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The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant-based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose-degrading ascomycetes for their capacity to react with G-type and S-type phenolic compounds. All six PPOs catalyze the ortho-hydroxylation of G-type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy-ortho-diphenols. Remarkably, a subset of these PPOs is also active towards S-compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy-ortho-diphenols. Assays with 18 O 2 demonstrate that these PPOs in particular catalyze ortho-hydroxylation and ortho-demethoxylation of S-compounds and generate methanol as a co-product. Oxidative (ortho-) demethoxylation of S-compounds is a novel reaction for PPOs, which we propose occurs by a distinct reaction mechanism as compared to aryl-O-demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy-ortho-diphenols from both G- and S-type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin-derived compounds., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Polyphenol oxidase (PPO) is the culprit behind the browning of fruits and vegetables. Therefore, how to reduce the thermal deactivation temperature of PPO or use as few safe reagents as possible to inhibit enzymatic browning has practical significance. Mesoporous silica nanoparticles (MSNs) and multiwalled carbon nanotubes (MWCNTs) are stable and have high biosafety. In the present study, efficient PPO inhibitors were developed based on MSNs and MWCNTs. It is found that after modification with a very small amount of dodecyl trimethylammonium bromide (DTAB, ≥60 μg/mL), MSNs can significantly inhibit the activity of PPO although single MSNs and single DTAB show very limited effect on PPO activity. After modification with a very small amount of sodium dodecyl sulfate (SDS, 5.7-9.5 μg/mL), MWCNTs almost completely inactivate PPO. However, SDS@MSN and DTAB@MWCNT cannot decrease PPO activity significantly.

Excessive tyrosinase expression leads to pigmented diseases in humans and browning in plants, necessitating effective tyrosinase inhibitors. This study investigated the inhibitory effect and mechanism of 7-hydroxycoumarin-3-carboxylic acid (7-HC-3-CA) on tyrosinase. Using UV-visible absorption spectroscopy, we found that 7-HC-3-CA effectively inhibited tyrosinase activity, with an IC 50 value of 364 ± 1.3 μM. Enzyme kinetics, fluorescence methods and molecular simulation techniques revealed that 7-HC-3-CA acted as a reversible and competitive inhibitor, forming a stable complex with tyrosinase through hydrophobic interactions and hydrogen bonding. This altered the microenvironment of Tyr and Trp residues, causing the structural stretching and conformational changes that diminish catalytic activity. Preservation experiments demonstrated that 0.5 mM 7-HC-3-CA significantly reduced mass loss and decreased browning of fresh-sliced apples. It also lowered polyphenol oxidase activity from 0.22 to 0.18 and delayed phenolic oxidation, enhancing total phenolic content from 0.34 to 0.54, thereby controlling browning and extending storage life. Cell assays indicated that 0.5 mM 7-HC-3-CA had no significant impact on cell proliferation, with viability over 80 %. Acute toxicity tests proved that 0.5 mM of 7-HC-3-CA is completely non-lethal to KM mice. In conclusion, this study confirmed 7-HC-3-CA was a viable and safe antibrowning agent and revealed its potential application in the field of food preservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The development of natural inhibitors of polyphenol oxidase (PPO) is crucial in the prevention of enzymatic browning in fresh foods. However, few studies have focused on the effect of subsequent sterilization on their inhibition efficiency. This study investigated the influence and mechanism of high hydrostatic pressure (HHP) on the inhibition of PPO by epigallocatechin gallate (EGCG), cyanidin-3-O-glucoside (C3G), and ferulic acid. Results showed that under the conditions of 550 MPa/30 min, the activity of EGCG-PPO decreased to 55.92%, C3G-PPO decreased to 81.80%, whereas the activity of FA-PPO remained stable. Spectroscopic experiments displayed that HHP intensified the secondary structure transformation and fluorescence quenching of PPO. Molecular dynamics simulations revealed that at 550 MPa, the surface interaction between PPO with EGCG or C3G increased, potentially leading to a reduction in their activity. In contrast, FA-PPO demonstrated conformational stability. This study can provide a reference for the future industrial application of natural inhibitors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present work evaluated kiwi juice addition alongside pasteurization (at 85 °C for 5 min) or microwave treatment (for 3 min) on the quality improvement of sugarcane juice. The juice was treated in the presence of kiwi juice (0-8%), and its physicochemical properties and microbial load were compared with raw juice. The study also highlighted the key enzymes causing sugarcane juice discoloration, peroxidase (POD) and polyphenol oxidase (PPO), by quantifying kiwi juice constituents using GC-MS and monitoring their effects by molecular docking. Kiwi addition considerably raised (p < 0.05) acidity, ascorbic acid (54.28%), and phenolic compounds (32%), and decreased the POD and PPO activity of raw cane juice. Pasteurization in the presence of kiwi, rather than microwave treatment, has significantly (p < 0.05) increased the phenolic compounds and reduced POD and PPO activities until barley was detected. Molecular docking revealed that heptacosane, oleic acid, and melezitose are the primary kiwi components responsible for enzyme inactivation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Laccase is an enzyme known for its eco-friendly uses in environmental cleanup and biotechnology. However, it has limitations such as low stability, high cost, and complex recycling. So, there is a need for laccase mimics that can effectively imitate its properties. Herein, we created copper formate-lysine nanoparticles (Cuf-Lys) that mimic laccase's activity. The developed Cuf-Lys demonstrated remarkable polyphenol oxidase-like activity, stability, and recyclability, making them suitable for the fabrication of efficient colorimetric sensors for the detection of epinephrine. These sensors had a specific response and could accurately measure epinephrine concentrations ranging from 2.5 to 50 μM, with a detection limit as low as 1 μM. Furthermore, the biosensor demonstrated high sensitivity and selectivity when applied to the detection of rutin. The limit of detection for rutin was determined to be 0.16 μM while in the linear concentration range of 0.25 to 150.0 μM. We believe that Cuf-Lys provide a new route for the design of laccase mimics, showing potential applications for biomedical diagnosis and environmental monitoring., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

