Control of Enzyme–Solid Interactions via Chemical Modification

Electrostatic forces could contribute significantly toward enzyme–solid interactions, and controlling these charge–charge interactions while maintaining high affinity, benign adsorption of enzymes on solids is a challenge. Here, we demonstrate that chemical modification of the surface carboxyl groups of enzymes can be used to adjust the net charge of the enzyme and control binding affinities to solid surfaces. Negatively charged nanosolid, α-Zr­(HPO₄)₂·H₂O (abbreviated as α-ZrP) and two negatively charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have been chosen as model systems. A limited number of the aspartate and glutamate side chains of these proteins are covalently modified with tetraethylenepentamine (TEPA) to convert these negatively charged proteins into the corresponding positively charged ones (cationized). Cationized proteins retained their structure and activities to a significant extent, and the influence of cationization on binding affinities has been tested. Cationized GO, for example, showed 250-fold increase in affinity for the negatively charged α-ZrP, when compared to that of the unmodified GO, and cationized Hb, similarly, indicated 26-fold increase in affinity. Circular dichroism spectra showed that α-ZrP-bound cationized GO retained native-like structure to a significant extent, and activity studies showed that cationized GO/α-ZrP complex is ∼2.5-fold more active than GO/α-ZrP. Cationized Hb/α-ZrP retained ∼75% of activity of Hb/α-ZrP. Therefore, enzyme cationization enhanced affinities by 1–2 orders of magnitude, while retaining considerable activity for the bound biocatalyst. This benign, chemical control over enzyme charge provided a powerful new strategy to rationally modulate enzyme–solid interactions while retaining their biocatalytic properties

Toward “Stable-on-the-Table” Enzymes: Improving Key Properties of Catalase by Covalent Conjugation with Poly(acrylic acid)

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, <i>M</i>_w 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(<i>x</i>)-<i>y</i>, where <i>x</i> is the protein to polymer mole ratio and <i>y</i> 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 <i>K</i>_m or <i>V</i>_max 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