Phenol and 2-naphthol production by toluene 4-monooxygenases using an aqueous/dioctyl phthalate system

Abstract

A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l(-1) (39 mM), and wild-type T4MO, a 51+/-9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems

“…The inhibitory effect of 1-naphthol was greater than that of naphthalene, and no growth was observed even with 0.5 g/liter 1-naphthol. These results are comparable to the results of similar work done previously (38). Therefore, maintaining low concentrations of the substrate and the product is critical for maintaining the cell viability and the activity for the reaction.…”

“…Oxidation of naphthalene has also been improved using biphasic reactions (13,23,35,38). Tao et al (38) used a biphasic system for 2-naphthol and phenol production using toluene 4-moooxygenase and its variant TmoA(I100A). They obtained 10-to 21-fold increases in 2-naphthol and phenol concentrations using dioctyl phthalate as the organic solvent.…”

“…However, this process is not economically feasible owing to the very low optimum concentration of naphthalene (0.1 mM [7], which is less than the solubility of naphthalene, 0.23 mM [28]) and the toxicity of both naphthalene and 1-naphthol (38,44). Substrate loading has to be increased, and the toxicities of both naphthalene and 1-naphthol have to be minimized to make the process feasible.…”

“…McIver et al (23) used naphthalene dioxygenase to oxidize naphthalene to cis-(1R,2S)-1,2-naphthalene dihydrodiol using dodecane as the organic solvent and obtained a productivity of 1.7 g/g (dry weight) cells/h in the first 6 h. In spite of the significant improvements achieved by using a biphasic system for various reactions, application of this strategy to 1-naphthol production has not been explored yet. Considering the high toxicities of naphthalene and 1-naphthol (38), biphasic reactions can enhance the productivities. In this work, a biphasic system was used to increase 1-naphthol productivities with whole cells of Escherichia coli expressing the TOM-Green enzyme.…”

“…Biocatalysis offers the advantage of performing reactions under mild conditions and provides an environmentally benign approach for chemical reactions (1,38). Oxygenases are a class of enzymes that have great potential and versatility for catalyzing reactions that are generally not accessible by chemical routes with high regio-, stereo-, and enantioselectivities (6,27,42,43).…”