Journal of Experimental Botany, Vol. 47, No. 304, pp. 1789-1795, November 1996
Journal of
Experimental
Botany
Purification and properties of a starch granule-degrading
a-amylase from potato tubers
Wolfgang Witt and Jorg J. Sauter1
Botanisches Institut der Universita't Kiel, Olshausenstr. 40, D-24098 Kiel, Germany
Received 6 March 1996; Accepted 28 June 1996
Abstract
An a-amylase (EC 3.2.1.1) was purified to apparent
electrophoretic homogeneity from potato [Solanum
tuberosum L.) tubers by affinity chromatography on a
starch granule column, Q-Sepharose chromatography,
and gel filtration. The enzyme was purified 24 300-fold
over the crude extract of soluble proteins with a yield
of 13.2% to a specific activity of 824 //mol min" 1 mg 1.
The classification as a-amylase was verified by substrate specificity and identification by HPLC of the
degradation products. This amylase migrated on
SDS-PAGE gels at 44 kDa, while it was detected by
native PAGE as a band on top of separation gels comprising amylopectin. The optimum pH value was
between 7.2 and 8.0. The increase of the pronounced
heat-lability in the presence of CaCI2 as well as the
lack of response to EDTA indicated that the activity
was not stabilized, but rather inhibited by Ca 2+ . The
purified a-amylase from potato tubers was reversibly
bound to starch granules from the same source, and
the enzyme was able to catalyse the degradation of
native granules in vitro. The observation that an amylase activity with the same characteristics as the soluble form was also associated to freshly prepared
granules indicated that the enzyme was at least partially localized in plastids. The results suggest that
this starch hydrolase is involved in the initiation of
reserve starch dissolution in potato tubers.
Key words: a-Amylase, enzyme purification, potato tuber,
Solanum, starch degradation.
Introduction
Conflicting results were reported on the content of amylolytic enzymes in potato tubers and on the mechanism of
1
To whom correspondence should be addressed. Fax: +49 431 880 1527.
& Oxford Univeraty Press 1996
starch dissolution (reviewed by Steup, 1988). In most
cases, it was assumed that the main pathway is phosphotolytic because high activities of glucan phosphorylase (EC
2.4.1.1) were found while the starch hydrolase content
was low or not detectable (Morrell and ap Rees, 1986;
Sowokinos, 1990). In contrast, amylase activity in protein
extracts from tubers and the electrophoretic separation
of several amylolytic bands has also been reported
(Nowak, 1977; Bailey et al., 1978; Davies and Ross, 1987;
Wegrzyn and MacRae, 1995), and Davies and Ross
(1987) showed that the a-amylase activity clearly
increased in the phase of maximal starch breakdown
during sprouting, whereas the phosphorylase activity
remained on a constant level. A sharp increase of starchhydrolysing enzymes was also observed during the first
weeks of cold-induced sweetening (Cottrell et al., 1993),
but the further characterization of these enzymes has not
yet been reported.
Recently, an endoamylase with some unusual properties
was described in another starch-storing tissue, the parenchyma cells of poplar wood (Witt et al., 1995). This
enzyme was hardly detectable in extracts of soluble protein due to its tight association to starch granules, and
the activity was only released by incubating the granules
with maltose or malto-oligosaccharides in high concentrations or by increasing the incubation temperature.
Evidence was found that both effects may contribute to
the initiation of cold-induced starch breakdown in poplar
wood. In addition to its affinity to native starch granules
this enzyme differed with respect to electrophoretic mobility and kinetic properties from the numerous other starch
hydrolases in this material (Witt et al,, 1995).
The objectives of the present investigation were to look
for a similar starch granule-bound hydrolase in potato
tubers, a storage organ with a completely different physiological background from the taxonomically non-related
species (poplar wood), and to develop a purification
1790
Witt and Sauter
procedure so as to study the role of this amylase in the
breakdown of granular starch without interference by
other amylolytic enzymes. The presence in tubers of an
enzyme with similar properties to the granule-degrading
amylase in poplar might indicate that these enzyme forms
are widely distributed among starch-storing tissues.
