Biosci. Biotechnol. Biochem.
, 69 (1), 189196, 2005
Purication, Characterization, and Antifungal Activity of Chitinases
from Pineapple (Ananas comosus) Leaf
Toki T AIRA,y Noriko T OMA, and Masanobu ISHIHARA
Department of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu University, Okinawa 903-0213, Japan
Received September 16, 2004; Accepted October 22, 2004
Three chitinases, designated pineapple leaf chitinase
(PL Chi)-A, -B, and -C were puried from the leaves of
pineapple (Ananas comosus) using chitin anity column
chromatography followed by several column chroma-
tographies. PL Chi-A is a class III chitinase having a
molecular mass of 25 kDa and an isoelectric point of 4.4.
PL Chi-B and -C are class I chitinases having molecular
masses of 33 kDa and 39 kDa and isoelectric points of
7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and
the others are simple proteins. The optimum pHs of
PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4,
and 9 respectively. The chitin-binding ability of PL Chi-
C is higher than that of PL Chi-B, and PL Chi-A has
lower chitin-binding ability than the others. At low ionic
strength, PL Chi-B exhibits strong antifungal activity
toward Trichoderma viride but the others do not. At high
ionic strength, PL Chi-B and -C exhibit strong and
weak antifungal activity respectively. PL Chi-A does not
have antifungal activity.
Key words: chitinase; chitin-binding protein; pineapple;
Ananas comosus; antifungal activity
There are several types of chitinase in plants. On the
basis of their amino acid sequences, plant chitinases
have been classied into four classes: class I chitinases
consisting of an N-terminal chitin-binding domain and a
catalytic domain, class II chitinases with only a catalytic
domain homologous to that of class I chitinases,
class III chitinases sharing no homology with class I
or class II chitinases, and class IV chitinases sharing
homology with class I chitinases but smaller due to four
deletions.13) Even in the same class, there are various
isoforms which have low homology and/or a dierent
isoelectric point. Why do various structures of chitinase
exist in plants? Chitinases having dierent structures
should play dierent roles or the same roles in dierent
manners. Their biochemical characters, depending on
their structures, are expected to relate to their physio-
logical roles. One of the physiological roles of plant
chitinases is to protect plants against fungal pathogens
y
To whom correspondence should be addressed. Fax: +81-98-895-8802; E-mail: tokey@agr.u-ryukyu.ac.jp
Abbreviations: PL Chi, pineapple leaf chitinase; CAM, Crassulacean acid metabolism; RSC, rye seed chitinase; PAGE, polyacrylamide gel
electrophoresis; CBB, Coomassie brilliant blue; PAS, periodic acid Schi; PDA, potato dextrose broth with agar
by degrading chitin, a major component of the cell walls
of many fungi,2,3) but researchers poorly understand
what characters of chitinases have high antifungal
activity.4) In this study, we attempted to determine both
the biochemical characters and the antifungal abilities of
chitinases derived from a tropical plant, and we discuss
the relationship.
Tropical plants are exposed to many stresses such as
ultraviolet rays, high temperatures, and osmotic stresses.
PR (pathogenesis-related) proteins containing chitinase
are induced not only by pathogenesis but also by these
stresses.3,58) Therefore, it is to be expected that
chitinases derived from tropical plant are expressed in
large quantities and/or exhibit unique characters as
compared to those derived from other plants. We
screened various tropical plants for chitinase activity.
Pineapple (Ananas comosus) showed high chitinase
activity relative to the other tropical plants assayed.
Considering that pineapple is a CAM (Crassulacean acid
metabolism) plant9) adapted to strong light and dry
climate, it is to be expected that the chitinases derived
from leaves of pineapple exhibit unique characters.
In this paper, we describe the purication, biochem-
ical characterization, and antifungal activity of three
chitinases from the leaves of pineapple.
Materials and Methods
Materials. Pineapples (Ananas comosus) were har-
vested at Nago Agricultural experiment station. Chitin
and chitin beads were purchased from Seikagaku and
New England BioLabs respectively. All other reagents
were of analytical grade.
