Production of tripeptide from gelatin using collagenase-immobilized porous hollow-fiber membrane

Tripeptide was produced during the permeation of a gelatin solution through the pore of a collagenase-immobilized porous hollow-fiber membrane. Gelatin was obtained via hydrolysis of fish collagen. First, an epoxy-group-containing monomer was graft-polymerized onto an electron-beam-irradiated porous hollow-fiber membrane. Second, the 2-hydroxyethylamino group was introduced into the epoxy group to bind collagenase on the basis of electrostatic interaction. Third, adsorbed collagenase was cross-linked with glutaraldehyde to prevent leakage of the enzyme. Gelatin solution (10-50 g/L) was forced to permeate across the collagenase-immobilized porous hollow-fiber membrane with a density of immobilized collagenase of 52 mg/g at various residence times of the gelatin solution ranging from 0.13 to 20 min. Fourteen percent in weight of 10 g/L gelatin solution was hydrolyzed into tripeptide at a residence time of 20 min.     

Multilayer binding of proteins to polymer chains grafted onto porous hollow-fiber membranes containing different anion-exchange groups

Various anion-exchange groups were introduced into the polymer chains grafted onto a porous hollow-fiber membrane for protein recovery by radiation-induced graft polymerization and subsequent functionalization of a monomer containing an epoxy group. The graft chains extended from the pore surface toward the pore interior, resulting in the multilayer binding of proteins to the graft chains. Combinations of three anion-exchange groups, namely, amino (AM), ethylamino (EA), and diethylamino (DEA) groups, and three proteins, namely, beta-lactoglobulin, bovine serum albumin, and urease, were examined to evaluate the degree of multilayer binding of protein to the graft chains in the permeation mode. Multilayer binding was observed for hollow-fiber membranes containing EA and DEA groups, with conversions of epoxy groups to EA or DEA groups of higher than 80%. The amount of adsorbed protein remained constant irrespective of the conversion for the hollow-fiber membrane containing an AM group. The dependence of the flux on the conversion was consistent with that of the degree of multilayer binding to the graft chains.     

Highly multilayered urease decomposes highly concentrated urea

Urease was immobilized at a density of 1.2 g of urease per gram of a matrix via ion-exchange binding of urease to an anion-exchange polymer chain grafted onto a pore surface of a porous hollow-fiber membrane and subsequent cross-linking of urease with transglutaminase. Urea was hydrolyzed during the permeation of a urea solution, the concentration of which ranged from 2 to 8 M, through the pores of the resultant membrane with a thickness of approximately 1 mm. Quantitative hydrolysis of 4 M urea was achieved at a permeation rate lower than 1 mL/h, i.e., a residence time longer than 5.1 min, at ambient temperature. This performance is ascribed to convective transport of urea through the pores rimmed by the urease-immobilized polymer chains at a high density. Urease was denatured in the presence of urea at concentrations higher than 6 M while hydrolyzing urea.     

Adsorption characteristics of an immobilized metal affinity membrane.

An immobilized metal affinity (IMA) hollow-fiber membrane was prepared by radiation-induced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group and its chelation with copper(II) ion. The IDA hollow fiber, whose degree of GMA grafting was 120%, was found to retain 0.42 mol of Cu ion/kg of dry weight of the resulting IMA hollow fiber. The pure water flux of the affinity membrane was 0.90 m/h at a filtration pressure of 1 x 10(5) Pa. The 0.1 g/L L-histidyl-L-leucine (His-Leu) solution permeated across the IMA hollow fiber, whose inner diameter and thickness were 0.78 and 0.365 mm, respectively, at a prescribed filtration pressure ranging from 0.2 x 10(5) to 1.0 x 10(5) Pa. The adsorption of His-Leu during permeation of the solution showed that the overall adsorption rate was independent of the filtration pressure, i.e., the residence time, because of the negligible diffusional resistance of His-Leu to the pseudobioaffinity ligand located on the pore surface of the membrane. No deterioration in the adsorption capacity was observed after five cycles of His-Leu adsorption, its elution, and reimmobilization of copper. The adsorption isotherm of bovine serum albumin (BSA) on the IMA hollow fiber was measured and compared with that for the conventional agarose-based bead containing the IDA-Cu ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilization of an Esterase Inhibitor on a Porous Hollow-Fiber Membrane by Radiation-Induced Graft Polymerization for Developing a Diagnostic Tool for Feline Kidney Diseases

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.     

Protein binding to polymer brush,based on ion-exchange,hydrophobic, and affinity interactions

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.     

Mathematical model of microporous hollow-fiber membrane extractive fermentor

A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize product inhibition.     

