R-phenylacetylcarbinol (PAC), an intermediate in the production of ephedrine and pseudoephedrine, is currently produced by the controlled addition of benzaldehyde to an actively growing culture of yeast (usually Saccharomyces cerevisiae). A decarboxylation/condensation biotransformation is effected by pyruvate decarboxylase (PDC) between pyruvate produced by the yeast and added benzaldehyde (see Fig. 9). Using this traditional process, 12-15 gL-1 PAC is usually produced in 10-12 h with a yield of 70% theoretical based on benzaldehyde [100].

Confirmation of PAC production from benzaldehyde and pyruvate using purified PDC from various sources, including Z. mobilis, S. carlsbergenis, S. cerevisiae, S. fermentati and S. delbrueckii, was demonstrated by sev­eral groups during the late 1980s to mid 1990s [101-105]. Bringer-Meyer et al. [106] isolated and characterized PDC obtained from Z. mobilis. By comparison with yeast PDC (Saccharomyces sp., Candida sp.), bacterial PDC (Zymomonas sp.) had a lower benzaldehyde affinity and was inhibited more strongly by benzaldehyde, even though its affinity for pyruvate was similar or higher than that of yeast PDC.

However, interest in PDC from Z. mobilis continued due to its greater sta­bility than yeast PDC at room temperature with an enzyme half-life in the absence of benzaldehyde of over 100 h [107,108]. Unlike yeast PDC, it is also able to utilize the lower cost acetaldehyde as an alternative substrate to pyru­vate for production of PAC [109]. Advances in site-directed mutagenesis tech­niques have facilitated the production of mutant PDC from Z. mobilis with greater carboligase activity and higher stability towards acetaldehyde [110]. This mutant enzyme, designated PDCW392M, resulted from replacement of the bulky tryptophan residue 392 with methionine. A continuous process with PDCW392M was used in a biotransformation process for conversion of acetaldehyde and benzaldehyde to PAC in an enzyme membrane reactor. A volumetric productivity (space-time yield) of 81 gl-1 day-1 was reported with final PAC concentration of 22 mM and molar yields of 45% (initial sub­strates), based on 50 mM reaction mixture of both aldehydes [111,112].

In further studies by Rosche et al. [113], a biphasic enzymatic biotransfor­mation system for production of PAC from acetaldehyde and benzaldehyde with Z. mobilis PDCW392 was evaluated. Higher concentrations of benzalde — hyde and PAC in the organic phase (octanol) provided protection for the aqueous phase PDC. As a result, a specific PAC production of 11 mg PAC U PDC-1 was achieved compared with 1.2 mg PAC U PDC-1 in the absence of an organic phase. A similar two-phase system has been developed sub­sequently for conversion of pyruvate and benzaldehyde to PAC using PDC from yeast (C. utilis) with higher concentrations and productivities being at­tained [114,115].

A similar aqueous/organic two-phase system has been used also to screen a number of yeasts and bacteria for the enantio-specific reduction of the al­pha, beta-unsaturated carbon bond in citral to produce citronellal [116]. In comparison to the bacteria tested, the eukaryotes showed at least 5-fold lower citral reductase activities. Bacterial strains were found to produce the (S)- enantiomer of citronellal preferentially with ee values > 99% for Z. mobilis and 75% for Citronella freundii. The possible use of a Z. mobilis biofilm biore­actor for production of other fine chemicals has been proposed also [117] as it has been demonstrated that increased tolerance to aromatic substrates such as benzaldehyde can occur with such a bioreactor.

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