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.
An example of a highly stable and functional bienzyme-polymer conjugate triad assembled on a topologically orthogonal support, layered graphene oxide (GO), is reported here. Glucose oxidase (GOx) and Horseradish Peroxidase (HRP) catalytic dyad were used as the model system for cascade biocatalysis. Poly (Acrylic Acid) (PAA) was used to covalently conjugate these enzymes and then the conjugate has been subsequently adsorbed on to GO. The resultant nanobiocatalysts are represented as GOx-HRP-PAA/GO. Their morphology and structural characteristics were examined by transmission electron microscopy (TEM), agarose gel electrophoresis, circular dichroism (CD) and zeta potential. These robust conjugates remarkably functioned as active catalysts under biologically challenging conditions such as extreme pH's, high temperature (65°C) and in the presence of a chemical denaturant. In one example, GOx-HRP-PAA/GO presented doubling of the K cat (TON) (68 × 10 -2 s -1 ) at pH 7.0 and room temperature, when compared to the corresponding physical mixture of GOx/HRP (32 × 10 -2 s -1 ) under similar conditions. In another case, at 65°C, GOx-HRP-PAA/GO displayed ~120% specific activity, whereas GOx/HRP showed only 16% of its original activity. At pH 2.0 and in the presence of 4.0 mM SDS as the denaturant, GOx-HRP-PAA/GO presented greater than 100% specific activity, while GOx/HRP was completely deactivated under these conditions. Thus, we combined two concepts, enzyme-polymer conjugation followed by adsorption on to a 2D nanolayered material to obtain enhanced substrate channeling and excellent enzyme stability under challenging conditions. These features have never been attained by either traditional enzyme-polymer conjugates or enzyme-GO hybrids. This, general, modular and powerful approach may also be used to produce environmentally benign, biologically compatible (edible) and efficient cascade biocatalysts.
We report a general and modular approach for the synthesis of multi enzyme–polymer conjugates (MECs) consisting of five different enzymes of diverse isoelectric points and distinct catalytic properties conjugated within a single universal polymer scaffold. The five model enzymes chosen include glucose oxidase (GOx), acid phosphatase (AP), lactate dehydrogenase (LDH), horseradish peroxidase (HRP) and lipase (Lip). Poly(acrylic acid) (PAA) is used as the model synthetic polymer scaffold that will covalently conjugate and stabilize multiple enzymes concurrently. Parallel and sequential synthetic protocols are used to synthesise MECs, 5-P and 5-S, respectively. Also, five different single enzyme–PAA conjugates (SECs) including GOx–PAA, AP–PAA, LDH–PAA, HRP–PAA and Lip–PAA are synthesized. The composition, structure and morphology of MECs and SECs are confirmed by agarose gel electrophoresis, dynamic light scattering, circular dichroism spectroscopy and transmission electron microscopy. The bioreactor comprising MEC functions as a single biocatalyst can carry out at least five different or orthogonal catalytic reactions by virtue of the five stabilized enzymes, which has never been achieved to-date. Using activity assays relevant for each of the enzymes, for example AP, the specific activity of AP at room temperature and 7.4 pH in PB is determined and set at 100%. Interestingly, MECs 5-P and 5-S show specific activities of 1800% and 600%, respectively, compared to 100% specific activity of AP at room temperature (RT). The catalytic efficiencies of 5-P and 5-S are 1.55 × 10−3 and 1.68 × 10−3, respectively, compared to 9.11 × 10−5 for AP under similar RT conditions. Similarly, AP relevant catalytic activities of 5-P and 5-S at 65 °C show 100 and 300%, respectively, relative to native AP activity at RT as the native AP is catalytically inactive at 65 °C The catalytic activity trends suggest: (1) MECs show enhanced catalytic activities compared to native enzymes under similar assay conditions and (2) 5-S is better suited for high temperature biocatalysis, while both 5-S and 5-P are suitable for room temperature biocatalysis. Initial cytotoxicity results show that these MECs are non-lethal to human cells including human embryonic kidney [HEK] cells when treated with doses of 0.01 mg mL−1 for 72 h. This cytotoxicity data is relevant for future biological applications.
We report the synthesis of linear‐ and brush‐type poly(ɛ‐caprolactone) (PCL) networks and investigate their thermal, mechanical, and shape memory behavior. Brush‐PCLs are prepared by ring‐opening metathesis polymerization (ROMP) of a norbornenyl‐functionalized ɛ‐caprolactone macromonomer (MM‐PCL) of different molecular weights. The linear analog, diacrylate end‐functionalized PCL (linear‐PCL), having comparable molecular weight of side chain of brush‐PCL is also synthesized. These polymers are thermally cured by a radical initiator in the presence of poly(ethylene glycol) diacrylate crosslinker. Thermal and linear viscoelastic properties as well as shape memory performance of the resulting PCL networks are investigated, and are significantly impacted by the PCL architecture. Therefore, our work highlights that tailoring macromolecular architecture is useful strategy to manipulate thermal, mechanical, and resulting shape memory properties. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 3424–3433
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