Tyrosinase-catalyzed Reaction Research Articles

Light Emission in Betalains: From Fluorescent Flowers to Biotechnological Applications.

The discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.

Efficient Synthesis of Theaflavin 3-Gallate by a Tyrosinase-Catalyzed Reaction with (-)-Epicatechin and (-)-Epigallocatechin Gallate in a 1-Octanol/Buffer Biphasic System.

Theaflavins, the orange-red pigments contained in black tea, have attracted attention as a result of their health-promoting effects. However, their synthetic preparation, in which the enzymatic oxidation of catechol-type catechin is followed by the quinone-induced oxidative dimerization of selectively combined catechol- and pyrogallol-type catechins, provides only a low yield. In the present study, we found that a 1-octanol/buffer biphasic system improved the yield of theaflavin 3-gallate in a tyrosinase-catalyzed synthetic reaction with (-)-epicatechin and (-)-epigallocatechin gallate. When the enzymatic reaction proceeded in a buffer solution, oxidized (-)-epigallocatechin gallate was preferentially used for self-dimerization. However, self-dimerization was suppressed in the octanol phase, allowing oxidized (-)-epigallocatechin gallate to participate in coupling with (-)-epicatechin quinone, leading to effective production of theaflavin 3-gallate. Furthermore, the preferential localization of theaflavin 3-gallate in the octanol phase prevented (-)-epicatechin-quinone-induced degradation.

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity

The leading cause of synthetic graft failure includes thrombotic occlusion and intimal hyperplasia at the site of vascular anastomosis. Herein, we report a co-immobilization strategy of heparin and potent anti-neointimal drug (Mitogen Activated Protein Kinase II inhibitory peptide; MK2i) by using a tyrosinase-catalyzed oxidative reaction for preventing thrombotic occlusion and neointimal formation of synthetic vascular grafts. The binding of heparin–tyramine polymer (HT) onto the polycarprolactone (PCL) surface enhanced blood compatibility with significantly reduced protein absorption (64.7% decrease) and platelet adhesion (85.6% decrease) compared to bare PCL surface. When loading MK2i, 1) the HT depot surface gained high MK2i-loading efficiency through charge-charge interaction, and 2) this depot platform enabled long-term, controlled release over 4weeks (92–272μg/mL of MK2i). The released MK2i showed significant inhibitory effects on VSMC migration through down-regulated phosphorylation of target proteins (HSP27 and CREB) associated with intimal hyperplasia. In addition, it was found that the released MK2i infiltrated into the tissue with a cumulative manner in ex vivo human saphenous vein (HSV) model. This present study demonstrates that enzymatically HT-coated surface modification is an effective strategy to induce long-term MK2i release as well as hemocompatibility, thereby improving anti-neointimal activity of synthetic vascular grafts.

Tyrosinase-Mediated Surface Coimmobilization of Heparin and Silver Nanoparticles for Antithrombotic and Antimicrobial Activities.

Thrombus and infections are the most common causes for the failure of medical devices, leading to higher hospitalization costs and, in some cases, patient morbidity. It is, therefore, necessary to develop novel strategies to prevent thrombosis and infection caused by medical devices. Herein, we report a simple and a highly efficient strategy to impart antithrombotic and antimicrobial properties to substrates, by simultaneously immobilizing heparin and in situ-synthesized silver nanoparticles (Ag NPs) via a tyrosinase-catalyzed reaction. This consists of tyrosinase-oxidized phenolic groups of a heparin derivative (heparin-grafted tyramine, HT) to catechol groups, followed by immobilizing heparin and inducing the in situ Ag NP formation onto poly(urethane) (PU) substrates. The successful immobilization of both heparin and in situ Ag NPs on the substrates was confirmed by analyses of water contact angles, XPS, SEM, and AFM. The sustained silver release and the surface stability were observed for 30 days. Importantly, the antithrombotic potential of the immobilized surfaces was demonstrated by a reduction in fibrinogen absorption, platelet adhesion, and prolonged blood clotting time. Additionally, the modified PU substrates also exhibited remarkable antibacterial properties against both Gram-positive and Gram-negative bacteria. The results of this work suggest a useful, effective, and time-saving method to improve simultaneous antithrombotic and antibacterial performances of a variety of substrate materials for medical devices.