Preparation of crude chitinase. Pineapple leaves
(500 g) were homogenized with 2,500 ml of deionized
water and then left for 4 h at 4
C. After centrifugation,
the supernatant was saturated with ammonium sulfate
and the precipitate produced was collected. The precip-
itate was resolved with 10 mM TrisHCl buer (pH 7.5)
and the solution was put on a Sephadex G-25 column
190 T. T AIRA et al.
(12  44 cm) previously washed with the same buer,
and developed with the same buer. The fraction that
contained chitinase activity was collected as crude
chitinase.
Protein measurement. Absorbance was measured at
280 nm to monitor the proteins during chromatographic
separation. Protein concentration was measured by the
bicinchoninic acid method10) using bovine serum albu-
min as the standard.
Assay of chitinase activity. Chitinase activity was
assayed colorimetrically using glycolchitin or chitin
beads. Ten ml of the sample solution was added to 250 ml
of 0.2% (w/v) glycolchitin or 1% (w/v) chitin beads in
0.1 M buer. After incubation at 37
C for 15 min, the
reducing power of the reaction mixture was measured
using ferri-ferrocyanide reagent by the method of Imoto
and Yagishita.11) The chitinase activity of each fraction
in the chromatography was expressed by the absorbance
of the reaction mixture at 420 nm (
A420). One unit of
enzyme activity was dened as the amount of enzyme
releasing 1 mmol of GlcNAc per min at 37
C.
Chitin anity column chromatography. The crude
chitinase was put on a chitin column (3:0  30 cm)
previously equilibrated with 10 mM TrisHCl buer,
pH 7.5. After washing out the unadsorbed protein with
the same buer, the adsorbed protein was eluted with
1 M NaCl in the same buer. Then still adsorbed protein
was eluted with 0.5 M acetic acid.
Ion-exchange column chromatography. Resource Q
and SP-Sepharose HP column chromatographies were
done by putting the proteins in 10 mM sodium acetate
buer, pH 5.0, on 1:6  3 cm and 0:5  5 cm columns
respectively and then eluting the adsorbed proteins with
a liner gradient of NaCl from 0 to 0.2 M in the same
buer.
Hydrophobic column chromatography. Butyl-Toyo-
pearl 650M was done by putting the proteins in 1.5 M
ammonium sulfate-20 mM TrisHCl buer, pH 7.5, on a
1:5  16 cm column. Then the adsorbed proteins were
eluted with a linear gradient of ammonium sulfate from
1.5 to 0 M in the same buer. Phenyl superose column
chromatography was done by putting the proteins in
0.75 M ammonium sulfate-20 mM TrisHCl buer,
pH 7.5 on 0:5  5 cm, and then the adsorbed proteins
were eluted with a linear gradient of ammonium sulfate
from 0.75 to 0 M in the same buer.
SDSpolyacrylamide gel electrophoresis. SDSPAGE
was done by the method of Laemmli12) using a 15%
acrylamide gel. Proteins on the gel were stained with
Coomassie brilliant blue R-250. Carbohydrates on the
gel were detected using periodic acid Schi staining
(PAS staining).13) The molecular mass was measured by
SDSPAGE in the presence of 2-mercaptoethanl using a
MW-marker Kit (Apro Science).
Isoelectric point. The isoelectric point of chitinase
was measured using electrofocusing on a 5% polyacryl-
amide gel slab containing a 2% carrier ampholyte of
pH 3.510.0 (Sigma), as described by Vesterberg.14)
Proteins on the gel were stained with Coomassie
brilliant blue R-250. The isoelectric point was deter-
mined using an IEF-marker Kit (Sigma).
N-terminal amino acid sequence analysis. N-terminal
amino acid sequence analysis was done by Edman
degradation with a protein sequencer (Applied Biosys-
tems, Model 473A).
