Food Sci. Technol. Res., 16 (6), 523–530, 2010

Different Distributions of α−Glucosidases and in Milling Fractions of Rice Grains

1 2* 1 1 Mika Tsuyukubo , Tetsuya Ookura , Yuka Mabashi and Midori Kasai

1 Department of Nutrition and Food Science, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo- ku, Tokyo 112-8610, Japan 2 Food Function Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kan-nondai, Tsukuba, Ibaraki 305-8642, Japan

Received February 24, 2010; Accepted July 29, 2010

The distribution of -degrading in dormant rice grains was investigated with specific antibodies against α-glucosidases, α-amylases, and β-amylases. The α-glucosidases were predominantly localized in the inner endosperm. The α-amylases were mainly localized in the outer layers, and the β-amylases were distributed in whole grains. We propose a model to demonstrate how these enzymes de- grade starch during rice cooking.

Keywords: α-glucosidase, α-, β-amylase, immunoblot, rice cooking

Introduction (Takahashi et al., 1971; Matsui et al., 1988). The α-amylase Rice (Oryza sativa) is a major food in Asian countries, is an endoamylase that catalyzes the random hydrolysis of including Japan. Extensive studies have been carried out internal 1,4-α-glucans of starch and releases oligosaccha- to determine how a preferable taste can be achieved in rice rides. Two studies demonstrated that rice grains contained cooking. It was reported that physical properties such as more than 10 types of α-amylase isoforms (Daussant et al., stickiness and hardness (Okabe, 1979; Maruyama, 1991; 1983; Mitsui et al., 1996). These isoforms are classified Kainuma, 1992; Okadome et al., 1996; Okadome et al., into two groups on the basis of their optimum temperatures: 1999), and chemical components such as sugars and amino α-amylase I (37℃) and α-amylase II (70℃) (Mitsui et al., acids contribute to the taste of cooked rice (Matsuzaki et al., 1996). These α-amylase isoforms are remarkably induced 1992; Tajima et al., 1992; Ikeda, 2001). The amounts of sug- during germination (Alpi and Beevers, 1983; Perata et al., ars (reducing sugars and oligosaccharides) and amino acids 1992; Perata et al., 1993) and play a major role in degrading correlate with the activities of starch-degrading enzymes and native starch to oligosaccharides during rice seed develop- proteases, which are activated during cooking (Maruyama et ment. The β-amylases catalyze the liberation of maltose al., 1981; Kasai et al., 2000; Maruyama, 2002). However, it from the non-reducing ends of 1,4-α-glucans in starch. remains unclear which isoforms play a pivotal role β-Amylase activity is found in dormant and germinating rice in the sequential degradation of starch granules during rice grains (Shinke et al., 1973; Matsui et al., 1977; Okamoto cooking. and Akazawa, 1978). α-Glucosidases are enzymes that pro- The isoforms that exist in dormant rice and degrade starch duce from the non-reducing ends of 1,4-α-glucans granules during germination are α-amylases (EC 3.2.1.1) in starch. At least three isoforms of α-glucosidases (ONG1, (Mitsui et al., 1996; Yu et al., 1996), β-amylases (EC 3.2.1.2) ONG2-I, and ONG2-II) are present in the rice cultivar Nip- (Matsui et al., 1975; Yamaguchi et al., 1999), ponbare (Nakai et al., 2007). Nakai et al. also demonstrated (EC 3.2.1.68) (Fujita et al., 1999), (EC 3.2.1.41) that ONG2-II is the major isoform in dormant rice and is a (Takeuchi et al., 1999), and α-glucosidases (EC 3.2.1.20) post-translational isoform of ONG2-I. The existence of an ONG2-II-like α-glucosidase is implicated in Koshihikari *To whom correspondence should be addressed. (Awazuhara et al., 2000; Mabashi et al., 2009) and Yama- E-mail: [email protected] danishiki (Iwata et al., 2003). 524 M. Tsuyukubo et al.