Materials and methods
granules, aliquots of the granule suspension comprising 100 mg
starch were centrifuged, and the supernatant was discarded
before 3.5 units of purified a-amylase in 0.5 ml buffer A, but
supplemented with 0.2% (w/v) BSA and 0.02% (w/v) Na-azide,
were added to the pellets. The reaction vials were agitated at
25 °C with an overhead rotator to avoid sedimentation of the
granules. Samples of granules without amylase were incubated
in parallel. The sum of the released glucose, maltose, and
malto-oligosaccharides was determined as Glc after digestion
with AMG (amyloglucosidase, EC 3.2.1.3) as previously
reported (Witt and Sauter, 19956).
Plant material
Tubers of potato (Solarium tuberosum L.) were purchased from
local markets and stored at 20-22 °C in the dark. The cv.
Sirtema was used for most enzyme purifications, but largely the
same results were obtained with cvs Bintje, Erstling and Prior.
Extraction and purification of amylase activity
Tubers (4-6 cm in diameter) were peeled and, if necessary, sprouts were removed. This material (400 g) was sliced
with a coarse grater and transferred into 800 ml of buffer
A (50 mM HEPES/KOH, pH 8.0, 5 mM MgCl2, lOmM
2-mercaptoethanol, 0.2 mM Na-EDTA, 0.05% (w/v) defatted
BSA). The tissue was disrupted for 30 s with an Ultra-Turrax
homogenizer (Ika-Werk) at full speed. The cell debris was
removed by filtration through Miracloth and centrifugation at
16 000 xg for 20 min. The supernatant was pumped through a
column (1.6 x 15 cm) packed with starch granules from potato
(ICN Biomedicals) at a flow rate of 2 ml min" 1 . The granules
had been washed twice in water and twice in buffer A before
they were packed into the column. The column was further
washed at 0.2 ml min" 1 for 12-14 h with buffer A and
subsequently for 1 h at 1 ml min~' with buffer B (same as A,
but BSA was replaced by 0.05% (w/v) Triton X-100). Bound
endoamylase activity was subsequently eluted with 100 ml of
0.7 M maltose in buffer B at a flow rate of 1 ml min" 1 . The
maltose eluate was not collected, but it was directly passed
through a column (0.8 x 9 cm) of Q Sepharose ff (Pharmacia).
Fractions of the ion exchanger eluate which showed amylase
activity were combined and concentrated to approximately
10 ml by ultrafiltration using YM10 membranes (Amicon). The
filtrate was desalted (PD-10 columns, Pharmacia) in buffer C
(same as B, but without Triton X-100) and further concentrated
by ultrafiltration (YM 10) to about 1.5 ml. Finally, traces of
carbohydrates and other contaminants were removed by gel
filtration (Toyopearl HW-55S from Tosohaas, column size
1.6 x 48 cm). The column was equilibrated and eluted at a flow
rate of 0.25ml min" 1 in buffer D (same as B, but the
concentration of the detergent was 0.1%).
All steps were carried out at 0—4 °C. In some cases, buffers B
and D were replaced by buffer C in order to obtain a detergentfree enzyme.
Starch granule preparation and amylolytic degradation
Peeled and sliced potato tubers (40 g) were homogenized by
mortar and pestle in 80 ml buffer A. The homogenate was
filtered and centrifuged as above to obtain the crude starch
pellet. This pellet was resuspended in 20 ml buffer A and filtered
a second time through Miracloth. The granules were left to
settle for 30 min. The supernatant was then removed, and the
grains were washed twice more by settling and resuspending in
buffer A. Contamination by tissue debris or by organelles were
not detectable by light microscopy. The starch content was
determined as described elsewhere (Witt et al, 1995).
