Isolation and characterization of a cysteine protease of freesia corms

Article abstract of DOI:10.1271/bbb.66.448

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M_r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromer-curibenzoic acid but not by phenylmethane- sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.

Full text of DOI:10.1271/bbb.66.448

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Isolation and Characterization of a Cysteine Protease
of Freesia Corms
Tetsuya UCHIKOBAa, Michiko OKUBOb, Kazunari ARIMAc & Hiroo YONEZAWAc
a Kagoshima University Musium 1-21-30, Korimoto, Kagoshima 890-0065, Japan
b Food and Nutrition, Kagoshima Immaculate Heart College 4-22-1, Toso, Kagoshima
890-8525, Japan
c Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kagoshima
University 1-21-35, Korimoto, Kagoshima 890-0065, Japan
Published online: 22 May 2014.
To cite this article: Tetsuya UCHIKOBA, Michiko OKUBO, Kazunari ARIMA & Hiroo YONEZAWA (2002) Isolation and
Characterization of a Cysteine Protease of Freesia Corms, Bioscience, Biotechnology, and Biochemistry, 66:2, 448-452,
DOI: 10.1271/bbb.66.448
To link to this article: http://dx.doi.org/10.1271/bbb.66.448
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Biosci. Biotechnol. Biochem., 66 (2), 448–452, 2002
Note
Isolation and Characterization of a Cysteine Protease of Freesia Corms
1,
,
† Michiko OKUBO,2 Kazunari ARIMA,3 and Hiroo YONEZAWA
3
Tetsuya UCHIKOBA
1Kagoshima University Musium, 1-21-30, Korimoto, Kagoshima 890-0065, Japan
2Food and Nutrition, Kagoshima Immaculate Heart College, 4-22-1, Toso, Kagoshima 890-8525, Japan
3Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kagoshima University,
1-21-35, Korimoto, Kagoshima 890-0065, Japan
Received August 15, 2001; Accepted October 9, 2001
A protease, freesia protease (FP)-A, was puriˆed to
electrophoretic homogeneity from regular freesia
of proteases toward reserved proteins in corms. In
our previous paper, two cysteine proteases (termed
FP-A and FP-B) had been found from freesia corms,
(
Freesia re‰acta) corms in harvest time. The Mr of FP-A
was estimated to be 24 k by SDS-PAGE. The optimum
pH of the enzyme was 8.0 using a casein substrate.
and then the latter had been isolated.16) The
Mr of FP-
B was estimated to be 26 k by SDS-PAGE. The opti-
These enzymes were strongly inhibited by
p
-chloromer-
mum pH of FP-B was 6.0–7.0 at 30 C using a casein
9
curibenzoic acid but not by phenylmethane-sulfonyl-
‰uoride and EDTA. These results indicate that FP-A
belongs to the cysteine proteases. The amino terminal
sequence of FP-A was similar to that of papain, and the
sequences was regarded to the conservative residues of
cysteine protease. From the hydrolysis of peptidyl-p-
NAs, the speciˆcity of FP-A was found to be broad. It
was thought that FP-A was a new protease from freesia
corms.
substrate. It was thought that the enzyme was a typi-
cal papain-like cysteine protease.
In this paper, we have isolated FP-A from freesia
corms in harvest time and described the properties
and substrate speciˆcity of this protease.
All procedures for puriˆcation of the enzyme were
done at 79C. Freesia corms, Freesia re‰acta ssp.
`White Mary' (200 g, Takii Co., Kyoto, Japan), were
homogenized with a domestic mixer in 400 ml of
20 m
M
Na, K-Pi buŠer at pH 7.0. The homogenate
Key words: plant corm; cysteine protease; plant en-
dopeptidase; freesia; Freesia re‰acta
was ˆltered through a cotton cloth and was cen-
×
trifuged (4,000
sulfate was added to the supernatant to 50
tion and kept for 24 h. After centrifugation (4,000
, for 30 min), the pellet was dissolved in 10 m Na,
K-Pi buŠer at pH 7.0. The solution was put directly
g
, for 30 min). Solid ammonium
z
satura-
×
During germination and the early growth stage of
plants, storage proteins are degraded by proteolysis.
Cysteine proteases of higher plants appear to have
many proteolytic functions in intracellular and ex-
tracellular processes such as degradation of storage
proteins in germinating seeds.1–6) Aleurain from the
aleurone layer of barley (Hordeum vulgare) is a well-
characterized seed cysteine protease.7,8) In the investi-
gation of plant endopeptidase, many enzymes have
been found in latex, fruits, and seeds,9) however, the
endopeptidases from the underground parts of the
plants have been poorly understood. Some enzymes
were reported as follows: an endopeptidase
from maize roots,10) a protease from Arabidopsis
roots,11,12) proteases of sprouting potato (Solanum
tuberosum) tubers,13,14) and a cysteine protease of
ginger (Zingiber o‹cinale Roscoe) rhizomes.15) We
have been interested in measuring the protease activi-
ty of plant parts in order to understand the function
g
M
×
a column (5.0 50 cm) of DEAE-cellulose
on
equilibrated with the same buŠer. The column was
washed with buŠer A, and the protease was eluted
with 0.2
sulfate was added to the eluate from the column to
50 saturation. After 24 h, the mixture was cen-
trifuged (10,000 , for 15 min), the pellet was sus-
pended in 10 m Na, K-Pi buŠer, pH 7.0, and then
the suspension was dialyzed against the same buŠer
M
Na, K-Pi buŠer, pH 7.0. Solid ammonium
z
×
M
g
×
g,
overnight. The dialysate was centrifuged (10,000
for 15 min), and the supernatant was put on a
DEAE-Sepharose column (1.8 26 cm) equilibrated
with 10 m Na, K-Pi buŠer, pH 7.0, and then the
proteins adsorbed to the column were eluted with a
linear gradient from 10 m Na, K-Pi buŠer, pH 7.0
(1 liter) to 0.2 Na, K-Pi buŠer, pH 7.0 (1 liter).
×
M
M
M
†
To whom correspondence should be addressed. Fax: +81-99-285-8117; E-mail: uchik
-dithiobis (2-nitrobenzoic acid); freesia protease, FP; MIA, monoiodoa-
-leucyl-agmatine; PCMB, -chloromercuribenzoic acid; PCMPS, -chloromer-
curiphenylsulfonic acid; Pi, phosphate; PMSF, phenylmethanesulfonyl‰uoride; NA, -nitroanilide; STI, soybean trypsin inhibitor; Tos-
Lys-CH2Cl, -Tosyl- -lysine chloromethylketone; Tos-Phe-CH2Cl, -Tosyl- -phenylalanine chloromethyl ketone
＠
sci.kagoshima-u.ac.jp
Abbreviations: DFP, diisopropyl ‰uorophosphate; DTNB, 5,5
cetic acid; E-64, ( -3-trans-carboxyoxirane-2-carbonyl)-
?
L
L
p
p
p
p
N
L
N
L

