Exo-modeofactionofcellobiohydrolase Cel48C
from
Paenibacillus
sp. BP-23
A uniquetypeofcellulaseamong Bacillales
Marta M. Sa
´
nchez, F. I. Javier Pastor and Pilar Diaz
Department of Microbiology, Faculty of Biology, University of Barcelona, Spain
Sequence analysis ofaPaenibacillussp.BP-23 recombinant
clone coding for a previously described endoglucanase
revealed the presence of an additional truncated ORF with
homology to family 48 glycosyl hydrolases. The corres-
ponding 3509-bp DNA fragment was isolated after gene
walking and cloned in Escherichia coli Xl1-Blue for expres-
sion and purification. The encoded enzyme, a cellulase
of 1091 amino acids with a deduced molecular mass of
118 kDa and a pI of 4.85, displayed a multidomain organ-
ization bearing a canonical family 48 catalytic domain, a
bacterial type 3a cellulose-binding module, and a putative
fibronectin-III domain. The cloned cellulase, unique among
Bacillales and designated Cel48C, was purified through
affinity chromatography using its ability to bind Avicel.
Maximum activity was achieved at 45 °C and pH 6.0 on
acid-swollen cellulose, bacterial microcrystalline cellulose,
Avicel and cellodextrins, whereas no activity was found
on carboxy methyl cellulose, cellobiose, cellotriose, pNP-
glycosides or 4-methylumbeliferyl a-
D
-glucoside. Cellobiose
was the major product of cellulose hydrolysis, identifying
Cel48C as a processive cellobiohydrolase. Although no
chromogenic activity was detected from pNP-glycosides,
TLC analysis revealed the release of p-nitrophenyl-glyco-
sides and cellodextrins from these substrates, suggesting that
Cel48C acts from the reducing ends of the sugar chain.
Presence of such acellobiohydrolase in Paenibacillus sp.
BP-23 would contribute to widen up its range ofaction on
natural cellulosic substrates.
Keywords: cellobiohydrolase; cellulase; cellulose; family 48;
Paenibacillus.
The semicrystalline character of cellulose, one of the most
abundant renewable polymers on earth, makes its degra-
dation a problem of considerable proportions. In nature,
cellulose is mostly degraded by cellulolytic microorgan-
isms, including fungi and bacteria froma variety of
groups [1,2]. Breakage of cellulose seldom occurs as an
isolated process, but is instead part ofa concerted attack
on the complex constituted by cellulose, lignin and
hemicellulose. For this purpose, the combined action of
several extracellular enzymes bearing complementary
activities is essential [3,4].
Most cellulolytic microorganisms produce a battery of
cellulases which act synergistically to solubilize crystalline
cellulose [5]. Cellulases have traditionally been grouped into
endoglucanases and exoglucanases, sharing a common
specificity for 1,4-b-glucans, but differing in their mode of
action [1,3]. Efficient hydrolysis of cellulose depends on the
simultaneous actionof nonprocessive endo-1,4-b-glucanases
(EC 3.2.1.4), which produce new ends at random within the
polysaccharide chain, and processive exo-1,4-b-glucanases
(cellobiohydrolases; EC 3.2.1.91), which remain attached to
the substrate and split off cellobiose from such free ends [4,6].
On the basis of sequence homology and hydrophobic
clustering, the catalytic domains of known cellulases have
been assigned to different families in the glycosyl hydrolase
group of enzymes ([7] http://afmb.cnrs-mrs.fr/cazy/
CAZY/index.html). Among them, families 5, 6, 7, and 48
contain cellobiohydrolases. These enzymes display an exo-
mode ofaction by means of the shape of their active site
pocket, which is blocked by a bulky extension of the protein
that covers the catalytic amino acids and adopts a tunnel-
like structure [4,8]. Thus, cellulose can only access the active
site through one of its ends, where the enzyme acts
processively releasing cellobiose units by sliding along the
substrate chain [4,8]. Although the activity of most cello-
biohydrolases occurs at the nonreducing end of the glucose
polymer, certain processing enzymes acting from the
reducing end of the carbohydrate chain have been identified
[9,10,11]. Existence of both types of processive enzymes with
specificity for either chain-end would account for a
productive and complete degradation of cellulose [9,12].
