title: Toward "stable-on-the-table" enzymes: improving key properties of catalase by covalent conjugation with poly(acrylic acid).
abstract: Several key properties of catalase such as thermal stability, resistance to protease degradation, and resistance to ascorbate inhibition were improved, while retaining its structure and activity, by conjugation to poly(acrylic acid) (PAA, Mw 8000) via carbodiimide chemistry where the amine groups on the protein are appended to the carboxyl groups of the polymer. Catalase conjugation was examined at three different pH values (pH 5.0, 6.0, and 7.0) and at three distinct mole ratios (1:100, 1:500, and 1:1000) of catalase to PAA at each reaction pH. The corresponding products are labeled as Cat-PAA(x)-y, where x is the protein to polymer mole ratio and y is the pH used for the synthesis. The coupling reaction consumed about 60-70% of the primary amines on the catalase; all samples were completely water-soluble and formed nanogels, as evidenced by gel electrophoresis and electron microscopy. The UV circular dichroism (CD) spectra indicated substantial retention of protein secondary structure for all samples, which increased to 100% with increasing pH of the synthesis and polymer mole fraction. Soret CD bands of all samples indicated loss of ∼50% of band intensities, independent of the reaction pH. Catalytic activities of the conjugates increased with increasing synthesis pH, where 55-80% and 90-100% activity was retained for all samples synthesized at pH 5.0 and pH 7.0, respectively, and the Km or Vmax values of Cat-PAA(100)-7 did not differ significantly from those of the free enzyme. All conjugates synthesized at pH 7.0 were thermally stable even when heated to ∼85-90 °C, while native catalase denatured between 55 and 65 °C. All conjugates retained 40-90% of their original activities even after storing for 10 weeks at 8 °C, while unmodified catalase lost all of its activity within 2 weeks, under similar storage conditions. Interestingly, PAA surrounding catalase limited access to the enzyme from large molecules like proteases and significantly increased resistance to trypsin digestion compared to unmodified catalase. Similarly, negatively charged PAA surrounding the catalase in these conjugates protected the enzyme against inhibition by negatively charged inhibitors such as ascorbate. While Cat-PAA(100)-7 did not show any inhibition by ascorbate in the presence of 270 μM ascorbate, unmodified catalase lost ∼70% of its activity under similar conditions. This simple, facile, and rational methodology produced thermostable, storable catalase that is also protected from protease digestion and ascorbate inhibition and most likely prevented the dissociation of the multimer. Using synthetic polymers to protect and improve enzyme properties could be an attractive approach for making "Stable-on-the-Table" enzymes, as a viable alternative to protein engineering.
authors.affiliation: Department of Chemistry, ‡Department of Molecular and Cell Biology, and §Institute of Materials Science, University of Connecticut , Storrs, Connecticut 06269-3060, United States.
authors.forename: Caterina M
authors.initials: CM
authors.lastname: Riccardi
authors.forename: Kyle S
authors.initials: KS
authors.lastname: Cole
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Benson
authors.forename: Jessamyn R
authors.initials: JR
authors.lastname: Ward
authors.forename: Kayla M
authors.initials: KM
authors.lastname: Bassett
authors.forename: Yiren
authors.initials: Y
authors.lastname: Zhang
authors.forename: Omkar V
authors.initials: OV
authors.lastname: Zore
authors.forename: Bobbi
authors.initials: B
authors.lastname: Stromer
authors.forename: Rajeswari M
authors.initials: RM
authors.lastname: Kasi
authors.forename: Challa V
authors.initials: CV
authors.lastname: Kumar
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D002374:Catalase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature
chemical_list: D000180:Acrylic Resins; D004791:Enzyme Inhibitors; C006903:carbopol 940; D002374:Catalase