Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase

Recombinant Escherichia coli (pATBX 1.8) secreting extracellular xylanase was used as a model system to study the application of an aqueous two phase system for extractive cultivation. An increase in the polymer concentrations from 6 to 20% in the polyethylene glycol phosphate aqueous two phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of xylanase in the top phase. However, varying phosphate concentrations from 8 to 16% decreased both the phase volume ratio and the partition coefficient of xylanase. The polyethylene glycol (6%) and phosphate (12%) system was found to be optimum for extracellular cultivation of E. coli, where extracellular xylanase was selectively partitioned to the top phase giving a purification ratio of above 1.0. The process was extended to a semicontinuous operating mode at the optimal condition, wherein the top phase containing xylanase was recovered and the surviving cells were recycled together with the new top phase. The maximum recovery of xylanase was obtained after 12 h in the top phase with a twofold increase in the specific activity as compared to the one obtained in the reference fermentation. In the present work, we report for the first time the use of the two phase system for the extractive cultivation of recombinant E. coli (pATBX 1.8) with the purpose of obtaining a simple and inexpensive separation procedure and achieving the maximal extraction of xylanase to one phase.     

Release of periplasmic enzymes fromEscherichia coli using tangential flow filtration

Summary Escherichia coli cells producing the periplasmic enzymes nuclease, -lactamase and alkaline phosphatase were exposed to osmotic shock treatment in a closed system consisting of a tank, a pump and a filtration unit. The enzymes were released by circulating the cell suspensions in the filtration system and separated from cells and spheroplasts by filtration. This novel releasing method was shown to be equally effective as osmotic shock treatments performed by agitation and centrifugation. Since the extraction is performed in a closed system, aerosol formation is avoided. In addition the method may easily be scaled up.     

Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

Predicting the partition coefficients of a recombinant cutinase in polyethylene glycol/phosphate aqueous two-phase systems

A model for the prediction of protein partition coefficients in aqueous two-phase systems has been developed. This model accounts for both charge-independent and electrostatic effects. The determination of nonelectrostatic effects was based on the model of Eiteman and Gainer for uncharged solutes while the electrostatic contribution was computed using TITRA, a program that uses a continuum electrostatic model to treat charge interactions in proteins and considers the effect of pH and ionic strength. The partition coefficients of Fusarium solani pisi recombinant cutinase have been satisfactorily predicted in polyethylene glycol (PEG) 1000 and phosphate aqueous two-phase systems at a pH range of 6.0-9.0. The model failed to predict the enzyme partitioning behavior at pH 4.5. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 248-257, 1997.     

Purification and characterization of enolase fromEscherichia coli

A method for the purification of enolase (EC 4.2.1.11) from an overproducing strain ofEscherichia coli JA 200 pLC 11–8 is described. The procedure included treatment of the crude sonic extract with protamine sulfate, followed by ammonium sulfate fractionation, hydrophobic interaction chromatography with phenyl Sepharose, HPLC ion exchange chromatography with a DuPont Sax column, and HPLC hydrophobic interaction chromatography with a Bio-Rad 5-PW column. The enzyme was purified to homogeneity as determined by silver staining of 10% sodium dodecylsulfate polyacrylamide gels. The native molecular weight ofE. coli enolase was found to be 92 kilodaltons and consisted of two subunits of identical molecular weight, 46 kilodaltons each. The isoelectric point was found to be 4.9.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Shape of phosphofructokinase fromEscherichia coli in solution