A Cysteine Protease of Freesia Corms
449
Protease (FP-A)-containing fractions were collected,
and solid ammonium sulfate was added to the eluate
was observed to be about 8. This proˆle was similar
to that of melain, a cysteine protease from bead tree
fruit under the same conditions.19) The pH stability of
FP-A was examined by incubating at various pHs at
from the column to 50
sulfate precipitate of the eluate from the DEAE-
Sepharose column was centrifuged (10,000 , for
z
saturation. The ammonium
×
g
37
80
9
C for 10 min, before an assay at pH 7.0. At least
15 min), then the pellet was dissolved in distilled
water and the solution being put on a Bio-gel P-60 gel
z of the activities of FP-A remained after incuba-
tion between pH 7–10.5, (Fig. 2(B)).
×
ˆltration column (2.4 95 cm) equilibrated with
10 m Na, K-Pi buŠer, pH 7.0. The eluate from the
gel ˆltration was diluted with distilled water, and was
The eŠects of temperature on the proteolytic activ-
ities of FP-A are shown in Fig. 2(C). FP-A had an
optimal activity in the range of 50 C. The thermal
9
stability of the enzyme was examined by incubating it
at various temperatures for 30 min, before an assay
M
×
put on
a
Q-Sepharose column (2.0 6.0 cm)
equilibrated with the above buŠer. The proteins
adsorbed to the column were eluted with a linear
at pH 7.0. At least 80
of FP-A remained after incubation at 45
shown in Fig. 2(D).
z
of the proteolytic activities
gradient from 10 m
(500 ml) to 0.08 Na, K-Pi buŠer, pH 7.0 (500 ml).
The pooled active fraction was dialyzed against
10 m Na, K-Pi buŠer, pH 7.0, overnight.
M
Na, K-Pi buŠer, pH 7.0
9C, as
M
The eŠects of various compounds on the enzymatic
activity are represented in Table 2. The enzyme
(0.5 ml) was added to 0.5 ml of the inhibitor solution
M
An enzyme, FP-A, was isolated from the freesia
corms in harvest time. The peak size of the caseino-
lytic activity of FP-B was smaller than that of the
previous study.16) The freesia corms contained a rela-
tively large amount of protein. The greater part of an
undesired protein was e‹ciently removed at the ini-
tial treatment of a DEAE-Sepharose column (Fig. 1).
The yields of each puriˆcation step are summarized
in Table 1. FP-A was ˆnally puriˆed from the Q-
in 67 m
35 C for 60 min. The remaining activity of protease
was assayed using casein as a substrate. The activity
of FP-A was completely inactivated by 0.1 m
PCMPS. The protease activity of FP-A was strongly
inactivated by 1.0 m DTNB, 0.01 m E-64, and
1.0 m PCMB. The eŠects of antipain (1 m ) and
M
K-Pi buŠer at pH 7.2 and incubated at
9
M
M
M
M
M
Sepharose column chromatography with 6
z recov-
ery. From 200 g of the freesia corms, 13 mg of the
puriˆed enzyme was obtained.
The puriˆed FP-A showed as a single band on
SDS-PAGE by the method of Laemmli,17) having a
Mr of 24 k as shown in Fig. 1 (box).
Isoelectric focusing of FP-A was done with an
acrylamide slab gel containing Ampholine (pH
3.5–10.0) by the procedure of Westermeier18) using
7
z
gel. The pI of FP-A (6.9) was estimated by isoe-
lectric focusing on an acrylamide slab gel. The data
was consistent with the elution point of FP-A, frac-
tion number 50 to 60 on a DEAE-Sepharose chro-
matography (Fig. 1).
Fig. 1. Elution Proˆle of Protease Activity of Freesia Corms on
DEAE-Sepharose Ion-exchange Chromatography and SDS-
PAGE of Puriˆed FP-A from a Q-Sepharose Column.
The ‰ow rate of the column was 0.9 ml min. Each fraction
W
FP-A from corms was characterized by measuring
these protease activities with casein as a substrate at
various pHs (Fig. 2(A)). Proteolytic activity was
measured with casein as a substrate by the method
described previously.16) The optimum pH of FP-A
was 15 ml. Solid bars, absorbance at 280 nm; (
activity. Box: SDS-PAGE of puriˆed FP-A. The samples were
electrophoresed in a 15 polyacrylamide gel. The gel was

