A quantitative starch–iodine method for measuring alpha-amylase and glucoamylase activities Zhizhuang Xiao ¤,1, Reginald Storms, Adrian Tsang Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal H4B 1R6, Quebec, Canada Alpha-amylase (EC 3.2.1.1), which cleaves internal -1,4glycosidic linkages in starch to produce glucose, maltose, or dextrins, and glucoamylase (EC 3.2.1.3), which cuts -1,4and -1,6-glycosidic linkages to release glucose from the nonreducing ends of starch, are widely used in the industrial conversion of starch into sugars. The characterization of -amylases and glucoamylases generally needs to use diVerent chromatography techniques such as paper chromatography [1,2], high-performance liquid chromatography [3,4], and thin-layer chromatography [5,6]. There are mainly two types of assays that are used to determine the activity of -amylase and glucoamylase. One is based on measuring the amount of reducing sugars by the dinitrosalicylic acid (DNS)2 assay [2,4,5,7–10] or the Nelson–Somogyi [1,11,12] method, whereas the other is based on the decreased staining value of blue starch–iodine complexes [13]. The second method, which was developed by Fuwa [13] and is widely used [10–12,14–16], is based on color development that results from iodine binding to starch polymers. However, the starch–iodine assays reported by diVerent researchers are quite diverse with iodine concentrations ranging from 3 M [12] to 0.25 mM [15] and with the wavelength used to measure color development varying from 550 nm [15] to 700 nm [13]. Moreover, -amylase activity is calculated as relative activity according to the following equation. Dextrinizing activity D (D0-D) ¥ D0 £ 100 ¥ 10, where D is the absorbance of the enzyme sample and D0 is the absorbance of the amylose control without the addition of enzyme [13]. Dextrinizing activity * Corresponding author. Fax: +1 514 496 6265. E-mail address: zhizhuang.xiao@cnrc-nrc.gc.ca (Z. Xiao). 1 Present address: Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. 2 Abbreviations used: DNS, dinitrosalicylic acid. calculated using this formula is therefore not expressed in units that are related to the amount of substrate consumed. In addition, since the assay has a Wnal volume of 20–200 ml, it is not suitable for screening a large number of samples. In this paper, we describe a microplate-based starch–iodine assay that measures the amount of starch degraded to determine the activity of -amylase and glucoamylase. We also propose a new strategy to distinguish between -amylase and glucoamylase enzymes by comparing the results obtained from the DNS and starch–iodine assays. In the Fuwa method [13], 2.5 ml of buVered -amylase is mixed with 2.5 ml amylose (2 g/L) and incubated at 37 °C for 30 min. The reaction is then terminated by adding 5 ml of 1 N acetic acid. Following reaction termination, the mixture is then transferred into a 250-ml Xask and diluted to nearly 200 ml with H2O, followed by the addition of 5 ml of iodine reagent (0.2% iodine and 2% potassium iodide). Finally, the volume is adjusted to 200 ml with H2O and the amount of color development is determined by measuring the absorbance at 700 nm. During the development of our microplate-based starch–iodine assay, we made the following four observations. (i) the wavelength of maximum absorbance for the starch–iodine complexes is 580 nm (Fig. 1A); (ii) concentrations of at least 2.5 mM iodine are required for complete color development when the starch concentration is 2.0 g/L; (iii) the presence of maltose, which is one of the main end products produced when starch is hydrolyzed by -amylase, does not change the spectral properties of the iodine reagent nor those of the starch–iodine complexes (Fig. 1A); and (iv) greater than 95% of the enzyme activity is detected with the starch–iodine amylase assay even in the presence of 250 mM maltose (Table 1). Our microplate-based starch–iodine assay was carried out as follows. Assay reactions were initiated by adding 40 l of starch (Sigma S-2630) solution (2.0 g/L) and 40 l of A 2.5 Blank Maltose Maltose+Starch 2g/l Starch 2g/l Starch 1g/l Absorbance 2 1.5 1 0.5 0 400 450 500 550 600 650 700 Wavelength (nm) Absorbance at 580 nm B 2.5 y = 0.0273x R2 = 0.9998 2 1.5 1 0.5 0 0 20 40 60 80 100 Starch (microgram per assay) Fig. 1. Absorbance of the starch–iodine complexes. (A) Absorbance spectra of the starch–iodine complexes; (B) standard curve for the microplatebased starch–iodine assay. enzyme in 0.1 M phosphate buVer at pH 7.0 to microplate wells. To minimize evaporative loss during incubation, a plastic mat was used to cover the microplate in combina- tion with using a temperature block