Phenolase Activity Research Articles

Physiological, metabolome, and transcriptome analysis revealed the effect of ethyl 3-amino-3-thioxopropanoate on the browning of fresh-cut banana during storage

The quality of fresh-cut bananas is often affected by surface browning, which does not meet consumer expectations. The ethyl 3-amino-3-thioxopropanoate (EAT) is an ethyl ester organic compound screened from Maillard reaction products that may have browning inhibition effects. However, there is still a lack of research on the practical application of EAT. This study investigated the impact of exogenous EAT on the browning process and the underlying mechanisms in fresh-cut banana through a comprehensive analysis involving physiological, metabolomic, and transcriptomic approaches. Treatment with EAT effectively mitigated the discoloration of the cut surface of slices and decreased the browning index by 74 % at the end of storage. Additionally, EAT application significantly reduced polyphenol oxidase activity, increased phenylalanine ammonia-lyase activity and total phenolic content, inhibited lipoxygenase activity, decreased malondialdehyde accumulation, enhanced antioxidant enzyme activity, and improved DPPH free radical scavenging rate to 68.49 %. Moreover, 839 metabolites were identified through non-targeted metabolomics analysis. Metabolomic and transcriptomic analysis further highlighted the importance of lipid metabolism, amino acid metabolism and secondary metabolites in influencing the sensitivity to post-cutting browning. EAT treatment also influenced the expression levels of key genes related to browning. Thus, it could be speculated that the mechanism by which EAT produces its anti-browning effect may be by reducing phenolase activity, improving levels of phenols and amino acids, enhancing the antioxidant capacity of the fruit, and alleviating membrane lipid peroxidation. In summary, this study provides valuable insights into the response mechanism employed by exogenous EAT to inhibit browning in freshly cut bananas. However, the application security and mass production methods of EAT still need further clarification.

Effects of microwave drying on color change, phenolic substance content and phenolase activity of different parts of persimmon slices

Effects of microwave drying on color change, phenolic substance content and phenolase activity of different parts of persimmon slices

Transcriptomics and metabolomics provide insight into the anti-browning mechanism of selenium in freshly cut apples.

Enzymatic browning has a considerable negative impact on the acceptability and marketability of freshly cut apples. However, the molecular mechanism by which selenium (Se) positively affects freshly cut apples in this regard is not yet clear. In this study, 0.75 kg/plant of Se-enriched organic fertilizer was applied to "Fuji" apple trees during the young fruit stage (M5, May 25), the early fruit enlargement stage (M6, June 25), and the fruit enlargement stage (M7, July 25), respectively. The same amount of Se-free organic fertilizer was applied as a control. Herein, the regulatory mechanism by which exogenous Se exerts its anti-browning effect in freshly cut apples was investigated. The results showed that the M7 treatment applied in Se-reinforced apples could remarkably inhibit their browning at 1h after being freshly cut. Additionally, the expression of polyphenol oxidase (PPO) and peroxidase (POD) genes treated with exogenous Se was significantly reduced compared to controls. Moreover, the lipoxygenase (LOX) and phospholipase D (PLD) genes, which are involved in membrane lipid oxidation, were expressed at higher levels in the control. The gene expression levels of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and ascorbate peroxidase (APX) were upregulated in the different exogenous Se treatment groups. Similarly, the main metabolites measured during the browning process were phenols and lipids; thus, it could be speculated that the mechanism by which exogenous Se produces its anti-browning effect may be by reducing phenolase activity, improving the antioxidant capacity of the fruits, and alleviating membrane lipid peroxidation. In summary, this study provides evidence regarding and insight into the response mechanism employed by exogenous Se to inhibit browning in freshly cut apples.

Multielectron Redox Catalysis with Efficient Tyrosinase Activity Based on a Visible‐Light Controlled Artificial Photoenzyme

A stepwise non‐enzymatic conversion of the natural amino acid L‐tyrosine into physiologically relevant intermediates L‐DOPA, dopaquinone and dopachrome has been demonstrated in aqueous solution under ambient conditions. The photocatalytic redox process applied involves controlled activation of molecular oxygen forming water and hydrogen peroxide as further reaction products. Substrate conversion can be switched on and off conveniently by regulating the degree of visible‐light or sunlight exposure of an immobilized multielectron transfer sensitizer, which is very easy to apply and can be separated again from the supernatant reaction medium. Quantitative analysis of the six‐electron redox conversion of L‐tyrosine catalyzed by the artificial oxidoreductase enzyme system reveals a remarkable degree of abiotic phenolase activity which nearly approaches the benchmark value of the native metalloenzyme tyrosinase under substrate saturation conditions.

