Melanin, an important biological pigment synthesized by tyrosinase, possesses broad application potential. However, traditional production approaches have suffered from low efficiency and high cost, limiting large-scale manufacturing. To overcome these challenges, this study aimed to identify a previously uncharacterized tyrosinase (MelC2) from a melanin-producing actinomycete and establish a biocatalytic system for DOPA-melanin biosynthesis. A melanin-producing actinomycete, designated LY-1, was isolated and identified as Streptomyces griseorubiginosus based on morphology, pigment formation, and 16S rRNA phylogeny. Genomic analysis revealed a melC gene cluster encoding the tyrosinase (MelC2) and tyrosinase metallochaperone (MelC1). Phylogenetic analysis, multiple-sequence alignment, and structure prediction demonstrated that MelC2 contained six conserved histidine residues coordinating a binuclear copper center, while MelC1 possessed a signal peptide for secretion. MelC2 was heterologously expressed in Escherichia coli, purified, and biochemically characterized. The enzyme exhibited maximal activity at pH 6.0 and 40 °C, moderate pH stability, and typical Michaelis-Menten kinetics toward L-DOPA. Spectroscopic analyses (UV-Vis, FT-IR, 1H NMR) confirmed the formation of DOPA-melanin. Whole-cell catalysis was subsequently optimized by adjusting reaction temperature, pH, Cu2+ concentration, cell density, and substrate loading. Constant alkaline conditions (pH 10.0) yielded the highest DOPA-melanin production. Under optimized conditions, the engineered whole-cell system achieved a DOPA-melanin titer of 7.49 g/L. This study provides the first biochemical characterization of a previously uncharacterized tyrosinase (MelC2) from S. griseorubiginosus and establishes a whole-cell strategy for sustainable DOPA-melanin biosynthesis.
Keywords: DOPA-melanin; Tyrosinase; Whole-cell catalysis.
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