Expression, secretion and functional characterization of three laccases in E. coli

Abstract

Endocrine Disrupting Chemicals (EDCs) are a group of molecules that can influence hormonal balance, causing disturbance of the reproductive system and other health problems. Despite the efforts to eliminate EDC in the environment, all current approaches are inefficient and expensive. In previous research, studies revealed that laccase-producing microorganisms may be a potential candidate for EDC degradation, as laccases have been found to be able to degrade many kinds of EDCs effectively and steadily. Here, we created two recombinant laccases, each fused with secretion peptide, Novel Signal Peptide 4 (NSP4), and expressed them in Escherichia coli ( E. coli, BL21), together with one laccase without secretion peptide. We first optimized the culture condition of expressing these laccases. Then, we test the activity of the recombinant laccases of decolorizing of a synthetic dye, indigo carmine. Finally, we confirmed the secreted can degrade one of the EDCs, β-estradiol, showing the potential of using the laccase secretion system to degrade toxic compounds.

Fungal Laccases: Fundamentals, Engineering and Classification Update

“…However, laccase expression was only detected by zymogram, and no activity values were provided. Recently, the use of the Novel Signal Peptide 4 has contributed to the functional expression of a laccase from T. versicolor in E. coli, but the enzyme was secreted into the cell medium in much lesser amounts than other non-fungal laccases [154]. A possible explanation for the poor expression levels of fungal laccases in bacteria is related to differences between the host and the fungal post-translational modification machineries, which could lead to the formation of non-functional aggregates of the recombinant protein [155].…”

Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae

“…Owing to the difficulty in culturing and manipulating the genes and genomes of basidiomycetes, even with CRISPR, the use of model organisms is strongly considered. Prokaryotic model organisms such as Pseudomonas putida , Escherichia coli , and Streptomyces species have been used to secrete laccase and peroxidase [ 8 - 12 ]. Although prokaryotic model microorganisms are good at producing enzymes, eukaryotes, especially yeasts, such as Saccharomyces cerevisiae and Pichia pastoris , are better at secretory protein production [ 13 , 14 ].…”