Microbial Enzymes and Biotransformations Microbial Enzymes and ...

226 Weckbecker and Hummel
Glucose dehydrogenase accepts NADP + as well as NAD + as a cofactor.
Therefore, this enzyme is a good candidate for the regeneration of NADPH or
NADH. This is of interest in asymmetric reductions of prochiral ketone compounds
to produce chiral hydroxy or amino acids or alcohols, which are valuable
building blocks and starting materials for the synthesis of important drugs.
The majority of the currently applied oxidoreductases depend on nicotinamide
coenzymes. For NADH, the regeneration based on formate/formate dehydrogenase
(FDH) has reached the most advanced stage of development (13).
Nevertheless, the only moderate stability and low specific activity of FDH are
severe limitations, especially for industrial applications. For the regeneration of
NADPH, there is no established method available.
One area of application of GDH is the regeneration of NADH. A few examples
are known in literature. In the synthesis and conversion of 2-keto-6-
hydroxyhexanoic acid to L-6-hydroxynorleucine by reductive amination using
beef liver glutamate dehydrogenase, the GDH from Bacillus sp. regenerates
NADH (14). The same purpose serves the GDH from Bacillus sp. during the
production of L-carnitine from 3-dehydrocarnitine by L-carnitine dehydrogenase
from Pseudomonas putida IAM12014 (15).
A further application of GDH is the regeneration of NADPH. For the asymmetric
reduction of ethyl 4-chloro-3-oxobutanoate, an aldehyde reductase from
Sporobolomyces salmonicolor was coupled and coexpressed with the GDH
from Bacillus megaterium for use as an NADPH regenerator (16). GDH from
Gluconobacter scleroides KY3613 was used for cofactor recycling in the production
of L-leucovorin (17).
Glucose-6-phosphate dehydrogenase can also regenerate NADPH.
Commercially available glucose-6-phosphate dehydrogenase was used to
regenerate NADPH (18), as well as glucose-6-phosphate dehydrogenase from
Leuconostoc mesenteroides (19).
We coupled the glucose dehydrogenase from Bacillus subtilis with an
NADP + -dependent (R)-specific alcohol dehydrogenase from Lactobacillus kefir
overexpressed in E. coli for the stereoselective production of alcohols. Since E.
coli cells do not have a sufficient cofactor regeneration system we developed a
coexpression system to express both genes in E. coli and to perform whole-cell
biotransformations.
2. Materials
1. pET-21a(+) expression system (Novagen, Madison, WI).
2. E. coli strains Tuner(DE3), BL21(DE3), Origami(DE3) (Novagen).
3. Oligonucleotide primers.
4. Restriction enzymes, T7 DNA polymerase, T4 DNA ligase.
5. Agarose.