Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase |
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Authors: | Coulombel Lydie Nolan Louise C Nikodinovic Jasmina Doyle Evelyn M O'Connor Kevin E |
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Institution: | (1) School of Biomolecular and Biomedical Sciences, Ardmore House, University College Dublin, Belfield, Dublin 4, Ireland; |
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Abstract: | Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols
as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation
of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was
undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In
a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The
rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and
4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation
decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved
within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed
to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold
decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol
products followed by column chromatography resulted in the production of purified products with a final yield of between 33%
and 38%. |
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