Phenolic Substrates Research Articles

In situ Generation of Cyclohexanone Drives Electrocatalytic Upgrading of Phenol to Nylon‐6 Precursor

AbstractCoupling in situ generated intermediates with other substrates/intermediates is a viable approach for diversifying product outcomes of catalytic reactions involving two or multiple reactants. Cyclohexanone oxime is a key precursor for caprolactam synthesis (the monomer of Nylon‐6), yet its current production uses unsustainable carbon sources, noble metal catalysts, and harsh conditions. Herein, we report the first work to synthesize cyclohexanone oxime through electroreduction of phenol and hydroxylamine. The Faradaic efficiency reached 69.1 % over Cu catalyst, accompanied by a corresponding cyclohexanone oxime formation rate of 82.0 g h−1 gcat−1. In addition, the conversion of phenol was up to 97.5 %. In situ characterizations, control experiments, and theoretical calculations suggested the importance of balanced activation of water, phenol, and hydroxylamine substrates on the optimal metallic Cu catalyst for achieving high‐performance cyclohexanone oxime synthesis. Besides, a tandem catalytic route for the upgrading of lignin to caprolactam has been successfully developed through the integration of thermal catalysis, electrocatalysis, and Beckmann rearrangement, which achieved the synthesis of 0.40 g of caprolactam from 4.0 g of lignin raw material.

Site-Directed Mutagenesis of Two-Domain Laccase ScaSL for Obtaining a Biocatalyst with Improved Characteristics

Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution.

High-Valence Mn MOF Inspired by Laccase Mediators Enables Versatile Nature-Mimicking Catalysis.

In nature, active Mn3+ -ligand complexes produced by laccase catalyzed oxidation can act as the low-molecular mass, diffusible redox mediators to oxidize the phenolic substrates overcoming the limitations of natural enzymes. Learning from the metal-ligand coordination of natural functional units, high-valence Mn metal-organic framework (Mn MOF) is constructed to simulate the catalysis in natural mediator system. Benefiting from the characteristics of nanoscale size, rich metal coordination unsaturated sites, and mixed valence state dominated by Mn(III), Nano Mn(III)-TP exhibits superior laccase-mimicking activity, whose Vmax (maximal reaction rate) is much higher than that of natural laccase. Referring to natural systems, relevant free radical experiments prove that the material induces the production of active oxygen species with the assistance of carboxylic acid, and active oxygen species further oxidize phenolic substrates. Based on its robust performances, the primary oxidative degradation of an emerging pollutant triclosan (TCS) is creatively applied, an important antiasthmatic medicine terbutaline sulfate (TBT) detection, and the synthesis of non-toxic and black near-natural dyes for dyeing. By simulating the essential mediators of natural enzymatic catalysis, an Mn MOF-based material that demonstrates multiple novel applications is successfully developed, which introduces a new reliable strategy for achieving versatile nature-mimicking catalysis.

Characterization and Degradation of Triphenylmethane Dyes and Their Leuco-Derivatives by Heterologously Expressed Laccase From Coprinus cinerea.

Laccase is a copper-containing polyphenol oxidase that can oxidize phenolic and non-phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L-1protein. The highest production level of the recombinant laccase was 2760 U L-1. The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as the substrate, the Km and Vmax values were 0.136 mM and 9778 μM min-1 mg-1, respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco-crystal violet (LCV) and leuco-malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution.

Identification of Plant Peroxidases Catalyzing the Degradation of Fluorinated Aromatics Using a Peroxidase Library Approach

ABSTRACTIn this work, the degradation of mono‐ and polyfluorinated phenolic compounds was demonstrated by a series of crude plant peroxidases, including horseradish root (HRP) and six members of the Cucurbita genus. Highly active samples were identified using a library screening approach in which more than 50 crude plant samples were initially evaluated for defluorination activity toward 4‐fluorophenol. The highest concentrations were observed in the HRP, pumpkin skin (PKS), and butternut squash skin (BNS), which consistently gave the highest intrinsic rates of decomposition for all the substrates tested. Although HRP exhibited a significant decrease in activity with increased fluorination of the phenolic substrate, PKS showed only minor reductions. Furthermore, in silico studies indicated that the active site of HRP poorly accommodates the steric bulk of additional fluorines, causing the substrate to dock farther from the catalytic heme and thus slowing the catalysis rate. We propose that the PKS active site might be larger, allowing closer access to the perfluorinated substrate, and therefore maintaining higher activity compared to the HRP enzyme. However, detailed kinetic characterization studies of the peroxidases are recommended. Conclusively, the high catalytic activity of PKS and its high yield per gram of tissue make it an excellent candidate for developing environmentally friendly biocatalytic methods for degrading fluorinated aromatics. Finally, the success of the library approach in identifying highly active samples for polyfluorinated aromatic compound (PFAC) degradation suggests the method may find utility in the quest for other advanced catalysts for PFAS degradation.

