Whole-Cell Processes

Reducing the temperature has been another strategy to cope with the need for high substrate and product concentrations. Operating at a lower temperature reduces the toxic effects of the substrate and product on the biocatalyst. A very 

---

Enzyme preparations versus whole cell processes 13... 

Disadvantages of Whole-Cell Process Compared with the Isolated... 

C—H activation has perhaps the highest potential of all enzyme-catalyzed transformations for synthetic applications. At the same time, these transformations are often the most difficult processes to be carried out on a practical scale. Currently, they require whole-cell processes and the outcome is often unpredictable. The discovery of new oxygenases and efficient hosts for protein expression remain keys to further expanding the synthetic applications of enzymatic hydroxylation. 

An example of a whole-cell process is the two-step synthesis of an enantiopure epoxide by asymmetric reduction of an a-chloro ketone (Scheme 6.4), catalyzed by recombinant whole cells of an Escherichia coli sp. overexpressing an (R)-KRED from Lactobacillus kefir and GDH from Thermoplasma acidophilum, to the corresponding chlorohydrin, followed by non-enzymatic base-catalyzed ring closure to the epoxide [17]. 

Figure 1.44 Double ketone reduction in a whole cell process.

Membrane reactors can be considered passive or active according to whether the membrane plays the role of a simple physical barrier that retains the free enzyme molecules solubilized in the aqueous phase, or it acts as an immobilization matrix binding physically or chemically the enzyme molecules. Polymer- and ceramic-based micro- and ultrafiltration membranes are used, and particular attention has to be paid to the chemical compatibility between the solvent and the polymeric membranes. Careful, fine control of the transmembrane pressure during operation is also required in order to avoid phase breakthrough, a task that may sometimes prove difficult to perform, particularly when surface active materials are present or formed during biotransformahon. Sihcone-based dense-phase membranes have also been evaluated in whole-cell processes [55, 56], but... 

Two types of immobilization are used for immobilizing glucose isomerase. The intracellular enzyme is either immobilized within the bacterial cells to produce a whole-cell product, or the enzyme is released from the cells, recovered, and immobilized onto an inert carrier. An example of the whole-cell process is one in which cells are disrupted by homogenization, cross-linked with glutaraldehyde, flocculated using a cationic flocculent, and extruded (42). 

Cyclohexanone oxygenase from Acinetobacteria converts a variety of alicyclic ketones into lactones in a regio- and enantioselective manner (Table 12). The reaction can be carried out by a whole-cell process (181) or with the isolated enzyme (178). For example, 2-norbomanone [497-38-1] (112) is converted to the corresponding lactone in 81% yield (178). The enzyme, however, is not selective both enantiomers react equally well. The oxidation rate of camphor [76-22-2] (113), is about one-third that of (112) nevertheless, given sufficient amount of time, the product yield reaches 89%. Substituted cylohexanones (114) and cyclopentanones (115) are converted into the corresponding lactones in moderate to good yield and selectivity (179—181). 

Enzymatic processes also advance in the area of large-scale pharma intermediates /flactam antibiotics can now be produced in a fully biotechnological process, including the semi-synthesis from the /flactam core to the penicillin or cephalosporin. A precursor to ephedrine, long produced by a whole-cell process in yeast, can be obtained from benzaldehyde and acetaldehyde with the help of pyruvate dehydrogenase acting as a carboligase. 

The reaction was already being used in the 1930s by Knoll (Ludwigshafen, Germany) (later BASF, since 2001 Abbott) (German Patent DE 546459, 1932) to produce ephedrine via (R)-phenylacetylcarbinol, (R)-PAC. Recently, efforts have been renewed to obtain an isolated enzyme process or a whole-cell process based on recombinant DNA technology. It was found that pyruvate could be substituted by the less expensive acetaldehyde, which did not produce C02 as a side product. 

However from the standpoint of green chemistry, the use of isolated enzymes (or dead whole cells) is highly preferred because it avoids the generation of copious amounts of biomass. It must be emphasized that the productivity of microbial conversions is usually low, since non-natural substrates are only tolerated at concentrations as low as 0.1-0.3% [106]. The large amount of biomass present in the reaction medium causes low overall yields and makes product recovery troublesome. Therefore the E-factors for whole cell processes can be extremely high. Moreover the use of wild-type cells often causes problems because an array of enzymes is present which can interfere in the reduction of a specific ketone (giving opposite selectivities). The use of recombinant techniques, however, which only express the desired enzyme can overcome this problem [108]. 

The problems encountered with the bakers yeast whole cell process may be overcome by the appUcation of isolated enzymes. Pohls group applied site-directed mutagenesis strategies to tailor PDC for fhe synfhesis of (P)-PAC. They... 

Not only the enzymes but also the cellular components such as coenzymes and carbohydrates are conserved in the cell, which makes the whole cell processes favorable. For example, the addition of an expensive coenzyme and an auxiliary enzyme for coenzyme regeneration is not necessary, which makes the system simple and economical when comparing with the equivalent isolated enzyme process. 

Another advantage of the isolated enzyme system is that the reaction pathway can be understood and predictions made. For example, for HLADH, the crystal structure [40 421 and the active site (diamond lattice) model113, 141 are available to understand the reduction, whereas, in a whole cell process, even the catalytic species itself may not be clear. 

---