Biocatalyst format

Basis of separation Methods Examples of technologies available 

Volatility differences Evaporation Stripping Vacuum distillation Pervaporation Transmembrane distillation 

Solubility differences Extraction into Diphasic with organic solvent 

Permeability differences Permeation Dialysis Electrodialysis Reverse osmosis Nanofiltration 

Adsorption differences Immobilization Hydrophobic adsorption Ion exchange Affinity adsorption 

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Perhaps the first decision to be made in process development is the difficult decision of whether the enzymes to be used should be used in an integrated format. Such a question does not arise with conventional single biocatalytic steps but is highly important in multienzyme processes. One of the key criteria here is whether the enzymes can be operated together without compromise to any of the individual enzyme s activity or stability. An interaction matrix (see Section 10.6) can be used to assist such decision making. In cases where the cost of one or more of the enzyme(s) is not critical, it will be possible to combine in a one-pot operation. In other cases, where the cost of an individual enzyme becomes critical, then it may be necessary to separate the catalysts, such that each can operate under optimal conditions. Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell, cell-free extract, soluble enzyme, or combinations thereof) in combination with the basic reactor type (packed bed, stirred tank, or combinations thereof) and biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof) will determine the structure of the process flowsheet and therefore is an early consideration in the development of any bioprocess. The criterion for selection of the final type of biocatalyst and reactor combination is primarily economic and may best be evaluated by the four metrics in common use to assess the economic feasibility of biocatalytic processes [29] ... 

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

Applications of peroxide formation are underrepresented in chiral synthetic chemistry, most likely owing to the limited stability of such intermediates. Lipoxygenases, as prototype biocatalysts for such reactions, display rather limited substrate specificity. However, interesting functionalizations at allylic positions of unsaturated fatty acids can be realized in high regio- and stereoselectivity, when the enzymatic oxidation is coupled to a chemical or enzymatic reduction process. While early work focused on derivatives of arachidonic acid chemical modifications to the carboxylate moiety are possible, provided that a sufficiently hydrophilic functionality remained. By means of this strategy, chiral diendiols are accessible after hydroperoxide reduction (Scheme 9.12) [103,104]. 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

The use of molecular biology methods, described in Section 5.3 seems to be especially worthwhile as it offers novel possibilities of optimization on process adjustment. Directed evolution leads to the formation of new biocatalysts with improved characteristics (selectivity, activity, stability, etc.). Incorporation ofnon-proteinogenic amino acids makes it possible to reach beyond the repertoire of building blocks used by nature. The prospect of bioconjugate preparation offers the possibility to form functional clusters of enzymes and to perform multiple synthetic steps in one pot. 

Catalytic transformations can be divided on the basis of the catalyst-type - homogeneous, heterogeneous or enzymatic - or the type of conversion. We have opted for a compromise a division based partly on type of conversion (reduction, oxidation and C-C bond formation, and partly on catalyst type (solid acids and bases, and biocatalysts). Finally, enantioselective catalysis is a recurring theme in fine chemicals manufacture, e.g. in the production of pharmaceutical intermediates, and a separate section is devoted to this topic. 

Despite advent of theoretical methods and techniques and faster computers, no single theoretical method seems to be capable of reliable computational studies of reactivities of biocatalysts. Ab initio quantum mechanical (QM) methods may be accurate but are still too expensive to apply to large systems like biocatalysts. Semi-empirical quantum methods are not as accurate but are faster, but may not be fast enough for long time simulation of large molecular systems. Molecular mechanics (MM) force field methods are not usually capable of dealing with bond-breaking and formation... 

Over the years of evolution, Nature has developed enzymes which are able to catalyze a multitude of different transformations with amazing enhancements in rate [1]. Moreover, these enzyme proteins show a high specificity in most cases, allowing the enantioselective formation of chiral compounds. Therefore, it is not surprising that they have been used for decades as biocatalysts in the chemical synthesis in a flask. Besides their synthetic advantages, enzymes are also beneficial from an economical - and especially ecological - point of view, as they stand for renewable resources and biocompatible reaction conditions in most cases, which corresponds with the conception of Green Chemistry [2]. 

In addition to the enzymatic hydrolysis of esters, there also ample examples where an epoxide has been cleaved using a biocatalyst. As described by the Faber group [19], reaction of the ( )-2,3-disubstituted ds-chloroalkyl epoxide roc-8-40 with a bacterial epoxide hydrolase (BEH), led to the formation of vie-diol (2 ,3S)-8-41 (Scheme 8.11). The latter underwent a spontaneous cydization to give the desired product (2i ,3i )-8-42 in 92 % ee and 76 % yield. The same strategy was used with the homologous molecule rac-8-43, which afforded the THF derivative (2R,3R)-S-4S in 86% ee and 79% yield. 

Acryl amide is an important bulk chemical used in coagulators, soil conditioners and stock additives. The chemical synthesis has several drawbacks because the rate of acryl amide formation is lower than the formation of the by-product acrylic acid [54]. Further, the double bonds of the reactants and products cause by-product formations as well as formation of polymerization products. As a result of optimization with methods of molecular engineering, a very high activity of the biocatalyst nitrile hydratase at low temperature is yielded, enabling a successful biotransformation that is superior to the chemical route. Here, the synthesis is carried out at a low temperature of about 5°C, showing a conversion of 100%. 

Kaup, B., Bringer-Meyer, S. and Sahm, H. (2004) Metabolic engineering of Escherichia coir, construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation. Applied Microbiology and Biotechnology, 64 (3), 333-339. 

By screening 53 Rhodococcus and Pseudomonas strains, an NHase-amidase biocatalyst system was identified for the production of the 2,2-dimethylcyclopropane carboxylic acid precursor of the dehydropeptidase inhibitor Cilastatin, which is used to prolong the antibacterial effect of Imipenem. A systematic study of the most selective of these strains, Rhodococcus erythropolis ATCC25 544, revealed that maximal product formation occurs at pH 8.0 but that ee decreased above pH 7.0. In addition, significant enantioselectivity decreases were observed above 20 °C. A survey of organic solvent effects identified methanol (10% v/v) as the... 

The high sulfur-containing feedstock and the biocatalyst, usually suspended in the aqueous phase have to be contacted with each other in a bioreactor. A homogeneous, continuous phase would be preferred, which would imply formation of an emulsion, preferably a microemulsion. Several bioreactor designs have been suggested for biodesulfurization of petroleum feedstocks including impeller-mixed systems [65,202], electro-spray bioreactor [220,261,262], and draft tube air-lift bioreactor [263],... 

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