Biotransformations cell-containing

Since there is no commercially available D-aminoacylase, the production process of D-amino acids involves cloning of the D-aminoacylase and the whole cells containing the recombinant d-aminoacylase are used in biotransformation of /V-acetyl-D-amino acid, d-Amino acids can be generated in large quantities at low cost using whole-cell biotransformation [23]. 

Because of the possible effects of active and carrier-mediated processes and metabolic biotransformation, the issue of tissue viability is important for in vitro buccal mucosal experiments. The barrier nature of the buccal mucosa resides in the upper layers of the epithelium, where unlike in the stratum corneum, the cells contain a variety of functional organelles [119, 122, 125, 150], and so tissue viability may be an important component of the barrier function of the tissue. Various methods have been employed to assess the viability of excised buccal mucosa, including measurement of biochemical markers, microscopic methods, and linearity of transport data [42], While biochemical methods, including measurement of adenosine 5 -triphosphate (ATP) levels and utilization of glucose, provide information on the metabolic activity of the tissue, this does not necessarily relate to the barrier function of the tissue. In excised rabbit buccal mucosa, levels of ATP were measured and found to decline by 40% in 6 h, and this correlated well with transmission electron microscopic evaluation of the tissue (intact superficial cells) [32], In addition, the permeability of a model peptide was unaltered up to 6 h postmortem, but at 8 h, a significant change in permeability was observed [32], These investigators therefore claimed that excised rabbit buccal mucosa could be used for diffusion studies for 6 h. 

Figure 17.1 Sequence of events in the overall process of biotrans-formations (1) bacterial cell containing enzymes takes up organic chemical, /, (2) i binds to suitable enzyme, (3) enzyme / complex reacts, producing the transformation product(s) of /, and (4) the product(s) is(are) released from the enzyme. Several additional processes may influence the overall rate such as (5) transport of / from forms that are unavailable (e.g., sorbed) to the microorganisms, (6) production of new or additional enzyme capacity [e.g., due to turning on genes (induction), due to removing materials which prevent enzyme operation (activation), or due to acquisition of new genetic capabilities via mutation or plasmid transfer], and (7) growth of the total microbial population carrying out the biotransformation of /. ...

The biocatalytic addition of ammonia to trans-cinnamic acid, 28, proceeds enantio-selectively in the presence of whole-cells containing L-phenylalanine ammonia lyase. Of the suitable strains, wild-type strains of Rhodococcus rubra as well as Rhodotorula rubra were found to be very efficient. The biotransformation is carried out in basic media at pH 10.6. In addition, analogous reactions furnishing non-natural substituted derivatives of L-phenylalanine starting from substituted trans-cinnamic acid derivatives were reported by Mitsui, and Great Lakes, respectively [38 c]. 

Enzymes are made of proteins and are the organic catalysts produced by hving cells. Each cell contains a multienzyme system, and this enzyme system is complex and interrelated in substrates and products - that is, the product of one enzymatic reaction may become the substrate (reactant) for another enzyme reaction. The protein structure of each enzyme is stereochemically specific and forms complexes with substrates to carry out transformations - an enzyme-substrate complex. This complex is then transformed with a coenzyme or cofactor to the biotransformed product. 

Such isolated enzyme approaches for deracemization have a clear disadvantage in that they require two operational manipulations with an intermediate recovery step. A one-pot strategy is offered by employing whole-cell biotransformations with strains containing set(s) of complementary dehydrogenases operating in both biooxidative and bioreductive modes. Trace amounts of the intermediate ketone species can be isolated in several cases. In order to lead to an efficient deracemization... 

Cyclic dithioketals and acetals represent another important class of sulfur containing chiral auxiliaries, which are available in chiral form by biooxidation. Biotransformations were performed on a preparative scale using whole-cells (wild type and recombinant) and isolated enzyme. Again, enantiocomplementary oxidation of unsubstituted dithianes (linear and cyclic, R = H) was observed when using and CPMOcomo (Scheme 9.28) [211,212]. Oxygenation of functionalized substrates (R = substituted alkyl) with gave preferably trans... 

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

These results may be viewed in the wider context of interactions between potential ligands of multifunctional xenobiotics and metal cations in aquatic environments and the subtle effects of the oxidation level of cations such as Fe. The Fe status of a bacterial culture has an important influence on synthesis of the redox systems of the cell since many of the electron transport proteins contain Fe. This is not generally evaluated systematically, although the degradation of tetrachloromethane by a strain of Pseudomonas sp. under denitrifying conditions clearly illustrated the adverse effect of Fe on the biotransformation of the substrate (Lewis and Crawford 1993 Tatara et al. 1993). This possibility should therefore be taken into account in the application of such organisms to bioremediation programs. 

Analogous to the KRED reductions they can be performed as whole-cell biotransformations [48, 49] (baker s yeast, for example, contains a number of EREDs) or with isolated enzymes [50-52]. In the latter case the nicotinamide cofactor can... 

The oximes contain a quaternary ammonium group that contributes to their acidity and their strong binding to the inhibited enzyme. This appears to be a key structural element in known reactivators, but it tends to make them poorly soluble in lipids. Practically, this means that the drugs are slowly absorbed from the gastrointestinal tract, have difficulty entering the brain, do not easily enter hepatic cells to be biotransformed, and are not reabsorbed from the renal tubular urine. 

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