Báo cáo khoa học: Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide ppt

Directed evolution of formate dehydrogenase from
Candida boidinii for improved stability during entrapment
in polyacrylamide
Marion B. Ansorge-Schumacher
1
, Heike Slusarczyk
2
, Julia Schu
¨
mers
1
and Dennis Hirtz
1
1 Department of Biotechnology, RWTH Aachen University, Germany
2 Institute of Enzyme Technology, Heinrich-Heine-University Du
¨
sseldorf, Research Center Ju
¨
lich, Germany
Entrapment in polymeric matrices has long since
become an important technique to improve recycling,
handling, and mechanical strength of delicate biocata-
lysts during application in large-scale organic synthesis.
It also enables reactions with otherwise instable or
cofactor-dependent enzymes in organic solvents [1,2]
and can thus enlarge the scope of industrial biocataly-
sis. However, while entrapment matrices can in princi-
ple be formed from many monomeric or polymeric,
natural or synthetic compounds [3], only a few are
strong enough to withstand the chemical stress and
mechanical forces in a technical process. Very suitable

properties can be found in polyacrylamide (PAA) gels
which combine stability under almost all relevant
reaction conditions [4] with high elasticity and low
abrasion in stirred-tank reactors [5,6]. The network
structure of PAA can easily be adapted to ensure opti-
mal retention of any biocatalyst [4], while no ionic
interaction with entrapped enzymes occurs [7,8]. In
spite of this, PAA gels have rarely been applied as
entrapment matrices for biocatalysts [9] because of the
detrimental effect that the entrapment process can
exert on the activity of enzymes [5,6,10,11]. An exact
explanation for this effect is not known to date. How-
ever, irreversible changes of amino acid residues caused
by some of the compounds participating in matrix for-
mation are indicated [12–16].
In this study, we explored the possibility to increase
the stability of isolated enzymes during entrapment in
PAA gels. This was done by means of error-prone
PCR and a screening method which employed selected
components of PAA gels instead of the gel itself.
As an investigation system, formate dehydrogenase
[(FDH) E.C.1.2.1.2] from Candida boidinii was chosen
for its outstanding importance as a cofactor regener-
ation system in biocatalyzed syntheses [17].
Keywords
directed evolution; entrapment; formate
dehydrogenase; polyacrylamide; stabilization
Correspondence
M. B. Ansorge-Schumacher, Department of
Biotechnology, RWTH Aachen University,

D-52056 Aachen, Germany
Fax: +49 241 8022387
Tel: +49 241 8026620
E-mail:
Website: />biokat
(Received 4 April 2006, revised 19 June
2006, accepted 26 June 2006)
doi:10.1111/j.1742-4658.2006.05395.x
In two cycles of an error-prone PCR process, variants of formate dehy-
drogenase from Candida boidinii were created which revealed an up to
4.4-fold (440%) higher residual activity after entrapment in polyacrylamide
gels than the wild-type enzyme. These were identified in an assay using sin-
gle precursor molecules of polyacrylamide instead of the complete gel for
selection. The stabilization resulted from an exchange of distinct lysine,
glutamic acid, and cysteine residues remote from the active site, which did
not affect the kinetics of the catalyzed reaction. Thermal stability increased
at the exchange of lysine and glutamic acid, but decreased due the
exchange of cysteine. Overall, the variants reveal very suitable properties
for application in a technical synthetic process, enabling use of entrapment
in polyacrylamide as an economic and versatile immobilization method.
Abbreviations
APS, ammonium peroxodisulfate; FDH, formate dehydrogenase; PAA, polyacrylamide; TEMED, N,N,N¢,N¢-tetramethylethylenediamine.
3938 FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS
Results and Discussion
The success of error-prone PCR in the directed evolu-
tion of biocatalysts strongly depends on the screen
applied to identify improved properties. Best results
are usually obtained when screening conditions resem-
ble the conditions during application as closely as
possible [18]. Consequently a screen involving entrap-

