Difference between revisions of "Part:BBa K5398600"
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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 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.
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         <img src="https://static.igem.wiki/teams/5398/mfp6-picture/.webp" width="400" height="auto" alt="Protein purification">
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         <p class="caption"><b>Fig. 1 Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b><br></p>
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         <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p>
 
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===Usage and Biology===
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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.
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===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 hours, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.  
 
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.  
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         <p class="caption">Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).<br></p>
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         <p><b>Fig. 3 | Expression of recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
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        Lane 7: TyrVs-Whole Cell Lysate(+IPTG). Lane 8: TyrVs-Supernatant(+IPTG). Lane 9: TyrVs-Pellet(+IPTG). Lane 10: TyrVs-Whole Cell Lysate(CK). Lane 11: TyrVs-Supernatant(CK). Lane 12: TyrVs-Pellet(CK).
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<p>Lane 1: Marker. lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively;lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
 
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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.
 
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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         <p class="caption"><b>Fig. 3 Protein expression of SUMO-TyrVs(52.2 kDa).</b></p>
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         <p><b>Fig. 5 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
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        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.
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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>
 
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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.   
 
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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            width: 800px; /* 模块宽度 */
 +
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 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 
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         <p class="caption"><b>Fig. 4 Tyrosinase TyrVs kinetic parameters</b></p>
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         <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
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      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.
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<p>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. </p>
 
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<br><br><br><br>
 
<br><br><br><br>
 
==== Reference ====
 
==== Reference ====

Revision as of 04:05, 29 September 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 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.

Protein purification

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

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.

Protein purification

Fig. 3 | Expression of recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

Lane 1: Marker. lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively;lanes 5 to 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. 5 | 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°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.
Protein purification

Fig. 7 | 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.





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


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]