Decomposer faecal food web and C sequestration in soil. Can near infrared spectroscopy describe transfers and transformations from fresh organic inputs to protected forms in soil aggregates?

This study sought to characterize the transfer of agricultural organic inputs by macrofauna to the biogenic aggregate compartments of the soil and test the existence of a faecal food web process based on coprophagic feeding behaviours. In an experimental plantain crop field of Colombia, we applied the agroecological FBO (Fertilisation Bio Organique ®) technology, a nucleation technique which consists in planting perennial plants in 1.0 x 0.4 × 0.4 m deep trenches where low- and high-quality organic materials are added in a specific design, and endogeic earthworms are inoculated. We evaluated the effectiveness of Near Infrared Spectroscopy (NIRS) to track this flow of organic matter through different natural stages of decomposition represented by soil macroaggregates produced by soil macroinvertebrates as faecal pellets and casts. After one year, great part of the organic inputs had been either mineralized or incorporated into macroaggregates of different sizes and origins. Using NIRS and physicochemical laboratory analyses, we assessed the quality and quantity of organic matter in the different types of aggregates separated manually according to their origin: large biogenic aggregates (LBA), medium sized biogenic aggregates (MBA) and small biogenic aggregates (SBA); physical aggregates (PA), root aggregates (RA) and residual soil (RS).Earlier studies had shown that these structures were presumably formed by the activity of macroinvertebrates of three different functional groups, Diplopoda and Isopoda litter transformers for SBA, epidendogeic earthworms for MBA and mesohumic endogeic earthworms for LBA, organized in a feeding succession.A Coinertia analysis between showed significant covariation among the two sets of physico chemical (22 variables) and NIR spectral (100 different wavelength absorbances) characterization of 135 samples representative of the three classes of biogenic macroaggregates, physical aggregates and residual non macroaggregated soil, (RV = 0.55; p < 0.001). This analysis clearly separated biogenic structures and ranked them according to their size, from small SBA to medium sized MBA and large LBA. Physical PA aggregates and RS residual soil were projected close to the LBAs in the coinertia factorial plan. Multiple combinatorial data analysis CDA, associated 5 specific wavelength absorbance ranges with aggregate types and residual soil. Along the sequence from small to large biogenic aggregates, residual soil and physical aggregates, wavelengths associated to easily decomposed substrates (in ranges 1708–1716 nm; 1796–1948 and 2164–2316 nm) had progressively decreasing absorbances. Substrates associated to slowly decomposing aromatic, alkane and phenolic substrates either increased (1420–1436) or decreased (1284–1380) along this sequence.These results are compatible with the hypothesis of a progressive transformation and transfer of organic residues first into small biogenic aggregates that are presumably faecal pellets of Isopoda and Diplopoda, then to medium sized biogenic aggregates identified as casts of epiendogeic earthworms and finally to large biogenic aggregates expected to be the casts of the large endogeic earthworms present in these soils. A definitive verification of the hypothesis and the involvement of specific macroinvertebrates will require direct experimentation in controlled laboratory conditions.

19F NMR Reveals the Dynamics of Substrate Binding and Lid Closure for Iodotyrosine Deiodinase as a Complement to Steady-State Kinetics and Crystallography.

Active site lids are common features of enzymes and typically undergo conformational changes upon substrate binding to promote catalysis. Iodotyrosine deiodinase is no exception and contains a lid segment in all of its homologues from human to bacteria. The solution-state dynamics of the lid have now been characterized using 19F NMR spectroscopy with a CF3-labeled enzyme and CF3O-labeled ligands. From two-dimensional 19F-19F NMR exchange spectroscopy, interconversion rates between the free and bound states of a CF3O-substituted tyrosine (45 ± 10 s-1) and the protein label (40 ± 3 s-1) are very similar and suggest a correlation between ligand binding and conformational reorganization of the lid. Both occur at rates that are ∼100-fold faster than turnover, and therefore these steps do not limit catalysis. A simple CF3O-labeled phenol also binds to the active site and induces a conformational change in the lid segment that was not previously detectable by crystallography. Exchange rates of the ligand (130 ± 20 s-1) and protein (98 ± 8 s-1) in this example are faster than those above but remain self-consistent to affirm a correlation between ordering of the lid and binding of the ligand. Both ligands also protect the protein from limited proteolysis, as expected from their ability to stabilize a compact lid structure. However, the minimal turnover of simple phenol substrates indicates that such stabilization may be necessary but is not sufficient for efficient catalysis.

