Cell Surface Display Fungal Laccase as a Renewable Biocatalyst for Degradation of Persistent Micropollutants Bisphenol A and Sulfamethoxazole

Abstract

Fungal laccases have high activity in degrading various persistent organic pollutants. However, using enzymes in solution for water treatment has limitations of nonreusability, short enzyme lifetimes, and high cost of single use. In this study, we developed a new type of biocatalyst by immobilizing fungal laccase on the surface of yeast cells using synthetic biology techniques. The biocatalyst, referred to as surface display laccase (SDL), had an enzyme activity of 104 ± 3 mU/g dry cell (with 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS)). The SDL retained over 90% of the initial enzyme activity after 25 days storage at room temperature, while, in contrast, activity of free laccase declined to 60% of its initial activity. The SDL could be reused with high stability as it retained 74% of initial activity after eight repeated batch reactions. Proof-of-concept evaluations of the effectiveness of SDL in treating contaminants of emerging concern were performed with bisphenol A and sulfamethoxazole. Results from contaminant degradation kinetics and the effects of redox mediator amendment provided insights into the factors affecting the efficacy of the SDL system. This study reports, for the first time, the development of a surface display enzyme biocatalyst as an effective and renewable alternative for treating recalcitrant organic micropollutants.

Biocatalytic properties of cell surface display laccase for degradation of emerging contaminant acetaminophen in water reclamation

“…A codon‐optimized LAC3 gene (coding for laccase) from Trametes sp. was cloned into the pCTcon2 plasmid (Addgene, Cambridge, MA) and then transformed into the S. cerevisiae EBY100 strain using the LiAc/PEG method (Chen et al, ). The SDL biocatalyst was prepared by a two‐stage cultivation and induction process as detailed in the Supporting Information.…”

“…A novel class of cell surface display enzyme biocatalysts has emerged with the advancement of synthetic biology technology to address the challenges of existing enzyme immobilization systems (Chen, Stemple, Kumar, & Wei, ; Zhu, Chen et al, ; Zhu & Wei, ). We have constructed a surface display laccase (SDL) biocatalyst, where the laccase from white‐rot fungi Trametes sp.…”

Laccases: Production, Expression Regulation, and Applications in Pharmaceutical Biodegradation

“…Antibiotics that are not metabolized enter the environment (Larsson, 2014). Conventional water treatment processes cannot effectively remove antibiotics (Oulton et al, 2010), while more efficient advanced treatment methods have disadvantages such as high costs and secondary pollution (Chen et al, 2016). Antibiotics pose health risks by selecting for antibiotic-resistance bacteria (ARB).…”

“…and Myceliophthora thermophila (recombinantly expressed in Aspergillus oryzae ) and from actinobacteria Streptomyces ipomoeae (expressed in E. coli ; Table 4). Laccases immobilized by different methods have been used for antibiotic degradation, including enzymatic membrane reactors (Nguyen et al, 2014b; Becker et al, 2016), granular activated carbon (Nguyen et al, 2016a), silica beads (Rahmani et al, 2015), oriented immobilization (Shi et al, 2014), magnetic cross-linked enzyme aggregates (Kumar and Cabana, 2016; Yang et al, 2017), and cell surface display (Chen et al, 2016). In particular, enzymatic membrane reactors (gelatin-ceramic membranes grafted with commercial T. versicolor laccase) in tetracycline degradation have been evaluated in depth with respect to membrane preparation, efficiency, kinetics, and economics (de Cazes et al, 2014, 2015; Abejón et al, 2015a,b).…”

Laccase removal of 2‐chlorophenol and sulfamethoxazole in municipal wastewater

“…An alternative approach is to mitigate the amount of laccase required by immobilizing it on a carrier or by recovering it and recycling it within the treatment system. Immobilizing laccase onto a carrier can yield high removals (Chen, Stemple, Kumar, & Wei, ; Le et al., ) although immobilization can sometimes lower activity relative to free laccase (Becker et al., ). Recently, several systems are under development to recycle laccase within wastewater treatment systems (Arca‐Ramos, Eibes, Feijoo, Lema, & Moreira, ; Lloret et al., ).…”