Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase.

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%.

Dioxygenase- and monooxygenase-catalysed synthesis of cis-dihydrodiols, catechols, epoxides and other oxygenated products.

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.

Use of Pseudomonas mendocina, or recombinant Escherichia coli cells expressing toluene-4-monooxygenase, and a cell-free tyrosinase for the synthesis of 4-fluorocatechol from fluorobenzene.

The transformation of fluorobenzene (FB) by whole cell expressing toluene-4-monooxygenase (T4MO) resulted in the formation of various hydroxylated products. The predominant product was either 4-fluorophenol (4FP) or 4-fluorocatechol (4Fcat) depending on the ratio of biocatalyst to substrate concentration. The transformation of 1 mM FB by whole cells (1.5 mg CDW/ml) gave a 52% yield of 4Fcat as a single product. The yield of 4Fcat was improved 1.6-fold (80%) by adding 10 mM ascorbic acid to the biotransformations. A combination of two biocatalysts (whole cells expressing T4MO and cell free mushroom tyrosinase) also resulted in the transformation of FB (5 mM) to higher concentrations of 4Fcat (1.8 mM) compared to a whole cell biotransformation alone. However, mixed products were formed and the yield of 4Fcat from FB was lower using the two-step (tandem) method (27%) compared to the use of whole cells of P. mendocina KR1 alone (80%).

A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: determination of toluene-4-monooxygenase activity.

A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (epsilon) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was determined experimentally to be 12,827 M(-1) cm(-1) with a linear range of quantification between 2.5 and 75 microM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as determined spectrophotometrically, were 61.8+/-4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9+/-4.6 nmol/min/mg P. mendocina KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6+/-1.4 nmol/min/mg P. mendocina KR1 protein.