Pharmaceuticals are a class of emerging contaminants that have been widely detected in wastewater treatment facilities’ influent and effluent. They threaten the environment and non-target life. Thus, a promising treatment method, soybean peroxidase (SBP; EC 1.11.1.7), which catalyzes the oxidation of phenolic and anilino donors in the presence of hydrogen peroxide, was investigated as a treatment method. The aim was to remove two non-steroidal anti-inflammatory drugs, diclofenac (DCF) and aceclofenac (ACF), from synthetic wastewater via enzymatic oxidation, oligomerization, and precipitation. SBP can be extracted from soybean hulls, a byproduct of the soybean industry. DCF (0.10 mM) and ACF (0.10 mM) were amenable to SBP-catalyzed removal under the optimal operational parameters of pH 5 and 4; hydrogen peroxide: 0.40 and 0.45 mM; and minimum effective enzyme concentration: 0.15 and 0.60 U/mL, respectively. The initial first-order rate constant and half-life of each substrate were also determined under the established optimum conditions. Under these optimum conditions, the half-lives for DCF and ACF were 1.43 ± 0.01 and 0.84 ± 0.05 min, respectively. The results demonstrated that SBP is a robust enzyme that can achieve more than 95% removal for both compounds. Mass spectrometric analysis of the enzymatic treatment products of DCF revealed the formation of an oxidative tetramer. The SBP-catalyzed reaction is a highly effective method for removing DCF and ACF from synthetic wastewater, highlighting its potential for environmental cleanup of pharmaceutical contaminants.

Sulfa drugs are a broad family of antibiotics widely used in the treatment of a range of infections. They have been found in surface and groundwater, as well as in sewage and effluent (treated sewage and sludge) of municipal or industrial wastewater treatment plants in concentrations of ng/L to >g/L. The continued presence of these so-called emerging contaminants (ECs) and their metabolites can cause adverse ecological effects, including bacterial resistance, even at very low concentrations. In this study, the first aim was to explore the feasibility of oxidation processes catalyzed by soybean peroxidase as an eco-friendly and economically advantageous alternative method for the conversion of the sulfonamides, sulfamethoxazole, and sulfamerazine. Optimum conditions were determined for 0.2 and 0.1 mM of the respective substrates. Optimum pHs were 1.6 and 3.6, respectively. Optimum molar peroxide ratios were 3.0 and 2.5 for the respective substrates. Enzyme activities of 4 and 2 U/mL showed 83 and 76

Soybean peroxidase (SBP) is a promising enzyme for wastewater treatment due to its ability to catalyze the oxidation of aromatic compounds, leading to the formation of precipitable polymers. This study investigates SBP's efficacy in removing p-, o-, and m-cresol from water. Experimental optimization using a continuously stirred batch reactor achieved 98, 97, and 100

Enzyme activity on a synthetic substrate (p-nitrophenyl-beta-D-glucopyranoside (pNPG)) ranked by specificity (kcat/ Km), placed Bgl-3 (a recombinant P. carotovorum subsp. carotovorum-ss-glucosidase expressed in Escherichia coli) in a group of ss-glucosidases, whereas ranking according to turnover number (kcat) placed Bgl-3 at the top of the group, implying its potential for high activity in biomass processing. The role of Lys211, His212, Arg172, and Asp114, in substrate recognition and stabilization of a glucose unit in catalysis is proposed here due to in-teractions with the substrate C1-C6 hydroxyls through hydrogen bonding, as well as the role of two methionines, Met255, Met322, in hydrophobic stabilization. However, enzyme inactivation due to ion pair dissociation, at pH range >8 and <5, of the enzyme nucleophile and the acid-base, can influence enzyme-intermediate complex formation, suggesting the latter as rate limiting in Bgl3 catalysis. Asp290 and Glu517 in the substrate binding clefts, are the nucleophile and the catalytic acid/base suggested, respectively. The close proximity of His212 and His522 stabilizes the enzyme glycosyl-intermediate through hydrogen bonding.

