Part:BBa_K5398610

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. </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

Plasmid Construction

We considered cloning TyrVs into the pET-PC-SUMO vector to explore the potential for enhancing its expression level. We constructed the pET-PC-SUMO-TyrVs vector and transformed it into E. coliBL21(DE3).

Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.

a.Expression plasmids of TyrVs. b.PCR results of pET-PC-SUMO-TyrVs. Line 1: Marker. Lines 2-3:Vector;Lines 4-5:Gene.

Protein expression

a single colony from a freshly streaked plate of the cells was cultured in 5 mL of LB medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were then incubated at 37℃ and 200 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells were collected by centrifugation at 6000 ×g at 4℃ for 20 min.The recombinant cells were harvested by centrifugation and re-suspension in lysis buffer(10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, pH 8.0)and lysed on ice by sonication.Sonicated samples were centrifuged at 12,000 ×g at 4 ◦C for 20 min to obtain insoluble and soluble fractions.After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.

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.

Western blotting

Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.

Fig. 4 | Western blotting analysis recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG 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.

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.

Enzyme activity test

We dialyzed the extracted SUMO-TyrVs for 24 hours, followed by diluting it 10,000-fold for enzymatic activity assays. In a 96 Well Cell Culture Plates, we prepared different concentrations of tyrosine and L-DOPA solution, added the diluted SUMO-TyrVs, and measured the change in OD475 over the first 5 minutes using a microplate reader.

Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.

a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone.

The data were processed to generate a Michaelis-Menten curve and a Lineweaver-Burk plot. 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^-1·s^-1, 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^-1·s^-1, respectively.

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]