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A Bioelectrochemical Methodfor the Determination ofAcetate with ImmobilizedAcetate KinaseXiao-Jing Tang a & Gillis Johansson aa Department of Analytical Chemistry , University ofLund , P.O. Box 124, S-22100, LundPublished online: 22 Aug 2006.

To cite this article: Xiao-Jing Tang & Gillis Johansson (1997) A BioelectrochemicalMethod for the Determination of Acetate with Immobilized Acetate Kinase, AnalyticalLetters, 30:14, 2469-2483, DOI: 10.1080/00032719708001758

To link to this article: http://dx.doi.org/10.1080/00032719708001758

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ANALYTICAL LEITERS, 30(14), 2469-2483 (1997)

A BIOELECTROCHEMICAL METHOD FOR THE DETERMINATION

OF ACETATE WITH IMMOBILIZED ACETATE KINASE

Key words Enzyme reactor; flow injection, acetate kinase; pyruvate kinase,

L-lactic dehydrogenase

Xiao-Jing Tang and Gillis Johansson

Department of Analytical Chemistry, University of Lund,

P.O.BOX 124, S-22100 Lwd

ABSTRACT

Flow injection determinations of acetate were carried out using immobilized

acetate lunase, pyntvate b a s e and lactic dehydrogenase with an amperometric

method. Two acetate kinases from E. coli and B. stearothermophilus were tested.

It was found that the immobilized acetate kinase from B. stearothermophilus was

more stable than that from E. coli., but it is much more expensive and less

available. Acetate b a s e coupling at pH 7.4 using CPG aminopropyl and

glutaraldehyde seems to be superior to other immobilization methods. A high

immobilization yield can be obtained by immobilization of the three enzymes

2469

Copyright 0 1997 by Marcel Dekker. Inc.

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2470 TANG AND JOHANSSON

separately giving high conversions of all the three. Plots of current versus

concentdon show a useful operating range from 0.3 to 2 mM acetate with a linear

response. The detection limit was 0.2 mM at a flow rate of 0.3 d m i n with 200 pl

injections. The method is therefore well suited for monitoring of the level of acetate

in fermentations with acetate as the carbon source.

INTRODUCTION

Classical methods for determination of carboxylic acids rely on pre-

punfcation by extractions followed by ion-exchange and gas chromatography. Thin

layer chromatography, although mainly qualitative, can resolve a great number of

acids ' . Samples from biological sources will usually also contain amino acids

wluch makes the separation more complex. Acetate determinations by means of gas-

liquid chromatography2 exhibit very low detection limits, but the need for

preliminary treatment and the use of propionic acid as an internal standard3 make

these methods less attractive. Ion chromatography has rapidly become a method of

choice since about 1975. It can be used to determine many inorganic and organic

ionic compounds, as well as ionizable compounds. Ion chromato-graphy detection

has been developed by using a self-regenerating suppressor which provides high performance, high sensitivity, good baseline stability, and wide applicability.

A variety of enzymatic methods for determination of acetate in biological

fluids have previously been de~eloped"~, but the methods lack sensitivity and

selectivity because of interferences from various compounds in the sample.

Determination of acetate with acetate kinase was reported before, it relied on

monitoring the NADH-consumption by spectrophotometric method^^-^. Because of

the insufficient purity of the reagent, especially the enzymes, the acetate values

were in error. A slow decrease in the absorbance after the end of the reaction

indicates interferences.

The immobilization of acetate kinase has been reported before. The best

immobilization was obtained with the glutaraldehyde and glutaraldehyde succinate

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IMMOBILIZED ACETATE KINASE 2471

dihydrazide activated glass beads'". The chosen camer. controlled pore glass

(CPG), offers many advantages. The glass beads are inert against microbial

contamination and are mechanically stable permitting high flow rates and

pressures".

