Phenolic Substrates Research Articles

Exceptional Substrate Diversity in Oxygenation Reactions Catalyzed by a Bis(μ-oxo) Copper Complex.

The enzyme tyrosinase contains a reactive side‐on peroxo dicopper(II) center as catalytically active species in C−H oxygenation reactions. The tyrosinase activity of the isomeric bis(μ‐oxo) dicopper(III) form has been discussed controversially. The synthesis of bis(μ‐oxo) dicopper(III) species [Cu2(μ‐O)2(L1)2](X)2 ([O1](X)2, X=PF6 − , BF4 −, OTf−, ClO4 −), stabilized by the new hybrid guanidine ligand 2‐{2‐((dimethylamino)methyl)phenyl}‐1,1,3,3‐tetramethylguanidine (L1), and its characterization by UV/Vis, Raman, and XAS spectroscopy, as well as cryo‐UHR‐ESI mass spectrometry, is described. We highlight selective oxygenation of a plethora of phenolic substrates mediated by [O1](PF6)2, which results in mono‐ and bicyclic quinones and provides an attractive strategy for designing new phenazines. The selectivity is predicted by using the Fukui function, which is hereby introduced into tyrosinase model chemistry. Our bioinspired catalysis harnesses molecular dioxygen for organic transformations and achieves a substrate diversity reaching far beyond the scope of the enzyme.

Laccase and catecholoxidase activities contribute to innate immunity in slipper limpets, Crepidula fornicata

The slipper limpet Crepidula fornicata is an invasive, non-native, marine species found throughout the coastal waters of southern England and Wales, UK. These limpets are considered to blight commercial shellfish banks, notably oysters, yet little is known about their disease-carrying capacity or their immunobiology. To address the latter, we isolated haemolymph (blood) from limpets and tested for the presence of the immune-enzyme phenoloxidase. Invertebrate phenoloxidases produce melanic polymers from simple phenolic substrates, which are deployed in the presence of pathogens because of their potent microbicidal and microbiostatic properties. We used a series of established substrates (e.g., tyrosine, hydroquinone) and inhibitors (e.g., 4-hexylresorcinol, benzoic acid) to target three distinct enzymes: laccase (para-diphenoloxidase), catecholoxidase (ortho-diphenoloxidase) and tyrosinase (monophenoloxidase). We confirmed laccase and catecholoxidase activities and characterised their kinetic properties across temperature and pH gradients (5–70 °C and 5–10, respectively). Crucially, we demonstrated that products derived from such laccase and catecholoxidase activities reduced significantly the numbers of colony-forming units of both Gram-positive and Gram-negative bacteria in vitro. We further screened limpet tissues for signs of melanin using wax histology, and found cells replete with eumelanin-like pigments and lipofuscin in the digestive gland, connective tissues, barrier epithelia and gills. Our data represent the first account of enzyme-based antibacterial defences, notably laccase, in C. fornicata.

Aspartic Acid Can Effectively Prevent the Enzymatic Browning of Potato by Regulating the Generation and Transformation of Brown Product

Abstract Enzymatic browning is a crucial factor affecting the quality of fresh-cut potatoes. In this study, the effects of aspartic acid (Asp) on browning, polyphenol oxidase (PPO) activity, and phenolic substrates were investigated. The results showed that all the Asp solution treatments significantly reduced the browning of fresh-cut potato chips. At 2–4 °C, control chips had lost saleability on day 1, while 1.0 % Asp treated chips were still acceptable on the day 7 evaluation. Asp can prevent the discoloration of potato pulp completely and also partly decolorize the brown color. Addition of Asp decreased the PPO activity of potato pulp. At the same pH, the PPO activities of potato pulps treated with Asp were lower than those treated with phosphoric acid (H3PO4). With the recovery of pH to the control level, the activity of the Asp-treated pulp did not improve completely like those treated with H3PO4. Moreover, the addition of copper acetate solution to the Asp-treated pulp significantly increased PPO activity. As browning progressed, tyrosine and chlorogenic acid content in the Asp-treated potato pulp was higher than that of the control, but caffeic acid, gallic acid, and protocatechuic acid content did not increase. In vitro simulation experiments demonstrated that Asp prevented the discoloration of tyrosine and chlorogenic acid solution, and also caused the fading of the discoloration to a certain degree. These results indicated that Asp is a promising anti-browning agent for fresh-cut potato and the mechanism involves the reduction of PPO activity via a decrease in pH and chelating Cu2+ by Asp, inhibiting the formation of brown color from tyrosine and chlorogenic acid solution and enhancing the fading of the brown color.

