Multicopper oxidases (MCOs) are a family of enzymes that use copper

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. electron microscopy. To our knowledge, this is the 1st report that provides the direct observation of Mn(II) oxidation with the heterologously indicated protein CotA, Consequently, this novel getting not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal. Intro Manganese is the second most abundant transition in the Earths crust and the fifth most abundant element within the Earths surface. Generally, manganese offers three environmentally relevant oxidation claims, Mn(II), Mn(III) and Mn(IV) [1], [2]. Among these, Mn(II) is definitely thermodynamically favored at low pH and Eh, whereas Mn(III) and Mn(IV) oxides are stable at high pH and Eh [3]. The soluble form of Mn(II), serves as a crucial micronutrient for organisms, while the insoluble form of Mn(III/IV) oxide, is definitely a highly reactive mineral phase that participates in a wide range of redox and adsorptive reactions, playing a significant part in CT19 the bioavailability and geochemical cycling of many essential or harmful elements [1]. In many environments, the chemical oxidation of Mn(II) MK 0893 by O2 in the pH range of 6.0C8.5 is at a considerably low level while in the presence of Mn(II)-oxidizing microorganisms, including a variety of bacteria and fungi, the oxidation rate can be accelerated by as much as five orders of magnitude [4], [5]. Consequently, microbial processes are considered to be primarily responsible for the formation of Mn oxides [1], [6]. Although many microorganisms capable of oxidizing Mn(II) have been isolated and belong to varied phyla, the biochemical mechanism of Mn(II) oxidation is still enigmatic [3]. As fresh insights are gained regarding some proteins involved in Mn(II) oxidation, several enzymes have been gradually recognized from some varieties of bacteria and most of them belong to a family of multicopper oxidases (MCOs). MCOs are a class of copper proteins that utilize copper like a cofactor to catalyze four one-electron oxidations of various substrates concomitantly with the reduction of O2 to water [7], [8]. MCOs have been found in a wide range of organisms including bacteria, fungi (laccase), vegetation, bugs and vertebrates (ceruloplasmin) [7], [9]. So far, three model bacteria, sp. strain SG-1 MK 0893 [6], [10], [11], SS-1 [12] and sp. ACM3067 [13], [14], have been demonstrated to require the MCOs in Mn(II) oxidation by disruption of the related genes (and SI85-9A1 and sp. strain SD21 [4], [15] have uncovered a second class of enzymes involved in Mn(II) oxidation: heme-binding peroxidase named MopA. Another putative MCO (CumA) proposed in GB-1 [16], [17], however, has been proven to not be a Mn(II) oxidase by in-frame deletion of GB-1 [18]. To day, no bacterial Mn(II) oxidase has been purified to a large quantity adequate for detailed biochemical study. In addition, no MCO gene thought to encode a Mn(II) oxidase has been successfully indicated inside a heterologous sponsor to yield an active enzyme [1], [5], [19], let alone the enzymological properties of the Mn(II) oxidase. To conquer these unsettled problems, our particular emphasis is placed on overexpression, purification and biochemical characterization of plentiful recombinant MCOs in varieties, which naturally improve our focus on the CotA (endospore coating protein A), a previously reported MCO from ATCC 7061 [21] and ATCC 14580 [22]. CotA was a classical bacterial laccase, which was able to oxidize 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP) [20]C[22]. To test whether the CotA is responsible for Mn(II) oxidation from a highly active Mn(II)-oxidizing strain WH4 isolated by our collaborators [23], [24], the gene was cloned and the N-terminal His-tagged CotA was overproduced and purified. Its Mn(II) oxidase activity and enzymological properties offered the most direct evidence between the MCO and the Mn(II) oxidation. Furthermore, the Mn(II) oxidizing activities from the recombinant strain cultured both in Mn-containing K liquid medium system MK 0893 and on agar plates were also investigated comprehensively. Materials and Methods Materials Taq DNA polymerase, restriction endonucleases and additional modifying enzymes were from Takara Biotechnology Co., MK 0893 Ltd. (Dalian, China) and Fermentas (St. Leon-Rot, Germany). Ampicillin, kanamycin, isopropyl–D-thiogalactoside (IPTG) and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic.