A highly efficient, multifunctional, bioderived white-emitting hydrogel (biophosphor) consisting of crosslinked bovine serum albumin and three fluorescent dyes, Coumarin 460, fluorescein, and 5(6)-carboxy-x-rhodamine, is reported here. White emission is obtained upon excitation of the biophosphor at 365 nm with appropriate mole ratios of the above dyes. The CIE 1931 chromaticity coordinates of white emission with 365 nm excitation are (0.36, 0.37), and the correlated color temperature is 5300 K. Multifunctional nature of the biophosphor is also demonstrated. A UV-light-emitting-diode (361 nm) coated with this biophosphor, for example, indicates white emission (CIE 0.28, 0.31) with a half-life of 106 (±5) h. The white emission is also highly sensitive to pH over a broad range (pH 1-11). Incorporation of glucose oxidase and peroxidase in the biophosphor allows for the detection of glucose over a physiologically relevant range of 1.8-288 mg dL −1 . This is a unique, advanced biophosphor with LED and sensing applications, and it is the first example of a multifunctional, proteinaceous white emitter. molar absorptivity and quantum efficiency of emissive components is a significant challenge for systems that combine direct emission and sensitized emission via Förster resonance energy transfer (FRET) to produce white light. [6,7] Hydrogels are hydrophilic polymer networks [8] and are emerging as versatile new matrices for high-efficiency generation of white light using intermolecular energy transfer processes. The gel matrix improves energy transfer efficiency by rigidifying the orientation of the donor (D)-acceptor (A) pairs [9] and preventing their aggregation, which can lead to quenching. [10] Despite the advantageous properties of white-emitting hydrogels, implementation in a functional device and photostability were not systematically studied, and biodegradability has not been demonstrated. Additionally, white-emitting proteinbased hydrogels are not known other than a report of a gelatin hydrogel with chromaticity coordinates [0.26, 0.33], far from being coordinates of pure white emission [0.33, 0.33]. [2] To the best of our knowledge, there are no reports of a multifunctional, nontoxic, biodegradable, white-emitting protein hydrogel.BSA is inexpensive and readily available as a waste product of the meat industry. BSA has a large number of primary amines (59 lysine) and carboxylic acids (99 aspartic acid/glutamic acid), [11] which can be crosslinked under controlled conditions by carbodiimide chemistry to form a network of amide bonds without disrupting the intricate secondary structure of the protein. [12,13] The protein's secondary structure plays an important role for dye binding at the intended site and for enzyme activity retention, when enzymes are incorporated in the matrix for sensing or catalytic applications. We envisioned that this molecular network of BSA would result in a water-rich hydrogel with discrete sites for dye binding which would be suitable for the construction of a white-emitting gel.Previou...

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.

Cytochrome c–poly(acrylic acid) conjugates with 34-fold enhanced peroxidase activity due to acidification of enzyme microenvironment and suppression of wasteful intermediates.