Difference between revisions of "Part:BBa K5398610"
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+ | ===Introduction=== | ||
+ | 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. | ||
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+ | <meta charset="UTF-8"> | ||
+ | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
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+ | .module { | ||
+ | border: 1px solid #ccc; /* 边框 */ | ||
+ | padding: 20px; /* 内边距 */ | ||
+ | margin: 20px auto; /* 外边距,自动居中 */ | ||
+ | width: 800px; /* 模块宽度 */ | ||
+ | text-align: center; /* 内容居中 */ | ||
+ | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */ | ||
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+ | <img src="https://static.igem.wiki/teams/5398/mfp6-picture/.webp" width="400" height="auto" alt="Protein purification"> | ||
+ | <p><b>Fig. 1 Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p> | ||
+ | </div> | ||
+ | </body> | ||
+ | </html> | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA. | In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA. |
Revision as of 00:42, 29 September 2024
A tyrosinase enzyme TyrVs
Contents
Introduction
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. </p>
Fig. 1 Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.
Usage and Biology
In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA.
Characterization
Protein expression
We considered cloning TyrVs into the pET-SUMO vector to potentially increase its expression levels. So 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.
Enzyme activity test
We dialyzed the extracted SUMO-TyrVs for 24 hours and then diluted it 10,000 times for the enzyme activity assay. Given that tyrosinase exhibits dual catalytic properties, capable of catalyzing the conversion of tyrosine to L-DOPA and L-DOPA to dopaquinone, we aimed to develop a model to determine how to maximize the oxidation of tyrosine to L-DOPA. Therefore, 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.
==== Reference ====
#TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized Verrucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules [J]. Appl. Microbiol. Biotechnol., 2019, 103(14): 5663-78.
#YAO L, WANG X, XUE R, et al. Comparative analysis of mussel foot protein 3B co-expressed with tyrosinases provides a potential adhesive biomaterial [J]. Int. J. Biol. Macromol., 2022, 195: 229-36. Sequence and Features