Báo cáo khoa học: Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris ppt

Purification, characterization, cDNA cloning and nucleotide sequencing
of a cellulase from the yellow-spotted longicorn beetle,
Psacothea
hilaris
Masahiro Sugimura
1
, Hirofumi Watanabe
1
, Nathan Lo
1
and Hitoshi Saito
2
1
National Institute of Agrobiological Sciences, Ibaraki, Japan;
2
Department of Applied Biology, Faculty of Textile Science,
Kyoto Institute of Technology, Japan
A cellulase (endo-b-1,4-glucanase, EC 3.2.1.4) was purified
from the gut of larvae of the yellow-spotted longicorn beetle
Psacothea hilaris by acetone precipitation and elution from
gels after native PAGE and SDS/PAGE with activity
staining. The purified protein formed a single band, and the
molecular mass was estimated to be 47 kDa. The purified
cellulase degraded carboxymethylcellulose (CMC), insoluble
cello-oligosaccharide (average degree of polymerization 34)
and soluble cello-oligosaccharides longer than cellotriose,
but not crystalline cellulose or cellobiose. The specific
activity of the cellulase against CMC was 150 lmolÆ
min
)1
Æ(mg protein)

)1
. TLC analysis showed that the cellu-
lase produces cellotriose and cellobiose from insoluble cello-
oligosaccharides. However, a glucose assay linked with
glucose oxidase detected a small amount of glucose, with a
productivity of 0.072 lmolÆmin
)1
Æ(mg protein)
)1
.The
optimal pH of P. hilaris cellulase was 5.5, close to the pH in
the midgut of P. hilaris larvae. The N-terminal amino-acid
sequence of the purified P. hilaris cellulase was determined
and a degenerate primer designed, which enabled a 975-bp
cDNA clone containing a typical polyadenylation signal to
be obtained by PCR and sequencing. The deduced amino-
acid sequence of P. hilaris cellulase showed high homology
to members of glycosyl hydrolase family 5 subfamily 2, and,
in addition, a signature sequence for family 5 was found.
Thus, this is the first report of a family 5 cellulase from
arthropods.
Keywords: cDNA cloning; cellulase; endoglucanase; insect;
purification.
Cellulase (endo-b-1,4-glucanase) is a widespread enzyme in
micro-organisms such as bacteria and fungi [1,2]. Until
recently it was believed that cellulose digestion in animals
was mediated by microbial cellulase activity in their
intestine, and that no animals possessed endogenous
cellulase. This traditional view of cellulase activity in
animals was challenged by two reports of endogenous

animal cellulase genes from plant-parasitic nematodes and
a termite [3,4]. Since these discoveries, a number of other
animal cellulase genes have been reported (summarized in
[5]).
Glycosyl hydrolases are categorized into 90 families
according to amino-acid sequence similarity and hydropho-
bic cluster analysis, and among them, cellulases are found
in 14 families [6,7] (refer also to: />CAZY/index.html). Known animal cellulases belong to
three glycosyl hydrolase families (GHFs): GHF 5 (plant-
parasitic nematodes), GHF 9 (termites, cockroaches and
crayfish) and GHF 45 (mussel and beetle). These three
families are structurally unrelated and their evolutionary
origins are likely to be independent.
Larvae of the yellow-spotted longicorn beetle, Psacothea
hilaris, feed on mulberry and fig trees, tunneling inside the
stems and ingesting the living wood. The major constituent
is cellulose (44.6%), followed by hemicellulose (28.5%);
soluble sugars constitute only 4.7% of the dry weight of the
wood [8]. The habitat of P. hilaris larvae suggests that they
possess the ability to digest cellulosic materials. In fact, a
variety of carbohydrase activities, including endo-b-1,
4-glucanase and b-glucosidase, have been detected in the
gut of P. hilaris larvae and adults [8].
To clarify further the cellulase activity in P. hilaris,we
purified, characterized and obtained the cDNA sequence of
a protein from this species with cellulolytic activity.
Materials and Methods
Measurement of the pH in the gut juice of
P. hilaris
larvae

