Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato

Article abstract of DOI:10.1271/bbb.67.1609

Purple acid phosphatase (PAP) was purified from sweet potato dry powder, which is used as a food additive. Spectrometric and enzymatic analyses, and analysis of the amino-terminal sequence indicated that the purified purple acid phosphatase was PAP1. High

Full text of DOI:10.1271/bbb.67.1609

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Purification and Characterization of Purple Acid
Phosphatase PAP1 from Dry Powder of Sweet Potato
Tatsuya KUSUDOa, Toshiyuki SAKAKIa & Kuniyo INOUYEa
a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
UniversityKitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
Published online: 22 May 2014.
To cite this article: Tatsuya KUSUDO, Toshiyuki SAKAKI & Kuniyo INOUYE (2003) Purification and Characterization of Purple
Acid Phosphatase PAP1 from Dry Powder of Sweet Potato, Bioscience, Biotechnology, and Biochemistry, 67:7, 1609-1611,
DOI: 10.1271/bbb.67.1609
To link to this article: http://dx.doi.org/10.1271/bbb.67.1609
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Biosci. Biotechnol. Biochem., 67 (7), 1609–1611, 2003
Note
Puriˆcation and Characterization of Purple Acid Phosphatase PAP1 from
Dry Powder of Sweet Potato
Tatsuya KUSUDO, Toshiyuki SAKAKI,† and Kuniyo INOUYE
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
Received March 12, 2003; Accepted April 8, 2003
Purple acid phosphatase (PAP) was puriˆed from
sweet potato dry powder, which is used as a food addi-
tive. Spectrometric and enzymatic analyses, and analy-
sis of the amino-terminal sequence indicated that the
puriˆed purple acid phosphatase was PAP1. High activ-
ity in neutral and acidic conditions, broad substrate
speciˆcity, and good thermal stability of PAP1 suggest
the possibility of practical applications of PAP1.
z
natant was put on a non-denaturing 4 to 20 linear
gradient polyacrylamide gel at 4
9
C. After staining
with Fast Blue RR and -naphthyl phosphate, four
a
distinct bands were observed on the gel in fresh sweet
potato (data not shown). On the other hand, only
one major band was detected in the dry powder,
suggesting that only one isoform of the acid phos-
phatases retained the activity during the processes for
production of dry powder.
Key words: purple acid phosphatase (PAP); sweet
potato PAP1; dry powder of sweet pota-
to; binuclear Fe–Mn center
The purple acid phosphatase was puriˆed as
follows. The sweet potato dry powder (250 g) was
suspended in 500 ml of 50 m
taining 0.5 NaCl, and ˆltered through ˆlter paper
to yield a crude extract. After addition of ammonium
sulfate at 25 saturation, the crude extract was cen-
trifuged at 10,000 for 30 min. The supernatant
M
acetate, pH 5.6, con-
M
Purple acid phosphatases (PAPs) are widely dis-
tributed in animals, plants, and microorganisms, and
catalyze the hydrolysis of activated phosphoric acid
ester and phosphoric acid anhydrides in the pH range
4–7. Recently, the reaction mechanism of PAP from
kidney beans was proposed on the basis of its
crystal structure.1) This enzyme has a binuclear
Fe(III)–Zn(II) center, and shows speciˆcally high
activity toward ATP as an ATPase. In contrast, PAP
from sweet potato has a binuclear Fe(III)–Mn(II)
center.2) Unfortunately, however, the tertiary struc-
ture of PAP from sweet potato has not been identi-
ˆed yet. Comparison of tertiary structure of PAP
between sweet potato and kidney beans would reveal
the structure-function relationship of the PAPs and
their physiological roles. A convenient method for
puriˆcation of PAP would allow us to obtain a large
amount of pure PAP for crystallization. In this
study, we attempted to establish a highly convenient
and reproducible method for puriˆcation of PAPs
from a dry powder of sweet potato.
z
×
g
was applied to a Toyopearl phenyl-650M column
×
(39 mm I.D. 17 cm) equilibrated with the same
buŠer. The column was washed with 1 l of equilibra-
tion buŠer and eluted with a linear gradient of am-
monium sulfate (1.0–0.5
acid phosphatase was dialyzed against 5 L of 50 m
acetate, pH 5.6, and put on a DEAE A‹-gel Blue
(27 mm I.D. 19 cm) equilibrated with the same
M
). The eluent containing
M
×
buŠer. After washing the column with 5 volumes of
the same buŠer, the acid phosphatase was eluted with
a linear gradient of NaCl (0–1.5
taining acid phosphatase was dialyzed against 50 m
acetate, pH 5.6, containing 0.5 NaCl, and applied
to a Con A Sepharose 4B column (16 mm I.D.
M
). The fraction con-
M
M
×
6 cm) equilibrated with the same buŠer. The column
was washed with 10 column volumes of the same
buŠer, and eluted with a linear gradient of glucose
concentration (0–0.2 ). The fraction containing acid
M
In order to examine the multiplicity of PAP in the
materials, non-denaturing PAGE followed by activi-
ty staining was done as follows. Fresh sweet potato
tissue was quickly frozen in liquid nitrogen, ground
phosphatase was concentrated by ultraˆltration
(Amicon YM-30) and put through gel permeation
chromatography on a Sephacryl S-200 HR column
×
M
(16 mm I.D. 55 cm) equilibrated with 50 m
in a cold mortar, and suspended in 1.0 ml 0.1
M
acetate, pH 5.6, containing 0.5 NaCl. Table I sum-
M
acetate buŠer (pH 6.0). After centrifugation of the
marizes the puriˆcation of the acid phosphatase. The
speciˆc content of acid phosphatase of the crude
×
g for 10 min at 49C, the super-
suspension at 10,000
†
To whom correspondence should be addressed. Tel:
81-75-753-6267; Fax: 81-75-753-6265; E-mail: tsakaki kais.kyoto-u.ac.jp
＋ ＋ ＠
Abbreviations: PAP, purple acid phosphatase; ALP, alkaline phosphatase; pNPP, p-nitro-phenyl phosphate