ment of FDH-variants in PAA would provide most
suitable conditions for the identification of variants
with an improved stability towards entrapment in this
matrix. Because of the high demands of this method in
terms of materials and time, and the inapplicability of
simple optical assays for the determination of activity
in opaque gel matrices, such an approach was not feas-
ible in this study. An alternative screen exerting the
effects of gel formation without employing gel forma-
tion itself was therefore developed.
Effects of PAA building blocks on wild-type FDH
Formation of PAA gels involves the cross-linking of
acrylamide and bis-acrylamide monomers in a radical
polymerization process employing ammonium peroxo-
disulfate (APS) and N,N,N¢,N¢-tetramethylethylene-
diamine (TEMED) as radical-forming agent and
enhancer, respectively [19,20]. The acrylamide concen-
tration for the entrapment of enzymes can range
between 5% (w ⁄ v) [10] and 30% (w ⁄ v) [21], depending
on the required network density [4].
When FDH was entrapped in 8% (w ⁄ v) PAA, the
activity of the immobilisates was only $10% of the
activity that had been expected from the amount of
enzyme introduced into the matrix. This was in accord-
ance with the many reports on the severe deactivation of
enzymes during entrapment in PAA [5,6,10,11]. The
incubation of FDH with only one or two building com-
pounds of PAA, allowing no polymerization, demon-
strated that this deactivation was to a large extent a
result of the mere presence of monomers and auxiliaries

(Table 1). Compared with the enzyme stored in plain
potassium phosphate buffer (pH 7.5), the half-life of
wild-type FDH (wt-FDH) in 8% (w ⁄ v) acrylamide (the
term ‘acrylamide’ always referring to a mixture of acryl-
amide and bis-acrylamide in a ratio of 37.5 : 1)
decreased by 91.8% to 111 min. While this observation
was still in accordance with the deactivation of enzymes
by acrylamide monomers reported by Dobryszycki et al.
[22,23], it was also observed that the deactivation of
FDH was enhanced when acrylamide was combined
with 0.1% (w ⁄ v) of APS or TEMED, decreasing half-
life by 96 and 97.2% to 45 and 38 min, respectively. The
half-life of FDH was also heavily affected in a mixture
of APS and TEMED, while neither of these components
alone exerted a strong effect on stability. This indicates
a cooperation of the compounds in the deactivation of
enzymes independent of the actual polymerization pro-
cess. With regard to the identification of FDH variants
with a higher stability towards the entrapment in PAA,
this finding means that complete gel formation is not
required for successful screening, but employment of as
many building blocks and auxiliaries as possible is
favourable.
FDH variants with improved stability in solutions
of acrylamide/TEMED
Error-prone PCR applied to the wt-FDH gene at a
Mn
2+
concentration of 0.05 mmolÆL
)1

yielded about
3500 recombinant clones, 70% of which expressed act-
ive FDH. This was estimated from a qualitative activ-
ity staining on agar plates. For quick identification of
active FDH variants with improved stability towards
PAA entrapment, their residual activity after 30 min of
incubation in a buffered solution of 8% (w ⁄ v) acryla-
mide and 0.1% (w⁄ v) TEMED in relation to their
activity in plain buffer was determined. The composi-
tion of this activity screen was based on the findings
about the effect of PAA building blocks on wt-FDH
described above. The incubation time was chosen on
the assumption that PAA formation is usually comple-
ted within 30 min [5,6,11], i.e. the presence of devasta-
ting monomers becomes negligible after this time.
Among 1764 FDH variants, three were considerably
less affected by acrylamide and TEMED than
wt-FDH. The residual activity of one of these variants
was 70%, the other two revealed a residual activity of
90%. Related to wt-FDH, which had a residual activ-
ity of only 44% under the same conditions, this was
an improvement of 59 and 105%, respectively. The
half-life of FDH increased from 38 min to 104 min
(2.7-fold), 150 min (3.9-fold), and 154 min (4.1-fold),
respectively. The mutations underlying these improve-
ments are given in Table 2.
Table 1. Half-life (t
1 ⁄ 2
) of wt-FDH in the presence of compounds
involved in PAA formation. The term ‘acrylamide’ refers to a mix-