This study was aimed to evaluate the potential of high-humidity hot air impingement cooking (HHAIC) on Penaeus vannamei, focusing on its drying characteristics, microstructure, water distribution, enzyme activity, astaxanthin content, antioxidant capacity, color, and Maillard reaction. Results demonstrated that a 3 min HHAIC significantly improved the shrimp's color and optimized astaxanthin content with a notable increase in scavenging capacity based on an in-vitro as antioxidation activity evaluation. Compared to the untreated samples, HHAIC could significantly inactivate polyphenol oxidase by 95.76%. Also, it suppressed the Maillard reaction by decreasing 5-hydroxymethylfurfural content and shortened the drying time by 40%. In addition, the low-field nuclear magnetic resonance and microstructure analysis showed alterations in the shrimp muscle fiber structure and water distribution. This study indicated that HHAIC could elevate quality, enhance appearance, and reduce the processing time of dried shrimp, presenting valuable implications for industry progress., Competing Interests: Declaration of competing interest The authors confirm no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Copper metalloenzymes ascorbate oxidase (AOase), amine oxidase (AmOase), and catechol oxidase (COase) possess copper(II) sites of coordination, which are trimeric, homodimeric, and dimeric, respectively. Two newly mononuclear copper(II) complexes, namely, [Cu(L)(bpy)](ClO 4 ) (1) and [Cu(L)(phen)](ClO 4 ) (2) where HL = Schiff base, have been synthesized. UV-visible, EPR and single-crystal X-ray diffraction examinations were used to validate the geometry in solution and solid state. For complex 1, the metal exhibits a coordination sphere between square pyramidal and trigonal bipyramidal geometry (τ, 0.49). A positive Cu II / I redox potential indicates a stable switching between Cu II and Cu I redox states. Despite the monomeric origin, both homogeneous catalysts (1 or 2) in MeOH were found to favor three distinct chemical transformations, namely, ascorbic acid (H 2 A) to dehydroascorbic acid (DA), benzylamine (Ph-CH 2 -NH 2 ) to benzaldehyde (Ph-CHO), and 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) [k cat : AOase, 9.6 (1) or 2.0 × 10 6  h -1 (2); AmOase, 13.4 (1) or 9.4 × 10 6  h -1 (2); COase, 2.0 (1) or 1.9 × 10 3  h -1 (2)]. They exhibit higher levels of AOase activity as indicated by their k cat values compared to the AOase enzyme. The k cat values for COase activity in buffer solution [5.93 (1) or 2.95 × 10 5  h -1 (2)] are one order lower than those of the enzymes. This is because of the labile nature of the coordinated donor, the flexibility of the ligand, the simplicity of the catalyst-substrate interaction, and the positive Cu II / I redox potential. Interestingly, more efficient catalysis is promoted by 1 and 2 concerning that of other mono- and dicopper(II) complexes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bael (Aegle marmelos) beverage was pasteurized using continuous-microwave (MW) and traditional thermal processing and the activity of native enzymes, pulp-hydrolyzing enzymes, bioactive, physicochemical, and sensory properties were analyzed. First-order and linear biphasic models fitted well (R 2  ≥ 0.90) for enzyme inactivation and bioactive alteration kinetics, respectively. For the most resistant enzyme, polyphenoloxidase (PPO), the inactivation target of ≥ 90 % was achieved at 90 °C T MW (final temperature under MW) and 95 °C for 5 min (conventional thermal). MW treatment displayed faster enzyme inactivation and better retention of TPC and AOC. MW treatment at 90 °C T MW showed 5.3 min D-value, 90% total carotenoid content, 3.42 crisp sensory score (out of 5), and no or minor change in physicochemical attributes. Thermal and MW treatment caused the loss of 14 and 10 bioactive compounds, respectively. The secondary and tertiary structural modifications of PPO enzyme-protein revealed MW's lethality primarily due to its thermal effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Anthocyanins are bioactive compounds responsible for various physiological processes in plants and provide characteristic colors to fruits and flowers. Their biosynthetic pathway is well understood; however, the enzymatic degradation mechanism is less explored. Anthocyanase (β-glucosidase (BGL)), peroxidase (POD), and polyphenol oxidase (PPO) are enzymes involved in degrading anthocyanins in plants such as petunias, eggplants, and Sicilian oranges. The aim of this work was to investigate the physicochemical interactions between these enzymes and the identified anthocyanins (via UPLC-MS/MS) in cranberry ( Vaccinium macrocarpon ) through molecular docking to identify the residues likely involved in anthocyanin degradation. Three-dimensional models were constructed using the AlphaFold2 server based on consensus sequences specific to each enzyme. The models with the highest confidence scores (pLDDT) were selected, with BGL, POD, and PPO achieving scores of 87.6, 94.8, and 84.1, respectively. These models were then refined using molecular dynamics for 100 ns. Additionally, UPLC-MS/MS analysis identified various flavonoids in cranberries, including cyanidin, delphinidin, procyanidin B2 and B4, petunidin, pelargonidin, peonidin, and malvidin, providing important experimental data to support the study. Molecular docking simulations revealed the most stable interactions between anthocyanase and the anthocyanins cyanidin 3-arabinoside and cyanidin 3-glucoside, with a favorable ΔG of interaction between -9.3 and -9.2 kcal/mol. This study contributes to proposing a degradation mechanism and seeking inhibitors to prevent fruit discoloration.

Polyphenol oxidase (PPO) plays a key role in the enzymatic browning process, and this study employed Gaussian-accelerated molecular dynamics (GaMD) simulations to investigate the catalytic efficiency mechanisms of lotus root PPO with different substrates, including catechin, epicatechin, and chlorogenic acid, as well as the inhibitor oxalic acid. Key findings reveal significant conformational changes in PPO that correlate with its enzymatic activity. Upon substrate binding, the alpha-helix in the Q53-D63 region near the copper ion extends, likely stabilizing the active site and enhancing catalysis. In contrast, this helix is disrupted in the presence of the inhibitor, resulting in a decrease in enzymatic efficiency. Additionally, the F350-V378 region, which covers the substrate-binding site, forms an alpha-helix upon substrate binding, further stabilizing the substrate and promoting catalytic function. However, this alpha-helix does not form when the inhibitor is bound, destabilizing the binding site and contributing to inhibition. These findings offer new insights into the substrate-specific and inhibitor-induced structural dynamics of lotus root PPO, providing valuable information for enhancing food processing and preservation techniques.

