Difference between revisions of "Part:BBa K5398600"
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| − | Tyrosinase is a copper-containing oxidoreductase that possesses two catalytic activities, and is involved in the first few steps of melanin synthesis from l-tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA via its monophenolase (MP) activity, and consecutively oxidizes l-DOPA to l-dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. l-dopaquinone is not stable and will be further non-enzymatically oxidized to l-dopachrome (a red-colored product) in the presence of O<sub>2</sub>.TyrVs refers to a tyrosinase enzyme derived from <em>Verrucomicrobium spinosum</em>, which plays a critical role in the hydroxylation of tyrosine residues into L-Dopa. This enzyme has shown efficient activity, particularly in the context of biological adhesion, as demonstrated in studies co-expressing mussel foot protein 3 with TyrVs. | + | Tyrosinase is a copper-containing oxidoreductase that possesses two catalytic activities, and is involved in the first few steps of melanin synthesis from l-tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA via its monophenolase (MP) activity, and consecutively oxidizes l-DOPA to l-dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. l-dopaquinone is not stable and will be further non-enzymatically oxidized to l-dopachrome (a red-colored product) in the presence of O<sub>2</sub>.TyrVs refers to a tyrosinase enzyme derived from <em>Verrucomicrobium spinosum</em>, which plays a critical role in the hydroxylation of tyrosine residues into L-Dopa. This enzyme has shown efficient activity, particularly in the context of biological adhesion, as demonstrated in studies co-expressing mussel foot protein 3 with TyrVs. |
| + | |||
| + | To validate the functionality of the tyrosinase TyrVs, we designed bacteria expressing TyrVs.We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 hours, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis. | ||
| + | |||
| + | We purified SUMO-TyrVs using a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE and was found to be present in the 50 mM imidazole elution fraction. | ||
| + | |||
| + | We conducted tests on the reactions from tyrosine to dopaquinone and from L-DOPA to dopaquinone. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37°C with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 456.8 μmol/L and 0.31 μmol/L·s, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 8787 μmol/L and 0.86 μmol/L·s, respectively. | ||
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Revision as of 12:00, 10 September 2024
A tyrosinase enzyme TyrVs
Tyrosinase is a copper-containing oxidoreductase that possesses two catalytic activities, and is involved in the first few steps of melanin synthesis from l-tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA via its monophenolase (MP) activity, and consecutively oxidizes l-DOPA to l-dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. l-dopaquinone is not stable and will be further non-enzymatically oxidized to l-dopachrome (a red-colored product) in the presence of O2.TyrVs refers to a tyrosinase enzyme derived from Verrucomicrobium spinosum, which plays a critical role in the hydroxylation of tyrosine residues into L-Dopa. This enzyme has shown efficient activity, particularly in the context of biological adhesion, as demonstrated in studies co-expressing mussel foot protein 3 with TyrVs.
To validate the functionality of the tyrosinase TyrVs, we designed bacteria expressing TyrVs.We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 hours, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.
We purified SUMO-TyrVs using a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE and was found to be present in the 50 mM imidazole elution fraction.
We conducted tests on the reactions from tyrosine to dopaquinone and from L-DOPA to dopaquinone. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37°C with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 456.8 μmol/L and 0.31 μmol/L·s, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 8787 μmol/L and 0.86 μmol/L·s, respectively.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 309
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]