Full Papers
such as an IL offers an innovative strategy to achieve enhanced
performance for enzyme-catalyzed reactions.
tivity of the thermostable TKgst were investigated. Notably, the
influence of ILs on this family of enzymes has never been de-
scribed. After an initial screening, a more thorough study was
conducted on 1-butyl-3-methylimidazolium chloride ([BMIm]
[Cl]) aqueous solution to define the optimal proportion of IL in
the medium and the substrate specificity of TKgst toward differ-
ent hydrophilic aldehydes. The immobilization of TKgst on
a MgAl-LDH matrix was performed by direct coprecipitation. To
further develop an efficient, reusable biocatalyst to produce
chiral polyols, TKgst was immobilized on an inorganic LDH. The
chemical composition of MgAl-NO3 was chosen because this
matrix had been shown previously to give the best results for
the immobilization of TKs from other sources (S. cerevisiae,
E. coli).[49,50] These TKs, like TKgst, are homodimers and have
Our study focuses on transketolase (TK; E.C. 2.2.1.1) an
enzyme dependent on thiamine pyrophosphate (ThDP) that
catalyzes the synthesis of chiral polyols in one step by the ste-
reospecific formation of a CÀC bond. For synthetic purposes,
hydroxypyruvate (HPA) is used as a ketol donor substrate,
which renders the reaction irreversible through the release of
CO2 (Scheme 1).
a
strong homology of protein sequences and active
Scheme 1. Irreversible reaction catalyzed by TK in the presence of Li-HPA as
the donor substrate and an a-hydroxyaldehyde as the acceptor substrate.
sites.[36,54,55] MgAl-NO3, which has a point of zero net charge of
approximately 10–11, displays platelets with a net positive
charge under experimental conditions (pH 9), whereas TK has
an overall negative charge. The activity of the as-prepared bio-
hybrid in IL/water medium was compared with the activity of
free TKgst, and the recyclability of the biocatalyst was assessed.
Finally, the application of the MgAl-NO3@TKgst biohybrid for the
synthesis of the chiral polyol sugar d-sedoheptulose from
d-ribose in aqueous [BMIm][Cl] media was studied. Notably,
d-sedoheptulose is rare and, though commercially available,
very costly. In plants, the occurrence of free d-sedoheptulose
remains a matter of conjecture, in contrast to the well-docu-
mented roles of its mono- and bisphosphate esters.[56] In
humans, d-sedoheptulose is a key marker of various diseases
such as cystinosis, an autosomal recessive lysosomal storage
disease,[57] and transaldolase deficiency, a disorder of the car-
bohydrate metabolism with multisystem involvement discov-
ered recently.[58]
Previous studies[26–30] performed with TKs from yeast (TKyst)
and E. coli (TKeco) showed that TK accepts preferentially, as
non-natural acceptor substrates, (2R)a-hydroxylated aldehydes
with a short carbon chain (up to C4). The TK products are
d-threo ketoses (3S, 4R). The TK reaction has been used for the
synthesis of various compounds of biological interest, such as
amino alcohols,[31] thiosugars,[32] azasugars,[33] and phosphory-
lated sugars.[34,35] These properties lend TK a chemical synthetic
potential that makes it an attractive biocatalyst for further in-
dustrial applications. A thermostable TK from Geobacillus
stearothermophilus (TKgst) was recently identified and overex-
pressed, which offers an optimum temperature of 658C and
a thermostability of five days at 508C.[36] To broaden the sub-
strate specificity and modify the stereoselectivity, some inter-
[37–39]
esting TKeco
and TKgst variants[40] have been obtained. As
often reported with other enzymes, thermostability is associat-
ed with a high tolerance towards unusual media such as or-
ganic solvents and ILs.[41] In parallel, to further optimize the
process and to improve storage, thermal and/or pH stability of Results and Discussion
the enzyme, immobilization on supports is of great inter-
TKs in IL/water solution
est.[42,43] TK immobilization has been investigated on organic or
semiorganic supports, such as activated sepharose,[44] Eupergit-
In a preliminary step, the influence of different ILs based on
C, Amberlite XAD-7 and Nylon,[45–47] and polyacrylamide gel.[48]
imidazolium and anions such as ClÀ, CH3CO2À, BF4À, and am-
monium bromide on TK stability was tested (Figure 1a). Two
sources of TK, a new thermostable TK from G. stearothermophi-
lus[36] (TKgst) and TKeco, used commonly in biocatalysis, were
studied and compared. These TKs, as the other TKs described
in the literature, are dimeric enzymes and each individual mon-
omer is not active. The residual activities observed in different
aqueous buffered media that contained 0–60% of ILs are re-
ported in Figure 1a and b.
[49]
We have also reported the immobilization of TKyst
and
[50]
TKeco
on an inorganic support, layered double hydroxides
(LDHs). LDHs are synthetic materials with positively charged
brucite-like layers of mixed metal hydroxides that are de-
scribed by the abbreviated formula [M2+M3+A], in which M2+
and M3+ are divalent and trivalent metals, respectively, and A is
the interlayer anion that compensates for the positive charge
of the metal layers.[51,52] These layered compounds constitute
a support that is easy to prepare in a cost-effective way in soft
conditions suitable for biomolecule immobilization to maintain
the biological activity. The efficient immobilization of amino
acids, adenosine 5’-triphosphate (ATP), DNA, and many en-
zymes on LDH have been reported.[51,53]
Except for [BMIm][Cl], a drastic alteration of TKgst activity
was observed systematically. TKgst preserved its total activity at
up to 50% of [BMIm][Cl], whereas for TKeco, a progressive de-
crease in activity was observed above 20% of [BMIm][Cl]. Inter-
estingly, even if the incubation time of TKgst in 50% [BMIm][Cl]
was extended to 16 h, 80% of the enzyme activity was main-
tained. This resistance of TKgst to high concentrations of
[BMIm][Cl] can be correlated with the thermostable properties
of TKgst, which make it more robust and stabilize the dimer. A
The purpose of our study was to improve and extend the
applications of TK in biocatalysis to a broader series of sub-
strates and products under nonconventional conditions. To
this end, the effects of various ILs on both the stability and ac-
ChemCatChem 2015, 7, 3163 – 3170
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