A facile and high yield centrifugal spinning technique known as Forcespinning® (FS) was used to develop unique microstructures consisting of PLA microbeads along alginate fibers. Morphological variation and structural features appeared in the field-emission scanning electron micrographs for the PLA-alginate composites and dried PLA-alginate films from precursor emulsions at constant PLA and varied alginate contents. Shrunk and deflated microbeads were observed for composites whilst spherical beads were evident for the PLA control. Furthermore, PLA was found surrounding the alginate when the alginate was present at 0.24 wt% or lower, while alginate (mushroom-like structures), were seen protruding through the PLA layer at ≥0.34 wt% alginate. Rheological characterization of the composite emulsions revealed that the filler (alginate) provided shear thinning properties including pseudoplasticity, desirable for printing and other related applications in contrast to the Newtonian flow shown by the PLA control. Along with infra-red spectroscopy, the nanocomposites were further characterized using thermal gravimetry and differential scanning calorimetry featuring reversible events influenced by heat capacity and irreversible kinetic/thermodynamic counterparts. The work provides a comprehensive investigation of biocompatible networks of PLA-alginate microbeads embedded in nano-sized fibers and the prospective application of these microbeads as a drug delivery system.

Enzyme activity on a synthetic substrate (p-nitrophenyl-β-D-glucopyranoside (pNPG)) ranked by specificity (kcat/Km), placed Bgl-3, a recombinant ß-glucosidase from Escherichia coli (E. coli), in the midst of a group of several ß-glucosidases, whereas ranking according to turnover number (kcat) placed Bgl-3 at the top of the group, implying its potential for high activity in biomass processing. The role of two highly conserved histidines, His212, and His522, in Bgl3, in stabilizing the aglycone intermediate burst and the glycosyl-enzyme intermediate complex, was proposed for the enzyme low turnover number equivalent to/proportional to the enzyme net mass sizes. The role of these two histidines in stabilizing the electrostatic interactions and, hence, loss of activity at low pHs closely correspond with other GH-3 ß-glucosidases. The enzyme’s multi-functional activity on model cellulose polymers, and on biomass-derived lignocellulose degradation onto value added compounds (e.g., glucose, cellobiose) account for the broader impact of this work, that Bgl-3 is well-suited for biorefinery applications, which will be presented in future research.

Soybean peroxidase effectively transformed selected amino- and hydroxyl-azoles by radical coupling to dimers and trimers, showing feasibility for wastewater treatment.

Two simple methods for the determination of eugenol were developed. The first depends on the oxidative coupling of eugenol with p-amino-N,N-dimethylaniline (PADA) in the presence of K-3[Fe(CN)(6)]. A linear regression calibration plot for eugenol was constructed at 600 nm, within a concentration range of 0.25-2.50 mu g.mL(-1) and a correlation coefficient (r) value of 0.9988. The limits of detection (LOD) and quantitation (LOQ) were 0.086 and 0.284 mu g.mL(-1), respectively. The second method is based on the dispersive liquid-liquid microextraction of the derivatized oxidative coupling product of eugenol with PADA. Under the optimized extraction procedure, the extracted colored product was determined spectrophotometrically at 618 nm. A linear plot within a concentration range of 0.05-1.65 mu g.mL(-1) (r = 0.9997) was constructed. The LOD and LOQ were 0.053 and 0.177 mu g.mL(-1), respectively. Both methods were tested for the analysis of eugenol in commercial personal-care products, and the results confirmed that the procedures are accurate, precise, and reproducible (RSD < 1%).

A procedure, depending on the mutual derivatization and determination of thymol and Dapsone was developed and validated in this study. Dapsone was used as the derivatizing agent for the determination of thymol, and thymol was used as the derivatizing agent for the determination of Dapsone. An optimization study was performed for the derivatization reaction, i.e., the diazonium coupling reaction. Linear regression calibration plots for thymol and Dapsone in the direct reaction were constructed at 460 nm, within the concentration range of 0.3-7 μg.mL-1 for thymol and 0.3-4 μg.mL-1 for Dapsone, with limits of detection 0.086 and 0.053 μg.mL-1, respectively. Corresponding plots for the cloud point extraction of thymol and Dapsone were constructed at 460 nm, within the concentration range of 0.1-2 μg.mL-1 for thymol and 0.1-1.8 μg.mL-1 for Dapsone, with limits of detection 0.0445 and 0.023 μg.mL-1, respectively. Correlation coefficients and molar absorptivities, were improved using cloud point extraction. The proposed method can be applied for their trace detection in different matrices.

