Báo cáo Y học: Comparison of starch branching enzyme I and II from potato ppt

Comparison of starch branching enzyme I and II from potato
Ulrika Rydberg
1
, Lena Andersson
2
, Roger Andersson
2
, Per A
˚
man
2
and Ha
˚
kan Larsson
1
1
Department of Plant Biology, and
2
Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden
The in vitro activities of purified potato starch branching
enzyme (SBE) I and II expressed in Escherichia coli were
compared using several assay methods. With the starch–
iodine method, it was found that SBE I was more active than
SBE II on an amylose substrate, whereas SBE II was more
active than SBE I on an amylopectin substrate. Both
enzymes were stimulated by the presence of phosphate. On a
substrate consisting of linear dextrins (chain length 8–200
glucose residues), no significant net increase in molecular
mass was seen on gel-permeation chromatography after
incubation with the enzymes. This indicates intrachain
branching of the substrate. After debranching of the

products, the majority of dextrins with a degree of
polymerization (dp) greater than 60 were absent for SBE I
and those with a dp greater than 70 for SBE II. To study the
shorter chains, the debranched samples were also analysed
by high-performance anion-exchange chromatography. The
products of SBE I showed distinct populations at dp 11–12
and dp 29–30, whereas SBE II products had one, broader,
population with a peak at dp 13–14. An accumulation of dp
6–7 chains was seen with both isoforms.
Keywords: gel-permeation chromatography (GPC); high-
performance anion-exchange chromatography (HPAEC);
Solanum tuberosum; starch branching enzyme; starch.
Starch is composed of linear and branched chains of
a-
D-glucose residues. The starch branching enzymes
(EC 2.4.1.18), which are responsible for forming
a-1,6-linkages in the glucan, can be divided into two
classes, class A (e.g. potato and maize SBE II, pea SBE I)
and class B (e.g. potato and maize SBE I, pea SBE II). The
A and B isoforms have highly similar amino-acid sequences
but usually differ by an N-terminal extension of the B form
and a C-terminal extension of the A form [1,2]. In vitro
studies of the maize isoforms have shown that SBE I
preferentially branches amylose, whereas SBE II preferen-
tially branches amylopectin [3]. Furthermore, SBE I
transfers longer chains than SBE II in vitro, and it has
been suggested that SBE I takes part in the synthesis of long
and intermediate chains during amylopectin biosynthesis
[4]. This model is supported by the observation of an
increased average chain length in amylopectin of amylose-

extender maize mutants that lack SBE II [5]. There is no
known mutant with reduced SBE I; however, the chain
length distribution in amylopectin was not significantly
affected in transgenic potato plants with a reduced level of
SBE I [6,7]. Interestingly, the physical properties of the
starch from transgenic potato with reduced SBE I levels are
clearly changed [6–8].
SBE I from potato was first characterized as having a
relative mass of 80/85 kDa [9,10]. In 1991 it was shown that
intact potato SBE I had a relative molecular mass of
103 kDa [11]. The active 80/85-kDa form present in potato
tubers was isolated and shown to have an almost intact
N-terminus and thus thought to result from proteolytic
cleavage in the C-terminal part [12]. Both intact SBE I and
the 85-kDa form have been shown to transfer chains from a
donor chain to an acceptor chain (interchain branching)
[13,14]. The occurrence of intrachain branching, i.e. transfer
within one and the same chain, could not be excluded in
those experiments.
Thorough studies of the activity of maize SBE I and II
isolated from endosperm [3] or expressed in Escherichia
coli [15,16] have been performed on various substrates.
Potato SBE II was first observed to be present as a granule-
bound protein in tuber starch [17]. SBE II seems to be less
abundant in potato tubers than SBE I and has not been
isolated from potato in amounts required for activity
analysis. Recently, however, both isoforms of potato SBE
have been expressed in E. coli [18–20], and the present
paper reports the activity of potato SBE I and SBE II with
amylose, amylopectin and linear dextrins as substrates.