Acetate is an important carbon source in fermentations and monitoring of the

acetate levels could be used either to follow the progress of the reaction or to

control the addition of more substrate. Methods that can be used for automatic

process monitoring are particularly attractive. Unless resources are available for

sterile monitoring which is seldom the case, a flow-injection off-line analysis is the

best available option.

The objective of this study was to develop a flow-injection method for the

determination of acetate in bioprocess monitoring. A reaction using immobilized

acetate kinase and amperometric detection was selected for further work.

THEORY

The method used in this work relies upon the acetate kinase reaction

according to the following equation sequence:

AK

ACETATE + ATP ACETYLPHOSPHATE + ADP ( 1 )

PK

ADP + PEP -+ ATP + PYRUVATE (2)

LDH

PYRUVATE + NADH LACTATE + NAD' (3)

(ATP, Adenosine 5' triphosphate; PEP, phosphoenolpyruvate; ADP, Adenosine 5'

diphosphate; AK, Acetate kinase; PK, Pyruvate kinase; NADH, Nicotinamide

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2472 TANG AND JOHANSSON

adenine dinucleotide, reduced; LDH, Lactate dehydrogenase. The equilibrium

constants for reactions (1) are 8 x 10” or 8.6 x 10” 15,21, for reaction (2) 6 . 4 5 ~ Id at pH 7.4, 30 ‘C, tris buffer and for (3) 3 . 6 ~ lo5 at pH 7.0,25 O C , tris buffer’5,

respectively . Injected solutions of acetate react with ATP to give ADP, which react further

with PEP to give pyruvate, which is reduced by NADH. So the acetate was

determined by the depletion of NADH which was monitored mperometrically with

a graphite electrode modified with mediator. The electrochemical oxidation of

NADH around 0 mV is extremely slow under normal conhtions. The mediator

which itself can undergo a fairly fast redox reaction with NADH reacts readily with

the electrode. Meldola Blue which was a mediator, can be adsorbed on graphite to

give a chemically modified electrode. The electrode is most stable in acid

soi~tion’~. The mediator can mediate the electron transfer from NADH in solution

to the electrode. The reaction sequence is

NADH + MB+ * NADH.MB+ + NAD+ + MBH (4)

MBH + MB+ + 2e- + H+ Eappi. ’ E’’ ( 5 )

NADH combines with the mehator to form a complex, which decomposes to

NAD+ and the reduced form of the mediator in a rate-determining step. MBH is

reoxidized rapidly electrochemically when the applied potential is larger than the

formal potential.

EXPERIMENTAL

Instrumentahn

The experiments were done in a flow injection system with a peristaltic

pump (Gilson Minipuls 2) and a pneumatic injection valve with a sample loop.

Amperometric measurements were carried out by means of a potentiostat connected

to an electrochemical three-electrode cell of the wall-jet type. The working

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IMMOBILIZED ACETATE KINASE 2473

electrode was pressed into a Teflon holder, with the modified surface exposed, and

inserted into the flow-through cell. A platinum wire in the cell is an auxiliary

electrode and a saturated calomel electrode (SCE) is a reference. A voltage of 0 mV

vs. SCE was applied to the working graphite electrode.

The graphite rods were cut, polished on wet, tine emery paper and washed

with deionized water, dried at 60°C for 30 min, they were then heated in muffle

h a c e at 700°C for 90 seconds. The treated electrodes were stored in a desiccator.

An electrode was chemically modified by dropping 2 - 3 drops of an aqueous

solution of the mediator (about 2 x 10” M) to the end surface, waiting for 4 min

and rinsing carefilly with millipore water. The surface coverage (r) was determined

with cyclic voltammetry. r > 2 - 3 n moles/cm2 is sufficient for maximal response

to NADH. The cyclic voltammograms were recorded with deaerated 0.1 M

imidazole buffer, pH 7.5, as supporting electrolyte.

