Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Trypsin concentration by ultrafiltration]

Criteria for the selection of membranes and optimal conditions for the trypsin concentration by ultrafiltration through acetylcellulose membranes have been developed. The possibility of a repeated concentration of trypsin by means of these membrances has been shown.     

Kinetic properties of gel-immobilized acid phosphatase in a tubular membrane reactor

Summary Acid phosphatase has been immobilized onto the internal surface of tubular ultrafiltration membranes by two different methods, namely copolymerization/gelation and co-gelation. Rate parameters for p-nitrophenyl phosphate hydrolysis by the enzyme in both gel-immobilization conditions have been determined and compared to the corresponding values obtained in previous work using a flat ultrafiltration membrane. Results indicate that the kinetic properties of the enzyme seems not substantially modified by the membrane geometry; however, for industrial purposes an enzyme reactor equipped with tubular membranes should be preferred.     

Studies on Box-Behnken design experiments: cellulose acetate-polyurethane ultrafiltration membranes for BSA separation

Ultrafiltration membranes were prepared from mixtures of cellulose acetate-polyurethane blend membranes. During the last 1 or 2 decades, the concentration purification and separation of Albumin by ultrafiltration through semipermeable membranes have been put into practice and hence membrane separation is considered as the unit operation. The blend solution was prepared from cellulose acetate and polyurethane in polar solvent in presence of polyvinylpyrrolidone as additive. The performance of modified blend membranes applied for Bovine Serum Albumin (BSA) separation by ultrafiltration technique using Box-Behnken design with three variables: additive, time and pressure. Three different levels was studied to identify a significant correlation between the effect of these variables on the amount of separation of BSA. The methodology identifies the principal experimental variables, which have the greatest effect on the separation process. The experimental values are in good agreement with predicted values, the correlation coefficient was found to be 0.9871.     

Difficulties encountered with an ultrafiltration method for measuring the binding of ligands to protein

The ultrafiltration method of Paulus has been used to study the binding of aspartate and ITP to the catalytic subunit of aspartate transcarbamylase. Markedly different estimates of dissociation constants and of the number of moles of ligand bound per mole of enzyme have been obtained using different batches of UM 10 Diaflo membranes. Estimates derived using Visking membranes are presented for comparison.For both an amino acid and a nucleotide invalid results have been obtained using UM 10 Diaflo membranes in conjunction with this enzyme. The importance of excluding artifacts in any system studied by this method is emphasized.     

Retention of small charged impurities during ultrafiltration

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.     

Effect of membrane charge on flow and protein transport during ultrafiltration

Although several recent studies have demonstrated the importance of electrostatic interactions in ultrafiltration, there have been few quantitative studies of the effects of membrane charge density on protein transport and membrane hydraulic permeability. Data were obtained using a series of charge-modified cellulose membranes, with the surface charge density controlled by varying the extent of addition of a quaternary amine functionality. The membrane charge was evaluated from streaming potential measurements. Protein transmission decreased by a factor of 100 as the membrane zeta potential increased from 0.3 to 6.6 mV. The protein sieving data were in good agreement with a partitioning model accounting for electrostatic effects, while the hydraulic permeability data were consistent with a flow model accounting for the effects of counter-electroosmosis. The results provide the first quantitative analysis of the effects of membrane charge density on the performance of ultrafiltration membranes.     

Purification of densonucleosis virus by tangential flow ultrafiltration

Purification at commercial scale of viruses and virus vectors for gene therapy applications and viral vaccines is a major separations challenge. Tangential flow ultrafiltration has been developed for protein purification. Here tangential flow ultrafiltration of parvoviruses has been investigated. Because these virus particles are small (18-26 nm), removal of host cell proteins will be challenging. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow some virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the 30, 50, and 100 kDa membranes, indicating possible entrapment of virus particle in the membrane pores. The permeate flux and level of protein rejection is strongly affected by the cell culture growth medium. The results indicate that when developing a new process, it is essential that the cell culture and purification operations be developed in parallel.     

Purification of lysozyme using ultrafiltration

This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.     

Advances in S-layer nanotechnology and biomimetics

Two-dimensional crystalline bacterial S-layers composed of identical protein or glycoprotein subunits turned out to be ideal materials for the development of biomimetic membranes and new approaches in molecular nanotechnology. These isoporous protein lattices have already been used as (i) structure for producing isoporous ultrafiltration membranes with very precisely defined molecular sieving properties, (ii) matrices for immobilizing monolayers of functional molecules, (iii) stabilizing structure for LB-films and liposomes, and (iv) patterning elements in molecular nanotechnology.     

Ultrafiltration membranes as carriers for lipase immobilization.

