Harvey W. Blanch - US grants

This project is concerned with two areas of importance in the development of bioprocesses for the production of high and intermediate-value biochemicals. The first concerns the use of aqueous two-phase polymer systems for the recovery of fermentation products. The research is directed at obtaining the molecular thermodynamic properties of these solutions and developing predictive models for phase behavior and partitioning of solute molecules between phases. The second area concerns the use of non-aqueous solvents for enhancing substrate solubility in enzymatically catalyzed reactions. Supercritical carbon dioxide, alone with selected entrainers, is used as a solvent for studying the kinetics of several commercially important reactions: cholesterol oxidation, interesterification of lipids, and various hydrolytic reactions which can be run "backwards".

This renewal project is concerned with two areas of importance for increasing the impact of biological processes on production of specialty chemicals: use of non-aqueous solvents for enzyme catalyzed reactions and development of novel separations processes based on aqueous two-phase systems. The first aspect will focus on enzyme reactions in supercritical solvents: In the previous grant period, the stability of enzymes, both free and immobilized in dense carbon dioxide, has been demonstrated, and kinetic data have been obtained for the oxidation of cholesterol and the interesterification of triacylglycerides. This research expands the possible range of substrate concentrations which can be used with supercritical carbon dioxide by exploring its use with entrainers; it also provides a more detailed study of the kinetic and mass transfer effects of importance for both enzymes studied. In addition, the conformation of enzymes in this non-aqueous medium is examined by the use of EPR spectroscopy. A nitroxide spin-labelled cholesterol probe is employed to monitor the spatial coordinates at the active site of cholesterol oxidase. The second aspect will focus on aqueous two-phase systems for product recovery: This research is directed toward establishing the fundamental molecular thermodynamics of aqueous two-phase systems. An important objective is to determine what polymer-polymer systems may be best suited for bioprocessing. Toward that end, the PIs continue their measurements using light scattering and differential vapor pressure to obtain thermodynamic parameters for aqueous mixtures containing dextran, polyethyleneglycol, proteins and combinations of polymer-polymer and polymer protein pairs. These data, combined with information on electrical-potential effects due to salt partitioning and a molecular-thermodynamic model based upon the osmotic virial equation, allow the PI's to predict polymer-polymer-water phase diagrams and protein partition coefficients for process design and optimization.

This proposal requests funding for the purchase of a low-angle laser light-scattering (LALLS) photometer and a high performance liquid and size exclusion chromatography (HPLC-SEC) system. This equipment is to be used to determine molecular-thermodynamic parameters and experimental ternary polymer-polymer-water phase diagrams required to develop a predictive model for a promising new separation technique in biotechnology, i.e., two-phase aqueous polymer-polymer systems for selective extraction of biomolecules. The PIs are considered to be very well qualified to effectively utilize the proposed equipment. Funding of this proposal is being recommended and the costs are to be shared by the Interfacial, Transport and Thermodynamic Processes and the Biotechnology Programs of the Engineering Directorate within NSF, and the University of California at Berkeley. Biotechnology Program $ 50,932 Interfacial, Transport and $ 30,000 Thermodynamic Processes Program University of California at $ 40,466 Berkeley

This proposal describes a comprehensive program aimed at developing new methods for the production of speciality biochemicals and advanced biomaterials. These materials will be formed by reversing the normal hydrolytic action of two proteases. These proteases will be modified to alter their selectivity and to enhance their stability under reaction conditions. Trypsin or chymotrypsin specificity will be altered by several techniques; site-directed mutagenesis, screening for mutants which produce enzymes with the ability to act on the substrates of interest, refolding partially denatured enzyme around novel substrates and a new approach which will permit the insertion of synthetic amino acids at the active site of the enzyme. Cathepsin C will be modified by a refolding procedure, and immobilized by a variety of techniques. Methods for enhancing the reversal of the hydrolytic reaction for dipeptide and oligomer synthesis with non-natural amino acids will be developed. These include the use of non-aqueous solvents (both water-miscible and immiscible) to increase substrate solubility and the use of directed methods for enzyme immobilization to enhance enzyme stability under reaction conditions. Changes in enzyme conformation induced by non-aqueous solvents and by immobilization will be probed by several techniques, including EPR.

