Production of a Recombinant Catechol 2,3-Dioxygenase for the Degradation of Micropollutants

FH-HES Universities of Applied Sciences

Authors

  • Domenico Celesia Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Universita degli Studi di Palermo, Viale delle Scienze, Ed. 16, 90100, Palermo, Italy
  • Isabelle Salzmann Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland
  • Emanuel Vaz Porto Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland
  • Floriane Walter Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland
  • Cindy Weber Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland
  • Rémy Dufresne Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland
  • Simon Crelier Institute of Life Technologies, University of Applied Sciences Western Switzerland, Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland. simon.crelier@hevs.ch

DOI:

https://doi.org/10.2533/chimia.2017.734

Keywords:

Bioconversion, Catechol, Catechol 2,3-dioxygenase, Enzyme, Micropollutants

Abstract

Phenolic compounds such as catechol represent a particular type of micropollutant whose high stability prevents rapid decay and metabolization in the environment. We successfully cloned a catechol 2,3-dioxygenase (C2,3O) from Pseudomonas putida mt-2 and expressed it in Escherichia coli BER2566. The biomass isolated from shake-flask fermentations was used to partially purify the enzyme. The enzyme proved unstable in clarified liquid fractions (50 mM Tris buffer, pH 7.6) and lost more than 90% of its activity over 7 h at 25 °C. In the presence of 10% acetone, the process was slowed down and 30% residual activity was still present after 7 h incubation. Storage of the enzyme in clear liquid fractions also proved difficult and total inactivation was achieved after 2 weeks even when kept frozen at –20 °C. Lowering the storage temperature to –80 °C preserved 30% activity over the same period. Only minor reactivation of the affected enzyme could be achieved after incubation at 20 °C in the presence of FeSO4 and/or ascorbic acid. Activity loss seems to be due mostly to Fe2+ oxidation as well as to subunit dissociation in the tetrameric structure. However, complete degradation of 1.0 mM catechol could be achieved at 20 °C and pH 7.6 over a 3 h period when using a suspension of whole cells or alginate-encapsulated cells for the biotransformation. Contrary to the clear liquid fractions, these forms of biocatalyst showed no significant sign of inactivation under the working conditions.

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Published

2017-10-25

How to Cite

[1]
D. Celesia, I. Salzmann, E. V. Porto, F. Walter, C. Weber, R. Dufresne, S. Crelier, Chimia 2017, 71, 734, DOI: 10.2533/chimia.2017.734.