Toxic substrates

The most limiting factor for enzymatic PAC production is the inactivation of PDC by the toxic substrate benzaldehyde. The rate of PDC deactivation follows a first order dependency on benzaldehyde concentration and reaction time [8]. Various strategies have been developed to minimize PDC exposure to benzaldehyde including fed-batch operation, immobilization of PDC for continuous operation and more recently an enzymatic aqueous/octanol two-phase process [5,9,10] in which benzaldehyde is continuously fed from the octanol to the enzyme in the aqueous phase. The present study aims at optimal feeding of benzaldehyde in an aqueous batch system. 

A high level of poly(3HB) accumulation is also obtained if the cells are grown under carbon substrate limitation, and the cultivation in the second fermenter is also carried out under carbon limitation. In this case, a substrate flow rate (F2) below that corresponding to the maximum specific poly(3HB) formation rate should be chosen [114]. This cultivation strategy is especially convenient when using toxic substrates like acetic acid. Low substrate concentrations are more conveniently maintained in continuous cultivation than in fed-batch cultivation. The only additional equipment needed is a system to ensure constant working volumes and flow rates. 

The continuous regime of poly(3HB) production has some important advantages. High and constant productivities are obtainable because high rates of growth and product formation can be maintained over long periods. Continuous cultivation methods are also convenient for the use of toxic substrates, and constant product quality should be easily obtained. 

Straightforward. We have therefore employed XAD-4 to combine biocatalytic synthesis with simultaneous product extraction. The system (Figure 15.8) comprises a continuously stirred tank reactor, a starting material feed pump, a product recovery loop with a (semi-) fluidized bed of XAD-4, and a pump to circulate the entire reaction mixture through the loop." ° Preliminary studies indicated that XAD-4 had no detrimental effects on E. coli JMlOl (pHBP461), hence, separation of biomass and reaction liquid prior to catechol extraction was not required. The biocatalytic reaction was carried out at very low concentrations of the toxic substrate and product. This was achieved by feeding the substrate at a rate lower than the potential bioconversion rate in the reactor. 

One of the most important quality characteristics of advanced oxidation processes is their ability to reduce the toxicity of an industrial wastewater and to enhance its biodegradability (see Chapter 7.1.5). This criterion can be established by several standardized procedures using different test organisms ranging from microbes to intact animals (cf Tab. 5-1). An easy to perform variant is the bioluminescence assay that uses the inhibition of the bioluminescence intensity of the test organism Vibrio fischeri in the presence of toxic substrates. This is a bioassay used worldwide for the evaluation of toxicity data of individual chemicals or of industrial wastewaters (Froehner et al, 2000, DIN, 1991). Commercial systems are... 

ALS is also inhibited by a number of compounds which are structurally unrelated to the sulfonylureas. These include two other classes of herbicides the imidazolinones (5) and the triazolopyrimidines (Hawkes, T.R. Howard, J.L. Pontin, S.E. In Herbicides and Plant Metabolism, in press). LaRossa et al. have speculated on why ALS is such an effective target for so many inhibitors (6). Blocking ALS leads to the buildup of the toxic substrate a-ketobutyrate. The elevated levels of this metabolite combined with the reduced levels of the branched chain amino acids appear to make the inhibition of ALS a particularly lethal event. 

ALS has also been shown to be inhibited by two other structurally unrelated classes of herbicides, the imidazolinones (8,9) and the triazolopyrimidines (10,11). It has been shown that the toxicity of the sulfonylurea herbicides to bacteria is due, in part, to the accumulation of an ALS substrate a-ketobutyrate, which is itself toxic. It has been suggested that the dual effects of the accumulation of a toxic substrate and the inability to synthesize isoleucine, leucine and valine make ALS a particularly good target for herbicides (12). 

The hydrations were carried out either at low substrate concentrations with slow feeding of the substrate (for example benzonitrile, 2,6-difluorobenzonitrile and 3-indoleacetonitrile) or, in the case of less toxic substrates, by direct incubation at high substrate concentrations (for example 3-indolylacetonitrile and 2-cyanopyr-azine[2, 76]). 

Morasch, B. Annweiler, E. Warthmann, R. J. Meckenstock, R. U. (2001) The nse of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites degradation of naphthalene, o-, and m-xylene by sulfate-reducing bacteria. J. Microbiol. Meth. 44,183-191. 

A few other studies in two-stage continuous systems were conducted with other microorganisms. In the case of P(3HB4HB) production, Delftia acidovorans P4a was cultivated on mixtures of acetic acid and y-butyrolactone here, P(3HB4HB) copolymers with a molar fraction of 2.7-19% 4HB were obtained. The authors state that, especially in the case of toxic substrates like acetic acid and y-butyrolactone, the two-stage continuous production strategy is very convenient [132]. 

Straathof AJJ (2003) Auxiliary phase guidelines for microbial biotransformations of toxic substrate into toxic product. Biotechnol Prog 19(3) 755-62 Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production a review. Bioresour Technol 83(1) 1-11... 

The use of biphasic aqueous-organic systems in biocatalytic reductions is of great interest because the enzyme and its cofactor are dissolved in the aqueous phase, where the reaction takes place, while the hydrophobic substrate and product are mostly located in organic solvent layer and partitioned into the aqueous phase. This distribution reduces the concentrations of toxic substrate and product around the enzyme in aqueous layer and relieves the enzyme from substrate and product inhibition. Other distinctive features of this biphasic system are simple separation, easy regeneration of the enzyme, and easy recovery of the products. However, in this system, the reaction rates are relatively low because of a low rate of mass-transfer across the interface. Although this hindrance can be eliminated by intensive agitation, the increased interface often results in faster denaturation and inactivation of the enzyme. 

Toxic substrates and products to whole-cell biocatalysts. Finally, in whole-cell format, the substrate and/or product of the bioreduction can be toxic to the cells, preventing cofactor regeneration. Such irreversible loss of regeneration capacity is, of course, catastrophic for the process. In principle, this can be overcome by maintaining a low substrate concentration, but this will ultimately prevent a sufficiently high product concentration for an effective process. In some cases, dependent upon the water-solubility (and if the substrate is a liquid), it may be possible to feed the substrate, such that a low concentration is provided to the cells in the reactor, but at the end of the reaction a high product concentration is achieved. However, in nearly all cases at the required concentration for an... 

Organic Solvents Organic solvents have been used for the reaction by oxidoreductase to solubilize hydro-phobic substrates, to construct a two-layer system using hydrophobic solvents to reduce the concentration of toxic substrate and product around enzymes in an aqueous layer, and to simplify the work-up procedure. Water miscible and immiscible solvents have been used. For example, sol-gel-encapsulated alcohol dehydrogenase (WHOA mutant of Thermoanerobacter ethanolicus) was used for asymmetric reduction of 4-phenyl-2-butanone to (5)-4-phenyl-2-butanol in hexane. ... 

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