Laccases in Food Industry: Bioprocessing, Potential Industrial and Biotechnological Applications.

Laccase is a multi-copper oxidase that catalyzes the oxidation of one electron of a wide range of phenolic compounds. The enzyme is considered eco-friendly because it requires molecular oxygen as co-substrate for the catalysis and it yields water as the sole by-product. Laccase is commonly produced by fungi but also by some bacteria, insects and plants. Due it is capable of using a wide variety of phenolic and non-phenolic substrates, laccase has potential applications in the food, pharmaceutical and environmental industries; in addition, it has been used since many years in the bleaching of paper pulp. Fungal laccases are mainly extracellular enzyme that can be recovered from the residual compost of industrial production of edible mushrooms as Agaricus bisporus and Pleurotus ostreatus. It has also been isolated from microorganisms present in wastewater. The great potential of laccase lies in its ability to oxidize lignin, one component of lignocellulosic materials, this feature can be widely exploited on the pretreatment for agro-food wastes valorization. Laccase is one of the enzymes that fits very well in the circular economy concept, this concept has more benefits over linear economy; based on “reduce-reuse-recycle” theory. Currently, biorefinery processes are booming due to the need to generate clean biofuels that do not come from oil. In that sense, laccase is capable of degrading lignocellulosic materials that serve as raw material in these processes, so the enzyme’s potential is evident. This review will critically describe the production sources of laccase as by-product from food industry, bioprocessing of food industry by-products using laccase, and its application in food industry.

Study on the browning mechanism of betel nut (Betel catechu L.) kernel.

Taking betel nut (Betel catechu L.) kernel as raw materials, to analyze the browning mechanism of betel nut kernel. First, we extract and separate the phenolic substances and polyphenol oxidase (PPO) from the betel nut kernel and then find out how O2 penetrates into the kernel, study the above 3 key factors of enzymatic browning so as to prove the possibility of enzymatic browning, and further study the browning products to clarify the mechanism of browning of betel nut kernel during storage process. The results showed that 11 kinds of phenolic compounds were isolated and identified from the betel nut kernel by liquid chromatography–mass spectrometry, among which chlorogenic acid was the highest, followed by dopamine and L‐epicatechin, and other contents were relatively low. Meanwhile, PPO was also separated from the betel nut kernel by DEAE–Sepharose Fast Flow and Phenyl–Sepharose 6 Fast Flow column chromatography. On this basis, scanning electron microscopy showed that the damage of the betel nut tissue was aggravated with the prolonged storage time, the wax was gradually decomposed, and the stratum corneum of the peel is destroyed in a honeycomb‐shaped; the lignification of the flesh was aggravated, and the interstitial space was increased; and the crack of the kernel membrane was also enlarged. These changes of structure contribute to increase gas exchange within and outside the organization, including the entry of O2. Finally, the oxidation products generated from simulated reaction of chlorogenic acid, dopamine, and epicatechin with purified PPO under aerobic conditions in vitro were compared with the products extracted from naturally brown betel nut, and the same absorption spectra were found. Therefore, it indicates that the browning of betel nut kernel is an enzymatic browning caused by the reaction of phenolic substrates were oxidized by PPO.