Phenolic compounds (PCs) are diverse in nature and undergo complex migration and transformations in the environment, making it challenging to use techniques such as chromatography and other traditional methods to determine the concentration of PCs by separation, individual monitoring and subsequent addition. To address this issue, a facile and on-site strategy was developed to measure the concentration of PCs using a novel nanozyme with polyphenol oxidase-like activity. First, the nanozyme was designed by coordinating the asymmetric ligand nicotinic acid with copper to mimic the structure of mononuclear and trinuclear copper clusters of natural laccases. Subsequently, by introducing 2-mercaptonicotinic acid to regulate the valence state of copper, the composite nanozyme CuNA10S was obtained with significantly enhanced activity. Interestingly, CuNA10S was shown to have a broad substrate spectrum capable of catalyzing common PCs. Building upon the superior performance of this nanozyme, a method was developed to determine the concentration of PCs. To enable rapid on-site sensing, we designed and prepared CuNA10S-based test strips and developed a tailored smartphone sensing platform. Using paper strip sensors combined with a smartphone sensing platform with RGB streamlined the sensing process, facilitating rapid on-site analysis of PCs within a range of 0-100 μM. Our method offers a solution for the quick screening of phenolic wastewater at contaminated sites, allowing sensitive and quick monitoring of PCs without the need for standard samples. This significantly simplifies the monitoring procedure compared to more cumbersome large-scale instrumental methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Sugarcane juice, which has a short shelf life, is a popular thirst-quenching and rejuvenating beverage worldwide. The limited shelf life is a result of changes in polyphenol oxidase (PPO) activity, total plate count (TPC) and color attributes (L*, a* and b*-values). We hypothesized that chemical kinetics and thermodynamics of blanched sugarcane cane juice causing alterations in PPO, TPC, and L, a* and b*-values will address the challenges of sugarcane juice preservation., Results: Sugarcane billets were blanched at variable time-temperature combinations in the range 0-20 min and 70-90 °C. Reaction rates increased with increasing temperature; PPO activity, TPC and colour followed first-order kinetics. PPO activity had an activation energy (Ea) of 81 kJ mol -1 . The half life (t ½ ) dropped from 16.5 to 3.47 min and decimal reduction time (D-values) dropped from 54.83 to 11.52 min. Thus reactions were temperature-sensitive. Thermodynamic studies indicated an endothermic (positive enthalpy values, ΔH > 0; 78.10 kJ mol -1 ) and reversible process (negative entropy values ΔS < 0; -0.044 kJmol -1  K -1 ). Michaeli-Menten constant (K m ) and maximum velocity (V max ) of PPO activity were determined by adding variable lemon juice concentrations in sugarcane juice. As the Km values increased (from 5.53 to 15.81 mm) and V max values decreased (from 666.67 to 384.61 UmL -1 ), a Lineweaver-Burk plot suggested decreased PPO affinity of sugarcane juice., Conclusion: The results of the present study indicate that studies on chemical kinetics and thermodynamics (PPO, TPC and L, a* and b*-values) of blanched sugarcane cane juice shall mitigate challenges of sugarcane juice preservation. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

