Tyrosinase from the pulps of local cultivars of Musa spp: Purification, characterization, immobilization, and application in the batch production of l-3,4-dihydroxyphenylalanine.

Tyrosinase, an enzyme involved in browning reactions in plants/crops exposed to mechanical injury, was isolated from the pulp of some different locally available bananas (M. cavendish, M. acuminata, and M. paradisiaca). Tyrosinase from the pulps was extracted, purified, immobilized, and characterized. Thereafter, the potentials of the immobilized tyrosinase in the possible production of l-3,4-dihydroxyphenylalanine (L-DOPA) in an improvised batch reactor was exploited using tyrosine and ascorbate as the substrates. L-DOPA production was monitored via thin-layer chromatography and spectrophotometry (Arnow's method). L-DOPA is a drug that is used in the treatment of Parkinson's disease. Hence, this study exploited a non-chemical route for its synthesis using the tyrosinase obtained from the banana pulps. The purified tyrosinase had an optimum pH and temperature of 6.5 and 7.0, respectively. The molecular weight of the purified tyrosinase was 45 kDa. Quercetin and resorcinol both competitively inhibited the purified tyrosinase from the three cultivars. Immobilized M. cavendish tyrosinase produced the highest concentration (0.60 mM) of L-DOPA after 8 h in an improvised batch reactor. The tyrosinase in the banana pulps serves as a cheap and readily available green route for the possible production of L-DOPA.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction.

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Laccase and Tyrosinase Biosensors Used in the Determination of Hydroxycinnamic Acids.

In recent years, researchers have focused on developing simple and efficient methods based on electrochemical biosensors to determine hydroxycinnamic acids from various real samples (wine, beer, propolis, tea, and coffee). Enzymatic biosensors represent a promising, low-cost technology for the direct monitoring of these biologically important compounds, which implies a fast response and simple sample processing procedures. The present review aims at highlighting the structural features of this class of compounds and the importance of hydroxycinnamic acids for the human body, as well as presenting a series of enzymatic biosensors commonly used to quantify these phenolic compounds. Enzyme immobilization techniques on support electrodes are very important for their stability and for obtaining adequate results. The following sections of this review will briefly describe some of the laccase (Lac) and tyrosinase (Tyr) biosensors used for determining the main hydroxycinnamic acids of interest in the food or cosmetics industry. Considering relevant studies in the field, the fact has been noticed that there is a greater number of studies on laccase-based biosensors as compared to those based on tyrosinase for the detection of hydroxycinnamic acids. Significant progress has been made in relation to using the synergy of nanomaterials and nanocomposites for more stable and efficient enzyme immobilization. These nanomaterials are mainly carbon- and/or polymer-based nanostructures and metallic nanoparticles which provide a suitable environment for maintaining the biocatalytic activity of the enzyme and for increasing the rate of electron transport.

Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera.

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity.

Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van 't Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van 't Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation.

Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives.

A convenient enzymatic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.

Efficient purification of a highly active H-subunit of tyrosinase from Agaricus bisporus.

A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 °C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity. The known H subunit of tyrosinase from Agaricus bisporus (45 kDa, H-AbTyr) and another protein of 50 kDa were present in the supernatant. Sodium phosphate buffer was added to adjust the ionic strength of the solution up to 100 mM and Triton X-100 was added (final concentration of 0.07% v/v) to control the hydrophobicity effect for both proteins. This solution was offered again to fresh octadecyl-Sepabeads support, immobilizing selectively the H-AbTyr and leaving exclusively the 50 kDa protein as a pure sample in the supernatant. This tyrosinase isoform of 50 kDa was almost 4-fold more active than the known H-TyrAb, with a specific tyrosinase activity of more than 38,000 U/mg.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold nanoparticles by using purified fungal phenol oxidases.

The work shows the ability of cultured Basidiomycetes of different taxonomic groups-Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa-to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl4 to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

The evaluation of the synergistic effect of 3-(2,4-dihydroxyphenyl)propionic acid and l-ascorbic acid on tyrosinase inhibition.

3-(2,4-Dihydroxyphenyl)propionic acid (DDPA) and l-ascorbic acid (vitamin C) show tyrosinase inhibition activity. A synergistic effect on tyrosinase inhibition was observed when the two compounds were mixed. The effect significantly decreased the IC50 value of both compounds.

Integrated kinetic studies and computational analysis on naphthyl chalcones as mushroom tyrosinase inhibitors.