The wider use of enzymes in organic syntheses, where their high degree of regio- and stereo-selectivity is difficult to achieve with conventional catalysts, is severely hampered by the low aqueous solubility of many substrates of interest. The encapsulation of enzymes in reverse micelles, which provides a method for stabilization of the enzyme, is a new and particularly promising approach for biological synthesis or transformation of organic compounds in organic solvents. This project concerns fundamental studies for the development of new bioreactors employing such encapsulated enzymes. It includes a study of the effect of encapsulation of dopamine beta- monooxygenase, a non-specific enzyme which hydroxylates ring- substituted phenethylamines, alkenes, aldehydes, amides and sulphides. The effect of micelle structure on the conformation of the enzyme is studied by use of ESR and kinetic measurements. A molecular- thermodynamic model, relating the properties of the aqueous miniphase to prevailing conditions, is also developed. The use of reverse micelles to protect the enzyme from the often denaturing influence of organic solvents is a new approach to enzyme stabilization. Current research has focussed on the use of reverse micelles for protein purification. The inner water pool of a reverse micelle can solubilize large protein molecules and the micelle provides a large surface area for transport of reactant and substrate to and from the enzyme. Little is understood of the behavior of enzymes within such micelles or of how their kinetic properties differ from enzymes in solution. The investigators create micelles using non-polar surfactants, so that transport of polar organic substrates can be enhanced by the addition of carrier molecules in the organic phase, such as quaternary alkyl amines. There is current interest in describing the behavior of water in reverse micelles from a phase equilibrium thermodynamic viewpoint. Of even greater importance is the development of a molecular-thermodynamic model to relate the properties of the aqueous miniphase to the external conditions, so that a predictive model can be employed for bioreactor design.

A novel fiber optic probe will be developed and then used to investigate: (1) the movement of solutes through a support matrix and (2) the intrinsic kinetics of solute binding by immobilized ligands. The probe is an optical fiber employing an evanescent wave to excite molecules at the fiber surface; the electromagnetic field which is the wave penetrates only 2000 angstroms into the surrounding medium, and can be used to study the molecules at the surface of the optical fiber. In the second part of the project, the waveguide will be coated with a very thin film of a support matrix and the rate of movement of solutes of interest through an affinity column will be determined. This will allow separation of mass transfer and equilibrium effects by direct measurement. Thus, the influence of the nature of the support matrix and the method of ligand attachment can be unambiguously determined. This knowledge will be of considerable importance in affinity, chromatography, a separation technique with applications in biotechnology. Affinity-based separations are an important technique for the recovery of biological products. Though extensively studied, most investigations have been at the macroscopic level; for example, some studies have used frontal analysis or pulse techniques from chromatography theory. This project will attempt a study of factors affecting binding constants and mass transfer rates from a microscopic perspective.

This grant is for purchase of a supercritical fluid chromatograph (SFC). It will provide versatile analytical capabilities for an ongoing NSF supported projects on novel bioreactor configurations and on development of novel enzyme- catalyzed reactions to produce new materials which cannot be manufactured by conventional chemical methods. SFC offers significant advantages over other methods for chemical analysis such as gas-liquid chromatography and high- performance liquid chromatography. SFC will enable the investigators to obtain essential quantitative data efficiently and rapidly and will also enable them to make quantitative measurements at very low concentrations that cannot be made with conventional equipment.

The proposed research is concerned with separating a mixture of proteins in aqueous solution through selective precipitation, i. e., by formation of a phase rich in the target protein in equilibrium with a second phase where the concentration of the target protein is very low. Precipitation is achieved by salts or nonionic polymers or both. To obtain a quantitative understanding of protein- precipitation equilibria, a molecular-thermodynamic model based on an extended Guggenheim/McMillan-Mayer osmotic viral expansion is presented. The goal of this research is to establish an engineering-oriented correlation for rational design of protein-precipitation processes. A long-range benefit of this research is that it is likely to provide useful knowledge for better understanding of protein crystallization.

This collaborative project between workers at Berkeley and UCSF is designed to produce new polymeric peptides with useful properties. The investigators are all excellent scientists, although all well supported by other grants. In particular Dr. Craik has another NSF grant which duplicates his effort in this proposal. The P.I. has developed enzymatic methods for reacting amino acid esters in organic solvents with free amino acids to generate dipeptides. The approach has been extended to a tetra peptide, but the strategy seems unlikely to produce useful polymers, although it might be practical for short peptides. It should be noted that carboxypeptidase cannot be employed in such syntheses because it does not have and acyl enzyme intermediate. There are some interesting ideas in this proposal, but the practicality of synthesizing polypeptides by these approaches seems minimal.

