Parts

Laccases can be found among many different organisms such as fungi, bacteria and even plants. Fungal laccases come up with a higher activity, higher redox potentials and wider substrate range compared to bacterial laccases. They are also more active at lower temperatures which can be found, for instance, in wastewater. On the other hand, bacterial laccases are more stable regarding high salt concentrations and are especially easier to produce.^1,2

In order to investigate the degradation of diclofenac and carbamazepine within our approach, two laccases were evaluated. The first laccase tested was an eukaryotic fungal laccase of Trametes versicolor which has already been shown to degrade diclofenac.³ Therefore, a Biobrick contributed by the iGEM-Team Stockholm 2018 (the laccase with a his-Tag) was further improved by adding a poly- Lys-Tag which should facilitate the immobilization within the Mesocellular Silica Foam (MCF). The second laccase we used in this study was a less known bacterial laccase derived from Streptomyces cyaneus. Its performance regarding degradation of the substrates of interest was assessed, since a prokaryotic laccase would provide benefits regarding costs and duration of expression compared to an eukaryotic laccase. Additionally, we added a his-Tag for clean-up and the poly-Lys-Tag for improved immobilization conditions.

New BioBrick

BBa_K3379001

We want to contribute another laccase to the iGEM-Biobricks. This one is derived from Streptomyces cyaneus and is not as well studied as the laccase of Trametes versicolor. It showed good potential regarding activity and stability during ABTS-Assays, so we assessed its capability of degrading our substrates of interest. It showed the ability to degrade diclofenac. Hence, this Biobrick could be a suitable addition to wastewater treatment approaches through immobilized enzymes. Furthermore, a bacterial host allows cheaper and faster expression compared to expression in eukaryotic hosts like Pichia pastoris.

We ordered the sequence from IDT and obtained a pET28a-plasmid by the IMB of the University of Stuttgart. After a XbaI and NotI digestion and subsequent ligation, the ligation product was transformed in competent DH5α-E.coli cells. We screened for positive clones, isolated the pET28a- lac_Sc vector and verified successful cloning via sequencing. The expression was performed in the E. coli strain BL21. The resulting protein lysate showed good potential regarding stability and activity for our immobilization approach.

This Biobrick is the DNA-sequence of the laccase of S. cyaneus. In addition, there is a 6xhis-tag at the C-terminus of the sequence and a 6xlys-tag at the N-terminus of the protein. The his-tag should allow purification of the laccase while the lys-tag could possibly improve the immobilization process through providing free amino-groups.

Improved BioBrick

BBa_K3379002

It is an alteration of an already existing part. It´s a laccase of T. versicolor which was contributed by the iGEM-Team Stockholm 2018. We added a poly-lysine-tag at the C-terminus of the laccase sequence to investigate its influence on the immobilization process.

Link to Improvement of BioBrick

Summary

Name	Type	Description	Size
BBa_K3379001	Protein domain	Laccase of Streptomyces cyaneus with 6xHis Tag and 6xLys Tag	1941 bp
BBa_K3379002	Protein domain	Laccase Trametes versicolor with 3xlys tag and 6xhis tag	1530 bp

Literature

Zhang, Y. et al. Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects. Appl. Microbiol. Biotechnol. 102, 10409–10423 (2018).
Margot, J. et al. Bacterial versus fungal laccase: Potential for micropollutant degradation. AMB Express 3, 1–14 (2013)
Alharbi, S. et al. Degradation of diclofenac, trimethoprim, carbamazepine, and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent. Biocatalysis and Biotransformation 37(6), 399-408 (2019)