Whole-cell transformation

Triclosan is a broad spectrum antibacterial agent with antifungal and antiviral properties, widely employed in personal care products such as soaps, shampoos, toothpastes, and cosmetics [40]. Fungal-mediated degradation studies have been mainly performed by means of enzymatic processes, although a couple of whole cell transformation reports are available. 

Scheme 2.2.7.3 Reduction of diketo ester la by baker s yeast whole-cell transformation.

Scheme 15.1 Rhodococcus nitrile hydratase (i)-amidase (ii) whole cell transformation of alicyclic nitriles ( )-1a-( )-4a to amides 1b-4b and carboxylic acids 1c-4c n = 1,2.

Figure 15.2 fi-Amino nitriles for whole cell transformations with Rhodococci (only one enantiomer is depicted). 

The structures of N-tosylated amino nitriles for enantioselective hydrolysis are depicted in Figure 15.2. The results listed in Tables 15.1-15.3 are isolated yields of the whole cell transformations after extraction and chromatographic purification. The enantiomeric excesses (e.e.s) are listed in parentheses. AH reactions were... 

Catalysis is one of the most important and rapidly expanding areas in modem organic chemistry. Catalytic reactions can be achieved by either chemocatalysis or biocatalysis. The former field is dominated by transition metal catalysis, whereas in biocatalysis the use of isolated enzymes dominates over whole-cell transformations. 

H. G. Davies, R. H. Green, D. R. Kelly, and S. M. Roberts, Bio-Transformations in Preparative Organic Chemisty The Use of Isolated Enymes and Whole-Cell Systems in Synthesis, Academic Press, London, 1989. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

The term biotransformation or biocatalysis is used for processes in which a starting material (precursor) is converted into the desired product in just one step. This can be done by use either of whole cells or of (partially) purified enzymes. Product examples range from bulk chemicals (such as acrylamide) to fine chemicals and chiral synthons (chiral amines or alcohols, for example). There are several books and reviews dealing with the use of bio transformations either at laboratory or at industrial scales [1, 10-13]. 

Malic add has a limited use in the food industry as an addifying agent where it is an alternative to dtric add. In nature, only L(-) malic add is found whereas the relatively cheap, chemical synthetic methods yield D/L mixtures. The favoured industrial way to produce the L(-) add is by enzymic transformation from fumaric add. Either whole cells or isolated and immobilised enzymes can be used, with high conversion effidendes. 

As illustrated in Figure A8.3 nitrilases catalyse conversions of nitriles directly into the corresponding carboxylic adds (route A), while other nitrile converting enzymes, die nitrile hydratases, catalyse the conversion of nitriles into amides (route B) which, by the action of amidases usually present in the whole cell preparations, are readily transformed into carboxylic adds (route C). 

Almost all types of cell can be used to convert an added compound into another compound, involving many forms of enzymatic reaction including dehydration, oxidation, hydroxyla-tion, animation, isomerisation, etc. These types of conversion have advantages over chemical processes in that the reaction can be very specific, and produced at moderate temperatures. Examples of transformations using enzymes include the production of steroids, conversion of antibiotics and prostaglandins. Industrial transformation requires the production of large quantities of enzyme, but the half-life of enzymes can be improved by immobilisation and extraction simplified by the use of whole cells. 

Enantioselective transformations of several cyclopropane or oxirane-containing nitriles were studied using nitrile-transforming enzymes [78]. Microbial Rhodococcus sp. whole cells containing a nitrile hydratase/amidase system hydrolyzed a number... 

Whole cells are grown for a variety of reasons. The cells may perform a desired transformation of the substrate, e.g., wastewater treatment the cells themselves may be the desired produce, e.g., yeast production or the cells may produce a desired product, e.g., penicillin. In the later case, the desired product may be excreted, as for the penicillin example, and recovered in relatively simple fashion. If the desired product is retained within the cell walls, it is necessary to lyse (rupture) the cells and recover the product from a complex mixture of cellular proteins. This approach is often needed for therapeutic proteins that are created by recombinant DNA technology. The resulting separation problem is one of the more challenging aspects of biochemical engineering. However, culture of the cells can be quite difficult experimentally and is even more demanding theoretically. 