In situ Generation of Cyclohexanone Drives Electrocatalytic Upgrading of Phenol to Nylon-6 Precursor.

Coupling in situ generated intermediates with other substrates/intermediates is a viable approach for diversifying product outcomes of catalytic reactions involving two or multiple reactants. Cyclohexanone oxime is a key precursor for caprolactam synthesis (the monomer of Nylon-6), yet its current production uses unsustainable carbon sources, noble metal catalysts, and harsh conditions. Herein, we report the first work to synthesize cyclohexanone oxime through electroreduction of phenol and hydroxylamine. The Faradaic efficiency reached 69.1 % over Cu catalyst, accompanied by a corresponding cyclohexanone oxime formation rate of 82.0 g h-1 gcat -1. In addition, the conversion of phenol was up to 97.5 %. In situ characterizations, control experiments, and theoretical calculations suggested the importance of balanced activation of water, phenol, and hydroxylamine substrates on the optimal metallic Cu catalyst for achieving high-performance cyclohexanone oxime synthesis. Besides, a tandem catalytic route for the upgrading of lignin to caprolactam has been successfully developed through the integration of thermal catalysis, electrocatalysis, and Beckmann rearrangement, which achieved the synthesis of 0.40 g of caprolactam from 4.0 g of lignin raw material.

Palladium‐Catalyzed Sc(OTf)3/Cs2CO3‐Assisted C−O Cross‐Coupling of Vinylidenecyclopropane‐Diesters with Phenolic Compounds

AbstractWe herein describe a method in the synthesis of aryl ether derivatives from a cross‐coupling reaction of vinylidenecyclopropane‐diesters (VDCP‐diesters) with commercially available phenolic compounds for the formation of C−O bond under palladium catalysis and assisted by Sc(OTf)3/Cs2CO3. This developed transformation proceeds through a nucleophilic addition at the 2‐position of allenic moiety at 50 °C for 4.0 h and is compatible with substituents such as bromine, iodine and borate on the phenolic substrates via a zwitterionic π‐propargyl palladium species, affording the desired products in 29% to 95% isolated yields. A plausible reaction mechanism has been also proposed on the basis of control, deuterium labeling experiments and density functional theory (DFT) calculations.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 μM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

Computational Insights into Enzyme‐Substrate Binding Interplay Exhibit Variable Binding Attributes: A Framework for Implementing Oxidoreductase‐Based Applications

AbstractLaccase (LAC) is a potent multicopper oxidase that relies on O2 for its catalytic activity. LAC has been affirmed as an environmentally friendly biocatalyst that often catalyzes a wide array of phenolic substrates. Bacterial‐derived LACs have been less investigated for non‐phenolic substrates in contrast to fungi‐derived LAC. To comprehend the substrates (3,4‐Dimethoxybenzyl alcohol, and Dimer (Guaiacyl 4‐O‐5 guaiacyl) binding interactions of LAC (Thermus thermophilus HB27) was carried out and contrasted with fungal‐derived Lignin peroxidase (LiP) (Trametes cervina) exploiting computational methods, including physicochemical properties, Sequence Annotated by Structure (SAS), Extra precision docking (Glide), and DESMOND‐directed MD‐ simulation. Protein structures exhibited by LAC, and LiP have diverse dissimilar component architects. The XP docking suggested LiP‐Dimer seems to have a comparatively lowest binding affinity (−8.413 kcal/mol), with an MMGBSA score of −33.249 kcal/mol. Further, docked complexes were validated leveraging 50 ns NPT system‐based MD‐simulation for structural and functional stability. The system achieved equilibrium and stability at the end of the simulation, with only the LiP‐3,4‐Dimethoxybenzyl alcohol complex maintained stability. The results of this study offer a framework for improving the binding ability of substrates by way of the use of in‐silico protein engineering, which might eventually result in more effective catalytic applications.