An optimized multi-stage separation strategy was developed to purify lily bulb polyphenol oxidase (PPO) for revealing its molecular structure. The PPO was purified 14.64-fold with high specific activity of 153,900 U/mg via optimized conditions of phosphate buffer pH (6.5), solid-liquid ratio (1:3), PVPP content (2 %), extraction time (4 h), followed by 30 %-50 % ammonium sulfate, diethylaminoethyl ion-exchange chromatography (0.1 M NaCl), and size exclusion chromatography. The PPO was identified as a dimeric protein with molecular weight of 135 kDa, containing 58.79 % random coil, 20.78 % α-helix, 17.41 % β-folding, and 3.02 % β-corner. The three-dimensional structure via homology modeling suggested that active center CuA bound to His151, His172, and His181, CuB bound to His307, His311, and His341. Furthermore, molecular docking indicated that its Phe337 and Tyr312 residues were catalytic cavity gates of catechol and 4-methylcatechol, respectively. Therefore, this study successfully analyzed purified PPO structure and further provided a theoretical foundation for its browning mechanism., Competing Interests: Declaration of competing interest No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication. The work described was original research that has not been published previously, and not under consideration for publication elsewhere. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed to elucidate the changes of browning-related metabolite in fresh-cut potato and to identify anti-browning agents. Metabolomics and weighted correlation network analysis (WGCNA) were used to identify metabolites and correlate them with potato browning traits. A total of 79 browning trait-positive-related metabolites and 19 browning trait-negative-related metabolites were obtained from four key modules via WGCNA. The accumulation of metabolites with rich reducing groups and acidic groups were found to enhance anti-browning activity in potatoes. Among these metabolites, only L-pyroglutamic acid (L-PA) and ascorbic acid had variable importance for the projection (VIP) values greater than 1.5. In addition, it was found that L-PA inhibited polyphenol oxidase (PPO) activity by lowering pH and interacting with amino acid residues of PPO. L-PA also inhibited the growth of microorganisms in fresh-cut potato. Our results show that L-AP is an effective novel anti-browning agent with antibacterial activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Whole white button mushrooms (WWBM) exhibit a limited shelf-life owing to the oxidative enzymatic browning. Inactivation of polyphenol oxidase-PPO and peroxidase-POD in WWBM and its kinetic behavior were studied using pulsed light(PL) treatment (0.13-1.11 J/cm 2 ). The first-order kinetics explained PL-induced enzyme inactivation. Rate constants(k) for PPO and POD were 3.84 and 2.55 cm 2 /J. FTIR-analysis revealed secondary-structural changes in partially-purified enzyme. PL-treatment retarded browning, retained phenolics and enhanced vitamin D 2 . PL-treatment at 1.11 J/cm 2 rendered WWBM both microbially and enzymatically stable. The PL-treated WWBM's shelf-life at 4, 20, and 37 °C were 5, 3, and 1 day. At 4 °C, browning increased by 6.1 %; firmness decreased by 55.2 %, while PL-treated mushrooms retained 90.6 % phenolics, 78.9 % antioxidant capacity, and 64.2 % D 2 after 5 days. Higher activation energy value confirmed phenolics were most sensitive during storage. PL-technology supports UN Sustainable Development Goals by reducing chemical use, lowering carbon-footprints, minimizing pollution, and enhancing shelf-life, promoting sustainable global trade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fruit derivatives are commonly obtained by applying processing operations deemed responsible for the loss of phenol compounds, but very little information is available on the fate of phenols upon digestion of these products. The present study evaluated the effect of thermal and mechanical treatments, commonly applied to turn apple pulp into puree and homogenate, on phenolic bioaccessibility and antioxidant activity. Despite a 20 % decrease in polyphenols due to processing, their bioaccessibility was higher in apple derivatives (>20 %) compared to pulp (∼2 %). Polyphenol oxidase (PPO), inactivated by thermal treatments in apple derivatives but not in the pulp, was hypothesized to be responsible for this difference. Results acquired on an unprocessed PPO-free apple model, only featuring quercetin-3-glucoside and pectin, actually exhibited similar bioaccessibility as processed derivatives. The radical scavenging capacity was unaffected by the structural integrity of apples, indicating independence from the plant tissue's hierarchical arrangement. After digestion, radical scavenging capacity decreased in the real apple matrices, correlating with phenolic content, while it was retained in the apple model, further suggesting the pivotal food matrix role in modulating polyphenols bioaccessibility and subsequent biological activity. Translating these results to an industrial scale, processing conditions can be optimized not only to guarantee that the quality requirements are met, but also to achieve desired nutritional benefits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Theaflavins are beneficial to human health due to various bioactivities. Biosynthesis of theaflavins using polyphenol oxidase (PPO) is advantageous due to cost effectiveness and environmental friendliness. In this review, studies on the mechanism of theaflavins formation, the procedures to screen and prepare PPOs, optimization of reaction systems and immobilization of PPOs were described. The challenges associated with the mass biosynthesis of theaflavins, such as poor enzyme activity, undesirable subproducts and inclusion bodies of recombinant PPOs were presented. Further strategies to solve these challenges and improve theaflavins production, including enzyme engineering, immobilization enzyme technology, water-immiscible solvent-water biphasic systems and recombinant enzyme technology, were proposed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Accurate and quick judgement of the food quality can protect the legitimate rights of consumers. Currently, nanozymes are widely employed in the rapid detection of food due to their stability and economy. The contents of bisphenol A and antioxidant can be used to measure the quality of beverages. However, due to the complexity of the actual samples, it is still challenging to achieve the sensitive detection of both at the same time. The development of nanozyme with high enzyme activity is essential for sensitive detection of targets in complex foods., Results: In this work, a novel nanomaterial (ZrTGA) was synthesized based on thioglycolic acid-modified Metal-Organic Framework (MOF-818). The interaction between Cu-S bonds and increase in the proportion of Cu 1+ resulted in ZrTGA exhibiting higher peroxidase-like and polyphenol oxidase-like activities. These enzyme activities were 317 % and 200 % of the original values, respectively. With high enzyme activity can sensitively detect two important indicators of bisphenol A and antioxidants in beverages. The increased enzyme activity of ZrTGA enabled the content of both substances to be detected by smartphone extraction of RGB. Finally, through the output of the ''0″ and ''1″ signals of the logic gates, it is possible to quickly determine the level of the two substances and thus directly assess the quality of the beverages., Significance: The modification of nanozyme enables the detection of substances at low concentrations based on enhancing dual-enzyme activity. The combination of mobile phone photography and logic gate technology enables the continuous detection of two important indicators in beverages, overcoming the limitations of traditional large-scale instruments. It also provides an alternative strategy for food quality detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Apostichopus japonicus (A. japonicus) has rich nutritional value and is an important economic crop. Due to its rich endogenous enzyme system, fresh A. japonicus is prone to autolysis during market circulation and storage, resulting in economic losses. In order to alleviate this phenomenon, we investigated the effect of polyphenol oxidase (PPO) mediated (-)-epigallocatechin gallate (EGCG) on the activity and structure of endogenous cathepsin series protein (CEP) from A. japonicus. Research on cathepsin activity showed that PPO mediated EGCG could significantly reduce enzyme activity, resulting in a decrease in enzymatic reaction rate. SDS-PAGE and scanning electron microscopy results showed that PPO mediates EGCG could induce CEP aggregation to form protein aggregates. Various spectral results indicated that EGCG caused changes in the structure of CEP. Meanwhile, the conjugates formed by PPO mediated EGCG had lower thermal stability. In conclusion, PPO mediated EGCG was an effective method to inhibit the endogenous enzyme activity., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Polyphenol oxidase (PPO) is an industrially important enzyme associated with browning reactions. In the present study, a set of ten new dihydropyridine [2,3-d] pyrimidines (TD-Hid-1-10) were synthesized and was found to be proven characteristically by 1 H NMR, 13 C NMR, IR, elemental analysis, and assessed as possible PPO inhibitors. PPO was purified from banana using three-phase partitioning, achieving an 18.65-fold purification and 136.47% activity recovery. Enzyme kinetics revealed that the compounds TD-Hid-6 and TD-Hid-7 are to be the most potent inhibitors, exhibiting mixed-type inhibition profile with IC 50 values of 1.14 µM, 5.29 µM respectively against purified PPO enzyme. Electronic structure calculations at the B3LYP/PBE0 level of theories using def-2 SVP, def2-TZVP basis sets with various molecular descriptors characterized the electronic behavior of studied derivatives TD-Hid-1-10. Molecular electrostatic potential (MEP) and reduced density gradient analyses of RDG-NCI provided insights into charge distributions and weak intermolecular interactions. Docking study simulations predicted binding poses within crucial amino acid sequence in the 2y9x enzyme's active site, which is typically similar in sequence to the PPO form is not allowed. Ligands were analysed in terms of binding energies, inhibitor concentrations (mM) and various molecular interactions such as H-bonds, H-carbon, π-carbon, π-sigma, π-sigma, π-π T-shaped, π-π stacked, π-alkyl, Van der Waals and Cu interactions. The lowest binding energy (-7.83 kcal/mol) and the highest inhibitory effect (1.83 mM) were shown by the ligand Td-Hid-6, which forms H-bonds with Met280 and Asn260, exhibits π-sigma interactions with His61 and π-alkyl interactions with Val283. Other ligands also showed different interactions with various amino acids; for example, the Td-Hid-1 ligand formed H-bonds with His244 and showed π-sigma interactions with His244 and Val283., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fabrication of nanozyme with catecholase-like catalytic activity faces the great challenge of merging outstanding activity with low cost as well as simple, rapid, and low-energy-consumed production, restricting its industrial applications. Herein, an inexpensive yet robust nanozyme (i.e., DT-Cu) via simple one-step coordination between diaminotriazole (DT) and CuSO 4 within 1 h in water at room temperature is constructed. The asymmetric dicopper site with CuN 3 O configuration for each copper as well as Cu─O bond length of ≈1.83 Å and Cu···Cu distance of ≈3.5 Å in DT-Cu resemble those in catechol oxidase (CO), which ensure its prominent intrinsic activity, outperforming most CO-mimicking nanozymes and artificial homogeneous catalysts. The use of inexpensive DT/CuSO 4 in this one-pot strategy endows DT-Cu with only ≈20% cost of natural CO per activity unit. During catalysis, O 2 experienced a 4e-dominated reduction process accompanied by the formation of 1 O 2 and H 2 O 2 intermediates and the product of H 2 O. Benefiting from the low cost as well as the distinctive structure and superior intrinsic activity, DT-Cu presents potential applications ranging from biocatalysis to analytical detection of biomolecules such as epinephrine and beyond., (© 2024 Wiley‐VCH GmbH.)