The feasibility of directly incorporating lipase from Rhizopus in the interior of poly(vinyl chloride) ultrafiltration membranes during the phase inversion process for their manufacturing has been demonstrated. The obtained membranes used for plant oil hydrolysis have shown better time stability as compared with those of lipase immobilized by adsorption. The specific activity of entrapped lipase achieves values higher than those of the soluble one. The activity of immobilized lipase is strongly affected by the rate of removing fatty acids from the interior of the membrane.     

THE PREPARATION OF THE GRADED COLLODION MEMBRANES OF ELFORD AND THEIR USE IN THE STUDY OF FILTERABLE VIRUSES

1. The method described by Elford for the preparation of graded collodion membranes suitable for ultrafiltration was found to give excellent results, and his findings are fully confirmed. 2. A formula is given for the preparation of collodion from which satisfactory membranes of graded porosity can be prepared. 3. The technique and apparatus used in the preparation, and standardization of membranes are described in detail. 4. The technique and apparatus required for ultrafiltration experiments are described, and some drawbacks encountered in the experiments are discussed. 5. The results of ultrafiltration experiments show that the pores of the membranes are remarkably uniform in size.     

Alginate ultrafiltration membranes

Summary A simple method of producing ultrafiltration membranes, using calcium and barium alginates, has been developed. Although restricted in their flux and rejection characteristics these membranes offer a cheap model system for studying the fouling of hydrophilic membrane surfaces. The method is reproducible, and with suitable modification allows the membranes to be dried and reconstituted with little effect on performance.     

Challenging protein purification from anammox bacteria

The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.     

Production of antihypertensive and antioxidant activities by enzymatic hydrolysis of protein concentrates recovered by ultrafiltration from cuttlefish processing wastewaters

This work focuses on the production of antihypertensive and antioxidant activities using enzymatic hydrolysis of protein concentrates recovered by ultrafiltration of different wastewaters from the industrial processing of cuttlefish (Illex argentinus). The effluents were produced in the processes of thawing (E1), softening (E2), boiling (E3) and gelation (E4). Our results showed that membranes with cut-off at 100, 30 and 10 kDa were an effective resource to protein concentration of E2 and E3 but limited for E1 and E4. In addition, E2 and E3 retentates led to remarkable antihypertensive and antioxidant activities, further improved by enzymatic hydrolysis. Also sequential ultrafiltration revealed the enrichment of these protein concentrates in peptides with high angiotensin-converting enzyme (ACE)-inhibitory activity. Thereby, UF-fractionation followed by proteolysis of protein concentrates from cuttlefish wastewaters offers new opportunities for the development of bioactive hydrolysates with application in the food industry. In addition, this approach contributes to an improved depuration of industrial wastewaters, reducing the treatment costs and leading to a decrease in its contaminating effect.     

A novel membrane based process to isolate photosystem-I membrane complex from spinach

The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300 kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O₂ electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.     

Salt-induced changes in plasmid DNA transmission through ultrafiltration membranes

Recent studies have demonstrated the feasibility of using ultrafiltration for the purification of plasmid DNA, but there is still little understanding of the factors governing DNA transmission. Experimental data were obtained for the transmission of a 3.0 kbp supercoiled plasmid DNA through composite regenerated cellulose ultrafiltration membranes as a function of solution ionic environment in a stirred ultrafiltration cell. The dependence on salt concentration was quite dramatic, with the sieving coefficient increasing by more than 80-fold as the NaCl concentration increased from 1 to 150 mM at a fixed filtrate flux. At the same total ionic strength, the sieving coefficient in an MgCl2 solution was significantly larger than that evaluated in NaCl. The sieving results are consistent with independent studies showing a reduction in the effective plasmid size due to salt specific shielding of intramolecular electrostatic interactions. DNA transmission was also a strong function of the filtrate flux, with negligible transmission below a critical value of the flux. The predicted values of the critical filtrate flux determined using a modified elongational flow model were in excellent agreement with the experimental data. These results clearly demonstrate that salt-induced changes in plasmid DNA structure have a significant effect on plasmid DNA transmission through ultrafiltration membranes.     

Parameter scanning ultrafiltration: rapid optimisation of protein separation

High-resolution fractionation of proteins using ultrafiltration is feasible only at highly optimised conditions. Conventional process optimisation methodology demands both time and material. Pulsed sample injection ultrafiltration has been suggested as a rapid process optimisation technique. In the present work the scope of this technique is further extended by "parameter scanning ultrafiltration," which involves continuous change of a process parameter (e.g., pH, salt concentration). The time and material consumption are thus further reduced. The technique was validated using different proteins and membranes. Sieving coefficients at different pH and salt concentration were compared to those obtained in fixed parameter ultrafiltration experiments. As fractionation case studies the separation of monoclonal antibody from bovine serum albumin and separation of human IgG from human serum albumin were examined.     

Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors

The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 x 10(4) D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell-side convective flow and its influence on the inoculum and high-molecular-weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.