Extracellular Tyrosinase Research Articles

Skin Anti-Aging Potential through Whitening and Wrinkle Improvement Using Fermented Oil Derived from Hermetia illucens Larvae.

As the aging population increases, so has interest among emerging seniors in anti-aging ingredients that enhance functionality by incorporating fermentation with natural materials. In this study, fermentation conditions for enhancing the functionality of Hermetia illucens larvae oil (HIO) were established, and its anti-aging potential was evaluated. First, the lipase activity and amount of lipid degradation products of the fermentation strains were measured in order to select Lactobacillus gasseri and Lactiplantibacillus plantarum as the strains with high fermentation ability. A fermentation period of 28 d and a fermentation method that uses only the strain culture medium were established by evaluating the fermentation degree after fermenting HIO with the selected strains. The whitening functionality test results of fermented HIO (FHIO) showed an increase of approximately 20% in extracellular tyrosinase inhibition activity compared with HIO. Additionally, within melanocytes, there was a 12% increase in tyrosinase inhibition activity and a 26% enhancement in melanin production inhibition ability. For wrinkle-improving functionality, it was observed that, for fibroblasts, there was a 10% increase in collagen production, a 9% increase in collagenase inhibition ability, and an 8% increase in elastase inhibition ability. Therefore, FHIO was confirmed to be an effective cosmetic raw material, with high functionality for anti-aging within the senior generation. This is achieved through increased whitening and wrinkle-improving functionality.

A Systematic Study of Yiqi Qubai Standard Decoction for Treating Vitiligo Based on UPLC-Q-TOF/MS Combined with Chemometrics, Molecular Docking, and Cellular and Zebrafish Assays.

The Yiqi Qubai (YQ) formula is a hospital preparation for treating vitiligo in China that has had reliable efficacy for decades. The formula consists of four herbs; however, the extraction process to produce the formula is obsolete and the active ingredients and mechanisms remain unknown. Therefore, in this paper, fingerprints were combined with the chemometrics method to screen high-quality herbs for the preparation of the YQ standard decoction (YQD). Then, the YQD preparation procedure was optimized using response surface methodology. A total of 44 chemical constituents, as well as 36 absorption components (in rat plasma) of YQD, were identified via UPLC-Q-TOF/MS. Based on the ingredients, the quality control system of YQD was optimized by establishing the SPE-UPLC-Q-TOF/MS identification method and the HPLC quantification method. Network pharmacological analysis and molecular docking showed that carasinaurone, calycosin-7-O-β-d-glucoside, methylnissolin-3-O-glucoside, genkwanin, akebia saponin D, formononetin, akebia saponin B, and apigenin may be the key active components for treating vitiligo; the core targets associated with them were AKT1, MAPK1, and mTOR, whereas the related pathways were the PI3K-Akt, MAPK, and FoxO signaling pathways. Cellular assays showed that YQD could promote melanogenesis and tyrosinase activity, as well as the transcription and expression of tyrosinase-associated proteins (i.e., TRP-1) in B16F10 cells. In addition, YQD also increased extracellular tyrosinase activity. Further efficacy validation showed that YQD significantly promotes melanin production in zebrafish. These may be the mechanisms by which YQD improves the symptoms of vitiligo. This is the first systematic study of the YQ formula that has optimized the standard decoction preparation method and investigated the active ingredients, quality control, efficacy, and mechanisms of YQD. The results of this study lay the foundations for the clinical application and further development of the YQ formula.

Pupation inhibition and larvicidal activity of tyrosinase on Culex pipiens third-instar larvae

BackgroundMosquitoes are considered to be the main vector of a variety of diseases in both humans and domesticated animals. The development of insecticide resistance and the limitations of traditional insecticide-based strategies have resulted in significant efforts to develop eco-friendly, alternative methods.Main bodyIn this study, nine fungi species were screened to produce tyrosinase enzyme. This was done in order to evaluate its inhibitory activity against Culex pipiens third-instar larvae. The extracellular tyrosinase was produced by five strains. Aspergillus tamarii NRC3 was found to possess the highest tyrosinase activity and was therefore used in this study. Some factors were studied to enhance the production of the enzyme. The enzyme was partially purified using ammonium sulfate at a 70% saturation, giving 1.861 purification fold. The toxicity on Culex pipiens varied, depending upon the tyrosinase concentration and the period of exposure. In addition, notable histological effects were seen in the midgut region.ConclusionsA concentration of 80% on third-stage larvae showed 90% inhibition in the formation of pupae at 72 h post-treatment. In addition, a significant cellular microvillus disruption was seen in the midgut region at 24 h post-treatment.

Pseudoalteromone A, a Ubiquinone Derivative from Marine Pseudoalteromonas spp., Suppresses Melanogenesis.

