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PRBI-9803; No. of Pages9
ARTICLE IN PRESS
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the nonspecific adsorptions are limited, and the stability of the
biomolecule is usually improved. Recent examples, specifically on
alcohol dehydrogenases, are those reported by Bolivar et al. [13,18]
and Rocha-Martin et al. [19].
2.2. Expression and purification of His-HLADH-EE
purified as previously reported [38]. Crude protein concentration was determined
by Bradford protein assay dye reagent (Bio-Rad Laboratories GmbH, Germany)
with bovine serum albumin as a standard. Pure protein concentrations were deter-
mined by UV absorption at 280 nm using the absorbitivity reported in the literature
(0.441 mL/(mg cm) for His-HLADH-EE) [39].
a zinc-
containing enzyme, successfully used in biocatalysis [20–22].
Native HLADH is found in two isoforms, E and S, which leads in
vivo to the formation of a dimeric enzyme of mixed composition
(EE, ES and SS) [23]. While the E subunit is specific for ethanol, the
S subunit recognizes steroidal substrates. Following our interest in
the use of enzymes for the synthesis of bioactive molecules [24–28]
we applied HLADH to the enantioselective synthesis of (2S)-2-
arylpropanols starting from the parent racemic aldehydes via the
activation of an efficient dynamic kinetic resolution (DKR) process
[29–31]. A DKR process combines a selective kinetic resolution and
allows for the conversion of both enantiomers of the racemic sub-
strate into a single enantiomer of the product and overcomes the
50% yield limitation in classical kinetic resolution whenever the
racemization rate successfully competes with that of the resolution
reaction [32]. In this chemo-enzymatic reaction the commercial
HLADH preparation and a recombinant EE-enzyme were used with
similar results in enantioselectivities and yields. The products (2S)-
arylpropanols are useful intermediates in flavour manufacture and
(NSAIDs).
2.3. Immobilization of His-HLADH-EE
Sepabeads EC-EP/S (a commercially available rigid methacrylic polymer matrix
with diameter ranging between 100 to 300 m, activated with epoxy groups in a
ratio of 100 mol/g of wet resin) were derivatized with iminodiacetic acid (IDA) and
CoCl2 following the procedure reported in literature by Guisan, allowing a modifi-
cation of around 5% of the epoxy-groups in the support [17], 1 g of beads was shaken
at room temperature for 2 h in 2 mL of support modification buffer (0.1 M sodium
borate, 2 M iminodiacetic acid, pH 8.5). The derivatized resin, rinsed with double
distilled water, was then re-suspended in 5 mL of metal containing solution (0.05 M
sodium phosphate buffer pH 6.0, 1.0 M NaCl and 5 mg/mL of CoCl2) and shaken at
room temperature for 2 h. The resin, rinsed again with double distilled water, was
then put in contact with His-HLADH-EE (1 mg of enzyme per 1 g of resin, in storing
buffer pH 8.5, room temperature if not otherwise stated) and the mixture was gently
shaken at room temperature over 24 h. The resin was then thoroughly washed using
a desorption buffer (20 mM Na2HPO4-NaH2PO4, 50 mM EDTA, 0.5 M NaCl, pH 7.4, as
per IMAC purification procedure) [38] and water to achieve complete removal of the
cobalt first and of the residual EDTA after. The un-reacted epoxides were neutral-
ized using a blocking buffer (3 M glycine, pH 8.5, 4 mL per g of beads) over 20 hrs at
room temperature, with gentle shaking. The immobilized enzyme was thoroughly
washed and routinely stored in buffer (Tris–HCl, pH 8.5). To achieve immobilization
using different metals the following salts were used: NiCl2 × 6H2O, CuSO4 × 5H2O
(5 mg/mL, 5 mL for 1 g of beads, allowing a 2 hrs contact with the derivatized beads).
The metal solutions were prepared using buffer 50 mM Na2HPO4-NaH2PO4, pH 6.0.
Immobilization has been performed on purified enzyme, if not otherwise stated.
Native HLADH has been immobilized in the past onto different
supports with various degree of success by co-polymerization [34]
adsorption [35] and more recently by covalent linkage [36,37].
Recently, we reported on the production and characterization of
His(6)-tagged Horse Liver Alcohol Dehydrogenase (His-HLADH-EE)
[38].
Here we describe a method for its immobilization, which
took advantage of the increased metal affinity due to the poly-
histidine tag. The immobilized His-HLADH-EE (imHis-HLADH-EE)
thus obtained showed enhanced properties in terms of stability,
pH and temperature profiles, presence of organic co-solvents, and
reusability. Finally we report its application in the bio-reduction of
aliphatic and aromatic aldehydes; in particular it showed excellent
results in the enantioselective reduction of some racemic Profenals
yielding, with a complete unprecedented enantioselectivity, enan-
tiopure (S)-Profenols such as (S)-ibuprofenol, and (S)-naproxenol,
valuable intermediates in the synthesis of (S)-Ibuprofen and (S)-
Naproxen, benchmark drugs in the class of NSAIDs.