Experimental and theoretical bio-processing studies are directed toward the development of a molecular-thermodyamic model for separationg a target protein from a mixture of proteins in aqueuous solution. Protein separation is achieved by precipitation induced by salts, nonionic polymers, or both. The resulting system consists of two liquid phases in equilbrium, with one enriched in the target protein. A molecular-thermosynamic model is outlined for describing protein-precipitation phase equilibria. This model is derived from an understanding of the intermolecular forces in the precipitating solutions; it uses the mean-spherical- approximation to define the reference system as a mixture of charged hard spheres. Pertubations to this reference system result from the dispersion forces, dipole-dipole forces and perhaps, specific association forces. Osmotic pressures calculated from the model will be compared to osmotic-pressure data obtained from membrane osmometry and to osmotic second virial coefficients measured by low-angle laser-light scattering. Experimental phase-equilibrium (precipitation) measurements will provide data against which the molecular- thermodynamic model can be compared. Protein aggregate size distribution data from light scattering will be used to augment the theory when significant aggregation occurs. The goal of this reech is to establish an engineering-oriented correlation for the rational design of protein-precipitation processes. The long-range benefit of this research is that it is likely to provide useful knowledge for a better understanding of protein crystallization bioprocesses.

The University of California Berkeley will receive ARI Facilities support to create modern facilities for researchers working in a unique, integrated Program in Biological Chemistry and Engineering within the College of Chemistry. The renovated facilities will promote a strong interaction among bioinorganic chemists, biochemical engineers, and bioorganic chemists working in the areas of biotechnology and environmental research. The renovations activity funded by this award will be directed at the improvement of 1,305 square meters in 30-year old Latimer Hall and will consolidate researchers. Program activity will be enhanced as researchers are currently located in geographically distributed campus laboratories that are between 32 and 77 years old. A 7 month- long space assessment precedes the project which will involve gutting much of the interior space, installation of modular laboratory units and new fume hoods and upgrades to the mechanical, electrical and plumbing services for increased safety and improved efficiency. Provision of adequate facilities will enable the basic discovery processes to be linked more closely with the development process. Biochemical engineers will study separation techniques for the recovery of biological products, biomimetic adsorbents for metal removal and recovery and new approaches to bioremediation. Bioinorganic chemists will study metal ion transport that is essential to life processes, the use of chelating agents in the sequestration of heavy metals, and lanthanide complexes for possible use in enhancing MRI. A total of 8 professors and 117 graduate students and postdoctoral fellows will benefit from these improvements.

Increasing the rate of DNA restriction mapping and sequencing is one of the major obstacles to be overcome in efforts to map the human genome. Capillary electrophoresis provides a means of enhancing the speed of a separation compared to conventional slab gel electrophoresis, but is presently restricted to small DNA fragments. We have developed a capillary electrophoresis technique using uncrosslinked hydroxyethyl cellulose which has shown the ability to separate DNA fragments up to l,400 bp in length with a resolution of 10 bp, and larger DNA fragments up to 23,000 bp with more limited resolution. Based on our present theoretical model of the mechanism of separation, this upper base-pair limit can be considerably extended, and resolution much improved. The first set of objectives of this proposal is thus: (l) To determine the mechanism of DNA separations in dilute, uncrosslinked polymer solutions. (2) To investigate the upper limit of DNA size which can be separated by capillary electrophoresis in uncrosslinked polymer solutions, and to extend the usefulness of CE to separations of DNA > 50 kbp. (3) To correlate sieving polymer properties, such as molecular weight, stiffness, and hydrophilicity, with the efficiency of DNA restriction mapping separations. (4) To develop a model of DNA electrophoretic mobility in uncrosslinked polymer solutions, which allows prediction of the appropriate polymer molecular weight and concentration required to separate a given mixture of DNA restriction fragments, as a function of temperature and electric field strength. We also propose to develop a new sieving matrix coupled with a new high- frequency alternating crossed-field-gradient gel electrophoresis technique (HFAC-GE). The novel matrix is a polyacrylamide-based, interpenetrating polyampholyte gel with the gel fibers individually charged positively or negatively. The gel fibers will move in the alternating crossed-field- gradient thus altering the pore structure of the gel as the DNA migrates. This technique is designed to improve the resolution of DNA sequencing and to extend the range of restriction fragment mapping beyond that available with steady field methods.

