Whole cells acids

Scheme 8.5 A biocatalytic (whole-cell) acid reduction scheme. AOR aldehyde oxidoreductase, ADH alcohol dehydrogenase.

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. 

Since 1978, several papers have examined the potential of using immobilised cells in fuel production. Microbial cells are used advantageously for industrial purposes, such as Escherichia coli for the continuous production of L-aspartic acid from ammonium fur-marate.5,6 Enzymes from microorganisms are classified as extracellular and intracellular. If whole microbial cells can be immobilised directly, procedures for extraction and purification can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. Whole cells are used as a solid catalyst when they are immobilised onto a solid support. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Chadha et al, have published a series of papers on the deracemization of P-hydroxyesters using whole cells of Candida parapsilosis. For example, deracemization of racemic ethyl 2-hydroxy-4-phenylbutanoic acid (22 R = H) yielded the (S) enantiomer in 85-90% yield and >99% ee (Figure 5.15) [26]. 

Both cis- and trans-chrysanthemic nitriles and amides were resolved into highly enantiopure amides and acids by Rhodococcus sp. whole cells [85]. The overall enantioselectivity of reactions of nitriles originated from the combined effects of a higher (lJ )-selective amidase and a (IJ )-selective nitrile hydratase (Figure 6.29). Chrysanthemic acids are related to constituents of pyrethrum flowers and insecticides. 

In case of primary alcohol substrates, biooxidation can also proceed to the carboxylic acid, enabling a facile separation of the chiral products by simple extraction. Whole-cells of Gluconobacter oxydans were utilized to produce S-2-phenylpro-panoic acid and R-2-phenylpropionic alcohol in excellent yields and optical purities (Scheme 9.4) [46]. 

In contrast to 2,3-dioxygenases, the related ipso/ortho oxygenation of aryl carbox-ylates has received considerable less attention and has hardly been utilized by the synthetic community, so far. Biooxidation of benzoic acid and P-naphthalene carboxylate provide access to corresponding 1,2-dihydroxylated dihydroaryl compounds in excellent stereoselectivity (Scheme 9.35), analogous to TDO- and NDO-mediated ortho/meta oxygenations. Whole-cell-mediated biotransformations were performed with mutant strains of Rahtonia and Pseudomonas and enable access to preparative quantities in >5 gl titers [261,262]. 

FIG. 8 Electron micrographs of freeze-etched preparations of whole cells from (a, b) Bacillus sphaericus CCM 2120 exhibiting a square S-layer lattice or from (c, d) Thermoanaerobacter ther-mohydrosulfuricus Llll-69 carrying a hexagonally ordered S-layer lattice, (a, c) Native S-layer lattices (b, d) S-layer lattices after covalent binding of ferritin to carbodiknide-activated carboxylic acid groups of the S-layer protein. Bars, 100 nm. 

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

The carboxylafion of indole into indole-3-carboxylate was observed by the purified indole-3-carboxylate decarboxylase as well as by the whole cells. For the carboxylafion reaction, temperatures over 30°C were not appropriate. The activities at 10, 20, and 30°C were about the same. The activity was maximal at pH 8.0 (Tris-HCl buffer, 100 mM). As shown in Fig. 10, the resting cells of A. nicotianae F11612 also catalyzed the carboxylafion of indole efficiently in the reaction mixture containing 20 mM indole, 3M KHCO3, 100mM potassium phosphate buffer (pH 6.0) in a tightly closed reaction vessel. By 6h, 6.81 mM indole-3-carboxylic acid accumulated in the reaction mixture with a molar conversion yield of 34%. Compared to the carhoxylation of pyrrole by pyrrole-2-carboxylate decarboxylase, the lower value compared might derive from the lower solubility of indole in the reaction mixture. 

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. 

The recent synthesis of (—)-tetracycline by Myers and co-workers incorporates a biocatalytic step in the first stage which oxidizes benzoic acid aerobically to an a,f)-dihydroxy derivative in the presence of a whole-cell mutant strain of Alcaligenes eutrophus. Figure 4.61 shows a reduced tree diagram for the synthesis and Table 4.28 summarizes the metrics parameters. 

DuPont has developed a process for the manufacture of glyoxylic acid by aerobic oxidation of glycolic acid (Fig. 2.33) mediated by whole cells of a recombinant methylotrophic yeast (Gavagnan et al, 1995). The glycolic acid raw material is readily available from the acid-catalysed carbonylation of formaldehyde. Traditionally, glyoxylic acid was produced by nitric acid oxidation of acetaldehyde or glyoxal, processes with high E factors, and more recently by ozonolysis of maleic anhydride. 

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. 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

Despite the large number whole-cell MALDI protocols that have been tested a single approach has not yet been widely adopted. There remain different and sometimes conflicting reports in the literature regarding optimum methodologies.3 In addition to the ferulic acid matrix mentioned above sinapinic acid, a-yano-4-hydroxycinnamic acid, 2,4-hydroxyphenyl benzoic... 

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