ture of acrylamide and bis-acrylamide in a ratio of 37.5 : 1.
Composition t
1 ⁄ 2
(min)
Buffer 1346
0.1% APS 763
0.1% TEMED 890
0.1% APS, 0.1% TEMED 84
8% acrylamide 111
8% acrylamide, 0.1% APS 54
8% acrylamide, 0.1% TEMED 38
M. B. Ansorge-Schumacher et al. Stabilisation of FDH for entrapment
FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS 3939
The considerable stabilization of FDH-K35T
towards deactivation in the presence of acrylamide
and TEMED by replacement of a lysine residue in
position 35 confirms the findings of Cavins and
Friedman [12] and Bordini et al. [16], who reported
an interaction of acrylamide with the e-amino func-
tion of lysine residues. Even more effective was the
replacement of glutamic acid in position 53, which
was detected in both the variants with the best resid-
ual activity. This was an interesting result, as gluta-
mic acid had not been recognized as a special target
of acrylamide or TEMED before. It was also surpri-
sing that such good results were obtained with valine
as replacement for glutamic acid, given that Perez
et al. [15] had found a strong interaction between
valine residues and acrylamide. Possibly, an even
higher stability towards acrylamide would be created

if this randomly inserted valine was exchanged for a
more inert amino acid by site-directed mutagenesis.
In contrast, no obvious effect on stability was
achieved when additional to the replacement of
glutamic acid in position 53, a lysine residue in posi-
tion 56 was replaced by arginine. As the 3D struc-
ture of FDH from C. boidinii has not been solved to
date, the distinct locations of the mutations were not
directly accessible. However, homology modelling
according to Slusarczyk et al. [26], implies that all
mutations are peripheral to the protein and remote
from the active site. A salt bridge that is probably
formed by the residues E53 and K56 in the wild-
type FDH would be destroyed in the variants FDH-
E53V and FDH-E53V ⁄ K56R. As a consequence, the
flexibility of the enzyme could be increased.
The gene coding for variant FDH-E53V ⁄ K56R was
used as template for a second error-prone PCR, con-
sidering that the replacement of the lysine residue had
no negative effect on the residual activity of FDH in
the presence of acrylamide and TEMED, and might
exert a positive effect under reinforced screening condi-
tions. This second error-prone PCR induced a higher
mutation rate by using a Mn
2+
concentration of
0.15 mmolÆL
)1
[24,25] and yielded 50% of recombinant
clones expressing active FDH. For identification of

FDH variants with further improved stability towards
PAA entrapment, the concentration of acrylamide in
the activity screen was increased to 15% (w ⁄ v). At this
concentration, the template FDH-E53V ⁄ K56R had a
residual activity of 22%.
Among 1092 FDH-variants of the second genera-
tion, again three with improved stability were found.
All of them exerted an activity of 120% when incuba-
ted in 15% (w ⁄ v) acrylamide ⁄ 0.1% (w ⁄ v) TEMED
(Table 2), which was an improvement of 5.5-fold
compared with the template FDH-E53V ⁄ K56R. The
half-life of these variants in a solution of 8% (w ⁄ v)
acrylamide and 0.1% (w ⁄ v) TEMED was about
1600 min, which was 42.1-fold more than that of wt-
FDH (38 min). In all three variants, this considerable
improvement was caused by an identical mutation, the
exchange of cysteine in position 23 for serine. Consid-
ering that Chiari et al. [13] observed an at least two-
fold stronger effect of acrylamide on cysteine than on
other amino acids, this is quite intelligible. It is an
interesting result, however, that the randomly inserted
mutation C23S should be identical to the one that
Slusarczyk et al. [26] had identified as being most
effective for stabilizing FDH during technical applica-
tion. This finding indicates that the cysteine residue in
this position might play a general role in FDH-stabil-
ity. It is again a residue located rather at the periphery
of the enzyme molecule than anywhere near the active
site [26].
Biochemical properties of mutant FDHs

