engineering Journal 13 (2003) 35-45"

WOS: 000183023700010

Epoxy-derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system

WOS: 000087900100016Poly(2-hydroxyethylmethacrylate) (pHEMA) membranes were prepared by W-initiated photopolymerization of HEMA in the presence of an initiator (alpha-alpha '-azobisisobutyronitrile, AIBN). The epoxy group, i.e., epichlorohydrin, was incorporated covalently, and the urease was immobilized onto pHEMA membranes by covalent bonding through the epoxy group. The retained activity of the immobilized enzyme was found to be 27%. The K-m values were 18 and 34 mM for the free and the immobilized enzymes, respectively, and the V-max values were found to be 59.7 and 16.2 U mg(-1) for the free and the immobilized enzyme. The optimum pHs was 7.2 for both forms, and the optimum temperature for the free and the immobilized enzymes were determined to be 45 and 50 degrees C, respectively. The immobilized urease was characterized in a continuous system and during urea degradation the operational stability rate constant for the immobilized enzyme was found to be 5.83 x 10(-5) min(-1). (C) 2000 John Wiley & Sons, Inc

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

WOS: 000088071900021Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 mu g g(-1) bead and the retained activity of the immobilized enzyme was found to be 44%. The K-m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V-max values were found to be 1890 and 760 U mg(-1) for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 degrees C and for the immobilized enzyme was 45 degrees C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. (C) 2000 Society of Chemical Industry

Performance of different metal-dye chelated affinity adsorbents of poly(2-hydroxyethyl methacrylate) in lysozyme separation

WOS: 000168274700005The triazine dye Cibacron Blue F3GA was covalently immobilized as an affinity ligand onto microporous poly(2-hydroxyethyl methacrylate) (pHEMA) membranes. Three different metal ions [i.e.. Fe(III), Zn(II), or Cu(II)] were then chelated with the immobilized Cibacron Blue F3GA molecules. Lysozyme adsorption onto these affinity adsorbents from aqueous solutions containing different amounts of lysozyme at different pH was investigated in a batch system. Lysozyme adsorption capacity of all of the metal-dye-immobilized membranes was greater than that of the dye-immobilized membranes. The nonspecific adsorption of the protein on the pHEMA membranes was negligible. The adsorption phenomena appeared to follow a typical Lang muir isotherm. The maximum capacity (q(m)) of the Fe(III)-Zn(II),-or Cu(II)-dye chelated membranes for lysozyme adsorption (384, 326, and 306 mug/cm(2)) was greater than that of the dye-immobilized membrane (224 mug lysozyme/cm(2)), respectively. The dissociation constant (k(d)) values were found to be 2.51 x 10(-7) M with dye-immobilized membrane, and 2.32 X 10(-7), 2.38 X 10(-7), and 2.40 x 10(-7) M with the Fe(III)-Zn(II),-and Cu(II)-dye-chelated membranes, respectively. More than 95% of the adsorbed lysozyme was desorbed in 60 min in the desorption medium containing 0.5 M KSCN at pH 8.0

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA-MMA) beads for sucrose hydrolysis

WOS: 000236238800003The epoxy group containing poly(glycidyl methacrylate-co-methyl methacrylate) poly(GMA-MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA-MMA)-PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA-MMA)-PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K-m) and the maximum rate of reaction (V-max) were calculated from the Lineweaver-Burk plot. The K-m and V-max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 degrees C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA-MMA)-PEI beads can be easily regenerated and reloaded with the enzyme for repeated use. (c) 2005 Elsevier B.V. All rights reserved

Purification of lysozyme from egg white by Reactive Blue 4 and Reactive Red 120 dye-ligands immobilised composite membranes

