Difference between revisions of "Part:BBa K5398600"
 
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__NOTOC__
 
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<partinfo>BBa_K5398600 short</partinfo>
 
<partinfo>BBa_K5398600 short</partinfo>
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__TOC__
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===Introduction===
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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 tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of tyrosine to L-DOPA via its monophenolase (MP) activity, and consecutively oxidizes L-DOPA to dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. dopaquinone is not stable and will be further non-enzymatically oxidized to 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.
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<html lang="zh">
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<head>
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <style>
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        .module {
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            border: 1px solid #ccc; /* 边框 */
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            padding: 20px; /* 内边距 */
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            margin: 20px auto; /* 外边距，自动居中 */
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            width: 800px; /* 模块宽度 */
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            text-align: center; /* 内容居中 */
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            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
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        }
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  </style>
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</head>
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<body>
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    <div class="module">
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        <img src="https://static.igem.wiki/teams/5398/design/design-new/part-fig1.png" width="800" height="auto" alt="Protein purification">
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        <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product dopachrome.</b></p>
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    </div>
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</body>
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</html>
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===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===
 +
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 h, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.
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<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .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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        }
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  </style>
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</head>
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<body>
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    <div class="module">
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        <img src="https://static.igem.wiki/teams/5398/tyrvs/pre-expression.webp" width="400" height="auto" alt="Protein purification">
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        <p><b>Fig. 2 | Expression of recombinant TyrVs in <i>E. coli</i> BL21(DE3) with pET-PC-SUMO-TyrVs.</b></p>
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<p>Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
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    </div>
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</body>
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</html>
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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.
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<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .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 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs-mizuo-new.webp" width="400" height="auto" alt="Protein purification">
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        <p><b>Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
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<p>Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole. </p>
 +
    </div>
 +
</body>
 +
</html>
 +
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℃ with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (V<sub>max</sub>) 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 (V<sub>max</sub>) were 8787 μmol/L and 0.86 μmol/L·s, respectively. 
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .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 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/new-abcd.webp" width="600" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 4 | The activity assay results of tyrosinase TyrVs</b></p>
 +
<p><b>a-b.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. <b>c-d.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments. </p>
 +
    </div>
 +
</body>
 +
</html>
 +
<br><br><br><br>
  
−
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. 
 
  
 
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<partinfo>BBa_K5398600 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5398600 SequenceAndFeatures</partinfo>
  
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=== Reference ===
 +
<br>[1] TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized <em>Verrucomicrobium spinosum</em> tyrosinase on polyhydroxyalkanoate nano-granules [J]. <em>Appl. Microbiol. Biotechnol.</em>, 2019, 103(14): 5663-78.
 +
<br>[2] 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]. <em>Int. J. Biol. Macromol.</em>, 2022, 195: 229-36.
 +
<i>Biomacromolecules</i>, 2014, 15(9): 3278-3289.</p>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 09:08, 2 October 2024


A tyrosinase enzyme TyrVs

Introduction

Tyrosinase is a copper-containing oxidoreductase that possesses two catalytic activities, and is involved in the first few steps of melanin synthesis from tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of tyrosine to L-DOPA via its monophenolase (MP) activity, and consecutively oxidizes L-DOPA to dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. dopaquinone is not stable and will be further non-enzymatically oxidized to 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.

Protein purification

Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product 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

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 h, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.

Protein purification

Fig. 2 | Expression of recombinant TyrVs in E. coli BL21(DE3) with pET-PC-SUMO-TyrVs.

Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.

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.
Protein purification

Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.

Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole.

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℃ 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.
Protein purification

Fig. 4 | The activity assay results of tyrosinase TyrVs

a-b. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. c-d. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments.






Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference


[1] 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.
[2] 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. Biomacromolecules, 2014, 15(9): 3278-3289.</p>