For the quantification of the degradation of native starch
Enzyme assays
The tests on amylase activity using soluble polysaccharides
(starch, amylose, amylopectin, /Mimit dextrin from amylopectin,
pullulan) as substrates by the dinitrosalicylic acid procedure
and on /3-amylase with the blocked PNP-malto-oligosaccharides
of the Testomar test kit (Behringwerke) as well as the assays
on the degradation of maltose and malto-oligosaccharides were
carried out as outlined in previous communications (Witt and
Sauter, 1994a, b; Witt and Sauter, 1995a), except that CaCl2
was not included in the reaction mixtures and that the buffer
was HEPES/KOH, pH 8.0. Amylase activity was also determined using the chromogenic substrate starch azure (Sigma) as
described elsewhere (Witt et al, 1995), but at a final substrate
concentration of 0.9% (w/v) and without DTT. An activity unit
is defined as the increase of absorbance at 595 nm of 1 unit per
minute. All tests were run in duplicate at 30 °C. Slight
modifications of these procedures are detailed in the legends of
Figures and Tables.
Other analytical methods
Proteins were separated by SDS-PAGE under denaturing,
reducing conditions using 12% gels under a Laemmli-buffer
system (Laemmli, 1970). The isoenzyme profile of amylase
samples was analysed by the same system, but on gels without
SDS (6% monomer concentration) containing 0.05% (w/v)
amylopectin as described in previous communications (Witt
and Sauter, 1994a; Witt et al., 1995). Briefly, the samples were
electrophoresed at 30 mA for 4 h at 2-4 °C. The gels were
incubated on ice for 30 min in buffer (lOOmM MOPS/KOH,
pH 7.0, 5 mM DTT, 5 mM CaCl 2 ) and then, after a change of
the solution, for 2 h at 30 °C in the same medium. Amylolytic
bands were visualized by negative staining with Ii/KI. In some
cases, gels without amylopectin were used for native PAGE.
The incubation medium to detect the bands therefore contained
soluble starch from potato in a concentration of 1% (w/v).
Protein was determined by the Coomassie dye-binding assay
(Bradford, 1976). Samples of the purified enzyme in Tritonbuffer were extracted and concentrated with phenol/ether (Sauve
et al, 1995) and freeze-drying before the tests were run.
Standard solutions of BSA were treated in the same way.
Maltose and malto-oligosaccharides in fractions of chromatographic separations were detected by the H2SO4/phenolprocedure (Dubois et al, 1956). For the detection of amylase
digestion products, the purified enzyme in Triton-buffer (1.1
units) was incubated at pH 8.0 for 2 h at 30 °C with 27 mg
soluble potato starch (Sigma) in a total volume of 1.5 ml.
Blanks without enzyme were run in parallel. Carbohydrates
were then extracted and analysed by HPLC as described
elsewhere (Witt and Sauter, 1995a), except that the flow rate
was increased to 1.5ml min"'. The peaks were identified by
comparison with authentic linear malto-oligoglucans (Sigma).
Amylase in potato tubers
Results
Purification of a potato endoamylase
Electrophoretic separations of crude potato tuber protein
extracts using amylopectin-containing gels revealed several
bands of amylolytic activity (Fig. 1, lane A/a). Affinity chromatography on a column of starch granules from a commercial
source and selective desorption with maltose was used as a first
and most efficient step to separate these enzymes. Only one
band, migrating on top of the separation gel (Fig. 1, lane A/c),
was bound to the granules under appropriate conditions (pH 8,
low temperature), whereas all other bands were recovered in
the effluent (Fig. 1, lane A/b). The further purification by a
passage through Q Sepharose and gel filtration yielded an
enzyme preparation which showed a single band of 44 kDa in
SDS-PAGE (Fig. 1, lane B).
It has to be noted that the purified amylase entered native
gels which did not contain substrate in the gel matrix in a
similar way as a recently described endoamylase from poplar
wood (Witt et al, 1995). The band on top of the gels (Fig. 1 A)
was obviously only retarded by interaction with the immobilized
amylopectin (results not shown).
The results of a typical purification run are summarized in
Table 1. The enzyme was purified about 24 OOO-fold over the
crude extract with a recovery of 13%. The specific activity
measured with starch azure was 8090 U mg" 1 , being equivalent
to 824 /xmol mg" 1 min" 1 with /3-limit dextrin as substrate. The
B
kDa
-66
-45
-36
1791
yield is certainly underestimated because the substrate, starch
azure, was also attacked by the other amylolytic enzymes,
which were not bound to the affinity column. This fraction
amounted to up to 50% of the total activity (results not shown).