Mechanisms of interference of p-diphenols with the Trinder reaction.

p-Diphenols, such as homogentisic acid, gentisic acid, etamsylate, and calcium dobesilate, interfere with diagnostic tests utilizing the Trinder reaction but the mechanisms of these effects are not fully understood. We observed substantial differences both in oxidation of p-diphenols by horseradish peroxidase and their influence on oxidation of 4-aminoantipyrine and various phenolic substrates. Homogentisic acid was rapidly oxidized by the enzyme and completely blocked chromophore formation. Enzymatic oxidation of the remaining p-diphenols was slow and they only moderately inhibited chromophore formation. However, in the presence of standard substrates all tested p-diphenols were rapidly converted to p-quinones. Hydrogen peroxide consumption was significantly accelerated by homogentisic acid but not much affected by the other p-diphenols. The magnitude and mechanisms of interference caused by p-diphenols therefore depend on their structure which determines their electrochemical properties - while for homogentisic acid with an electron-donating substituent and a lower reduction potential both enzymatic oxidation and reduction of the peroxidase-generated radicals occur, for p-diphenols with electron-withdrawing substituents and higher reduction potentials only the second mechanism is significant. Correlation of the effects on the Trinder reaction with reduction potentials of interfering compounds allows prediction of such properties for a wide range of other reducing compounds based on this parameter. It also explains why compounds with very different structures but strong reducing properties show such effects.

Rational synthesis of TS-1 zeolite to direct both particle size and framework Ti in favor of enhanced catalytic performance

Abstract Simultaneous control over particle size and framework Ti content are the major challenges of synthesis of efficient TS-1 catalysts. Here we firstly report control over the particle size and framework Ti content of TS-1 catalysts in a wide range through conventional hydrothermal approach, evaluate catalytic performances of obtained TS-1 catalysts, and then rationally design a novel procedure of synthesis. The characterization of TS-1 catalysts by several techniques revealed that synthesis conditions through conventional hydrothermal approach to reach minimum particle size is different than that to reach maximum framework Ti content. The catalytic study of TS-1 catalysts showed that conversion of phenol (kinetic diameter of 0.57 nm) increased with increase in the particle size, reached a maximum of 29.9% for the TS-1 with particle size of 270 nm, and then dropped significantly for the TS-1 with biggest particle size. The conversion of dibenzothiophene (kinetic diameter of 0.9 nm), however, decreased with increase in the particle size and maximum conversion of 69.0% was achieved by the TS-1 with smallest particle size of 130 nm. No TS-1 catalyst synthesized through conventional approach was efficient in oxidation of the both substrates as the catalyst lacks either small particle size or enhanced framework Ti. Based on the property-function results from conventional synthesis, we rationally designed a novel synthesis of TS-1 with minimum water and optimum pH by (NH4)2CO3 addition to minimize the particle size and maximize framework Ti content, respectively. The resultant TS-1 with both features of small particle size of 185 nm and enhanced framework Ti resulted in full conversions of phenol and bulky substrate of dibenzothiophene and generates the least amount of wastewater. The new approach applied here is promising and applicable to make efficient Lewis-acid zeolites.

INDUCED PHENOLOXIDASE PROFILES IN SILKWORM Bombyx mori (L.) UNDER BIOTIC STRESS

Phenol oxidase (PO) is one of the stress enzyme protein in living organism. The conversion of Pro-PO into an activation form of PO required a stress protein. In the present study has emerged with the novel finding of induced phenoloxidase was identified under bacterial endotoxin viz., Lipopolysaccharide (LPS) activity using silkworm Bombyx mori as an animal model. The PO enzyme plays an important role for insect survival during pathophysiological conditions. The enzyme activity was analyzed from ten different silkworm races with two phenolic substrates viz., L-Dopa and Dopamine by Native-PAGE. The bacterially induced PO was found in hemolymph and midgut of silkworm, PO3 were induced by LPS injection. In control PO1 & PO2 are non-bacterially induced protein having the molecular weight of 72 and 71. The results shown that there is no substrate specificity and similar functional activity was found in hemolymph and midgut under pathogenic condition. It was observed that bacterially inducible PO clearly differed from non-inducible PO (control). At final observation of induced isozymes of PO in the haemolymph and midgut system of tolerant silkworm races points out the existence of biochemical immunity against biotic stress of LPS. This is the first report to document the silkworm immunity under the LPS toxin in different silkworm races to identify the tolerant and susceptibility against a biotic stress.