WOS: 000225723800057A composite membrane was synthesized from 2-hydroxyethylmethacryl ate (HEMA) and chitosan (pHEMA/chitosan) via UV initiated photo-polymerisation. Reactive Blue 4 (Blue-4) and Reactive Red 120 (Red-120) were immobilised onto pHEMA/chitosan membranes. In the first part of this study, the binding characteristics of lysozyme to different dye-ligand immobilized membranes have been studied from aqueous solution using the plain membrane as a control system. The polarity of the investigated membranes was determined by contact angle measurement. The adsorption capacities of both dye-ligand immobilised membranes increased with increasing temperature but decreased with increasing NaCl concentration. The adsorption isotherm fitted both the Langmuir and the Freundlich models. A theoretical analysis has been conducted to estimate the thermodynamic contributions (changes in enthalpy, entropy and Gibbs free energy) for the adsorption of lysozyme to different dye-ligand immobilised composite membranes. In the second part, their purification efficacy of lysozyme from egg white was investigated. The purity of the eluted lysozyme was analysed by HPLC. The purity of lysozyme extracted from egg white was 84% with Red-120 immobilised membrane. This was 21% for Blue-4 immobilised membrane. The recovery yields were 72% and 16% for Red-120 and Blue-4 immobilised membranes, respectively. The Red 120 immobilised membrane provided an efficient method to purify lysozyme from egg white. showing high adsorption capacity and high selectivity for the lysozyme. On the other hand, the Blue-4 immobilised composite membrane had a lower adsorption capacity and selectivity than that of the Red-120 immobilised one. Purification was monitored by determination of lysozyme activity using Micrococcus lysodeiktictis as substrate. The dye-ligand immobilised composite membranes are stable when subjected to sanitization with sodium hydroxide after repeated separation-elution cycles. (C) 2004 Elsevier Ltd. All rights reserved

Surface energy components of a dye-ligand immobilized pHEMA membranes: Effects of their molecular attracting forces for non-covalent interactions with IgG and HSA in aqueous media

WOS: 000235269500005PubMed: 16405992In the present paper, we report the study of the adsorption behaviour of human immunoglobulin G (IgG), human serum albumin (HSA) and polyethylenimine (PEI) onto surfaces of Procion Green HE-4BD (PG) immobilized poly(hydroxyethylmethacrylate) (pHEMA) membranes. The adsorption behaviour of the IgG and HSA onto surfaces of the PG-PEI complexed membrane was also studied. Surface wettability and hydrophilicity of all the membranes were investigated by static contact angle measurements. The measurements of the contact angle to various test liquids, i.e., water, glycerol, formamide, diiodomethane (DIM) and ethylene glycol on the investigated membranes were made by sessile drop method. In accordance to the Young equation, the smaller the surface tension of the test liquid, the smaller becomes the contact angles measured on all the investigated membranes surfaces. The highest contact angles were obtained with water, whereas ethylene glycol gave the lowest contact angles for all the tested membranes. Component and parameters of the surface free energy of all the investigated membranes were calculated from measured contact angle values using two methods (the geometric mean by Fowkes and acid-base by van Oss). HSA adsorption was enhanced after complexation of PEI with the immobilized dye-ligand. The adsorption of proteins and PEI significantly changed both the contact angles and component of surface free energies of the investigated membranes. (c) 2005 Elsevier B.V. All rights reserved

Procion Green H-E4BD-immobilized porous poly(hydroxyethylmethacrylate) ion-exchange membrane: preparation and application to lysozyme adsorption

WOS: 000171103500006Lysozyme adsorption onto Procion Green HE-4BD-immobilized poly(2-hydroxyethylmethacrylate) (pHEMA) membrane were investigated. The membrane were prepared by ultraviolet-initiated photopolymerization of HEMA in the presence of an initiator (alpha-alpha'-azoisobutyronitrile; AIBN). The water content of the dye-immobilized membrane was 69%, the amount of immobilized dye on the membrane was 0.544 mu mol ml(-1) and it used in the lysozyme adsorption studies. Lysozyme adsorption on these membranes from aqueous solutions containing different amounts of lysozyme at different pH was investigated in batch reactors. Lysozyme adsorption capacity of the dye-immobilized membrane was 13.33 mg ml(-1). The maximum lysozyme adsorption capacity (q(m)) of the dye-immobilized wet membrane was 14.14 mg ml(-1) and the dissociation constant (kd) value was found to be 0.707 mg ml(-1) lysozyme. More than 95% of the adsorbed lysozyme were desorbed in 120 min in the desorption medium containing 0.5 M KCl at pH 6.0. (C) 2001 Elsevier Science B.V. All rights reserved