Most known cellobiohydrolases display a multidomain
structure, including a catalytic domain, one or more
cellulose-binding modules (CBMs), cell interaction motifs,
Correspondence to P. Diaz, Department of Microbiology,
Faculty of Biology, University of Barcelona., Avenue. Diagonal 645,
08028-Barcelona, Spain.
Fax: + 34 93 4034629, Tel.: + 34 93 4034627,
E-mail: pdiaz@bio.ub.es
Abbreviations: CBM, cellulose-binding modules; Fn3, central type III
fibronectin; LB, Luria–Bertani; CMC, carboxy methyl cellulose;
ASC, acid swollen cellulose; IPTG, isopropyl thio-b-
D
-galactoside;
BMCC, bacterial microcrystalline cellulose.
Enzymes: endo-1,4-b-glucanases (EC 3.2.1.4); exo-1,4-b-glucanases
(cellobiohydrolases; EC 3.2.1.91).
(Received 13 March 2003, revised 30 April 2003,
accepted 15 May 2003)
Eur. J. Biochem. 270, 2913–2919 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03673.x
linker or repeat regions, and central type III fibronectin
(Fn3) modules [7,13–15]. Presence of these motifs has been
proposed to provide efficiency and stability to the enzyme
during catalysis [2,16]. In fact, CBMs significantly contri-
bute to the activity of the enzymes against cellulo-
sic substrates by increasing enzyme–substrate proximity,
enhancing accessibility, and modifying the surface of the
cellulose crystals [16,17]. As for catalytic domains, a classi-
fication of CBMs based on sequence homology (http://
afmb.cnrs-mrs.fr/cazy/CAZY/index.html), hasbeen estab-
lished [18].
Strain Paenibacillussp.BP-23 (formerly Bacillus sp. BP-
23) [19] shows a multienzymatic glycanase system, including
several cellulases [20,21], xylanases [22,23], or pectinases
[24]. In this study we report the cloning, purification and
characterization ofPaenibacillussp.BP-23 cellobiohydro-
lase Cel48C, auniquetypeofcellulaseamong Bacillales,
bearing a multidomain structure and showing the properties
of a processive enzyme acting from the reducing ends of the
sugar chain.
Materials and methods
Strains, plasmids and growth conditions
Paenibacillus sp.BP-23 (CECT 4592) [19] was routinely
grown in nutrient broth at 30 °C. Escherichia coli Xl1-Blue
[25], used as the recipient strain for recombinant plasmids,
was grown in Luria–Bertani (LB) medium at 37 °C. Plasmid
pUC19 (Boehringer Mannheim) was used as cloning vector.
Detection of activity on carboxy methyl cellulose (CMC,
Sigma) or acid swollen cellulose (ASC) [26] was performed
by incubation of grown cultures, cell suspensions, cell
extracts or culture supernatants either on LB-agar plates
supplemented with 1% CMC (w/v), or on thin agarose gels
supplemented with 2% ASC (w/v), for 1–24 h at 37 °C.
Activity was detected by staining with Congo red (Sigma),
as described [20].
Nucleic acid procedures
Plasmid and genomic DNA were purified and mani-
pulated essentially as described [25]. Restriction nucleases
and DNA-modifying enzymes were obtained from Roche
(Boehringer Manheim) and used according to the manu-
facturer’s specifications. Primer oligonucleotides were
purchased at Invitrogen, and pfu polymerase was from
VWR Int. The nucleotide sequence of both strands of the
isolated DNA fragments was determined [20], and
homology analysed through
BLAST
[27] and
FASTA
3
(http://www.ebi.ac.uk/fasta33). Sequence alignments were
done using
CLUSTALW MULTALIGN
program (http://
www2.ebi.ac.uk/clustalw), and signal peptide identifica-
tion was performed through
SIGNALP
V2.0 software [28],
according to the criteria described for Gram-positive
signal sequence identification [29]. Presence of defined
protein patterns, the physico-chemical parameters and the
three-dimensional structure of the deduced amino acid
sequence were determined using
PRODOM
,P
ROSITE
and S
WISS
-M
ODEL
at ExPASY (http://www.expasy.org).