Biomedical and Pharmaceutical-Related Applications of Laccases.

The oxidation of a vast range of phenolic and non-phenolic substrates has been catalyzed by laccases. Given a wide range of substrates, laccases can be applied in different biotechnological applications. The present review was conducted to provide a broad context in pharmaceutical- and biomedical- related applications of laccases for academic and industrial researchers. First, an overview of biological roles of laccases was presented. Furthermore, laccase-mediated strategies for imparting antimicrobial and antioxidant properties to different surfaces were discussed. In this review, laccase-mediated mechanisms for endowing antimicrobial properties were divided into laccase-mediated bio-grafting of phenolic compounds on lignocellulosic fiber, chitosan and catheters, and laccase-catalyzed iodination. Accordingly, a special emphasis was placed on laccase-mediated functionalization for creating antimicrobials, particularly chitosan-based wound dressings. Additionally, oxidative bio-grafting and oxidative polymerization were described as the two main laccase-catalyzed reactions for imparting antioxidant properties. Recent laccase-related studies were also summarized regarding the synthesis of antibacterial and antiproliferative agents and the degradation of pharmaceuticals and personal care products.

Dinuclear μ‐Phenoxo and μ‐Hydroxo Bridged Copper Complexes Exhibiting Oxidation of Phenols and Isoelectronic Compounds: Cytotoxicity and Evidences for Cellular Apoptosis

AbstractThe compartmental pentadentate ligands HLR (where R=Me, OMe, tBu) have been utilized for the synthesis of dinuclear hydroxo and phenolato bridged copper complexes viz. [Cu2(LMe)(μ‐OPh)(μ‐OH](ClO4)2]CH3CN; (1), [Cu2(LOMe)(μ‐OPh)(μ‐OH)(μ‐OH2)](ClO4)2.CH3CN; (2) and [Cu2(LtBu)(μ‐OPh)(μ‐OH)(OH2)](ClO4)2; (3). All the three complexes were characterized by various spectroscopic techniques. Tyrosinase activity was studied by insitu reaction of Cu(I) and ligands exploiting 2,4‐di‐tert‐butyl phenol as substrate. The phenolic substrate (2,4‐DTBP) underwent radical mediated C−C coupling reaction with no insertion of molecular oxygen in contrast to the oxidative aromatic hydroxylation reaction. The catalytic activities of complexes 1–3 on diphenols and related substrate molecules were investigated and all three complexes efficiently oxidize 3,5‐di‐tert‐butylcatechol. Treatment of copper complexes on o‐aminophenol and o‐phenylene diamine resulted in the oxidative coupling of substrate molecules, leading to the formation of phenoxazinone and diaminophenazine respectively. Furthermore, oxidative properties of reported copper complexes were studied in relevance to their ability in inducing apoptotic cell death. Remarkably, all three copper complexes (1, 2 and 3) exhibited excellent cytotoxic activity towards MCF‐7 cell lines (human breast cancer cell lines) with IC50 values of 1.6 μM, 2.0 μM and 0.65 μM respectively and carried out self activated DNA cleavage of plasmid pUC18 DNA.

Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System.

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato (Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family.

Activity adaptability of a DhHP-6 peroxidase-mimic in wide pH and temperature ranges and solvent media

Deuterohemin-β-Ala-His-Thr-Val-Glu-Lys (DhHp-6): peroxidase with high activity.

