2 research outputs found
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(HPO<sub>4</sub>)<sub>2</sub>·H<sub>2</sub>O (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><sub>w</sub> 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><sub>m</sub> or <i>V</i><sub>max</sub> 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