Molecular design of oxidoreductases for the biosynthesis of carbohydrate-based industrial polyols

1
Molecular design of oxidoreductases for the
biosynthesis of carbohydrate-based industrial
polyols
Research Objectives
TmMtDH properties
Production of mannitol from glucose in an
electrochemical reactor combining TmMtDH with
Thermotoga neapolitana xylose isomerase
Research objectives Our two objectives in this
project are to develop industrial catalysts for
the enzymatic productions of mannitol and
sorbitol from glucose. Objective 1 Mannitol is
produced enzymatically from fructose by mannitol
dehydrogenase (MtDH) using NAD(P)H as the
cofactor. It is theoretically possible to
stoichiometrically convert glucose to mannitol in
a single electrochemical reactor containing both
immobilized thermostable MtDH and glucose
isomerase. While thermostable glucose isomerases
are commercially available, all known MtDHs are
mesophilic enzymes. Our goal in this project was
to clone a thermostable MtDH, characterize it,
and engineer it for industrial application.
Objective 2 Sorbitol can be produced from
fructose by sorbitol dehydrogenase (SDH), but it
can also be produced directly from glucose by
aldose reductase (AR). Because no gene was
identified in the genomes of hyperthermophiles
that potentially encodes an SDH or an AR, our
goals here were to express a fungal AR in
Escherichia coli, characterize the enzymes
catalytic and stability properties, and engineer
this AR for high activity on glucose and high
thermostability.
Effect of temperature on TmMtDH activity
TmMtDH is most active around 90C
Effect of pH on TmMtDH activity
First reactors run at 60C, pH 6.0, with 300 mM
glucose produce 130 mM mannitol in 5h. Why the
reaction stops halfway is being investigated.
Possible reasons include pH increase (up to 9.0).
TmMtDH optimally reduces fructose at pH 5.5, and
it optimally oxidizes mannitol at pH 8.5 (assays
performed at 80C)
Electrode design
Objective 2 Expression of the Candida boidinii
aldose reductase in Escherichia coli and enzyme
characterization.
Objective 1 Characterization of a thermostable
MtDH
Effect of temperature on TmMtDH kinetic
inactivation
In 100 mM phosphate buffer (pH 7.0), TmMtDH has
half-lives of 91 min at 75C, 57 min at 80C, 32
min at 85C, 15 min at 90C, and 6 min at 95C.
Of all protein sequences showing significant
similarity to Leuconostoc mesenteroides MtDH
(Genbank AAM09029), a single one was from a
hyperthermophile. Thermotoga maritima TM0298 is
annotated as an alcohol dehydrogenase. When
TM0298 was used as the query sequence for a
BLASTp search of GenBank, though, the best scores
against proteins of known function were against
the Lactobacillus mesenteroides and L. reuteri
MtDHs. TM0298 shares 31 identity and 52
similarity with these mesophilic MtDHs (see
below). For this reason, we cloned TM0298 to
characterize its substrate specificity.
C. Boidinii AR properties
Effect of pH on CbAR activity



CbAR kinetic parameters at 37C, pH 6.5
V max (unit/mg protein) Km (mM) Vmax / Km
Glucose 7.90 0.16 0.449 0.026 17.575
NADPH 7.68 0.23 0.05 0.004 153.6
NADH N.A. N.A. -
Xylose 38.7 0.59 0.054 0.004 716.67
NADPH 39.83 0.64 0.031 0.002 1284.84
NADH 0.5 0.18 2.78



Partial alignment of TM0298 with selected
dehydrogenases. LrMtDH L. reuteri MtDH (Genbank
AY090766) TmMtDH T. maritima MtDH (Genbank
TM0298) HLADH horse liver alcohol dehydrogenase
(Genbank P00328) TeSADH Thermoanaerobacter
ethanolicus secondary alcohol dehydrogenase
(Genbank U49975). Red residues involved in
catalytic Zn2 binding blue residues involved
in structural Zn2 binding green consensus
cofactor binding region.
TmMtDH zinc content
Approximate values
Zn2 in TmMtDH was titrated spectrophotometrically
with ?-hydroxymercuriphenyl sulfonate (PMPS) in
the presence of 4-(2-pyridilazo)resorcinol. The
?OD500 of 0.43 reached at the plateau corresponds
to 0.69 mol of Zn2/subunit of enzyme. This
result agrees with our atomic emission
spectroscopy results that gave mol/mol TmMtDH. a
Zn2 content of 0.73 Despite the fact that it
contains four cysteines that could be involved in
structural Zn2 binding, TmMtDH only contains a
single, catalytic Zn2. Zn2, Mn2, and Co2
restore full activity to the EDTA-treated TmMtDH.
?OD250, ?OD500
Expression and purification of TmMtDH
The T. maritima mtdh gene was cloned in pET24a.
From this construct, TmMtDH is expressed in
Escherichia coli at high level with a C-terminal
His-tag. TmMtDH has a specific activity of 85.2
unit/mg protein at 80C on fructose with NADH as
the cofactor (100 activity). It shows no
detectable activity on glucose, xylose,
threonine, acetaldehyde, or 2-butanone, but it
shows 18 activity on D-xylulose, 29 on
D-tagatose, and 5 on L-sorbose. In alcohol
oxidation assays, TmMtDH is active on mannitol,
but it shows no activity on sorbitol, xylitol,
ethanol, or 2-butanol.
Publications in preparation Hassler, B.L., Song,
S.H., Vieille, C., Zeikus, J.G., and R.M. Worden.
Coupling multiple enzymes to interfaces for
bioelectronic applications. In preparation. Puttic
k, P., C. Vieille, S.H. Song, M.N. Fodje, P.
Grochulski and L.T.J. Delbaere. Crystallization,
preliminary X-ray diffraction and structure
analysis of Thermotoga maritima mannitol
dehydrogenase. Submitted to Acta
Crystallog. Song, S.H., N. Ahluwalia, and C.
Vieille. Thermotoga maritima TM0298 is a highly
thermostable mannitol dehydrogenase. In
preparation.
Treatment Relative activity ()
Control 100
Plus 10 mM EDTA 3.5
Plus 20 mM metals ZnCl2 MnCl2 CoCl2 MgCl2 CaCl2 80.1 93.9 132.4 3.8 4.1
? Markers ? Soluble crude extract ? Heat-treated
extract ? TmMtDH after Ni-NTA column
This project was supported by the National
Research Initiative of the USDA Cooperative State
Research, Education and Extension Service, grant
number 2005-35504-16239.
TmMtDH is the first thermostable mannitol
dehydrogenase.