Column buffers comprising Triton X-100 in a final concentration of 0.1 % were used in the experiment of Table 1 in order to
stabilize the activity (see below) and to achieve higher yields
and specific activities. The purification was also carried out in
the absence of the detergent to study the effects of other
activating compounds and to evaluate the ability of this enzyme
to degrade starch granules. An about 10800-fold purified
enzyme preparation with a specific activity of 420 U mg" 1 was
obtained in this way in a typical run.
Properties and classification of the purified enzyme
The potato amylase shows the same substrate specificity
as a recently described starch-bound enzyme from poplar
wood (Witt et al., 1995). Some common polyglucan
substrates—soluble starch, amylose, amylopectin, /Mimit
dextrin—were readily degraded at nearly the same rate.
Pullulan, maltose, maltotriose, maltotetraose, and the jSamylase specific PNP-malto-oligosaccharides of the
Testomar test kit, were not at all attacked (results not
shown). The separation by HPLC of the reaction products
of the degradation of soluble starch (Fig. 2) shows several
prominent peaks which co-migrated with authentic maltooligosaccharides of the chain length G3-G9, G6-G8
being the main components, while glucose and maltose
were hardly detectable. These bands were not found with
control samples without amylase. Taken together, these
results are consistent with the classification of this enzyme
as a-amylase.
The purified enzyme shows a broad pH-response with
maximum activity in the slightly alkaline range between
-29
-24
-20
0.4
-TD
0.2
G6
G7
G4
o
fig. 1. Separation by native PAGE (A) and by SDS-PAGE (B) of
amylolytic enzymes from potato tubers. Aliquots of 20 ;J in 20%
glycerol of the crude protein extract (lane A/a), the crude extract after
the passage through the starch affinity column (lane A/b), the maltoseeluate from the starch column (lane A/c), and the maltose-released
amylase from native starch granules (lane A/d) were applied to the gel
containing amylopectin. Another sample of the purified a-amylase was
analysed by SDS-PAGE (lane B). The protein band was detected by
silver staining. The positions of marker proteins of known molecular
mass are indicated on the right; TD, tracking dye.
0
©
Q
-0.2
/\
"1
J
10
20
Elution time (min)
30
Rg. 2. Product analysis by HPLC of the degradation of soluble potato
starch by purified potato a-amylase. The peaks labelled G4-G9
co-eluted with authentic linear oligoglucans of chain lengths 4-9.
Table 1. Purification of a-amylase from tubers o/Solanum tuberosum L., cv. Sirtema
The purification was run as outlined in Materials and methods in buffers comprising Triton X-100. The activity was tested with starch azure.
Purification step
Total activity
(units)
Specific activity
(units mg" 1 )
Crude extract
Gel filtration
442
58.2
0.333
8090
Purification
Recovery
(%)
24300
13.2
1792
Witt and Sauter
Table 2. Activity of a-amylase from potato tubers as affected by
various activating and inhibiting agents
The purified enzyme in buffer containing Tnton X-100 (right column)
or without detergent (left column) was incubated at 25°C for 10min
in the presence of the substances in two concentrations (lOmM and
50 mM, or 0.1% and 0.5%, w/v) before the reaction was started by
addition of the substrate, starch azure. The relative activity refers to
the activity (as %) of assays with 0.5% BSA (left column) or without
additives (right column).
Reagent
5
6
6.5
7
7.5
8
Concentration
Relative activity
— Triton
+ Triton
4.6±3.1
3.1 ± 1-7
4.4±3.7
4.1 ± 1.5
5.9±2.7
5.2±3.6
7.0 ±5.3
88.1 ±16.2
100
67.9±21.8
80.0 ±9.1
13.7±11.4
25.5 ±20.7
6.2 ±4.4
10.1 ± 6 0
100
8.5
pH
Fig. 3. The pH-effect on potato tuber a-amylase. The activity of the
purified enzyme (O) and of the non-bound protein fraction of the
starch column ( • ) was tested with amylopectin from potato as
substrate. The buffer substances used were MES/K.OH (5.5-6.2),
MOPS/KOH (6.5-7.0), and HEPES/KOH (7.2-8.5)
CaCl2
EDTA
DTT
BSA
Triton
pH 7.2 and 8 (Fig. 3). High amounts of enzyme were
used in these assays to reduce the incubation time to
5 min, and the concentration of BSA in the assay medium
was increased to 0.2% (w/v) in order to avoid the inactivation at low pH. In contrast, the amylolytic enzymes which
were not bound to the affinity column were active over
the entire pH range, with maximum activity at pH 6.7
(Fig. 3).
