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

Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was used for this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened for biotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing 0.72 ± 0.03 g/liter 1-naphthol with a productivity of 0.46 ± 0.02 g/g (dry weight) cells after 48 h. The effects of the organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest 1-naphthol concentration (1.43 g/liter) and the highest 1-naphthol productivity (0.55 g/g [dry weight] cells) were achieved by optimization of the organic phase. The ability to recycle both free cells and cells immobilized in calcium alginate was tested. Both free and immobilized cells lost more than ∼60% of their activity after the first run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvement in 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueous media.Biocatalysis has emerged as an important technology in industrial organic synthesis for the production of chemical synthons and high-value products (29, 34, 37). 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). Oxygenases introduce either one or two atoms of molecular oxygen into organic molecules using NADH or NADPH as a cofactor. To eliminate the addition of a costly cofactor, whole cells expressing oxygenases are generally used (34, 43).One of the potential applications of biocatalysis utilizing oxygenases is the oxidation of naphthalene to 1-naphthol. 1-Naphthol has wide applications in the manufacture of dyes, drugs, insecticides, perfumes, and surfactants (2, 7, 17). Tao et al. (39) have compared the reaction rates and regioselectivities of various wild-type and modified monooxygenases for the oxidation of naphthalene to 1-naphthol. Of the monooxygenases tested, the best enzyme for the oxidation of naphthalene to 1-naphthol was a toluene ortho-monooxygenase (TOM) variant, TomA3(V106A), also known as TOM-Green. TOM was isolated from Burkholderia cepacia G4 and consists of an α₂β₂γ₂ hydroxylase (encoded by tomA1, tomA3, and tomA4) with two catalytic oxygen-bridged binuclear iron centers, an NADH-oxidoreductase (encoded by tomA5), a protein (encoded by tomA2) involved in electron transfer between oxidoreductase and hydroxylase, and a relatively unknown subunit (encoded by tomA0) (26, 36). TOM-Green was produced by directed evolution of TOM with one amino acid change in the alpha-subunit of the hydroxylase (7, 33). TOM-Green retained high regioselectivity (98%) and was sevenfold faster than wild- type TOM.There has been considerable effort to identify and characterize oxidative biocatalysts for 1-naphthol production (7, 11, 26, 33, 36, 40, 41). 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. As a consequence, biotransformations in water-organic solvent biphasic media have been developed (8, 9, 12, 21, 45, 46). The use of a second phase consisting of an organic solvent not only increases substrate loading but also maintains low concentrations of toxic compounds in the aqueous phase (4). The organic solvent chosen is critical for achieving the benefits of biphasic media. Two main criteria for solvent selection are a high distribution coefficient for the product and biocompatibility with microorganisms (3, 4). Biocompatibility is generally correlated with the logP of the solvent, which is the logarithm of the partition coefficient in an octanol-water system, and organic solvents with logP values greater than 4 are generally biocompatible with microorganisms (19). However, the correlation of activity with logP is specific to the microorganism, and the critical logP above which solvents are biocompatible has to be identified for each microorganism (8, 16).Biphasic systems have been widely used for reactions involving a toxic substrate and/or product to enhance productivity or to improve recovery of the product (22-25, 31, 38). 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. 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. Organic solvents were screened, and solvents suitable for high 1-naphthol productivity were identified. The organic phase was optimized by studying the effects of the naphthalene concentration and the organic phase ratio. The stability of the biocatalyst for recycling was also tested.