Enzyme assays in the dormant rice revealed the distribu- the mixtures for 15 h at 4℃. The solution was centrifuged tion of enzyme activity: α-glucosidases in the inner endo- at 12,000 rpm for 30 min at 4℃, and the supernatants were sperm, α-amylases in the outer layers, and β-amylases in then filtered through filter paper (No. 2, Advantec, Tokyo, whole grains (Awazuhara et al., 2000; Iwata et al., 2002). Japan). Next, 100 μL of 100% (w/v) ice-cold trichloroacetic However, the particular distribution of each enzyme isoform acid (TCA) was added to 900 μL of the filtered supernatants, in dormant grains is unclear. and the mixtures were kept on ice for 30 min. The mixtures In this study, we focused on the distribution of were then centrifuged at 12,000 rpm for 15 min at 4℃. The α-glucosidases, α-amylases, and β-amylases. Additionally, pellets were washed twice with ethanol, and the precipitated we attempted to visualize the localization of these enzymes proteins were dissolved into 50 mM sodium phosphate buf- in dormant grains with a specific antibody against each en- fer (pH 7.0) and used as crude enzyme extracts. Purified zyme. We analyzed how the localization of enzymes and α-glucosidase was obtained as previously described (Mabashi their sequential activation affect the production of reducing et al., 2009). sugars during rice cooking. Preparation of germinating rice seeds and their crude enzyme extracts Crude enzyme extracts from germinat- Materials and Methods ing seeds were prepared as follows. Brown rice seeds were Materials Reagents and chemicals were purchased soaked in a 5% NaClO solution for 15 min. After extensive from the following commercial sources: acrylamide and washing in distilled water, the seeds were germinated on protein marker from Daiichi Pure Chemicals (Tokyo, Ja- filter paper moistened with distilled water in a plastic dish in pan), bis-acrylamide from Sigma (St. Louis, MO, USA), a the dark at 30℃ for 8 days. The germinating seeds were ho- protein assay kit from Bio-Rad (Richmond, CA, USA), and mogenized in PBSD medium using a polytron homogenizer. horseradish peroxidase-labeled anti-rabbit IgG antibody Concentrated crude enzyme extracts were obtained using (HRP) and chemiluminescence reagent from GE Healthcare the procedure described above. These extracts were used as (Buckinghamshire, UK). Others were purchased from Wako positive controls for α-amylases and β-amylases in immu- Pure Chemicals (Osaka, Japan) unless otherwise noted. noblots. Polyclonal antibodies against α-amylase H, α-amylase A + B, Protein assay The total protein of the crude extract was and β-amylase were kindly provided by Dr. Mitsui (Niigata assayed using a Bio-Rad protein assay kit with bovine serum University, Japan) and Dr. Yamaguchi (Hokkaido University, albumin solution as a standard (Bradford, 1976). Japan). Polyclonal antibody production An antibody against Preparation of rice flour Brown rice (Oryza sativa L., α-glucosidase was raised in rabbits with a synthetic peptide cv. Koshihikari) was purchased from Uonuma in Niigata in (QDVIPRPSPDSFLA), which corresponded to the 99th 2008 and stored at 4℃ until use. Brown rice flour samples through 112th amino acid residues for ONG2 α-glucosidase (hereafter designated as whole rice flour) were prepared (Nakai et al., 2007). This peptide was conjugated to keyhole by grinding rice grains with an electric mill (WB-1, Osaka limpet hemocyanin through an extra cystein residue at its N- Chemical, Japan). The brown rice was fractionated into four terminus. The antibody was purified by affinity chromatog- parts (100 to 90% rice flour, 90 to 80% rice flour, 80 to 70% raphy with the immobilized synthetic peptide onto Sepharose rice flour, and 70 to 0% rice flour) by dry-milling. The 100 (T-K craft, Gunma, Japan). to 90% rice flour was obtained by milling brown rice up to Sodium dodecyl sulfate polyacrylamide gel electrophore- 90% with a polishing machine (MC-90A, Toyo Rice Clean- sis (SDS-PAGE) and immunoblot Sodium dodecyl sulfate ing Machine, Japan). The remaining polished rice