Technical aspects of extractive enzyme purification.

We see the advantages of extraction processes for large-scale enzyme isolation and purification in the high capacity of the method, savings in process time and energy, high activity yields and the possibilities for continuous processing at all stages. Commercially available equipment can be used for separation of aqueous two-phase systems with minor modifications. It is hoped that, through using such technology, intracellular enzymes will become available in large quantities and at lower cost.

Extractive purification of enzymes from animal tissue using aqueous two phase systems: pilot scale studies.

Pilot scale trials have been carried out to assess the feasibility of using PEG-salt aqueous phase systems for extraction and purification of enzymes from animal tissue on an industrial scale. Comminuted bovine liver was used as a starting material, and it was easy to separate a clear upper phase containing proteins of interest from a mixture containing 20% biomass, 15% PEG and 8% phosphate using a disc separator. Similar attempts with decanters were unsuccessful. Second-phase separation was simply accomplished by the addition of salt to the separated, clear upper layer and standing or allowing passage through a disc separator. The method was tested using continuous mixing on the GBF continuous mixing aqueous phase extraction plant, with and without computer control. Good separations were achieved. The enzyme superoxide dismutase was purified using this method yielding a 4-fold purification factor with respect to soluble protein and a recovery rate of 83%, with the enzyme in a clarified solution suitable for further processing by chromatographic methods. The general applicability of this method, its economics and its potential application in industry are discussed.

Indications for computer-assisted treatment of cranio-maxillofacial tumors.

OBJECTIVE: Ablative tumor surgery requires detailed planning using computed tomography (CT) or magnetic resonance imaging (MRI). Reconstruction following tumor resection is dependent on reliable information for choosing the correct type and volume of grafts and predicting the outcome. This study evaluates the benefit of and the indications for computer-assisted surgery in the treatment of cranio-maxillofacial tumors. MATERIALS AND METHODS: Based on a CT or MRI data set, the STN Navigation System (Stryker-Leibinger) was used for preoperative planning, intraoperative navigation, and postoperative control of radical tumor resection and primary and secondary reconstruction. Tumor resection was preoperatively planned and intraoperatively navigated. Preoperatively, the required soft and hard tissue were measured using the mirrored data set of the unaffected side of the facial skeleton; the size and location of the graft were chosen virtually. Intraoperatively, contours of transplanted tissues were navigated in accordance with the preoperatively simulated reconstructive result. RESULTS: Computer-assisted treatment was successfully completed in all cases of radical tumor resection, and safety margins outlined preoperatively could be precisely controlled during tumor resection. Reconstruction was designed and performed exactly as virtually planned. CONCLUSIONS: Image-guided treatment improves preoperative planning by visualization of the individual anatomy and the intended reconstructive outcome, and by objectivation of the effect of adjuvant chemo-/radiotherapy. Intraoperative navigation makes radical tumor surgery more reliable by showing the determined safety margins, preserving vital structures, and guiding reconstruction to preplanned objectives.

Continuous aqueous phase extraction of proteins: automated on-line monitoring of fumarase activity and protein concentration.

Automated assay techniques are described for on-line measurements of fumarase activity and total protein concentration, including in-line sample dilution and sample multiplexing during continuous aqueous phase extraction. Fumarase was determined by following the conversion of L-malate to fumurate at a wavelength of 250 nm, while the protein assay was based on the Biuret reaction. Actual assay times of 2 and 4 min were achieved for the fumarase and protein measurements, respectively, with an effective measurement cycle time of 2 min. Standard deviations of c. 3.2 and 2% of the measured values were calculated for the enzyme and protein values, respectively. The assay system was coupled to a computer to allow on-line data visualization and storage.

Studies of the interaction between aminoacyl-tRNA synthetase and transfer ribonucleic acid by equilibrium partition.

The partition behavior of isoleucyl-tRNA synthetase, leucyl-tRNA synthetase and tRNA in aqueous two-phase systems composed of the polymers poly(ethyleneglycol) and dextran was investigated. From the results of this investigation a two-phase system could be derived which can be employed for the study of the interactions between synthetases and their cognate tRNAs by equilibrium partition. These measurements show that in each case one molecule of cognate tRNA is bound per molecule of enzyme. The binding constants were in the range 1-5micronM-1. It could be demonstrated that equilibrium partition is a useful method for the study of interactions between macromolecules.