Before investigating the performance of the new
FDH-variants in PAA, their overall suitability for
application was checked by comparing their kinetics
and thermal stability to the properties of wt-FDH.
The results of this study are summarized in Table 3. It
should be noted that discrepancies of the values pre-
sented herein for wt-FDH from formerly published
data is due to the use of a different analysis software:
In contrast to scientist for windows, which was
used by Slusarczyk et al. [26], excel for windows,
employed herein, takes into account the inhibition
Table 2. Characteristics of FDH variants with increased activity
towards acrylamide (AA) ⁄ TEMED. Percentage values of half-life
(t
1 ⁄ 2
) are related to the half-life of wt-FDH under the same condi-
tions (38 min, see Table 1).
Codon
exchanges
8% AA ⁄ 0.1%
TEMED
Fold
increase
Residual
activity t
1 ⁄ 2
(min)
Variants of first generation
K35T aaa fi aca 70% 104 2.7
E53V gaa fi gta 90% 150 4

E53V ⁄ K56R gaa fi gta 90% 154 4.1
aaa fi aga
Variants of second generation
E53V ⁄ K56R ⁄ C23S gaa fi gta 120%
a
1600 42
aaa fi aga
tgt fi agt
a
Residual activity was measured in 15% AA ⁄ 0.1% TEMED.
Stabilisation of FDH for entrapment M. B. Ansorge-Schumacher et al.
3940 FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS
constants for formate and NAD
+
and thus leads to
slightly different, but probably more exact results.
It was found that none of the mutations in FDH
exerted a considerable effect on its kinetics. FDH-
E53V ⁄ K56R and FDH-E53V ⁄ K56R ⁄ C23S had a
slightly decreased K
M
of formate, while K
M
of
NAD
+
increased slightly in FDH-E53V ⁄ K56R ⁄ C23S.
Inactivation by NADH remained almost unchanged
in all variants, and temperature optima were in the
same range as of wt-FDH. The same holds true for

the inactivation temperature. Thus, the catalytic
properties of all FDH-variants are comparable to
those of wt-FDH.
Thermal half-life of all three first-generation FDH-
variants at 50 °C increased by 12–27% compared
with wt-FDH. The best improvement was found in
FDH-E53V ⁄ K56R, which had also revealed the best
stability towards the presence of acrylamide ⁄
TEMED. Thus, the possibly higher molecular flexi-
bility of this variant due to the disruption of a per-
ipheral salt bridge (see above) had no negative
influence on the thermostability of the enzyme. In
contrast to the stabilization towards acrylamide ⁄
TEMED, however, thermal stabilization was slightly
affected by the exchange of lysine in position 56 for
arginine. This can be concluded from the additional
improvement in thermostability of FDH-E53V ⁄ K56R
compared to FDH-E53V (Table 3). Mutation C23S,
which dominated the second generation of FDH var-
iants and had a highly beneficial effect regarding the
stability in acrylamide ⁄ TEMED, had a detrimental
effect on thermal half-life of the enzyme. Compared
with wt-FDH, half-life decreased by 2.5-fold; further-
more, in the template enzyme, FDH-E53V ⁄ K56R the
decrease was 3.1-fold. This result is in accordance to
the findings of Slusarczyk et al. [26], who observed a
decrease in half-life of their mutant FDH-C23S of
80% at 50 °C, and confirms the special relevance
of C23 for FDH stability. The better performance of
our FDH variant compared with FDH-C23S is obvi-

ously a result of the stabilizing effects of the addi-
tional mutations E53V and K56R which were
introduced during the first step of evolution.
Stability of mutant FDHs in PAA
Finally, the performance of the FDH variants after
entrapment in PAA was investigated by measuring the
activity in comparison to equally entrapped wt-FDH.
For entrapment, acrylamide concentrations of 8% (w ⁄ v)
as well as 15% (w ⁄ v) were used to ensure a close rela-
tionship to the formerly employed screening conditions.
However, the concentrations of TEMED and APS in
the polymerization mix had to be increased to 1% (w ⁄ v)
and 5% (w ⁄ v), respectively, in order to obtain stable
and reproducible gel beads within a polymerization time
of 30 min. Activity was measured after a reaction time
of 60 min.
The FDH variants of both generations had a clearly
increased residual activity after entrapment in PAA
compared to wt-FDH (Fig. 1). The improvement was
more pronounced in the variants of the second evolu-
tionary step, and the difference in stability between
variants from the first and second generation increased
with increasing concentration of PAA. These findings
were in very good accordance with the behaviour of
the variants under screening conditions. Of course,
based on the observed half-lives in a solution of 8%
Table 3. Kinetic constants, temperature optima and half-life of FDH-variants.
FDH variant
K
M,formate