Microporous carbon fibers as electroconductive immobilization matrixes: Effect of their structure on operational parameters of laccase-based amperometric biosensor.

In this study, we describe the fabrication of sensitive biosensor for the detection of phenolic substrates using laccase immobilized onto two types of microporous carbon fibers (CFs). The main characteristics of microporous CFs used for preparation of biosensors are given. Two CFs were characterized by different specific surface area, CFA (<1m2·g-1) and CFB (1448m2·g-1), but with comparable size of the micropores estimated by positron annihilation lifetime spectroscopy. The structural analysis was shown that CFA is formed by thin interwoven fibers forming a highly porous structure, as well as CFB - by granular formations with uneven edges that shape a cellulose membrane of lower porosity. The results of amperometric analysis revealed that the laccase-bound CFs possesses better electrochemical behavior for laccase than non-modified rod carbon electrodes (control). Using chronoamperometric analysis, the operational parameters of the CFs-modified bioelectrodes were compared to control bioelectrodes. The bioelectrodes based on CFs have demonstrated 2.4-2.7 folds enhanced maximal current at substrate saturation (Imax) values, 1.2-1.4 folds increased sensitivity and twice wide linearity compared with control bioelectrodes. The sensitivity of the developed CFs-based bioelectrodes was improved compared with the laccase-bound electrodes, described in literature. The developed biosensor was tested for catechol analysis in the real communal wastewater sample.

Transcriptional Changes on Blight Fruiting Body of Flammulina velutipes Caused by Two New Bacterial Pathogens.

A blight disease of Flammulina velutipes was identified with symptoms of growth cessation of young fruiting bodies, short stipe, and brown spots on the pileus. The pathogenic bacteria were identified as Arthrobacter arilaitensis and Pseudomonas yamanorum by Koch’s postulate, gram staining, morphological and 16S ribosomal RNA gene sequence analyses. Either of the pathogenic bacteria or both of them can cause the same symptoms. Transcriptome changes in blighted F. velutipes were investigated between diseased and normal samples. Compared to the control group, 1,099 differentially expressed genes (DEGs) were overlapping in the bacteria-infected groups. The DEGs were significantly enriched in pathways such as xenobiotic metabolism by cytochrome P450 and tyrosine metabolism. Based on weighted correlation network analysis (WGCNA), the module most correlated to the pathogen-treated F. velutipes samples and candidate hub genes in the co-regulatory network were identified. Furthermore, a potential diseased mechanism involved in cell wall non-extension, phenolic substrate oxidation, and stress defense response was proposed based on the up-regulation of differentially expressed genes encoding chitin deacetylase, tyrosinase, cytochrome P450, MFS transporter, and clavaminate synthase-like protein. This study provides insights into the underlying reactions of young fruiting body of F. velutipes suffering from blight disease and facilitates the understanding of the pathogenic procedure of bacteriosis in edible mushrooms.

Phenolic Compound Biotransformation by Trametes versicolor ATCC 200801 and Molecular Docking Studies.

The filamentous fungus Trametes versicolor is a rich source of laccase (Tvlac). Laccases catalyze reactions that convert substituted phenol substrates into diverse derivatives through aromatic oxidation. We investigated methyl p-coumarate, methyl ferulate, and methyl caffeate biotransformation by Trametes versicolor ATCC 200801. Despite substrate similarity, the biotransformation reactions varied widely. Only methyl p-coumarate was converted into three derivatives. We isolated and identified the chemical structures of such derivatives by NMR and IR analysis. Hydroxylation, methylation, and hydrolysis were the main reactions resulting from the studied biotransformation. We also analyzed the interactions between Tvlac (PDB ID: 1GYC) and the three phenolic substrates by molecular docking simulations. The substituents in the phenol ring influenced substrate conformation and orientation in the Tvlac site. The biotransformation reaction selectivity correlated with the different binding energies to the Tvlac site. Our results demonstrated that docking studies successfully predict the biotransformation of cinnamic acid analogs by T. versicolor.

