Difference between revisions of "Part:BBa K5398610"
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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.
 
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.
 
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.
 
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.
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        <p class="caption"><b>Fig. 1 Plasmid profile of pET-PC-SUMO</b><br></p>
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<p style="text-align: center!important;"><b>Fig. 1 Plasmid profile of pET-PC-SUMO</b>
 
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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. The SUMO-TyrVs (52.2 kDa) was primarily present in the supernatant, indicating that it was expressed in a soluble form.
 
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. The SUMO-TyrVs (52.2 kDa) was primarily present in the supernatant, indicating that it was expressed in a soluble form.
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        <img src="https://static.igem.wiki/teams/5398/tyrvs/pre-expression.webp">
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        <p class="caption"><b>Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).</b><br></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 style="text-align: center!important;"><b>Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).</b>
 
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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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Subsequently, 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.
 
Subsequently, 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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            width: 562.5px;
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            border: 1px solid #000;
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            padding: 10px;
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        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-mizuo.webp">
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        <p class="caption"><b>Fig. 3 Protein expression of SUMO-TyrVs(52.2 kDa).</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 style="text-align: center!important;"><b>Fig. 3 Protein expression of SUMO-TyrVs(52.2 kDa).</b>
 
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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>
 
 
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.
 
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.
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<p style="text-align: center!important;"><b>Fig. 4 Tyrosinase TyrVs kinetic parameters</b>  
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(a) Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. (b) Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments.
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        <img src="https://static.igem.wiki/teams/5398/tyrvs/abcd-3.webp">
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        <p class="caption"><b>Fig. 4 Tyrosinase TyrVs kinetic parameters</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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==== Reference ====
 
==== Reference ====
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#Tan D , Zhao J P , Ran G Q ,et al.Highly efficient biocatalytic synthesis of l -DOPA using in situ immobilized V errucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules[J].<em>Appl. Microbiol. Biotechnol.</em>, 2019, 103(4).
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#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]. <em>Appl. Microbiol. Biotechnol.</em>, 2019, 103(14): 5663-78.
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<br>#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.
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#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.
+
<br>#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.
  
 
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<!-- Add more about the biology of this part here

Revision as of 03:10, 18 September 2024

A tyrosinase enzyme TyrVs

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. 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. Image with Caption

Fig. 1 Plasmid profile of pET-PC-SUMO

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. The SUMO-TyrVs (52.2 kDa) was primarily present in the supernatant, indicating that it was expressed in a soluble form. Image with Caption

Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).

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).
Subsequently, 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. Image with Caption

Fig. 3 Protein expression of SUMO-TyrVs(52.2 kDa).

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 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. Image with Caption

Fig. 4 Tyrosinase TyrVs kinetic parameters

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 BBa_K5398600 SequenceAndFeatures