Transfer rate measurement of lysozyme by liquid-liquid extraction using reverse micelles with dense CO2 (original) (raw)
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Biotechnology Progress, 2011
Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100-300 lm, mean pore size: 1-3 lm, pore volume: 57-59%, and bulk density: $1,438 kg/m 3) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for highthroughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads-CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high-throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m 3 h) and specific activity (2.2 Â 10 5 U/ mg) were higher than that obtained with traditional particulate resins. V
Excess adsorption of lysozyme and water at solid-liquid interfacesα
Colloids and Surfaces B: Biointerfaces, 1994
isotherms of lysozyme at solid-water interfaces have been studied as a function of protem con~nt~atlon~ iomc strength of the medium, pH and tem~~atur~ using silica, a~urn~na, carbon, chromium and Sephadex as solid surfaces. Ad~o~~tjo~ of lysozyme is affected strongly by change of pH, tem~rat~re and ionic strength. In mast cases
Journal of Molecular Liquids, 2018
Silica based SBA-15 nanoparticles functionalized with aminopropyl-trimethoxysilane (AP) groups were prepared, and then Cu 2+ ions were decorated onto them via AP (Cu +2-SBA-15), and calculated by ICP-OES as 137.9 mg/g nanoparticles. The characterizations of SBA-15 and SBA-15-AP were examined by XRD, FTIR and BET analysis. Prepared Cu +2-SBA-15 nanoparticles were embedded into cryogel column (Cu +2-SBA-15 NEC) photographed by SEM, and tried to investigate some adsorption parameters against to lysozyme selected as model protein. Lysozyme adsorption studies were conducted in a continuous system. The maximum adsorption capacity (1275.2 mg/g particles) of Cu +2-SBA-15 nanoparticles was obtained at phosphate buffer of pH 7.0 with initial lysozyme concentration of 1 mg/mL. According to examined Langmuir and Freundlich adsorption isotherms, Langmuir isotherm was fitted the best to this study. Composite cryogels were also used for 30 times in adsorption-desorption experiments. Purification capability of matrix was expressed as 88.5% with 91.3 purification yield from hen egg white (HEW) by means of SDS-PAGE.
Study of the interaction between lysozyme and sodium octanoate in aqueous solutions
Colloids and Surfaces A-physicochemical and Engineering Aspects, 2004
The interactions of sodium octanoate with lysozyme as a function of temperature have been investigated by a combination of UV absorbance and electrical conductivity to detect and to characterize the conformational transitions of lysozyme. By using difference spectroscopy, the transition was followed as a function of surfactant concentration and the data were analysed to obtain the Gibbs energy of the
Influence of some formulation parameters on lysozyme adsorption and on its stability in solution
International journal of pharmaceutics, 2002
According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.
Factors Influencing Recombinant Human Lysozyme Extraction and Cation Exchange Adsorption
Biotechnology Progress, 2006
Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milksources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5.
Lysozyme adsorption at the air/water interface
Journal of Colloid and Interface Science, 1990
The adsorption of reductively methylated chicken egg white lysozyme (EC 3.2.1.17) to the air/water interface has been measured by the radiotracer technique. This method enables direct determination of surface excess concentration as well as relative rates of adsorption and desorption. Improvements in calibration and radiolabeling techniques led to differences between the present results and previous lysozyme isotherms reported in the literature. The isotherm indicates monolayer saturation at low concentrations of bulk protein (below 2 × 10-5 wt%) and multilayer adsorption at high concentrations (above 10-3 wt%). At intermediate concentrations, an abrupt increase in surface concentration with increasing bulk concentration indicates a change in orientation of the adsorbed protein molecules. Adsorption experiments performed by sequential addition of protein to the bulk solution provide evidence that lysozyme molecules adsorbed at concentrations below the multilayer region do not exchange significantly with lysozyme molecules in the bulk solution. A kinetic model which incorporates the monolayer plateau, the multilayer adsorption, and the change of orientation of adsorbed lysozyme is presented.
Reverse Micelle Liquid-Liquid Extraction of Bovine Serum Albumin and Lysozyme
Jurnal Teknologi, 2012
Reverse micelle extraction by using Sodium bis (2-ethylhexyl) Suffoccinate (AOT) of protein bovine serum albumin (BSA) and lysozyme was investigated in this research. Study of factors affecting the surfactant concentration and pH of aqueous for both forward and backward extraction process was performed in the research. The BSA concentrations were characterized by using the UV- spectrophotometer at wavelength, λ = 280 nm. The result indicated that the extraction percentage of lysozyme was higher than BSA in forward transfer for both parameters; however BSA demonstrated a better extraction performance in backward extraction process. The maximum lysozyme extracted in the forward extraction process was at 60 mM of surfactant concentration while for BSA was 100 mM since BSA is a bulky molecule and the size is larger than of lysozyme. Pengekstrakan misel terbalik dengan menggunakan Sodium bis (2-ethylhexyl) Suffoccinate (AOT) untuk protein bovine serum albumin (BSA) dan lysozyme disiasat ...
A Surfactant Copolymer Facilitates Functional Recovery of Heat-Denatured Lysozyme
Annals of the New York Academy of Sciences, 2005
The triblock copolymer poloxamer 188 is a non-cytotoxic, nonionic surfactant with both hydrophobic and hydrophilic domains. We show that P188 is able to facilitate the recovery of catalytic activity of heat-denatured lysozyme in dilute solution at low molar ratios of P188:enzyme. Heat-denatured enzyme retained 55% of native activity. After treatment with P188, the enzyme's activity was 85% of native. Because of the low molar ratios used and the non-cytotoxic nature of the compound, P188 may be of potential use in burn therapy.
Diffusivity of lysozyme in undersaturated, saturated and supersaturated solutions
Journal of Crystal Growth, 1994
The diffusion coefficient of lysozyme, a globular protein, was measured at various conditions as functions of lysozyme concentration, salt concentration, and solution "age" in concentrated, saturated, and supersaturated solutions, employing Gouy interferometry. Distilled water, 0.05M potassium phosphate buffer, and 0.1M sodium acetate buffer solutions with 0, 2, 4, and 5 wt% NaC1 were used as solvents. The pH of lysozyme solutions in distilled water was 4.75 due to the self-buffering capacity of lysozyme. The pH values of the lysozyme solutions in the potassium phosphate and sodium acetate buffers were adjusted to 6.8 and 4.0, respectively. The experimental temperature was 25°C. In a salt-free system, the concentration dependent diffusion of lysozyme showed typical electrolyte diffusion behavior, while a salt-polyelectrolyte system exhibited the behavior of a non-electrolyte. Diffusion results in the supersaturated region showed little effect of concentration or solution "age" at a fixed NaC1 concentration. A rapid decline in diffusion coefficient with increasing NaCI concentration in the supersaturated region, however, was observed.