Like enzymes, whole cells are sometime immobilized by attachment to a surface or by entrapment within a carrier material. One motivation for this is similar to the motivation for using biomass recycle in a continuous process. The cells are grown under optimal conditions for cell growth but are used at conditions optimized for transformation of substrate. A great variety of reactor types have been proposed including packed beds, fluidized and spouted beds, and air-lift reactors. A semicommercial process for beer used an air-lift reactor to achieve reaction times of 1 day compared with 5-7 days for the normal batch process. Unfortunately, the beer suffered from a mismatched flavour profile that was attributed to mass transfer limitations. 

Biocatalysts these are essential for life and play a vital role in most processes occurring within the body as well as in plants. In the laboratory biocatalysts are usually natural enzymes or enzymes produced in situ from whole cells. They offer the possibility of carrying out many difficult transformations under mild conditions and are especially valuable for producing enantiomerically pure materials. Their huge potential is currently largely untapped, partially due to the time and expense of isolating and screening enzymes. 

The results presented in Tables 3 and 4 deserve some comments. First, a variety of enzymes, including whole-cell preparations, proved suitable for the resolution of different hydroxyalkanephosphorus compounds, giving both unreacted substrates and the products of the enzymatic transformation in good yields and, in some cases, even with full stereoselectivity. Application of both methodologies, acylation of hydroxy substrates rac-41 and rac-43 or the reverse (hydrolysis of the acylated substrates rac-42 and rac-44), enables one to obtain each desired enantiomer of the product. This turned out to be particularly important in those cases when a chemical transformation OH OAc or reverse was difficult to perform. As an example, our work is shown in Scheme 3. In this case, chemical hydrolysis of the acetyl derivative 46 proved difficult due to some side reactions and therefore an enzymatic hydrolysis, using the same enzyme as that in the acylation reaction, was applied. Not only did this provide access to the desired hydroxy derivative 45 but it also allowed to improve its enantiomeric excess. In this way. 

Conventional use has been made of the radioisotope C, and details need hardly be given here. Illustrative examples include the elucidation of pathways for the anaerobic degradation of amino acids (Chapter 7, Part 1) and purines (Chapter 10, Part 1). Some applications have used C with high-resolution Fourier transform NMR in whole-cell suspensions, and this is equally applicable to molecules containing the natural or the synthetic P nuclei. As noted later, major advances in NMR have made it possible to use natural levels of C. 

Whole cells of Rhodococcus opacus strain Icp were used to study the metabolism of fluorophenol isomers (Finkelstein et al. 2000), in which both fluorocatechols and fluoro-pyrogallols were produced (Figure 9.35a). Both 3- and 4-fluorophenol produced 5-fluoro-pyrogallol, which was transformed into 2-pyrone-4-fluoro-6-carboxylate (Figure 9.35b). 

The techniques and different approaches used in microbial transformations have been mentioned and reviewed by several authors [1,4,11,13-17]. The compounds can be metabolized by using either different pure enzymes or simply cultivated microbial whole cells [18-22]. A more general scheme is shown in Fig. 2 for the development of a standard system. 

The same 2,2 -bimorphine has been further transformed into 10-o -S-mono-hydroxy-2,2 -bimorphine and 10,10 -a,a -S,S -dihydroxy-2,2 -bimorphine by the same group of researchers, again utilizing C. didymum whole-cell suspensions [50]. These compounds were confirmed using classical spectroscopic... 

Another example of a biocatalytic transformation ousting a chemical one, in a rather simple reaction, is provided by the Lonza nitotinamide process (Fig. 2.34) (Heveling, 1996). In the final step a nitrile hydratase, produced by whole cells of Rh. rhodoccrous, catalyses the hydrolysis of 3-cyano-pyridine to give nitotinamide in very high purity. In contrast, the conventional chemical hydrolysis afforded a product contaminated with nicotinic acid. 

Stump, M. J. Jones, J. J. Fleming, R. C. Lay, J. O., Jr. Wilkins, C. L. Use of double-depleted 13C and 15N culture media for analysis of whole cell bacteria by MALDI time-of-flight and Fourier transform mass spectrometry. J. Am. Soc. Mass Spectrom. 2003,14,1306-1314. 

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