1610
T. KUSUDO et al.
juice from the dry powder was nearly the same as
that from fresh sweet potato (data not shown). SDS-
PAGE analysis under reducing conditions indicated
activity of the puriˆed PAP1 was calculated to be 608
±
20 U mg protein from ˆve separate experiments.
W
SDS-PAGE analysis under reducing conditions
demonstrated that a molecular mass of the puriˆed
acid phosphatase was 60,000. On the other hand, gel
permeation chromatography with Sephacryl S-200
HR indicated that the puriˆed acid phosphatase had
a molecular mass of 120,000. These results strongly
that the extract of the dry powder contained b-amy-
lase with a molecular weight of 53,000 and a protein
with a molecular weight of 24,000 as major proteins
(Fig. 1). The eluent from the DEAE A‹-gel blue
column chromatography showed a clear band corre-
sponding to purple acid phosphatase (Fig. 1). The
speciˆc activity was dramatically increased after
DEAE A‹-gel blue column chromatography. Con
A-Sepharose and Sephacryl S-200 column chro-
matography also increased the speciˆc activity. The
suggest that the puriˆed acid phosphatase exists as a
4)
dimer as described by Uehara et al
.
The puriˆed
acid phosphatase was a purple color and its absorp-
tion spectrum had an absorption maximum at
550 nm. The A280 550 ratio was calculated as 30, in
W
ˆnal sample showed the speciˆc activity of 609 U mg
good agreement with the previous studies.4,5) The N-
terminal sequence of the puriˆed PAP was
LPNAEDVDMPWVS, which was identical with that
of PAP1.2) The advantages of the dry powder for the
puriˆcation of PAP are summarized as follows.
First, dry powder is much more stable than fresh
sweet potato, and can be used readily all the year.
Secondly, a good reproducibility with a good yield
was obtained on the puriˆcation of the acid phos-
phatase. Thirdly, the dry powder appears to contain
a single form of acid phosphatase, PAP1, although
fresh sweet potato contained multiple forms of acid
phosphatase.
W
protein, which was nearly the same as that reported
3)
±
by He‰er et al. The mean SD value of the speciˆc
For the measurement of phosphatase activity
toward pNPP, the amount of p-nitrophenol was esti-
mated on the basis of the absorbance change at
410 nm. The phosphatase activity toward glucose-6-
phosphate,
fructose-6-phosphate,
sucrose-6-
phosphate, ATP, ADP, AMP, and NADP was esti-
mated by measuring the phosphate concentration as
described by Heinonen and Lahti.6) The kinetic
constants Km and kcat were calculated by nonlinear
regression analysis using the Kaleida-Graph (Synergy
Software, Readings, PA). Table II summarizes the
phosphatase activity of the puriˆed PAP1 toward 8
substrates. Among the substrates, the puriˆed PAP1
showed the lowest Km and the highest kcat toward
Fig. 1. SDS-PAGE Analysis of the Puriˆed Samples from Each
Step.
Linear gradient polyacrylamide gel (4–20
z) from Daiichi
Pure Chemicals (Tokyo, Japan) was used. Lanes 1 and 7
molecular markers (rabbit phosphorylase, Mw. 97,400; bovine
serum albumin, Mr 66,267; rabbit aldolase, Mr 42,400; bovine
carbonic anhydrase, Mr 30,000; soy bean trypsin inhibitor, Mr
21,000; egg white lysozyme, Mr 14,400); lane 2, crude extract;
lane 3, eluate from TOYOPEARL phenyl-650M; lane 4, eluate
from DEAE A‹-gel blue; lane 5, eluate from Con A-Sepharose
4B; lane 6, eluate from Sephacryl S-200. Each well of lanes 2–6
pNPP at pH 5.6. The PAP1 showed similar
k
K
cat Wm
toward glucose 6-phosphate and fructose 6-
phosphate, while it showed a signiˆcantly lower value
toward sucrose 6-phosphate. Among ATP, ADP,
and AMP, PAP1 showed the lowest
and the highest kcat toward ATP. Judging from the
, NADP was considered to be a better sub-
Km toward ADP
contained 1.0 mg of protein.
k
K
cat Wm
Table 1. Puriˆcation of PAP1 from Sweet Potato Dry Powder
Total
protein
(mg)
Total
activity
(unit)
Speciˆc
activity
Puriˆcation
(fold)
Recovery
Step and fraction
Crude juice
TOYOPEARL Phenyl-650M
DEAE A‹-gel Blue
(
z)
(unitWmg)
2200
452
17
4276
2419
1996
1669
1335
1.9
5.3
117
301
609
1
3
60
155
313
100
60
42
37
29
Con A Sepharose 4B
Sephacryl S-200 HR
5.5
2.2