The flow injection system consisted of three channels (Fig.1). Teflon

tubings, i.d. 0.5 mm, were used to connect the various parts. The flow rates used for

the canier (Millipore water: channel l), 0.1 M imidaz.de buffer (PH 7.6) containing

0.1 M KNO,, 0.015 M of Mg(NO&, 6 mM of ATP, 2 mM of PEP and 0.25 mM

NADH (channel 2) and 0.1 M sodium acetate buffer @H 4.5) in channel 3 were all

equal. The carrier and the imidazole buffer with the reagents were mixed in a

knitted tubing before reaching the reactor. A sodium acetate buffer was introduced

between the enzyme reactor and detector in order to decrease the pH to a level

compatible with the operating range of the mediator. The reagents solution was

made fresh each day.

enzvmes and re-

The working electrode material was spectrographic graphite (RWOO 1,

Ringsdorff Werke GmbH), diameter 3.05 mm. The mediator, Meldola blue. 7-

dimethylamino- 1,2-bemphenoxazine (No. 258504, Boehringer Mannheim GmbH,

Germany), was used as an electron transfer mediator. CPG (controlled pore glass),

Aminopropyl(200-400 mesh) with pore size 500 A (G-4643) from Sigma was used.

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2414 TANG AND JOHANSSON

t electrode I inlet I

\

Pump Inj. \

Fig. 1. Schematic set-up of the flow injection analysis. M, mixing chamber;

W, waste; D, flow-though electrochemical detector; P, three-

electrode potentiostat; R, enzyme reactor. Solution 1 was millipore

water, solution 2 was imidazole buffer with reagents. Solution 3 was

acetate buffer, pH 4.5. The injected volume was 100 pl.

The enzymes acetate kinase, EC 2.7.2.1. either from Escherichra colr, or

from Bacillus stearothemzophilus. pyruvate kinase. EC 2.7.1.40. from rabbit muscle

and L - lactic dehydrogenase, EC 1.1.1.27. from rabbit muscle, lyophilized salt-free

powder, were all from Sigma. ATP, ADP, PEP, NADH, pyruvic acid and

glutaraldehyde were of the highest purity offered by Sigma. All other chemicals

were of analyhcal grade.

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IMMOBILIZED ACETATE KINASE 2475

Immobilization of enzymes o n controlled pore & Prior to dilution of the commercial 25% glutaraldehyde solution it was

purified with activated carbon and centrifuged to remove polymeric material and

the clear supernatant was used. The glutaraldehyde is acceptable if the absorbance

A 235/ A,,, is less than 0.23.

PK (3943 Units) and LDH (4350 Units) were dissolved in 0.5 ml of 0.1 M,

pH 7 phosphate buffer, and 80 mg glutaraldehyde-activated CPG added according

to Weetal122 in each enzyme solution. The mixtures were allowed to react at

reduced pressure for 30 min and then in a refngerator over night, and then the

immobilized glass was washed extensively with Millipore water before the

enzymes-linked glass was packed in a reactor. The immobilization yield for PK and

LDH were 68% and 66%, respectively, as estimated from the absorbance of the

enzyme solutions at 280 nm before and after immobilization. An enzyme reactor,

150 pl. was packed with the mixture of immobilized PK and LDH and stored in

phosphate b a a in a rehgerator, when not in use. AK (870 units, from E. coli) was

dissolved in 0.3 ml of 0.1 M, pH 7.4 [ 1 11 phosphate buffer and added to 105 mg

glutaraldehyde-activated CPG. The immobilization procedures were the same as the

described above. The immobilization yield was 73%. A reactor, 100 pl, was packed

with immobilized AK. Reactor AK and PWLDH were connected in series in the

flow system (they were used for running experiments, unless otherwise stated).

