Biosci. Biotechnol. Biochem., 71 (2), 583–586, 2007 Note A New Colorimetric Method for Determining the Isomerization Activity of Sucrose

Sang-Eun PARK,1 Mee-Hyun CHO,1 Jin Kyu LIM,2 Jong-Sang KIM,2 y Jeong Hwan KIM,3 Dae Young KWON,4 and Cheon-Seok PARK1;

1Institute of Life Science and Resources, Graduate School of Biotechnology, KyungHee University, Yongin 449-701, Korea 2Department of Animal Science and Biotechnology, Kyungpook National University, Daegu 702-701, Korea 3Department of Food Science and Technology, Gyeongsang National University, Jinju 660-701, Korea 4Korea Food Research Institute, Sungnam 463-746, Korea

Received September 15, 2006; Accepted November 14, 2006; Online Publication, February 7, 2007 [doi:10.1271/bbb.60509]

A new colorimetric method for determining the to make glucose and fructose as end-products. This isomerization activity of sucrose isomerase was devel- means that sucrose isomerase performs two catalytic oped. This colorimetric method is based on the enzy- activities, the hydrolysis and the isomerization of matic reactions of invertase and glucose oxidase-perox- sucrose (Fig. 1). The enzymatic properties of several idase (GOD-POD). The main scheme for assaying microorganisms containing sucrose , such as sucrose isomerase activity is to degrade sucrose in the Erwinia rhapontici,8) Klebsiella sp. LX3,7) Pantoea reaction mixture to glucose and fructose by invertase dispersa UQ68J,9) and Serratia plymuthica,10) have been and to detect the concentration of glucose generated extensively characterized to date. using GOD-POD. The concentrations of and A number of methods are currently available to isomaltulose, reaction products of sucrose isomerase, measure sucrose isomerase activity, including HPLC are calculated from the concentration of glucose. This and dinitrosalicylic acid (DNS).7,11) HPLC is the most method allows rapid and accurate determination of the accurate, sensitive, and reliable method for the detection isomerization activity of sucrose isomerase without of various sugars. However, this procedure requires the inhibition by hydrolysis activity. application of relatively expensive analytical instru- ments and comparatively longer times than the simple Key words: glucose oxidase-peroxidase; invertase; iso- colorimetric method. Furthermore, it is not easy to maltulose; sucrose; sucrose isomerase separate five mono- and disaccharides (glucose, fruc- tose, sucrose, trehalulose, and isomaltulose) in the Isomaltulose (-D-glucosylpyranosyl-1,6-D-fructofra- reaction mixture with typical carbohydrate analysis nose, also designated palatinose) is a functional and columns and conditions.7) Hence, the simple DNS structural isomer of sucrose with very similar physico- method is generally used for assaying sucrose isomer- chemical properties.1) Due to its non-carcinogenic nature ase.11,12) Specifically, a sample of is mixed with and low calorific value, the compound is considered an solution containing sucrose, and incubated ideal sugar substitute for use in the food industry.2) After under appropriate conditions with gentle agitation. The isomaltulose intake the rate of hydrolysis is very slow reaction is terminated, and the total reducing sugar is compared to that of sucrose and maltose. Thus the assayed using DNS solution.13) This procedure can be glucose and insulin levels in humans after oral admin- used to quantify the presence of a free carbonyl group istration rise more slowly and reach lower maxima than (C=O) or so-called reducing sugars, and is based on the those after sucrose ingestion, supporting its potential as oxidation of the aldehyde or ketone functional groups a parenteral nutrient acceptable for both diabetics and present in reducing sugars, such as glucose and fructose. non-diabetics.3–5) This potential sugar substitute is Simultaneously, 3,5-dinitrosalicylic acid (DNS) is re- produced by an enzyme known as sucrose isomerase duced to 3-amino,5-nitrosalicylic acid under alkaline or isomaltulose synthase (EC 5.4.99.11).6,7) The enzyme conditions. After boiling, the change in color is catalyzes the isomerization of sucrose to produce mainly determined with a spectrophotometer, and enzyme isomaltulose and trehalulose (-D-glucosylpyranosyl- activity is calculated based on the standard curve. 1,1-D-fructofranose), as well as the hydrolysis of sucrose Notably, there are a number of limitations in using this

y To whom correspondence should be addressed. Tel: +82-31-201-2631; Fax: +82-31-204-8116; E-mail: [email protected] 584 S.-E. PARK et al.

