Résumé:
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.
Keywords: Bioconversion · Catechol · Catechol 2,3-dioxygenase · Enzyme · Micropollutants
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