Graphene oxide microstrip antenna-based sensor for oxygen gas sensing in medical applications

In this work, we report the development of a new graphene oxide (GO) sensor based on a microstrip antenna for detecting and measuring O2 concentration. GO was prepared from natural graphite powder using a modified Hummers procedure and then deposited upon a phenolic substrate to act as the sensing element of the designed device. Detection tests employing the sensor to evaluate oxygen gas (O2) in higher concentrations were performed, whereas it was observed that the two operating frequencies of the device was displaced to higher values as the concentration oxygen gas increased. The effect of relative humidity (RH) in the response sensor too was observed, whereas the 9.704 GHz mode demonstrating to be the most sensitive to O2 variation after sensitivity values have been corrected, presenting limit of detection (LOD) equal to 0.38%O2. Evaluating the results, it is possible to note that the device studied could be used as sensor in medical equipment operating at elevated oxygen concentrations, such as ventilators, O2 concentrators, and other medical equipment. Moreover, evaluated sensor would be a promisor candidate for healthcare monitoring applications for the reason that this device could act as both an O2 sensor and antenna, and thus transmitting their information to other devices.

A Robust Pyrazolate Metal-Organic Framework for Efficient Catalysis of Dehydrogenative C-O Cross Coupling Reaction.

Construction of robust heterogeneous catalysts with atomic precision is a long-sought pursuit in the catalysis field due to its fundamental significance in taming chemical transformations. Herein, we present the synthesis of a single-crystalline pyrazolate metal-organic framework (MOF) named PCN-300, bearing a lamellar structure with two distinct Cu centers and one-dimensional (1D) open channels when stacked. PCN-300 exhibits exceptional stability in aqueous solutions across a broad pH range from 1 to 14. In contrast, its monomeric counterpart assembled through hydrogen bonding displays limited stability, emphasizing the role of Cu-pyrazolate coordination bonds in framework robustness. Remarkably, the synergy of the 1D open channels, excellent stability, and the active Cu-porphyrin sites endows PCN-300 with outstanding catalytic activity in the cross dehydrogenative coupling reaction to form the C-O bond without the "compulsory" ortho-position directing groups (yields up to 96%), outperforming homogeneous Cu-porphyrin catalysts. Moreover, PCN-300 exhibits superior recyclability and compatibility with various phenol substrates. Control experiments reveal the synergy between the Cu-porphyrin center and framework in PCN-300 and computations unveil the free radical pathway of the reaction. This study highlights the power of robust pyrazolate MOFs in directly activating C-H bonds and catalyzing challenging chemical transformations in an environmentally friendly manner.

After 50 Years of Hepatic Clearance Models, Where Should We Go from Here? Improvements and Implications for Physiologically Based Pharmacokinetic Modeling.

There is overwhelming preference for application of the unphysiologic, well-stirred model (WSM) over the parallel tube model (PTM) and dispersion model (DM) to predict hepatic drug clearance, CLH , despite that liver blood flow is dispersive and closer to the DM in nature. The reasoning is the ease in computation relating the hepatic intrinsic clearance ( CLint ), hepatic blood flow ( QH ), unbound fraction in blood ( fub ) and the transmembrane clearances ( CLin and CLef ) to CLH for the WSM. However, the WSM, being the least efficient liver model, predicts a lower EH that is associated with the in vitro CLint ( Vmax / Km ), therefore requiring scale-up to predict CLH in vivo. By contrast, the miniPTM, a three-subcompartment tank-in-series model of uniform enzymes, closely mimics the DM and yielded similar patterns for CLint versus EH , substrate concentration [S] , and KL / B , the tissue to outflow blood concentration ratio. We placed these liver models nested within physiologically based pharmacokinetic models to describe the kinetics of the flow-limited, phenolic substrate, harmol, using the WSM (single compartment) and the miniPTM and zonal liver models (ZLMs) of evenly and unevenly distributed glucuronidation and sulfation activities, respectively, to predict CLH For the same, given CLint ( Vmax and Km ), the WSM again furnished the lowest extraction ratio ( EH,WSM = 0.5) compared with the miniPTM and ZLM (>0.68). Values of EH,WSM were elevated to those for EH, PTM and EH, ZLM when the Vmax s for sulfation and glucuronidation were raised 5.7- to 1.15-fold. The miniPTM is easily manageable mathematically and should be the new normal for liver/physiologic modeling. SIGNIFICANCE STATEMENT: Selection of the proper liver clearance model impacts strongly on CLH predictions. The authors recommend use of the tank-in-series miniPTM (3 compartments mini-parallel tube model), which displays similar properties as the dispersion model (DM) in relating CLint and [ S ] to CLH as a stand-in for the DM, which best describes the liver microcirculation. The miniPTM is readily modified to accommodate enzyme and transporter zonation.

