However, these distinct advantages have to be taken into consideration alongside
some significant drawbacks:
• The productivity of microbial conversions is usually low since the majority of
nonnatural substrates are toxic to living organisms and are therefore only
tolerated at low concentrations (~0.1–0.3% per volume).
• The large amount of biomass present in the reaction medium causes low
recovery, particularly when the product is stored inside the cells and not excreted
into the medium. Since only a small fraction (typically 0.5–2%) of the auxiliary
cosubstrate is used for coenzyme recycling, while the bulk is metabolized
forming polar byproducts, product purification is troublesome and monitoring
of the reaction becomes difficult.
• Finally, different strains of a microorganism most likely possess different
specificities; thus it is important to use exactly the same culture to obtain
comparable results with the literature [921].
• Stereoselectivities may vary to a great extent due to the presence of multiple
enzymes. If two enzymes, each with high but opposite stereochemical preference, compete for the same substrate, the optical purity of the product is
determined by the relative rates of the individual reactions. The latter, in turn,
depend on the substrate concentration. At concentrations below saturation, the
relative rates are determined by the ratio V max /K M for each enzyme. On the other
hand, when saturation is reached using elevated substrate concentrations, the
relative rates mainly depend on the ratio of k cat of the two reactions. Consequently, when two (or more) enzymes are involved in the transformation of
enantiomeric substrates, the optical purity of the product becomes a function of
the substrate concentration, because the values of K M and k cat for the substrate
enantiomers are different for both competing enzymes. With yeasts, it is a
well-known phenomenon that lower substrate concentrations often give higher
e.e. p s [922].
The following general techniques can be applied to enhance the selectivity of
microbial reduction reactions:
• Substrate modification, e.g., by variation of protecting groups which can be
removed after the transformation [923–925]
• Variation of the metabolic parameters by immobilization [926–928]
• Using cells of different age [929]
• Variation of the fermentation conditions [930–932]
• Screening of microorganisms to obtain strains with the optimum properties
(a hard task for nonmicrobiologists) [933, 934]
• Selective inhibition of one of the competing enzymes (see below)
Reduction of Aldehydes and Ketones by Baker’s Yeast
Asymmetric Reduction of Ketones Baker’s yeast (Saccharomyces cerevisiae) is
by far the most widely used microorganism for the asymmetric reduction of ketones
[935–939]. It is ideal for nonmicrobiologists, since it is readily available at a very
reasonable price and its use does not require sterile fermenters but can be handled
2.2 Reduction Reactions
147
some significant drawbacks:
• The productivity of microbial conversions is usually low since the majority of
nonnatural substrates are toxic to living organisms and are therefore only
tolerated at low concentrations (~0.1–0.3% per volume).
• The large amount of biomass present in the reaction medium causes low
recovery, particularly when the product is stored inside the cells and not excreted
into the medium. Since only a small fraction (typically 0.5–2%) of the auxiliary
cosubstrate is used for coenzyme recycling, while the bulk is metabolized
forming polar byproducts, product purification is troublesome and monitoring
of the reaction becomes difficult.
• Finally, different strains of a microorganism most likely possess different
specificities; thus it is important to use exactly the same culture to obtain
comparable results with the literature [921].
• Stereoselectivities may vary to a great extent due to the presence of multiple
enzymes. If two enzymes, each with high but opposite stereochemical preference, compete for the same substrate, the optical purity of the product is
determined by the relative rates of the individual reactions. The latter, in turn,
depend on the substrate concentration. At concentrations below saturation, the
relative rates are determined by the ratio V max /K M for each enzyme. On the other
hand, when saturation is reached using elevated substrate concentrations, the
relative rates mainly depend on the ratio of k cat of the two reactions. Consequently, when two (or more) enzymes are involved in the transformation of
enantiomeric substrates, the optical purity of the product becomes a function of
the substrate concentration, because the values of K M and k cat for the substrate
enantiomers are different for both competing enzymes. With yeasts, it is a
well-known phenomenon that lower substrate concentrations often give higher
e.e. p s [922].
The following general techniques can be applied to enhance the selectivity of
microbial reduction reactions:
• Substrate modification, e.g., by variation of protecting groups which can be
removed after the transformation [923–925]
• Variation of the metabolic parameters by immobilization [926–928]
• Using cells of different age [929]
• Variation of the fermentation conditions [930–932]
• Screening of microorganisms to obtain strains with the optimum properties
(a hard task for nonmicrobiologists) [933, 934]
• Selective inhibition of one of the competing enzymes (see below)
Reduction of Aldehydes and Ketones by Baker’s Yeast
Asymmetric Reduction of Ketones Baker’s yeast (Saccharomyces cerevisiae) is
by far the most widely used microorganism for the asymmetric reduction of ketones
[935–939]. It is ideal for nonmicrobiologists, since it is readily available at a very
reasonable price and its use does not require sterile fermenters but can be handled
2.2 Reduction Reactions
147