New Insoluble supports for Protein Immobilization for Use in Metalloprotein Affinity Metal Chromatography

EPA Grant Number: R822721C661
Subproject: this is subproject number 661 , established and managed by the Center Director under grant R828598
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Gulf Coast HSRC (Lamar)
Center Director: Ho, Tho C.
Title: New Insoluble supports for Protein Immobilization for Use in Metalloprotein Affinity Metal Chromatography
Investigators: Vincent, John , Woski, Stephen
Institution: University of Alabama
EPA Project Officer: Lasat, Mitch
Project Period: September 1, 1998 through August 31, 1999
RFA: Gulf Coast Hazardous Substance Research Center (Lamar University) (1996) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

Recently these laboratories have demonstrated that it is possible to use metalloproteins immobilized on insoluble supports as efficient, selective, and recyclable materials for heavy metal and radionuclide removal and recovery. This novel patented process has been termed metalloprotein affinity metal chromatography (MAMC). Using the protein transferrin (which is inexpensive and remarkably stable when immobilized), transition metal and actinide ions (initially present at ppm levels or lower) can be removed from aqueous solution to ppb levels or less with very high to quantitative recovery. The use of isolated metalloproteins results in a high degree of selectivity (especially when compared against supported biomass) and benign conditions for metal recovery (the product is not contaminated biomass or strongly acidic, dilute metal ion solutions). However, it is not yet economically feasible to apply this process to real-world applications (or even pilot scale testing). Such scaling up will require the development of a new type of solid support which is inexpensive can withstand high flow rates, has a low potential to bind metal ions in the absence of the linked protein, and is not biodegradable. The solution of this problem will require the application of techniques from organic chemistry, biochemistry, and separation sciences.

Recent studies which examined the potential of a variety of insoluble supports for use in transferrin MAMC indicate that polystyrene copolymers are excellent candidates. While polystyrene itself is quite inexpensive, the chemistry required to attach proteins to the surface is not well developed. MAMC applications require linkers which 1) can be prepared inexpensively in bulk and with a high density of protein on the polymer surface, 2) are capable of maintaining the protein at a distance from the hydrophobic surface of the polymer to prevent denaturation of the protein, and 3) lack moieties capable of non-specifically coordinating metal ions. The use of rigid linear linker arms is an attractive approach for meeting these requirements. Oligophenyl chains and mixed phenyl/acetylene chains can be utilized in design of such linkers. The extended pi-systems of these linkers enforce a fixed geometry (180° in the cases of para-substituted phenyl and acetylene units) with high barriers to bending in order to maintain the proteins at a fixed distance from the polystyrene bead. In addition, linkers of this type should show little if any metal ion binding with hard Lewis acids such as actinide ions, especially in competition with the metal-binding sites of proteins. Finally, the combination of a polystyrene resin with these linkers should provide a robust and biologically inert support for the immobilization of proteins. The starting materials for the syntheses of these novel supports will be unmodified polystyrene/divinylbenzene copolymer.

To identify alternative procedures for activating hydrophobic polymers such that 1) proteins (most notably transferrin) can be covalently attached in high yield without appreciably affecting the metal binding properties of the biomolecule. 2) The polymer does not possess metal-binding capacity, and 3) The expenses of producing the activated support is minimized. The synthesis, evaluation and optimization of these materials should lead to the development of new Metalloprotein Affinity Metal Chromatography (NAMC) media that can be used in practical scale remediation applications for the recovery and/or removal of metals from contaminated water.

Approach:

The phenyl groups of the polystyrene can be initially derivatized by several different methods, and the linkers can then be synthesized using a sequence of metal catalyzed cross coupling reactions and further derivatization. This approach takes advantage of the nature of the polymer to effect a solid phase synthesis of the linker. Repetition of the reaction sequence allows for the formation of linkers of any desired length. Chemistry will also be developed to attach the protein molecules to the support. The protein conjugation chemistry and other factors such as linker length will be optimized to produce supported proteins with high loading and good long-term stability.

Expected Results:

These efforts should lead to the development of viable methods for removal of hazardous metals from water for waste minimization and remediation.

Publications and Presentations:

Publications have been submitted on this subproject: View all 1 publications for this subprojectView all 359 publications for this center

Supplemental Keywords:

Scientific Discipline, INTERNATIONAL COOPERATION, Waste, Water, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Chemical Engineering, Physics, Remediation, Wastewater, Environmental Chemistry, Chemicals, Analytical Chemistry, Chemistry and Materials Science, Engineering, Chemistry, & Physics, Water Pollution Control, hazardous waste treatment, wastewater treatment, hazardous liquid waste, heavy metal recovery, wastewater remediation, radionuclides, metalloprotein affinity metal chromatography, metalloproteins, contaminated waters, protein immobilization, hazardous waste, metal recovery, alternative technology, hazadous waste streams, metal extraction, aqueous waste, metalloprotein, heavy metal contamination, water treatment, extraction of metals, metals removal, green chemistry, heavy metals, aqueous waste stream, aqueous waste streams

Progress and Final Reports:

  • Final

  • Main Center Abstract and Reports:

    R828598    Gulf Coast HSRC (Lamar)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
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