(mmolÆL
)1
)
K
M,NAD
(lmolÆL
)1
)
K
I,NADH
(lmolÆL
)1
)T
opt
(°C) T
m
(°C)
t
1 ⁄ 2
at 50 °C
(min)
wt-FDH 5.9 50 4 60 62 365
FDH-K35T 6.2 49 5 55 58 408
FDH-E53V 5.2 49 3 55–60 55 433
FDH-E53V ⁄ K56R 5.0 52 8 60 60 462
FDH-E53V ⁄ K56R ⁄ C23S 4.5 58 5 55–60 59 148
Fig. 1. Activity of wt-FDH and FDH-variants in 8% (w ⁄ v) PAA (grey
columns) and 15% (w ⁄ v) PAA (black columns). The activity was
determined after 1 h of reaction at 30 °C, activity of wt-FDH was
set to 100% after determination in the respective gel.

M. B. Ansorge-Schumacher et al. Stabilisation of FDH for entrapment
FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS 3941
acrylamide and 0.1% TEMED, a lower activity of
FDH-K35T and a more similar performance of vari-
ants FDH-E53V and FDH-E53V ⁄ K56R in PAA had
been expected. The differences could be explained by
inaccuracies in determination of CO
2
. Also, however,
it is possible that the presence of APS, the increased
concentrations of APS and TEMED, and the polymer-
ization process itself affect the FDH variants in differ-
ent ways, depending on their respective mutations.
Indeed, an additional effect of these factors is indica-
ted by the overall lower stabilization of all variants in
PAA than in polyacrylamide ⁄ TEMED.
Conclusions
In principle, screening with a mixture of acrylamide and
TEMED turned out to be well suited to resembling the
deactivating forces of PAA formation on FDH, and
thus enable the identification of variants with consider-
ably improved stability. Thus, the adaptation of syn-
thetically valuable enzymes for a technically and
economically reasonable immobilization in PAA is now
possible. Further improvements could probably be
achieved when additional combinations of PAA forming
compounds, such as acrylamide ⁄ APS or TEMED ⁄ APS,
and higher concentrations of both APS and TEMED
were employed. Also, combinations of a variety of
favourable mutations by in vitro recombination methods

such as DNA-shuffling [27] or staggered extension [28]
could lead to considerably improved stability.
Experimental procedures
Materials
Buffer salts and chemicals were of analytical grade and pur-
chased from Fluka (Neu-Ulm, Germany), Roth (Karlsruhe,
Germany), or Merck (Darmstadt, Germany). Restriction
enzymes, DNA-modifying enzymes, and dNTPs were
obtained from Roche Diagnostics (Mannheim, Germany).
Markers for DNA and protein analysis, and PCR buffer
were purchased from Invitrogen (Karlsruhe, Germany).
Error-prone PCR and cloning
Ten nanograms of the expression plasmid pBTac-FDH [26]
and 20 pmol of each of the primers pBTacF1 (5¢-TG
CCTGGCAGTTCCCTACTC-3¢) and pBTacR2 (5¢-CGA
CATCATAACGGTTCT GG-3¢) were added to a mixture
of 10 lL mutagenesis buffer [0.1 molÆL
)1
Tris ⁄ HCl, pH 8.3;
0.5 molÆL
)1
KCl, 70 mmolÆL
)1
MgCl
2
, 0.1% (w ⁄ v) gelatine],
0.05 or 0.15 mmolÆL
)1
of MnCl
2