equipped with a hot lid [17]. After 30 min of incubation at 50 °C, where the assayed enzymes were most active, 20 l of 1 M HCl was added to stop the enzymatic reaction, followed by the addition of 100 l of iodine reagent (5 mM I2 and 5 mM KI). Following color development, 150 l of the iodine-treated sample was transferred to a transparent Xat-bottomed 96-well microplate and the absorbance at 580 nm (A580) was measured using microplate reader (Bio-TEK Instruments, Winooski, VT, USA). Fig. 1B shows that the microplate format assay accurately measured the amount of starch in 40-l samples containing 10–80 g of starch. We also compared the microplate format starch–iodine assay and the DNS reducing sugar assay [9,17] using sets of enzyme samples prepared with an Aspergillus oryzae -amylase (Sigma A-6211) and an Aspergillus niger glucoamylase (Sigma A-1602), respectively. The results presented in Table 1 demonstrate that both assays were highly reproducible. Furthermore, both methods generated equivalent values for the number of enzyme units present in the set of glucoamylase samples. In other words, the amount of starch consumed in mg/min as measured by the iodine method was equal to the amount of glucose produced in mg/min as measured by the DNS assay. In contrast, the amount of -amylase activity in the samples, as determined with the two assays, was very diVerent. The units of -amylase activity in samples measured with the iodine assay (mg of starch equivalents consumed/ min) was Wve times higher than the units of activity (mg of glucose equivalents produced/min) measured with the DNS method (Table 1). Apparently, equivalent units of glucoamylase but not alpha-amylase activity were obtained using the iodine and DNS assays for the following reasons. Glucoamylases degrade starch by removing glucose units from the non- Table 1 Activity of -amylase and glucoamylase as determined with the microplate-based starch–iodine assay and the DNS assay Sample Glucoamylase from Aspergillus niger (U/ml)c Alpha-amylase from Aspergillus oryzae (U/ml) DNS assaya Iodine assayb Iodine assayb2 DNS assay Iodine assay 1 2 3 4 5 6 7 8 1.02 1.05 1.07 1.05 1.04 1.03 1.04 1.05 4.85 4.96 4.82 4.94 4.88 4.88 5.02 4.97 4.67 4.68 4.77 205 214 208 203 199 201 206 206 197 202 203 200 201 203 204 205 Averaged 1.04 §0.02 4.92 § 0.07 4.71 § 0.06 205 § 5 202 § 3 All data are averages of triplicate determinations. a One unit (U) of activity as determined by the DNS assay is deWned as an average of 1 mg of glucose equivalents released per min in the assay reaction. b One unit (U) for the microplate-based starch–iodine assay is deWned as the disappearance of an average of 1 mg of iodine binding starch material per min in the assay reaction. U/ml was calculated using the formula: U/ml D (A580 control ¡A580 sample) ¥ A580/mg starch ¥ 30 min ¥ 0.04 ml, where A580 control is the absorbance obtained from the starch without the addition of enzyme, A580 sample is the absorbance for the starch digested with enzyme, A580/mg starch is the absorbance for 1 mg of starch as derived from the standard curve in Fig. 1B, 30 min is the assay incubation time, and 0.04 ml is the volume of the enzyme used in the assay. b2 Enzyme assays were performed in the presence of 250 mM maltose. c The calculated t value for glucoamylase is 1.61, below the critical t value of 2.14 at p D 0.05, df D 14, when we compare the activity determined by the DNS assay with that by the starch–iodine assay. d The calculated F values of the microplate-based starch–iodine assay to the DNS assay for both enzymes are below the critical F value of 3.79 at 95% conWdence level with df D 7, 7. reducing ends, thereby reducing the mass of starch available for iodine binding and producing an equivalent mass of glucose. In contrast, endo-acting -amylases reduce the concentration of starch polymers that are able to bind iodine (mg consumed/min) much more quickly than they produce reducing sugar ends (due to the production of sugar oligomers that are too short to eYciently bind iodine). Comparing the results obtained with the iodine-binding assay and the DNS assay can therefore provide a simple alternative to the various chromatography techniques currently used to distinguish between -amylase and glucoamylases. In summary, we have developed an accurate microplatebased starch–iodine assay for quantifying starch–iodine complexes. Furthermore, this assay can serve as a platform to screen large populations of amylase mutants—for example, mutants generated by directed evolution—to identify variants with improved characteristics for industry-speciWc applications. 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