Whole toxic substrates, products

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... 

LY300164, an orally administered benzodiazepine developed by Eli Lilly (India-napolis/IN, USA), is efficaceous against amylotropic lateral sclerosis (ALS, Lou Gehrig s disease). In an early key step, a 3,4-disubstituted phenylacetone is reduced to the (S)-alcohol by yeast whole cells from Zygosacchamyces rouxii (ATCC 14462) in > 99.9% e.e. and 96% yield. The subsequent steps encompass a series of chemical steps (Figure 13.30). Volumetric productivity is optimized in remarkable fashion the ketone substrate is introduced adsorbed on an XAD-7 resin to keep the concentration in solution (2 g L-1) below the toxicity limit (6 g L-1). Product alcohol likewise is adsorbed so that a loading of 80 g L-1 of substrate or product is achieved. 

The whole-cell biocatalysis approach is typically used when a specific biotransformation requires multiple enzymes or when it is difficult to isolate the enzyme. A whole-cell system has an advantage over isolated enzymes in that it is not necessary to recycle the cofactors (nonprotein components involved in enzyme catalysis). In addition, it can carry out selective synthesis using cheap and abundant raw materials such as cornstarches. However, whole-cell systems require expensive equipment and tedious work-up because of large volumes, and have low productivity. More importantly, uncontrolled metabolic processes may result in undesirable side reactions during cell growth. The accumulation of these undesirable products as well as desirable products may be toxic to the cell, and these products can be difficult to separate from the rest of the cell culture. Another drawback to whole-cell systems is that the cell membrane may act as a mass transport barrier between the substrates and the enzymes. 

The main disadvantage of this whole cell biotransformation is the high dilution more than 500 L aqueous medium was used for 1 kg of triketone substrate. Such problems can be circumvented by the application of two-phase systems, where the substrate is solubilized in high concentration in the organic phase. Furthermore, the substrate and product concentration in the aqueous phase is lowered, circumventing toxicity problems. 

Industrial applications of P450s have so far been restricted to whole-cell systems, which mostly solve the problem of cofactor delivery and regeneration. In such instances, however, physiological effects such as limited substrate uptake and reduced efflux of products out of cells, substrate or product toxicity, product degradation, as well as elaborate downstream processing are additional limiting factors that must be taken into account and often require optimization [34]. 

Hydrophobic ILs have been considered as a solvent for biocatalytic reactions. It was shown that the reaction of 2-octanone reduction to 2-octanol catalysed by alcohol dehydrogenase is faster when IL BMIM (Cp3S02)2N is present in comparison to organic solvent applied so far (Eckstein et al., 2004). Additional beneficial effects of ILs is reduction of the toxicity towards the cell and possibility of their application in whole-cell biocatalysis (Weuster-Botz, 2007 Pfruender et al,. 2004). This advantageous effect comes from excellent solvent properties of ILs extracting either products or reaction substrates. 

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