Copper selenide nanoparticles (CuSeNP) were synthesized using histidine, ethylenediamine, and sodium selenate as precursors by one-step microwave digestion methods. The as-prepared CuSeNPs exhibit excellent catechol oxidase mimic enzyme and catalase (CAT)-like activities. Dopamine (DA) can be oxidized to aminochrome with H 2 O 2 by CuSeNPs, and the intermediate product aminochrome can further react with α-naphthol to yield a highly fluorescent derivative. It was confirmed that Cr(III) could adsorb on the surface of CuSeNPs and inhibit the production of semiquinone radicals in the reaction system, and the catalytic activity of CuSeNPs was inhibited. The detection mechanisms, kinetics, and catalytic properties of CuSeNPs were systematically investigated. As a result, a novel fluorescence method for the assay of Cr(III) was established. The feasibility of CuSeNP nanozyme in detecting speciation Cr(III) in food samples was explored with satisfactory results. It showed the obvious potential for developing effective and dependable fluorescent detection method for protecting food safety., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A new nanozyme (CuGaa) with switchable enzyme-like activity of peroxidase and polyphenol oxidase was successfully prepared based on guanidinoacetic acid and copper. The two enzyme-like activities can be easily switched by changing temperature or adding MnCl 2 . At 4 °C, polyphenol oxidase-like activity decreased to nearly 1%, and the material is mainly characterized by peroxidase-like activity at this point. However, at 60 °C in the presence of 20 mM MnCl 2 , the peroxidase-like activity decreased to nearly 10%, and the polyphenol oxidase-like activity of the materials increased to 140%. Based on the switchable enzyme-like activity of CuGaa, detection methods for thymol and hydrogen peroxide were developed. In addition, a rapid combination strategy was further established combined with logic gate technology for the facile identification of complex contamination in honey, which provided new ideas for low-cost and rapid honey identification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (K i : 0.07 ± 0.03 μM), mushroom (K i : 0.7 ± 0.3 μM), and eggplant (K i : 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (K i : 1112 ± 324 μM), mushroom (K i : 567 ± 81 μM), and eggplant (K i : 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this study, inactivation of mushroom polyphenol oxidase (PPO) by low intensity direct current (DC) electric field and its molecular mechanism were investigated. In the experiments under 3 V/cm, 5 V/cm, 7 V/cm and 9 V/cm electric fields, PPOs were all completely inactivated after different exposure times. Under 1 V/cm, a residual activity of 11.88 % remained. The inactivation kinetics confirms to Weibull model. Under 1-7 V/cm, n value closes to a constant about 1.3. The structural analysis of PPO under 3 V/cm and 5 V/cm by fluorescence emission spectroscopy and molecular dynamics (MD) simulation showed that the tertiary structure was slightly changed with increased radius of gyration, higher potential energy and rate of C-alpha fluctuation. After exposure to the electric field, most of the hydrophobic tryptophan (TRP) residues turned to the hydrophilic surface, resulting the fluorescence red-shifted and quenched. Molecular docking indicated that the receptor binding domain of catechol in PPO was changed. PPO under electric field was MD simulated the first time, revealing the changing mechanism of the electric field itself on PPO, a binuclear copper enzyme, which has a metallic center. All these suggest that the low intensity DC electric field would be a promising option for enzymatic browning inhibition or even enzyme activity inactivation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'-OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one-pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6-fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5-100 %, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhG biosynthesis, but also serves as a proof-of-concept for cell-free enzymatic construction of diverse natural products., (© 2024 Wiley-VCH GmbH.)

Microbial biofilm accumulation poses a serious threat to the environment, presents significant challenges to different industries, and exhibits a large impact on public health. Since there has not been a conclusive answer found despite various efforts, the potential green and economical methods are being focused on, particularly the innovative approaches that employ biochemical agents. In the present study, we propose a bio-nanotechnological method using magnetic cross-linked polyphenol oxidase aggregates (PPO m-CLEA) for inhibition of microbial biofilm including multidrug resistant bacteria. Free PPO solution showed only 55-60% biofilm inhibition, whereas m-CLEA showed 70-75% inhibition, as confirmed through microscopic techniques. The carbohydrate and protein contents in biofilm extracellular polymeric substances (EPSs) were reduced significantly. The m-CLEA demonstrated reusability up to 5 cycles with consistent efficiency in biofilm inhibition. Computational work was also done where molecular docking of PPO with microbial proteins associated with biofilm formation was conducted, resulting in favorable binding scores and inter-residual interactions. Overall, both in vitro and in silico results suggest that PPO interferes with microbial cell attachment and EPS formation, thereby preventing biofilm colonization.

Polyphenol oxidase (PPO) is critical due to enzymatic browning in fruits and vegetables, developing economic impact in fruits industry. Metal-Organic Frameworks (MOF) have shown interesting characteristics such as water stability, low toxicity, and good adsorption yield, making them good candidates for PPO inactivation. Al-based-MOFs, MIL-53(Al), DUT-5, and MIL-110 were tested as PPO inactivators in apple juice by enzyme-MOF interactions at r.t. through two possible mechanisms, i) substrate scavengers (substrates:catechol and 4-methylcatechol) or ii) enzyme activity modifiers. The scavenging behavior of Al-based-MOFs was moderate, in the same magnitude, being catechol adsorption better than 4-methylcatechol. PPO activity was reduced by at least 70% by MIL-53(Al)/DUT-5 in 10/30 min respectively, and MIL-110 inactivated PPO in 50 min with some structural modifications. Enzyme-MOF interactions are major responsible for PPO inactivation. This could be a new applicability of MOFs, as an alternate PPO inactivation process, easily included in juice processing, retaining sensorial/nutritional properties, developed at r.t thus energy-cost-effective., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sugarcane juice is a popular beverage and is also processed to produce sugar. The polyphenol oxidase (PPO) in sugarcane juice causes enzymatic browning and makes the process of sugar production complex and cumbersome. Storage of sugarcane juice is also hampered by the high sugar content and rapid microbial fermentation. The present research assessed the potential of lemon juice (LJ) and ginger extract (GE) as natural inhibitors of PPO. Enzyme kinetics and the mechanism of inhibition of LJ and GE were studied. Primary investigation was carried out using molecular docking approach to assess the inhibitory potential of LJ and GE and to determine the nature of interaction between the enzyme and inhibitors. Extracts were used as inhibitors and studies revealed that both reduced the PPO activity. Subsequently, pure bioactive inhibitors such as ascorbic acid, citric acid, and 6-shogaol present in these natural extracts were used to study the mode of inhibition of PPO. Citric acid decreased PPO activity by lowering pH, while ascorbic acid was found to be a competitive inhibitor of PPO with a K i of 75.69 µM. The proportion of LJ and GE required in sugarcane juice was optimized on the basis of browning index and sensory acceptance. Further, the sugarcane cane juice after inhibition of PPO under optimized conditions was spray dried and evaluated for reconstitution properties. The product formulated in the present study is a new and effective approach to address quality-compromising issues associated with long-term storage of cane juice., (© 2024 Institute of Food Technologists.)