P. hilaris larvae were anesthetized by immersion in ice water
for 10 min and their guts, including their contents, were
removed by dissection. The removed guts were washed in
ice-cold distilled water to prevent contamination by body
fluid and blotted on filter paper. Then, the guts were cut into
three parts (anterior midgut, posterior midgut and hindgut)
and transferred into 1.5 mL plastic centrifuge tubes. The
samples in the tubes were centrifuged, and insoluble
materials were discarded. The pH values of the recovered
Correspondence to H. Watanabe, National Institute of
Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan.
Tel./Fax: + 81 29 8386108,
E-mail: hinabe@affrc.go.jp
Abbreviations: GHF, glycosyl hydrolase family; CMC, carboxy-
methylcellulose; CBB, Coomassie Brilliant Blue.
Enzymes: endo-b-1,4-glucanase (EC 3.2.1.4).
(Received 30 January 2003, revised 22 May 2003,
accepted 30 June 2003)
Eur. J. Biochem. 270, 3455–3460 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03735.x
supernatants were measured with a pen-type pH meter
(model B-212; Horiba, Kyoto, Japan).
Enzyme assay
A carboxymethylcellulase (CMCase) assay was performed
by measuring the amount of reducing sugars after incuba-
tion of 100 lL 1% (w/v) CMC (standard molecular mass,
250 kDa; degree of carboxymethyl substitution, 0.7; Sigma-
Aldrich) in 0.1
M
sodium acetate (pH 5.5) with 20 lL
sample at 37 °C for an appropriate time period. Reducing

sugars were measured with tetrazolium blue (Sigma-
Aldrich) as a chromogenic reagent, with glucose as a stand-
ard, as described by Jue & Lipke [9]. Glucose production
from insoluble cello-oligosaccharide by the P. hilaris cellu-
lase was investigated with a commercial glucose assay kit
(Glucose C-test; Wako Pure Chemicals), which utilizes
glucose oxidase in an enzyme-linked colorization step,
according to the instructions. Insoluble cello-oligosaccha-
ride was prepared by the method of Sawano et al. [10], and
the average degree of polymerization was  34. To test the
degradation activity of P. hilaris cellulase against crystalline
cellulose, Avicel (Merck) was used under the same condi-
tions as the CMCase assay. Optimal pH for P. hilaris
cellulase activity against CMC was determined with 0.1
M
sodium acetate (4–6), sodium phosphate (5.5–7.5) or Tris/
HCl (7–9.5). Hydrolytic products from cello-oligosaccha-
rides (cellobiose, cellotriose, cellotetraose and cellopentaose)
were analyzed by TLC.
Electrophoresis and activity staining
SDS/PAGE was performed as described by Laemmli [11],
and native PAGE was carried out in the same way except
that SDS was excluded. Proteins were stained with CBB,
and cellulase was visualized by activity staining on the gel. In
the case of activity staining, the gel contained 0.1% (w/v)
CMC and the conditions of sample treatment for SDS/
PAGE were changed from boiling for 5 min to incubating at
33 °C for 30 min. After being run, the gel was twice rinsed in
distilled water with gentle shaking for 5 min, and soaked in
0.1

M
sodium acetate buffer (pH 5.5) for 5–20 min. The gel
was briefly rinsed with distilled water before staining with
0.2% (w/v) Congo red (Sigma-Aldrich) in water for 30 min.
The excess dye was removed in 1
M
NaCl with gentle
shaking and replacement of the solution several times.
Purification procedure
P. hilaris larvae reared on a commercial artificial diet (Insecta
LF; Nihon Nosan Kogyo, Yokohama, Japan) were dissec-
ted, and whole guts were obtained. The guts from six larvae
were homogenized in 4.5 mL sodium acetate buffer (0.1
M
,
pH 5.5) using a glass homogenizer, and centrifuged at
10 000 g for 10 min. The supernatant was mixed with 3 vol.
cold ()20 °C) acetone by gentle stirring and then kept in a
freezer ()35 °C) for 15 min. The supernatant was decanted
after centrifugation at 20 000 g for 5 min, and the pellet was
dissolved in a minimum volume of 62.5 m
M
Tris/HCl
(pH 6.8). The suspension was centrifuged at 20 000 g for
5 min to remove insoluble materials, and then the super-
natant was loaded on a gel for native PAGE and activity
staining. After electrophoresis, the gel was rinsed twice in
distilled water with gentle shaking for 5 min and divided into
two parts: (a) a gel strip for activity staining; (b) the remaining
gel sheet for elution of proteins. The gel sheet was stored at