MATERIALS AND METHODS
Branching enzyme isoforms
Potato SBE I and II were expressed in E. coli and purified
by ammonium sulfate precipitation, starch affinity chroma-
tography, and anion-exchange chromatography, as described
by Khoshnoodi (SBE I) [18] and Larsson (SBE II) [19]. The
preparations of potato SBE I and SBE II expressed in
E. coli were judged to be highly pure as SDS/PAGE
followed by Coomassie blue staining revealed only one
additional, faint band for SBE II and none for SBE I [19].
The protein concentration was measured by the Bradford
method with BSA as standard. Aliquots of 1 m
M in 50 mM
Correspondence to H. Larsson, Department of Plant Biology, SLU, PO
Box 7080, SE-750 07, Uppsala, Sweden. Fax: 1 46 18 673279,
Tel.: 1 46 18 673396, E-mail:
Enzymes: starch branching enzyme (EC 2.4.1.18); isoamylase
(EC 3.2.1.68).
Note: a wep page is available at />(Received 4 July 2001, revised 27 September 2001, accepted
28 September 2001)
Abbreviations: dp, degree of polymerization; GPC, gel-permeation
chromatography; HPAEC-PAD, high-performance anion-exchange
chromatography with pulsed amperometric detection; SBE, starch
branching enzyme.
Eur. J. Biochem. 268, 6140–6145 (2001) q FEBS 2001
Tris/HCl, pH 7.5, containing 1 mM dithiothreitol and 10%
glycerol buffer were stored at 270 8C until use.
Determination of branching enzyme activity on amylose
and amylopectin with the starch–iodine assay
Amylose (type III, Sigma) and amylopectin (Sigma) from

potato were typically dissolved at 10 mg
:
mL
21
in 0.5 M
NaOH. The solutions were buffered with 1 M KH
2
PO
4
and
pH adjusted to 7.5 with NaOH. The reaction mixtures
contained 0.6 mg
:
mL
21
substrate, 90 mM KH
2
PO
4
, and
0.01 m
M branching enzyme. Incubations were performed at
room temperature (22 8C), and aliquots were withdrawn at
several intervals between 5 and 180 min after the addition of
the SBE and terminated by heating at 95 8C for 5 min. A
100-mL sample of each aliquot was mixed with 900 mL
iodine solution (0.0125% I
2
and 0.04% KI, freshly made
from a 100 Â stock solution), and the absorbance between

400 and 800 nm was measured immediately on a Beckman
PU-70 spectrophotometer. The control did not contain
branching enzyme, but was otherwise treated as the other
samples. The experiments were repeated at least twice with
essentially the same results. When phosphate stimulation
was investigated, Tris/HCl, pH 7.5 (final concentration
50 m
M) was used to buffer the reaction mixtures and
KH
2
PO
4
, pH 7.5, was added to obtain increasing concen-
trations of phosphate. Incubations were terminated at
120 min and analysed by the starch –iodine assay.
Incubation of linear dextrins with branching enzyme for
gel-permeation chromatography (GPC) and
high-performance anion-exchange chromatography
(HPAEC)
Linear dextrins with a relatively narrow weight range
were produced by enzymatic degradation of retrograded
starch by the method of Andersson et al. [21]. The linear
dextrins were dissolved in a small volume of 2
M KOH and
diluted with Tris buffer to a final concentration of
4mg
:
mL
21
dextrins and 50 mM Tris/HCl, pH 7.6. To

900 mL of this solution was added 100 mL1m
M branching
enzyme or water (control sample). The samples were
incubated at room temperature for 16 h, and the reactions
terminated by heating at 100 8C for 5 min. After addition of
150 mL1
M acetate buffer, pH 3.6, the samples (1 mL)
were debranched with 295 U isoamylase (Hayashibara
Biochemical Laboratories Inc., Okayama, Japan) for 5 h at
38 8C. Before injection on to a column, the reaction was
terminated by heating to 100 8C for 5 min, and the pH
adjusted to . 10 with NaOH as described by Andersson
et al. [21].
Chromatographic methods
GPC was conducted as previously described [22] using a
Sepharose CL-6B column eluted with 0.25
M KOH. The
relative amounts of carbohydrate in the collected fractions
were measured by the phenol/sulfuric acid method [23].
HPAEC-PAD and a CarboPac PA-100 column was used as
described by Koch et al. [24]. In this method, correction for
detector response is performed. All experiments were run in
duplicate with only small differences between the samples.
RESULTS AND DISCUSSION
Comparison of SBE I and SBE II on the amylose and
amylopectin substrates
To compare the activity properties of potato SBE I and
SBE II, commercially available amylose and amylopectin
were used as substrates in a kinetic study using the starch –
iodine method (Fig. 1). SBE I was more active than SBE II

