Abstract

Manganese pollution has become a global environmental issue in aquatic systems. Bacterially driven Mn(II) oxidation can convert Mn(II) into manganese oxides, thereby reducing the concentration of soluble Mn(II). Previous proteomics of the Mn(II)-oxidizing bacterium Pseudomonas sp. AN-1 revealed a multicopper oxidase McoA potentially involved in Mn(II) oxidation. However, the enzymatic evidence still lacked. In this study, McoA was successfully heterologously expressed in Escherichia coli BL21(DE3). The purified McoA has a molecular weight of approximately 124.02 kDa, with a characteristic absorption peak (T1Cu) at 607 nm. It was identified as a bifunctional enzyme (laccase and Mn(II) oxidase). Kinetic analysis revealed that under pH 4.0 with 0.5 mM Cu(II), McoA exhibited high catalytic efficiency toward ABTS ( K ₘ = 105.1 μM, k cat  = 60.17 s −1 , V max  = 8.448 μM·min −1 ). At pH 7.0 with 0.5 mM Cu(II), it showed significant oxidation activity toward Mn(II) ( K ₘ = 7.45 mM, k cat  = 12.10 min −1 , V max  = 23.41 μM·min −1 ). The effect of Cu(II) for Mn(II) oxidation displayed concentration-dependent modulation, with maximum activity observed at 0.75 mM Cu(II). The enzyme exhibited remarkable thermal stability, retaining over 50% residual activity even at 70 °C, with an optimum reaction temperature of 50 °C. Mn(II) transport and oxidation mechanism of McoA was supposed based on predicted structure. Additionally, McoA was also capable of oxidizing solid-phase manganese phosphate, with lower k cat and higher K m than soluble Mn(II), Finally, although the McoA-engineered E. coli showed slower initial Mn(II) oxidation rate compared to the wild-type strain, it achieved comparable Mn(II) removal efficiency after 7 days.