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Food Science and Technology Research, 24 (2), 363 368, 2018

Copyright © 2018, Japanese Society for Food Science and Technology
doi: 10.3136/fstr.24.363

http://www.jsfst.or.jp

Note

Binding Interaction of Porcine Pancreatic α-Amylase with waxy/amylose

extender Double-mutant Rice Starch Granules Does Not Determine Their
Susceptibility to Hydrolysis

Hiroki Takagi1,2, Akiko Kubo1, Mei Inoue1, Makoto Nakaya1, Shiho Suzuki1 and Shinichi Kitamura1*

1
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan
2
Nihon Shokuhin Kako Co., Ltd., Fuji 417-8530, Japan

Received September 16, 2017 ; Accepted December 14, 2017

The starch from waxy/amylose extender (wx/ae) double-mutant rice is almost indigestible by porcine pancreatic
α-amylase (PPA) and behaves like resistant starch, even though PPA digests waxy mutant (wx) and wild-type (WT)
rice starches easily. To clarify the relationship between binding ability and susceptibility to PPA hydrolysis, we
studied the PPA adsorption capacity of wx/ae starch. Results showed that the wx/ae starch adsorbed 80% more PPA
than wx and WT starches, consistent with the ratio of their specific surface areas. This suggests that PPA adsorption
capacity is determined by the specific surface area of starch granules. Thus, the resistance to digestion of the wx/
ae starch is not due to adsorption capacity, but to the higher-order nanostructure of the starch granules. The
morphological features of digested wx/ae starch granules observed by scanning electron microscopy (SEM) suggest
that the higher-order nanostructure of wx/ae starch is similar to that of resistant starches, such as high-amylose rice
and potato starches.

Keywords: waxy/amylose extender, porcine pancreatic α-amylase, adsorption, digestibility

Introduction of RS. RS1 is physically inaccessible to digestion due to the

Starch is a digestible carbohydrate that also has components
resistant to digestion called resistant starch (RS). RS is defined as
the portion of the starch that is not broken down by digestive
enzymes, mainly pancreatic α-amylase, in the small intestine. Some
native starches, isolated from plants and without any structural
modification, are resistant to digestion. Potato and high-amylose
maize starches show greater resistance to hydrolysis by amylolytic
enzymes than other native starches (Banks and Greenwood, 1975).
We found that starch from the rice double-mutant waxy and
amylose extender (wx/ae) is almost indigestible by α-amylase in
vitro (Kubo et al., 2010). The wx/ae starch, along with potato and
high-amylose maize starches, is classified as RS2, one of four types
presence of intact cell walls in the grains, seeds or tubers. RS2 is
composed of native starch granules that are protected from
digestion by the higher-order nanostructure of the starch granules.
RS3 is composed of non-granular starch-derived materials that
resist digestion and is usually formed during the retrogradation of
starch granules. RS4 is generated by chemical modification to
reduce digestibility (Sajilata et al., 2006).
Digestibility by amylases is affected by the composition and
structure of starch granules, including amylose content,
amylopectin double helices, crystalline domains, pores and other
characteristics (Qi and Tester, 2016a). The wx/ae starch consists of
pure amylopectin with relatively long unit chains (fewer with

Abbreviations: wx/ae: waxy/amylose extender; wx: waxy; WT: wild-type; PPA: porcine pancreatic α-amylase

*To whom correspondence should be addressed. E-mail:skita@bioinfo.osakafu-u.ac.jp