grain polyacrylamide gel electrophoresis (SDS-PAGE) was carried was scraped off up to 70% using a grain testing mill (TM05, out with 7.5% and 10% gels (Laemmli, 1970), and proteins Satake Engineering Co., Ltd., Tokyo, Japan). The first 10% were stained with 0.2% (w/v) Coomassie Brilliant Blue fractions were designated as 90 to 80% rice flour, and the R-250 in 40% (v/v) methanol and 10% (v/v) acetic acid. later 10% fractions were designated as 80 to 70% rice flour. For immunoblots, the electrophoresed gels were equili- The 70 to 0% rice flour was prepared by milling the remain- brated with a blotting buffer (50 mM Tris, 5 mM glycine, ing endosperm of the grains with the electric mill. Each ma- 0.037% SDS). The proteins were then transferred to nitro- terial was sieved through a 500 μm mesh. membranes (Bio-Rad) with a transblotting system Preparation of crude enzyme extracts Each rice flour (Nihon Eido, Tokyo, Japan) (Towbin et al., 1979). The (3 g) was suspended in 30 mL of 50 mM sodium phosphate transferred membrane was incubated in 20 mM Tris buffered buffer (pH 7.0) containing 5 mM dithiothreitol (DTT) and saline (TBS, pH 7.5) containing 0.1% Tween-20 and 5% 100 mM NaCl (PBSD medium), and extracted by shaking skim milk for blocking. The transferred membrane was re- Distributions of Enzymes in Rice Grains 525 acted with specific antibodies: anti-rice α-glucosidase ONG2 in each fraction (Fig. 1B), and the amount gradually de- (1:5000 dilution), anti-α-amylase isoform A + B (1:300 dilu- creased toward the outer layers in grains. In the outer layer tion) and isoform H (1:500 dilution) (Mitsui et al., 1996), of 100 to 90% rice flour (rice bran), α-glucosidases were and anti-β-amylase (1:100 dilution) (Yamaguchi et al., 1999). hardly detected in this experiment. An HRP-labeled antibody and a chemiluminescence reagent Localization of α-amylases in milling fraction of rice were used to detect immunoreactive bands with a LAS-4000 grains The distribution of α-amylases in rice grains was molecular imager (Fujifilm, Tokyo, Japan). analyzed with anti-α-amylase antibodies. For this analysis, 15 mg of enzyme extracts from each fraction of rice flour Results was loaded and separated on 10% polyacrylamide gels. Fig- Localization of α-glucosidases in milling fractions of rice ure 2 presents the results of SDS-PAGE and the immunoblot grains The distribution of α-glucosidases in rice grains was experiment with α-amylase antibodies (anti-A + B and anti- analyzed with SDS-PAGE and immunoblot. For this analy- H). The α-amylases, which are hardly detected in dormant sis, 10 mg of crude enzyme extracts from each fraction of rice grains, are reportedly induced during germination (Mit- rice flour was loaded and separated on 7.5% polyacrylamide sui et al., 1996). We thus prepared positive control samples gels. Figure 1 presents the results of SDS-PAGE and the im- from germinating seeds. The anti-α-amylase antibodies rec- munoblot experiment with an anti-α-glucosidase antibody. ognized the band from the germinating seeds (lane 7), which The purified anti-α-glucosidase antibody recognized 1 μg of had an estimated molecular weight of 44 kDa. Bands of the the purified α-glucosidase (lane 7), which had an estimated same molecular weight were detected in the lanes of crude molecular weight of 95 kDa. Bands of the same molecular enzyme extracts from each fraction (arrows in Figs. 2B and weight were detected in the lanes of crude enzyme extracts 2C). The visualized bands on each crude enzyme extract from each fraction (arrows in Fig. 1B). The visualized bands were consequently identified as α-amylases in rice grains. on each crude enzyme extract were consequently identified The α-amylase isoforms recognized by anti-α-amylase as α-glucosidases in rice grains. isoforms A + B and isoform H were localized in the outer We found that the inner endosperm layer of rice grains (70 layers of 100 to 90% and 90 to 80% rice flours. The to 0% rice flour) had the highest amounts of α-glucosidases α-amylase isoforms recognized by anti-α-amylase isoforms