OH HO O H H H H O HO OH H O HOHO OH H HO Sucrose

Sucrose Isomerase

Isomerization Hydrolysis

HO HO H O H H H H O H OH OH HO H O O HO OH HO H HO HO OH H H H HO OH Isomaltulose Glucose

OH HO O H H H O OH OH H H HO H O O HOHO HO H H HO HO H OH HO OH Trehalulose Fructose

Fig. 1. Reaction Scheme for Sucrose Isomerase. method to assay the isomerization of sucrose isomer- glucose and fructose.18) In this reaction scheme, one ase.14,15) In general, one mole of reducing sugar will mole of sucrose generates one mole of glucose and react with one mole of DNS. However, there may be fructose. Glucose oxidase (-D-glucose:oxygen 1-oxi- several additional side reactions, and thus the actual doreductase, EC 1.1.3.4), which catalyzes the oxidation reaction stoichiometry is more complex. Moreover, the of -D-glucose to D-glucono-1,5-lactone and hydrogen type of side reaction depends on the exact nature of the peroxide using molecular oxygen as the electron accep- reducing sugars. Different reducing sugars generally tor, is widely used for the determination of glucose in yield distinct color intensities. The color changes for body fluids.19) The main scheme for assaying sucrose glucose, fructose, isomatulose, and trehalulose obtained isomerase activity is to degrade sucrose in the reaction by the DNS method were considerably different from mixture to glucose and fructose with invertase, and to each other (data not shown). Unlike other carbohydrate detect the concentration of glucose generated using hydrolyzing , which produce a single reaction GOD-POD. Finally, the concentrations of trehalulose , sucrose isomerase produces multiple reducing and isomaltulose, the reaction products of sucrose sugar molecules, including glucose, fructose, trehalu- isomerase, are calculated from the concentration of lose, and isomaltulose (Fig. 1),7,16) This enzyme is of glucose. The isomerization activity of sucrose isomerase considerable interest to researchers due to its main can be separated from its hydrolysis activity by this products, trehalulose and isomaltulose,7,17) but the procedure, and this is yet to be achieved with other reaction mixture always contains substantial amounts analytical methods. of glucose and fructose as by-products.9) Therefore, the The specificity of invertase (Sigma Chemical, St. detection of reducing sugar in the reaction mixture for Louis, MO) for various disaccharides, including treha- assaying sucrose isomerase is basically inaccurate. Here lulose and isomaltulose, was examined by thin layer we propose a novel colorimetric method for the sucrose chromatography (TLC) and high performance anion isomerase assay that allows rapid and accurate determi- exchange chromatography (HPAEC; Dionex BioLC nation of the isomerization activity of sucrose isomerase system, Sunnyvale, CA) (Fig. 2). The reaction mixture without inhibition by hydrolysis activity. was composed of invertase (0–33 units) and substrate Our colorimetric method is based on the enzymatic (7.3–73 mmol) in a total reaction volume of 500 ml. The reactions of invertase and glucose oxidase-peroxidase reaction was performed at 55 C for 10 min and (GOD-POD). Invertase (-fructofuranosidase, EC immediately boiled to inactivate invertase. Our data 3.2.1.26) hydrolyzes sucrose into the monosaccharides show that invertase is specific to sucrose. Thus treha- A Novel Enzymatic Assay for Sucrose Isomerase 585 Tokyo). The glucose colorimetric assay was linearly detected within a range of 10 to 350 nmol. Analogous to invertase, the GOD-POD reaction was specific to glucose, but not fructose, sucrose, trehalulose, or iso- maltulose. Moreover, the colorimetric reaction of glu- cose was not inhibited by other sugars. The concentrations of sucrose isomerization products (trehalulose and isomaltulose) were calculated using the following equation: ½Conc of trehalulose and isomaltulose ¼½Conc of glucose without SI ½Conc of glucose with SI 1 2 3 4 5 6 7 8 9 10 Trehalulose and isomaltulose are sucrose isomerization (A) products generated by sucrose isomerase. Both concentrations calculated after treatment with invertase and GOD-POD. SI refers to sucrose isomerase. 16

