Laccases are a prominent class of multi-copper oxidases (MCOs) found in species of fungi and bacteria. They are versatile enzymes that are proven to be capable of degrading phenolic compounds and are a top interest in their different industrial applications as they can use a wide range of substrates.
Bacillus licheniformis is one bacterium that contains a laccase which has been tested to degrade textile dyes in wastewater by cloning the laccase gene into the host
Pichia pastoris, a yeast, in a paper by Lu et al. (2013).
For the purpose of the UConn iGEM 2023 project, the phenolic compound of interest to degrade was the synthetic estrogen, 17a-ethynylestradiol or otherwise known as EE2. Considering the structure of EE2 also makes it a phenolic compound, it is a prime target for the action of laccases. The fact that other bacterial laccase such as from B. licheniformis have shown promise in the degradation of dyes, which are also phenols, provides the reasoning behind the choice of using a bacterial laccase against EE2. The laccase gene of
B. licheniformis was cloned into the pET22b(+) vector and transformed into Escherichia coli DH5-alpha bacterial cells.
The part codes for a laccase that is 513 amino acids in length and is a part that can be customized for compatbility, such as with RFC10. The restiction enzymes EcoRI and XbaI were utilized to insert the synthesized laccase sequence into the multiple cloning site of the pET22b(+) vector, which is a vector commonly applied for cloning and expression of recombinant proteins in
E. coli.