The purified potato amylase was rapidly inactivated in
buffers without Triton and in the absence of other stabilizing agents, especially at low concentration of the enzyme.
A loss of activity of 40% was observed within 1 d of
storage in buffer C at 4°C, and the activity of a 10-fold
diluted sample was reduced by 75% under the same
conditions. Bovine serum albumin was therefore added
to enzyme solutions immediately upon completion of a
purification run in buffers without detergent to stabilize
the activity. The instability was much more pronounced
at elevated temperature; the activity of the 10-fold diluted
enzyme solution in buffer C was reduced to 5.4 ±3.1%
(mean ±SD, n = 4) within 10 min at 25 °C.
The result of a survey of several compounds that might
affect the activity is presented in Table 2. In addition to
Triton, only BSA could prevent the thermal inactivation
of the enzyme in buffer C (Table 2, left column). Other
neutral or zwitterionic detergents with a high critical
micelle concentration, NOGA w-octanoyl-jS-D-glucosylamine) and SB 12 (;V-dodecyl-yV,./V-dimethyl-3-ammonio1-propanesulphonate), showed no significant effects. The
activity of amylase preparations which were obtained by
purification in the presence of Triton was not or only
slightly affected by the tested substances, with two exceptions: CaCl2 and the zwitterionic detergent SB 12, both
used in high concentration, were clearly inhibitory
(Table 2, right column).
The results in Table 2 with CaCl2 and EDTA are
SB12
NOGA
10 mM
50 mM
10 mM
50 mM
10 mM
50 mM
0.1%
0.5%
0.1%
0 5%
0.1%
0.5%
0.1%
0.5%
62.1 ±8.7
102±5.0
111.7±5.6
117.4 ±5.7
115.6±7.8
110.8 + 48
123.4±16.7
116.8±122
1169±10.5
111.6+12.5
74.8 ±7.8
I5.6±7.8
94.6±2.5
80 9± 11.5
especially noteworthy because endoamylases are widely
considered as Ca2 + -containing proteins (MacGregor and
Svensson, 1989). Aliquots of the Triton-enzyme were
therefore depleted of divalent cations by addition of
EDTA and a passage through a desalting column in order
to investigate the effect in more detail. Figure 4 shows
that the exposure of this enzyme preparation to 25 °C in
the presence of CaCl2 led to a rapid reduction of the
activity; about 50% was lost after 30 min and 94% after
5 h. Inactivation at this temperature was also observed in
0
1 2
3
4
5
Incubation time (h)
Fig. 4. Effect of Ca 2+ on the heat-lability of potato tuber amylase. A
sample of the purified enzyme in Triton-buffer (0.6 units in 2.5 ml) was
depleted of MgG 2 by addition of EDTA to a concentration of 10 mM
and by a passage through a desalting column in buffer D, but without
MgCl2 . EDTA ( • ) or CaCl2 ( • ) was then added to 10 mM. These
enzyme samples and another one without additions (O) were kept on
ice for 5 h before they were incubated at 25 r C. Aliquots were then
removed at the indicated times and tested for amylase activity with
starch azure as substrate.
Amylase in potato tubers
the presence of EDTA or without any additives, but at a
clearly lower rate (Fig. 4). No activity was preserved
after 24 h at 25 °C irrespective of the composition of the
incubation medium. In contrast, no losses of activity were
observed with Ca2+-free enzyme samples during storage
at 4°C for up to 5 d (results not shown).