Widespread occurrence of various heterocyclic aromatic compounds is reported in concentrations from 1 to 20 μg/L in surface and groundwater as well as influents and effluents of wastewater treatment plants around the world. These so-called emerging contaminants and their metabolites can cause adverse effects on the environment and humans, even at very low concentration, hence raised environmental concerns. In this study, feasibility of soybean peroxidase-catalyzed removal of three selected heterocyclic aromatics from water was investigated, including sensitivity to the most important operational conditions, pH (range 3.6–9.0), H2O2 concentration (range 0.10–1.50 mM), and enzyme activity (range 0.001–5.0 U/mL). 3-Hydroxycoumarin and 2-aminobenzoxaozle were found to be substrates for the enzyme, having ≥95% and 45% removal efficiency with most effective pHs of 7.0 and 6.0, respectively. Time course study was also conducted to determine the initial first-order rate constants and half-lives; half-lives normalized for enzyme activity (0.0257 and 452 min for the respective substrates) are compared with those of 21 other compounds reactive with soybean peroxidase. High-resolution mass spectrometry was employed to characterize the plausible oligomerization products of enzymatic treatment, which revealed formation of dimers and trimers of the two substrates.

BACKGROUND The presence of azo dyes in wastewater from the textile industry is a major environmental concern. The dyes not only make water aesthetically unacceptable, but also have severe toxicological concerns. Research into treatment processes for removal of dyes has focused primarily on decolourization and little attention has been focused on analysis of the degradation products, that could plausibly be more toxic than the parent compound. This study focusses on soybean peroxidase (SBP)-catalyzed treatment of two azo dyes, Methyl Orange (MO) and CI Direct Yellow 12 (DY12) in water, chosen because they lack phenolic and primary anilino functional groups, which are usually expected to form free radicals under peroxidase catalysis. RESULTS DY12 was found not to be a substrate of SBP, but optimized reaction conditions for 0.50 mmol L-1MO and 1.0 mmol L(-1)p-anisidine (structurally analogous to 4-ethoxyaniline, a possible azo-cleavage product of DY12) achieved >= 95% conversion at exceptionally low minimum effective SBP activities (0.0070 and 0.0018 U mL(-1)) at pH optima of 4.0 and 5.5 and [hydrogen peroxide]/[substrate] of 2 and 1, respectively. CONCLUSIONS Mass spectrometric (MS) analysis for the substrates revealed formation of dimers and trimers forp-anisidine. For MO, high-performance liquid chromatography, UV-visible spectrophotometry and MS provided evidence of azo-bond cleavage, hetero-coupling of the dye with the cleavage product and also self-coupling of the dye through tertiary amine activation. (c) 2020 Society of Chemical Industry (SCI)

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

Background. Some industrial manufacturing processes generate and release dyes as water pollutants, many of which are toxic and hazardous materials. There is a need for milder, greener methods for dye treatment. Objectives. The objective of the present study was to investigate and optimize azo dye decoloration by a crude soybean peroxidase (SBP), based on two dyes that have widespread industrial use, but that differ greatly in structural complexity, Acid Black 2 and Acid Orange 7, and to investigate the effects of specific parameters on the removal process. Methods. Batch reactors were used to remove 95% of the dyes' color and to produce substantial precipitates. Results. The optimum pH for enzymatic decoloration of Acid Black 2 was in the acidic region, pH 4.4, and that of Acid Orange 7 occurred under neutral conditions, pH 6.9. The minimum enzyme activity needed for sufficient removal was 1.2 U/mL for both dyes at 0.5 mM. The minimum molar hydrogen peroxide/substrate ratio was 3 for Acid Orange 7 and 2.5 for Acid Black 2 to achieve approximately 95% removal. First-order fitting of progress curve data collected under the respective optimum conditions gave half-lives of 23.9 and 28.9 minutes for Acid Orange 7 and Acid Black 2, respectively. Conclusions. The feasibility of SBP-catalyzed treatment of industrial dyes Acid Black 2 and/or Acid Orange 7, or dyes that resemble them, as they might occur in industrial effluents, was successfully demonstrated. Competing Interests. The authors declare no competing financial interests