Cloning procedure
The DNA fragment coding for cellobiohydrolase Cel48C
was isolated by PCR after sequence determination by gene
walking. The DNA insert of recombinant clone E. coli
5K/pC7 coding for endoglucanase Cel9B [21] contained an
additional truncated ORF with homology to family 48
cellulases (Fig. 1). Primer FWC48A, designed from the
known sequence of the truncated cellulase gene, and a
second degenerated primer, BKC48B, designed in the
opposite direction from the consensus C-terminal sequence
of the catalytic domains from previously described family
48 cellulases, were used for isolation ofa 2.2-kb DNA
fragment, using a cell suspension fromPaenibacillus sp.
BP-23 as a template. Complete sequencing of the whole
gene was performed by gene walking through the
Fig. 1. Physical map of the cel 9B region of the Paenibacillussp.BP-23 chromosome containing the truncated ORF found in plasmid pBRC7 (A) and
complete ORF and multidomain structure ofCel48C (B). SP, signal peptide; GHF9, family 9 catalytic region of endoglucanase Cel9B; CBM_3,
carbohydrate-binding module, type 3; Fn3, Fibronectin like domain; ORF?, putative truncated ORF coding for a new cellulase; GHF48, family 48
catalytic domain ofcellobiohydrolase Cel48C. Transcription orientation is indicated by thick black arrows. The small arrows indicate the positionof
the primers used for sequencing by gene walking. FW48A corresponds to the first primer used, starting at the known region of the truncated ORF.
2914 M. M. Sa
´
nchez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
consecutive use of primers FWC48B to FWC48G, until the
complete nucleotide sequence of the new ORF was obtained
(Fig. 1). The known DNA coding sequence was used to
design a new set of primers (FWC48I, BKC48I) for
isolation of the complete gene (Fig. 1). Both strands of
the resulting DNA fragment were sequenced and cloned in
E. coli Xl1-Blue, using pUC19 as a vector for expression of
the encoded protein. The recombinant clone obtained,
designated E. coli/pUCel48C, was used for further enzyme
production and purification.
Enzyme activity
E. coli/pUCel48C cell extracts were prepared after induc-
tion with isopropyl thio-b-
D
-galactoside (IPTG) (0.4 m
M
)
of late exponential growth cultures from E. coli/pU-
Cel48C, followed by an additional 2 h of incubation for
gene expression. Induced cultures were centrifuged and
cells recovered and suspended in 100 m
M
phosphate
buffer pH 6.0 prior to disruption through French Press
(1000 psi, SLM Instruments), essentially as described [30].
Cellulase activity was assayed as described previously
[21], by measuring the amount of reducing sugars
released after an 18 h incubation at 45 °C with different
cellulosic substrates [31]. Specific activity was calculated
using a calibration curve for glucose. One activity unit
was defined as the amount of enzyme capable to release
1 lmol of reducing sugar equivalentÆmin
)1
under the
assay conditions used. Liberation of p-nitrophenol from
p-nitrophenyl-glycosides (Sigma) was measured by
absorbance at 400 nm in alkaline solution. One unit of
enzyme activity was defined as the amount of enzyme
producing 1 lmol of p-nitrophenolÆmin
)1
. Activity at
different pH and temperature was determined after
incubation of the reaction mixtures at different condi-
tions, and measuring the release of reducing sugars as
described above.
Binding assays
Concentrated cell extracts of recombinant E. coli/pU-
Cel48C were mixed with an equal volume of 5% solutions
of Avicel (Fluka), bacterial microcrystalline cellulose
(BMCC, Monsanto) or ASC [26] in water, and incubated
for 1 h at 4 °C with gentle rotatory shaking. Samples
were then centrifuged (16 000 g,Beckman),andthe
corresponding pellets washed for three times with the
same buffer. For analysis of bound proteins, the last
pellets were eluted using 0.2
M
glucose, 1
M
NaCl, 1.5
M
urea or H
2
O before loading onto SDS–polyacrylamide
gels (10% acrylamide) for protein analysis and binding
determination.