Purification of docosahexaenoic acid ethyl ester using a silver-ion-immobilized porous hollow-fiber membrane module

Docosahexaenoic acid ethyl ester (DHA-Et) was purified by adsorption on Ag-ion-immobilized membranes via selective interaction between silver ion and carbon-carbon double bonds of DHA-Et. Silver ions were immobilized onto sulfonic-acid-group-containing porous hollow-fiber membranes at an Ag ion density of 1.4 mol/kg of membrane, and 30 membranes were housed in one module (inner diameter = 18 mm and effective length = 80 mm). The adsorption isotherms of DHA-Et in various organic solvents revealed that DHA-Et was adsorbed on the immobilized Ag ions with a DHA-Et/Ag ion molar binding ratio of 1/5 in methanol, and that acetonitrile was the solvent of choice for the elution of the adsorbed DHA-Et. Permeation of bonito oil ethyl ester solution in methanol through the Ag-ion-immobilized hollow-fiber membrane module demonstrated that the displacement adsorption of other lower unsaturated fatty-acid ethyl esters by DHA-Et proceeded along the membrane thickness. The purity of DHA-Et was improved to 99 wt % by permeating first bonito oil ethyl ester containing 95 wt % DHA-Et and then acetonitrile through the module.     

Protein binding to amphoteric polymer brushes grafted onto a porous hollow-fiber membrane

Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 microm. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins.     

Production of cycloisomaltooligosaccharides from dextran using enzyme immobilized in multilayers onto porous membranes

Anion-exchange porous hollow-fiber membranes with a thickness of about 1.2 mm and a pore size of about 0.30 microm were used as a supporting matrix to immobilize cycloisomaltooligosaccharide glucanotransferase (CITase). CITase was immobilized to the membrane via anion-exchange adsorption and by subsequent enzymatic cross-linking with transglutaminase, the amount of which ranged from 3 to 110 mg per gram of the membrane. The degree of enzyme multilayer binding was equivalent to 0.3-9.8. Dextran, as the substrate, was converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides (CI-7, -8, and -9) at a maximum yield of 28% in weight at a space velocity of 10 per hour during the permeation of 2.0% (w/w) dextran solution across the CITase-immobilized porous hollow-fiber membrane. The yield of CIs increased with increasing degree of CITase multilayering.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

On the performance of a hollow-fiber bioreactor for acidolysis catalyzed by immobilized lipase

The present communication describes the chemical modification of anhydrous butterfat by interesterification with oleic acid catalyzed by a lipase of Mucor javanicus. Two reactor configurations were tested, a batch-stirred tank reactor containing suspended lipase and a batch-stirred tank reactor in combination with a hollow-fiber membrane module containing adsorbed lipase. The goal of this research was to assess the advantage of using a (hydrophobic) porous support to immobilize the lipase in attempts to engineer butterfat with increased levels of unsaturated fatty acid residues (oleic acid) at the expense of medium-to-long chain saturated fatty acids (myristic and palmitic acids). Reactions were carried out at 40 degrees C in the absence of solvent under controlled water activity, and were monitored by chromatographic assays for free fatty acids. The results obtained indicate that the rate of interesterification using the proposed reactor configuration is enhanced by a factor above 100 relative to that using suspended lipase, for the same protein mass basis. Although hydrolysis of butterfat occurred to some degree, the enzymatic process that uses the hollow-fiber reactor was technically superior to the stirred tank system. Copyright 1998 John Wiley & Sons, Inc.     

Treatment of trichloroethylene (TCE) in a membrane biofilter

This article reports on the biodegradation of trichloroethylene (TCE) in a hollow-fiber membrane biofilter. Air contaminated with TCE was passed through microporous hollow fibers while an oxygen-free nutrient solution was recirculated through the shell side of the membrane module. The biomass was attached to the outside surface of the microporous hollow fibers by initially supplying toluene in the gas phase that flows through the fibers. While studies on TCE biodegradation were conducted, there was no toluene present in the gas phase. At 20-ppmv inlet concentration of TCE and 36-s gas-phase residence time, based on total internal volume of the hollow fibers, 30% removal efficiency of TCE was attained. At higher air flow rates or lower gas-phase residence times, lower removal efficiencies were observed. During TCE degradation, the pH of the liquid phase on the shell side of the membrane module decreased due to release of chloride ions. A mathematical model was developed to describe the synchronous aerobic/anaerobic biodegradation of TCE. (c) 1996 John Wiley & Sons, Inc.     

