In-situ product removal

For the aforementioned reasons the use of isolated enzymes is often preferred due to reduction in side reactions and higher productivities (see Ref. [12] for a review of this topic). However this brings other challenges such as the need for effective cofactor regeneration. The choice between enzyme and whole-cell biocatalysts is complex and requires more work in the future to establish a clearer strategy to help the process design and implementation of bioreductions. 

Substrate and product (and sometimes even coproduct) often have relatively similar properties, meaning that selective separation is really not possible using simple technology. This means that in many cases ISPR is at best very challenging, and at worst unsuitable for shifting equilibrium, for example, where selectivity is 

An interesting and well-cited review [16] of recent developments in ISPR integrated with whole-cells (fermentation and resting cells) cites 250 examples. Interestingly, only a few have been implemented at an industrial scale. Often the system is too complex, but in many cases it is also the lack of a suitable method for evaluation of the technology alongside alternatives that makes implementation so difficult. 

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Biocatalysts in nature tend to be optimized to perform best in aqueous environments, at neutral pH, temperatures below 40 °C, and at low osmotic pressure. These conditions are sometimes in conflict with the need of the chemist or process engineer to optimize a reaction with respect to space-time yield or high product concentration in order to facilitate downstream processing. Furthermore, enzymes and whole cells are often inhibited by products or substrates. This might be overcome by the use of continuously operated stirred tank reactors, fed-batch reactors, or reactors with in situ product removal [14, 15]. The addition of organic solvents to increase the solubility of substrates and/or products is a common practice [16]. 

Next to reactions catalyzed by transaminases, hydrolase-catalyzed reactions also lead to limitations regarding the equilibrium. This problem occurs during ester synthesis, because this condensation reaction produces water. The equilibrium is shifted by high amounts of water towards the reactants therefore, an efficient removal is necessary to reach high conversions. Here, two process setups of Unichema Chemie B V will be discussed illustrating in situ product removal [41]. The first setup is based on azeotropic distillation of the water produced... 

Vicenzi, J.T., Zmijewski, M.J., Reinhard, M.R. et al. (1997) Large-scale stereoselective enzymatic ketone reduction with in-situ product removal via polymeric adsorbent resins. Enzyme and Microbial Technology, 20, 494-499. 

Woodley, J.M., Bisschops, M., Straathof, A.J.J. and Ottens, M. (2008) Future directions for in-situ product removal (ISPR). Journal of Chemical Technology and Biotechnology (Oxford, Oxfordshire 1986), 83 (2), 121-123. 

Recently, an industrial process development for nootkatone production from valencene by microbial transformation (bacteria, fungi) was mentioned [199, 200]. Although no details were given, the author claimed the development of an in situ product-removal technique by which an extremely selective recovery of nootkatone from the reaction mixture and the excess precursor during the proceeding production was achieved and which was said to be essential for an economically viable bioprocess. 

Let us assume that turbulence in the tank keeps the suspended particle concentration homogeneous, but that at the bottom of the tank the particles can sink through some screen below which no water currents exist (Fig. 23.2 b). In the absence of any external particle fluxes or in situ production/removal of particles, the mass balance equation for suspended particle mass is given by equating the rate of change of particle mass in the water volume V with time with the rate of loss due to settling ... 

Separation of products from the reaction mixture In situ product removal from enzymatic reactor via a nanofiltration or ultrafiltration membrane Removal of selected enantiomer via a liquid membrane Removal of water in esterification reactions via a pervaporation membrane... 

Fig. 3.51). The chiral alcohol is a key intermediate in the synthesis of an anticonvulsant drug. The yeast Zygosaccharomyces rouxii employed for this process is hampered by substrate and product toxicity at levels of >6gL-1. This was solved by using in situ adsorption on Amberlite XAD-7. In this way in situ product removal could be achieved. At the end of the process (8-12 h) 75-80 g of the alcohol is found on the resin and about 2 g L-1 remains in the aqueous phase. Finally an isolated yield of 85-90% could be obtained with an ee>99.9%.

Lye G.J., Woodley J.M., Application of in situ product-removal techniques to biocatalytic processes, Trends Biotechnol. L7, 395-402 (1999)... 

The (1S,5R)-2-oxabicydo-octenone (see Fig. 5) is formed with 96% ee from one ketone enantiomer of racemic bicyclo[3.2.0]hept-2-en-6-one, which is synthesized in a cycloaddition reaction from cyclopentadiene and dichloroacetylchloride with subsequent Zn-reduction. The key limitation to the biocatalytic Baeyer-Villiger process is product inhibition [26]. In order to overcome product inhibition problems, in situ product removal was required. 

One elegant way of in situ product removal is to use the product of a first dehydrogenase reaction as substrate for a subsequent enzymatic reaction, thus recycling the oxidized nicotinamide coenzyme (Fig. 16.2-3). Various NAD(P)-de-pendent enzymes can be applied as regeneration enzymes in this cascade reaction. 

Benzaldehyde can be produced from benzoyl formate with whole cells of Pseudomonas putida ATCC 12633 as biocatalyst119 201 (Fig. 16.6-5). Alternatively, but less effectively, mandelic acid can be used as starting material. A pH of 5.4 was found to be optimal for benzaldehyde accumulation. At this proton concentration, partial inactivation of the benzaldehyde dehydrogenase isoenzymes and activation of the benzoyl formate decarboxylase are reported. Fed-batch cultivation prevented substrate inhibition. In situ product removal is necessary to prevent product inhibition. 

Biocatalytic processes for the manufactming of complex or sensitive molecules require highly selective sepai ation and purification methods. Tlie in situ separation of inhibitory or toxic byproducts or the shifting of unfavourable equilibria aie additional aims of bioprocess conhol technologies. In situ product removal advances both the conhol of biocatalytic processes and the recovery of target molecules. 

Wei D, Yang L, Song Q (2003) Effect of temperature on the enzymatic synthesis of cefaclor with in situ product removal. J Mol Catal B Enzym 26 99-104... 

Yang L, Wei Z (2003) Enhanced enzymatic synthesis of a semi-synthetic cephalosporin, cefaclor, with in situ product removal. Biotechnol Lett 25 1195-1198... 

Zhang Y, Wei D, li D et til. (2007) Optimisation of enzymatic synthesis of cefaclor with in situ product removal and continuous acyl donor feeding. Biocatal Biotiansform 25(l) 59-64... 

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