14 To assert the effectiveness of the method developed,

12 the dependence of enzyme concentration on the rate of product (isomaltulose and trehalulose) formation was 10 confirmed by the novel colorimetric method (Fig. 3). 8 The product (isomaltulose and trehalulose) formation mole µ 6 was calculated by subtracting the glucose concentration in the sucrose isomerase reaction from the glucose 4 concentration without sucrose isomerase (Fig. 3A). The 2 reaction rate (initial velocity) was linear up to a protein concentration of 1 mg/1 ml in the case of Erwinia 0 rhapontici sucrose isomerase (Fig. 3B). To further 0 5 10 15 20 25 30 35 ensure the accuracy and applicability of the novel Units colorimetric method described here, the reaction mixture (B) of sucrose isomerase of Erwinia rhapontici was assayed by both high performance anion exchange chromatog- Fig. 2. Analysis of Invertase Reactions with Various Substrates. raphy (HPAEC; Dionex BioLC system, Sunnyvale, CA) A, TLC analysis: Lanes 1, 3, and 5 represent sucrose, isomaltu- and the new procedure. The isomerization reaction of lose, and trehalulose respectively; lanes 2, 4, and 6 are similar to Erwinia rhapontici lanes 1, 3, and 5, but come after the invertase reaction; lane 7, sucrose isomerase was performed glucose; lane 8, fructose; lane 9, the reaction mixture of sucrose with 1% sucrose (29 mM) as a substrate in 50 mM sodium isomerase of Erwinia rhapontici; lane 10, similar to lane 9, but after citrate buffer (pH 6.0) in a final volume of 0.5 ml at the invertase reaction. B, Specificity of invertase for various 30 C for 30 min. After the reaction was terminated by disaccharides, including sucrose, trehalulose and isomaltulose. boiling for 5 min, the invertase reaction was stimulated Sugars were examined by HPAEC. The symbols are sucrose ( – by adding 450 ml of 100 mM sodium citrate (pH 4.5) and ), trehalulose ( – ), and isomaltulose ( – ). 50 ml of invertase (150 units) at 55 C for 10 min. Inactivation of invertase was not required. The glucose lulose and isomaltulose, the two sucrose isomer prod- concentration in the reaction mixture was determined ucts, are not degraded by invertase. Sucrose (1%, with the GOD-POD method, as described above. approximately 15 mmol) was completely hydrolyzed by Comparable results were obtained by both methods. 15 units of invertase under the above reaction con- The estimated concentrations of isomerization products ditions. of the sucrose isomerase reaction were 12:1 0:2 mmol/ Next, the reactivity of GOD-POD to various sugars ml and 11:9 0:4 mmol/ml by HPAEC and the colori- was examined. For both convenience and consistency, a metric method, respectively. Thus the novel method glucose detection kit (Yongdong Pharmaceutical, Yon- appears to be easy, efficient, and accurate for assaying gin, Korea) employing the GOD-POD method was sucrose isomerase. applied, following the manufacturer’s protocol. The In conclusion, the colorimetric procedure using sample solution (0.1 ml) was mixed with GOD-POD invertase and GOD-POD provides a powerful technique enzyme solution (0.9 ml) and incubated at 37 C for 5 for evaluating the isomerization reaction of sucrose min. Optical density was immediately monitored at 505 isomerase with the advantage of eliminating side nm using a spectrophotometer (UV-1201, Shimadzu, hydrolysis activity. 586 S.-E. PARK et al.

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