Insight into the glycosylation and hydrolysis kinetics of alpha-glucosidase in the synthesis of glycosides.

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.

Phenolic and non-phenolic substrates oxidation by laccase at variable oxygen concentrations: Selection of bisubstrate kinetic models from polarographic data

Using laccase as a case study, an alternative methodology for discrimination of bisubstrate mechanism types, useful for Bi-Bi reactions when water is one of the products, is presented. A purified enzyme from fungal cultures of Ganoderma resinaceum and two reducing substrates: 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) are used. Kinetic assays of ABTS or DMP oxidation using variable initial concentrations of dioxygen are performed in a closed system and depletion rates of dioxygen are measured using polarography. Seven bisubstrate models based on Michaelis-Menten Bi-Bi mechanisms are applied to the experimental data. Akaike information criterion, a penalized likelihood analysis, and Akaike weights are used for model selection. Results show that the more likely bisubstrate models for ABTS and DMP oxidation are ping-pong and Theorell-Chance, respectively. However, the catalytic efficiency of oxygen reduction to water exhibits similar values, and is compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. Finally, Michaelis-Menten constants towards dioxygen, 233 μM with ABTS and 700 μM with DMP, indicate that the soluble electron acceptor available for laccase-catalyzed reactions, is far removed from saturation.

Promoting microbial utilization of phenolic substrates from bio-oil.

The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70wt% Tween® 20 and 30wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Production of Melanins With Recombinant Microorganisms.

The melanins constitute a diverse group of natural products found in most organisms, having functions related to protection against chemical and physical stresses. These products originate from the enzyme-catalyzed oxidation of phenolic and indolic substrates that polymerize to yield melanins, which include eumelanin, pheomelanin, pyomelanin, and the allomelanins. The enzymes involved in melanin formation belong mainly to the tyrosinase and laccase protein families. The melanins are polymeric materials having applications in the pharmaceutical, cosmetic, optical, and electronic industries. The biotechnological production of these polymers is an attractive alternative to obtaining them by extraction from plant or animal material, where they are present at low concentrations. Several species of microorganisms have been identified as having a natural melanogenic capacity. The development and optimization of culture conditions with these organisms has resulted in processes for generating melanins. These processes are based on the conversion of melanin precursors present in the culture medium to the corresponding polymers. With the application of genetic engineering techniques, it has become possible to overexpress genes encoding enzymes involved in melanin formation, mostly tyrosinases, leading to an improvement in the productivity of melanogenic organisms, as well as allowing the generation of novel recombinant microbial strains that can produce diverse types of melanins. Furthermore, the metabolic engineering of microbial hosts by modifying pathways related to the supply of melanogenic precursors has resulted in strains with the capacity of performing the total synthesis of melanins from simple carbon sources in the scale of grams. In this review, the latest advances toward the generation of recombinant melanin production strains and production processes are summarized and discussed.

Impact of Intramolecular Hydrogen Bonding on the Reactivity of Cupric Superoxide Complexes with O-H and C-H Substrates.

The dioxygen reactivity of a series of TMPA-based copper(I) complexes (TMPA=tris(2-pyridylmethyl)amine), with and without secondary-coordination-sphere hydrogen-bonding moieties, was studied at -135 °C in 2-methyltetrahydrofuran (MeTHF). Kinetic stabilization of the H-bonded [( TMPA)CuII (O2.- )]+ cupric superoxide species was achieved, and they were characterized by resonance Raman (rR) spectroscopy. The structures and physical properties of [( TMPA)CuII (N3- )]+ azido analogues were compared, and the O2.- reactivity of ligand-CuI complexes when an H-bonding moiety is replaced by a methyl group was contrasted. A drastic enhancement in the reactivity of the cupric superoxide towards phenolic substrates as well as oxidation of substrates possessing moderate C-H bond-dissociation energies is observed, correlating with the number and strength of the H-bonding groups.