[816, 817]. The equilibrium is shifted towards the product because the
gluconolactone formed is spontaneously hydrolyzed to give gluconic acid. The
glucose dehydrogenase from Bacillus cereus is highly stable [818] and accepts
either NAD
+ or NADP
+ with high specific activity. Like FDH, however, GDH is
expensive and product isolation from polar gluconate may complicate the workup.
In the absence of purification problems, this method is attractive for laboratory use,
and it is certainly a convenient way to regenerate NADPH.
Similarly, glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation
of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, which spontaneously
hydrolyzes to the corresponding phosphogluconate (Scheme 2.110). The enzyme
from Leuconostoc mesenteroides is inexpensive, stable and accepts both NAD
+ and
NADP
+ [582, 819], whereas yeast-G6PDH accepts only NADP
+
. A major disadvantage of this system is the high cost of G6P. Thus, if used on a large scale, it may be
enzymatically prepared from glucose using hexokinase and this involves the regeneration of ATP using kinases (see pp. 107–109). Alternatively, glucose-6-sulfate and
G6PDH from Saccharomyces cerevisiae may be used to regenerate NADPH
[820]. The sulfate does not act as an acid catalyst for the hydrolysis of NADPH
and is more easily prepared than the corresponding phosphate [821]. Overall, the
G6P/G6PDH system complements glucose/GDH as an excellent method for
regenerating NADPH and is a good method for regenerating NADH.
More recently, phosphite dehydrogenase has been shown to offer a promising
alternative [822, 823]: The equilibrium is extremely favorable, both phosphite and
phosphate are inoccuous to enzymes and act as buffer. The wild-type enzyme from
Pseudomonas stutzeri accepts only NAD
+ [824], but thermostable mutants were
generated which are also able to reduce NADP
+ [825–827].
Ethanol and alcohol dehydrogenase (ADH) have been used in the past to
regenerate NADH and NADPH [828, 829]. The low to moderate cost of ADH
and the volatility of both ethanol and acetaldehyde make this system attractive for
lab-scale reactions. An alcohol dehydrogenase from yeast reduces NAD
+
, while an
ADH from Leuconostoc mesenteroides is used to regenerate NADPH (Scheme
2.110). However, due to the low redox potential, only activated carbonyl substrates
such as aldehydes and cyclic ketones are reduced in good yields. With other
substrates, the equilibrium must be driven by using ethanol in excess or by
removing acetaldehyde. The latter may be achieved by sweeping with nitrogen
[830] or by further oxidizing acetaldehyde to acetate [831], using aldehyde dehydrogenase thereby generating a second equivalent of reduced cofactor. All of these
methods, however, give low TTNs or involve complex multi-enzyme systems.
Furthermore, even low concentrations of ethanol or acetaldehyde inhibit or deactivate enzymes. Alternatively, a crude cell-free extract from baker’s yeast has been
recommended as an (unspecified) enzyme source for NADPH recycling by using
glucose as the ultimate reductant [832].
A particularly attractive alternative for the regeneration of NADH makes use of
hydrogenase enzymes, so called because they are able to accept molecular hydrogen directly as the hydrogen donor [833, 834]. The latter is strongly reducing,
innocuous to enzymes and nicotinamide cofactors, and its consumption leaves no
138
2 Biocatalytic Applications
gluconolactone formed is spontaneously hydrolyzed to give gluconic acid. The
glucose dehydrogenase from Bacillus cereus is highly stable [818] and accepts
either NAD
+ or NADP
+ with high specific activity. Like FDH, however, GDH is
expensive and product isolation from polar gluconate may complicate the workup.
In the absence of purification problems, this method is attractive for laboratory use,
and it is certainly a convenient way to regenerate NADPH.
Similarly, glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation
of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, which spontaneously
hydrolyzes to the corresponding phosphogluconate (Scheme 2.110). The enzyme
from Leuconostoc mesenteroides is inexpensive, stable and accepts both NAD
+ and
NADP
+ [582, 819], whereas yeast-G6PDH accepts only NADP
+
. A major disadvantage of this system is the high cost of G6P. Thus, if used on a large scale, it may be
enzymatically prepared from glucose using hexokinase and this involves the regeneration of ATP using kinases (see pp. 107–109). Alternatively, glucose-6-sulfate and
G6PDH from Saccharomyces cerevisiae may be used to regenerate NADPH
[820]. The sulfate does not act as an acid catalyst for the hydrolysis of NADPH
and is more easily prepared than the corresponding phosphate [821]. Overall, the
G6P/G6PDH system complements glucose/GDH as an excellent method for
regenerating NADPH and is a good method for regenerating NADH.
More recently, phosphite dehydrogenase has been shown to offer a promising
alternative [822, 823]: The equilibrium is extremely favorable, both phosphite and
phosphate are inoccuous to enzymes and act as buffer. The wild-type enzyme from
Pseudomonas stutzeri accepts only NAD
+ [824], but thermostable mutants were
generated which are also able to reduce NADP
+ [825–827].
Ethanol and alcohol dehydrogenase (ADH) have been used in the past to
regenerate NADH and NADPH [828, 829]. The low to moderate cost of ADH
and the volatility of both ethanol and acetaldehyde make this system attractive for
lab-scale reactions. An alcohol dehydrogenase from yeast reduces NAD
+
, while an
ADH from Leuconostoc mesenteroides is used to regenerate NADPH (Scheme
2.110). However, due to the low redox potential, only activated carbonyl substrates
such as aldehydes and cyclic ketones are reduced in good yields. With other
substrates, the equilibrium must be driven by using ethanol in excess or by
removing acetaldehyde. The latter may be achieved by sweeping with nitrogen
[830] or by further oxidizing acetaldehyde to acetate [831], using aldehyde dehydrogenase thereby generating a second equivalent of reduced cofactor. All of these
methods, however, give low TTNs or involve complex multi-enzyme systems.
Furthermore, even low concentrations of ethanol or acetaldehyde inhibit or deactivate enzymes. Alternatively, a crude cell-free extract from baker’s yeast has been
recommended as an (unspecified) enzyme source for NADPH recycling by using
glucose as the ultimate reductant [832].
A particularly attractive alternative for the regeneration of NADH makes use of
hydrogenase enzymes, so called because they are able to accept molecular hydrogen directly as the hydrogen donor [833, 834]. The latter is strongly reducing,
innocuous to enzymes and nicotinamide cofactors, and its consumption leaves no
138
2 Biocatalytic Applications