on the amylose substrate, whereas SBE II was more active
than SBE I on the amylopectin substrate. For both enzymes,
the greatest effect was observed on the amylose substrate
where the A
655
with SBE I decreased to 27% of that of the
control, and with SBE II it decreased to 46% (Fig. 1A). On
the amylopectin substrate, the A
520
with SBE I decreased to
74% of the control and with SBE II to 64% (Fig. 1B). Thus,
the potato isoforms differed in that SBE I was more active
than SBE II on amylose and SBE II was more active than
SBE I on amylopectin, which is in accordance with the
results obtained with maize SBE I and II [3,15,16].
The l
max
of the amylose substrate shifted from 616 nm to
543 nm after incubation for 180 min with SBE I and to
574 nm after incubation with SBE II for the same time
Fig. 1. Activity of SBE I and SBE II over time. Absorbance of the
starch–iodine complex after incubation of amylose substrate (A),
measured at 655 nm, or amylopectin substrate (B), measured at 520 nm,
for different periods of time with SBE I (O) or SBE II (Â)in90m
M
phosphate buffer. The absorbance of the control samples without
enzyme (set to 100%) was 1.09 for the amylose substrate and 0.53 for
the amylopectin substrate.
q FEBS 2001 Comparison of SBE I and II from potato (Eur. J. Biochem. 268) 6141
(Fig. 2A). Incubation overnight did not notably further

change the l
max
(data not shown). The difference in final
l
max
values and a comparison of the shapes of the two
spectra indicate that SBE I reduced more efficiently than
SBE II the long linear chains that mainly give rise to
absorbance above 600 nm. Similar differences between the
final l
max
values with amylose as a substrate were
previously observed with maize SBE I and II [3,15,16].
The l
max
values after incubation with the amylopectin
substrate shifted from 551 nm to 522 nm with SBE I, and to
538 nm with SBE II, after 180 min of incubation (Fig. 2B).
Similar values were obtained after incubation overnight (not
shown). These results differed from those with the maize
isoforms, which both reduced the l
max
from 530 nm to
about 490 nm [15,16]. Although this suggests that there may
be a difference between the enzymes from maize and potato,
the divergent results could also be due to a difference
between the substrates, with relatively long and linear chains
in potato amylopectin as indicated by the relatively high
l
max

of 551 nm as compared with a l
max
of 530 nm with the
maize amylopectin [15,16].
Effect of phosphate on the activity of potato SBE I and II
Phosphate has been reported to increase the branching
activity of SBE I and II from wheat [25] and SBE I from
potato [26]. The activity assay shown in Fig. 1 was
performed in 90 m
M phosphate. To investigate the effect of
phosphate, increasing concentrations of phosphate from 0 to
135 or 180 m
M were included in the iodine-activity assay
with commercially available amylose and amylopectin as
substrates. The delta absorbance at 655 nm for the amylose
substrate and 520 nm for the amylopectin substrate, after
120 min of incubation, as a function of phosphate
concentration is shown in Fig. 3. Phosphate concentration
did not affect the absorbance of the starch–iodine complex
in samples without enzyme (data not shown).
Close to maximal activation of both SBE I and II was
obtained at 90 m
M phosphate with the amylose substrate as
well as with the amylopectin substrate. With the
amylopectin substrate, the stimulatory effect was 130%
and 40% for SBE I and II, respectively. Half-maximal
Fig. 2. Activity of SBE I and SBE II after 180 minutes. Absorbance
spectra of the starch–iodine complex of the amylose substrate (A) and
the amylopectin substrate (B) incubated for 180 minutes with SBE I
(dashed lines), SBE II (dotted lines), or control sample without enzyme

