Int. J. Electrochem. Sci.

, 8 (2013) 9163 - 9170

International Journal of
ELECTROCHEMICAL
SCIENCE
www.electrochemsci.org

Short Communication
A Simple and Rapid Method for Probing of Isomerization of
Glucose to Fructose with Ferroceneboronic Acid
Lin Liu*, Ning Xia, Jimin Du, Qige He, Yun Qin, Huihui Li, Caicai Li

College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan 455000,
People's Republic of China
*
E-mail: liulin@aynu.edu.cn

Received: 28 May 2013 / Accepted: 25 June 2013 / Published: 1 July 2013

Isomerization of glucose to fructose is used in a variety of industrially relevant processes and in
glycolysis. Currently used methods for probing of the isomerization need complicated instruments and
are time-consuming. In this work, we reported a simple and rapid method for probing of the
isomerization with electrochemically active ferroceneboronic acid (FBA) probe. Specifically, fructose
resulting from the isomerization of glucose induced the shift of the FBA potential through the
formation of FBA-fructose complex. To demonstrate the feasibility and application, the isomerization
mediated by glucose isomerase (GI) was investigated. The effect of experimental environmental (e. g.
pH, reaction time and temperature, enzyme concentration) on the conversion ratio of glucose to
fructose was demonstrated. We believe that the method will find many applications in both food
industry and laboratory research for probing of the activity of enzymes (e.g. isomerase, phosphatase
and kinase) and screening of new catalysts for isomerization of sugars.

Keywords: Isomerization; glucose isomerase; fructose; ferroceneboronic acid; enzyme activity;
electrochemistry

1. INTRODUCTION

The transformations of biomass into a wide variety of products have attracted extensive
attention in chemical and food industry. Isomerization of glucose to fructose is of commercial
importance in food industry, such as the production of high-fructose corn syrup (HFCS), since fructose
is the most sweet natural sugar and glucose is the cheapest and the only abundant monosaccharide
available in nature [1]. In the present commercial process, the isomerization is carried out by glucose
isomerase (GI, EC 5.3.1.5), one of the three highest tonnage value enzymes (GI, amylase and
protease). Very recently, metalloenzyme-like chemical catalysts have been shown to be promising

However. such as narrow pH operation window. the binding affinity of fructose to boronic acid is 1~2 orders higher than that of glucose [18. which was used as received. these methods need the use of expensive and less-stable enzymes [12]. RNA. Currently used methods for monitoring the isomerization activity is to determine the amount of produced fructose with high-performance liquid chromatography (HPLC). these methods need complicated instruments and are time-consuming.8]. glycoproteins.. fluorometric and electrochemical detection sensors for determination of diol-containing compounds (e. All other reagents are of the AR grade. Electrochem. fructose produced from glucose is first conversed into the form of ketose with concentrated acid under heating for reaction with resorcinol.or 1. inhibition by some essential elements (e.g.3-diols. sucrose and lactose were purchased from Sangon Biotech. can bind to diol- containing compounds and have been used for design of electrochemical sensors because of its well- defined redox reaction [17-26]. Scheller’s group reported an amperometric biosensor for the determination of glycated hemoglobin in human with FBA [19. maltose. China).and six-membered cyclic diesters between boronic acids and 1. 2013 9164 alternative to GI in the production of fructose in view of some drawbacks of GI-mediated isomerization. 8. In addition. It is well known that boronic acids can form boronate ester covalent bonds with cis-diols. Ferroceneboronic acid (FBA). Co. which makes the experiments complex and needs the use of hazardous chemical. Sci. 2.g.28]. Based on this interaction.2. In the present work. Hampton) from Streptomyces rubiginosus was obtained from Seebio (Shanghai. requirement of Co2+ for the enzyme activity. FBA stock solution was prepared with . China). mannose. (Shanghai. an electrochemically active ferrocene derivative. For example. fructose. in the classical Seliwanoff’s test.27]. Glucose. and reported a simple and rapid electrochemical method for probing of the isomerization of glucose to fructose. investigated the activity of the enzymes (tyrosinase and thrombin) based on the interaction of FBA and enzyme- generated catechol ligand on electrode [21]. J. Li et al. Ca and Zn) and low efficiency from an economic point of view [2-7]. GI (HR7-100. For example. extensive efforts have been focused on the development of colorimetric. Activity of enzymes and chemical catalysts is the most important factor in evaluation of their suitability for industrial application. gas chromatography (GC) and Seliwanoff’s test (Scheme 1) [7. the diols must be in the form of cis-configuration. xylose. However. and the molecule of diols should possess syn-periplanar torsion angle between the neighboring hydroxyls for effective complexation interaction [16]. Therefore. EXPERIMENTAL 2. we distinguished glucose and fructose using FBA probe. a variety of optical and electrochemical techniques have also been reported for the selective detection of fructose [9-11]. Because of the difference in the structure of fructose verse glucose. The principle of the recognition is the formation of five. Ltd. there remains significant room for the development of a theoretically and technically simple approach for probing of the isomerization of glucose to fructose. sugars. Note that. Vol..1 Chemicals and reagents Ferroceneboronic acid (FBA) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from Sigma–Aldrich.Int. catechol) [13-15].