Eects of pH and temperature on activity and
stability. The optimum pH of the chitinases was
measured after incubation with glycolchitin in the buer
at various pHs (112) at 37
C for 15 min. The pH and
thermal stabilities of the enzymes were measured from
the residual activities after incubation in the buer at
various pHs at 37
C for 24 h and in 0.1 M sodium
phosphate buer, pH 7.0, at various temperatures for
1 h, respectively. The residual activities of PL Chi-A,
-B, and -C were measured at pH 3.0, 4.0, and 9.0
respectively. The buers used were: 0.1 M HCl (pH 1),
glycineHCl buer (pH 23), sodium acetate buer
(pH 45), sodium phosphate buer (pH 67), TrisHCl
buer (pH 89), and glycineNaOH buer (pH 1013).
Chitin-binding assay. The chitin-binding ability of the
chitinase was examined using a chitin column (0:7 
2:5 cm), as described by Yamagami et al.15) Twenty-ve
mg of the chitinase was dissolved in 10 mM sodium
phosphate buer, pH 7.0, and put on a chitin column
previously equilibrated with the same buer. After
washing unadsorbed protein with the same buer, the
chitin-binding protein was eluted rst with 1 M NaCl in
the same buer, second with 0.1 M acetic acid, and third
with 1 M acetic acid solution.
HPLC analysis of the hydrolysis products of
(GlcNAc)n by chitinases. The hydrolysis products of
(GlcNAc)n by chitinases were analyzed by HPLC on a
Tosoh TSK-Gel amide-80 column (0:46  25 cm) using
the method of Koga et al.16) The reaction mixture,
consisting of 10 pmol of chitinase and 10 nmol of
(GlcNAc)n in 100 ml of 4 mM buer after incubation
for 30 minutes at 25
C, was immediately cooled in an
ice bath, and a 5 ml potion was applied onto the column.
Elution of the hydrolysis products was done with 70%
acetonitrile at a ow rate of 0.7 ml/min.
Antifungal assay.17) An agar disk (6 mm in diameter)
with the fungus Trichoderma viride, which was derived
from the fungus in an actively growing state previously
cultured on a potato dextrose broth with 1.5% (w/v)
 Characterization of Pineapple Leaf Chitinases
agar (PDA), was placed in the center of a Petri dish
containing PDA. In the assay to examine the eects of
ionic strength, the PDA plates contained 0, 0.05, 0.1, or
0.15 M NaCl at the nal concentration. The plates were
incubated at room temperature for 12 h. Wells were
subsequently punched into the agar at a distance of
15 mm from the center of the Petri dish. The samples to
be tested were placed into the wells in 10 ml of sterile
water. The plates were incubated for 24 h at room
temperature and then photographed.
Results
Purication of pineapple leaf chitinases
All procedures were done in a cold room. Since the
optimum pH of crude chitinase was 3, chitinase activity
was measured at pH 3 during purication.
The elution prole of the crude chitinase in chitin
anity column chromatography is shown in Fig. 1.
Chitinase activities were found in the 1 M NaCl eluted
fraction (A) and the 0.5 M acetic acid eluted fraction (B),
but not in an unadsorbed fraction.
The chitinase in fraction A was partially puried
using Butyl-Toyopearl 650M column chromatography
followed by Resource Q column chromatography. The
partially puried chitinase derived from fraction A was
designated pineapple leaf chitinase-A (PL Chi-A). Final
purication of PL Chi-A was achieved by HPLC with a
Phenyl superose column in 0.75 M ammonium sulfate-
20 mM TrisHCl buer, pH 7.5.
The chitinases in fraction B were dialyzed against
0.75 M ammonium sulfate-20 mM TrisHCl buer,
pH 7.5, and put on a Phenyl superose column. Chitinase
activity was found mainly in two fractions. The former
fraction and the latter were designated PL Chi-B and -C
respectively. PL Chi-B was further puried. Final
Fig. 1. Chitin-Anity Column Chromatogram of Crude Extracts
from Pineapple Leaves.
The crude chitinase was put on a chitin anity column (3:0 
30 cm) previously equilibrated with 10 mM TrisHCl buer, pH 7.5.
After washing out the non-adsorbed protein with the same buer, the
adsorbed protein was eluted with 1 M NaCl in the same buer. Then
the still adsorbed protein was eluted with 0.5 M acetic acid.