Application of continuous zone electrophoresis to preparative separation of proteins.

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s.

Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.

Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.

Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale.

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T-500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran-rich phase and a significant shift in the macroscopic structure of these phases, observed as the "gel-forming" properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T-500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T-500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two-phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.

Isolation and binding properties of leucyl-tRNA synthetase from Escherichia coli MRE 600.

A procedure for the large-scale isolation of leucyl-tRNA synthetase from E. cole MRE 600 is described: The enzyme was purified about 320-fold to homogeneity by precipitation with cetyl-trimethyl-ammonium bromide, two consecutive chromatographies on DEAE-cellulose and three on hydroxyapatite with an over-all yield of 4%. The molecular weight of leucyl-tRNA synthetase from E. coli MRE 600 was found to be 99 000 daltons. Bindings studies by ultracentrifugation and equilibrium partition showed that the enzyme binds leucine, leucyl-adenylate and tRNA Leu, each in a 1 : 1 stoichiometry. For ATP only a very weak binding to the enzyme could be observed, which did not allow the evaluation of the complex stoichiometry. The presence of ATP was not required for the binding of leucine or tRNA to leucyl-tRNA synthetase from E. coli MRE 600.

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve.

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.

Separation of enzymes from microorganism crude extracts by free-flow zone electrophoresis.

Continuous, single-step, state-of-the-art preparative separations of enzymes from microorganism crude extracts by free-flow zone electrophoresis are presented. In the first example, the enzymes formate dehydrogenase, formaldehyde dehydrogenase, and methanol oxidase were continuously separated from Candida boidinii crude extract. Yields of 85% to 95% and purification factors between 3 and 7 were obtained along with a simultaneous separation of the finer cell debris from the enzymes. Using multiple injections of sample, a throughput of 46.2 mg protein/h was recorded. In the second example, a fivefold purification of beta-galactosidase from Escherichia coli was achieved along with complete, simultaneous cell debris separation from the enzyme. The yield of the enzyme was greater than 90%. The preparative free-flow zone electrophoresis experiments were run continuously for a period of 12 h and the separations were found to be stable; i.e., the enzymes and the cell debris eluted at their respective fraction numbers during the entire period. In both examples, choice of the type of buffer played a critical role and had to be investigated and optimized experimentally. Scale-up aspects of the separations are also discussed. Recently, by comparison of free-flow zone electrophoresis with ion-exchange chromatography, we have presented evidence that free-flow electrophoresis separations are governed by net surface charge (S. Nath et al., Biotechnol. Bioeng. 1993, 42: 829-835). Here, we offer further confirmation of this evidence by comparison of preparative free-flow zone electrophoresis experiments at various pHs on a mixture of two model proteins with analytical electrophoretic titration curves of the proteins. We are thus in a position to predict separations in free-flow zone electrophoresis. (c) 1996 John Wiley & Sons, Inc.

Procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae involving liquid-liquid separations.

A procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. The pullulanase is solubilized from the cell wall by cholate treatment; cells and cell debris are removed by partition in a poly(ethylene glycol) (PEG)-dextran two-phase system and from the upper (PEG) phase of this system the pullulanase is isolated by ultrafiltration and precipitation with N-cetyl,N-,N-,N-trimethyl ammonium bromide to a purity of about 80% with a yield of 70%. The preparations are free of alpha-amylase activity. The cell containing dextran-rich phase is passed through a Manton-Gaulin homogenizer. Then the phosphorylase is separated from the cell debris by partition in a second PEG-dextran system. From the top phase of this system the phosphorylase is isolated by distribution in a PEG-salt two-phase system followed by batch adsorption on carboxymethyl-Sephadex in a yield of 55%, a purity of around 90%, and nearly free of glycosyltransferase activity. All steps in the isolation of the two enzymes can be performed easily in a large scale.

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).