. Electrochem. GI was incubated with 1. 200 mM. Vol. 10 μL of the GI/glucose mixed solution was mixed with 3 mL FBA solution for 30 s before the electrochemical measurement. 2. Assay methods for probing of the isomerization of glucose to fructose and the activity of enzymes and chemical catalysts.).8]. and then sonicated in ethanol and water. Voltammetric determination in phosphate-buffered saline solution (PBS buffer. Sci.5 mM Co 2+.2 Electrochemical measurements The glass carbon (GC) electrode with a diameter of 3 mm was polished with alumina power. All aqueous solutions were prepared with a Millipore system (Simplicity Plus. As mentioned in Introduction.1 Feasibility for probing of isomerization of glucose to fructose with FBA probe HOH2C O HO OH Glucose HO OH Enzymes or chemical catalysts CH2OH CH2OH CH2OH CHO O CH2OH H2C O O O B Concentrated acid H HO FBA O H HO Fe H OH Heat H Ketose OH H H OH Fructose Fructose-FBA Resorcinol Seliwanoff's test HPLC or GC assay Electrochemical assay Scheme 1. Millipore Corp. J. more convenient is to measure the produced fructose by HPLC or GC assay and Seliwanoff’s test (Scheme 1) [7. In general laboratory practice for monitoring the isomerization of glucose to fructose. 3. Then.8 M glucose in 50 mM HEPES solution in the presence of 0.0) containing 1% DMSO. China) using a homemade plastic electrochemical cell room temperature. these methods are time-consuming and need complicated . respectively. 200 mM) was performed on a uECS-PRO electrochemical workstation (Changchun Institute of Applied Chemistry Chinese Academy of Science. 2013 9165 phosphate-buffered saline solution (PBS buffer.Int. For the assay of enzyme activity. A platinum wire and a Ag/AgCl electrode were used as the auxiliary and the reference electrodes. pH 7. 8. RESULTS AND DISCUSSION 3.

We also investigated the interaction between FBA and other sugars.0 FBA + Glucose FBA + Glucose + GI Current / A 0. maltose.Int. glucose did not cause apparent change in the voltammetric response of FBA at the same condition. and found that these sugars did not induce the appearance of the new peak (data not shown). glucose and GI were 100 μM.5 Potential / V vs.16 V decreased. fructose. 6 mM. The result is acceptable since these sugars have low binding affinity to boronic acids [18.0 -0. However. xylose. .0 HEPES buffer for 30 min at 80 °C.2 0. We first compared the voltammetric response of FBA in the absence and presence of glucose and fructose.3 0. the immobilization form of enzyme for reusability and the storage time [1]. 8. Vol. sucrose and lactose.28].2 -0.5 -1.1 0. the current of original reduction peak at 0. As shown in Fig. For the assay of isomerization. probing of the isomerization of glucose to fructose with FBA as redox probe is possible. Thus. Electrochem. The analysis principle in the present work is based on measurements of the change of electrochemical response of FBA caused by the formation of boronate ester covalent bond between FBA and fructose. Ag/AgCl Figure 1. 6 mM and 33 µg mL-1. The final concentrations of FBA. such as mannose. we tested commercial GI. this is because the reduced form of FBA has poor affinity to diol than the oxidized one [24]. FBA exhibits a couple of redox waves with the oxidation potential at 0. Expectedly. Sci. the isomerization activity is strictly dependent upon the type of organism producing the enzyme. after addition of GI to the glucose solution.0 -0. glucose was incubated with GI in pH 7. Notice that no apparent shift in the oxidation potential of FBA was observed. (A) Cyclic voltammograms (CVs) of FBA in different systems. which is attributed to the complex between FBA and fructose resulting from the isomerization of glucose.5 FBA FBA + Fructose 1.4 0. 1. In the commercial production of HFCS.23 V and the reduction potential at 0. 1.16 V.. After the addition of fructose. which is accompanied by the appearance of a new reduction peak at around 0 V.0 0. 2013 9166 instruments or use of concentrated acid. J.5 0.1 0. the electrochemical response reveals a reduction peak at 0 V. To demonstrate the feasibility and application of our method.