4 °C until use, and the gel strip was subjected to activity
staining. The position of cellulase activity was identified, and
the corresponding position of the gel sheet was cut out, sliced
into small sections ( 1 mm cubes) and transferred to
1.5 mL centrifugal micro tubes. The micro tubes were filled
with distilled water and kept at 4 °C overnight to allow
elution of the proteins from the gel sections. The tubes were
centrifuged at 10 000 g for 5 min to sediment the gel sections,
and the supernatants were transferred to a commercial
disposable device for ultrafiltration (UltrafreeÒ-MC 10 000
NMWL centrifugal filter unit, Millipore), which had a
polysulfone membrane with a 10-kDa exclusion size. The
filter unit was centrifuged at 5000 g until the remaining
volume decreased below 40 lL. The remaining solution, in
which proteins were concentrated, was mixed with an equal
volume of Laemmli Sample Buffer (Bio-Rad) without the
addition of 2-mercaptoethanol before incubation at 33 °C
for 30 min. Then, proteins were subjected to SDS/PAGE.
After electrophoresis, proteins including the cellulase activity
were eluted from the gel and concentrated as for native
PAGE. The purity of the eluted proteins was checked by
further SDS/PAGE with CBB staining.
Analysis of N-terminal amino-acid sequence
The purified cellulase from P. hilaris larvae was subjected
to SDS/PAGE and transferred to a poly(vinylidene difluo-
ride) membrane (Bio-Rad) in transfer buffer [48 m
M
Tris,
39 m
M

glycine, 0.0375% (w/v) SDS, pH 9.2] by using a
TRANS-BLOTÒ SD Semi-dry Transfer Cell (Bio-Rad).
Proteins were visualized with CBB, cut out, and subjected
to gas phase protein sequencing (model LF-3400 DT;
Beckman).
cDNA cloning, genomic PCR and sequencing
A QuickPrepÒ Micro mRNA Purification Kit (Amersham
Bioscience) was used for isolation of mRNA from P. hilaris
larval midguts. First-strand cDNA synthesis from the
isolated mRNA and the following amplification of the
target cDNA were performed with a SMART
TM
RACE
cDNA Amplification Kit (Clontech) according to the
manufacturer’s instruction except that SuperScript II
reverse transcriptase (Invitrogen) was used. A degenerate
oligonucleotide primer (5¢-GTICARGGIGTITGYATHG
TIGAYG-3¢) was designed based on the N-terminal amino-
acid sequence to amplify the cDNA for P. hilaris cellulase.
RACE amplification of the 3¢-end was performed with this
degenerate primer and an anchor primer corresponding to
the anchor sequence combined to the 3¢-end of the oligo-
dT primer for first-strand synthesis of cDNA [12], and
5¢-RACE amplification was performed with a gene-specific
primer based on the sequence of the 3¢-fragment. The
nucleotide sequence was determined by using a BigDyeTer-
minator cycle sequencing kit and an ABI3700 automated
DNA sequencer (Applied Biosystems). Sequence similarities
were determined by a
BLAST