ULASAN Masalah Pencoklatan pada Kultur Jaringan

<p>Several<br />tropical plant species contain high concentrations of phenolic<br />compounds, which become oxidised when their cells<br />are wounded or when the plant parts become senescences.<br />In tissue culture, the phenolic compounds usually leach into<br />the medium from the cut surfaces of explants. The phenolic<br />compounds caused the culture medium turns to dark brown<br />in colour due to oxidation. This is detrimental to the culture,<br />because it causes the isolated tissue fails to grow. The<br />browning of tissue culture and the medium can often be<br />prevented by one of the several different approaches, such<br />as by removing the phenolic compounds produced, modifying<br />the redox potential, inactivating phenolase enzymes,<br />reducing phenolase activity and substrate availability, as well<br />as pre-treatments by soaking and preconditioning on a basal<br />medium.</p>

Controlling Dicopper Protein Functions

Abstract Maturation processes of dinuclear copper proteins such as tyrosinase, catechol oxidase, and hemocyanin have been a long-standing mystery in copper protein chemistry. Until now, several crystal structures have revealed that these copper proteins share a similar dinuclear copper active site, where each copper ion is ligated by three histidine imidazoles, and binds molecular oxygen in a side-on fashion to form a (µ-η2:η2-peroxido)dicopper(II) species not only as the dioxygen-adduct in oxy-hemocyanins but also as the key reactive intermediate for the hydroxylation of phenols to catechols (phenolase reaction) and the oxidation of catechols to o-quinones (catecholase reaction) in tyrosinases and catechol oxidases. Recently, we have succeeded in determining the high-resolution crystal structures of the recombinant pro-form of yellow koji mold tyrosinase to find the existence of a distinct C-terminal domain containing a –CXXC– unit, that is the common sequence motif of the copper chaperons. Thus, the C-terminal domain apparently acts as a copper chaperon, helping construction of the dinuclear copper active site of tyrosinase. Furthermore, we have found that the proteolytic cleavage of the C-terminal domain from the pro-form (inactive-form) of tyrosinase greatly enhances the tyrosinase activity, thus suggesting that the C-terminal domain also acts as a shielding domain to regulate the enzymatic activity. In fact, overall structure of the pro-form resembles the structure of one of the functional units of octopus hemocyanin (oxygen carrier protein), which also has a similar C-terminal domain prohibiting the monooxygenase activity. On the basis of these results together with the detailed kinetic and spectroscopic analyses, the maturation process of the dinuclear copper proteins is discussed to provide new insights into the regulation mechanism of the dicopper protein functions; dioxygen binding and activation. We have also succeeded in evolving phenolase activity from molluscan and arthropod hemocyanins by treating them with a hydrolytic enzyme or an acid, and demonstrated that the reaction mechanism of their phenolase activity is the same to that of tyrosinase itself, that is the electrophilic aromatic substitution mechanism. Furthermore, we have developed an artificial dicopper protein exhibiting catecholase activity using metallo-β-lactamase, a dinuclear zinc enzyme, as a metal binding platform.

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

ChemInform Abstract: Designer Ligands: The Search for Metal Ion Selectivity

AbstractReview: 32 refs.

Influence of Pseudomonas fluorescens–Induced Plant Defenses on Efficacy of Nucleopolyhedrovirus of Helicoverpa armigera in Okra and Tomato

Baculovirus-based biopesticides like nucleopolyhedrovirus (NPV) offer management opportunities against Helicoverpa armigera (Hübner). The plant growth–promoting rhizobacteria Pseudomonas fluorescens imparts induced systemic resistance due to enhanced phenolase activity, which could regulate disease expression by baculoviruses. The possibility of integrated use of H. armigera nucleopolyhedrovirus and P. fluorescens on okra [Abelmoschus esculentus (L.) Moench], cv. Arka Anamika, and tomato (Solanum lycopersicum L.), cv. PKM 1, against H. armigera was investigated. The Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was applied to fruit from pot-cultured okra and tomato previously treated with P. fluorescens as a seed treatment (ST), foliar application (FA), or in combination and bioassayed against third instar H. armigera. Application of P. fluorescens as ST and FA, alone or in combination with HearNPV, reduced larval and pupal weights of H. armigera that fed on okra and tomato fruits. Lower percentage mortality (31.25% and 36.25%) due to NPV was noticed in okra and tomato plants, respectively, treated with P. fluorescens through ST + FA. Increased levels of phenol, tannin, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase were recorded in P. fluorescens–treated plants than in untreated checks. Reduced consumption by H. armigera larvae and inactivation of baculoviruses due to defense-related enzyme induction in P. fluorescens–treated plants could be responsible for the reduction in NPV-induced mortality of H. armigera larvae.