Enzyme purification
The ability ofCel48C to bind Avicel strongly was used for
enzyme purification in a simplified affinity chromato-
graphy system developed in our laboratory. Cell extracts
from 5-L cultures of recombinant E. coli/pUCel48C, were
mixed with an equal volume ofa 5% suspension of Avicel
in water. Binding to Avicel was performed in batch for
1h at 4°Cin50m
M
phosphate buffer pH 6.0, using a
rotatory shaker (12 r.p.m.). After binding, the suspensions
were washed three times by centrifugation with the same
buffer and gently re-suspended for removal of unbound
proteins. A final wash was performed with 10 m
M
phosphate buffer pH 6.0. Elution of bound proteins was
achieved by addition of 1 vol. water, followed by vigorous
agitation and centrifugation (16 000 g, Beckman) to
remove Avicel. The resulting supernatants were collected,
filtered through a 22-lm MillexÒ GP filter (Millipore),
and concentrated through a 50-kDa BiomaxÒ filter
(Millipore) prior to loading onto SDS/PAGE gels (10%
acrylamide). The purified protein was lyophilized and
stored for further assays.
TLC
Reaction mixtures prepared as above were analysed on
silica gel plates (60 F
254
, Merck) for detection of the
hydrolysis products. A mixture of chloroform, acetic acid
and water (6 : 7 : 1, v/v) was used as eluent for long
polysaccharides and cellodextrins, while the hydrolysis
products of pNP-glycosides were eluted with a mixture of
ethyl acetate, acetic acid and water (2 : 1 : 1, v/v). After
separation, sugars were detected by spraying the plates with
a freshly prepared mixture of ethanol/concentrated sulphu-
ric acid (95 : 5, v/v).
Nucleotide sequence accession number
The DNA sequence ofPaenibacillussp.BP-23 (cel48C_
PAE23) cellobiohydrolase coding gene was submitted to the
EMBL under accession number AJ488933 (Q8KKF7).
Results and discussion
Isolation of recombinant clone
E. coli
/pUCel48C
Sequence analysis ofPaenibacillussp.BP-23 recombinant
clone E.coli/pBRC7 revealed the presence of the complete
ORF coding for endoglucanase Cel9B, described else-
where [21]. An additional truncated ORF, designated
cel48C, was found 151 nucleotides downstream from
cel9B on the same strand, the deduced product of which
(161 amino acids) was highly homologous to bacterial
family 48 cellulases [7]. Fig. 1 shows a schematic repre-
sentation of the physical map of the region, including gene
cel9B and the known region of the truncated cel48C gene,
where both genes appear to be arranged as part ofa gene
cluster.
The complete DNA sequence ofcel48C ORF was
obtained by gene walking as described in Materials and
methods and used to isolate the whole coding region
(Fig. 1). The 3509-bp DNA fragment obtained was
sequenced for confirmation, cloned in E. coli Xl1-Blue
using pUC19 as a vector, and transformants were
selected in the absence of IPTG, as no recombinant
clones could be obtained when IPTG was present in the
growth medium. This fact suggests that the cloned
enzyme is somewhat toxic to E. coli cellsandwouldhelp
to explain why family 48 cellulases are more difficult to
clone, with only 12 family members identified up to now
[11].
Ó FEBS 2003 CellobiohydrolaseCel48CfromPaenibacillus (Eur. J. Biochem. 270) 2915
Sequence analysis
Analysis of the complete nucleotide sequence of both
strands ofcel48C showed the presence ofa ribosome-
binding site placed nine nucleotides upstream of the ATG
start codon, plus two putative )35 and )10 promoter
sequences, suggesting that indeed cel48C can be transcribed
from its own promoter while being part ofa cluster
constituted by the two contiguous genes coding for cellu-
lases Cel9B and Cel48C. A palindromic 18 nucleotide
(GTGCAG)
3
repeat with the appearance ofa rho-inde-
pendent terminator and with no similarity to previously
described operators was found 20 nucleotides downstream
the stop codon of cel9B and 28 nucleotides upstream the
hypothetical promoter region of cel48C. Presence of such a
structure could account for a regulatory region controlling
the differential expression ofCel48C under certain growth
conditions, as described for several Avicel-inducible cellu-
lases [32]. An additional palindromic sequence with the
appearance ofa terminator was found after the stop codon
of cel48C, acting as a signal structure for protein synthesis
termination.
