Patrick L. Coleman

Immobilization of Protein A at high density on azlactone-functional polymeric beads and their use in affinity chromatography

Abstract This paper presents the results of the use of highly cross-linked, porous, hydrophilic copolymer beads with protein immobilized on their surface for affinity chromatography. Copolymer beads composed of vinyldimethyl azlactone (oxazolone) and methylene-bis-acrylamide in various ratios, with up to 3/5 mequiv./g azlactone functionality, will undergo nucleophilic attack by amines, as well as by thiols and alcohols. The ring-opening reaction of a nucleophile-containing ligand (e.g., a protein) resulted in covalent attachment to the support. The reaction was rapid, half-complete in about 5 min, yielding proteins immobilized at very high densities, recombinant Protein A at 397 mg/g, and human immunoglobulin G at 225 mg/g. The reaction proceeded at significant levels from pH 4 to 9. There was a marked enhancement in the amount of protein coupled, its rate of reaction, and its biological activity when Protein A was made to react in the presence of high concentrations of sodium sulfate. Evaluatioin of affinity columns, prepared with Protein A immobilized at over 200 mg/g, gave molar ratios f immunoglobulin G to immobilized Protein A of 1:1 or greater. Up to 56 mg of immunoglobulin G was recovered per ml of column bed volume. The support combined high flow-rates with low back-pressures and nobed-volume changes upon changing mobile phases, including highly ionic aqueous solvents and ethanol.

Crosslinked, hydrophilic, azlactone-functional polymeric beads : a two-step approach

Abstract Crosslinked, hydrophilic, azlactone-functional polymeric beads are readily prepared via a two-step approach involving: (1) reverse phase suspension copolymerization of N-acryloyl-amino acids with water-soluble crosslinkers and, optionally, dimethylacrylamide, followed by (2) cyclodehydration of pendant acylamino acid groups to azlactones using acetic anhydride. Azlactone functionalities of 0.3 to 3.0 meq/g (typically greater than 70% of the theoretical value) were achieved by this procedure. The azlactone-functional group in these beads was found to be quite reactive towards amine nucleophiles, even in aqueous solution where little competition from hydrolysis was observed. Rapid, covalent coupling of protein could be accomplished from aqueous media under mild conditions, and indicated a potential for extremely high coupling densities (up to 245 mg protein/g of beads).

Immobilization of manganese peroxidase fromLentinula edodes on azlactone-functional polymers and generation of Mn3+ by the enzyme-polymer complex

Manganese peroxidase (MnP) purified fromLentinula edodes was covalently immobilized on 3M’s azlactone-functional copolymer, 3M EmphazeTM AB1 Biosupport Medium. Tethered MnP is capable of generating Mn3+ from Mn2+ and H2O2. Mn3+, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn3+ -generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn2+ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations < 2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn2+. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40–50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies > 65% with 100% of the coupled enzyme active have been measured.

Immobilization of manganese peroxidase fromLentinula edodes on alkylaminated emphazeTM AB 1 polymer for generation of Mn3+ as an oxidizing agent

Manganese peroxidase (MnP) is secreted by white-rot fungi and participates in the degradation of lignin by these organisms. MnP uses H2O2 as an oxidant to oxidize MnII to MnIII as the manganic ion Mn3+. The Mn3+ stabilized by chelation, is a highly reactive nonspecific oxidant capable of oxidizing a variety of toxic organic compounds. Previous attempts at immobilization of MnP, purified fromLentinula edodes through reactive amino groups, have been hindered by the protein’s low lysine content of only 1% and its instability above pH 6.0. As an alternative to amine coupling, the enzyme has now been covalently immobilized through its carboxyl groups, using an azlactonefunctional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The immobilization reaction was performed under acidic (pH 5.25) conditions, and 90% coupling efficiency was achieved within 2 h. Net immobilization efficiencies, expressed as the product of protein coupling efficiency and enzyme activity, have been measured at > 95% within 4 h. The MnP-NH-polymer and the free soluble protein were characterized and compared for their pH, temperature, and storage stabilities, as well as their H2O2 dependence and kinetics. The tethered MnP, employed in an immobilized enzyme bioreactor for generation of chelated Mn3+ may have industrial applications as a nonspecific oxidant of organopollutants.