Enzyme-Triggered Hydrogels for Pharmaceutical and Food Applications

Enzyme-mediated polymeric hydrogels are drawing considerable attention in pharmaceutical and food sectors owing to their superior biocompatibility and process controllability under physiological conditions. Enzymes play a significant role in polymeric hydrogel formation through different mechanisms. Oxidases (e.g., horseradish peroxidase and tyrosinase) have demonstrated to drive the crosslinking of gel precursors by oxidizing the phenolic or acrylic moieties to free radicals. Transferases and hydrolases catalyze elongation of biopolymer chains which gradually self-assemble into hydrogels. Still more certain enzymes also participate in hydrogel formation by releasing gelation factors. Enhancement of the desired properties of certain hydrogels through the interior and exterior post-modifications has also been demonstrated by certain enzymes. Hence, in this chapter, the authors explore the different mechanisms of enzyme-mediated hydrogels preparations and its fabrication towards pharmaceutical and food sectors along with the discussion of recent trends and further prospects.

Kinetic changes in activity of HR-peroxidase, induced by very low doses of phenol

The allosteric protein of horseradish peroxidase (HRP) shows two main types of activity, peroxidase and oxidase, depending on the kind of low molecular effectors. The effects of very low doses of phenol, prepared by successive dilutions in water or in 75% ethanol, on initial HRP activity in oxidation of o-dianisidine or luminol were tested in a systematic manner by colorimetric and luminometric methods. Results showed that phenol dilutions, including those below Avogadro’s number, could activate or inhibit HRP in peroxidase and oxidase type reactions with a sinusoidal pattern. Km values for the studied substrates changed parallel to HRP peroxidase/oxidase activity and the maximum activity in the peroxidase reaction corresponded to the minimum activity in the oxidase reaction and vice versa. The effect also depended on the type of dilutor. The observations of the peroxidase/oxidase oscillations in the sinusoidal pattern of HRP activity, dependent on the rate of phenol dissolution and the time of preincubation, point out to the conclusion that HRP might be a good model for high dilutions research. The experiments provide strong evidence that horseradish peroxidase (HRP) is a very sensitive detector of subtle changes in the concentration of phenol used as a cofactor in the peroxidase/oxidase reaction. Keywords: HR-peroxidase, peroxidase-oxidase, phenol, hormesis, homeopathy, high dilutions.  Mudanças cinéticas na atividade da HR-peroxidade induzidas por doses muito baixas de fenol Resumo A proteína alostérica da peroxidase do rabano (HRP) mostra dois tipos principais de atividade, peroxidase e oxidase, de acordo com o tipo de efetores de baixa molecularidade. Os efeitos de doses muito baixas de fenol, preparadas através de diluições sucessivas em água ou etanol 75% na atividade inicial da HRP sobre a oxidação da o-dianisidina ou luminol for testados de modo sistemático através de métodos colorimétricos e luminométricos. Os resultados mostram que as diluições de fenol, incluindo aquelas por baixo do número de Avogadro, foram capazes de ativar ou inibir a HRP em reações de tipo peroxidade e oxidase com um padrão sinusoidal. os valores Km dos substratos estudados variaram paralelamente àatividade peroxidase/oxidase da HRP; a atividade máxima da reação peroxidase correspondeu àatividade mínima na reação oxidase e vice-versa. O efeito também se mostrou dependente do tipo do solvente. A observação das oscilações sinusoidais na atividade da HRP, dependentes da taxa de dissolução do fenol e do tempo de pré-incubação, permitem concluir que a HRP pode ser um bom modelo na pesquisa das altas diluições. Os experimentos oferecem fortes evidéncias a favor da HRP como detector muito sensível de mudanças mínimas na concentração do fenol, utilizado como cofator na reação peroxidase/oxigenase. Palavras-chave: HR-peroxidase, peroxidase-oxidase, fenol, hormese, homeopatia, altas diluições.  Cambios cinéticos en la actividad de la HR-peroxidasa inducidos por dosis muy bajas de fenol Resumen La proteína alostérica de la peroxidasa del rábano (HRP) muestra dos tipos principales de actividad, peroxidasa y oxidasa, dependiendo del tipo de efectores de baja molecularidade. Los efectos de doses muy bajas de fenol, preparadas mediante diluciones sucesivas en agua o etanol al 75% sobre la actividad inicial de la HRP sobre la oxidación de o-dianisidina o luminol fueron testados de modo sistemático mediante métodos colorimétricos y luminométricos. Los resultados muestran que las diluciones de fenol, incluyendo aquellas abajo del número de Avogadro, pudieron activar o inhibir la HRP en reacciones de tipo peroxidasa y oxidasa con un patrón sinusoidal. Los valores Km de los sustratos estudiados variaron paralelamente a la actividad peroxidasa/oxidasa de la HRP; la actividad máxima de la reacción peroxidasa correspondió a la actividad mínima en la reacción oxidasa y viceversa. El efecto también se mostró dependiente del tipo de solvente. La observación de las oscilaciones sinusoidales en la actividad de la HRP, dependientes de la tasa de disolución del fenol y del tiempo de preincubación, llevan a concluir que la HRP puede ser un buen modelo para la investigación de las altas diluciones. Los experimentos ofrecen fuertes evidencias a favor de la HRP como detector muy sensible de cambios mínimos en la concentración de fenol, utilizado como cofactor en la reacción peroxidasa/oxigenasa. Palabras-clave: HR-peroxidasa, oxidasa-peroxidasa, fenol, hormesis, homeopatía, altas diluciones.  Correspondence author: Elzbieta Malarczyk, [email protected] How to cite this article: Malarczyk E. Kinetic changes in the activity of HR-peroxidase induced by very low doses of phenol. Int J High Dilution Res [online]. 2008 [cited YYYY Mmm DD]; 7(23): 48-55. Available from: http://journal.giri-society.org/index.php/ijhdr/article/view/37/349. ÂÂÂ