RESULTS AND DISCUSSION

The conversion in the enzyme reactors

Injections of NADH, pyruvate, ADP and acetate at different flow rates can

be used to evaluate the efficiencies of reactions (1) - (3). The conversions for

pyruvate, ADP and acetate were 100,93 and 16 %, respectively, at flow rate of 0.4

mumin, see Table 1. The conversion of acetate was low due to the unfavorable

equihbrium of equation (1). It may be seen from the constants that the equilibrium

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2416

Flow rate (ml/min)

Pyruvate

ADP

Acetate

TABLE 1

0.1 0.2 0.3 0.4 0.5 0.6

85 94 100 100 100 100

77 85 93 93 93 93

14 15 16 16 15 14

TANG AND JOHANSSON

Percentage conversion of substrates to products by the reactions given by eqn. (3), (2) and (I), respectively.

position of Reaction 1 markedly favors the direction to the left. In addition, the K,

of acetate is high, 300 mMI5. The higher the K,, the lower the affinity, thus the

high K, values of acetate means that the affinity of acetate b a s e for acetate is

low.

We have made efforts to improve the conversion for acetate using different

immobilization procedures including azo coupltng, but the conversion was still very

low. It is known that hydroxylamine in large quantities can shift the equilibrium of

reaction I to the right2', but a test using cyclic voltammetry showed that the

mediator was destroyed after keeping the electrode in a solution of the amine for 10

min, and this approach is therefore out of question.

The effect of flow rate

A comparison between injection of NADH, pyruvate, ADP and acetate (Fig.

2) shows that the response increased with flow rate because of the increased

hydrodynamic transport rate to the electrode surface as observaed in earlier work

with a similar electrochemical cellsI6. The equilibrium may be estimated from a

comparison of the current for NADH (0.25 mM), pyruvate (0.20 mM), ADP (0.20

mM) and acetate (0.20 mh4) injections, and the conversions are shown in Table 1.

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IMMOBILIZED ACETATE KINASE 2477

c a

5 300 200

\ .-

100 I t l

0 " " " " " " ' " 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Flow rate / ml min-'

Fig. 2. Effect of flow rate on the current for 0.25 mM of NADH (o), 0.20

mM of pyruvate (a), 0.20 mM of ADP (A) and 0.25 mM of acetate

(v).

lZHxfh3 Acetate kinase has an optimum activity at pH 7.4 [21] (AK from E. coli).

The rate optimum for soluble PK is at pH 7.4 - 8.412 and for LDH in the direction

towards lactate at pH 7.4 - 7.913. Considering all these factors, an imidazole buffer

of pH 7.5 was used as carrier in the enzyme reaction. Variations of the pH showed

that the conversion was almost independent of pH in the range pH 6.5 - 8.5 for ADP

and acetate, see Fig. 3. However, the optimum pH is less than 6 for eqn. (4)14 and

the outlet from the reactor was therefore mixed with an acetate buffer of pH 4.5 to

decrease the pH to a level compatible with the operating range of the mediator used

in the detection. The pH then became about 5 in the solution flowing through the

electrochemical cell.

. . of unmobllized AK The storage properties of the immobilized enzymes was investigated by

comparing the current for NADH (0.25 mM) and acetate (10 mM) injections at 0.3

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2478

I 2000 .-

I000

4000 r

-

- - TANG AND JOHANSSON

0 k 6.0 6.5 7.0 7.5 8.0 8.5

PH

Fig. 3. Plots of steady-state current response to 1 mM of NADH (o), 1 mM

of ADP (A) and 10 mM of acetate (v) as a function of pH at 0.5

ml/min. The response for NADH was in the positive direction,

whereas those for ADP and acetate were in the negative direction.

The imidazol buffer contained 1 mM ATP, 1 mM PEP and 0.5 mM

NADH. AK: 750 units, PK: 2350 units, LDH: 2550 units were co-

immobilized and the immobilization yield was 40 %. A reactor, 150

pl, was packed with immobilized enzymes.

ml/min. The stability of the immobilized PK and LDH were investigated by G.