191
Fig. 2. SDSPAGE of Pineapple Leaf Chitinases.
SDSPAGE was done by the method of Laemmli. Lane M,
marker proteins; lane 1, PL Chi-A; lane 2, PL Chi-B; lane 3,
PL Chi-C; lane 4, PL Chi-Bf. The left gel was stained with
Coomassie Brilliant Blue R250 (CBB), and the right gel was stained
with periodic acid-Schi reagent (PAS).
purication of PL Chi-B was achieved by HPLC with
a SP-Sepharose HP column in 10 mM sodium acetate
buer, pH 5.0. The puried PL Chi-A, -B, and -C gave
a single band having dierent molecular masses on
SDSPAGE (Fig. 2, left panel, lanes 13). 8.4 mg of
PL Chi-A, 0.2 mg of PL Chi-B, and 3.2 mg of PL Chi-C
were obtained from 500 g of pineapple leaves (data not
shown). A small amount of polypeptide, which has
chitinolytic activity and a smaller molecular mass than
PL Chi-B, was obtained from stored PL Chi-B solution
(Fig. 2, lane 4 of left panel). Therefore we concluded
that the polypeptide was a fragment of PL Chi-B. This
fragment was designated PL Chi-Bf and was used in
subsequent experiments as well as PL Chi-A, -B, and
-C.
Physico-chemical properties of puried pineapple
leaf chitinases
By SDSPAGE in the presence of 2-mercaptoethanol,
the molecular masses of PL Chi-A, -B, -C, and -Bf were
found to be 25, 33, 39, and 25 kDa respectively (Fig. 2,
left panel). PL Chi-C was detected by PAS staining
(Fig. 2, right panel). The isoelectric points of PL Chi-A,
-B, -C, and -Bf were 4.4, 7.9, 4.6, and 8.2 respectively
(data not shown).
N-terminal amino acid sequences of pineapple leaf
chitinases
The N-terminal sequences of PL Chi-A, -B, -C, and
-Bf, which were reduced and S-pyridylethylated, were
analyzed up to the 20th position with a protein
sequencer. But the N-terminal amino acid of PL Chi-C
was not detected. PL Chi-C was treated with pyroglu-
tamate aminopeptidase, and then its N-terminal se-
quence was determined. It was estimated that the N-
terminal amino acid of PL Chi-C is pyroglutamate. The
N-terminal sequences of pineapple leaf chitinases are
192 T. T AIRA et al.

Fig. 3. Comparison of the N-Terminal Amino Acid Sequences of Pineapple Leaf Chitinases and PL Chi-B Fragment with Those of Other Plant
Chitinases and Hevein.
Identical residues are shown in white with a black background. a, plant class III chitinase from rubber tree;18) b, plant class I chitinase from
rye seeds;19) c, chitin-binding peptide from rubber tree;20) d, plant class II chitinase from rye seeds.21) <Q indicates pyroglutamate residue.
X indicates unidentied residue.

shown in Fig. 3 in comparison with those of other plant

chitinases and hevein, a chitin-binding peptide derived
from the rubber tree. Of the 20 amino acid residues, 15
residues were common to PL Chi-A and Hevamine, a
class III chitinase derived from the rubber tree (Fig. 3,
top). Of the 20 amino acid residues, 13 residues were
common to PL Chi-B, PL Chi-C, rye seed class I
chitinase, and hevein (Fig. 3, middle). These results
suggest that PL Chi-A, B, and -C are to be classied into
plant class III, I, and I chitinase respectively. The amino
acid sequence of 824th from the N-terminal of PL Chi-
Bf was similar to that of 6177th of rye seed class I
chitinase and that of 218th of rye seed class II chitinase
(Fig. 3, bottom).
Fig. 4. Eect of pH and Temperature on Chitinase Activity and
Stability of Pineapple Leaf Chitinases.