Degradation of starch granules and the starch-binding of ctamyiase
In order to investigate the ability of the purified potato amylase
to catalyse the breakdown of native starch granules, the BSAstabilized enzyme and starch from the same batch of tubers
were incubated, and the released carbohydrates were quantified
as Glc after digestion with AMG (Fig. 5A). An almost linear
progression of starch degradation (77 nmol h" 1 was observed
during the first 2 h of incubation. The release of Glc units then
slowed down to a rate of about 20 nmol h ' 1 during the next
6 h. Further incubation for up to 3 d showed that starch
breakdown thereafter was still detectable, but at a much lower
rate (results not shown). The slowing down of starch degradation
after 2 h was probably not caused by the concomitant
inactivation of the amylase because the losses of total activity
were only about 9% within 8 h. Instead, a pronounced reduction
of the affinity of the granules for the enzyme was detected,
especially within the first 2 h of the incubation. The ratio of
bound to free amylase activity changed from about 1.5 at the
beginning to 0.9 at 2 h and to 0.5 at 8 h (Fig. 5B).
Figure 5A also shows that starch granules released a small
but clearly detectable amount of Glc units in the absence of
exogenously added amylase. This degradation may have been
caused at least partially by the amylase activity which was
adsorbed to the surface of freshly prepared granules or enclosed
within the starch matrix. The major part of the amylolytic
activity in the crude filtrate of the tuber homogenate was
recovered in the 'soluble' form, but 11.4±2.9% (mean±SD,
« = 6) was found in the centrifugation pellet. Further washing
Incubation time (h)
Fig. 5. Degradation of native starch granules. Starch granules from
potato tubers were prepared and incubated with (O) or without ( • )
purified o-amylase as detailed in the methods section. The incubations
were terminated at the indicated times, and the released glucans were
determined as Glc units after digestion with AMG (panel A). The
activity of the granule-bound ( • ) and the free amylase activity ( • )
was determined in parallel using starch azure (panel B).
1793
of the granules reduced this percentage, but amylase activity
was always detectable with exogenously added substrates. This
activity was released almost completely by incubating the
granules at 22 °C in the presence of maltose (0.5 M) or a
mixture of malto-oligosaccharides (Dextrin 20, Fluka) in a
concentration of 10% (results not shown). The released enzyme
exerts the same electrophoretic mobility (Fig. 1, lane A/d) and
kinetic properties (results not shown) as the 'soluble' form.
Another main factor to affect the starch binding is temperature.
When starch granules were incubated with purified a-amylase
under the same conditions as in the experiment of Fig. 5, 96%
of the activity was starch-bound at 1 °C, 78% at 15°C, and 33%
at30°C.
Discussion
The specific activity of the purified potato a-amylase
compares well to other highly purified endoamylases
(Ziegler, 1988), but the value of 824 ^mol m i n t i n g " 1
is about 50% lower than the highest reported specific
activities of enzymes from pea shoots (Beers and Duke,
1990) and pea cotyledons (Hirasawa and Yamamoto,
1991). The apparent Mr of 44 kDa as detected by
SDS-PAGE is in the same range of 42-45 kDa of most
other so far identified endoamylases from various other
higher plant sources (references in Witt and Sauter, 1996).
In contrast, the potato tuber endoamylase shows some
other unusual attributes, some of which may be of
physiological relevance. The activity was increased by
chelation of divalent cations, and the addition of Ca 2+
led to the accelerated inactivation at 25 °C rather than to
a protection from thermal inactivation as observed with
many other endoamylases (Steup, 1988; Beck and Ziegler,
1989). These enzymes belong to a large family of structurally related starch-degrading hydrolases (MacGregor and
Svensson, 1989; MacGregor, 1993; Svensson, 1994). All
a-amylases contain a Ca2 """-binding domain of major
importance for the stabilization of the tertiary structure
(MacGregor and Svensson, 1989). It therefore appears
to be reasonable that the activity is readily lost in most
cases upon an exposure to Ca2+-complexing agents.