, and 10 lL of dNTP-mix
(10 mmolÆL
)1
dCTP, 10 mmolÆL
)1
dTTP, 2 mmolÆL
)1
dATP, 2 mmolÆL
)1
dGTP), 99 lL double distilled water and
1 lL (1 U) Taq-polymerase. Amplification was conducted in
a Biometra 500 PCR-cycler (Biometra, Go
¨
ttingen, Germany)
applying one cycle at 95 °C for 5 min, 25 cycles of 5 min at
94 °C, 1 min at 50 °C, and 1 min at 72 °C each, and at last
one cycle at 72 °C for 5 min. The PCR fragments were puri-
fied, ligated into expression vector pBTac2 and transformed
into Escherichia coli JM101. The analysis of the nucleotide
sequence was done by Sequiserve (Vaterstetten, Germany).
Pre-selection of active clones
Colonies of E. coli were fixed on agar plates by overlaying
them with a solution of agar (1.6% w ⁄ v in 0.1 molÆL
)1
potas-
sium phosphate buffer, pH 7.5, containing 0.2% v ⁄ v Triton-
X-100 and 10 mmolÆL
)1
EDTA) at a maximum temperature
of 65 °C. When the layer of agar had cooled down to room

temperature and became solid, the plates were treated three
times with a solution of 0.2% (v ⁄ v) Triton-X-100 and
10 mmolÆL
)1
of EDTA in potassium phosphate buffer
(0.1 molÆL
)1
, pH 7.5) and another three times with potas-
sium phosphate buffer (0.1 molÆL
)1
, pH 7.5). Each treatment
was performed for 10–15 min. The plates were then overlaid
with a first dyeing solution (1.25 molÆL
)1
sodium formate,
0.2 gÆL
)1
phenazinethosulfate, and 2 gÆL
)1
nitrotetrazolium-
blue chloride in 0.1 molÆL
)1
of potassium phosphate buffer,
pH 7.5) and incubated in the dark for 10 min. A second dye-
ing solution (50 mmolÆL
)1
NAD
+
in 0.1 molÆL
)1

potassium
phosphate buffer, pH 7.5) was added in a ratio of 1 : 100
(v ⁄ v) and the plates were gently moved in the dark until vio-
let spots became visible. These spots marked colonies expres-
sing active FDH. The dyeing mixture was removed, the
plates were washed with water and left to dry.
Determination of FDH activity
Activity of native FDH was determined in a spectropho-
tometer (Beckmann DU
Ò
series; Beckmann, Fullerton, CA,
USA) or in a microtitre plate reader (ThermoMax; Molecu-
lar Devices, Ismaning, Germany) at 340 nm and 30 °C. For
measurements in the spectrophotometer, the assay mixture
contained 0.25 molÆL
)1
sodium formate and 1.7 mmolÆL
)1
NAD
+
in potassium phosphate buffer (0.1 molÆL
)1
,
pH 7.5). For measurements in the microtitre plate reader,
the assay mix was composed of 0.6 molÆL
)1
sodium formate
and 3.6 mmolÆL
)1
NAD

+
in 0.1 molÆL
)1
potassium phos-
phate buffer (pH 7.5). The reaction was started by adding
FDH and was monitored over 2 min. From the increase in
extinction, activity was calculated using the equation
A½U=ml¼
DE Ã V
total
V
enzyme
à e à d
with DE being the extinction increase within 1 min, V
total
the total volume of the assay mixture, V
enzyme
the volume
Stabilisation of FDH for entrapment M. B. Ansorge-Schumacher et al.
3942 FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS
of added enzyme solution, d the layer thickness of the
cuvette and e the extinction coefficient of NADH + H
+
(6.22 mLÆlmol
)1
Æcm
)1
). One unit is defined as the amount
of enzyme that catalyses the reduction of 1 lmol of NAD
+