9530793 Blanch Recovery and purification of proteins is of vital importance in biotechnology. Design and optimization of separation and purification processes requires an understanding of the behavior of proteins in complex aqueous solutions, where salts, polymers or other solutes may be dilute or concentrated. The objective of this proposal is to develop a separation processes. We are concerned in particular with protein precipitation by salts and with protein crystallization; however, protein solution behavior is also important in stability and formulation of therapeutic proteins, and in understanding intermolecular reactions. This proposal is for continued support of a two-year NSF project OThermodynamics of Protein Precipitation in Aqueous SolutionsO. The approach is to describe protein solution behavior on a molecular level, in terms of a two-body potential of mean force (PMF), which describes the overall interactions between two protein molecules. The PMF is determined by the physicochemical properties of the protein, the nature of the solution and temperature. From the PMF, thermodynamic solution properties may be obtained for predicting protein-precipitation phase-equilibrium properties. It is planned to employ liquid-state integral-equation theory to develop new expressions for the compressibility and Helmholtz energy of protein solutions; from these expressions phase equilibria are obtained. This modeling approach is coupled with an experimental program to determine the phase equilibria of a number of model proteins with particular attention given to selective precipitation of a target protein from a mixture. In addition, specific protein interactions will be experimentally quantified. Low-angle laser-light scattering and membrane osmometry will provide information on intermolecular forces and aggregation. Specific protein-protein interactions will be examined by dynamic light scattering. Electrostatic and hydrophobic contributions to the PMF will be probed by sy stematically changing amino-acid residues on the model proteins. Specific ion-protein interactions will be quantified using differential refractometry Cl NMR and protein titrations. *** A further component of the proposed research is to examine solution conditions which favor protein crystallization. The hypothesis that a crystallizing solvent results in weak attractive interactions between proteins, while strongly attractive conditions favor amorphous precipitation, will be examined by determining the osmotic second virial coefficients for a number of proteins in crystallizing solvents. It is planned to investigate critically an idea supported by preliminary data, viz. that crystallization is most likely when the osmotic second virial coefficient of a protein is slightly, but not strongly, negative.

Purpose and Program Characteristics The purpose of this training program is to provide a curriculum and research environment that will prepare trainees in chemical engineering, chemistry and cell and molecular biology at the predoctoral level for careers in biotechnology. Specifically, this program focuses on research training in areas relevant to the needs of biotechnology, pharmaceutical and chemical companies currently involved in the manufacture of products using biological routes and includes areas such as molecular genetics, fermentation, mammalian cell culture, enzyme technology, bioproduct recovery, combinatorial biocatalysis, combinatorial chemistry. The graduate program includes laboratory and classroom instruction in cell culture techniques (mammalian and bacteria cell culture), protein and enzyme isolation, purification and immobilization, mutagenesis and gene expression, protein and nucleic acid chemistry and biochemistry, and separation and purification methods. This will be supplemented with seminar courses on current topics in industrial and scientific areas, including fermentations involving recombinant organisms, bioproduct recovery (including macromolecular separations), site-directed mutagenesis techniques, metabolic regulation, enzyme inhibitor design and the application of enzymes and antibodies as catalysts in organic syntheses. Lectures from internationally known guests in a variety of related disciplines will further enrich the training program. Research will be conducted in the various area described above under the supervision of the faculty mentors. Trainees Predoctoral students will enter the program with varying backgrounds, all holding Baccalaureate degrees in engineering or science (some with Masters degrees). They will be selected on the basis of undergraduate scholastic performance, scores on GRE tests and recommendations from faculty at their undergraduate institutions. Support for 12 trainees is requested. The average duration of the doctoral program in the participating departments is 5.5 years.

The objective of this project is to use a combination of experiment and theory/simulations to study protein aggregation as a competitive process to folding at high protein concentrations. One strategy will be to study, by experiment, the influence of specific interactions between amino acid residues and secondary structure on the stability of aggregates vs. the native three-dimensional structure. A secondary strategy will be to determine, from computer simulations and theory, the general characteristics of minimal models of heterogeneous polymers that can either aggregate or fold.