(solid lines) in 90 m
M phosphate buffer. The vertical lines denote the
l
max
of the spectra.
Fig. 3. Effect of phosphate on the activity of SBE I and SBE II. The
delta absorbance of the amylose substrate (A), measured at 655 nm, and
the amylopectin substrate (B), mesured at 520 nm, after 120 min of
incubation with SBE I (K) or SBE II (Â) in increasing concentrations of
phosphate. Delta absorbance is defined as the difference between the
absorbance of the starch–iodine complex of the control and the
samples.
6142 U. Rydberg et al.(Eur. J. Biochem. 268) q FEBS 2001
activation was obtained for both isoforms at 15–20 mM
phosphate with both substrates, which is similar to that
reported for wheat SBE I and II [25]. A fivefold activation
by 10 m
M phosphate of potato and wheat SBE I has been
reported previously [25,26]. The effect of phosphate is
dependent on the buffer conditions [26], which could
explain the divergent results for potato SBE I. From the
studies performed by us and others, it cannot be excluded
that the observed stimulatory effect is a consequence of the
phosphate ions interacting with the substrate, and thereby
changing its structure, leading to enhanced enzyme
reactions. Further investigations are required to clarify this
and whether the effect of phosphate is of relevance in vivo.
Branching of linear dextrins
To obtain a more detailed comparison of the mode of action
of SBE I and SBE II, the branching products were further

examined using linear dextrins, prepared from commer-
cially available retrograded high-amylose maize starch [21],
as substrate. The majority of the chains of this substrate were
longer than 8 but shorter than 200 glucose residues and had a
peak maximum at degree of polymerization (dp) < 60.
These dextrins were less complex than commercially
available amylose or amylopectin and therefore more
suitable as substrates for the analysis of the branching
properties of SBEs by chromatographic methods.
The molecular mass distribution of the dextrin substrate
and the products formed after the branching process were
analysed by GPC. The elution profiles of the dextrins after
incubation with SBE I or SBE II for 16 h revealed only
small changes compared with the original substrate
(Fig. 4A). The absence of an increase in molecular mass
indicates that both enzymes mainly produced intrachain
branches, as we have previously reported for SBE I [21].
However, interchain branching cannot be excluded. Inter-
chain transfer of chains by 80/85-kDa potato SBE I has been
demonstrated by Borovsky et al. [14]. In a more recent
study, materials with higher molecular mass were formed,
possibly by multiple chain-transfer reactions, from linear
dextrins with relatively low molecular masses (dp 30 –40)
when incubated with 103-kDa SBE I from potato [13]. The
results show that potato SBE I has the ability to incorporate
glucans into starch in an interchain catalytic reaction,
although intrachain reactions could not be excluded. Thus,
in contrast with these previous studies, the results in Fig. 4A
suggest that potato SBE I and SBE II also produce
intrachain branches. The discrepancies between the studies

may be explained by differences in molecular masses and
phosphorylation of the substrates [13] or by differences
between the enzymes used. The experiments of Viksø-
Nielsen et al. [13] and Borovsky et al. [14] were performed
in 50 m
M phosphate and 100 mM citrate, respectively. The
results shown in Fig. 4 were obtained in the absence of
phosphate, but the same elution pattern was obtained in the
presence of 90 m
M phosphate (data not shown). Thus it
seems that SBE can produce branches by both intrachain
and interchain branching, depending on external factors.
After debranching with isoamylase, the GPC elution
profiles were shifted to lower molecular masses compared
with the original substrate, showing that extensive branching
had taken place (Fig. 4B). A more pronounced effect was
seen for SBE I than for SBE II. It is notable that, for both
enzymes, essentially all high-molecular-mass material had
disappeared. For SBE I, the majority of the dextrins with a
dp greater than 60 were missing and for SBE II those greater
than 70. At the same time, the proportion of short chains was
slightly increased for both enzymes and some new chains
shorter than those in the original substrate were detected.
These results are in agreement with the results from the
starch–iodine assay. Similarly, the product of maize SBE II
contained a higher amount of the longest chains than the
SBE I product [4].
To obtain a more detailed picture of the individual chains
produced by the enzymes, quantitative analyses of the
shorter unit chains (dp 6–47) were performed by HPAEC.