Background: Potato is an important non-cereal crop. It provides carbohydrates, a major source of energy in the human diet. Blanching during the processing of fresh fruits and vegetables is essential for their preservation. High-humidity hot-air impingement blanching (HHAIB) is a promising emerging technology for pretreating different food materials. This research aimed to identify the optimum HHAIB conditions for the inhibition of potato-browning enzymes, maintaining their nutritional and physical quality, and to compare this with conventional hot-water blanching (HWB)., Results: Polyphenol oxidase (PPO) inactivation, total phenol content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, color, textural attributes, thermal properties, microstructure, and particles crystallinity were evaluated. The relative humidity (RH), temperature, and duration of HHAIB required for PPO inactivation (2.59%) were 50%, 105 °C, and 4 min, respectively, which resulted in a complete gelatigination of potato starches, based on the thermal properties and the microstrcture of the blanched potatoes. These conditions led to improvements in TPC to 312.54 μg GAE.g -1 FP, DPPH scavenging to 1.99 μmol TE.g -1 FP, as well as enhancements in color and crystallinity. When HHAIB was conducted at lower temperatures (85 and 95 °C) there were negative effects on the blanched potatoes' color and crystallinity, along with a non-safe level of PPO activity., Conclusion: High-humidity hot-air impingement blanching was superior to HWB, inhibiting PPO, maintaining nutrients, and preserving physical properties, especially under the optimum conditions revealed by the principal component analysis. It provides an excellent technique for blanching and pretreating potatoes, preserving them, and maintaining their quality. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

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 V max (517.55 U·mL -1 ·min -1 ) and low K m (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. Cu 2+ and Ca 2+ 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.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase ( Oe PPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated Oe PPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. Oe PPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, Oe PPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six Oe PPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature Oe PPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

Phenolic acids are promising inhibitors of polyphenol oxidase (PPO), but the effects of carboxyl group and pH on their inhibition effects are still unclear. In this study, methyl cinnamate, cinnamic acid and 4-carboxycinnamic acid were investigated by their inhibitory effects with pH varied from 6.8 to 5.0. Results showed that 4-carboxycinnamic acid had the strongest inhibitory effect on PPO, followed by cinnamic acid and methyl cinnamate. Acidic pH enhanced the inhibitory effects of cinnamic acid and its derivatives on PPO, and the enhancement degree, IC 50 and K i declining degree were followed as 4-carboxycinnamic acid > cinnamic acid > methyl cinnamate. Methyl cinnamate exhibited competitive inhibition on PPO, while cinnamic acid and 4-carboxycinnamic acid exhibited mixed-type inhibition. Inhibitors induced slight changes in the secondary and tertiary structures of PPO, which were enhanced by acidic pH. Molecular docking results showed that 4-carboxycinnamic acid exhibited the strongest binding ability, and the main interaction forces were around carboxyl groups, and acidic pH enhanced the binding effect through more interactions and lower binding energy. This study could provide new insights into industrial application of cinnamic acid and its derivatives for the control of enzymatic browning of fruits and vegetables., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The synergistic effect of the initial state of the enzyme and pressure level on the denaturation of PPO has not been clear yet, but it significantly affects the application of high hydrostatic pressure (HHP) in the enzyme-containing food processing. Solid (S-) and low/high concentration liquid (LL-/HL-) polyphenol oxidase (PPO) was used as the study object, and the microscopic conformation, molecular morphology and macroscopic activity of PPO under HHP treatments (100-400 MPa, 25 °C/30 min) were investigated by spectroscopic techniques. The results show that the initial state has a significant effect on the activity, structure, active force and substrate channel of PPO under pressure. The effec can be ranked as follows: physical state > concentration > pressure, S-PPO > LL-PPO > HL-PPO. High concentration has a weakening effect on the pressure denaturation of the PPO solution. Under high pressure, the α-helix and concentration factors play a crucial role in stabilizing the structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Through dioxygen activation, a tetranuclear Mn(II,III,III,II) diamond core, [Mn 4 (HPTP*) 2 (μ-O) 2 (H 2 O) 4 ](ClO 4 ) 4 (1) complex, has been synthesised using a suitably designed septadentate ligand framework (HPTP*H = 1,3-bis(bis((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)amino)propan-2-ol). The newly prepared complex 1 was characterised using multiple spectroscopic techniques and X-ray crystallography. 1 exhibits excellent catalytic oxidation reactivity for the model substrates, namely, 3,5-di- tert -butylcatechol (3,5-DTBC) and 2-aminophenol, efficiently mimicking the enzymes catechol oxidase and phenoxazinone synthase, respectively. Remarkably, we employed aerial oxygen to catalyze the oxidation of these model substrates, 3,5-DTBC and 2-aminophenol, with turnover numbers of 835 and 14, respectively. A tetranuclear Mn-diamond core complex that mimics both catechol oxidase and phenoxazinone synthase could pave the way for further research into its potential as a multi-enzymatic functional mimic.

Crustaceans are perishable with a short shelf-life. They are prone to deterioration after capture, particularly during handling, processing, and storage due to melanosis caused by polyphenoloxidase (PPO). Therefore, inhibitory effects of chitooligosaccharide (CHOS) in comparison with CHOS-catechin (CHOS-CAT), CHOS-epigallocatechin gallate (CHOS-EGCG), and CHOS-gallic acid (CHOS-GAL) conjugates on Pacific white shrimp cephalothorax PPO were studied. IC 50 of CHOS-CAT (0.32 mg/mL) toward PPO was less than those of all conjugates tested ( p < 0.05). CHOS-CAT exhibited the mixed-type inhibition. K ic (0.58 mg/mL) and K iu (0.02 mg/mL) of CHOS-CAT were lower than those of other conjugates ( p < 0.05). CHOS-CAT showed static fluorescence-quenching, suggesting a change in micro-environment around the active site of PPO. Moreover, CHOS-CAT was linked with various amino acid residues, including Tyr208 or Tyr209 of proPPO via van der Waals, hydrophobic interaction, and hydrogen bonding as elucidated by the molecular docking of proPPO. Although CHOS-CAT had the highest PPO inhibitory activity, it showed a lower binding energy (-8.5 kcal/mol) than other samples, except for CHOS-EGCG (-10.2 kcal/mol). Therefore, CHOS-CAT could act as an anti-melanosis agent in shrimp and other crustaceans to prevent undesirable discoloration associated with quality losses., Competing Interests: The authors declare no conflicts of interest.