), caseinolytic
z
stained in Coomassie Brilliant Blue R-250 for 15 min and then
destained.
Table 1. Puriˆcation of Freesia Protease A from Corms
Total
protein
(mg)
Total
activity
(units)
Speciˆc
activity
Recovery
Puriˆcation
factor
Puriˆcation step
(
z)
(units mg)
W
Extract
16,000
100,000
6.4
100
1
Ammonium sulfate
precipitation
DEAE-cellulose
DEAE-Sepharose
Bio-gel P-60
12,000
1,800
380
210,000
94,000
9,800
8,100
6,000
18
53
26
190
480
210
92
10
8
3
8
4
30
75
42
13
Q-Sepharose
6
One unit of activity was deˆned as the activity giving 0.001 A280 units of change per min under these conditions.

450
T. UCHIKOBA et al.
Fig. 2. The EŠects of pH and Temperature on the Proteolytic Activity and the Stability.
(A) The eŠects of pH on the caseinolytic activity. The incubation mixture consisted of 1.0 ml of an enzyme solution containing 10 m
M
cysteine (60 mg ml) and 1.0 ml of a 2
BuŠers: 0.1
z
(w v) casein in various pH buŠers. The assay solution was incubated at 37
9
C for 60 min.
glycine-NaOH buŠer (pH 8.0–12.3). The activity assay
could not be done at pH 2–5, because casein was insoluble in that range of pH. (B) The pH stability of the enzyme was assayed with the
incubation in various pHs for 30 min. BuŠers: 0.1 citric acid-HCl (pH 2.0), 0.1 Na-acetate buŠer (pH 3–6), 33 m Na, K-Pi (pH
5.0–8.0), 50 m glycine-NaOH buŠer (pH 8.0–12.9). (C) The eŠects of temperature on the caseinolytic activity. The incubation mixture
consisted in 1.0 ml of an enzyme solution containing 10 m cysteine and 1.0 ml of a 2 (w v) casein in 67 m Na, K-Pi buŠer, pH 7.0.
W
W
citric acid-HCl (pH 2.0), 67 m K, Na-Pi (pH 5.0–8.0), 0.1
M M M
M
M
M
M
M
z
M
W
The assay solution was incubated at various temperatures for 30 min. (D) Thermal stability of the enzyme. The protease solution was in-
cubated at various temperatures for 30 min and the residual activities were assayed.
chymostatin (1 m
had no eŠect on the enzymatic activity. The inactiva-
tion of Tos- -Lys-CH2Cl towards FP-A was more
eŠective than those of Tos- -Phe-CH2Cl. The
activity of FP-B was strongly inhibited by Tos- -Lys-
CH2Cl. All protease activities were weakly inactivat-
ed by 2.0 m ZnCl2. The enzyme activity were
M
) were weak. PMSF and EDTA
sites for FP-A were large hydrophobic residues at the
P1 position, like Val or Leu. The substrates having
Gly or Glu at the P1 position were barely cleaved by
FP-A. From the digestion of seven peptidyl sub-
strates, the speciˆcity of FP-A was found to be ap-
proximately broad. The speciˆcity of FP-A toward
L
L
L
M
peptidyl-pNA may be broader than for FP-B.
gradually decreased by an addition of DTNB. In
the course of inhibition, an addition of 2-mercap-
toethanol to this reaction mixture reversed this eŠect
and restored the initial activity (data not shown).
Therefore, these results indicated that FP-A belong
to the cysteine proteases. It was found that the eŠect
of compounds using in Table 2 against the activity of
FP-A was similar to those of FP-B.
The N-terminal sequence of the FP-A was identi-
ˆed. The sequence of FP-A is aligned with those of
other cysteine proteases from plant tissues for maxi-
mum similarity (Fig. 3). Some consensus sequences
were found in the sequences of these proteases. The
N-terminal sequence of FP-B was not identical with
