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A New Colorimetric Method for Determining the Isomerization Activity of Sucrose Isomerase

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A New Colorimetric Method for Determining the Isomerization Activity of Sucrose Isomerase Biosci. Biotechnol. Biochem., 71 (2), 583–586, 2007 Note A New Colorimetric Method for Determining the Isomerization Activity of Sucrose Isomerase 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 isomerases, 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 trehalulose 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 enzyme is mixed with and low calorific value, the compound is considered an substrate 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 enzymes, which produce a single reaction GOD-POD. Finally, the concentrations of trehalulose product, 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 isomaltuloseà ¼½Conc of glucose without SIÃà ½Conc of glucose with SIÃà 1 2 3 4 5 6 7 8 9 10 ÃTrehalulose and isomaltulose are sucrose isomerization (A) products generated by sucrose isomerase. ÃÃBoth 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
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