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.

Isolation and Partial Purification of Polyphenol Oxidase from Seed of Melon (Cucumeropsis edulis)

Polyphenol oxidase (PPO) from Cucumeropsis edulis was extracted and partially purified through (NH4)2SO4 precipitation, dialysis, and ion-exchange chromatography on DEAE-Sephadex-A50. The spectrophotometric method was used to assay the enzyme activity in C. edulis using L-DOPA as substrate, the physicochemical properties such as the effect of pH and temperature, substrate specificity, kinetic constants - maximum enzyme velocity (Vmax), and Michaelis - Menten constant (Km) for three substrates namely, L-Dopa catechol and tyrosine were determined. The effects of inhibitors and metal ions on PPO activity were also investigated. The optimum pH and temperature values were found to be pH 6.5 and 50 °C, and the inhibitory effects of inhibitors such as ascorbic acid, EDTA, SDS, and metal ions were enhanced positively with increased concentration except with divalent metals such as Cu2+, Fe2+, and Zn2+ reflecting an activating effect on C. edulis PPO. Moreover, the enzyme solution showed both monophenolase and diphenolase activity with L-DOPA having the highest Vmax/Km value. However, the data obtained in this research provided a theoretical basis for the prevention of enzymatic browning of C. edulis during processing.

Achillea millefolium L. and Achillea biebersteinii Afan. Hydroglycolic Extracts–Bioactive Ingredients for Cosmetic Use

Studies on hydroglycolic (HG) extracts of Achillea biebersteinii (AB)—a less investigated representative of the genus—were performed to determine their potential for cosmetic applications compared to the well-known Achillea millefolium (AM). Three types of water:polyethylene glycol extracts (1:1, 4:1, 6:1 v/v) were obtained from both species and analyzed for their composition by high performance liquid chromatography coupled with mass spectrometry (HPLC-ESI-Q-TOF-MS) and assayed for their biological activities. The study led to the identification of 11 metabolites from different natural product classes with the highest share corresponding to 5-caffeoylquinic acid, axillarin, coumaroylquinic acid isomers and 3-caffeoylquinic acid. The highest antiradical capacity in DPPH and ABTS scavenging assays was shown for HG 4:1 of AB and AM extracts. HG 1:1 extracts from both species inhibited monophenolase and diphenolase activity of tyrosinase, whereas AB HG 4:1 extract showed significant monophenolase inhibition. The highest sun protection factor (SPF) was determined for AM HG 4:1 extract, equal to 14.04 ± 0.17. The AB extracts were cytotoxic for both human keratinocytes HaCaT and A375 melanoma, however HG 1:1 and 4:1 extracts were more cytotoxic for cancer than for noncancerous cells. In conclusion, AB HG 1:1 and 4:1 extracts display significant potential as active cosmetic ingredients.

Simultaneous Determination of Six Isoflavones from Puerariae Lobatae Radix by CPE-HPLC and Effect of Puerarin on Tyrosinase Activity

Tyrosinase inhibitors with excellent inhibitory activities and lower side effects have promising applications in the fields of medicine, agriculture, food sciences and cosmetics. In this study, a method for simultaneous separation and determination of six target compounds (puerarin, daidzin, genistein, daidzein, genistin, and formononetin) in Puerariae Lobatae Radix was established by cloud point extraction (CPE) and concentration combined with high performance liquid chromatography (HPLC). To achieve high extraction yields, an ultrasound-assisted extraction method was developed based on a salt-modified Triton X-100 system. The optimal extraction conditions are: surfactant Triton X-100 concentration 0.07 g/mL, liquid-solid ratio 80:1 (mL/g), NaCl addition amount 0.6 g, equilibrium time 40 min, equilibrium temperature 70 °C. Under the optimal conditions, the total maximum extraction yield of the six target isoflavones reached 8.92 mg/g. Using l-tyrosine and l-dopa as substrates, the effects of puerarin on the monophenolase and diphenolase activity of tyrosinase activity were investigated by the enzyme kinetics method. The results showed that puerarin inhibited monophenolase activity with an IC50 of 0.537 mg/mL and activated diphenolase activity. The inhibition type of puerarin on monophenolase and the activation type of puerarin on diphenolase were analyzed by Lineweaver-Burk plots which show that puerarin showed mixed inhibition on monophenolase and mixed activation on diphenolase. Therefore, puerarin can be used as both a tyrosinase inhibitor and a tyrosinase activator.

Arbutin Content and Tyrosinase Activity of Bergenia Extracts

The total arbutin content in the leaves of all the studied Bergenia plants ( B. crassifolia, B. ciliata and B. x ornata) was determined. The highest values of the arbutin content have been established for B. crassifolia (58.9 ± 0.7 mg.g−1 DW) and B. x ornata (51.0 ± 1.21 mg.g−1 DW), and the lowest for B. ciliata (5.9 ± 0.6 mg.g−1 DW). Arbutin concentration in the Bergenia leaves was the lowest in spring, in the autumn, on the contrary it increased. All the tested aqueous extracts caused a dose-dependent increase in diphenolase activity of fungal tyrosinase in a similar way as arbutin. On the other hand, all the ethanol extracts inhibited the diphenolase activity of tyrosinase.