An ubiquinone derivative, pseudoalteromone A (1), has been isolated from two marine-derived Pseudoalteromonas spp., APmarine002 and ROA-050, and its anti-melanogenesis activity was investigated. The anti-melanogenic capacity of pseudoalteromone A was demonstrated by assessing the intracellular and extracellular melanin content and cellular tyrosinase activity in the B16 cell line, Melan-a mouse melanocyte cell line, and MNT-1 human malignant melanoma cell line. Treatment with pseudoalteromone A (40 μg/mL) for 72 h reduced α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin production by up to 44.68% in B16 cells and 38.24% in MNT-1 cells. Notably, pseudoalteromone A induced a concentration-dependent reduction in cellular tyrosinase activity in B16 cell, and Western blot analyses showed that this inhibitory activity was associated with a significant decrease in protein levels of tyrosinase and tyrosinase-related protein 1 (Tyrp-1), suggesting that pseudoalteromone A exerts its anti-melanogenesis activity through effects on melanogenic genes. We further evaluated the skin-whitening effect of pseudoalteromone A in the three-dimensional (3D) pigmented-epidermis model, MelanoDerm, and visualized the 3D distribution of melanin by two-photon excited fluorescence imaging in this human skin equivalent. Collectively, our findings suggest that pseudoalteromone A inhibits tyrosinase activity and expression and that this accounts for its anti-melanogenic effects in melanocytes.

Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols

BackgroundTyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols.ResultsTwenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL− 1) and laccase (2.01 U.mL− 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase.ConclusionsThe polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Bacillus aryabhattai TFG5-mediated synthesis of humic substances from coir pith wastes

BackgroundHumic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment.ResultIn this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL−1) and laccase (2.1 U mL−1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV–Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS.ConclusionThe results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Application Studies of Purified Tyrosinase from Isolated Aeromonas sp. SNS with Detailed Characterization and Kinetic Studies

Tyrosinase is the key enzyme accountable for the bioconversion of L-tyrosine to L-DOPA and, further, in melanin. The extracellular tyrosinase from bacterium Aeromonas sp. SNS was purified by ionexchange chromatography using DEAE cellulose as a matrix. The enzyme was eluted with 0.2M NaCl, showing 20.93×10-3 U mg-1 specific activity with a 12.91 % yield and 17.01 purification fold. PAGE estimated the molecular weight of the purified enzyme at 120 and 150 KDa. The zymography of polyacrylamide gel with 5 mM L-DOPA as a substrate confirmed the protein was tyrosinase. Purified tyrosinase shows maximum activity at pH 7 and 40°C temperature. The screening of different inhibitors shows maximum (100 %) inhibition by 1 mM L-ascorbic acid, followed by kojic acid with 91.57 % inhibition. The Km and Vmax for L-DOPA were found to be 0.488 mM and 1.386×10-4 U mg-1, respectively, while for L-tyrosine, 0.470 mM Km and 2.428×10-4 U mg-1 Vmax were recorded. To retain activity for the maximum time, immobilization was performed using sodium alginate and CuCl2. The immobilized beads of tyrosinase were further used for the production of L-DOPA (2.2 mg L-1) in a packed bead reactor and the degradation of toxic dyes. The purification and application study of immobilized tyrosinase for L-DOPA production and dye degradation confirms its role in therapeutic and industrial applications.

Melanin production as a visual indicator of conjugal transfer in Streptomyces.

To visualize transfer of plasmid in Streptomyces during conjugation, we constructed a conjugative plasmid that harbored melC operon encoding an extracellular tyrosinase and placed it in Streptomyces hosts which were defective in expressing the operon. Hyphae of these donors were mixed with hyphae of a plasmidless recipient, which could express melC, and plated on a solid medium supplemented with tyrosine. After 8 to 9h of incubation, melanin started to appear in the mating mixture, indicating that the plasmid had entered the recipient and started to synthesize tyrosinase, which in turn catalyzed the formation of melanin. This visual monitoring system allows quick demonstration of conjugal transfer without tedious genetic or biochemical procedure commonly used. It may be applied to most Streptomyces species and may also be used for monitoring chromosome transfer.