Water contamination by refractory and persistent organic aromatic compounds such as poly-substituted mono cyclic, 4-chloro-o-toluidine and bis-anilino compound, 4,4 '-methylenebis-(2-chlororaniline), is an emerging environmental concern and the compounds are considered priority pollutants by U.S. Environmental Protection Agency. Thus, there is a search for new, efficient and eco-friendly treatment methods for converting and eliminating these pollutants at the point of release. The crude form of soybean seedcoat peroxidase, was able to catalyze the oxidation of these pollutants by generating reactive radicals which couple to form insoluble products. The reaction parameters, pH, hydrogen-peroxide-to-substrate molar ratio and minimum effective enzyme concentration were optimized to achieve >= 95 % removal of these substrates in a 3-h reaction time. The pH optima for 4-chloro-o-toluidine and 4,4 '-methylenebis (2-chlororaniline) were 4.4 and 4.2, respectively. Consumption of H2O2 of both substrates was near the theoretical stoichiometric value (i.e. H2O2/substrate <= 1.0). For 95 % removal of 1.0 mM 4-COT and 0.1 mM MOCA, 0.009 and 0.10 U/mL of enzyme were required, respectively. The enzyme kinetic mechanism based on the Michaelis-Menten model was determined for a group of aromatic amines, p-cresidine, 4,4 '-oxydianiline, 4-chloro-o-toluidine and 4,4 '-methylenebis-(2-chlororaniline). The lowest KM was for 4,4 '-methylenebis (2-chlororaniline), 1.70 +/- 0.14 mu M, indicating highest affinity for the enzyme compared to other amines studied. In addition, product analysis by mass spectrometry revealed presence of "oxidative oligomers" and "oxidized oxidative oligomers" (azo compounds) after the enzymatic treatment. A pro-forma cost estimation emphasized the feasibility of commercialization of the enzymatic treatment, showing it to be 5-8 times lower than conventional methods.

Various aromatic compounds, in particular phenols and aromatic amines, are present in wastewaters of numerous industries such as coal conversion, petroleum refining, organic chemicals, and dyes. Most of these compounds are toxic and some have been determined to be human carcinogens; therefore, the removal of such compounds from industrial aqueous effluent is of great practical significance. An enzymatic method for the removal of phenols from industrial aqueous effluent has been developed in the past several years. In this method, peroxidase enzymes catalyze the oxidation of phenol with hydrogen peroxide, generating phenoxy radicals. These radicals diffuse from the active site of the enzyme into solution and react nonenzymatically to eventually form higher oligomers and polymers which can be removed from wastewater by sedimentation or filtration. In this study, Arthromyces ramosus peroxidase (ARP) was applied to remove 1.0 mM phenol in synthetic wastewater in a continuous flow system. The system consisted of a mixing tank where hydrogen peroxide was added to the mixture of phenol, enzyme, and polyethylene glycol (PEG) to initiate the reaction. A plug flow reactor where the reaction takes place was followed by a flocculation tank where the alum was added, and finally the polymers formed were settled in a sedimentation tank and removed from the system. The removal efficiency of the system was dependent on the enzyme dose, molar ratio between hydrogen peroxide and phenol, pH, and PEG concentration. These parameters were optimized first using batch experiments, and then applied to the continuous experiments.

Azo dyes are a water-pollution problem causing damage to ecosystems and human health. Soybean peroxidase-catalyzed reactions of azo dyes, Acid blue 113 (AB113) and Direct black 38 (DB38), were optimized for color removal using response surface methodology on a Box-Behnken design (BBD). Parameters optimized were H2O2 concentration (mM), pH, and enzyme concentration (U/mL; U is a standard unit of catalytic activity). Optimum conditions for AB113 were pH 4.49, 2.57 mM H2O2, and 1.52 U/mL of enzyme for a predicted 5.6% color remaining (experimental value of 8.1%) and R2 value of 99.68%; and for DB38 the conditions were pH 3.68, 2.92 mM H2O2, and 2.84 U/mL of enzyme for a predicted 3.6% color remaining (experimental value of 5.1%) and R2 value of 99.07%. In addition, the agreement with the one-factor-at-a-time approach was checked. The BBD is a less time-consuming approach that allows identification of interactions between parameters. Kinetic studies (Michaelis-Menten model) quantitatively confirmed the efficiency and effectiveness of enzymatic dye treatment.