4 0. The optimal pH for GI activity was found to be in the range of 7 to 8. and the relative standard deviations (RSDs) are shown as the error bars. 3. 2B.0 0 1 2 3 4 5 6 Potential / V vs.5 (Fig.3 Effect of experimental environmental on the conversion of glucose to fructose The activity of enzyme is commonly affected by temperature and chemical environmental (e. the III/II ratio increases linearly with the increase of fructose concentration from 0.1 6.5 -1.2 Dependence on fructose concentration Differential pulse voltammetry can decrease the background charging currents and in turn increase the detection sensitivity.25 to 6 mM.9 0. was used to evaluate the performance of the sensor. 3B).3 -2. the ratio of the reduction current of peak II (III) to that of peak I (II). Vol.00 mM 0. 2A depicts the voltammetric responses in FBA solutions containing different concentrations of fructose. g. This is understandable since GI is unstable and denatured at low and high pH [1].9 (B) -0.4 1.2 -0. Sci. The value increases with the elevation of temperature and reaches to the maximum at 70 °C. (A) Differential pulse voltammograms (DPVs) acquired in FBA solutions containing different concentrations of fructose and glucose.Int.2 0.5 0.7 I 0. (B) Plots of the III/II ratio against the fructose concentration. Fig. The total concentration of glucose and fructose kept at 6 mM.2 [Fructose] II 0.0 0. To further demonstrate the amenability of our method for monitoring the isomerization reaction. The dependence of the current change upon fructose concentration was evaluated with differential pulse voltammetry. Because glucose at the concentration above 6 mM would induce the appearance of peak II.0 -0. 3A shows the effect of reaction temperature on the III/II ratio. Ag/AgCl [Fructose] / mM Figure 2.00 mM -2.00 mM -3. The slight decrease at the temperature above 70 °C is probably due to the instability of the enzyme at higher temperature. which can be expressed using III/II = 0.8 0.4 4. Fig.1 0. Furthermore.8 -0.1 0.2 5. we investigated the effect of reaction temperature and time. III/II.00 mM 0.99).50 mM 0. J. pH and enzyme concentration on the conversion ratio of glucose and fructose.3 (A) 0.3 0. -0. 8. pH). Electrochem. The arrow indicates the scan direction.1 2.141 [Fructose] + 0.6 III / II 0.5 0.002 (R2 = 0.7 Current / A -1. The effect of pH was carried out at pH varying from 5. the relationship of III/II and incubation . 2013 9167 3.6 0.00 mM -1..00 mM -2. As show in Fig. the total concentration of glucose and fructose in the assay kept at 6 mM. Each point was averaged from at least three replicates.25 mM 0.5 to 8.