Melanin helps to protect skin from the damaging ultraviolet radiation of the sun. Tyrosinase, the key enzyme in melanogenesis is responsible for coloration of skin, hair and eyes. This enzyme is considered to have a critical role in governing the quality and economics of fruits and vegetables, as tyrosinase activity can lead to spoilage through browning. Development of tyrosinase inhibitors is a promising approach to combat hyperpigmentation conditions like ephelides, lentigo, freckles and post-inflammatory hyperpigmentation. In the present study, we have used a docking algorithm to simulate binding between tyrosinase and hydroxy-substituted naphthyl chalcone oxime compounds and studied the inhibition of tyrosinase. The results of virtual screening studies indicated that the estimated free energy of binding of all the docked ligands ranged between -19.29 and -9.12 kcal/mol. Two of the oximes synthesized were identified as competitive tyrosinase inhibitors and were found to be twice as potent as the control kojic acid with their IC50 values of 12.22 µM and 19.45 µM, respectively. This strategy of integrating experimental and virtual screening methods could give better insights to explore potent depigmentation agents.

Purification and characterization of a phenoloxidase in the hemocytes of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors.

A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71. The purified PO showed the highest activity at pH 4-5 and temperatures of 35-40 °C. Na(+), K(+), Mn(+), Zn(2+) and Mg(2+) decreased activity of the purified PO but Ca(2+) and Cu(2+) increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest Vmax when L-DOPA was used as substrate but no significant differences were observed in case of Km for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC50 of 96.41 and 38.59 µg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC50 concentrations of the two IGRs revealed the increase of Km value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.

First laccase in green algae: purification and characterization of an extracellular phenol oxidase from Tetracystis aeria.

MAIN CONCLUSION: A green algal phenol oxidase was firstly purified, confirmed to be a laccase, and a hetero-oligomeric quaternary structure is suggested. The operation of a laccase-mediator system is firstly described in algae. Laccases (EC 1.10.3.2) catalyze the oxidation of a multitude of aromatic substrates. They are well known in higher plants and fungi, while their presence in green algae appears uncertain. Extracellular laccase-like enzyme activity has previously been described in culture supernatants of the green soil alga Tetracystis aeria [Otto et al. in Arch Microbiol 192:759-768, (2010)]. As reported herein, the T. aeria enzyme was purified 120-fold by employing a combination of anion exchange and size exclusion chromatography. The purified enzyme was confirmed to be a laccase according to its substrate specificity. It oxidizes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (pH optima of pH ≤ 2.5, 7.0, and 6.5; K m values of 28.8, 40.5, and 1,830 µM; respectively), but not L-tyrosine or Fe(2+). ABTS is by far the most efficient substrate. Two polypeptides, A (~110 kDa) and B (71 kDa), were co-purified by the applied procedure, both being highly N-glycosylated (≥~53 and ≥ 27 %, respectively). As suggested by various gel electrophoretic analyses, the native enzyme (apparent molecular mass of ~220 kDa) most probably is a hetero-oligomer with the composition AB 2 , wherein A is the catalytic subunit and B forms a disulfide-linked homo-dimer B2. The decolorization of anthraquinone (Acid Blue 62 and Remazol Brilliant Blue R) and diazo dyes (Reactive Black 5) was studied in the presence of redox-mediating compounds (ABTS and syringaldehyde), demonstrating the operation of the laccase-mediator system in algae for the first time. Thus, laccases from green algae may participate in the biotransformation of a wide spectrum of natural and xenobiotic compounds.

In vitro antibacterial analysis of phenoloxidase reaction products from the sea cucumber Apostichopus japonicus.

Three phenoloxidases (POs) of Apostichopus japonicus, AjPOs (AjPO1, AjPO2 and AjPO3), were partially purified from the coelomocytes with an electrophoretic method, and then employed for the in vitro antibacterial analysis. Using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, AjPO1 and AjPO2-derived compounds inhibited the growth of Vibrio splendidus and Staphylococcus aureus, while AjPO3-derived compounds only inhibited the growth of V. splendidus. When dopamine was used as a substrate, AjPO1 and AjPO3-derived compounds inhibited the growth of V. splendidus and Vibrio harveyi, while AjPO2-derived compounds only inhibited the growth of V. splendidus. Moreover, AjPO1-derived compounds showed stronger inhibition in V. harveyi than AjPO3-derived compounds did. However, all of the three AjPO reaction products showed no inhibitions on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica, Micrococcus lysodeikticus, Streptococcus dysgalactiae and Nocardiopsis sp. with L-DOPA or dopamine as a substrate. Scanning electron microscope (SEM) observation of V. harveyi treated by AjPOs and dopamine showed that AjPO1-derived compounds resulted in massive bacteriolysis, AjPO2-derived compounds caused no obvious alteration on bacterial morphology, and AjPO3-derived compounds increased the ratio of spheroidal bacteria. All these results suggested that AjPO reaction products derived by L-DOPA and dopamine had different but limited antibacterial spectrum, and the different antibacterial effects observed among three AjPOs resulted from the different reaction products generated by AjPOs with the same substrate.

