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.

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

“…This strategy is developed based on the polymer’s ability to conjugate one or two enzymes and protect the enzymes from stressors including temperature, aggregation and proteases as shown in previous publication. 8,30,32,44 The novelty of this work arises due to the use of PAA scaffold, to conjugate multiple enzymes of varying isoelectric points, pH profiles, physical properties and catalytic properties. We also wish to investigate the temperature stability of MECs not only at room temperature but also at 65 °C compared to unmodified native enzymes.…”

A Modular Approach for Interlocking Enzymes in Whatman Paper

“…Potential universality of this approach was tested using lipase (Lip), GOx, and HRP . These enzymes have varying physical and chemical properties (Supporting Information, Table S3), and are therefore suitable candidates to test BSA‐Paper as a potential universal enzyme support. Relative activities, recyclability, and turnover number by single or two‐layer approaches were examined (Table ).…”

Polymer conjugation of proteins as a synthetic post-translational modification to impact their stability and activity

“…[167][168][169] Kumar and coworkers attempted to enhance the enzymatic activity of catalase through the conjugation of poly (acrylic acid) (pAA). 170 Various lengths of polymer (100kDa, 500kDa, and 1000kDa) were conjugated to the enzyme with the goal of encapsulating the protein, this synthesis was conducted at various pH values (pH 5, 6, and 7) in order to control the protonation of the pAA chain. The catalytic activity of the conjugated enzyme was measured by studying the rate of decomposition of H 2 O 2 and compared to the native enzyme (Fig 12).…”