The effect and mechanism of sodium hexametaphosphate (SHMP) on polyphenol oxidase (PPO) enzymatic browning in yellow alkaline noodles (YAN) were investigated. The browning degree and PPO activity in YAN or PPO solutions decreased with the SHMP concentrations increased. Variations in pH values (pH 7-8.5) adjusted by HCl or acetic acid slightly affected the PPO activity, but SHMP inhibited PPO activity more pronounced. The inhibition of SHMP on PPO activity was irreversible. SHMP formed coordinate covalent bonds with Cu 2+ to make PPO inactive. HPLC analysis revealed that SHMP reduced the browning products and led to the color of PPO-catechol systems being lightened. Furthermore, SHMP inhibited browning by hampering the auto-oxidization of intermediate products due to the change in pH value. Besides, the HPLC chromatogram, UV-vis spectrum, and mass spectrometry revealed that SHMP could convert melanin (m/z 248.97, 723.5, and 836.58) to light-colored substances (m/z 137.11)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that during evolution plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase (PPO) that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the PPO family. Of the six PPOs highly induced in the line overexpressing MpMyb14, one, MpAS1, was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the PPO family in M. polymorpha evolved independently from PPOs in higher plants, indicating that aureusidin synthases evolved in parallel in land plants., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Significant amounts of fresh and fresh-cut fruits and vegetables are wasted every year due to enzymatic browning. Polyphenol oxidase (PPO) is the key enzyme involved in the enzymatic browning. In the past decades, various methods have been developed to inhibit browning of various fresh produce items. However, for most fresh horticultural produce, ideal measures accepted by industries and consumers are still scarce. This review provides up-to-date knowledge of browning control technologies, including physical methods, chemical methods such as natural inhibitors, molecular biotechnology, and nanotechnology. In addition, we propose some ideas to improve the efficacies of these strategies with fewer side effects. To better inhibit tissue browning, new research directions are also discussed, for example, regulation of PPO substrate techniques., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Main Conclusion: PPO was purified from Cistanche deserticola, and its enzymatic characteristics were clarified. It was found that microwave treatment was an efficient way to inactivate PPO. Polyphenol oxidase (PPO) from Cistanche deserticola was obtained and purified through an acetone precipitation and anion exchange column, the enzymatic characteristics and inactivation kinetics of PPO were studied. The specific activity of PPO was 73135.15 ± 6625.7 U/mg after purification, the purification multiple was 48.91 ± 4.43 times, and the recovery was 30.96 ± 0.27%. The molecular weight of the PPO component is about 66 kDa by SDS-PAGE analysis. The optimum substrate of PPO was catechol (V max  = 0.048 U/mL, K m  = 21.70 mM) and the optimum temperature and pH were 30 °C and 7, respectively. When the temperature is above 50 °C, pH < 3 or pH > 10, the enzyme activity can be significantly inhibited. The first-order kinetic fitting shows that microwave inactivation has lesser k values, larger D values and shorter t 1/2 . It was found that microwave treatment is considered as an efficient and feasible way to inactive PPO by comparing the Z values and E a values of the two thermal treatments., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

It has been revealed that slightly acid electrolysed water (SAEW) could delay enzymatic browning and melanin formation in food. In this work, multi-spectroscopic methods and UHPLC-Q-TOF-MS were combined to study the underlying reason. The reversible mixed-type inhibition mode of HOCl (main components in SAEW) was determined. The ground state complex formation quenched the intrinsic fluorescence of polyphenol oxidase (PPO) and it was stable at lower temperature. The PPO conformational change (transformation from α-helix to β-sheet) induced by SAEW was confirmed by 3D fluorescence and Circular dichroism (CD) spectrum. Moreover, the driving force of the interaction between HOCl and PPO was hydrogen bond, which was validated by the molecular docking result. Besides, the formation of melanin related compounds including dihydroxyphenylalanine (DOPA), dopaquinone, dopachrome, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), 5,6-dihydroxyindole (DHI), and 5,6-indolequinone were significantly inhibited by SAEW treatment. These results demonstrated the potential of SAEW as a PPO inhibitor in the food industry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The catecholase activities were routinely modeled using transition metal complexes as catalyst and in some case basic pH were used as a reaction condition. In this article, the catalytic aerobic oxidation of proxy substrate 3,5-di-tert-butylcatechol (DTBC) in methanol using triethylamine/diethylamine as catalyst was demonstrated as a functional mimic of catecholase activity. The kinetic manifestation of DTBC oxidation was explained as enzymatic substrate inhibition pattern in Michaelis-Menten kinetic model. The mechanistic insight of the aerobic oxidation of DTBC was further validated using various spectroscopic techniques and DFT methods., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Polyphenol oxidase (PPO) is present in most higher plants, but also in animals and fungi. PPO in plants had been summarized several years ago. However, recent advances in studies of PPO in plants are lacking. This review concludes new researches on PPO distribution, structure, molecular weights, optimal temperature, pH, and substrates. And, the transformation of PPO from latent to active state was also discussed. This state shift is a vital reason for elevating PPO activity, but the activation mechanism in plants has not been elucidated. PPO has an important role in plant stress resistance and physiological metabolism. However, the enzymatic browning reaction induced by PPO is a major problem in the production, processing, and storage of fruits and vegetables. Meanwhile, we summarized various new methods that had been invented to decrease enzymatic browning by inhibiting PPO activity. In addition, our manuscript included information on several important biological functions and the transcriptional regulation of PPO in plants. Furthermore, we also prospect some future research areas of PPO and hope they will be useful for future research in plants.

Enzymatic browning greatly affects the quality of potato products. Polyphenol oxidase (PPO) is the enzyme mainly responsible for potato enzymatic browning. PPO has soluble polyphenol oxidase (sPPO) and membrane-bound polyphenol oxidase (mPPO) forms. In this study, the properties of sPPO and mPPO were investigated in potato tubers. The molecular weight of potato sPPO and mPPO were estimated to be 69 kDa in the form of homodimers in vivo. The mass spectrometry results showed that the purified sPPO and mPPO protein in potato tubers was mainly tr|M1BMR6 (Uniprot). The optimum pH for sPPO and mPPO was 6.5, and the optimum temperatures were 20 and 30 °C, respectively. The Michaelis constant (K m ) and maximum unit enzyme activity (V max ) of sPPO were 6.08 mM and 2161 U/S when catechol was used as the substrate, whereas those of mPPO were 2.95 mM and 2129.53 U/S, respectively. The mPPO had stronger affinity to the substrate catechol than sPPO, whereas pyrogallic acid was stronger affinity for sPPO. Ascorbic acid and sodium sulfite were inhibitors of sPPO and mPPO, respectively. After understanding the different binding states of polyphenol oxidase, different inhibitors and treatment methods can be used to treat the enzyme according to different enzymatic properties, so as to achieve a greater degree of Browning control. These results will provide a theoretical basis for regulating PPO activity to reduce enzymatic browning during potato processing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier Inc.)

