Enhancing Catecholase Activity of a Recombinant Human Tyrosinase Through Multiple Strategies

Document Type : Research Paper


1 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

3 Department of Human Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

4 Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran


Background: Tyrosinases are copper-containing enzymes that initiate the melanin synthesis. They catalyze the direct oxidation of L-tyrosine or L-DOPA into L-DOPAquinone.
Objectives: In present study, we aimed to obtain a recombinant tyrosinase with enhanced catecholase activity through site-directed mutagenesis.
Materials and Methods: The coding sequence of human tyrosinase along with native signal sequence was cloned into pET-28a (+). BL-21 was used as expression host and recombinant protein was purified by Ni-NTA resins. Site-directed mutagenesis was performed on M374 residue to achieve four mutants: M374D, M374T, M374K and M374R. Chloride ions (Clˉ) were removed from all solutions, and an extra amount of Cu2+ ions was added to recombinant tyrosinases by a novel technique during the purification process. Removal of Clˉ ions and addition of extra Cu2+ ions tripled catecholase activity of the recombinant protein. Therefore, all mutants were obtained under similar conditions.
Results: Although all the mutants presented higher catecholase activity in comparison to the wild-type enzyme, a significant increase in catecholase activity of the M374D mutant was observed ‒ 13.2-fold. In silico modeling suggested that a de novo hydrogen bond occurs between side chain carboxyl oxygens of D374 and H367 in M374D. In the wild-type tyrosinase, the peptide oxygen atom of M374 is responsible for hydrogen bonding with H367.
Conclusions: Our data suggests that M374D mutational variant has applications in different areas such as agriculture, industry, and medicine.


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