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32 2 New Trends in the In Situ Enzymatic Recycling of NAD(P)(H) Cofactors

than 2000 U mg −1 [46] and very good stability under operative conditions. Moreover,
the reduction of pyruvate is thermodynamically highly favored. Therefore, when
coupled to a dehydrogenase-catalyzed oxidation, only stoichoimetric amounts of
the co-substrate are required.
Up to now, the only limitation in the use of LDH for the recycling of nicotinamide
+
cofactors was related to the strict specificity of the wild-type enzymes toward NAD .
However, by using a combined approach of rational design and directed evolution,
an LDH variant of the enzyme from Bacillus subtilis capable of utilizing both NAD +
+
and NADP as cofactor was recently generated [47]. Specifically, target active-
site residues involved in interactions with the cofactor were first identified by
homology modeling of B. subtilis LDH on the 3D structure of the LDH from
Geobacillus stearothermophilus. A library of mutants was subsequently generated
by site-saturation mutagenesis of the selected amino acids, and screening for
+
activity in the presence of NADP as the cofactor resulted in the identification
of a significantly improved variant (Val39Arg). Further characterization of this
mutant enzyme showed a 250-fold improved catalytic efficiency with NADPH
+
compared to the wild-type LDH. The ability of performing NADP recycling was
then demonstrated in the coupled reaction with an ADH-catalyzed oxidation.
Noteworthy, this single amino acid substitution significantly also improved the
activity with NADH and did not affect any of the other biochemical parameters
investigated, for example, pH and temperature optimum. Therefore, this new LDH
variant is an attractive alternative for the recycling of both nicotinamide cofactors
in oxidation reactions.

2.2.2.2 NAD(P)H Oxidase
+
Among the enzymes that have been studied for the regeneration of NAD(P) ,
NAD(P)H oxidases (NOXs, EC 1.6.3.1) have gained significant attention as this
regeneration approach does not require other substrate than the molecular oxygen
provided by the atmosphere [48]. In fact, NOXs, which are members of the
flavoprotein disulfide reductase family, catalyze the oxidation of NAD(P)H by
reducing molecular O to either hydrogen peroxide via a two-electron transfer
2
process or directly to water via a four-electron transfer process. In particular, the
water-forming NOXs look highly suitable for cofactor regeneration because they
do not produce any harmful reactive oxygen species that might interfere with the
coupled enzymatic activities [49].
During the last decades, several NOXs have been purified from various microbial
strains belonging to the genera Streptococcus [50–52], Lactobacillus [49, 53, 54],
Methanocaldococcus [55], Brevibacterium [56], Eubacterium [57], Thermococcus [48,
58–60], Archaeoglobus [61], and Clostridium [62]. In particular, NOXs purified
from thermophilic microorganisms are of interest because they are very stable
biocatalysts, which enable biotransformations to be operated at high temperature
and at increased reaction rates [48].
Table 2.1 reports some features of the NOXs described so far. In particular,
the pH optimum varies significantly from enzyme to enzyme, and it is therefore