Enzymatic synthesis of glucuronides using lipophilic hollow fiber membranes

A lipophilic hollow fiber membrane preparation was used for the enzymatic glucuronidation of lipophilic aromatic compounds. A crude solubilized microsomal enzyme preparation was circulated on the external side of the lipophilic membrane while the phenol containing buffer solution was circulated through the internal side of the hollow fiber membrane. Phenols, which accumulate in and penetrate the lipophilic membrane, were converted by UDP-glucuronyltransferase to the corresponding glucuronides. During this process the lipophilic compounds are converted to hydrophilic substances, which are not able to rediffuse through the lipophilic membrane into the donor side of the hollow fiber module. The produced glucuronide is separated by means of a coupled dialysis with a module of hydrophilic surface (cellulose acetate), while the enzyme protein is retained.On the stripping side of the dialysing module the glucuronide can be separated by solid phase extraction (Lichroprep RP-18) while a continuous substitution of cofactor into this compartment is possible. UDPGA follows its own concentration gradient and migrates into the enzymatic mixture, where it is utilized. This new technique using hollow fiber modules offers completely new possibilities for long-term high-capacity, highly specific glucuronidation of phenolic compounds. Fields of application are not only the economical production of special glucuronides, but also the specific elimination of phenols from waste fluids or from serum and blood of patients.For the production of glucuronides by this technique the use of highly purified enzymes is not essential. Cheap crude enzyme preparations are quite adequate for an optimal reaction. Using a crude enzyme preparation with a specific batch activity of 13 nMol/min per mg of protein, the activity in the reactor system was observed to be 4.6 nMol/min of 2-naphtol glucuronide formed per mg of protein. This corresponds to 3.6 nMol/h of product formed per mg of protein per cm² of hollow fiber surface.Using a membrane surface of 0.5 m² the production of 18 mMol of glucuronide per h and mg protein can be achieved.     

Characterization of an anion-exchange porous polypropylene hollow fiber membrane for immobilization of ABL lipase

Hollow fiber membrane offers the advantage to integrate catalytic conversion, product separation and catalyst recovery into a single separation process compared to conventional systems. Polypropylene (PP) hollow fiber membrane is a chemically inert and stable membrane with high potential for enzyme immobilization. The surface properties of polypropylene have been modified by radiation induced graft polymerization. Samples were prepared by grafting of glycidylmethacrylate (GMA) using gamma radiation, at different monomer concentrations and irradiation dose. The resulting epoxy was converted into a diethylamino group as an anion-exchange medium to bind the lipase molecules. Surface properties of the grafted and amine treated samples were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM) and contact angle measurements. AFM revealed higher surface roughness for grafted samples than that of virgin polymer. SEM micrographs illustrated that the porous network was retained at high degree of grafting. Contact angle measurements showed excellent wetting properties with water for the grafted and amine treated membranes. Thermal properties were studied using differential scanning calorimeter (DSC) and thermogravimetic analysis (TGA). It was observed that grafting occurred mainly in the amorphous region of the membranes. Activity and operational stability of ABL lipase, isolated from Arthobacter sp. were assayed after immobilizing it to the modified PP hollow fiber. Immobilized lipase retained 20U/g activity after ten hydrolysis cycles and 68% residual activity after 12 weeks of storage.     

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 mug/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.     

Effect of Solute Adsorption Properties on Its Separation from Supercritical Carbon Dioxide with a Thin Porous Silica Membrane

A thin porous silica membrane (average pore size of 3.3 mm) was prepared by the sol–gel method and used to separate the solute from supercritical carbon dioxide. The characteristics of solute permeation were investigated in respect of the adsorption properties of the solute, the desorption rate of the solute from the membrane being measured and the potential energy of solute near the silica surface being calculated by the molecular modeling technique. It was found that caffeine was strongly adsorbed to the surface and then slowly desorbed to form an adsorption layer, making the pores narrower and causing a molecular-sieving effect. Therefore, the rejection value was positive. On the other hand, the rejection value of n-octanoic acid, which was well adsorbed and rapidly desorbed, was negative. It is presumed that the molecules filled the pores due to their potential energy and were then forced to flow through the pores by the transmembrane pressure.     

Membrane reactor coupled with electrophoresis for enzymatic production of aspartic acid

Aspartic acid production by aspartase reaction on ammonium fumarate was carried out in a membrane reactor coupled with electrophoresis. A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. An electric field was applied across the membrane to preferentially remove the product aspartate from the reactor into the permeate stream. The charged molecule, aspartate, is much smaller than the molecular-weight cutoff of the membrane (10(4)) so that the ions would move freely through pores of the membrane. The concentration of aspartate in the permeate stream is determined by the electromigration velocity of the ions and the permeation rate of solvent (water) through the membrane. The permeation rate of solvent could be controlled by the applied pressure, and the migration velocity of the ions could be controlled by the electric field strength applied. The equilibrium conversion of ammonium fumarate to the aspartate was 70%. In the presence of electric field, the aspartase activity was not disturbed. Also, it is shown that the aspartate concentration in the permeate stream was 20% higher than that in the reaction solution with the permeate flow rate of 0.7 mL/min. The steady-state conversion was 60%. Instead of aspartate, aspartic acid can be recovered directly from the permeate stream by controlling the circulation of buffer electrolyte in the anode compartment.     

Protein adsorption in polysulfone hollow fiber bioreactors used for serum-free mammalian cell culture

The recovery of serum-free medium proteins from poly-sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre-adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA-coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA-coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30-kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen. (c) 1993 John Wiley & Sons, Inc.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献