Developing highly active and selective advanced nanozymes for enzyme-mimicking catalysis remains a long-standing challenge for basic research and practical applications. Herein, we grafted a chiral histidine- (His-) coordinated copper core onto Zr-based metal-organic framework (MOF) basic backbones to structurally mirror the bimetal active site of natural catechol oxidase. Such a biomimetic fabricated process affords MOF-His-Cu with catechol oxidase-like activity, which can catalyze dehydrogenation and oxidation of o -diphenols and then transfer electrons to O 2 to generate H 2 O 2 by the cyclic conversion of Cu(II) and Cu(I). Specifically, the elaborate incorporation of chiral His arms results in higher catalytic selectivity over the chiral catechol substrates than natural enzyme. Density functional theory calculations reveal that the binding energy and potential steric effect in active site-substrate interactions account for the high stereoselectivity. This work demonstrates efficient and selective enzyme-mimicking catalytic processes and deepens the understanding of the catalytic mechanism of nanozymes.

Two catecholase-like biomimetic catalysts, namely, two dinuclear copper complexes [Cu 2 (L1)(OH)(H 2 O)(EtOH)][ClO 4 ] 2 (C1) and [Cu 2 Ac 2 O(L1)ClO 4 ] (C2) with the 2,6-bis(4-methyl piperazin-1-yl-methyl)-4-formyl-phenoxy ligand (L1) together with the mononuclear complex Cu(ClO 4 ) 2 (L2) (C3) containing ligand 1,2-(C 5 H 4 N-6-OCH 3 -2-CHN) 2 CH 2 CH 2 (L2), were synthesized. Their catalytic pathways were investigated and compared. The evaluation of the catalytic activity of compound C1 (and C2, C3) using the Michaelis-Menten model was represented by values of K M = 272.93 (223.02; 1616) μmol L -1 and V max of 0.981 (1.617; 1.689) μmol L -1 s -1 . The role of water content in the solvent is also discussed. The dinuclear complexes C1 and C2 were found to be more efficient catalysts than mononuclear complex C3. The mode of catalytic action was characterized via cyclic voltammetry, spectrophotometry, and UV-Vis spectroelectrochemistry. The catalytic mechanism of 3,5-di- tert butyl catechol oxidation in the presence of oxygen was proposed. The reaction circle was proved by the confirmation of the chemical reversibility of complex reduction. The advantage of the in situ spectroelectrochemical measurement enabled to control the reduction of quinone formed by the chemical reaction of catechol with oxygen in solution. At this step, the simultaneous change in the absorption spectrum indicated a change in the copper redox state of the catalyst.

Atmospheric cold plasma (ACP) is a novel nonthermal technology with potential applications in maintaining and improving food quality. The effect of ACP on the activity and structure of mushroom (Agaricus bisporus) polyphenol oxidase (PPO) was evaluated. Results demonstrated that the dielectric barrier discharge (DBD) based plasma technology could inactivate PPO (up to 69%) at 50 kV with the increased concentrations of H 2 O 2 and NO x . An obvious enhancement of surface hydrophobicity was observed, whereas a gradual reduction of total sulfhydryl content was recorded with the increasing exposure time. Data from circular dichroism, atomic force microscopy, particle size distribution and fluorescence spectra displayed the rearrangement of secondary structure and disruption of the tertiary structure. Red shifts of fluorescence spectra showed positive correlations with the inactivation rate of PPO. Therefore, ACP treatment could be served as an alternative approach to inactivate undesirable enzymes to minimize the loss of food nutrition and quality., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Polyphenol oxidase (PPO) is considered to have a key role in the food industry because it initiates enzymatic browning in the processing and storage of fruit and vegetables. Increasing numbers of benzoic and cinnamic acid derivatives have been found to be efficient inhibitors of polyphenol oxidase, but a comparison study on activity and action mechanism is lacking. In this study, 18 benzoic acid and cinnamic acid hydroxy derivatives were selected and investigated., Results: Three substrates, four activators and 11 inhibitors were identified from benzoic and cinnamic acid derivatives. 2,4-Dihydroxycinnamic acid and benzoic acid showed the strongest inhibitory effect on PPO, with IC 50 of 0.092 and1.425 mmol L -1 , respectively. Benzoic acid reversibly inhibited PPO in a competitive manner, while 2,4-dihydroxycinnamic acid showed a mixed-type inhibition. Both of them showed that static-type fluorescence quenching and electrostatic interaction were the main driving force in the bonding process. Compared with benzoic acid, 2,4-dihydroxycinnamic acid more easily formed hydrogen bonds in the active site of PPO, making the interaction more stable., Conclusion: Comparative analysis showed that the inhibition effect of cinnamic acid hydroxyl derivatives on PPO was stronger than that of benzoic acid derivatives. Benzoic acid and 2,4-dihydroxycinnamic acid were the strongest inhibitors. PPO inhibitors identified from benzoic and cinnamic acid derivatives are expected to be promising inhibitors for controlling fruit and vegetable browning. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

The impetus to modeling of enzyme mimics comes from their potential to provide insight to the alternate mechanistic pathways of the native enzymes. The present study demonstrates the syntheses and characterization of two different cobalt(II) complexes, [Co(pdm)(Phen)Cl]Cl·H 2 O ( 1 ) and [Co(pmmH) 2 (SCN) 2 ] ( 2 ) with the aminoalcohol ligands such as pyridine-2,6-dimethanol (pdmH 2 ) or 2-pyridinemonomethanol (pmmH) and their assessment as catechol oxidase (CO) enzyme mimic. Single Crystal X-ray diffraction and powder X-ray diffraction data suggest the octahedral environment around the Co(II) ion and the complexes form extensive 1D or 2D propagating network as a result of non-covalent interactions (O···H and C-H···π). TD-DFT calculations were used to explain the spectral bands obtained during the UV-Vis absorption studies and it is ascertained that the transitions were mainly of the intra-ligand charge transfer (ILCT) type. The catecholase biomimetic catalytic activity of the synthesized complexes has been investigated in detail and the kinetics is also performed. The results obtained show that both the complexes catalyze the aerobic oxidation of catechol to the corresponding o-quinone. The K cat value for 1 is 106.99 h ‒1 and for 2 is 90.32 h ‒1 in methanol. It may be mentioned here that 1 and 2 are effective catalysts, with the order of activity being 1  >  2 . The order of enzymatic activity is well justified by CV and DFT studies.Communicated by Ramaswamy H. Sarma.