8.40 (A) 0.0 8. As shown in Fig.5 8. Zn2+ and Cu2+.0 6.40 (C) 0.40 (D) 0. inhibited the isomerization activity to some extent (Fig.30 III / II III / II 0.15 0.35 0. the conversion ratios of glucose to fructose by GI depend on the type and immobilization form of the enzyme.30 III / II III / II 0. In practice.40 0. pH (B). and found that metal ions.10 0.00 0.5 6. The results.20 0. In panel D.25 0.20 0.05 0. The reported value varies in the range of 26 to 59 % [1]. Vol.05 0.00 0 20 40 60 80 100 0 10 20 30 40 50 60 -1 Time / min [GI] / g mL Figure 3.0 in panels A and C. Our results indicate that the equilibrium conversion of glucose to fructose under the optimal conditions is around 45 %.35 (B) 0.15 0. indicating that GI-catalyzed isomerization reaction is completed within 40 min. The incubate time was 30 min in panels A and B. III/II increases with the increase of GI concentration.30 0.5 Temperature pH 0. 2013 9168 time was investigated.10 0.Int. The pH value was 7. ..25 0.20 0. III/II increases with the increase of the incubation time. in agreement with that reported previously [1]. respectively.15 0.05 0. Moreover.0 7. we investigated the dependence of III/II upon GI concentration under the optimal conditions. Moreover. Ni2+. Effect of temperature (A).00 0. The concentrations of GI and glucose used were 1 mg mL-1 and 1.25 0. 3C.30 0. The incubation temperature was 80 °C in panels B and C.10 0. 0. 3D.0) at 70 °C for 40 min. 4B). incubation time (C) and final GI concentration (D) on the III/II ratio.25 0.15 0.8 M. Sci.05 0. Electrochem. 4A).00 30 40 50 60 70 80 90 5.10 0. we also demonstrated that Co2+ is required for the enzyme activity in this process (Fig. such as Ca2+. further indicating that the method is applicable to monitor the isomerization process and probe the enzyme activity.5 7. GI was incubated with glucose (pH 7. J.35 0. and begins to level off beyond 40 min.35 0. As shown in Fig.20 0.

we reported a simple and rapid electrochemical method for probing of the isomerization of glucose to fructose. Moliner. A. M. References 1. Angew.2 0.5 With Co2+ 0. Palsdottir.15 -2. B. Nat. 51 (2012) 11736. S. Román-Leshkov. Proc. E. Int. R. 2013 9169 -0. Sci. Curtiss and M. Davis.4 0. obviates the use of expensive and less-stable enzymes.3 0. D. Moliner and M. this is the first report in which the activity of isomerases was investigated based on the interaction of ferrocene boronic acids and sugars. 109 (2012) 9727. Hwang.2 -0.0 Without Co2+ 0. H. Acad. V. Bhosale. R. Sci. R. Bermejo-Deval. Y.1 0. Moreover.05 -3.35 (B) Current / A -1. L. 21205003.20 -2.. E. Nat.-J. ACKNOWLEDGMENTS Partial support of this work by the National Natural Science Foundation of China (Nos. Conrad and I. Vol. Davis. Lobo. E. E. Nikolla.g. Román-Leshkov. The other experimental conditions are the same as those in Figure 3D.. ACS Catalysis.25 III / II -1. our method requires very simple and security sample-handling-procedure and minimum instrumental investment. .5 o 2+ o 2+ o 2+ o + C 2 /C a + /C /C /C Potential / V vs.10 0. 107 (2010) 6164. Y. phosphatase and kinase) and screening of new catalysts for isomerization of sugars. Nikolla. S. Compared with the currently used methods.. A. We believe that the method will find many applications in both food industry and laboratory research for probing of the activity of enzymes (e. 5.00 o 2+ -0. Davis. 60 (1996) 280. S. 21273010) and the Science & Technology Foundation of Henan Province (122102310517) is gratefully acknowledged. M.5 0. 1 (2011) 408. Chem. Y. M. C. F. Hammond. (A) DPVs acquired in FBA/glucose/GI mixed solutions with and without addition of Co 2+. Moliner. USA. and can be conducted in the field with portable devices. M.1 0. Sci. Assary.0 0. Acad. isomerase.30 0. Rao and V. Ag/AgCl C 2 u 2+ + i + Zn 2 N 2 C Figure 4. Silverman. 2. The concentrations of GI and metal ions used were 33 µg mL-1 and 0. J. Electrochem. S. 3. CONCLUSION In summary.0 0.0 0. Deshpande. Ed. 4. Microbiolog.5 mM. Proc. USA. Román-Leshkov and M.Int. 8. E.5 (A) 0. Hermans. 4. Rev. respectively. (B) Effect of metal ions on the enzyme activity.

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