that of FP-A. FP-A and FP-B are diŠerent proteins,
judging from the N-terminal sequences. It can there-
fore be presumed that these proteases arise indepen-
dently from translation of diŠeent mRNAs.
From this study, it was thought that the enzyme
properties of FP-A were similar to those of FP-B, but
it was perhaps an other enzyme than FP-B, and no
mentioned similarity was detected between FP-A and
the known cysteine proteases from underground
The substrate speciˆcity of FP-A was investigated
with synthetic substrates of Ala-Ala-Pro-X-pNAs
＝
(X Val, Leu, Lys, Ala, Phe, Gly, and Glu). The
rate of enzymatic hydrolysis for peptidyl- NA sub-
p
strates was measured by the method described previ-
ously.19) As shown in Table 3, all substrates were
hydrolyzed by the protease. The preferential cleavage

A Cysteine Protease of Freesia Corms
451
parts such as potato13) and ginger.15) On the other
hand, the r and optimum pH of FP-A activity were
M
similar to those of asclepain A3 from the latex of
Asclepias syriaca L.20)
In our previous paper, FP-B was isolated from
freesia corms.16) This is because in the previous study,
the corms were stored at 4–79C for several months,
and then the corms were used for the puriˆcation of
FP-B. In this study, the corms using for puriˆcation
of proteases were obtained at harvest time. The
amount of FP-B eluted from the DEAE-Sepharose
column was observed to increase as a preservation
Fig. 3. The Comparison of the N-Terminal Amino Acid Se-
quence of FP-A and Other Plant Cysteine Proteases.
These sequences were aligned for maximum similarily. Num-
bering is according to that of papain. Abbreviations of amino
acids follow the alphabetical system.
9
period of freesia corms at 7 C (data not shown). We
thought that the chilling tolerance in the corms was
regulated, and some proteases (specially FP-B) ap-
peared in response to chilling impossible for germina-
tion of the corms.
We plan to study, for future characterization, the
roles of proteases in corm dormancy and in normal
growth.
Table 2. EŠects of Various Compounds on the Activity of Free-
sia Proteases
Relative activity (
z)
Concentration
(m
Compounds
None
MIA
PCMB
PCMPS
Antipain
E-64
M
)
Protease A
Protease B
Acknowledgment
—
100
22
16
0
78
25
0
100
56
13
—
—
10
15
2.0
1.0
0.1
0.001
0.01
1.0
We thank Kimihiro Inoue and Hiroshi Matsui for
their generous help in obtaining the freesia corms and
for their e‹cient work in protein sequencing.
b
b
DTNB
DFP
PMSF
2.0
2.0
1.0
2.0
70
123
82
12
30
89
116
104
143
97
92
91
81
72
100
93
—
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Tos-Lys-CH2Cl
Tos-Phe-CH2Cl
Chymostatin
Ovomucoid
STI
b
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a
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Ala-Ala-Pro-Leu-
Ala-Ala-Pro-Lys-
Ala-Ala-Pro-Ala-
Ala-Ala-Pro-Phe-
Ala-Ala-Pro-Gly-
Ala-Ala-Pro-Glu-
p
p
p
p
p
p
p
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0.25
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0.10
0.092
0.053
0.015
100a
56
44
40
37
21
6
2
NA
NA
NA
NA
NA
NA
9
100b
7
4
0
0
a
Activity with Ala-Ala-Pro-Val-
Activity with Ala-Ala-Pro-Lys-
p
p
NA was taken as 100
NA was taken as 100
z
.
b
z.

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