The relative distribution of the original substrate showed a
broad peak with no distinct populations with chains down to
dp 6 (Fig. 5C). By debranching the substrate with
Fig. 4. Activity of SBE I and SBE II on linear dextrins analysed
with GPC. Elution profiles of linear dextrins after incubation with
SBE I (K), SBE II (Â) or control samples without SBE added (W)in
50 m
M Tris buffer. Elution profiles were obtained before (A) and after
(B) debranching with isoamylase. Data for SBE I has previously been
published in Andersson et al. [21]. Dp values obtained after column
calibration with pullulan standards are shown on the upper axes.
q FEBS 2001 Comparison of SBE I and II from potato (Eur. J. Biochem. 268) 6143
isoamylase, the presence of 1,6-linkages in the substrate
could be excluded (not shown). After incubation with SBE I
and debranching with isoamylase (Fig. 5A), major popu-
lations were found around dp 11 –12 and 29–30,
respectively, as previously reported [21]. The unit chains
with a high dp were present in only small amounts.
Incubation with SBE II revealed a different picture
(Fig. 5B). The most abundant chains, on a weight basis,
had a dp around 13 –14 and a considerable quantity of
chains with dp 6 was produced. SBE II seems to be less
efficient in using the longer chains as a substrate than SBE I
as the longer unit chains were present in larger amounts in
the SBE II product. The original substrate had a broad range
of chains that to some extent interfered with the product
chains, making it difficult to interpret the results
quantitatively. The results from all three analyses show
that SBE I was capable of branching chains that were not
branched by SBE II.

The mechanism of chain transfer for maize branching
enzymes has previously been investigated using reduced
amylose (chain length 405) as substrate. The study of maize
SBE I showed populations of transferred chains with a dp
of 11 –14 and 31 after debranching of the enzyme products
[4]. A more detailed investigation of the shorter chains
(, dp 34) produced by maize SBE I revealed an increase in
chains of dp 11–12 as well as of dp 6 [27]. Maize SBE II
has been shown to transfer shorter chains than maize SBE I,
and the most abundant chains were reported to be around
dp 9 by Takeda et al. [4], whereas Guan et al. [27] reported
an increase in chains of dp 6 –7 with a smaller peak at dp
10–12. In accordance with this, incubation with potato
SBE I and II generated chains of dp 6–9, in decreasing
concentrations, which has been shown to be a general
feature for amylopectin in potato [28]. Thus, it is possible
that during biosynthesis of amylopectin the branching
enzymes produce a fraction of very short chains which are
normally elongated by starch synthase III, as indicated by
the interesting results of Edwards et al. [29] and work by
Abel, as reviewed in Kossmann & Lloyd [8], showing that
the relative amount of dp 6 chains in amylopectin was
significantly higher in transgenic potato lines with reduced
levels of starch synthase III.
The presence of phosphate interfered with the chroma-
tography of the carbohydrates on the HPAEC column.
Therefore the samples shown here were incubated in a Tris-
buffer. However, samples incubated in a phosphate buffer
gave the same elution patterns (not shown). The absence of
phosphate, which has been shown to influence branching

enzyme activity, did not qualitatively change the branching
patterns of the isoforms in our study.
This study was performed with purified potato SBE I and
II that had been expressed in E. coli. The specific activity of
expressed SBE I was about twofold higher than SBE I
isolated from potato tubers [18], indicating that the
expressed SBE I was fully active. We have failed to isolate
active SBE II from potato and to our knowledge it has not
been achieved. However, as the activity of expressed SBE II
was higher on the amylopectin substrate compared with that
of expressed SBE I, it is resonable to assume that the
expressed SBE II was also fully active. The results
presented here show that there are significant differences
in activity characteristics between potato SBE I and II.
Further studies are needed in order to fully understand the
functions of the two enzymes and the detailed structure of
the products obtained.
In conclusion we found that: (a) potato SBE I was more
active than SBE II on long linear substrates and SBE II was
more active than SBE I on an amylopectin substrate; (b) the
activity of both isoforms increased in the presence of
phosphate; (c) GPC results indicate that both SBE I and
SBE II mainly branched the linear dextrins used in this
study by intrachain branching; (d) debranching of the
products showed that both isoforms produced a small
fraction of dp 6–7 chains and a larger fraction of chains
< dp 11–14, and in addition SBE I produced a population
of dp 29–30 chains.
ACKNOWLEDGEMENTS
We are grateful to Dr E. Johansson who expressed and purified the

starch branching enzymes used in our experiments. This work was
funded by the Swedish Foundation for Strategic Research and the
Swedish Farmer’s Foundation for Agricultural Research.
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