search (.
nih.gov/BLAST/). Forward (F) and reverse (R) primers,
3456 M. Sugimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003
1–24 (F), 209–230 (F), 289–311 (R), 446–469 (R), 651–680
(R), 667–688 (F), 713–740 (F), 781–807 (R) and 995–1013
(R), were designed from the cDNA sequence and used in
various combinations of genomic PCRs of P. hilaris fat
body tissue, extracted as described previously [13]. The
conditions for PCR were 35 cycles of 94 °Cfor1min,52°C
for 1 min, 72 °C for 3 min. Care was taken with the
solutions, and pipettes were used to avoid potential
contamination from previously prepared cDNAs.
Protein assay
Protein concentration was determined by using a protein
assay kit (CoomassieÒ Plus Protein Assay Reagent; Pierce)
with BSA as a standard.
TLC analysis
TLC was performed with silica gel 60 (Merck) in a solvent
system of butan-1-ol/acetic acid/water (2 : 1 : 1, by vol.),
and sugars were visualized by a heat treatment at 120 °Cfor
10 min after the spraying of 50% (v/v) H
2
SO
4
in methanol.
Results
Purification of
P. hilaris
cellulase from larval guts
The gut homogenate of P. hilaris larvae was precipitated

with acetone. The acetone treatment was effective in
reducing sample volume while minimizing loss of cellulase
activity. On native PAGE, although a number of proteins
from the acetone-treated samples were detected by CBB
staining, only one band was detected by activity staining
(data not shown). After elution and concentration of the
protein solution containing the cellulase activity after native
PAGE, the solution was mixed with sample buffer for SDS/
PAGE without reducing agent and incubated at 33 °Cfor
30mintoloadanSDS/polyacrylamidegel.TheSDS/
PAGE analysis detected three proteins bands by CBB
staining and one activity band by activity staining. Proteins
including cellulase activity were eluted and concentrated
from the unstained gel, and the protein solution was
incubated with sample buffer for SDS/PAGE, adding
reducing agent at 100 °C for 5 min. Then, the protein
solution was again subjected to SDS/PAGE. A single
47-kDa protein band was detected by CBB staining, which
indicated a successful purification of cellulase (Fig. 1).
Optimal pH for
P. hilaris
cellulase activity and pH
in the gut juice of
P. hilaris
larvae
The effect of pH on P. hilaris cellulase activity was tested
with 0.1
M
sodium acetate, sodium phosphate and Tris/HCl
buffers. For a particular pH, the use of different buffers did

not markedly alter enzyme activity, nor did different
concentrations of these buffers. The optimal pH for cellulase
activity against CMC was 5.5. Although the cellulase
activity was greatly reduced at pH values above 7,
some activity remained at pH 9.5 (data not shown). No
cellulase activity was observed at pH values less than 4.0.
The digestive tract of P. hilaris larvae was found to be
folded, and its total length was  1.5 times its body length.
The boundary between the midgut and hindgut was
indistinct, and no specialized hindgut structure such as that
of symbiont-possessing insects such as termites and scarab-
aeid beetles was observed. The gut contents were semisolid
and scarcely flowed out upon dissection. The pH values
estimated in the anterior midgut, posterior midgut and
hindgut were 5.7, 5.9 and 7.7, respectively.
Enzymatic degradation of cellulose and its derivatives
by
P. hilaris
cellulase
To investigate the ability of P. hilaris cellulase to degrade
cellulose and its derivatives, CMC (soluble in water but
carboxymethylated), insoluble cello-oligosaccharide (aver-
age degree of polymerization 34), Avicel (crystalline cellu-
lose), cellobiose, cellotriose, cellotetraose and cellopentaose
were used as substrates. P. hilaris cellulase readily degraded
CMC, and the specific activity was determined to be
150 lmolÆmin
)1
Æ(mg protein)
)1