Fig. 1. α-Glucosidases are predominantly localized in the inner endosperm layers. (A) Crude extracts from each fraction of rice grains were separated with SDS-PAGE (lane 1, whole rice flour; lane 2, 100 to 90% rice flour; lane 3, 90 to 80% rice flour; lane 4, 80 to 70% rice flour; lane 5, 70 to 0% rice flour; lane 6, whole rice flour). As a positive control, 1 μg of purified α-glucosidase was loaded in lane 7. Molecular size markers are indicated on the left side. (B) Immunoblot analysis of the α-glucosidase in rice grain fractions. Lane 6 was probed with only a HRP-labeled antibody. An arrowhead indicates bands of α-glucosidases. 526 M. Tsuyukubo et al.

Fig. 2. α-Amylases are localized in the outer layers of rice grains. (A) Crude extracts from each fraction of rice grains were separated with SDS-PAGE (lane 1, whole rice flour; lane 2, 100 to 90% rice flour; lane 3, 90 to 80% rice flour; lane 4, 80 to 70% rice flour; lane 5, 70 to 0% rice flour; lane 6, whole rice flour). As a positive control, 1 μg of crude enzyme extract from germinated grains was used in lane 7. Molecular size markers are indicated on the left side. (B) Immunoblot analysis of the α-amylase isoforms recognized by an antibody against α-amylase isoforms A + B in rice grain fractions. (C) Immunoblot analysis of the α-amylase isoforms recognized by an antibody against α-amylase isoform H in rice grain fractions. Lane 6 was probed with only a HRP-labeled antibody. Arrowhead indicates bands of α-amylase isoforms.

A + B were predominantly localized in the 90 to 80% rice The immunoreactive bands of β-amylases were found in flour. These isoforms are classified as α-amylase I, which all fractions (Fig. 3B). The β-amylases were widely local- consists of isoforms A, B, Y, and Z (Mitsui et al., 1996). The ized from the outer layers to the inner layers of rice grains. α-amylase isoforms recognized by anti-α-amylase isoform The 100 to 90% rice flour contained more β-amylases than H were mainly localized in 100 to 90% and 90 to 80% rice other fractions. In Fig. 3B, the bands indicated by arrows are flours. These isoforms are classified as α-amylase II, which non-specific bands with the HRP-labeled antibody. consists of isoforms F, G, H, I, and J. Some α-amylase I and α-amylase II were detected in the 80 to 70% rice flour. The Discussion inner layer of rice grains (the 70 to 0% rice flour) contained During rice cooking, glucose is liberated from few α-amylases (Figs. 2B and 2C). starch granules by starch-degrading enzymes, including Distribution of β-amylases in milling fraction of rice α-glucosidases, α-amylases, and β-amylases. To visualize grains Localization of β-amylases in rice grains was ana- the distribution of those enzymes in rice grains, we conduct- lyzed with an anti-β-amylase antibody. For this analysis, 15 ed immunoblot analyses of α-glucosidases, α-amylases, and mg of enzyme extract from each fraction of rice flour was β-amylases. loaded and separated on 10% polyacrylamide gels. Figure Three isoforms of α-glucosidases (ONG1, ONG2-I, and 3 presents the results of SDS-PAGE and the immunoblot ONG2-II) were purified from dormant rice grains (Nakai et experiment of the anti-β-amylase antibody. The β-amylases, al., 2007). Nakai et al. also demonstrated that ONG2-II is which are hardly detected in dormant rice grains, are report- dominant in dormant grains (cultivar Nipponbare) and can edly induced during germination. Thus, for the positive hydrolyze the α-1,4-glucosidic linkage of maltose and starch control, we prepared enzyme samples from germinating to produce glucose. In addition, several studies have dem- seeds. The anti-β-amylase antibody recognized the band onstrated that other rice cultivars contained ONG2-II-like from germinating seeds (lane 7), which had an estimated α-glucosidase, on the basis of their substrate specificity and molecular weight of 53 kDa. Bands of the same molecular the N-terminal amino-acid sequence (Takahashi et al., 1971; weight were detected in the lanes of crude enzyme extracts Matsui et al., 1988; Takeuchi, 2002; Iwata et al., 2003). In from each fraction (arrows in Fig. 3B). The visualized bands our previous study, we purified ONG2-II from the cultivar on each crude enzyme extract were consequently identified Koshihikari (Mabashi et al., 2009). In the present study, we as β-amylases in rice grains. prepared an anti-α-glucosidase antibody against the N-termi- Distributions of Enzymes in Rice Grains 527