364
degree of polymerization (DP) 6-14 and more with DP > 15) and
has a B-type polymorphic form (Nishi et al., 2001, Kubo et al.,
2008, Kubo et al., 2010). The average mean diameter of wx/ae
starch granules is 3.96 ± 1.22 μm (Kubo et al., 2010). The double
helices of amylopectin cannot be digested unless they are untwisted
(Gallant et al., 1992), and starch granules with the B-type
polymorphic form are much more resistant to amylolysis than those
with the A-type polymorphic form (Planchot et al., 1997). On the
other hand, wx/ae starch has a larger specific surface area and more
pores on its surface than waxy (wx) and wild-type (WT) rice
starches (Takagi et al., 2017). The large surface area provides more
access for enzymes to attach, resulting in increased hydrolysis
(Tester et al., 2006, Qi and Tester, 2016b), and the pores on the
granule surface might be sites of initial enzyme attack, allowing
enzyme molecules access to the granule interior (Fannon et al.,
1992). The resistance to digestion of wx/ae starch for α-amylase
can be explained by its change in composition and crystal form
(Kubo et al., 2010); however, its large specific surface area and
many surface pores should increase its susceptibility to enzyme
binding and hydrolysis.
We focused on the relationship between enzyme adsorption
and digestibility of wx/ae starch using porcine pancreatic α-amylase
(PPA). We analyzed the amount of PPA adsorbed to determine
whether or not the resistance to digestion of wx/ae starch is due to
its PPA adsorption capacity, and also observed the starch granules
after reaction with PPA to elucidate the patterns of hydrolysis
involved.
Materials and Methods
Rice grain We used the amylose-free amylose extender rice
mutant line AMF18 (wx/ae), which is genetically defective in
granule-bound starch synthase I (GBSSΙ) and starch branching
enzyme IIb (BEΙΙb). It is derived from a cross between the waxy
mutant line EM21 and the amylose extender mutant line EM16
(Nishi et al., 2001, Kubo et al., 2008, Kubo et al., 2010). We also
used the waxy mutant line EM21 (wx), which is genetically
defective in GBSSΙ, and its wild-type japonica parental cv.
Kinmaze rice (WT) (Nishi et al., 2001, Kubo et al., 2008, Kubo et
al., 2010). The wx/ae mutant rice plants were grown in the summer
of 2010 in a field in Shiga Prefecture, Japan. The wx mutant and
WT rice plants were both grown in the summer of 2007 in an
experimental field at Osaka Prefecture University, Japan.
Starch granule isolation Starch granules were isolated
according to a modified previous method (Kubo et al., 2010).
Kernels (400 g) were polished to about 85% of their initial weight.
Polished kernels were soaked in 0.05% (w/w) sodium hydroxide
for 16 h and washed with distilled water several times until the
supernatant had a neutral pH. The wet starch was homogenized
using a mortar and pestle. The slurry was filtered with a 0.106 mm
mesh sieve and starch granules were suspended in a 1.0% sodium
dodecyl sulfate (SDS) solution and mixed by magnetic stirrer for
H. Takagi et al.
1 h, then they were collected by centrifugation at 620 G for 5 min
and washed with distilled water several times to completely remove
the SDS. Starch granules were then isolated by ethanol
precipitation with 70, 90 and 99.5% ethanol. The isolated starch
was dried in an oven at 50℃ under vacuum and filtered through a
0.106 mm mesh sieve before use.
Enzyme binding property of starch The adsorption procedure
is summarized in Fig. 1. Porcine pancreatic α-amylase (PPA,
Product No. A4268), an endo-type amylase that hydrolyzes α-1,4-
glucosidic linkages of starch randomly, was purchased from Sigma
Aldrich (St. Louis, MO, USA) and used without further
purification. The protein content was determined from the optical
density at 280 nm using a molar adsorption coefficient of 13.8 x 104
_ _1
M 1cm (Alkazaz et al., 1996). We also used bovine serum
albumin (BSA, Sigma Aldrich) for the SDS poly-acrylamide gel
electrophoresis (SDS-PAGE) analysis. Starch (10 mg) was placed
in a 1.5 mL microtube and washed with 2.0% SDS solution to
remove surface proteins and then washed with 20 mM phosphate
buffer (pH 7.0) to remove the SDS. The starch granules were
suspended in 200 μL of buffer, and 200 μL of enzyme solution was
then added to the suspension. After mixing well, the sample was
placed in an ice bath for 10 min. The suspension was separated by
centrifugation at 15,000 G for 10 min at 4℃, and then the
supernatant was collected as a soluble fraction (S fraction). The
separated starch granules were resuspended in 200 μL of buffer.
The supernatant was collected as a washed fraction (W fraction) by
centrifugation under the same conditions as described above. The
microtube was transferred to room temperature and 200 μL of 2.0%
SDS solution was added to the tube. The starch was suspended by
mixing, and the supernatant was collected as a bound fraction (B
fraction) by centrifugation at 15,000 G for 10 min at 20℃.
Protein assay The S and B fractions collected in the
adsorption step were analyzed by SDS-PAGE. The proteins
obtained in each fraction were detected using Coomassie Brilliant
Blue stain (CBB R250; Bio-Rad Laboratories, Inc., Hercules, CA,
USA). The protein contents of the S and W fractions were
measured by the Bradford protein assay (Bio-Rad Laboratories,
Inc.) with a micro-plate reader (iMarkTM, Bio-Rad Laboratories,
Inc.). The protein content of the B fraction is the value of the added
protein minus the S and W fractions.
In vitro digestibility of starches with α-amylase Starch
digestibility by PPA was analyzed by measuring the content of
reducing sugars produced in enzyme reactions. The starch (40 mg)
was suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.5) with
0.4 mg sodium azide. Then, 40 μg of PPA (50 U, according to the
Certificate of Analysis from Sigma Aldrich) was added to the
suspension, and the suspension was maintained at 37℃. The
supernatant of each reaction mixture was collected by
centrifugation at 10,000 G for 5 min at 4℃. The reducing sugar
content was measured by the Somogyi-Nelson method.
Digestibility is shown as the ratio (as a percentage) of reducing
PPA Binding and Hydrolysis of Rice Starch Granules
Fig. 1. The procedure of adsorption analysis.
sugar content to the total reducing sugar content obtained from a
reaction using gelatinized starch.
Scanning electron microscopy (SEM) Starch was collected
from the enzyme reaction mixture by centrifugation, washed with
distilled water twice and then dried with ethanol. The starch was
next placed on a sample stage using carbon tape and coated by Au-
Pd using Ion Sputter (E1010; Hitachi High Technology, Tokyo,
Japan). The starch surface was observed by SEM (SU1510; Hitachi
High Technology) at accelerating voltage from 10 kV and 15 kV.
Results and Discussion
Adsorption of amylases SDS-PAGE results and the ratio of S
and B fractions in densitometric analysis after treatment by PPA
are shown in Fig. 2. The same experiment was performed using
BSA as a control for the adsorption analysis. BSA was detected in
365
the S fractions of all starches, but not in the B fractions (data not
shown). PPA was detected in both the S and B fractions of all
starches. As shown in the densitometric result (Fig. 2 right), the B
fraction band was denser and the S fraction band was lighter for the
wx/ae starch than for the wx and WT starches. This indicates that
the wx/ae starch has a stronger affinity to PPA, but is less digestible
than the other two starches (Kubo et al., 2010). In addition, another
protein was detected with lower molecular weight than the main
PPA protein (Fig. 2 left), possibly a pancreatic α-amylase-like
isoform. In this study, we focused on the main PPA protein because
its isoform protein was slightly detected in S and B fractions and
the difference between starches was not clear.
The protein content adsorbed by wx/ae, wx and WT starches is
shown in Fig. 3. This experiment was performed twice and similar
results were obtained. Representative results are shown in Fig. 3.
366 H. Takagi et al.

