Microfiltration membranes were studied as alternative adsorbents to the usual spherical hydrogels beads for the purification of proteins. Assumption of monolayer adsorption onto the internal area allowed the capacity measured by adsorption of model proteins to be predicted. The membrane could be modified with triazine dyes to yield an affinity-like support. The capacity of the dyed membrane was similar to the capacity of dyed Sepharose and the membrane could be used repeatedly. Enzymes from a crude extract of bakers yeast were selectively adsorbed in both batch and continuous experiments, allowing purification factors of at least ten.
Highly purified proteins and enzymes are usually obtained using chromatographic separations which require long cycle times. To improve mass transport other types of chromatographic support than porous particles seem to be interesting. In this paper a fixed bed composed of about 100 stacked microporous membranes is investigated for protein chromatography. A mathematical model and its experimental verification indicate the condition under which nonlinear, kinetic and dispersive effects will occur. A scale-up of two protein purifications and their incorporation in the complete recovery procedure is also presented. The two proteins are formate dehydrogenase (FDH) from Candida bodinii and pyruvate decarboxylase (PDC) from Zymomonas mobilis.
The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP+-dependent formate dehydrogenase (EC 126.96.36.199, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purification process were analyzed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purification were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the purified FDH was determined as 44 078 Da by matrix-assisted laser desorption/ionisation time of flight mass spectrometry
DOI : 10.1016/S0168-1656(97)00149-1 Anahtar Kelimeler :
Capillary electrophoresis, SDS-Gel capillary electrophoresis, Proteins, Recombinant formate dehydrogenase, Rapid analysis, MALDI-TOF-MS, matrix-assisted laser desorption/ionisation time of flight mass spectrometry
Cilt: 58 Sayı: 3 Sayfa: 187-195 ISSN: 0168-1656
The formation of membrane sublayers during cross-flow filtration was studied with a standardized E. coli suspension both in a tubular and a flat channel module with different membrane materials. The height of the layers was calculated for different experimental conditions. Transmembrane pressure, cross-flow velocity, compressibility of the suspended particles, properties of the suspension, particle size and concentration were all found to have a significant effect on the formation of membrane sublayers. A decrease of the layer thickness and corresponding filtration resistance with increasing channel length was observed due to the longitudal transmembrane pressure gradient. The filtration resistance of the layer is found to be the dominant factor determining the flux rate.
The use of an aqueous two-phase system with the nonionic polyoxyethylene detergent C12EO5 (pentaethylene-glycol mono n-dodecyl ether) for direct solubilization and extraction of membrane-bound cholesterol oxidase from an unclarified culture of Nocardia rhodochrous was investigated. The potential of such nonionic detergent-based aqueous two-phase systems to simplify downstream processing of proteins is demonstrated. In only one step a clarification, preconcentration (up to 4-fold) and a partial purification (up to 5-fold) can be achieved. More than 90% of the enzyme could be extracted into the coazervate phase. Optimum conditions for the protein extraction from unclarified culture medium are shown in an analytical scale by investigation of the key parameters of the extraction process. These are detergent and biomass concentration as well as several additional agents for protein solubilization and stabilization.
The partition coefficients of lysozyme, chymotrypsinogen A, albumin and catalase in 64 polyethyleneglycol/dextran systems are reported. The measurements were performed at four pHs for each protein. The simultaneous effect of pH changes and polymer molecular weight and concentration on the partition coefficient of each protein is analyzed. The partition coefficient of lysozyme (IP = 10.5) has a minimum value at its isoelectric point and it takes its maximum value at acidic pHs. We observe a change in the aggregational state of lysozyme when the pH is shifted from the acidic to the alkaline ranges. The partition coefficient of chymotrypsinogen A (IP = 9.5) has a minimum at pH 5.6 and increases towards more alkaline or acidic pHs. The partition coefficient of albumin (IP = 4.6) takes its minimum value at pH 5.6. The partition coefficient of catalase (IP = 5.6) takes its maximum value at pH 5.6. The effect of the pH on the partition coefficient of lysozyme and chymotrypsinogen A at high polymer concentrations is larger than at low total polymer concentrations but the effect of the pH on the partition coefficient of albumin and catalase at high polymer concentrations is smaller than at low polymer concentrations. We find that the partition coefficient of the four proteins studied becomes less sensitive to changes in the pH at high PEG molecular weights. Close to the isoelectric point the partition coefficient is less sensitive to changes in the molecular weight of the polymers than at conditions far from the isoelectric point.
The purification of the hydroxynitrile lyase (EC 188.8.131.52, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.
An enzymatic method is described for the determination of l-phenylalanine or phenylpyruvate using l-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of l-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors l-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for l-phenylalanine determination, too. Standard solutions of l-phenylalanine in the range of 10–300 μm and of phenylpyruvate (5–100 μm) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.
Harvesting of microbial cells by tangential filtration was investigated with well defined Escherichia coli and bakers yeast media. Parameters responsible for the sublayer formation at the membrane surface were studied in detail using two self-constructed modules of tube and flat channel configurations respectively and different types of membranes. The deposited mass and filtration resistance of the sublayers formed were analysed. Results show that layer heights (amounts deposited) decrease with increasing channel length due to the pressure drop behaviour of the modules. Layer heights between 0.5 and 40 μm were determined. The filtration resistance of the layers was found to be higher than the resistance of corresponding ‘cakes’ formed in static filtration. A simple empirically derived model was developed which can be used to calculate filtration performance over the range of experimental conditions studied.