Fig. 3. β-Amylases are distributed in rice whole grains. (A) Crude extracts from each fraction of rice grains were separated with SDS-PAGE (lane 1, whole rice flour; lane 2, 100 to 90% rice flour; lane 3, 90 to 80% rice flour; lane 4, 80 to 70% rice flour; lane 5, 70 to 0% rice flour; lane 6, whole rice flour). As a positive control, 5 μg of crude enzyme extracts from germinated rice grains was used in lane 7. Molecular size markers are indicated on the left side. (B) Immunoblot analysis of β-amylase in rice grain fractions. Lane 6 was probed with only a HRP-labeled antibody. An arrowhead indicates bands of β-amylases. An arrow indicates non-specific bands with the HRP-labeled antibody. nal region peptide of an ONG2. With this purified antibody, α-glucosidases in dormant grains are predominantly distrib- we were able to detect 1 μg of the purified α-glucosidase (Fig. uted in the inner endosperm. 1B). Several studies have found that the outer layer contained Immunoblot analysis of α-glucosidases indicated that the a large amount of proteins and that storage compounds such 70 to 0% rice flour fraction contained more α-glucosidases as starch were predominantly located in the inner endosperm than other fractions, and that enzyme amounts gradually de- of rice grains (Barber, 1972; Kennedy et al., 1974; Koga creased toward the outer layers (Fig. 1B). In the 100 to 90% et al., 1996; Okuda et al., 2007). Abundant localization of rice flour fraction, corresponding to bran, α-glucosidases α-glucosidases in the inner endosperm might be required to were hardly detected. In a previous study, Awazuhara et effectively degrade starch to glucose upon germination. al. (2000) reported that enzyme activity for reducing sugar The α-amylases exhibited a different distribution from production was much higher in the inner endosperm than the α-glucosidases. The α-amylase isoforms were confined in the outer endosperm or bran, and that the activity had an to the outer layers of rice grains (Fig. 2). The α-amylase optimum temperature above 60℃. Moreover, Awazuhara isoforms recognized by anti-α-amylase isoforms A + B were et al. suggested that α-glucosidase might be a dominant en- predominantly localized in the 90 to 80% rice flour fraction. zyme in causing starch degradation in the inner endosperm, The α-amylase isoforms recognized by anti-α-amylase iso- since glucose was the major product of starch degradation form H were mainly localized in the 100 to 90% and the 90 in the inner endosperm. Iwata et al. (2002) purified a 95- to 80% rice flour fractions. The former isoforms are classi- kDa α-glucosidase from cultivar Yamadanishiki and dem- fied as α-amylase I, which consists of isoforms A, B, Y, and Z; onstrated that the inner endosperm of rice grains had higher the latter isoforms are classified as the α-amylase II, which α-glucosidase activity than the outer layers. Mabashi et al. consists of isoforms F, G, H, I and J (Mitsui et al., 1996). (2010) investigated the effect of the milling ratio on the The optimum temperature of α-amylase I is 70℃, and that amounts of reducing sugars during rice cooking, and dem- of α-amylase II is 28 to 37℃. Iwata et al. (2002) reported onstrated that more glucose accumulated in the 85 and 90% that the α-amylase activity of dormant grains was restricted milled rice than in the 100 and 95% milled rice. These pre- to the outer layers. Awazuhara et al. (2000) reported that the vious results indicated that large amounts of α-glucosidases α-amylase isoforms with high and low optimum tempera- exist in the inner endosperm layer. Combining previous tures were mainly localized in the outer layer. In addition, findings with our results, we demonstrated that the main they reported that the latter isoform was likely to be isoform 528 M. Tsuyukubo et al.