Lysozyme and defense peptides as suppressors of phenoloxidase activity in Galleria mellonella.

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

Effects of Bromelia pinguin (Bromeliaceae) on soil ecosystem function and fungal diversity in the lowland forests of Costa Rica.

BACKGROUND: Bromelia pinguin (Bromeliaceae) is a terrestrial bromeliad commonly found under forest stands throughout the Neotropics that has been shown to have antifungal activity in vitro. We have hypothesized that this bromeliad would also have an effect on the fungal populations in nearby soil by decreasing fungaldiversity and negatively impacting C and N cycle-related activities. A previous study in the lowland forest of Costa Rica showed the soil beneath these bromeliads had decreased fungal ITS DNA and differences in C and N levels compared to adjacent primary forest soils. RESULTS: In this follow-up study, we found that the bromeliad soils had lower rates of C and N biomass development and lower phenol oxidase activity (suggesting less decreased fungal decomposition activity). The results of T-RFLP and cloning-based taxonomic analyses showed the community level diversity and abundance of fungal ITS DNA was less in bromeliad soils. Sequence analysis of fungal ITS DNA clones showed marked differences in fungal community structure between habitats of Basidiomycota (Tremellales, Agricales, Thelephorales), Ascomycota (Helotiales), and Zycomycota populations. CONCLUSIONS: The data show there to be differences in the soil nutrient dynamics and fungal community structure and activity associated with these bromeliads, as compared to the adjacent primary forest. This suggests the possibility that the anti-fungal activity of the bromeliad extends into the soil. The bromeliad-dense regions of these primary forest habitats provide a unique natural micro-habitat within the forests and the opportunity to better identify the role of fungal communities in the C and N cycles in tropical soils.

Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid detection of phenol constituents.

Most of the phenol compounds are toxic and have been considered as hazardous pollutants. Several physicochemical and biological methods are available to detect and monitor the phenol pollutants in water and soil. In the present study, phenol constituents of winery, paper and plastic industrial effluents were successfully detected employing tyrosinase-gold nanoparticles bioconjugate. The synthesis of extracellular tyrosinase and gold nanoparticles was achieved by a single isolate of Streptomyces sp. DBZ-39. Enhanced production (369.41 IU) of tyrosinase was produced in submerged bioprocess employing response surface method with central composite design. Extracellular gold nanoparticles synthesized (12-18 nm) by Streptomyces sp. DBZ-39 were characterized with TEM, EDAX and FTIR analysis. A rapid detection (within 10 min) of phenol constituents from winery effluents was achieved by bioconjugate, when compared to tyrosinases and gold nanoparticles independently. Streptomyces tyrosinase could exhibit relatively a better performance than commercially available mushroom tyrosinase in the detection of phenol constituents. Winery effluent has shown much higher content (0.98 O.D) of phenol constituents than paper and plastic effluents based on the intensity of color and U.V absorption spectra.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.

Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

Phenoloxidase in the scallop Chlamys farreri: purification and antibacterial activity of its reaction products generated in vitro.

Phenoloxidase (PO) was purified from hemocytes of the scallop Chlamys farreri using native-PAGE and gel permeation column chromatography, and then substrate specificity and antibacterial activity generated from reaction products of purified PO were analyzed. The results showed purified PO had a molecular mass of 576 kDa in native-PAGE and 53 kDa in denatured PAGE, and could catalyze the substrates L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, catechol and hydroquinone suggesting it is a type of p-diphenoloxidase. Using dopamine as a substrate, PO reaction products significantly inhibited the growth of Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas salmonicida. No significant inhibition was found in Streptococcus dysgalactiae, Streptococcus iniae, Micrococcus lysodeikticus and Edwardsiella tarda. When L-DOPA was used as a substrate, significant inhibition occurred in A. salmonicida only.