Facile surface PEGylation via tyrosinase-catalyzed oxidative reaction for the preparation of non-fouling surfaces

The control of biological interactions that occur at material-cell/blood interfaces is of great importance to help maximize in vitro and in vivo performance of biomedical devices. PEGylation has been extensively used as an effective surface modification tool that can alter biological responses on device surfaces. Herein, we report a new surface PEGylation method using a tyrosinase-catalyzed oxidative reaction. Tyramine (TA), an enzymatically active phenolic compound, was chemically conjugated to methoxy poly(ethylene glycol) (mPEG). Surface immobilization of mPEG-TA onto various substrates was accomplished simply and rapidly by adding tyrosinase under mild conditions. It was shown that the water contact angles on all surfaces modified with mPEG-TA were decreased, indicating successful introduction of hydrophilic PEG. In addition, the X-ray photoelectron spectroscopy (XPS) spectra demonstrated the differences in the atomic composition on the TiO2 surface after treatment with mPEG-TA. Non-fouling surfaces prevent non-specific interactions with proteins and cells; consistently, the PEGylated TiO2 surface clearly showed a decrease in both levels of bovine serum albumin (BSA) adsorption and NIH3T3 cell attachment. Therefore, the facile surface PEGylation using a tyrosinase-catalyzed oxidative reaction should be useful for designing non-fouling surfaces of biomedical devices.

Facile surface immobilization of cell adhesive peptide onto TiO2 substrate via tyrosinase-catalyzed oxidative reaction

A facile method to immobilize bioactive molecules including phenol molecules onto TiO2 surfaces via tyrosinase-catalyzed oxidative reaction was developed for surface bio-functionalization of biomaterials.

Comparison of substrate specificity of tyrosinases from Trichoderma reesei and Agaricus bisporus

Understanding the substrate specificity of tyrosinases (EC 1.14.18.1) as well as their capability to oxidize peptide-bound tyrosine residues is important in a view of applicability of tyrosinases. In the present study, two fungal tyrosinases, an extracellular enzyme from the filamentous fungus Trichoderma reesei (TrT) and an intracellular enzyme from the edible mushroom Agaricus bisporus (AbT) were compared. Oxidation of various mono- and diphenolic compounds and tyrosine-containing tripeptides was examined and kinetic constants determined using spectrophotometric and oxygen consumption measurements. TrT and AbT were found to show notable differences in their substrate specificity. TrT generally showed 10-fold higher K m values than AbT. The presence of a carboxylic and amine group in the substrate influenced the enzymes differently. While the substrates with a carboxyl group were observed not to be effectively oxidized by AbT, the amine group seemed to hider the oxidation in the TrT-catalyzed reactions. Moreover, the UV–visible absorption spectra on the oxidation of catechol and hydrocaffeic acid showed that the product patterns were different between the enzymes. The result is interesting as the primary products from tyrosinase-catalyzed reactions were assumed to be identical with both enzymes. Furthermore, a nucleophilic 3-methyl-2-benzothiazolinone hydrazone (MBTH) affected differently on the activity of the tyrosinases: the lag period related to the oxidation of monophenols was prolonged by MBTH with TrT, whereas with AbT the lag was shortened.