Fig. 2. SDS-PAGE results and the ratio of each fraction in the densitometric analysis of S and B fractions of PPA.
The gel image and ratio of fractions of PPA are shown at left and right, respectively. The values of the left images
are molecular weights (x 103). The densitometric analysis was performed using Image J from NIH (Schneider et al.,
2012). The bright and dark colors in the histogram correspond to S and B fractions, respectively. PPA was added at
the ratio of 22 U per mg starch. About 10 μg PPA was applied to SDS-PAGE as a control.

Fig. 3. The amount of PPA adsorbed by each starch. ● , wx/ae; ♦, wx; ▲, WT.
PPA has 1,250 U of enzyme activity per mg protein.

The wx/ae starch adsorbed significantly more PPA at a PPA
concentration of greater than 10 mg per g starch. At 55 mg/g starch,
approximately 66% of PPA was adsorbed. The PPA was entirely
adsorbed when its concentration was less than 17 mg/g starch;
whereas, in the wx starch, the PPA was entirely adsorbed below
8.4 mg/g starch concentration. At 51 mg/g PPA concentration, the
wx starch showed 39% adsorption. The WT starch showed the
lowest adsorption; only 66% of PPA was adsorbed at low PPA
concentrations and 37% at the highest concentration (61 mg/g
starch).
Thus, the wx/ae starch adsorbed approximately 80% more PPA
than the other two starches, and showed 80% greater specific
surface area (Takagi et al., 2017). We suggest that these two results
are related. The amount of enzyme bound to the starch granule
surface depends on granule size and surface area (Warren et al.,
2011). Warren et al. concluded that large granules have lower
affinity for amylase than small granules. The greater capacity of
wx/ae starch for PPA shown in our results is consistent with this
previous report, with the exception of its digestibility. Our findings
may provide a basis for further study of α-amylase binding to
starch granules.
Digestibility of starches by PPA The digestibility of starches
by PPA is shown in Fig. 4. The wx and WT starches were entirely
digested by PPA in 8 h, whereas the wx/ae starch was digested by
approximately 32% after 24 h. As shown in a previous report
(Kubo et al., 2010), wx/ae starch is less digestible than the other
two starches. The interaction between the physical properties of
starch and its hydrolysis by amylases has been studied (Qi and
PPA Binding and Hydrolysis of Rice Starch Granules 367

Fig. 4. The digestibility of each starch by PPA. ○ , wx/ae; ◇, wx; △, WT.