A new method is described for the synthesis of PN bonds under mild conditions and in high yields: water-soluble carbodiimides are used to cyclize 3′-amino-3′-deoxyadenosine 5′-monophosphate and 3′-N-benzylamino-3′-deoxyadenosine 5′-monophosphate in aqueous soln at pH 7·5 and 37°. When 3′-amino-3′-deoxyadenosine 2′,5′-diphosphate is treated with carbodiimide the 3′,5′-cyclic derivative is formed rather than the 2′,3′-cyclic derivative. 13C NMR spectra are used for structure determination of the products. The chemical shifts and 13C-31P spin-spin coupling constants are discussed.
In an effort to understand the molecular weight and temperature dependence of protein partition coefficients, we have conducted experiments to determine how the binodal curve and the difference in polymer concentrations between the top and bottom phases (▵PEG [PEG]T — [PEG]B and ΔDx [Dx]B — [Dx]T); depend on polymer molecular weight and temperature. We report the equilibrium compositions at 4°, 25° and 40°C for 16 different aqueous two-phase PEG-Dx systems which consist of all possible combinations of the four PEG molecular weights (4000, 6000, 10 000, and 20 000) and the four Dx molecular weights (10 000, 40 000, 110 000 and 500 000). Four tie lines were measured for each system. We confirm that increasing polymer molecular weight increases the tie line length and depresses the binodals to lower polymer concentrations, and that this effect will be greater when the ratio of this molecular weight increase to the molecular weight of the other species is large. The increase in the tie line length and in ΔDx and ΔPEG with increasing polymer molecular weight tends to level off at high polymer molecular weight and at high total polymer concentrations. It is shown that the effect of temperature is not symmetric, i.e. changes in temperature produce a change in ΔDx while ΔPEG remains nearly constant. The effect of polymer molecular weight on both ΔDx and ΔPEG is magnified with increasing temperature. The results found here suggest a series of experiments which could be used to isolate (or highlight) the effect of the second virial coefficient on the partition coefficient.
Heat conditioning of cell homogenates of B. cereus and a recombinant E. coli was studied for the isolation of leucine dehydrogenase and alanine racemase, respectively. The strain of E. coli carried the gene of the thermostable alanine racemase from B. stearothermophilus. Activity loss can be minimized (<5%) and aggregation and flocculation of soluble proteins (70–80%) and other cell components can be achieved, depending on temperature, biomass concentration and pH-value. Thereby a 3–6 fold increase in specific activity was obtained. The resulting extract after solid-liquid separation showed lower viscosity and less turbidity than unheated controls, making it more suitable for chromatographic separations.
The application of thiophilic membranes for the purification of monoclonal antibodies from hybridoma culture media was studied. Affinity filtrations were performed with membrane stacks and also in a cross flow module with a spiral filtration channel. Purification factors up to five and concentration factors of about eight could be achieved. The flux behaviour was analysed and interpreted according to existing models of filtration. The results were confirmed by scanning electron microscopy. The binding capacity of the membranes differed considerably with the mode of operation. The main component responsible for membrane fouling was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid sequence analysis as bovine serum albumin or its fragments.
Suspensions of several yeast strains and bacterial species were disrupted in a continuously operating industrial agitator mill of 22.7 litre internal working volume. The influence of agitator speed, flow rate, concentration of microorganisms in the slurry, packing density of glass beads and bead diameter on the disruption process was studied using bakers yeast (Saccharomyces cerevisiae). Cell disintegration was followed by assaying the appearance of protein and the activities of d-glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate:NADP+ oxidoreductase, EC 184.108.40.206] and α-d-glucosidase [α-d-glucoside glucohydrolase, EC 220.127.116.11] in the soluble fraction. The best operating conditions for the disintegration of bakers yeast with respect to activity yield appeared to be at a rotational speed of 1100 rev/min, a flow rate of 100 litre h−1 and a cell concentration of 40% (w/v). The location of the desired enzyme in the cell is of importance for the choice of bead diameter and packing density of the glass beads. Temperature increase and power consumption during disintegration are also strongly influenced by the bead loading in the mill. With optimized parameters, 200 kg bakers yeast can be processed per hour with a degree of disintegration >85%. The disruption process in the mill was found to be very effective for several yeast species tested, e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Candida boidinii. The usefulness of the Netzsch LME 20-mill for the disruption of bacteria species was demonstrated with Escherichia coli, Brevibacterium ammoniagenes, Bacillus sphaericus and Lactobacillus confusus. As expected, the mill capacity for bacterial disruption was significantly smaller than for the yeast. Between 10 and 20 kg per h bacteria may be processed, depending on the organism.
The isolation and purification of α-lactalbumin and β-lactoglobulin from whey can be conducted continuously in a Graesser contactor using an aqueous two-phase system based on polyethylene glycol and potassium phosphate. Processing of more than 600 g of whey proteins per day was performed in the apparatus described. β-Lactoglobulin partitioned almost quantitatively into the salt-rich bottom phase, and α-lactalbumin was enriched in the top-phase of the system applied. The residence time distribution and the fractional hold-up as a hydrodynamic parameter were determined as the basis for a qualitative prediction of the extraction efficiency of the process described.
The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene (pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His6 tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala-L-Phe-NH2 is hydrolyzed in the absence of cofactors to L-Ala-L-Phe-OH and ammonia with V
max=194 U/mg and K
m <0.5 mM. The natural function of Pam remains unclear. Chymostatin (K
i<0.3 µM) and Pefabloc SC (K
i not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.
Extrachromosomal elements were found in a strain ofX. dendrorhous, and were characterized as linear DNA forming two well defined groups, pPh1 with 3 high-copy-number molecules, pPh11 (6.9 kb), pPh12 (5.7), pPh13 (4.7), and pPh2 with 2 low-copy-number molecules, pPh21 (3.6 kb), pPh22 (3.0). A 4077 bp fragment from pPh13 was cloned in pUC18 (pDK1) and sequenced (accession no. AJ 278 424). Seven putative ORF and some possible regulator sequences were defined.