Specific capture of target proteins by oriented antibodies bound to tyrosinase-immobilized Protein A on a polyallylamine affinity membrane surface

In this study, affinity membranes for the specific capture of a target protein to its antibody were fabricated in a three-step process: (1) coating of an amine-functional polymer layer (polyallylamine), (2) immobilization of Protein A by tyrosinase-catalyzed reaction to the amine groups and (3) binding of antibody, through its Fc tail, to the Protein A. The IgG binding isotherm was measured and compared to a membrane onto which Protein A was immobilized through amine-glutaraldehyde chemistry, as a control. The normalized amount of IgG bound, at monolayer saturation, was 30 times higher than that for the control, suggesting the Protein A was more accessible. The affinity constant was 100 times higher, indicating that the site-directed immobilization of Protein A through a remote tyrosine residue results in a more desirable orientation for antibody binding. Affinity membranes for protein capture were created by attaching either anti-BSA (bovine serum albumin) or anti-GFP (green fluorescent protein) to the Protein A. Dot blots were used as a function of concentration to assess the ability of each affinity membrane to capture its specific protein antigen in a layered stack arrangement. The membranes based on tyrosinase-immobilized Protein A were able to selectively bind their respective targets at much lower concentrations than the controls. The membranes could also reproduce blotted shapes, an important attribute for the analysis of proteins from tissue sections.

Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications

We compared the ability of two enzymes to catalyze the formation of gels from solutions of gelatin and chitosan. A microbial transglutaminase, currently under investigation for food applications, was observed to catalyze the formation of strong and permanent gels from gelatin solutions. Chitosan was not required for transglutaminase-catalyzed gel formation, although gel formation was faster, and the resulting gels were stronger if reactions were performed in the presence of this polysaccharide. Consistent with transglutaminase's ability to covalently crosslink proteins, we observed that the transglutaminase-catalyzed gelatin–chitosan gels lost the ability to undergo thermally reversible transitions (i.e. sol–gel transitions) characteristic of gelatin. Mushroom tyrosinase was also observed to catalyze gel formation for gelatin–chitosan blends. In contrast to transglutaminase, tyrosinase-catalyzed reactions did not lead to gel formation unless chitosan was present (i.e. chitosan is required for tyrosinase-catalyzed gel formation). Tyrosinase-catalyzed gelatin–chitosan gels were observed to be considerably weaker than transglutaminase-catalyzed gels. Tyrosinase-catalyzed gels were strengthened by cooling below gelatin's gel-point, which suggests that gelatin's ability to undergo a collagen-like coil-to-helix transition is unaffected by tyrosinase-catalyzed reactions. Further, tyrosinase-catalyzed gelatin–chitosan gels were transient as their strength (i.e. elastic modulus) peaked at about 5 h after which the gels broke spontaneously over the course of 2 days. The strength of both transglutaminase-catalyzed and tyrosinase-catalyzed gels could be adjusted by altering the gelatin and chitosan compositions. Potential applications of these gels for in situ applications are discussed.

In vitro protein-polysaccharide conjugation: tyrosinase-catalyzed conjugation of gelatin and chitosan.

The enzyme tyrosinase was used for the in vitro conjugation of the protein gelatin to the polysaccharide chitosan. Tyrosinases are oxidative enzymes that convert accessible tyrosine residues of proteins into reactive o-quinone moieties. Spectrophotometric and dissolved oxygen studies indicate that tyrosinase can oxidize gelatin and we estimate that 1 in 5 gelatin chains undergo reaction. Oxidized tyrosyl residues (i.e., quinone residues) can undergo nonenzymatic reactions with available nucleophiles such as the nucleophilic amino groups of chitosan. Ultraviolet/visible, (1)H-NMR, and ir provided chemical evidence for the conjugation of oxidized gelatin with chitosan. Physical evidence for conjugation was provided by dynamic viscometry, which indicated that tyrosinase catalyzes the sol-to-gel conversion of gelatin/chitosan mixtures. The gels formed from tyrosinase-catalyzed reactions were observed to differ from gels formed by cooling gelatin. In contrast to gelatin gels, tyrosinase-generated gels had different thermal behavior and were broken by the chitosan-hydrolyzing enzyme chitosanase. These results demonstrate that tyrosinase can be exploited for the in vitro formation of protein-polysaccharide conjugates that offer interesting mechanical properties.

Chitosan based water-resistant adhesive. Analogy to mussel glue.