per min at pH 7.5 and 30 °C.
Screening for improved FDH stability
E. coli colonies expressing active FDH were transferred from
agar plates into 1.2 mL LB medium (containing 100 lgÆmL
)1
ampicillin) in 96-well plates and incubated at 37 °Cona
rotary shaker. When OD
550
reached 0.4, expression of FDH
was induced by addition of 50 lL isopropyl thio-b-d-galacto-
side (20 mmolÆL
)1
) and the cultivation proceeded for 4 h.
Afterwards, the cells were harvested and cell lysis was per-
formed in 0.1 molÆL
)1
potassium phosphate buffer (pH 7.5)
with 0.1% (v ⁄ v) Triton-X-100 and 0.2 molÆL
)1
EDTA. The
cell debris was removed by centrifugation and 35 lLof
the resulting crude extract, which was of comparable FDH
activity for all variants, were transferred into a 96-well micro-
titre plate, mixed with 35 lL acrylamide (16% w ⁄ v and 30%
w ⁄ v, respectively) and TEMED (0.2% w ⁄ v) in potassium
phosphate buffer (0.1 molÆL
)1
, pH 7.5), and incubated at
30 °C for 30 min 70 lL FDH-assay mix for microtitre plate
readers were then added and the extinction at 340 nm was

monitored at 30 °C. The initial performance was determined
by replacing the acrylamide ⁄ TEMED solutions by 35 lL
potassium phosphate buffer (0.1 molÆL
)1
, pH 7.5). The resid-
ual activity was calculated by dividing activity after incuba-
tion in acrylamide ⁄ TEMED by the activity after incubation
in buffer.
For determination of half-life, the activity of crude
extracts was determined after incubation in the desired mix-
tures of acrylamide, TEMED, and APS at 30 °C for
5–180 min, according to the protocol described above.
The measured inactivation was adapted to the time law
A ¼A
0
*e
()kt)
, with A
0
being the activity after incubation
time t and k the inactivation constant. The half-life was
then calculated using the equation s ¼ ln 2/k.
Expression and purification of FDH
Recombinant E. coli were cultivated at 37 °C in LB med-
ium containing 100 lgÆmL
)1
of ampicillin. Expression was
induced by addition of 0.5 mmolÆL
)1
isopropyl thio-b-d-

galactoside when the OD
620 nm
was about 0.5. between 6
and 8 h after induction, the cells were harvested by centrif-
ugation, resuspended in potassium phosphate buffer
(0.1 molÆL
)1
, pH 7.5) to give a concentration of 50%
(w ⁄ v), and PEG 400 was added to a final concentration of
30% (w ⁄ v). This solution was then mixed and incubated at
37 °C for 2 h. The resulting crude extract was cooled down
to 20 °C and H
2
O and K
2
HPO
4
were added to final con-
centrations of 21% (w ⁄ w) and 5% (w ⁄ w), respectively.
After complete dissolution of K
2
HPO
4
, PEG 1550 and
NaCl were added to final concentrations of 7% (w ⁄ w) and
6% (w ⁄ w), respectively, and the solution was stirred for
30 min. The solution separated into two phases within a
settling time of 2 h. The upper phase of the system was
removed and mixed with PEG 6000 and H
2

O at final con-
centrations of 20% (w ⁄ w) and 10% (w ⁄ w), respectively.
FDH precipitated from this solution after 2–3 h, was collec-
ted by centrifugation, and redissolved in a 1 : 1 (v ⁄ v) solu-
tion of potassium phosphate buffer (0.1 molÆL
)1
, pH 7.5)
and glycerine. It was stored at )20 °C until use.
Determination of kinetic constants
Kinetic constants were derived from duplicate measure-
ments of initial velocities under conditions where only one
substrate (formate or NAD
+
) was limiting. The data
obtained were fitted to the mathematical model of a dou-
ble-substrate kinetic given below, using exel for windows
(Micromath. Inc., Salt Lake City, UT, USA) for analysis.
In this model, v
0
describes the initial velocity of the reac-
tion, v
max
the maximum reaction rate, [x] the concentration
of the two substrates, K
Mx
the Michaelis–Menten constant of
substrate x, and K
INADH2
the inhibition constant of NADH
2

