Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A−ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 μM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson’s disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

A novel tyrosinase from Armillaria ostoyae with comparable monophenolase and diphenolase activity suffers substrate inhibition

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyaestrain C18/9 (AoTyr) was characterized and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the two activities. In silico analysis and mutagenesis experiments showed that residue 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the substitution of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km) (1.63 folds) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than diphenolase activity. In the present study, a novel tyrosinase with a comparable monophenolase/diphenolase activity ratio was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residue 262 and 266 in the substrate inhibition and activities modulation of the enzyme, and the variant D262N showed an enhanced monophenolase/diphenolase catalytic efficiency ratio as compared to the wild type enzyme.

Anti-Tyrosinase and Antioxidant Activity of Proanthocyanidins From Cinnamomum Camphora

In this study, the contents of total phenols (TP) and proanthocyanidins (PAs) in the leaves and branches of Cinnamomum camphora were investigated, and isolated PAs were determined using reversed-phase HPLC-ESI-MS. The anti-tyrosinase and antioxidant activities were also evaluated. Furthermore, the scanning study and L-DOPA oxidation were performed to further analyzed the inhibition mechanism of PAs on tyrosinase catalytic activity. PAs had strong inhibitory effects on tyrosinase monophenolase activity, with effectively prolonged the delay time and decreased the steady-state of monophenolase activity. For diphenolase activity, the PAs both showed reversible and mixed inhibition. Moreover, the PAs showed strong antioxidant activities in scavenging 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 1,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid) (ABTS) and the ferric reducing antioxidant power (FRAP) assays. The PAs in leaves showed stronger anti-tyrosinase and antioxidant capacity, suggesting that C. camphora may be a good resource for tyrosinase inhibitors and antioxidants. This study could provide a scientific basis for the resource utilization of C. camphora and the development of new natural tyrosinase inhibitors and antioxidants in medical, cosmetic, food and agricultural industries.

Synthesis, Tyrosinase Inhibiting Activity and Molecular Docking of Fluorinated Pyrazole Aldehydes as Phosphodiesterase Inhibitors

A series of fluorinated 4,5-dihydro-1H-pyrazole derivatives were synthesized in the reaction of corresponding acetophenone and different aldehydes followed by the second step synthesis of desired compounds from synthesized chalcone, hydrazine hydrate, and formic acid. Structures of all compounds were confirmed by both 1H and 13C NMR and mass spectrometry. Antibacterial properties of compounds were tested on four bacterial strains, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Among synthesized compounds, the strongest inhibitor of monophenolase activity of mushroom tyrosinase (32.07 ± 3.39%) was found to be 5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde. The PASS program has predicted the highest probable activity for the phosphodiesterase inhibition. To shed light on molecular interactions between the synthesized compounds and phosphodiesterase, all compounds were docked into the active binding site. The obtained results showed that the compound with the dimethoxyphenyl ring could be potent as an inhibitor of phosphodiesterase, which interacts in PDE5 catalytic domain of the enzyme. Key interactions are bidentate hydrogen bond (H-bond) with the side-chain of Gln817 and van der Waals interactions of the dimethoxyphenyl ring and pyrazole ring with hydrophobic clamp, which contains residuals, Val782, Phe820, and Tyr612. Interactions are similar to the binding mode of the inhibitor sildenafil, the first oral medicine for the treatment of male erectile dysfunction.

Some characteristics of active and latent monophenolase of mushroom polyphenol oxidase

Latent form of monophenolase of mushroom polyphenol oxidase (PPO) was activated by 0,1% sodium dodecyl sulfate (SDS). The addition of increasing concentrations of 4-methylcatechol di minished lag period of active and total monophenolase activity, measured using p-cresol with L-proline as a substrate. Changes of lag period were described by equation of one phase exponential decay when concentration of substrate varied from l to 10 mM. Affinity ( 1/K_m) toward substrate of latent monophenolase was over two times higher than that of the active form, while the maximum velocity (V_max) was two times lower. The catalytic power (V_max/K_m) of both forms of monophenolase were almost equal. Electrophoretic analysis followed by scanning technique of the gels was used. Absorbancy of spots, determined from computer image of isoenzyme bands pattern allowed for qualitative and quantitative estimation of electrophoregrams. Presence of one additional clearly defined slow migrating isoenzyme for SDS activated monophenolase differed in this respect active (2 bands) and total (3 bands) forms of monophenolase.