The objective of this project is to investigate the problem of protein folding/unfolding, and in particular, the mediation of this process by the chaperone DnaK. This project involves both modeling and experimental studies. First, folding-aggregation competition will be studied. Measurements of interprotein interactions at varied external conditions, e.g. temperature, pH, and ionic strength, will help identify separate contributions to the overall interprotein potentials and their role in molecular association. The Principal Investigators (PIs) have developed a three dimensional periodic Monte Carlo simulator for capturing the competition between refolding and aggregation of initially unfolded protein-like chains. In this system various interactions on short length scales will be considered. Also, the PIs have predicted that the presence of pre-folded proteins improves the folding rate of unfolded proteins because of enhanced surface interactions. This provides a basis for modeling the chaperone-unfolded protein interaction so crucial in vivo. Other computational tasks include an understanding of the kinetics of the refolding process, since both folded and unfolded forms are thermodynamically feasible so that the kinetics may define the overall extent of folding.

Blanch
0337080
Sea sponges are a prolific source of compounds with high potential therapeutic value. Over the past decade, compounds have been isolated from sponge extracts with potent anti-mitotic, anti viral and antibacterial properties. However, due to environmental constraints and accessibility, insufficient sponge biomass is available from which to extract these compounds for testing and clinical trials. To date, there are no immortalized sponge cell lines and techniques for cell cultivation are still in their infancy. This project is to carry out exploratory studies on the adaptation of modern tissue engineering techniques, particularly the recent developments in three-dimensional scaffold materials, to cultivate cells of the sea sponge Axinella corrugata in vitro.

[unreadable] DESCRIPTION (provided by applicant): Because cellular enzymes, transcription factors, and their respective genes interact in a complex and interdependent manner, cellular physiology is not dictated by the genome alone. Targeting the genetic aberrations of transformed cells may therefore not be as effective as directly targeting the altered physiology of tumors. Metabolic profiling, by determining the substrate fluxes in major metabolic pathways, can reveal phenotypic transformations in cancer cells, and provide more detailed information than signal transduction and genetic studies alone. Transforming agents typically induce high pentose pathway activity. Rapidly proliferating breast cancer cells have high rates of aerobic glycolysis and glutaminolysis, and enhanced fatty acid synthesis activity. These altered metabolic fluxes suggest that enzymes and pathways identified as critical for cancer cell proliferation may be excellent targets for drug development. In the proposed project, the concentrations and fluxes of intracellular metabolites of human breast cancer cells will be determined from 13C NMR spectroscopy of cells grown on 13C glucose and glutamine. Biochemical systems analysis, by providing a quantitative description of metabolite flows, will be employed to reveal the link between growth signaling and metabolic processes. Metabolic profiling of MCF7 breast cancer cells will be accomplished by determination of intracellular fluxes using 13C NMR, under conditions mimicking those found in tumors. The hypothesis is that the crucial enzymes responsible for the altered metabolic fluxes in transformed human cells provide targets for new anti-cancer drugs. Specific aims are to: (1) Identify the primary metabolic pathways that are operative in estrogen receptor positive (ER+) and negative cells (ER-) breast cancer cells under conditions that mimic those of a tumor. (2) To quantify the primary and secondary metabolic fluxes and enzymes control coefficients in breast cancer cells in the presence and absence of tamoxifen (TAM) and estrogen, including those fluxes which may be reversed by increased concentrations of estrogen ("estrogen rescue"), and those effects of TAM that are not related to ER binding. (3) To examine modes of estrogen and tamoxifen action by investigating the relationship between fatty acid synthesis, pentose phosphate pathway activity, and other pathways requiring NADP+/NADPH, and to determine the effects of impaired fatty acid synthesis (resulting from the addition of the fatty acid synthase inhibitor, cerulenin) on other metabolic pathways in the presence of estrogen and tamoxifen. [unreadable] [unreadable]

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Blanch

The goal of this proposed research is investigate the roles of primary sequence and conformational characteristics in determining protein aggregation behavior. The specific objectives are directed toward experimental and theoretical investigations of the underlying mechanisms. The ultimate goal is to control aggregation. To assist in uncovering forces and mechanisms essential to manipulate protein aggregation properties, the proposed research focuses on the effects of sequence patterns and solution parameters that impact the competition between aggregation and refolding. The experimental work will be performed on multiple systems of interest, including short oligopeptides as well as specific regions of three different proteins, all of which are suitable candidates for studies of aggregation.