Page 49 - Cascade biocatalysis

2.2 Advances in Cofactor Regeneration 25

of the thermal stability of the biocatalyst [6], which was subsequently recovered by
applying a directed evolution strategy [8].
Recently, a rational design approach was also successfully used to improve the
thermostability of a plant FDH, the FDH from Glycine max (soybean), which shows
some potential for synthetic applications because of its higher chemical stability
and lower K M values for both substrate and cofactor when compared with that of
FDHs isolated from bacteria and yeasts [9].
Moreover, as most of the FDHs investigated up to now are highly specific for
NADH, several studies have also been devoted to the change of coenzyme specificity.
NADPH-accepting FDH variants were generated starting from both bacterial [10]
and yeast [11–13] wild-type enzymes. Interestingly, wild-type NADPH-dependent
FDHs were only recently identified from Burkholderia spp. strains [14]. On the
basis of the sequence analysis of these novel FDHs, engineered variants of the
Mycobacterium vaccae N10 FDH were subsequently designed, in order to combine
the capability of regenerating NADPH together with the stability toward the
α-haloketone ethyl 4-chloroacetoacetate [15].
Although quite nice results have been obtained in altering the cofactor specificity
and improving the stability of wild-type FDHs, very little improvements have been
obtained toward an enhancement of the catalytic activity, which in the best cases
amounted to around 10 U mg −1 [15, 16]. This limitation is only partly balanced
by the satisfactory expression levels of yeast and bacterial FDHs obtainable by
recombinant production in Escherichia coli [4].
−1
On the other hand, highly active (up to 550 U mg ) and stable GDHs have been
isolated from bacteria, in particular from different Bacillus species, such as Bacillus
megaterium [17]. The cofactor specificity of Bacillus GDHs is usually not very strict;
therefore, the same enzyme can be advantageously used for the regeneration of
both NADH and NADPH in different reduction processes. However, the GDH
from the archaeal Thermoplasma acidophilum [18] has recently found application in
multienzymatic one-pot processes where a higher selectivity toward the coenzyme
was desired [19]. In fact, this GDH shows a high specificity toward the NADPH
+
+
cofactor, with K M values of 0.113 mM for NADP but >30 mM for NAD .
The features of available GDHs appear satisfactory in terms of specific activ-
ity, stability, and driving force. Moreover, glucose is a cheap co-substrate also
for large-scale transformations. However, a recent study has demonstrated that
GDHs are strongly inhibited by both the intermediate product glucono-1,5-lactone,
which spontaneously hydrolyzes to gluconate during the biotransformation, and
NAD(P)H [20]. After a careful kinetic evaluation of GDHs-catalyzed processes,
useful guidelines concerning enzyme and substrate concentration, as well as the
reaction pH, have been suggested to achieve an optimized exploitation of this
recycling system.
The main drawback in the application of the glucose/GDH system for NAD(P)H
cofactor regeneration in a homogeneous aqueous medium is the need to separate
the desired product from the co-product gluconate. However, this problem can
be easily solved by performing the reduction reaction in a biphasic system, where
the substrate and the product of synthetic relevance are ‘‘compartmentalized’’