. Insoluble cello-oligosaccha-
ride was also readily degraded, and the degradation
products were investigated by TLC. Cellobiose and cello-
triose were detected but not glucose (Fig. 2A). However,
with the use of a glucose assay kit, a small amount of
glucose was detected as a degradation product from
insoluble cello-oligosaccharide, and the glucose productivity
was 0.072 lmolÆmin
)1
Æ(mg protein)
)1
. Both cellotetraose
Fig. 1. SDS/PAGE analysis of the purified cellulase from larval gut
juice of P. hilaris . Lane 1, molecular mass standards consisting of
myosin (200 kDa), b-galactosidase (116 kDa), phosphorylase B
(97.4 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
(31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and
aprotinin (6.5 kDa). Lane 2, purified cellulase. Proteins were stained
with CBB R-250.
Ó FEBS 2003 Cellulase of the yellow-spotted longicorn beetle (Eur. J. Biochem. 270) 3457
and cellopentaose were degraded to cellobiose and cellotri-
ose (Fig. 2B). Production of cellobiose and cellotriose from
cellopentaose by degradation is natural but production of
cellobiose and cellotriose from cellotetraose is abnormal.
Cellotetraose should be degraded to produce two cellobiose
molecules or a combination of a cellotriose and glucose.
To obtain information about the missing glucose, glucose-
digesting activity was tested. No glucose-digesting activity
was detected in the purified P. hilaris cellulase, suggesting
that contamination by enzymes that digest glucose was not

responsible for the missing glucose. Transglycosylation
activity, known as the reverse reaction of some endo-
glucanases, was examined. Very low activity was detected in
the purified P. hilaris cellulase (data not shown). Avicel and
cellobiose were not degraded by P. hilaris cellulase, and
cellotriose was partially degraded to cellobiose.
Analysis of N-terminal amino-acid sequence, cDNA
sequence and deduced amino-acid sequence
The N-terminal amino-acid sequence of the purified P. hi-
laris cellulase was analyzed. The sample blotted to a
poly(vinylidene difluoride) membrane after SDS/PAGE
was used, and 30 amino acids from the N-terminus were
determined as follows: KDAAL ETVSK HGQLS VQGVD
IVDES GEKVQ. A degenerate primer was designed based
on the amino acids determined, and an  0.9-kbp 3¢-frag-
ment was amplified. The flanking region for the 5¢-end of
the cDNA was obtained by 5¢-RACE with a gene specific
primer based on the sequence of the first fragment. A full-
length of cDNA clone encoding a cellulase gene was
obtained and sequenced. The nucleotide sequence was
deposited in GenBank (accession number is AB080266).
The cDNA encoding P. hilaris cellulase contained an ORF
975 bp long, starting with an ATG codon at position 38 and
ending with a TAA codon at position 1014. A poly(A) tail
and typical polyadenylation signal were found. Two poten-
tial N-glycosylation sites, N270 and N300, were detected in
the deduced amino-acid sequence. The ORF consisted of a
protein of 325 amino acids. The molecular mass of P. hilaris
cellulase was calculated to be 36.0 kDa, and the first 21
amino acids were predicted to be a signal sequence for

secretion. The molecular mass of the mature enzyme was
deduced to be 33.8 kDa. The P. hilaris cellulase consisted
of a single catalytic module only and no carbohydrate
binding module was found.
BLAST
searches with the deduced
amino-acid sequence indicated that P. hilaris cellulase was
closely related to nematode cellulases and some bacterial
cellulases, those belonging to GHF5 subgroup 2. The
overall identities and similarities of P. hilaris cellulase
to GHF 5 subgroup 2 members were: 49% and 67%
to Pseudomonas fluorescence CelE, 49% and 66% to
Meloidogyne incognita MI-ENG1, respectively. A GHF 5
signature sequence, [LIV]-[LIVMFYWGA](2)-[DNEQG]-
[LIVMGST]-x-N-E-[PV]-[RHDNSTLIVFY], was con-
served in the deduced amino-acid sequence of P. hilaris
cellulase (Fig. 3). The conserved potential proton donor and
Fig. 2. Substrate specificity and degradation
products of P. hilaris cellulase. TLC was
performed with the solvent system butan-
1-ol/acetic acid/water (2 : 1 : 1, v/v/v.). Sugars
were visualized by incubating the plate at
120 °C for 10 min after spraying with 50%
(v/v) H
2
SO
4
in methanol. (A) Lane 1, standard
sugars: glucose (G1), cellobiose (G2), cellotri-
ose (G3) and cellotetraose (G4). Lane 2,

insoluble cello-oligosaccharides. Lane 3,
purified P. hilaris cellulase. Lane 4, insoluble
cello-oligosaccharides treated with P. hilaris
cellulase at 37 °C for 8 h. (B) Lane 1, standard
sugars: G1, G2, G3, G4, cellopentaose (G5)
and cellohexaose (G6). Lanes 2–5, G2–G5
treated with P. hilaris cellulase at 37 °Cfor
2h.
Fig. 3. Comparison of amino-acid sequences for potential catalytic
proton donor and nucleophile regions of P. hilaris cellulase (AB080266),
Globodera rostochiensis ENG1 (GR-ENG1, AF004523), Meloidogyne
incognita ENG1 (MI-ENG1, AF100549), Pseudomonas fluorescence
CelE (CelE, X86798) and Erwinia chrysanthemi CelZ (CelZ, Y00540).
Alignment was performed with the computer program
CLUSTAL X
(version 1.81) using the catalytic module of each cellulase. Residue
numbers are given on the left of the sequences. Amino acids with
similar groups of side chains and identical amino acids in sequences are
indicated by Ô:Õ or Ô*Õ, respectively, above the sequence. ÔPÕ and ÔNÕ below
the sequence indicate potential catalytic proton donor and nucleophile
amino acids, respectively.
3458 M. Sugimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003
nucleophile amino acids are also found in the sequence
(Fig. 3). Genomic PCR experiments with a variety of
different primers designed from the cDNA sequence
resulted in the amplification of bands in each case.
Sequencing of these bands showed that they matched the
sequence of the cellulase cDNA completely; however, no
introns were found in any case.
Discussion

Although optimal pH values for cellulase activity vary from
acidic to alkaline [14–17], all animal cellulases reported until
now have optimal activity under weak acidic conditions
[4,18,19]. The optimal pH for the purified cellulase from the
larval gut of P. hilaris against CMC was also 5.5. This is
reasonable for the physiological function of cellulase activity
in larval guts as the pH values in the anterior midgut,
posterior midgut and hindgut were 5.7, 5.9 and 7.7,
respectively. These results suggest that P. hilaris cellulase
functions mainly in the midgut, which is generally thought
of as a digestive and absorptive organ in the insect
alimentary canal.
The purified P. hilaris cellulase showed no degradation
activity against crystalline cellulose, which suggests that
P. hilaris larvae may utilize only the amorphous parts of
cellulose materials ingested. The elongated (about 1.5 times
its body length) digestive tract of P. hilaris presumably
means that the ingested cellulose material is exposed to
enzymatic digestion for long periods.
Animals in general absorb sugars in monomeric forms
such as glucose and fructose [20,21]. Although the degra-
dation products of the P. hilaris cellulase were found to be
cellotriose and cellobiose, glucose would be produced by
b-glucosidase activity in the larval gut, which has been
previously demonstrated in P. hilaris [8].
TLC analysis detected cellotriose and cellobiose after
cellotetraose was treated with the purified P. hilaris cellulase
(Fig. 2B). A molecule of cellotetraose should be degraded
into two cellobiose molecules, or a cellotriose molecule and
a glucose molecule, by a single catalytic event. Therefore,