Five monomeric hemocyanin subunits from Portunus trituberculatus: Purification, spectroscopic characterization, and quantitative evaluation of phenol monooxygenase activity

Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [( μ- η 2: η 2-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5 M borate buffer (pH 9.0) and reacted with 4-methylphenol ( p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3 M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo.

Carbon felt-based biocatalytic enzymatic flow-through detectors: Chemical modification of tyrosinase onto amino-functionalized carbon felt using various coupling reagents

Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt (CF) surface via eight different coupling reagents. Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by the treatment with 3-aminopropyltriethoxysilane (APTES). The APTES modification of the CF surface was confirmed by XPS and SEM measurements. The terminal amino groups on the CF surface were cross-linked with protein lysine group (or cysteine group) using various coupling reagents. The resulting TYR-immobilized CF (TYR-CF) was utilized as a working electrode unit of a biocatalytic enzymatic flow-through detector. Catechol and 4-chlorophenol (4-CP) were used as model analytes for the evaluation of catecholase activity and phenolase activity, respectively, and flow injection peaks based on the electro-reduction of the enzymatically produced o-quinone species were monitored at −0.05 V vs. Ag/AgCl. Among eight coupling reagents, glutaraldehyde (GA) exhibited the best results on the sensitivity, the operational stability and the storage stability. The detection limits of catechol and 4-CP obtained by the GA-coupling method were found to be 6.0 × 10 −9 M and 1.5 × 10 −8 M, respectively with the sample through-put of 36 samples/h. No serious degradation of the peak current was observed over 30 consecutive samples injections on the GA-coupling method, while gradual decrease in the peak currents was observed on other seven coupling reagents. The GA-coupling method showed the best results on the storage stability, and 85% of original activity for catechol oxidation remained after 25 days storage.

Highly sensitive flow-biosensor for toxic phenolic compounds using tyrosinase and acridine orange-adsorbed carbon felt

Tyrosinase (TYR, EC 1.14.18.1) was physically adsorbed onto a carbon felt (CF) together with acridine orange (AO). Coadsorption of AO was essential to prevent the denaturation of the TYR at the CF surface. The resulting TYR and AO-coadsorbed CF (TYR/AO-CF) was successfully utilized as a detection unit of novel and highly sensitive amperometric flow-biosensor for toxic chlorophenol compounds. Standard solutions of phenolic compounds (200 μL) were injected, and the cathodic peak currents due to the reduction current of o-quinones produced by the TYR-catalyzed oxidation (phenolase activity) were detected at the applied potential of −50 mV vs. Ag/AgCl. In this reaction, the electrochemically generated catechol compounds from o-quinones are re-oxidized repeatedly by catecholase activity of the TYR, leading to a sufficient amplified signal. The TYR/AO-CF exhibited much higher selectivity toward p-chlorophenol as compared with other chlorophenol compounds. When 0.1 mol/L phosphate buffer (pH 7.0) was used as a carrier at flow rate of 3.0 mL/min, cathodic peaks for p-chlorophenol was linear in the concentration range between 0.1 and 10 [xmol/L (sensitivity: 1.41(mA-L)/mmol) with sampling rate (30 samples/h), and the detection limit of p-chlorophenol was found to be 2.13 − 10 8 mol/L (S/N = 3. The ratio of signal and noise is 3). The TYR/AO-CF kept more than 80% of original activity after the storage in 0.1 mol/L phosphate buffer (pH 7.0) containing 0.2 mmol/L AO at 4°C.

Theoretical Studies on ortho‐Oxidation of Phenols with Dioxygen Mediated by Dicopper Complex: Hints for a Catalyst with the Phenolase Activity of Tyrosinase

AbstractTheoretical studies on the chemo‐ and regioselective ortho‐oxidation reaction of phenols mediated by a biomimetic (μ–η2:η2peroxo)dicopper(II) complex were performed using unrestricted hybrid density functional theory (UB3LYP) calculations, with the aim of providing a guide for the development of new bio‐inspired catalysts with the phenolase activity of tyrosinase. Energetic, structural, and electronic analyses suggested the involvement of a side‐on (μ–η2:η2)‐Cu2O2 complex as an active intermediate, and a single electron transfer (SET)‐induced electrophilic aromatic substitution mechanism is proposed for the rate‐determining CO bond forming process; this is consistent with experimental observations. Moreover, the inherent roles of, and requirement for, two copper ions in this reaction have been elucidated.

Significant Enhancement of Monooxygenase Activity of Oxygen Carrier Protein Hemocyanin by Urea

Oxygenation of a series of p-substituted phenols to the corresponding catechols (phenolase activity) by the (mu-eta2:eta2-peroxo)dicopper(II) species of Octopus hemocyanin has been directly examined for the first time by using a UV-vis spectroscopic method in a 0.5 M borate buffer solution containing 8 M urea under anaerobic conditions. Preliminary kinetic studies have indicated that the reaction involves an electrophilic aromatic substitution mechanism as in the case of phenolase reaction of tyrosinase. The oxygenation of phenols by hemocyanin also proceeded catalytically when the reaction was carried out under aerobic conditions.

