Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence

Highlights

• First study on oxidation of PAHs by B. subtilis CotA.

• Laccase activity of CotA was higher and copper independence than CueO.

• PAHs transformation by CueO was promoted by copper.

Abstract

Bacterial laccase CueO from Escherichia coli can oxidize polycyclic aromatic hydrocarbons (PAHs); however, its application in the remediation of PAH-contaminated soil mainly suffers from a low oxidation rate and copper dependence. It was reported that a laccase with a higher redox potential tended to have a higher oxidation rate; thus, the present study investigated the oxidation of PAHs using another bacterial laccase CotA from Bacillus subtilis with a higher redox potential (525 mV) than CueO (440 mV). Recombinant CotA was overexpressed in E. coli and partially purified, exhibiting a higher laccase-specific activity than CueO over a broad pH and temperature range. CotA exhibited moderate thermostability at high temperatures. CotA oxidized PAHs in the absence of exogenous copper. Thereby, secondary heavy metal pollution can be avoided, another advantage of CotA over CueO. Moreover, this study also evaluated some unexplained phenomena in our previous study. It was observed that the oxidation of PAHs with bacterial laccases can be promoted by copper. The partially purified bacterial laccase oxidized only two of the 15 tested PAHs, i.e., anthracene and benzo[a]pyrene, indicating the presence of natural redox mediators in crude cell extracts. Overall, the recombinant CotA oxidizes PAHs with high laccase activity and copper independence, indicating that CotA is a better candidate for the remediation of PAHs than CueO. Besides, the findings here provide a better understanding of the oxidation of PAHs using bacterial laccases.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants and widely distributed in the environment. Because of their mutagenic and carcinogenic potential (Fujikawa et al., 1993), 16 PAHs have been specified by the US Environmental Protection Agency (EPA) as the priority pollutants in 1976 (Keith and Telliard, 1979). The toxicity and recalcitrance of PAHs to microbial degradation correlate with the number and angularity of the fused benzene rings. High-molecular-weight (HMW) PAHs are more recalcitrant than low-molecular-weight PAHs and may persist in the environment for a long time (Shuttleworth and Cerniglia, 1995). Previous studies showed that soil bacteria degraded PAHs with less than four rings, but could not degrade HMW PAHs with more than five rings completely (Peng et al., 2008, Lu et al., 2011). In contrast, fungi could degrade HMW PAHs including carcinogenic benzo[a]pyrene. However, they mainly transform the PAHs into detoxified or polar metabolites rather than mineralize them to CO₂ (Bogan and Lamar, 1996). The increase in water solubility of these metabolites enhances their bioavailability to bacterial degradation. It was previously reported that coculture of fungi and bacteria for the degradation of PAHs increased the benzo[a]pyrene mineralization (Boonchan et al., 2000, Kotterman et al., 1998), in which the fungi performed the initial oxidation step. However, the destabilization of coculture in the soil environment decreases PAH degradation.

Laccases (EC 1.10.3.2) are extracellular lignin-degrading enzymes directly linking to oxidation of benzo[a]pyrene by white-rot fungi (Potin et al., 2004). Thereby, we proposed that laccase production in PAHs-degrading bacteria can increase benzo[a]pyrene mineralization, resolving the drawback of co-culture (Zeng et al., 2011). The results in that work first confirmed oxidation of PAHs by bacterial laccase CueO with unique characteristics such as thermostability; the laccase CueO plays a role in copper tolerance in Escherichia coli (Outten et al., 2000, Tree et al., 2005). However, CueO has obvious disadvantages in practice, i.e., a low oxidation rate, narrow range of reaction conditions, and copper dependence. Moreover, some different performances of CueO in PAH oxidation than fungal laccase attract our interests. For example, the oxidation of 15 EPA PAHs with crude CueO still proceed well in the absence of a mediator; some PAHs that were not oxidized by fungal laccases can be oxidized by crude CueO (Zeng et al., 2011).

As far as the low oxidation rate of CueO was concerned, it was shown that a laccase with a higher redox potential tended to have a higher oxidation rate (Fabbrini et al., 2002, Xu et al., 2000). However, CueO has a low redox potential (E° ≈ 440 mV) (Cambria et al., 2008). In contrast, CotA, the best-studied bacterial laccase from Bacillus subtilis has a higher redox potential (525 mV) (Brissos et al., 2009); the laccase plays a role in UV resistance in vivo (Hullo et al., 2001). Thereby, we envisioned that CotA with a higher redox potential can oxidize PAHs at a higher degradation rate. For the different performances of CueO in PAH oxidation, it was hypothesized that the natural materials present in crude enzyme extracts and different experimental conditions for CueO may affect the degradation of PAHs (Johannes and Majcherczyk, 2000, Pozdnyakova et al., 2006). Thus, the recombinant laccases overexpressed in Escherichia coli were partially purified to remove most of the natural materials, and then used to investigate the effects of experimental conditions different from those used in traditional fungal PAH degradation. The present study aimed to (1) find a better bacterial laccase with a higher PAH degradation rate and (2) evaluate these unexplained interestingly phenomena of CueO.