after cellotetraose is degraded, glucose equivalent to cello-
triose should be detected. However, only cellotriose was
detected by TLC. Similarly, after cellotriose was degraded
by the P. hilaris cellulase, TLC detected cellobiose but not
glucose (Fig. 2B). It is known that some endoglucanases
possess transglycosylation activity as the reverse reaction
[22]. Oikawa et al. [23] reported that the addition of acetone
to the reaction buffer increased transglycosylation activity
of the Rhodotorula glutinis cellulase. Observation of trans-
glycosylation activity in the purified P. hilaris cellulase was
attempted under various conditions, including the addition
of acetone to the reaction mixture. Although transglycosy-
lation activity was detected, it was not enough to explain the
lack of glucose. However, transglycosylation is the most
probable explanation for the missing glucose, because there
are few other potential mechanisms for eliminating it.
ThemolecularmassofP. hilaris cellulase deduced from
its DNA sequence is 36.0 kDa. The apparent molecular
mass of the purified P. hilaris cellulase was, however,
estimated to be 47 kDa from its mobility on SDS/PAGE.
N-Terminal amino-acid sequencing analysis indicated that
thematureproteinofP. hilaris cellulase was a truncated
form, which lacked a signal peptide composed of the first 21
amino acids. Therefore, the deduced molecular mass of
mature cellulase protein is 33.8 kDa and the difference is
13.2 kDa. This inconsistency may be explained by a possible
post-transcriptional modification at two potential N-glyco-
sylation sites, N270 and N300, in the amino-acid sequence
of P. hilaris cellulase. Alternatively, the mobility of the
P. hilaris cellulase protein on SDS/PAGE may be different

from those of the standard proteins used in the experiment.
A cDNA encoding a cellulase, which belongs to GHF 45,
has been cloned from a gut library from the phytophagous
beetle, Phaedon cochleariae [24]. This GHF 45 cellulase has
been confirmed to be expressed in the gut of P. cochleariae.
P. cochleariae and P. hilaris are closely related species,
belonging to the same superfamily, Chrysomeloidea. There-
fore, a GHF 45 cellulase might have been expected from
P. hilaris. However, the cellulase purified from P. hilaris in
the current study was shown to belong to GHF 5. The
activity staining indicated that there were no other CMCase
activities except the GHF 5 cellulase in the larval gut of
P. hilaris. The sensitivity of the activity staining used is high,
and CMCase activity has been detectable with 5 lL of 1000
times diluted gut juice of P. hilaris larvae. Therefore, if a
GHF 45 enzyme is present, its level of expression would be
extremely low in P. hilaris larvae.
Glycosyl hydrolases have been categorized into 90
families according to homologies of their amino-acid
sequences, and cellulases are distributed into 14 families
( Most of these
14 families are composed of cellulases only, but some
include other enzymes, such as xylanase and mannanase.
P. hilaris cellulase was proposed to belong to GHF 5 and
the signature sequence of GHF 5 was found in the amino-
acid sequence (Fig. 3). GHF 5 is the largest GHF and
includes endoglycosylceramidase, b-mannanase, exo-b-1,3-
glucanase, endo-1,6-b-glucosidase, b-xylanase and some
other enzymes, in addition to cellulase (endo-b-1,4-gluca-
nase). On the basis of sequence homology, GHF 5 enzymes

can be further divided into five subfamilies [1,25]. P. hilaris
cellulase is closely related to subfamily 2 members, which is
composed only of cellulases from bacteria, fungi and
nematodes. It has been shown that the nematode cellulase
genes contain several introns, and the positions are
conserved in the deduced amino-acid sequences [26].
Genomic DNA of P. hilaris was amplified and sequenced
to determine whether the cellulase gene contained introns;
however, none were found, despite combinations with
several primers. As intronless genes appear to be fairly
common among insects [27,28], the lack of introns in the
P. hilaris cellulase gene does not provide evidence for its
recent horizontal transfer from a prokaryote. A discussion
of the evolutionary origins of the cellulase enzymes in
animals will be given elsewhere [29].
Acknowledgements
We thank Ms. Sanae Wada for advice about rearing P. hilaris larvae.
This work was supported by the Promotion of Basic Research
Activities for Innovative Biosciences Fund from the Bio-oriented
Technology Research Advancement Institution (BRAIN; Omiya,
Saitama, 331-8537 Japan; www.brain.go.jp) and by the Pioneer
Research Project Fund (No. PRPF-0022) from the Ministry of
Ó FEBS 2003 Cellulase of the yellow-spotted longicorn beetle (Eur. J. Biochem. 270) 3459
Agriculture, Forestry and Fisheries of Japan. N.L. is supported by a
Science and Technology Agency of Japan Postdoctoral Fellowship.
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