2.4. Activity assays
Spectrophotometric activity measurements were based on the substrate-
dependent absorbance change of NADH at 340 nm and routinely done in reaction
mixtures (1 mL for the soluble enzyme and 5 mL for the immobilized) at 25 ◦C, using
a Varian Cary 50 Scan UV-visible spectrophotometer equipped with a Cary single
cell Peltier temperature controller. For the immobilized enzyme the reaction mix-
ture was shaken at 25 ◦C, 250 rpm and the absorbance at 340 nm was recorder every
minute as single readings. To test the recyclability of the enzyme after each cycle
(9 min in total duration) the resin was washed thoroughly with buffer and a new
reaction was set up. Unless otherwise stated, the reaction mixture for the oxidative
step contained NAD+ (1 mM), ethanol (4 mM), enzyme sample (appropriate amount)
and up to 1 mL of 0.1 M sodium pyrophosphate buffer, pH 8.8. The buffer was equil-
ibrated at 25 ◦C prior to the assay. One unit of HLADH corresponded to the amount
of enzyme required to reduce 1 mol of NAD+ per min at 25 ◦C. For the pH stabil-
ity test the following buffers were used: 50 mM Na2HPO4–NaH2PO4 buffer pH 6.5,
50 mM Tris–HCl pH 8.5. To investigate the optimum pH of reaction the following
buffers were used: 50 mM Na2HPO4–NaH2PO4 buffer pH 6.5, 7.5, 50 mM Tris–HCl
buffer pH 8.5, 0.1 M pyrophosphate buffer or 50 mM Glycine–NaOH buffer pH 8.8,
50 mM Glycine–NaOH buffer pH 9.5, 10.5, 50 mM Na2HPO4–NaOH buffer pH 11.0,
11.5, 50 mM Glycine–NaOH buffer, 50 mM KCl–NaOH buffer pH 12.5.
A pH 9.5 was used to investigate the optimum reaction temperature for the
oxidation of EtOH (4 mM) to acetaldehyde and temperatures between 25 ◦C and
55 ◦C were tested. The pH values of the buffers were always adjusted at the tem-
perature at which the experiment was carried out. To test the stability at different
solvents the enzymes were incubated in 10 (free enzyme) and 20% (free and immo-
bilized enzymes) of solvent (CH3CN, THF, DMSO and methanol were used) in 50 mM
Tris–HCl, pH 8.5 buffer with a total volume of 0.2 mL in all cases. For the immobi-
lized enzyme, 50 mg of resins were used in each sample, while the free enzyme
was always tested in a concentration of 0.7 mg/mL. The activity was recorded at
time zero and after 24 h in the usual conditions. To test the inactivation upon con-
tact with metals, the free enzyme was incubated at room temperature for 24 h in
a 3.5 mM metal solution which is the equivalent ratio of metal to enzyme in the
immobilization step. Samples with different metals (CoCl2, NiCl2 and CuSO4) were
checked for activity over 24 h and compared to a control sample.
2. Materials and methods
2.1. Materials, strains, vectors and culture conditions
All chemical reagents, unless otherwise stated, were purchased as analytical
grade from Sigma–Aldrich or TCI and used without further purification. Aldehydes
were commercial or prepared as reported in ref. [31]. Alcohols were commer-
cial or prepared from the corresponding aldehydes by reduction with NaBH4
in methanol. All spectra were consistent with reported data. NAD+ was pur-
chased from Apollo Scientific Ltd, Stockport, U.K. Staining and de-staining was
performed using the Stain/DeStain-Xpress protein detection kit (Enzolve Tech-
nologies, Ltd., Ireland). Escherichia coli BL21 competent cells were purchased
from Novagen (Germany). Transformed E. coli strains were generally cultured in
Luria–Bertani (LB) agar and in LB broth, both containing ampicillin (100 g/mL)
at 37 ◦C, shaking at 250 rpm. E. coli strains harbouring a pRSETb-EqADH-E vec-
tor were cultured in PG (minimal media) agar and in Auto Induction broth, both
containing ampicillin (100 g/mL) at 37 ◦C, shaking at 250 rpm. Sepabeads EC-
EP/S were kindly donated by Resindion SRL (Binasco, Milan, Italy). Morphological
2.5. General procedure for enzymatic reduction of aldehydes
2.5.1. Synthesis
Method A: into a vial stirred on an orbital shaker (140 rpm) at room temperature,
all reagents were added in the following order: 0.5 mL of a 5 mM solution of the
starting aldehyde in CH3CN, 0.146 mL of EtOH (0.5 M), 0.5 mL of a 0.1 mM solution
of NADH freshly prepared in the appropriate 0.1 M buffer, then 0.1 M buffer to reach
a total final volume of 5 mL and the chosen amount of enzyme.
investigation was carried out with
scope operating at 15 kV coupled with Energy-Dispersive X-ray Spectrometer.
Samples were air-dried, then sputter-coated with carbon 60 s prior to examina-
tion.
a Philips XL-20 Scanning Electron Micro-
Please cite this article in press as: Quaglia D, et al. His-tagged Horse Liver Alcohol Dehydrogenase: Immobilization and application in the bio-based
Quaglia, Daniela
