Troubleshooting laccase production

A part of our project was to purify laccases previously produced and test their activity first against ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and then against microplastics

The main problem encountered was during purification because after production and cell lysis the laccases were found in the insoluble fraction instead of the soluble fraction as expected, this was observed in western-blot analysis. We then contacted Dr. Thierry TRON, who has worked on laccases, and he advised us to supplement with copper and also grow the cells in microaerophilic condition to have improved metal incorporation, and thus a correct folding, of our laccases.

Even following these indications and following a published protocol, "A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization - Saadia Basheer, Naeem Rashid, Muhammad Sohail Akram, Muhammad Akhtar Biosci Biotechnol Biochem . 2019", we did not manage to obtain our laccase in the soluble fraction. .

To overcome this we decided to try and refold the laccase from the insoluble fraction. To do this, we solubilized the insoluble fraction by adding 0.5 % SDS "How do surfactants unfold and refold proteins ? - Daniel E Otzen, Jannik Nedergaard Pedersen, Helena stergaard Rasmussen, Jan Skov Pedersen - Adv Colloid Interface Sci." Once solubilized, we recovered our laccases that we washed to test their activity. Unfortunately we found no activity after testing the activity against ABTS. So it is possible that the laccases cannot fold up.

We have several hypotheses that could explain our problems :