Effects of Galgeungyulpitang on Cellular Production of Melanin and Elastase

Background: This study was designed to investigate the potential effects of Galgeungyulpitang for whitening and elasticity treatment by examining its effect on melanoma cells. Methods: The effects of Galgeungyulpitang on B16/F10 melanoma cell viability, production of melanin, tyrosinase and elastase, were investigated. Cell viability was measured by colorimetric assay that assesses cell metabolic activity (MTT assay). Melanin was measured by Hosei’s method, tyrosinase was measured by Yogi’s method and elastase was measured by James’s method. Results: At concentrations higher than 500 μg/mL Galgeungyulpitang, cell viability was significantly reduced (p ≤ 0.05). At concentrations of 500 μg/mL and lower, morphological changes were not observed. The rate of melanin synthesis was significantly reduced to 73.49% ± 2.92% at a concentration of 500 μg/mL Galgeungyulpitang compared with untreated cells (p < 0.05). Extracellular tyrosinase production was not significantly decreased in vitro, however, intracellular tyrosinase production was significantly reduced to 76.06% ± 2.17% when treated with Galgeungyulpitang at a concentration of 500 μg/mL compared with the control (p < 0.05). Elastase Type 1 production was significantly reduced to 74.98% ± 3.24% and 69.62% ± 4.66% at concentrations of 250 and 500 μg/mL Galgeungyulpitang, respectively (p < 0.05). Elastase Type 4 production was significantly reduced to 72.77% ± 3.52% at concentrations of 250 and 500 μg/mL (p < 0.05). Conclusion: The results in this study showed that Galgeungyulpitang may inhibit melanin and tyrosinase synthesis, and inhibit elastase production, suggesting that Galgeungyulpitang may be potentially beneficial for skin whitening and loss of skin elasticity treatments.

Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production

BackgroundFilamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics.ResultsHere, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth.ConclusionsOur data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of streptomycetes.

Understanding the role of extracts from sea buckthorn seed residues in anti-melanogenesis properties on B16F10 melanoma cells.

The hydroalcoholic extract of sea buckthorn (Hippophae rhamnoides L.) seed residues (HYD-SBSR) is a potential skin whitening agent. To test this material as a potential skin whitening agent, we identified and quantified the main chemical constituents of HYD-SBSR by using ultra-performance liquid chromatography coupled with quadruple time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) and ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry (UPLC-QQQ-MS). The anti-melanogenesis properties of HYD-SBSR on B16F10 mouse melanoma cells were analysed and the mechanism was measured on both the transcriptional and translational levels. About 24 compounds were identified. Kaempferol and its derivatives were the main compounds with a concentration of about (2796.22 ± 31.55) μg per g DW. The following order among the detected compounds was observed: quercetin and its derivatives > isorhamnetin and its derivatives > procyanidins. HYD-SBSR has a strong antioxidant activity but with a slight cytotoxic effect on B16F10 when treated with 45.45 μg mL-1 and 4.55 μg mL-1 respectively, for 48 h. HYD-SBSR has been found to significantly decrease melanin content (P < 0.01) in 24 h, 48 h, and 72 h. Additionally, strong inhibitory extracellular tyrosinase activities and decreasing intracellular tyrosinase activities were also observed (P < 0.01). HYD-SBSR shows inhibitory effects on the expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1), and the secretion of TYR and TRP-1 proteins in cell lines. The protein levels of tyrosinase-related protein 2 (TRP-2) and microphthalmia-associated transcription factor (MITF) showed no significant difference. HYD-SBSR may inhibit melanin synthesis by decreasing the tyrosinase activity and down-regulating the expression of TYR and TRP-1 which were probably induced by other transcriptional factors rather than MITF.

Effect of Extracellular Tyrosinase on the Expression Level of P450, Fpr, and Fdx and Ortho-hydroxylation of Daidzein in Streptomyces avermitilis.

We have reported that the expression of CYP105D7 in Streptomyces avermitilis produces 112.5mgL-1 of 7,3',4'-trihydroxyisoflavone (3'ODI) in 15h of the reaction time, when 7,4'-dihydroxyisoflavone (daidzein) is used as a substrate. Although production is significant, rapid degradation of 3'ODI after 15h was observed in a whole-cell biotransformation system, suggesting the further modification of 3'ODI by endogenous enzymes. In this present study, the effect of deletion of extracellular tyrosinase (melC2) in S. avermitilis for 3'ODI production as well as the expressions of CYP105D7, ferredoxin (Fdx), and ferredoxin reductase (Fpr) were investigated. The result revealed that daidzein hydroxylation activity in the ∆melC2 mutant decreased by 40% compared with wild-type S. avermitilis. Further, melC2 deletion significantly affects the messenger RNA (mRNA) expression profile of CYP105D7 and its electron transfer counterparts. Real-time PCR analysis of 9 Fdx, 6 Fpr, and CYP105D7 revealed a significant decrease in mRNA expression level compared to wild-type S. avermitilis. The result clearly shows that the decrease in daidzein hydroxylation activity is due to the lower expression level of CYP105D7 and its electron transfer counterpart in the ∆melC2 mutant. Furthermore, melC2 deletion prevents the degradation of 3'ODI.

Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded fora predicted protein of 42.3kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

Effect of Light Wavelengths and Coherence on Growth, Enzymes Activity, and Melanin Accumulation of Liquid-Cultured Inonotus obliquus (Ach.:Pers.) Pilát.

To investigate effects of light wavelengths and coherence on growth of liquid-cultured Inonotus obliquus mycelia, melanin accumulation and enzymes activity, culture condition as light of different wavelengths and coherence were studied. Short-term exposure of the vegetative mycelium by low-intensity coherent blue light was optimal for stimulation of growth, melanin synthesis, and increase in extracellular and intracellular activities of tyrosinase and polyphenoloxidase and extracellular catalase. Red coherent light, in the same mode, can effectively be used to stimulate the growth of mycelium and to increase intracellular and extracellular activity of polyphenoloxidase, extracellular catalase and tyrosinase, and intracellular peroxidase. Low-coherent light had less stimulating effect on the biosynthetic activity of I. оbliquus. It should be used in the cultivation directed at the obtaining endomelanin, polyphenoloxidase, and extracellular tyrosinase.

Partial Purification and Characterization of a Novel Extracellular Tyrosinase from Auricularia auricula

Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 °C and retained most of its activity between 4 and 50 °C. A. auricula tyrosinase could oxidize L-tyrosine, L-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze L-phenylalanine and ferulic acid. In comparison with other substrates, L-tyrosine displayed the highest affinity (K m of 0.11 mM) and the maximal reaction velocity (V max of 102.58 μmol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO4 and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO4.

Purification and Characterization of RNA Allied Extracellular Tyrosinase from Aspergillus Species

Production of L-DOPA, an anti-Parkinson's drug, using biological sources is widely studied in which tyrosinase is known to play a vital role. Tyrosinase is an omnipresent type 3 copper enzyme participating in many essential biological functions. Understanding properties of tyrosinase is essential for developing useful tyrosinase-based applications. Hence, extracellular tyrosinase from Aspergillus flavus UWFP 570 was purified using ammonium sulphate precipitation and DEAE ion exchange chromatography up to 8.3-fold. Purified protein was a riboprotein in nature containing significant amount of RNA which was confirmed colorimetrically and by electrophoresis. Removal of RNA reduced the activity and altered the conformation of tyrosinase as suggested by spectroflurometric results. Optimum pH and temperature of this 140 kDa protein were 7 and 40 °C, respectively. Copper sulphate and magnesium chloride enhanced the activity whereas in contrast FeCl₃ inhibited the activity completely. Purified tyrosinase transformed L-tyrosine (5 mM) to L-DOPA within 5 h.

Regioselective Hydroxylation oftrans-ResveratrolviaInhibition of Tyrosinase fromStreptomyces avermitilisMA4680

Secreted tyrosinase from melanin-forming Streptomyces avermitilis MA4680 was involved in both ortho-hydroxylation and further oxidation of trans-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of melC(2) and melD(2) encoding extracellular and intracellular tyrosinase, respectively; the melC2 deletion mutant did not produce piceatannol as well as melanin, whereas the melD2 deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted trans-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of trans-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of trans-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of trans-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type Streptomyces avermitilis cells, the conversion yield of 100 μM trans-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h(-1) DCW mg(-1) specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

Extracellular and Intracellular Polyphenol Oxidases Cause Opposite Effects on Sensitivity of Streptomyces to Phenolics: A Case of Double-Edged Sword

Many but not all species of Streptomyces species harbour a bicistronic melC operon, in which melC2 encodes an extracellular tyrosinase (a polyphenol oxidase) and melC1 encodes a helper protein. On the other hand, a melC-homologous operon (melD) is present in all sequenced Streptomyces chromosomes and could be isolated by PCR from six other species tested. Bioinformatic analysis showed that melC and melD have divergently evolved toward different functions. MelD2, unlike tyrosinase (MelC2), is not secreted, and has a narrower substrate spectrum. Deletion of melD caused an increased sensitivity to several phenolics that are substrates of MelD2. Intracellularly, MelD2 presumably oxidizes the phenolics, thus bypassing spontaneous copper-dependent oxidation that generates DNA-damaging reactive oxygen species. Surprisingly, melC+ strains were more sensitive rather than less sensitive to phenolics than melC − strains. This appeared to be due to conversion of the phenolics by MelC2 to more hydrophobic and membrane-permeable quinones. We propose that the conserved melD operon is involved in defense against phenolics produced by plants, and the sporadically present melC operon probably plays an aggressive role in converting the phenolics to the more permeable quinones, thus fending off less tolerant competing microbes (lacking melD) in the phenolic-rich rhizosphere.