costs has been recognized for many years and a large part of the research effort
concerning dehydrogenases has been expended in order to solve the problem of
cofactor recycling [555, 787–790].
Cofactor recycling is no problem when whole microbial cells are used as
biocatalysts for redox reactions. In this case, inexpensive sources of redox equivalents such as carbohydrates can be used since the microorganism possesses all the
enzymes and cofactors which are required for metabolism. The advantages and
disadvantages of using whole-cell systems are discussed in Sect. 2.2.3.
Recycling of Reduced Nicotinamide Cofactors
The easiest but least efficient method of regenerating NADH from NAD
+ is the
nonenzymic reduction using a reducing agent such as sodium dithionite (Na 2 S 2 O 4 )
[791]. Since the corresponding turnover numbers of this process are very low
(TTN 100), this method has only historical interest. Similarly, electrochemical
[792–794] and photochemical regeneration methods [795–798] suffer from insufficient electron transport causing side-reactions and show low to moderate turnover
numbers (TTN
1000).
26 On the other hand, enzymic methods for NADH or
NADPH recycling have been shown to be much more efficient and nowadays these
represent the methods of choice. They may be conveniently subdivided into
coupled-substrate and coupled-enzyme types.
Coupled-Substrate Process Aiming at keeping things as simple as possible, the
cofactor required for the transformation of the main substrate is constantly
regenerated by addition of a second auxiliary substrate (H-donor) which is
transformed by the same enzyme, but into the opposite direction (Scheme 2.108)
[799–801]. To shift the equilibrium of the reaction in the desired direction, the
donor must be applied in excess [802]. In principle, this approach is applicable to
both directions of redox reactions [803] and it constitutes a biological variant of a
transfer-hydrogenation. Although the use of a single enzyme simultaneously catalyzing two reactions appears elegant, some significant disadvantages are often
encountered in coupled-substrate cofactor recycling:
• The overall efficiency of the process is limited since the enzyme’s activity is
distributed between both the substrate (hydrogen acceptor) and the auxiliary
hydrogen donor.
• Enzyme inhibition caused by the high concentrations of the auxiliary substrate –
cosubstrate inhibition – is common, in particular when highly reactive carbonyl
species such as acetaldehyde or cyclohexenone are generated in the recycling
process.
• The product has to be purified from large amounts of auxiliary substrate used in
excess.
26 For example, if the reduction of NAD(P)
+ to NAD(P)H is 95% selective for hydride transfer onto
the p-position of the nicotinamide ring, after 100 turnovers the residual activity of the cofactor
would be 0.95
100 being equivalent to only ~0.6%.
2.2 Reduction Reactions
135
concerning dehydrogenases has been expended in order to solve the problem of
cofactor recycling [555, 787–790].
Cofactor recycling is no problem when whole microbial cells are used as
biocatalysts for redox reactions. In this case, inexpensive sources of redox equivalents such as carbohydrates can be used since the microorganism possesses all the
enzymes and cofactors which are required for metabolism. The advantages and
disadvantages of using whole-cell systems are discussed in Sect. 2.2.3.
Recycling of Reduced Nicotinamide Cofactors
The easiest but least efficient method of regenerating NADH from NAD
+ is the
nonenzymic reduction using a reducing agent such as sodium dithionite (Na 2 S 2 O 4 )
[791]. Since the corresponding turnover numbers of this process are very low
(TTN 100), this method has only historical interest. Similarly, electrochemical
[792–794] and photochemical regeneration methods [795–798] suffer from insufficient electron transport causing side-reactions and show low to moderate turnover
numbers (TTN
1000).
26 On the other hand, enzymic methods for NADH or
NADPH recycling have been shown to be much more efficient and nowadays these
represent the methods of choice. They may be conveniently subdivided into
coupled-substrate and coupled-enzyme types.
Coupled-Substrate Process Aiming at keeping things as simple as possible, the
cofactor required for the transformation of the main substrate is constantly
regenerated by addition of a second auxiliary substrate (H-donor) which is
transformed by the same enzyme, but into the opposite direction (Scheme 2.108)
[799–801]. To shift the equilibrium of the reaction in the desired direction, the
donor must be applied in excess [802]. In principle, this approach is applicable to
both directions of redox reactions [803] and it constitutes a biological variant of a
transfer-hydrogenation. Although the use of a single enzyme simultaneously catalyzing two reactions appears elegant, some significant disadvantages are often
encountered in coupled-substrate cofactor recycling:
• The overall efficiency of the process is limited since the enzyme’s activity is
distributed between both the substrate (hydrogen acceptor) and the auxiliary
hydrogen donor.
• Enzyme inhibition caused by the high concentrations of the auxiliary substrate –
cosubstrate inhibition – is common, in particular when highly reactive carbonyl
species such as acetaldehyde or cyclohexenone are generated in the recycling
process.
• The product has to be purified from large amounts of auxiliary substrate used in
excess.
26 For example, if the reduction of NAD(P)
+ to NAD(P)H is 95% selective for hydride transfer onto
the p-position of the nicotinamide ring, after 100 turnovers the residual activity of the cofactor
would be 0.95
100 being equivalent to only ~0.6%.
2.2 Reduction Reactions
135