Puriˆcation of PAP1 from Dry Powder of Sweet Potato
Table 2. Substrate Speciˆcity of the Puriˆed PAP1
1611
0.1
loss of the activity was observed at 40
PAP1 or ALP. The residual activity of PAP1 and
ALP at 55 C was 60 and 69 , respectively, suggest-
M
Bis-Tris-HCl (pH 7.0) at 40
9
C and 55
9C. No
9
C for either
kcat
Km
(m
kcat
m
WKm
－
1
Substrate
-NPP
Glucose-6-phosphate
Fructose-6-phosphate
Sucrose-6-phosphate
ATP
ADP
AMP
NADP
(s－
)
M
)
(s－
・
M
)
1
1
9
z
p
531
218
171
35
155
50
0.21
2.17
1.93
1.27
1.50
0.53
0.83
0.97
2528
100
88
ing that the thermal stability of PAP1 is not very
diŠerent from that of ALP. These results strongly
suggest the possibility of practical applications of
PAP1, for example, an enzyme-linked immunoassay
using enzymes that are active in neutral or acidic con-
ditions.
27
103
95
45
295
53
303
References
1) Klabunde, T., Straä ter, N., Frohlich, R., Witzel, H.,
strate than ATP. The high activity toward NADP
indicates the possibility of application of PAP1 to a
synchronous enzyme-reaction system described in
our previous report.7) The substrate speciˆcity of
PAP1 observed in this study was nearly the same as
that of PAP reported previously.2,5)
and Krebs, B., Mechanism of Fe(III)–Zn(II) purple
acid phosphatase based on crystal structures. J. Mol.
Biol., 259, 737–748 (1996).
2) Schenk, G., Ge, Y., Carrington, L. E., Wynne, C. J.,
Searle, I. R., Carroll, B. J., Hamilton, S., and de
Jersey, J., Binuclear metal centers in plant purple acid
phosphatases: Fe–Mn in sweet potato and Fe–Zn in
soybean. Arch. Biochem. Biophys., 370, 183–189
(1999).
3) He‰er, S. K., and Arevill, B. A., The ``Manganese
(III)-containing'' purple acid phosphatase from sweet
potatoes in an iron enzyme. Biochem. Biophys. Res.
Commun., 146, 1173–1177 (1987).
4) Uehara, K., Fujimoto, S., and Taniguchi, T., Studies
on violet-colored acid phosphatase of sweet potato. I.
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and Ohara, A., Puriˆcation, enzymatic properties, and
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Mammalian and bacterial alkaline phosphatases
(ALPs) are used in immunoassays and DNA modiˆ-
cation. However, the activity of ALP at neutral pH is
much lower than that at optimal pH. In order to
examine the possibility of practical applications of
the purple acid phosphatase, we compared the en-
zymatic properties and thermal stability of the puri-
ˆed PAP1 with those of calf intestine ALP, which
showed its maximum activity at pH 10.5. The kinetic
parameter kcat of PAP1 for pNPP at pH 7.0 was
377 s－1, while the kcat of ALP at pH 7.0 was 93 s－
.
1
Thus, the kcat of PAP1 was 4 times higher than that
of ALP at pH 7.0. To examine temperature depen-
dence of the activity, PAP1 or calf intestine ALP was
taining and phosphatase. J. Biol. Chem.
10664–10670 (1981).
, 256,
added to 0.1
5.0 m pNPP incubated at each temperature. PAP1
and ALP showed the highest activity at 50 C and
60 C, respectively. Based on Arrhenius plots in the
temperature range 25–50 C, the activation energy of
M
Bis-Tris-HCl (pH 7.0) containing
6) Heinonen, J. K., and Lahti, R. J., A new and
convenient colorimetric determination of inorganic
orthophosphate and its application to the assay of
inorganic pyrophosphatease. Anal. Biochem., 113,
313–317 (1981).
M
9
9
9
PAP1 and ALP was calculated to be 41.6 and
7) Inouye, K., Ueno, I., Yokoyama, S., and Sakaki, T.,
49.3 kJ mol, respectively. To examine the thermal
W
Development of
a synchronous enzyme-reaction
stability of PAP1 and calf intestine ALP, the activity
system for a highly sensitive enzyme immunoassay. J.
Biochem., 131, 97–105 (2002).
was measured at 259C, after incubation for 30 min in