Eect of pH and temperature on chitinase activity A, The eect of pH on the activities was examined after
The eects of pH on chitinase activity in the incubation at 37
C for 15 min. B, The eect of pH on stabilities was
pineapple leaf chitinases were examined using glycol- examined after incubation at 37
C for 24 h. C, The eect of
chitin as substrate. As shown in Fig. 4A, PL Chi-A had temperature on the activities was examined after incubation for
an optimum at pH 3.0 with 90% of the maximal activity 15 min. D, The eect of temperature on stabilities was examined
after incubation in 0.1 M sodium phosphate buer, pH 7.0, for 1 h.
at pH 2.0 and with 80% of that at pH 4.0. PL Chi-B had Details are described in Materials and Methods. , PL Chi-A; ,
an optimum at pH 4.0 with 80% of the maximal activity PL Chi-B; , PL Chi-C.
at pH 3.0 and with 85% of that at pH 5.0. PL Chi-C had
an optimum at pH 9.0 with 95% of the maximal activity
at pH 8.0 and with 60% of that at pH 10.0. As shown in pH and temperature on the chitinase activity of PL Chi-
Fig. 4B, PL Chi-A, -B, and -C were stable between Bf was the same as in the case of PL Chi-B (data not
pH 3 and 12, but unstable below pH 2.0 and above shown).
pH 13.
As shown in Fig. 4C, PL Chi-A, -B, and -C had an Chitinase activity and chitin-binding ability of pine-
optimum at 60
C, 70
C, and 70
C respectively. As apple leaf chitinases
shown in Fig. 4D, PL Chi-A was stable to 80
C and As shown in Table 1, the molar specic activity of
unstable above 90
C, while PL Chi-B was stable to PL Chi-A was the highest among the three chitinases
50
C and unstable above 60
C, and PL Chi-C was toward glycolchitin. The molar specic activity of
stable to 60
C and unstable above 70
C. The eect of PL Chi-B toward glycolchitin was slightly lower than
 Characterization of Pineapple Leaf Chitinases
Table 1. Specic Activity of Pineapple Leaf Chitinases towards the
Glycolchitin and Chitin Beads
Glycolchitin Chitin Beads
(units/mol enzyme) (units/mol enzyme)
PL Chi-A 7:11  109 3:40  108
PL Chi-B 5:91  109 2:77  109
PL Chi-C 1:50  109 5:76  108
Fig. 5. Elution Proles of Pineapple Leaf Chitinases and PL Chi-B
Fragment in Anity Chromatography on a Chitin Column.
Protein was put on a chitin column (0:7  2:5 cm) equilibrated
with 10 mM sodium phosphate buer, pH 7.0. After washing
unadsorbed protein with the same buer, the chitin adsorbed protein
was eluted successively with 1 M NaCl, 0.1 M acetic acid, and 1 M
acetic acid. A, PL Chi-A; B, PL Chi-B; C, PL Chi-C; D, PL Chi-Bf.
that of PL Chi-A, and was approximately four-fold
higher than that of PL-Chi-C. On the other hand, toward
chitin beads, the molar specic activity of PL Chi-B was
approximately eight and ve-fold higher than that of
PL Chi-A and PL Chi-C respectively.
PL Chi-A, -B, and -C were adsorbed to a chitin
column, and then were eluted with 1 M NaCl, with 0.1 M
acetic acid and with 1 M acetic acid solution respectively
(Fig. 5). These results indicate that the chitin-binding
ability of PL Chi-C is higher than that of PL Chi-B and
that that of PL Chi-A is lower than that of PL Chi-B or
-C. PL Chi-Bf was not adsorbed to the chitin column.
The chitin-binding abilities and N-terminal amino acid
sequences of PL Chi-B and -Bf suggest that PL Chi-Bf
is a polypeptide that consists of the catalytic region and
part of the hinge region, and that it lacks the chitin-
binding region of PL Chi-B.
Action of pineapple leaf chitinases on (GlcNAc)n
The cleavage patterns of (GlcNAc)n by pineapple leaf
chitinases and the anomeric form of the products were
determined by the method of Koga et al. (Fig. 6). In the
case of PL Chi-A (Fig. 6A and D), the major hydrolysis
products from (GlcNAc)6 were (GlcNAc)4 and
(GlcNAc)2 , and those from (GlcNAc)5 were (GlcNAc)4 ,
193
Fig. 6. HPLC Analysis of the Hydrolysis Products of (GlcNAc)6 and
(GlcNAc)5 by Pineapple Leaf Chitinases.