Fig. 5. SEM images of digested starch. Top, starches before digestion; middle, approximately 20% digested starches;
bottom, approximately 35% digested starches. Left, wx/ae; middle, wx; right, WT. The bars in the images represent 5 μm.

Tester, 2016a). Starch amylolysis depends on the composition of
starch granules (amylose/amylopectin ratio and lipids), the
structural features of starch granules (amylopectin double helices,
crystalline domains, blocklets, pores and growth rings), molecular
structure (phosphorylation) and structural modification by chemical
or physical processes. The wx/ae starch consists of pure
amylopectin with relatively long unit chains, and thus has more
amylopectin double helices (Kubo et al., 2010). Further, it has a
B-type polymorph pattern like potato starch, which is an
indigestible native starch. These changes at the molecular and
crystal levels might explain its resistance to PPA hydrolysis. The
wx/ae starch granules have many pores on their surfaces (Takagi et
al., 2017). Surface pores enable hydrolysis by amylases, and may
act as sites of initial enzyme hydrolysis and the pathway to the
granule interior for enzymes (Fannon et al., 1992). Maize starch,
which contains surface pores, is hydrolyzed by PPA faster than rice
starch, which is characterized by relatively few pores (Kong et al.,
2003). As shown in previous reports, pores on the starch surface
affect starch digestibility; however, wx/ae starch is resistant to PPA
hydrolysis. This implies that the surface pores of wx/ae starch
differ from those of other starches, such as maize.
Morphology of partially digested starches SEM images of
approximately 20% and 35% digested starches are shown in Fig. 5.
Starch samples of the former correspond to 8, 0.5 and 0.5 h
reactions of wx/ae, wx and WT, respectively, and those of the latter
correspond to 24, 2 and 2 h reactions. Many pores were observed
on the surfaces of digested wx and WT starches, but not digested
wx/ae starch, which was instead characterized by numerous surface
cracks. The pores on the granule surface of wx and WT starches
shared the typical pattern of α-amylase hydrolysis, which is in good
agreement with previous studies (Gallant et al., 1992, Oates, 1997,
Zhang et al., 2006, Zhu et al., 2011, Man et al., 2013). We propose
that following hydrolysis, the surface pores have been enlarged by
PPA or that weak areas (e.g., the amorphous lamellae) were
368
degraded by PPA. The cracking and erosion observed on the
digested wx/ae starch surface are similar to the amylase hydrolysis
patterns of high-amylose rice and potato starches (Man et al., 2013,
Dhital et al., 2014), which, like the wx/ae starch, are B-type
polymorphs. Man et al. discuss differences in digestion from the
viewpoint of the higher-order structure of starch granules. They
concluded that high amylose rice starch is resistant to PPA
hydrolysis because of the B-type crystallinity and the higher-order
structure in the peripheral region of subgranules and the
surrounding band.
Perhaps differences in the hydrolysis pattern of wx/ae starch
might be attributable to its higher-order nanostructure, which is
related to the primary and crystal structures resulting from the
double mutation. It is necessary to explain why the wx/ae starch
shows greater resistance to digestion than the other two starches
despite having many surface pores. Some of the genes coding for
α-amylases are upregulated by more than 100% 15 days after
pollination (Takagi et al., 2017). Perhaps the digestible part of
starch granules is eroded during maturation and pore development.
Further research is needed to clarify the process of pore
development and the behavior of pores in hydrolysis by amylolytic
enzymes.
Conclusions
We revealed that wx/ae starch has 80% more capacity to bind
PPA than wx and WT starches. We attribute this large binding
capacity to the 80% larger surface area of the wx/ae starch. The wx/
ae starch showed only 32% digestion by PPA in 24 h, while the
other two starches were almost completely digested after 8 h. The
hydrolysis pattern of wx/ae starch also differed from that of the
other two starches, which showed the typical pattern of hydrolysis
by α-amylases, and was similar to that of other B-type starches,
such as high-amylose rice and potato.
Acknowledgements This study was partially supported by the
Promotion Project of Knowledge-based Industrial Clustering of
Okinawa Prefecture. The authors thank Dr. Joseph Rodrigue
(Osaka Prefecture University) for reviewing the English of this
manuscript.
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