Biotransformations using whole cells

Holland s group40 has shown that the most versatile biotransformation using whole cell biocatalyst is the one using the fungus species NRRL 4671. From analysis of the sulfoxidation of a large number of substrates (> 90), they recently proposed a predictive model for chiral sulfoxidation by the fungus. The model (Fig. 2), developed from energy-minimized (MM+) structures of substrates produced by Hyperchem, is able to explain the stereochemical inversion seen for sulfoxidation of some phenyl alkyl sulfides, such as phenyl vinyl and phenyl hexyl sulfide. 

Make a list of characteristics of an ideal medium for biotransformations using whole cells. (Try dus on a piece of paper before reading on). 

The low yield and/or optical purity achieved with the previous methodologies, as well as the possibility to stabilize the cells by immobilization, has opened the possibility of biotransformations using whole cells. In this way, aryhnalonic acids were transformed into 2-arylpropionic acids by incubation with Alcaligenes bronchisepticus [60]. 

Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations. 

The biotransformation should be carried out using whole cells because ... 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

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... 

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]. 

One advantage of whole-cell biotransformation that has not been addressed adequately in this chapter is the ability to modify compounds with complex structure, such as natural products. Natural products are ideal substrates for biotransformation reactions since they are synthesized in a series of enzymatic reactions by the whole cells. The modification of natural products by biotransformation has been reviewed recently by Azerad [ 13] and a majority of the modifications were carried out by whole-cell biotransformations. Additional examples of modification of natural products by whole-cell biotransformations can also be found in the review article by Patel [2]. Natural products are an important source of new drugs and new drug leads [53]. The use of biotransformation, especially whole-cell biotransformation, in modification of natural products for lead optimization and generating libraries of derivatives for S AR and screening studies is important for the pharmaceutical industry. 

In a similar vein, Wu et al. prepared a a-disubstituted-a-cyanoacetamides 19 from the corresponding a,a-disubstituted-malononitriles 18 by using whole cells of Rhodococcus sp. CGMCC 0497 (Scheme 2.8). The biotransformation proceeded with greater than 99%e.e. and yields up 53% [10]. 

Fourthly, biotransformations have been used for the synthesis of 3-deoxy-2-glyculosonic acids, using whole cells or purified enzymes. For instance, 3-deoxy-D-araZu rao-heptulosonic acid (DAH) and its 7-phosphate (DAHP, 122) have been produced directly from D-glucose by mutants of E. coli JB-5, that lack dehydroquinate synthase, the enzyme that converts DAHP into the cyclic intermediate dehydroquinic acid (DHQ, Scheme 14). Both DAH and DAHP are secreted into the medium. The dephosphorylated product could be generated in vivo by a phosphatase acting on DAHP.312... 

Most of these enzymatic ways to regenerate nicotinamide cofactors have been successfully established in a multitude of synthetic processes. The introduction of organic-water two-phase systems seems to be a useful method to overcome problems with the low solubility of many organic substrates. Bioconversions using whole cells have been well investigated because they show some decisive advantages like increased enzyme stability. The use of recombinant DNA techniques offers a wide field of application. Tailor-made cells can be created to perform whole cell biotransformations efficiently. 

One of the most useful characteristics of this work is the fact that these epoxides could be routinely produced at yields approaching (at best) 1 g L-1 after simple overnight shaking using whole-cell or even crude cell-free systems. Thus, these results clearly opened the way to a new type of biotransformation which should be very useful for organic synthesis. 

The biotransformation of bicydo[3.2.0]hept-2-en-6-one using whole cell suspensions of the fungus Cylindrocarpon destructans gave not only different ratios of both lactones depending on the degree of conversion, but also no enantioselectivity was... 

The examples presented here are taken from121. Only those biotransformations were chosen where a classical chemical step was replaced. The enzymes involved are mainly from the groups of oxidoreductases (E.C. class 1) and hydrolases (E.C. class 3). There are a few examples of lyases (E.C. class 4) and one example of an isomerase (E. C. class 5). The processes involving oxidoreductases mainly use whole cells because of the problem of cofactor regeneration. The examples are sorted in the order of the main classes of the Enzyme Commission (E. C.). The big letter E denotes the biotransformation in the syntheses schemes. 

A new industrial avenue to oligosaccharide synthesis may be opened by an approach using whole-cell biotransformations. Researchers from Kyowa Hakko Co. (Japan) have developed systems for the large-scale production of UDP-Gal and globotriose (Fig. 11) from inexpen-... 

A reasonable munber of biotransformation processes using lyases or transferases have been developed on an industrial scale, but this has not yet led to a general picture about the best process configuration. The main problems that seem to occur with these reactions (unfavourable equilibria and instability of substrates or products) have been solved in different manners. Substrates are fed slowly into the reactor or dissolved gradually, products are removed in situ by extraction or crystallization, or the biotransformation enzyme is incorporated in a cascade of reactions using whole cells. Thus, either of the aforementioned approaches seems to be feasible, given a specific biotransformation. 

In the near future, the use of solvent-tolerant strains will make the application of organic solvents in biotransformations by whole cells a more realistic option. 

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