S: As standards, (GlcNAc)1{6 . A and D: (GlcNAc)6 and
(GlcNAc)5 were reacted with PL Chi-A for 30 min in 4 mM
glycineHCl buer (pH 3.0) at 25
C. B and E: (GlcNAc)6 and
(GlcNAc)5 were reacted with PL Chi-B for 30 min in 4 mM sodium
acetate buer (pH 4.0) at 25
C. C and F: (GlcNAc)6 and (GlcNAc)5
were reacted with PL Chi-C for 30 min in 4 mM TrisHCl buer
(pH 9.0) at 25
C. Standard (GlcNAc)1{6 and the reaction mixtures
were analyzed by HPLC, as described in Material and Methods.
(GlcNAc)3 , (GlcNAc)2 , and GlcNAc. In this condition,
(GlcNAc)2{4 were not degraded by PL Chi-A (data not
shown). Since the newly created reducing end of the
products was largely
(e.g., (GlcNAc)4 in Fig. 6A and
D), it is indicated that PL Chi-A is a retaining enzyme.
The amounts and /
anomer ratio of the products
indicated that PL Chi-A hydrolyzed mainly the fourth
linkage and rarely the second linkage from the non-
reducing end-side of the substrate. In the case of
PL Chi-B and -C (Fig. 6B, C, E, and F), the major
hydrolysis products from (GlcNAc)6 were (GlcNAc)4 ,
(GlcNAc)3 , and (GlcNAc)2 , and those from (GlcNAc)5
were (GlcNAc)3 and (GlcNAc)2 . In this condition,
(GlcNAc)2{4 were not degraded by PL Chi-B and -C
(data not shown). Since the newly created reducing end
of the products was largely , it is indicated that PL Chi-
B and -C are inverting enzymes. The amounts and /

anomer ratio of the products indicated that PL Chi-B
and -C hydrolyzed the second and third linkages from
the reducing or non-reducing end-side of the substrate.
194 T. T AIRA et al.

Antifungal activity of pineapple leaf chitinases

In the case of no addition of NaCl to PDA, PL Chi-B
and -Bf strongly inhibited hyphal extension but the
others did not. The antifungal activity of PL Chi-Bf
became weaker with increasing concentrations of NaCl,
opposite to that of PL Chi-C, which became stronger
(compare Fig. 7 top one with the others). PL Chi-B
exhibited antifungal activity in all tested conditions. The
results at high ionic strength suggest that there is a
contribution of the chitin-binding domain to the anti-
fungal activity of class I chitinases.

Discussion

In this study, three types of chitinase, designated

PL Chi-A, -B, and -C, were puried to homogeneity
from the leaves of pineapple. Several biochemical
characters of these chitinases were very dierent one
from another. PL Chi-A is an acidic class III chitinase
having a molecular mass of 25 kDa and an optimum pH
of 3. PL Chi-B is a weakly basic class I chitinase having
a molecular mass of 33 kDa and an optimum pH of 4.
PL Chi-C is an acidic class I chitinase having a mo-
lecular mass of 39 kDa and an optimum pH of 9.
Especially, PL Chi-C is unique in its high molecular
mass, glycosylation, and high chitin-binding ability.
Almost all known chitinases derived from plants have
molecular masses of 2535 kDa.2,3,5) Nielsen et al.
puried an acidic class IV chitinase that has a molecular
mass of 35 kDa and is glycosylated with xyloses from
leaves of sugar beet.22) Zhao and Chye cloned B. juncea
cDNA encoding BjCHI1, an acidic chitinase with two
chitin-binding domains and a catalytic domain.23) The
expected molecular mass of matured BjCHI1 is
39.4 kDa. PL Chi-C is the protein that has the highest
molecular mass among puried chitinases from plants
and the rst discovered glycosylated class I chitinase.