Kinetic Evaluation of Catalase and Peroxygenase Activities of Tyrosinase

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.

Kinetic evaluation of phenolase activity of tyrosinase using simplified catalytic reaction system.

A very simple tyrosinase reaction system has been developed using borate anion as a trapping agent of catechols and hydroxylamine as an external reductant to evaluate the phenolase activity without the interference of catecholase activity. Reactivities of variously para-substituted phenols in this system were compared directly to those of the phenols in the model reactions, demonstrating that the enzymatic oxygenation reaction of phenols proceeds via the same mechanism as the model reaction, that is, electrophilic aromatic substitution mechanism.

Photometric assay for polyphenol oxidase activity in olives, olive pastes, and virgin olive oils

AbstractA photometric method is proposed that allows the determination of phenolase activity in olive fruits, olive pastes, and virgin olive oil. The method can also be used to quantify partially purified phenolase from olives, and is based on the coupling between 4‐methyl‐o‐benzoquinone, the reaction product of phenolase toward its substrate 4‐methylcatechol, and the aromatic amine 4‐amino‐N,N‐diethylaniline. The deep‐blue adduct arising from this reaction has been characterized by means of nuclear magnetic resonance and mass spectrometric techniques and identified as 4‐(4′‐diethylaminophenylimino)‐2‐hydroxy‐5‐methyl‐cyclohexa‐2,5‐dienone. This compound shows an absorption band, centered (in dichloromethane) at 617 nm, with an ε of 11,080 M−1cm−1. The main advantage of the proposed method resides in the high absorption coefficient of the adduct and its ultraviolet/visible absorption pattern, with a λmax in a spectral region void of significant interferences by the pigments that ultimately will probably be present in the extracts to be tested by this proposed method. The method has proven to be sensitive, specific, and reliable.

Synthesis, structure and oxidation studies of a dinuclear copper(I)–copper(II) complex: [Cu 2(bdpdz) 2](ClO 4) 3

A dinuclear copper complex containing the ligand 3,6-bis(di-2-pyridylmethyl) pyridazine, bdpdz, [Cu 2(bdpdz) 2](ClO 4) 3, was prepared and its structure determined by X-ray analysis. The compound consists of a dinuclear mixed valence copper(I)–copper(II) entity with localised valences, co-ordinated to two bridging ligand molecules. The geometry around the copper(I) centre is distorted tetrahedral, while that of the copper(II) centre is tetragonally distorted octahedral. Oxidation studies reveal that the compound does not exhibit phenolase activity.

Influence of induced plant defenses in cotton and tomato on the efficacy of baculoviruses on noctuid larvae

Constitutive phenolase activity of plants has a profound ability to modulate disease in insects caused by baculoviruses. We investigated the influence of damage-induced plant phenolic oxidases in cotton and tomato on mortality caused by two different baculoviruses in their respective hosts, Heliothis virescens (L.) and Helicoverpa zea (Boddie). For both plant species, peroxidase (POD) and phenolic levels were predictive of larval mortality caused by baculoviruses. The higher the POD activity, the lower the mortality in both hosts. Different classes of phenolics (e.g., monohydroxyphenolics vs. catecholic phenolics) in combination with POD activity had different effects on the severity of viral disease depending upon mixture, which implies that viral efficacy is predictable only if total chemical content of the plants is specified. Inhibition of baculoviral disease by plant phenolase activity has potential implications for the compatibility of baculoviruses with induced resistance in IPM programs.

Effects of Plant Identity and Chemical Constituents on the Efficacy of a Baculovirus Against Heliothis Virescens

Baculoviruses are arthropod-specific, dsDNA viruses primarily used to control lepidopteran pests. A limitation of the use of baculoviruses for pest control is that their efficacy is modifiable by host-plant chemicals. The levels of phenolic substrates and two foliar oxidative enzymes, peroxidase (POD) and polyphenol oxidase (PTO), were significant predictors of disease caused by a baculovirus in Heliothis virescens fed on either cotton or lettuce; POD was the more influential of the two enzymes. The higher the plant phenolase activity, the lower the percent mortality and the slower the insects died from viral infection. Whether a particular class of phenolic substrates was correlated with enhanced or attenuated baculoviral disease depended upon context, i.e., admixture. Diminution of viral efficacy by plant oxidative activity may compromise the compatibility of baculoviruses with other components of an integrated pest management system such as host plant resistance.