Biotransformation with Resting Cells

One of the best examples for discussing biotransformations in neat solvents is the enzymatic hydrolysis of acrylonitrile, a solvent, to acrylamide, covered in Chapter 7, Section 7.1.1.1. For several applications of acrylamide, such as polymerization to polyacrylamide, very pure monomer is required, essentially free from anions and metals, which is difficult to obtain through conventional routes. In Hideaki Yamada s group (Kyoto University, Kyoto, Japan), an enzymatic process based on a nitrile hydratase was developed which is currently run on a commercial scale at around 30 000-40 000 tpy with resting cells of third-generation biocatalyst from Rhodococcus rhodochrous J1 (Chapter 7, Figure 7.1). 

Use a biological total synthesis by fermentation or multi step biotransformation (with growing cells, resting cells, multi-enzyme systems, genetically or metabolically engineered biocatalysts, etc). 

Stereoselective biotransformation with growing cells, resting free or immobilized cells, or isolated enzymes has been demonstrated to be a useful preparative method for the synthesis of centrochiral optically active organosilicon compounds [1-3]. In continuation of our own studies in this field, we have investigated stereoselective microbial transformations of rac-1-(4-fluorophenyl)-l-methyl-l-sila-2-cyclohexanone (rac-1) and rac-(Si5,CR/SiR,C5)-2-acetoxy-l-(4-fluorophenyl)-l-methyl-1-silacyclohexane [rac-(Si5,C/ /Si/f,C5)-3a]. We report here on (i) the synthesis of rac-1 and rac- SiS,CR/SiR,CS)-3a, (ii) the diastereoselective microbial reduction of rac-1 [— (Si5,C/ )-2a, (SiR,C5)-2a], and (iii) the enantioselective microbial hydrolysis of rac-(SiS,CR/SiR,CS)-3a [- (SiR,C5)-2a],... 

Preparative scale biotransformation of betulinic acid (1) with resting-cell suspensions of Cunninghamella sp. NRRL 5695 resulted in the production in low yield of an acyl glucoside metabolite (Figure 28.49), which has been characterized as 28-0-p-o-glucopyranosyl-3p-hydroxy-lup-20(29)-en-28-oate (149) based on spectral and enzymatic... 

Initial scale-up of microbial biotransformation is conveniently run with multiple flasks without extensive reaction optimization. A typical flask fermentation is performed at 28 °C, 250 rpm with 100 mL culture in a 500 mL Erlenmeyer flask, although other settings will work fine too. Three parameters need to be investigated before scale-up the time for adding the substrate, the optimal substrate concentration and the time course of product formation. Optimization of other factors, such as medium composition and pH, growing cells versus resting cells [74], is helpful, if the timeline allows and if there is a sufficient amount of the substrate to support the screening. 

Recently, it has been demonstrated that the yeast Saccharomyces cerevisiae (DHW S-3) can also be used for the (R)-selective reduction of the acetylsilane rac-48. By analogy with the bioconversions illustrated in Scheme 10, incubation of rac-48 with resting free cells of this microorganism yielded a 1 1 mixture of the corresponding diastereomeric (1-hydroxyethyl)silanes (SiR,CR)-49 and (SiS,CR)-5057. Under preparative conditions, the biotransformation products were isolated in 43% yield (relative to rac-48). The enantiomeric purities of the silanes (SiR,CR)-49 and (SiS,CR)-50 were >98% ee. 

Biotransformations transform defined precursors in a series of defined (not always known) steps with enzymes or resting cells to a desired target product. 

The use of resting cells of microorganisms to biotransform organic compounds by specific reactions is a well-established technique. Thus, incubation of norbornanone (12) with a washcd-cell suspension of cyclopentanol grown Pseudomonas sp. NCIB 9872 resulted in the rapid oxidation of the substrate to a mixture of lactones 13 and 14398. 

The industrial use of bio catalysts has been reviewed in many excellent papers from industrial and academic experts in recent years [6-20]. These publications clearly show that immobilized systems find only limited use in present bioprocesses. Straathof et al. recently investigated 134 industrial biotransformations and came to the conclusion that only 20 confirmed processes rely on immobilized bio catalysts [15]. This is due to the fact that immobilization can be a considerable cost factor and is frequently used in combination with less common continuous reactors. In addition, many transformations belong to the class of redox reactions and require a cofactor for the reaction to occur. Such processes can in many cases be realized perfectly under fermentative conditions by the use of living or resting cells [17,21-25]. 

---