However, the resistance of some a-amylases against this
treatment was also reported (Bulpin and ap Rees, 1978;
Okita et al., 1979; Bertoft et al., 1989; Jacobsen et al.,
1986; Ziegler, 1988). This finding does not exclude the
possibility that these enzymes and the endoamylase
described here are not Ca2+-proteins. Calcium may be
tightly bound and be inaccessible to the chelator. Detailed
structural information would be required to decide this
question.
Another characteristic feature of the potato tuber amylase is the pH-profile, with maximum activity between
pH 7.2 and 8.0, in contrast to most typical endoamylases
from higher plants with an optimum pH around 6 (Beck
and Ziegler, 1989). Provided that the pH in the amyloplast
stroma is similar to the value in chloroplasts, i.e. 7.2-8.2
1794
Witt and Sauter
(Werdany et al., 1975), the potato enzyme would be well
adapted in this respect to a role in starch degradation.
The finding that this amylase is able to catalyse the
breakdown of native starch granules in vitro also supports
this assumption. The results of Fig. 5 demonstrate that
the initial rate of the degradation was only maintained
for a short period, and that the subsequent slowing down
was not due to a concomitant loss of enzyme activity
during the incubation. The effect was better correlated
with the desorption of the enzyme from the starch granules. Many starch-degrading enzymes contain a second
starch granule binding site different from the active site,
and the binding to the substrate via that second site is a
prerequisite for the degradation of intact granules
(MacGregor, 1993; Svensson, 1994). These enzymes can
be released from granules by the reaction products, by
malto-oligosaccharides and also maltose in high concentrations. However, it is highly improbable that this effect
takes part in the release of the amylase in the in vitro
experiments of Fig. 5 because the concentration of the
accumulating reaction products is far too low to affect
the binding (MacGregor and Morgan, 1986). Other
explanations would be based on the assumption that the
enzyme was preferentially bound to easily accessible binding sites, possibly on damaged granules, which were
rapidly degraded at the beginning of the incubation
(MacGregor and Ballance, 1980), or that nona-l,4-glucosidic bonds at the granule surface may block
further attack by the endoamylase (Sun and Henson,
1990) as well as the binding of the enzyme. Evidence was
presented that the pronounced synergistic effect of
endoamylases and a-glucosidases is due to the ability of
the latter enzyme to eliminate these bonds in native starch
granules of barley (Sun and Henson, 1990). If similar
effects also apply to starch degradation in potatoes, a
continuous degradation at high rate would require the
participation of other classes of starch-degrading enzymes
than endoamylases.
The view that the potato a-amylase under investigation
has a physiological role in starch degradation is furthermore supported by the observation that the enzyme is
associated to a significant extent to starch granules. It is
concluded that the amylase is at least partially localized
in amyloplasts. Another starch-bound endoamylase from
poplar wood, in contrast, is more tightly and nearly
completely bound to native starch granules at low temperature (Witt et al., 1995). This difference may not be
exclusively due to divergent properties of the enzyme;
structural differences among the starch granules may have
to be considered in this respect (Cottrell et al., 1995).
Both enzymes otherwise exhibit highly similar properties:
both are most active in the alkaline pH range, neither
respond to EDTA, they attack native starch granules,
they require similar conditions to stabilize the activity,
they show the same electrophoretic mobility, and they
are both released from starch granules at elevated temperature and by maltose and malto-oligosaccharides. Further
studies are required to show whether starch-bound amylases of this type are present in other reserve starchstoring organs.
In conclusion, the results presented here support the
view that an a-amylase takes part in the breakdown of
starch in potato tubers. The low thermal stability and the
high affinity to granules at low temperature might suggest
a role for the enzyme especially in cold-induced sweetening. More investigations, especially on the subcellular
distribution and the interaction with other enzymes, are
necessary to estimate the significance of the a-amylase in
comparison to other pathways of starch degradation. At
least, the widespread assumption that the starch breakdown in this material is exclusively phosphorolytic
(Morrell and ap Rees, 1986; Sowokinos, 1990) has to
be revised.
Acknowledgements
The skilful technical assistance of Mrs B Langhoff and Mrs S
Karg is gratefully acknowledged. This work was supported by
a grant of the Deutsche Forschungsgemeinschaft.
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