Yamagami and Funatsu showed class I chitinase
derived from rye seeds bound to a chitin column under
neutral pH conditions and completely eluted with 0.1 M
acetic acid.15) In this study, under the same conditions
PL Chi-B and -C bound to the chitin column and
PL Chi-B was completely eluted with 0.1 M acetic acid,
but PL Chi-C was not eluted with the same solution
(Fig. 5). These results indicate that PL Chi-C has higher
binding ability to a chitin than PL Chi-B. This high
chitin-binding ability of PL Chi-C probably inuences
its chitinolytic activity. The molar specic activity of
PL Chi-C was weaker than that of PL Chi-B toward
both soluble and insoluble substrates, glycolchitin and
chitin beads. The dierence in molar specic activity
between PL Chi-B and -C toward chitin beads was Fig. 7. Antifungal Activity of Pineapple Leaf Chitinases and Eect
larger than that toward glycolchitin (Table 1). These of Ionic Strength on Their Activity.
The samples to be tested were placed into wells in 10 ml of sterile
results suggest that the high chitin-binding ability of
water with 50 pmol of puried chitinases. 1, blank; 2, sterile water;
PL Chi-C retards its turnover rates, especially toward 3, PL Chi-A; 4, PL Chi-C; 5, PL Chi-B; 6, PL Chi-Bf. The rst,
insoluble substrate. Iseli et al. found that the maximal second, third, and fourth plates from the top contain culture medium
specic activity of Tob (tobacco class I chitinase) with 0, 0.05, 0.10, and 0.15 M NaCl respectively.
toward an insoluble substrate, colloidal chitin, was
 Characterization of Pineapple Leaf Chitinases
lower than that of Tob
H (Tob without the chitin-
binding domain).24) Arakane et al. showed that the
fusion proteins, consisting of the catalytic domain of
Manduca sexta chitinase and one or two insect or plant
chitin-binding domains linked by its part of the linker
region, had high binding ability and lower Vmax toward
colloidal chitin than the proteins, consisting of the
catalytic domain and a part of the linker.25) The high
chitin-binding ability of chitinases perhaps retards their
turnover rates.
PL Chi-Bf consists of the catalytic domain and a part
of the linker region but lacks the chitin-binding domain
of PL Chi-B. PL Chi-Bf showed antifungal activity to a
similar extent with PL Chi-B in the absence of NaCl, but
the antifungal activity of PL Chi-Bf decreased with
increasing NaCl concentration in the culture medium as
opposed to that of PL Chi-B (Fig. 7). PL Chi-C did not
exhibit antifungal activity in the absence of NaCl. In the
presence of NaCl, however, PL Chi-C exhibited weakly
antifungal activity, and the activity increased with
increasing NaCl concentration. Previously we suggested
that basic class I chitinase binds to fungal hypha by
ionic interaction of the catalytic domain and by hydro-
phobic interaction of the chitin-binding domain.17,26)
The results in this study support our previous suggestion,
since higher salt concentrations might decrease ionic
interaction and increase hydrophobic interaction. Con-
sidering that the pineapple is a CAM plant adapted to
strong light and a dry climate, the ionic strength of the
tissues of pineapple can be high, depending on the
environment. PL Chi-C is abundant in the leaves of
pineapple and might play a defense role against
pathogenic fungi under high ionic strength conditions.
PL Chi-A was most abundant in the leaves of pine-
apple and had the highest chitinolytic activity toward
soluble chitin among pineapple leaf chitinases, but did
not exhibit antifungal activity. The cleavage pattern of
N-acetylchitooligosaccharides by PL Chi-A is dierent
from those by PL Chi-B and -C. PL Chi-A might
indirectly aect the invasion of pathogens, as by elicitor
producing, or may have other functions. To estimate the
physiological roles of these chitinases in the pineapple
plant, their expression patterns and localization will be
discussed in our subsequent papers.
Acknowledgments
We thank M. Takeuchi (Nago Branch, Okinawa
Prefectural Agricultural Experiment Station) for supply-
ing pineapple leaves. This project was supported by
grants from the Urma Trust Fund for Research into
Science and Humanity.
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