Using analogies from nature, we investigated the possibility that tyrosinase-catalyzed reactions of 3,4-dihydroxyphenethylamine (dopamine) could confer water-resistant adhesive properties to semidilute solutions of the polysaccharide chitosan. Rheological measurements showed that the tyrosinase-catalyzed, and subsequent uncatalyzed, reactions lead to substantial increases in the viscosity of the chitosan solutions. Samples from these high-viscosity modified-chitosans were spread onto dry glass slides, the slides were lapped and clipped together either in air or after being submerged in water, and the bound slides were held under water for several hours. Adhesive shear strengths of over 400 kPa were observed for these modified chitosan samples, while control chitosan solutions conferred no adhesive strength (i.e., the glass slides separated in the absence of measurable forces). High viscosities and water-resistant adhesive strengths were also observed when semidilute chitosan solutions were treated with the known cross-linking agent, glutaraldehyde. Further studies indicate a relationship between the increased viscosities and water-resistant adhesion. These results demonstrate that the renewable biopolymer chitosan can be converted into a water-resistant adhesive.

Enzymatic modification of the synthetic polymer polyhydroxystyrene

In aqueous-methanolic solutions, mushroom tyrosinase was observed to catalyze the oxidation of phenolic moieties of the synthetic polymer poly(4-hydroxystyrene) (PHS). Although oxidation is rapid, on the order of minutes, it seems that only a small number of phenolic moieties of the PHS polymer (1 to 2%) undergo oxidation. Enzymatically oxidized PHS was observed to undergo a subsequent nonenzymatic reaction with aniline yielding an aniline-modified PHS polymer with a low degree-of-substitution. To gain insights into linkages between aniline and oxidized PHS, we characterized products resulting from tyrosinase-catalyzed reactions of p-cresol and aniline. A major product from these small-molecule studies was the Michael’s-type adduct, 4-anilino-5-methyl-1,2-benzoquinone. Based on similarities in ultraviolet-visible and 1H nuclear magnetic resonance spectra, we believe Michael’s-type adducts are present in the aniline-modified PHS polymer. To illustrate the potential utility of this enzymatic approach for grafting PHS onto other polymers, we contacted enzymatically oxidized PHS with the amine-containing biopolymer chitosan. Ultraviolet spectra of this chitosan film suggests oxidized PHS is grafted onto chitosan.

Inactivation of ornithine decarboxylase by intermediates of tyrosinase-catalyzed reaction

1. 1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway. 2. 2. The inactivation was dependent on the substrate used (dihydroxybenzylamine ⪢ l-3,4-dihydroxy-phenylalanine ⪢ l-tyrosine) and on the concentration of intermediate produced rather than on the rate of formation. 3. 3. Sulfhydryl compounds (dithiothreitol and glutathione) or quinone-reducing agents (ascorbic acid) prevented the inactivation of ODC; l-ornithine, but not other aminoacids, also protected partially ODC. The results suggest that different cysteine residues in ODC molecule are implicated in the inactivatory event. 4. 4. When 14C-labeled catechols were used, numerous polypeptides resulted labeled, showing that the reactive quinones formed as intermediates in the process of melanin biosynthesis bind covalently to many cellular proteins.

Synthesis of 5- S- l-cysteinyl-glycine- l-dopa, a natural substrate for serum and melanocyte dipeptidase

A method for synthesis of the phaeomelanin pigment intermediate compound 5- S- l-cysteinyl-glycine- l-dopa is presented. This thioether has been suggested as a precursor to 5- S- l-cysteinyl- l-dopa, the key intermediate compound in phaeomelanin pigment formation. 5- S-Glutathione- l-dopa is first synthesized by the tyrosinase-catalyzed reaction between l-dopa and glutathione. The 5- S-glutathione- l-dopa is then converted to 5- S- l-cysteinyl-glycine- l-dopa using the enzyme γ-glutamyl transpeptidase. The compound thus obtained was suitable as a substrate for melanoma cell and serum dipeptidase which converts the compound into 5- S- l-cysteinyl- l-dopa and glycine. The optimum pH for the dipeptidase from melanoma cells was 7.5 and the K m was 1.2 m m.