An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst

Held, Martin; Schmid, Andreas; Kohler, Hans‐Peter E.; Suske, Winfried; Witholt, Bernard; Wubbolts, Marcel

doi:10.1002/(sici)1097-0290(19990320)62:6<641::aid-bit3>3.0.co;2-h

Cited by 80 publications

(60 citation statements)

References 26 publications

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“…Phenol monooxygenases are of interest for bioremediation stategies as well as for biocatalytic applications, because the regioselective hydroxylation of phenols to catechols is notoriously difficult to achieve by means of chemical methods [6,7]. So far, structural and mechanistic studies mainly have focused on the strictly NADPH-dependent phenol hydroxylase from the basidiomycetous yeast Trichosporon cutaneum [8±21].…”

mentioning

confidence: 99%

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604

Eppink¹,

Cammaart²,

Wassenaar³

et al. 2000

Eur J Biochem

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The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1,4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1,4-dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD.Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 8C, this step does not limit the rate of overall catalysis.During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.

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mentioning

confidence: 99%

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604

Eppink¹,

Cammaart²,

Wassenaar³

et al. 2000

Eur J Biochem

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“…Due to difficulties and relatively low overall production yield from chemical synthesis (Held et al, 1999), 3MC production mainly relies on an economic and efficient biotechnological process, in which toluene is employed as a substrate for a bioconversion to 3MC using bacterial whole-cells as biocatalysts (Faizal et al, 2007;Wery et al, 2000). However, since toluene and 3MC, even at concentrations as low as 1% (v/v), detrimentally affect microbial survival and their catalytic activity, their toxicity, especially to 3MC generated and accumulated in the fermentation system, is the main constraint of the production process (de Smet et al, 1978;Fillet et al, 2012;Husken et al, 2001).…”

Section: +mentioning

confidence: 99%

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

Kongpol¹,

Kato²,

Tajima³

et al. 2014

J. Gen. Appl. Microbiol.

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In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/ energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg 2+ , Ca 2+, Mn 2+ and Fe 2+ ), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe 2+ as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.Key words: an aqueous-organic two-phase fermentation; bioproduction; 3-methylcatechol; Pseudomonas putida TODE1; toluene; toluene dioxygenase Introduction 3-Methylcatechol (3MC) is an important precursor for the production of valuable pharmaceutical compounds such as barbatusol and L-DOPA analogues, as well as synthetic food flavors (Husken et al., 2002;Shirai, 1986). Due to difficulties and relatively low overall production yield from chemical synthesis (Held et al., 1999), 3MC production mainly relies on an economic and efficient biotechnological process, in which toluene is employed as a substrate for a bioconversion to 3MC using bacterial whole-cells as biocatalysts (Faizal et al., 2007;Wery et al., 2000). However, since toluene and 3MC, even at concentrations as low as 1% (v/v), detrimentally affect microbial survival and their catalytic activity, their toxicity, especially to 3MC generated and accumulated in the fermentation system, is the main constraint of the production process (de Smet et al., 1978;Fillet et al., 2012;Husken et al., 2001). Two common approaches to overcoming the toxicity limitation are 1) the use of an organic solvent-tolerant (OST) bacteria, which can thrive in relative high concentrations of organic compounds, as a biocatalyst, and 2) the use of a two-liquid (organicaqueous) phase biotransformation system (referred to as a two-phase system hereafter), where the immiscible organic phase with microbial compatibility acts effectively as a source and a sink for the particular organic substrate and product (Heipieper et al., 2007).3MC is typically produced by a genetically engineered bacterial host from a direct bioconversion of tolue...

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“…The overall product concentrations (including product absorbed on the resin) reached 4 g/L, despite the fact that the substrate, phenylphenol, and the product phenylcatechol are both highly bactericidal at concentrations of only 0.2 g/L [15]. Given the broad spectrum of commercially available resins (ion exchange, hydrophobic interaction), this bioprocess technology has considerable potential as both hydrophobic and hydrophilic molecules can be extracted from process liquids under appropriate conditions [16].…”

Section: Reaction Products Can Poison the Biocatalystmentioning

confidence: 99%

Biocatalysis. Biological systems for the production of chemicals

Held

Schmid

Beilen

et al. 2000

Pure and Applied Chemistry

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An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst

Cited by 80 publications

References 26 publications

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

Biocatalysis. Biological systems for the production of chemicals

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