Difference between revisions of "Part:BBa K5398610"
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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. | 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. | 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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| + | ====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. | 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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Revision as of 08:39, 18 September 2024
A tyrosinase enzyme TyrVs
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.
Fig. 1 Plasmid profile of pET-PC-SUMO
Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).
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.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.
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