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

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 mixtur

Candida boidinii for improved stability during entrapment

in polyacrylamide

Marion B Ansorge-Schumacher1, Heike Slusarczyk2, Julia Schu¨mers1and Dennis Hirtz1

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: m.ansorge@biotec.rwth-aachen.de

Website: http://www.biotec.rwth-aachen.de/

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.

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-acrylacryl-amide 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

Mn2+ concentration of 0.05 mmolÆL)1 yielded about

3500 recombinant clones, 70% of which expressed act-ive FDH This was estimated from a qualitatact-ive 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 (t1⁄ 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.

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-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 Mn2+ 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

(t1⁄ 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

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.

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 KM of formate, while KM 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 50C 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 50C, 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)

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.

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 CO2 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)1Tris⁄ HCl, pH 8.3;

0.5 molÆL)1KCl, 70 mmolÆL)1MgCl2, 0.1% (w⁄ v) gelatine],

0.05 or 0.15 mmolÆL)1of MnCl2, and 10 lL of dNTP-mix

(10 mmolÆL)1 dCTP, 10 mmolÆL)1 dTTP, 2 mmolÆL)1

dATP, 2 mmolÆL)1dGTP), 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 95C for 5 min, 25 cycles of 5 min at

94C, 1 min at 50 C, and 1 min at 72 C each, and at last one cycle at 72C 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)1EDTA) at a maximum temperature

of 65C 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)1phenazinethosulfate, and 2 gÆL)1 nitrotetrazolium-blue chloride in 0.1 molÆL)1of potassium phosphate buffer,

pH 7.5) and incubated in the dark for 10 min A second dye-ing solution (50 mmolÆL)1NAD+in 0.1 molÆL)1potassium 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 DUseries; Beckmann, Fullerton, CA, USA) or in a microtitre plate reader (ThermoMax; Molecu-lar Devices, Ismaning, Germany) at 340 nm and 30C 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)1sodium 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 Vtotal

Venzyme e  d with DE being the extinction increase within 1 min, Vtotal

the total volume of the assay mixture, Venzyme the volume

of added enzyme solution, d the layer thickness of the

cuvette and e the extinction coefficient of NADH + H+

(6.22 mLÆlmol)1Æcm)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 30C

Screening for improved FDH stability

E colicolonies 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 37C on a

rotary shaker When OD550reached 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)1potassium phosphate buffer (pH 7.5)

with 0.1% (v⁄ v) Triton-X-100 and 0.2 molÆL)1EDTA The

cell debris was removed by centrifugation and 35 lL of

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

30C for 30 min 70 lL FDH-assay mix for microtitre plate

readers were then added and the extinction at 340 nm was

monitored at 30C 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 30C for

5–180 min, according to the protocol described above

The measured inactivation was adapted to the time law

A¼A0*e()kt) , with A0 being the activity after incubation

time t and k the inactivation constant The half-life was

then calculated using the equation s¼ ln 2/k

Expression and purification of FDH

Recombinant E coli were cultivated at 37C 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 OD620 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

37C for 2 h The resulting crude extract was cooled down

to 20C and H2O and K2HPO4 were added to final

con-centrations of 21% (w⁄ w) and 5% (w ⁄ w), respectively

After complete dissolution of K2HPO4, 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 H2O 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, v0describes the initial velocity of the reac-tion, vmaxthe maximum reaction rate, [x] the concentration

of the two substrates, KMxthe Michaelis–Menten constant of substrate x, and KINADH2the inhibition constant of NADH2 All experiments were performed with purified enzyme

Measurement of temperature influences

For determination of Tm (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 30C 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

Tmand 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 50C was monitored until inactivation reached 80% From the time course, half-life at 50C 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 80C 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 molÆL)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 15C within 30 min The average bead volume was 7.4 ± 2.5 mm3 For calibra-tion of the CO2measurement, solutions of NaHCO3 were

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)1NAD+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 CO2 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 30C 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 40C

(detector temperature at 140C; injector temperature at

65C; carrier gas helium 5.0 at 62.5 mLÆmin)1) on a

CTR-1-column (Alltech, Munich, Germany) The retention time

of CO2under this conditions was 1.25 min The peak areas

obtained were converted into CO2 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 NaHCO3in 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 CO2resulting from the

quantita-tive transformation of NaHCO3into CO2at 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 CO2 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

References

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