Personal care products and pharmaceuticals have been reported in various concentrations in the effluent of municipal sewage treatment plants (STP). Although they are generally found in the nanogram to microgram per liter range, many of them might have adverse health effects on humans at these concentrations. Conventional treatments applied at the STP are unable to effectively remove most of these recalcitrant compounds, thus there is a necessity for development of alternative treatment techniques. In this article, the efficiency of enzymatic treatment using soybean peroxidase in treating some commonly found micropollutants is discussed. The target compounds were, two phenolic surfactant breakdown products, nonylphenol and octylphenol, two antimicrobial agents, Triclosan and sulfamethoxazole and three phenolic steroids. The effects of the most important parameters pH, enzyme concentration and peroxide concentration have been evaluated for each compound. The treatment of synthetic wastewater was shown to be effective (≥95% removal), except for sulfamethoxazole, in concentration ranges of 10 s of µM at neutral pH with 2–5 mU/L of catalytic activity and 2–3 molar equivalents of hydrogen peroxide. The effectiveness of the treatment has also been determined for lower concentrations (6–9 nM) which approximate those in real wastewater. A matrix effect was found in the treatment of Triclosan in spiked real wastewater indicating that re-optimization of important parameters for STP treatment would be required to achieve high removal efficiency. A reverse-phase, solid-phase extraction technique was used to concentrate target analytes in real wastewater, enabling chromatographic detection by UV absorbance.

Although heterocyclic aromatics make up a vast group of water contaminants, the effect of enzyme on this class of recalcitrant compounds is largely unknown. Here, the feasibility of treatment of selected quinolines with soybean peroxidase is demonstrated and the effects of the most important parameters; pH, enzyme concentration, and peroxide concentration are optimized for each compound. 3-hydroxy and 3-aminoquinoline were found to be substrates amenable to >= 94% removal with pH optima of 8.6 and 5.6, respectively. In order to identify the transformation products of such treatment, the solution and precipitate after enzymatic treatment were analyzed by mass spectrometry to find that the dominant products were dimers and trimers from oxidative radical coupling. In addition, for 3-aminoquinoline azo-linked dimers and trimers were found. Computational techniques used to investigate the influence of redox potential and ionization energy of pollutants on their treatability by SBP showed a possible threshold between substrates and non-substrates. Computed spin densities were used to predict the regiochemistry of the radical coupling reaction.

The search for an effective and sustainable treatment method to remove the recalcitrant atom-bridged bis-anilino compounds, 4,4'-methylenedianiline (MDA) and 4,4'-thiodianiline (TDA) from water is a major challenge and focus of this study. The escalating discharge of these two toxic and carcinogenic pollutants from industrial sources may pose a serious threat to the environment. Crude soybean peroxidase (SBP), isolated from soybean seed hulls (coats), catalyzes the oxidative polymerization of these aqueous pollutants in the presence of hydrogen peroxide. The effects of several process parameters, i.e., pH, hydrogen peroxide-to-substrate concentration ratio and SBP concentration, were investigated to optimize the performance of enzymatic treatment. The minimum effective SBP concentration required for removal of MDA was 0.70 U/mL, which was higher than that of TDA (0.15 U/mL). The reaction time course to achieve ≥95% removal of these compounds from water was determined under those optimum conditions. Identification of the transformed products was performed by means of high-resolution electrospray ionization mass spectrometry. The products generally observed were protonated oxidized oxidative dimers and higher oligomers (most commonly azo-coupled products). Michaelis constant, KM, and maximum reaction velocity, Vmax, obtained from the Michaelis-Menten (M-M) model revealed that TDA had a 65-fold lower KM than MDA (indicating TDA's higher affinity for SBP), and almost 5-fold higher Vmax than MDA. A pro-forma cost analysis is presented to assess the possibility of commercialization of enzymatic treatment as an alternative to conventional/traditional treatment methods.