Moges et. al. The enzyme reactor was run for a total time of eight months giving

100% conversion ef€iciency16. The life-time of the immobilized AK from the E.

colr was investigated by comparing the current for NADH (0.25 mM) and acetate

(0.20 mM) injections. The conversion had decreased to 33% of the original value

after 10 days (Table 2). however the PK and LDH still had 100 and 93 YO conversion, respectively. AK from B. stearothemophilus was more stable retaining

most of its activity after 18 days. So far the conclusion is that the acetate kinase

from B. srearothermophrlus is more stable than that from E. coli. The former one

is expensive and available only in packages with few units. It became unavailable

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IMMOBILIZED ACETATE KINASE

Days

AKfrom B. stear.

AKfrom E.coli

2479

1st 2nd 4th 8th 11th 15th 18th

0.7 0.6 0.7 0.6 0.5

17.5 15.8 13.7 7.3 5.7

TABLE 2

commercially during the project. It is obviously better to take larger amounts of

enzyme from E. coli with limited stability than to use a little of an expensive but

more stable enzyme from B. stearothermophilus.

Selectivity

The selectivity of the system was examined with eight compounds. As shown

in Table 3, the acetate kinase is highly selective for its natural substrate, acetate.

There was some response to propionate, butyric acid and ethanol. Fortunately, the

letter three compounds are not generally present in fermentation broths. No

measurable response were obtained with glycolic acid, formate, glycerol or glycine.

Calibration curves for ace-

The response of the Meldola Blue-modified electrode in the present system

was investigated in the concentration range fiom 0.5 mM to 1.2 mM acetate at 0.4

d m i n with 100 p1 injections. Different electrodes yielded a response of 11 1s5

pA/M (n = 4). The repeatability was 3.4 % RSD.

Plots of the current versus the concentration have shown that the reactor

which has the higher conversion of acetate gave the calibration curve with narrower

linear range and the one with the low conversion of acetate gave the one with

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2480

Compounds

Acetate

(20 mM)

Propionate

Glycolic acid

Sutyric acid

TANG AND JOHANSSON

Relative response Compounds Relative response

100 Ethanol 2.7

8.2 Formate 0

(%) (20 mM) (W

0 Glycerol 0

4.1 Glycine 0

TABLE 3

Response of the system to various compounds.

160 r

0 ’ I

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Acetate I mM

Fig. 4. The calibration curve for acetate at flow rate of 0.3 mllmin. The

imidazol buffer contained 6 mM ATP, 2 mM PEP and 0.25 mM

NADH. 720 units of AK was in a 100 pl of reactor.

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IMMOBILIZED ACETATE KINASE 248 1

broader linear range, e.g., the calibration curve was linear from 0.1 to 0.5 mM of

acetate when the conversion of acetate was 16% or from 0.3 mM to 2.0 mM of

acetate when the conversion of acetate was 1.6%. It is not necessary (it is also very

difficulty) to reach a high sensitivity, because the acetic acid concentration in

fermentation broth is usually rather high. The linear rauge of a calibration curve can

also be changed by changing the sample loop.

A typical calibration curve is shown in Fig. 4. The fermentation medium was

used to make a standard acetate solution in this case. It was found that the

sensitivity of the electrode response increased about 2 times compared to the one

of the same electrode when the standard acetate solution was made in imidazole

buffer without magnesium. Previous work in our laboratory has shown that M$+ is the activator for the immobilized pyruvate lanase. So the fermentation medium

containing 2 mM of MgSO, played a role in the increase in the sensitivity of the

electrode response.

NADH is a reactant in eqn. (3) and it must be present in excess of the

produced pyruvate to force the equilibrium towards the lactate side. The

concentration of NADH should not be too high, otherwise the background becomes

high. Usually a low background is desirable for a high sensitivity setting to be able

to detect low concentrations of the substrate.

ACKNOWLEDGEMENT

Authors thank A. Tocaj for providing the fermentation medium. Financial

support was obtained from the Swedish Natural Research Council.

REFERENCES

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2482 TANG AND JOHANSSON

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IMMOBILIZED ACETATE KINASE 2483

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Received: May 15, 1997 Accepted: July 27, 1997

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