Removal of Persistent Sulfamethoxazole and Carbamazepine from Water by Horseradish Peroxidase Encapsulated into Poly(vinyl chloride) Electrospun Fibers

Enzymatic conversion of pharmaceutically active ingredients (API), using immobilized enzymes should be considered as a promising industrial tool due to improved reusability and stability of the biocatalysts at harsh process conditions. Therefore, in this study horseradish peroxidase was immobilized into sodium alginate capsules and then trapped into poly(vinyl chloride) electrospun fibers to provide additional enzyme stabilization and protection against the negative effect of harsh process conditions. Due to encapsulation immobilization, 100% of immobilization yield was achieved leading to loading of 25 μg of enzyme in 1 mg of the support. Immobilized in such a way, enzyme showed over 80% activity retention. Further, only slight changes in kinetic parameters of free (Km = 1.54 mM) and immobilized horseradish peroxidase (Km = 1.83 mM) were noticed, indicating retention of high catalytic properties and high substrate affinity by encapsulated biocatalyst. Encapsulated horseradish peroxidase was tested in biodegradation of two frequently occurring in wastewater API, sulfamethoxazole (antibiotic) and carbamazepine (anticonvulsant). Over 80% of both pharmaceutics was removed by immobilized enzyme after 24 h of the process from the solution at a concentration of 1 mg/L, under optimal conditions, which were found to be pH 7, temperature 25 °C and 2 mM of H2O2. However, even from 10 mg/L solutions, it was possible to remove over 40% of both pharmaceuticals. Finally, the reusability and storage stability study of immobilized horseradish peroxidase showed retention of over 60% of initial activity after 20 days of storage at 4 °C and after 10 repeated catalytic cycles, indicating great practical application potential. By contrast, the free enzyme showed less than 20% of its initial activity after 20 days of storage and exhibited no recycling potential.

Photothermal Detection of MicroRNA Using a Horseradish Peroxidase-Encapsulated DNA Hydrogel With a Portable Thermometer

MicroRNA (miRNA) detection has attracted widespread interest as a tumor detection marker. In this work, a miRNA-responsive visual and temperature sensitive probe composed of a horseradish peroxidase (HRP)-encapsulated DNA hydrogel was designed and synthesized. The biosensor converted the miRNA hybridization signal to a photothermal effect which was measured using a digital thermometer. The substrate DNA linker strand of the hydrogel hybridizes with different sequences of miRNA resulting in the collapse of the hydrogel and the release of HRP. HRP oxidizes 3,3′,5,5′-tetramethylbenzidine (TMB) resulting in a color change and a strong photothermal effect was observed after shining near-infrared light on the oxidized product. The thermometer-based readout method has a wide linear range (0.5–4.0 µM) and a limit of detection limit of 7.8 nM which is comparable with traditional UV-vis absorption spectrometry detection and quantitative real time polymerase chain reaction methods. The low cost, ease of operation, and high sensitivity shows that this biosensor has potential for point-of-care biomolecular detection and biomedical applications.

Engineered Stable 5-Hydroxymethylfurfural Oxidase (HMFO) from 8BxHMFO Variant of Methylovorus sp. MP688 through B-Factor Analysis

What is known as Furan-2,5-dicarboxylic acid (FDCA) is an attractive compound since it has similar properties to terephthalic acid. Further, 5-hydroxymethylfurfural oxidase (HMFO) is an enzyme, which could convert HMF to FDCA directly. Most wild types of HMFO have low activity on the oxidation of HMF to FDCA. The variant of 8BxHFMO from Methylovorus sp. MP688 was the only reported enzyme that was able to perform FDCA production. However, the stabilization of 8BxHMFO is still not that satisfactory, and further improvement is necessary for the industrial application of the enzyme. In this work, stability-enhanced HMFO from 8BxHFMO was engineered through employing B-factor analysis. The mutation libraries were created based on the NNK degeneracy of residues with the top ten highest B-factor value, and two of the effective mutants were screened out through the high throughput selection with the horseradish peroxidase (HRP)-Tyr assay. The mutants Q319K and N44G show a significantly increased yield of FDCA in the reaction temperature range of 30 to 40 °C. The mutant Q319K shows the best performance at 35 °C with a FDCA yield of 98% (the original 8BxHMFO was only 85%), and a half-life exceeding 72 h. Moreover, molecular dynamic simulation indicates that more hydrogen bonds are formed in the mutants, which improves the stability of the protein structure. The method could enhance the design of more stable biocatalysts; and provides potential for the further optimization and utilization of HMFO in biotechnological processes.