G, judging from the isoelectric point. In this study, our im- more β-amylase than other fractions. Combining previous munological data confirmed these previous results and visu- findings with our results, we conclude that β-amylase is alized the spatial distribution of α-amylase isoforms. widely distributed in dormant Koshihikari grains. The β-amylases in rice grains were detected in all frac- Summarizing these results, Fig. 4 describes the relation- tions (Fig. 3). This finding differed from the results for ship between enzyme activity and temperature during rice other starch-degrading enzymes (e.g., α-glucosidases and cooking. The α-amylase II in the outer layers (100 to 80% α-amylases). Past studies reported that β-amylases were fraction) and the ubiquitously distributed β-amylase are ac- hardly detected in dormant rice grains (cultivar Arborio) tivated at 37℃ (Mitsui et al., 1996; Awazuhara et al., 2000). with enzyme assay and with immunoblot analysis (Gug- Oligosaccharide and maltose are consequently produced lielminetti et al., 1995). Gulielminetti et al. also reported from starch granules. The α-glucosidases in the inner endo- that the amounts of β-amylases were clearly enhanced on sperm are then activated at 60℃ (Awazuhara et al., 2000), germination. Yamaguchi et al. (1999) identified at least four and glucose is liberated from starch and oligosaccharide. β-amylase-deficient cultivars (Nipponbare, Sachikaze, Chu- Moreover, α-amylase I in the outer layer is activated at 70℃ kyoasahi, and Shinriki) by amylolytic activity staining after (Mitsui et al., 1996), degrading starch into oligosaccharide. the separation of β-amylases by isoelectric focusing. Several In addition, gelatinization of rice starch occurs at 58℃ to reports suggested the presence of β-amylases in dormant rice 69℃ (Ayabe et al., 2006). The coincidence of gelatiniza- grains (e.g., cultivars Shinsetsu, Takanenishiki and Koshi- tion and enzyme activation facilitates starch degradation hikari) by assaying enzyme activity and zymogram pattern into reducing sugars. Additionally, Awazuhara et al. (2000) (Shinke et al., 1973; Matsui et al., 1975, Uyen et al., 2001). reported that high levels of α-glucosidase activity contribute Awazuhara et al. (2000) detected β-amylase activity in the to starch degradation into reducing sugars. Kasai et al. (2000) outer layer of dormant rice grains (cultivar Koshihikari). reported that the time required for the temperature to rise Shinke et al. (1978) reported that β-amylase was widely between 40 and 80℃ is about 5.5 min. This duration seems distributed in rice grains. In this study, we found that Koshi- sufficient to activate the enzymes, judging from the fact that hikari contained β-amylases in every fraction in dormant rice the amounts of reducing sugars increased 55 mg/(100 g raw grains and that the 100 to 90% rice flour fraction contained rice) during this period.

Fig. 4. Timing and location of starch-degrading enzyme activation during rice cooking. The starch-degrading enzymes in rice grains are activated at each optimum temperature during rice cooking and degrade starch into reducing sugars (e.g., oligosaccharide, maltose, and glucose). The activities of α-amylase II, localized in the outer layers (100 to 80%), and β-amylases, ubiquitously distributed (100 to 0%), are enhanced at 37℃. At this temperature, rice starch is degraded into reducing sugars, the main components of which are oligosac- charides. A large amount of reducing sugars (almost glucose) is then produced by α-glucosidases in the inner endo- sperm (70 to 0%) at 60℃ and α-amylase I in the outer layer (90 to 80%) at 70℃. The gelatinization of rice starch occurs from 58℃ to 69℃ *1 Ayabe et al., 2006; *2 Mitsui et al., 1996; *3 Awazuhara et al., 2000. Distributions of Enzymes in Rice Grains 529

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