Summary Small angle X-ray scattering measurements and electron microscopic studies were carried out onE. coli phosphofructokinase (E.C. 2.7.1.11; ATP: D-fructose-6-phosphate-1-phosphotransferase). The results suggest a tetrahedral arrangement of the protomers resulting in a radius of gyration of the enzyme of R=34.6 Å and a Stokes' radius of R₀=44.0 Å. The stereochemical arrangement of the four protomers, each of a molecular weight of 35,000, within theE. coli enzyme was further substantiated by a comparison of theoretical scattering functions with the experimental scattering measurements in dilute solutions of phosphofructokinase under physiological conditions. Moreover, from other hydrodynamic measurements,e.g., intrinsic viscosity and sedimentation coefficient, theMandelkern-Scheraga factor, , was calculated to be 2,095×10⁶, which is significantly lower than the ₀ for rigid spheres of 2,112×10⁶. This low -value might be due to a considerable porosity of the four protomers for mobile water molecules. The -value of 2,095×10⁶ is an indication of a porous sphere of almost uniform density at aDebye shielding ratio of 6.5, corresponding to a sphere radius of 22.0 Å for one protomer and an inverse hydrodynamic shielding length of 0.45 Å^–1.Fachrichtung Biochemie der Pflanzen undFachrichtung Feinstrukturforschung und Elektronenmikroskopie.     

Predicted secondary structure of maltodextrin Phosphorylase fromEscherichia coli as deduced using Chou-Fasman model

Secondary structure of maltodextrin Phosphorylase fromEscherichia coli has been predicted using Chou-Fasman model. The enzyme protein contains 28% α-helix, 27% β-pleated sheets and 20% reverse β-turns. The secondary structure predicted 4 regions showing Rossman-fold super secondary structure. Two regions, one from residue 268–361 and the another from residue 606–684, having 4 consecutive strands of parallel β-pleated sheets and 3 joining α-helix, are predicted. Two regions, one from residue 379–434 and the another from residue 496–573, having 3 consecutive strands of parallel β-pleated sheets and two joining α-helix, are predicted.     

The Crystal Structure of Glutamine-binding Protein fromEscherichia coli

The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.     

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems

A study was made to find out maximum partitioning of Bacillus licheniformis alkaline phosphatase in different ATPSs composed of different molecular weight of PEG X (X = 2000, 4000, 6000) with salts (magnesium sulphate, sodium sulphate, sodium citrate) and polymers (dextran 40, dextran T500). Physicochemical factors such as effect of system pH, system temperature and production media were evaluated for partitioning of alkaline phosphatase. PEG 4000 [9.0% (w/v)] and dextran T500 [9.6% (w/v)] were selected as most suitable system components for alkaline phosphatase production by B. licheniformis based on greater partition coefficient (k = 5.23). The two-phase system produced fewer enzymes than the homogeneous fermentation (control) in early stage of fermentation, but after 72 h the enzyme produced in the control system was less than that in the ATPS. Total alkaline phosphatase yield in ATPS fermentation was 3907.01 U/ml and in homogeneous fermentation 2856.50 U/ml.     

Overproduction and purification of Lon protease fromEscherichia coli using a maltose-binding protein fusion system

Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.     

Liquid-liquid partition chromatography of biopolymers in aqueous two-phase systems.

The theoretical and practical principles of liquid-liquid partition chromatography (LLPC) applying aqueous two-phase polymer systems are presented. The method is based on support materials which bind one of the two aqueous phases with high preference and reject the other. This selectivity is obtained by making use of incompatibilities between polymers grafted on support particles and polymers in solution. Applications of the separation technique to the fractionation of protein and nucleic acid mixtures are shown. For the DNA-fractionation according to base composition an affinity partition chromatography using polyethylene glycol-bound base-specific complexing agents has been developed which exhibits a resolution superior to all other methods known.     

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.     

Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins

Partition coefficients in poly(ethylene glycol)/dextran aqueous two-phase systems are reported for mixed-casein and its components, alpha, beta and kappa casein. Rates of casein proteolysis by alpha-chymotrypsin and by trypsin are reported in single-phase and aqueous two-phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.     

A catalytically inactive form of isocitrate dehydrogenase fromEscherichia coli

A protein exhibiting immunological cross-reactivity with NADP-specific isocitrate dehydrogenase, but containing no catalytic activity, has been isolated from nalidixic acid-resistantEscherichia coli. The two proteins have, within the limits of experimentation, identical molecular weight, subunit structure, and amino acid homology. The absence of catalytic activity in the protein isolated from nalidixic acid-resistant mutants may result from a mutation in the isocitrate dehydrogenase structural gene.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.