A study on heterologous expression vector construction and enzyme activity of tyrosine oxidase from Ralstonia solanacearum

Abstract

Tyrosinase (TYR) is a copper-containing metalloenzyme that plays a crucial role in melanin synthesis and the metabolism of phenolic compounds. Beyond its significance in catalyzing the production of L-DOPA (L-3,4-dihydroxyphenylalanine), TYR holds substantial application value across multiple industries, including food and fruit processing, wastewater treatment, agriculture, cosmetics, and pharmaceutical healthcare. Ralstonia solanacearum, also known as bacterial wilt pathogen, was first identified by Janse in tobacco plants from Indonesia. In this study, we successfully accomplished the vector construction, heterologous expression, and enzymatic characterization of TYR. Specifically, the gene encoding TYR from Ralstonia solanacearum was cloned into the pET-28a(+) vector to generate the recombinant plasmid pET-28a(+)-TYR, which was subsequently expressed in Escherichia coli BL21(DE3). The recombinant enzyme was purified via Ni-affinity chromatography, and its enzymatic properties were systematically analyzed. High-performance liquid chromatography (HPLC) was employed to identify the catalytic products. Enzymatic assays revealed that TYR exhibits a specific activity of 120 U/mg toward L-DOPA under optimal conditions (pH 7.0, 30°C), with a Km value of 0.5 mM. HPLC analysis confirmed that the primary catalytic product was dopaquinone. This study provides a theoretical foundation for the industrial utilization of Ralstonia solanacearum-derived tyrosinase (TYR), highlighting its potential in biotechnological and industrial applications.