.
All experiments were performed with purified enzyme.
Measurement of temperature influences
For determination of T
m
(midpoint of thermal inactiva-
tion), inactivation experiments were carried out by incuba-
ting purified FDH in potassium phosphate buffer
(0.1 molÆL
)1
, pH 7.5) at different fixed temperatures
between 30 °C and 71 °C for 20 min and then assayed for
residual activity and protein concentration. The tempera-
ture at which the residual activity was 50% was defined as
T
m
and was calculated from the point of inflection in a plot
of residual activity at different temperatures versus the
respective temperatures. Additionally, thermal inactivation
of FDH at 50 °C was monitored until inactivation reached
80%. From the time course, half-life at 50 °C was calcula-
ted analogous to the calculation of half-life in the presence
of acrylamide. Temperature optima were determined by
plotting the initial velocity of FDH activity at temperatures
between 15 and 80 °C versus the temperature.
Formation of PAA immobilisates
A solution of 8% (w ⁄ v) or 15% (w ⁄ v) acrylamide (concen-
tration of bis-acrylamide: 2.67% w ⁄ w) in potassium phos-
phate buffer (0.1 molÆ L
)1

, pH 7.5) was degassed for 15 min
1% TEMED and 5% APS were added, and the mixture
was dropped into silicone oil (M50; Roth, Karlsruhe, Ger-
many). Polymerization took place at 15 °C within 30 min.
The average bead volume was 7.4 ± 2.5 mm
3
. For calibra-
tion of the CO
2
measurement, solutions of NaHCO
3
were
M. B. Ansorge-Schumacher et al. Stabilisation of FDH for entrapment
FEBS Journal 273 (2006) 3938–3945 ª 2006 The Authors Journal compilation ª 2006 FEBS 3943
added before polymerization. For entrapment of FDH,
0.33 UÆmL
)1
of the purified enzyme were included before
polymerization and the beads were equilibrated in a solu-
tion of 50 mmolÆL
)1
NAD
+
in potassium phosphate buffer
(0.1 molÆL
)1
, pH 7.5) for 16 h after the polymerization pro-
cess was complete.
Determination of activity of entrapped FDH
The activity of entrapped FDH was determined by monit-

oring the formation of CO
2
. For this, 1 g PAA beads and
3 mL hexane were placed into an 8 mL GC-vial, which
thereafter was sealed with a rubber cap and left to equili-
brate to a temperature of 30 °C for 30 min. The reaction
was started by injecting 100 lmol of formate into the vial
and stopped after 60 min by injecting 1 mL of HCl
(1 molÆL
)1
). After another 30 min of incubation, a gas sam-
ple of 100 lL was withdrawn from the headspace with a
gas-tight syringe, injected into a GC ⁄ HCD (Perkin Elmar,
Connecticut, USA) and analysed isothermally at 40 °C
(detector temperature at 140 °C; injector temperature at
65 °C; carrier gas helium 5.0 at 62.5 mLÆmin
)1
) on a CTR-
1-column (Alltech, Munich, Germany). The retention time
of CO
2
under this conditions was 1.25 min. The peak areas
obtained were converted into CO
2
content by use of the
calibration curve illustrated in Fig. 2. This calibration was
obtained by entrapping defined concentrations (1–60 mmolÆ
L
)1
) of NaHCO

3
in PAA beads instead of FDH and per-
forming the same procedure as described for the FDH
immobilisates. The measured peak area was related to the
known concentrations of CO
2
resulting from the quantita-
tive transformation of NaHCO
3
into CO
2
at acidic pH.
Acknowledgements
We thank PD Dr. A. Eisentra
¨
ger and Dipl Ing. C.
Grundke, both at the Institute of Hygiene and Envi-
ronmental Medicine, Klinikum Aachen, for provision
of the gas chromatograph and expert help with the
determination of CO
2
concentration, and Professor
M R. Kula (Institute of Enzyme Technology Hein-
rich-Heine-University Du
¨
sseldorf) and Professor W.
Hartmeier (Department. of Biotechnology, RWTH.
Aachen University) for provision of laboratory space
and equipment.
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