Inhibition of enzymatic browning in freeze-thawed apricot fruit by combined chlorogenic acid and osmotic dehydration treatments

In this study, the inhibitors of apricot browning were analyzed by investigating the effects of osmotic dehydration (OD) pretreatment combined with chlorogenic acid (CA) on cell disruption, loss of phenolic substrates, and changes in key enzymes during freeze-thawing. Both OD and OD-CA mitigated the deterioration caused by ice crystal damage during freeze-thawing. OD and OD-CA significantly reduces the BI of apricots by 43.27 and 59.55 compared to CK. The PPO and POD activity in OD-CA apricots are only 61.11% and 42.13% of those in OD. OD-CA could also protect SOD and CAT activity, with their activity in the OD-CA being 1.41 and 1.14 times that of OD group. This indicated that OD-CA treatment protected against the activities of ROS-scavenging enzymes. Compared with OD, OD-CA could better protect the bioactive substance of apricot. The contents of ascorbic acid, total phenols, and total flavonoids in OD-CA were 1.32 times, 1.11 times, and 1.05 times higher than those in OD. OD-CA also effectively protected chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, catechins, and rutin. In summary, OD-CA primarily reduced the browning by protecting the integrity of cell structures, minimizing the potential for exposure to air, enzymes, and phenolic substrates, and inhibiting browning enzymes activity.

Purification and Biochemical Characterization of Polyphenol Oxidase Extracted from Wheat Bran (Wan grano).

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL-1·min-1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of -5.28 kcal/mol, -4.65 kcal/mol, -4.21 kcal/mol, and -5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea.

Mutability landscape guided engineering of a promiscuous microbial glycosyltransferase for regioselective synthesis of salidroside and icariside D2

Microbial glycosyltransferases efficiently synthesize glucosides and have garnered increasing interest. However, limited regioselectivity has impeded their broad application, particularly in the pharmaceutical industry. In this study, the UDP-glycosyltransferase YjiC from Bacillus licheniformis (BlYjiC) was engineered to achieve the bidirectional regioselective glycosylation of tyrosol and its derivatives. Initially, site-directed saturation mutagenesis was performed on two newly identified substrate-binding cavities in the acceptor pocket of BlYjiC to provide a comprehensive blueprint of the interplay between mutations and function (mutability landscape). Iterative saturation mutagenesis was performed, guided by the mutability landscape. Two highly regioselective mutants M6 (M112L/I325Y/L70R/Q136E/I67E/M77R) and M2’ (M112D/I62L) were generated, exhibiting >99 % regioselectivity toward the alcoholic and phenolic hydroxyl of tyrosol, respectively, compared with the wild-type (product mixture: 51:49 %). Both mutants exhibited excellent regioselectivity toward several dihydroxy phenolic substrates, offering valuable biocatalysts for the regioselective synthesis of glucosides. Their application was confirmed in a short synthesis of salidroside (3.6 g/L) and icariside D2 (2.4 g/L), which exhibited near-perfect regioselectivity. This study provides valuable insights into future protein engineering of similar enzymes and opens new avenues for their practical applications.

Almond By-Products Substrates as Sustainable Amendments for Green Bean Cultivation.

Almond processing generates a high quantity of by-products, presenting the untapped potential for alternative applications and improved sustainability in production. This study aimed to evaluate whether the incorporation of almond by-products (hulls/shells) can improve the biochemical characteristics of green bean pods when used as an alternative to traditional growing media in green bean plants. Four substrates were prepared: the Control substrate (C): 70% peat + 30% perlite; substrate (AS): 70% peat + 30% shells; substrate (AH): 70% peat + 30% perlite + 1 cm hulls as mulch; substrate (MIX): 70% peat + 15% shells + 15% hulls. Plants were grown in each of these substrates and subjected to two irrigation levels, 100% and 50% of their water-holding capacity. Biochemical parameters (photosynthetic pigments, total phenolics, flavonoids, ortho-diphenols, soluble proteins, antioxidant capacity) and color were evaluated in the harvested pods. Results showed that pods from plants growing in AH substrate presented statistically significant higher values in their total phenolic content, while AS and MIX substrates did not reveal significant benefits. Summarily, this study highlights the potential of almond hulls as a promising medium for green bean cultivation, particularly when employed as mulch. Further research is recommended to gain a more comprehensive understanding of the application of almond by-products as natural fertilizers/mulch.

Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalase.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal

Chia gum’s molecular structure with distinctive properties as well as the alginate-based hydrogel’s three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30–50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30–40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.