Optimization of the recombinant two-domain laccases producing process

Abstract

Laccases (EC 1.10.3.2) belong to the family of copper-containing oxidases and contain four copper
atoms in their active centre. Laccases can oxidise a broad range of organic and inorganic compounds. Enzymes of this class are used in different biotechnological processes (paper and pulp, textile, food industries) and can be structurally divided into two groups: two-domain (2D) and three-domain (3D) enzymes. Common properties of 2D laccases are resistance to specific inhibitors of copper-containing oxidases and high thermal stability. 2D laccases have lower redox potential as compared to three-domain enzymes. However, it can be increased by using redox mediators. High thermal stability and resistance to inhibitors are important criteria for the selection of enzymes for biotechnology. Another important criterion for the biotechnology-relevant enzymes is the cost of their production. If obtaining an enzyme requires significant costs, and the yield of the final product is low, the production of the enzyme is impractical.
Therefore, the aim of this work is to optimise the process of production of 2D recombinant laccases
expressed heterologously in the Escherichia coli strain, and to calculate the cost of the final product (using
the SgfSL, SvSL, and SaSL enzymes obtained in our laboratory as an example). Previously, three recombinant two-domain laccases were cloned and expressed in the Escherichia coli strain M15 (pRep4). In this study we examined the effect of various factors on the maximal yield of laccases: the effect of copper ions, the effect of inductor concentration, cultivation conditions, optical density of the culture, and other conditions.
It was shown that the optimal concentration of copper ions is 1 mM while the optimal concentration of
inductor IPGT is 0.1 mM (the laccase aggregation effect was absent and a high laccase yield was observed).
We confirmed the conclusions of our colleagues that microaerobic conditions are required for obtaining laccases maximally saturated with copper ions. Without the stage of microaerobic growth, the specific activity of the purified enzymes decreases by 2 times. It was found that if the rate of cell stirring during the induction of enzyme synthesis is too high, it leads to the aggregation of enzymes. The stirring rate at which laccases do not aggregate is 50-100 rpm. As a result, a process of production of two-domain bacterial recombinant laccases was developed and optimised. The maximum yield of enzymes was up to 180 mg of protein per liter of medium. The enzyme had a low production cost (16-32 euros per 1 g of protein).

References
1. Baldrian P., FEMS Microbiol Lett., 2006, Vol. 30, No 2, pp. 215-242. DOI:
10.1111/j.1574-4976.2005.00010.x. Available at: https://onlinelibrary.wiley.com/ (accessed
16.12.2019).
2. Claus H., Arch Microbiol., 2003, Vol. 179, No 3, pp. 145-150. DOI: 10.1007/s00203-002-
0510-7. Available at: https://link.springer.com/(accessed 16.12.2019).
3. Otto B., Schlosser D., Planta, 2014, Vol. 240, No 6, pp. 1225-1236. DOI:
10.1007/s00425-014-2144-9. Available at: https://link.springer.com/ (accessed 16.12.2019).
4. Lisov A.V., Zavarzina A.G., Zavarzin A.A., Leontievsky A.A., FEMS Microbiol Lett, 2007,
Vol. 275, No 1, pp. 46-52. DOI: 10.1111/j.1574-6968.2007.00858.x. Available
at: https://academic.oup.com/femsle (accessed 16.12.2019).
5. Thurston C.F., Microbiology, 1994, Vol. 140, pp. 19-26.
6. Sterjiades R., Dean J.F., Eriksson K.E., Plant Physiol., 1992, Vol. 99, No 3, pp. 1162-1168. DOI: 10.1104/pp.99.3.1162. Available at: http://www.plantphysiol.org/ (accessed 16.12.2019).
7. Endo K., Hosono K., Beppu T., Ueda K., Microbiology, 2002, Vol. 148, pp. 1767-1776.
DOI: 10.1099/00221287-148-6-1767. Available at: https://www.microbiologyresearch.org/ (accessed
16.12.2019).
8. Lu L., Zeng G., Fan C., Zhang J. et al., Appl Environ Microbiol, 2014, Vol. 80, No 11, pp.
3305-3314. DOI: 10.1128/AEM.00223-14. Available at: https://aem.asm.org/ (accessed 16.12.2019).
9. Minussi R.C., Pastore G.M., Duran N., Trends Food Sci Tech, 2002, Vol. 13, No 6-7, рр. 205-216.
10. Dominguez A., Couto S.R., Sanroman M.A., World J Microbiol Biotechnol., 2005, Vol. 21, No 4, pp. 405-409. DOI 10.1007/s11274-004-1763-x. Available at: https://link.springer.com/ (accessed 16.12.2019).
11. Couto S.R., Herrera J.L.T., Biotechnol Adv., 2006, Vol. 24, No 5, pp. 500-513. DOI:
10.1016/j.biotechadv.2006.04.003. Available at: https://www.sciencedirect.com/ (accessed
16.12.2019).
12. Trubitsina L.I., Tishchenko S.V., Gabdulkhakov A.G., Lisov A.V. et al., Biochimie, 2015, Vol. 112, pp. 151-159. DOI: 10.1016/j.biochi.2015.03.005. Available at: https://www.sciencedirect.com/ (accessed
16.12.2019).
13. Tishchenko S., Gabdulkhakov A., Trubitsina L., Lisov A. et al., Acta Crystallogr F Struct Biol Commun., 2015, Vol. 71, pp. 1200-1204. DOI: 10.1107/S2053230X15014375.
Available at: http://journals.iucr.org/f/ (accessed 16.12.2019).
14. Lisov A.V., Trubitsina L.I., Lisova Z.A., Trubitsin I.V. et al., Process biochemistry,
2019, Vol. 76, pp. 128-135. https://doi.org/10.1016/j.procbio.2018.11.001.
Available at: https://www.sciencedirect.com/ (accessed 16.12.2019).
15. Durao P., Chen Z., Fernandes A.T., Hildebrandt P. et al., J Biol Inorg Chem., 2008, Vol.
13, No 2, pp. 183-193. DOI 10.1007/s00775-007-0312-0. Available at: https://link.springer.com/ (accessed
16.12.2019).
16. Gunne M., Urlacher V.B., PLoS One, 2012, Vol. 7, No 12, pp. e52360. DOI:
10.1371/journal.pone.0052360. Available at: https://journals.plos.org/plosone/ accessed
16.12.2019).