Yeast cells phenylacetyl carbinol

Ephedrine was originally isolated as the active agent present in plant extracts used in ancient Chinese medicine for respiratoiy ailments. As long ago as 1921 the formation of optically active phenylacetyl carbinol (PAC) from benzaldehyde and pyravate by brewers yeast and cell-free yeast extracts was reported. The PAC can then be reductively animated to produce optically active L-ephedrine (Figure 4.18). L-Ephedrine is widely used in the treatment of asthma and hay fever as a bronchodilating agent and decongestant. 

Though use of isolated purified enzymes is advantageous in that undesirable byproduct formation mediated by contaminating enzymes is avoided [37], in many industrial biotransformation processes for greater cost effectiveness the biocatalyst used is in the form of whole cells. For this reason baker s yeast, which is readily available, has attracted substantial attention from organic chemists as a catalyst for biotransformation processes. One of the first commercialized microbial biotransformation processes was baker s yeast-mediated production of (R)-phenylacetyl carbinol, where yeast pyruvate decarboxylase catalyzes acyloin formation during metabolism of sugars or pyruvate in the presence of benzaldehyde [38]. 

Tripathi and co-workers [46] evaluated phenylacetyl carbinol biotransformation efficiency of harvested whole yeast cells, grown continuously under glucose-limited conditions at different dilution rates. They found that cells from increasing dilutions showed increasing specific rates of product formation. 

Immobilization of yeast cells was shown to reduce the toxic effect of benzaldehyde because of diffusional limitations and gradients of toxic compounds that are established within the immobilizing matrix [35,54,55]. Cells of S. cerevisiae immobilized in sodium alginate beads withstand higher concentrations of benzaldehyde (up to 6 g/L) and produce more phenylacetyl carbinol [54]. Phenylacetyl carbinol production by immobilized cells was significantly higher (1.4-, 2.5-, and 7.5-fold) than that by free cells, using initial benzaldehyde concentrations of 2, 4, and 6 g/L, respectively, in fermentation medium. 

However, with immobilization of yeast cells, it is not as easy to regulate metabolism with the same efficiency as can be done with free cells. In shake flasks, cells of C. utilis, immobilized in calcium alginate beads, exhibited enhanced resistance to benzaldehyde in comparison with free cells [35]. They also produced higher levels of phenylacetyl carbinol. But in experiments with programmed feeding of benzaldehyde in a controlled bioreactor the final phenylacetyl carbinol production by immobilized cells was 15 g/L, significantly less than the 22 g/L achieved with a free cell fed-batch system. This difference in phenylacetyl carbinol productivity was attributed to the inability to regulate yeast metabolism via RQ in immobilized cells. 

Phenylacetyl carbinol production by yeast cells immobilized on carriers other than sodium alginate did not meet with great success [57]. All the six immobilized yeast systems... 

Nikolova and Ward [72,73] studied production of phenylacetyl carbinol from benz-aldehyde and pyruvate by whole-cell yeast biotransformation in two-phase systems. For the biocatalyst preparation fresh pressed commercial baker s yeast (50 g) was suspended in 50 ml 0.05 M sodium citrate buffer (pH 6.0) and lyophilized. Aliquots of 300 mg of lyophilized cells were mixed witii 1 g celite and the mixture was resuspended in 0.05 M sodium citrate buffer (pH 6.0). The suspension was lyophilized again and stored at 4°C. Scanning electron micrographs of the carrier celite and yeast cells lyophilized on celite are given in Fig. 1. Prior to use, organic solvents purchased in anhydrous form were saturated with 0,05 M sodium citrate buffer (pH 6.0). The same buffer was used as an aqueous component of the biphasic systems. 

Smith and Hendlin [51] suggested that there are two systems competing for benzaldehyde in the yeast cell. The first is the phenylacetyl carbinol-synthesizing system and the second is alcohol dehydrogenase, catalyzing reduction of benzaldehyde to benzyl alcohol. These researchers found that the increase in phenylacetyl carbinol production is accompanied by a decrease in the formation of benzyl alcohol and vice versa [78]. 

When the phenylacetyl carbinol-generating biottansformations catalyzed by yeast whole cells were carried out in monophasic systems consisting of an aqueous phase and water-miscible organic solvents, a decrease in benzyl alcohol formation was observed [44,45]. 

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