Whole-cell biocatalysts advantage

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

It is worth noting that this whole-cell biocatalyst accepts a broad range of substrates, thus allowing access to numerous enantiomerically pure L-a-amino acids, l-2. Selected examples for successfully converted hydantoins are shown in Fig. 10. A further advantage of the hydantoinase-based technology is that hydantoins are readily accessible and economically attractive starting materials. 

Table 1-7. Advantages and disadvantages using isolated enzymes or whole cell biocatalysts.

An ionic liquid can be used as a pure solvent or as a co-solvent. An enzyme-ionic liquid system can be operated in a single phase or in multiple phases. Although most research has focused on enzymatic catalysis in ionic liquids, application to whole cell systems has also been reported (272). Besides searches for an alternative non-volatile and polar media with reduced water and orgamc solvents for biocatalysis, significant attention has been paid to the dispersion of enzymes and microorganisms in ionic liquids so that repeated use of the expensive biocatalysts can be realized. Another incentive for biocatalysis in ionic liquid media is to take advantage of the tunability of the solvent properties of the ionic liquids to achieve improved catalytic performance. Because biocatalysts are applied predominantly at lower temperatures (occasionally exceeding 100°C), thermal stability limitations of ionic liquids are typically not a concern. Instead, the solvent properties are most critical to the performance of biocatalysts. 

In general there are two principle possibilities using a biocatalyst in organic synthesis, namely as whole cells or as isolated enzymes - free or immobilized. The advantages and disadvantages of each can be intensively discussed, but the outcome of this consideration always depends on the whole system and the kind of application. There are numerous examples of both and thus there is no partitioning between whole cell biotransformation and isolated enzymes in this review. 

Describe in your own words the advantages and disadvantages of using whole cells compared to isolated enzymes as biocatalysts, with respect to catalyst immobilization, catalyst recovery, ease of use, and product selectivity and purification. 

There has been considerable effort directed toward the immobilization of both enzymes and whole cells in a wide array of formats.15 Initial attempts to immobilize enzymes on naturally derived supports such as charcoal were conducted early in the twentieth century and eventually led to the development of more robust biocatalysts immobilized on synthetic resins by the mid-1950s. Immobilization often confers a number of advantages relative to the free biocatalyst including ease of removal from the process stream, potential for reuse, improvements in stability, favorable alterations in kinetic parameters, suitability for continuous production and in some cases the ability to operate in organic solvents. The focus of this section is on the immobilization of enzymes, however, many of the same principles apply to whole cells, the primary difference being the fact that immobilized cells are often less stable than individual enzymes and may contain additional undesired enzyme activities. 

Whole cell catalysts do not need immobiUzation, especially when mycelial micro-organisms are involved, since their morphological structure allows for easy filtration and re-utihzation. Carboxylesterases bound to the mycelia of molds have been advantageously employed as biocatalysts in water and/or organic solvents the first report of the use of fungal myceha in organic solvent dates back to 1978... 

Immobilization is the process of adhering biocatalysts (isolated enzymes or whole cells) to a solid support. The solid support can be an organic or inorganic material, such as derivatized cellulose or glass, ceramics, metallic oxides, and a membrane. Immobilized biocatalysts offer several potential advantages over soluble biocatalysts, such as easier separation of the biocatalysts from the products, higher stability of the biocatalyst, and more flexible reactor configurations. In addition, there is no need for continuous replacement of the biocatalysts. As a result, immobilized biocatalysts are now employed in many biocatalytic processes. 

Enzymes in a pure form, in a partially purified form, and in the whole cell can be used for organic synthesis, and each has advantages and disadvantages131. The proper choice of the form of the biocatalyst is important because it affects the enantio-, regio- and chemo-selectivities, the requirement (or not) of a coenzyme and an auxiliary enzyme, the ease of catalyst preparation and work up procedures, etc. as shown in Table 15-1. 

Figure 15-21. Advantages and disadvantages of whole cell, isolated enzymes and recombinant cell as biocatalysts.

Though MBR offer advantages over the more conventional bioreactors, they, themselves, are not completely free of problems. One such key problem, as previously noted, relates to changes in biocatalyst activity. This is a serious concern for whole-cell MBR, when the cells are immobilized in the membrane s pore structure. Diffusional limitations for nutri-... 

When immobilized whole cells were first introduced many researchers hesitated to use them because of their non-specificity and the expected by-product formation. Efforts have been made to utilize as many as possible of the potential advantages offered by immobilized whole cells, and at the same time to make the biocatalyst specific. In such endeavours selective enzyme denaturation, cell membrane modification (9,32,33) and specific mutants for example, have been used. 

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