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ABSTRACT: 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 (Cl-) 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 Cl- 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.
SUBMITTER: Shahrisa A
PROVIDER: S-EPMC7856396 | biostudies-literature | 2020 Apr
REPOSITORIES: biostudies-literature
Iranian journal of biotechnology 20200401 2
<h4>Background</h4>Tyrosinases are copper-containing enzymes that initiate the melanin synthesis. They catalyze the direct oxidation of L-tyrosine or L-DOPA into L-DOPAquinone.<h4>Objectives</h4>In present study, we aimed to obtain a recombinant tyrosinase with enhanced catecholase activity through site-directed mutagenesis.<h4>Materials and methods</h4>The coding sequence of human tyrosinase along with native signal sequence was cloned into pET-28a (+). BL-21 was used as expression host and rec ...[more]