The protein deduced fromcel48C contained 1091 amino
acids and showed a predicted molecular weight and pI of
118 kDa and 4.85, respectively. As confirmed by SignalP
program, a 35-amino acid stretch with the features of a
signal peptide [29] was found at the N-terminal region of
the protein, indicating its extracellular location. Analysis of
Cel48C amino acid sequence revealed a modular structure
(Fig. 1) consisting ofa canonical family 48 catalytic domain
located at the N-terminal region of the protein (residues
51–748), a central Fn3 module (residues 757–850), and a
bacterial type 3a CBM located in the C-terminal portion of
the enzyme (residues 943–1087). All conserved residues of
CBM_3a were found in Cel48C [7]. According to the latest
nomenclature, the cloned enzyme was described as
Cel48C_PAE23, with the structural designation CD48/
Fn3/CBM_3a to indicate the type and location of the
different domains and providing information about the
organism of origin, Paenibacillussp.BP-23 [3,7].
The deduced amino acid sequence ofCel48C catalytic
domain showed 41–46% identity to the catalytic domains of
previously described family 48 cellulases (http://afmb.cnrs-
mrs.fr/cazy/CAZY/index.html) [3,7], while the noncata-
lytic regions ofCel48C showed the highest identity (63%)
to the C-terminal region of the preceding endoglucanase
Cel9B [21], both containing a highly conserved sequence at
their C-terminal portions. When analysed separately, the
CBM_3a contained in Cel48C showed homology
(36–40%) to other type 3 CBMs present in a large number
of bacterial glycosyl-hydrolases [7,27].
The theoretical three-dimensional structure of Cel48C
was generated based on those of Clostridium cellulolyticum
CelF [8] and Clostridium thermocellum CelS [6] family 48
cellulases. The overall model produced a good fit with both
of them, showing the proposed catalytic nucleophile and the
putative acid–base catalysts [8] at positions E45, E56, and
D235. The strictly conserved amino acids of subsites )7, )5,
)3and)2 lining the tunnel structure in family 48 cellulases
were found at positions W317, W319, Y304 and W158,
respectively [6,8]. The most important amino acid differ-
ences affecting the three-dimensional structure of the cloned
enzyme with respect to CelF and CelS consist of several
additional loops (V92–D96, L172–S175, I436–A438, L443–
F448, F467–Y479, R487–E504, A569–G571) placed at the
protein surface that seem not to interfere with the hydrolytic
functions of the enzyme. Nevertheless, a 4-amino acid loop
(V92–D96) located close to subsites )3and)5 of the tunnel
structure could account for differences in substrate speci-
ficity as a result ofa differential recognition capacity.
Purification and properties of Cel48C
For qualitative detection ofCel48C activity, cell extracts,
cell suspensions, or grown cultures from E. coli/pUCel48C
were assayed on CMC-supplemented agar plates as des-
cribed before [20]. No activity on CMC could be detected
under the different conditions assayed. In order to deter-
mine the ability ofCel48C to hydrolyse other insoluble
cellulosic substrates, a new method for detection of activity
on ASC was developed. The new system consists on the use
of thin agarose gels supplemented with ASC, prepared on
the surface ofa glass slide. As shown in Fig. 2, activity of
Cel48C could be detected on this substrate after an 18 h
incubation of concentrated E. coli/pUCel48C cell extracts
in the presence of 2% ASC. As expected, no activity was
observed for control E. coli/pUC19 cell extracts, while low
activity was shown by E. coli/pBRC7 cell extracts. No
activity was found for Paenibacillussp.BP-23 supernatants,
probably due to the low concentration ofCel48C protein in
the samples.
Fig. 2. Simple activity assay developed to detect cellobiohydrolase deg-
radation of ASC (A) and SDS/PAGE (15% polyacrylamide, B; 10%
polyacrylamide, C) analysis of cell extracts from E. coli/pUCel48C (1)
and E. coli/pUC19 (2). (A) A thin agarose gel supplemented with 2%
ASC was prepared on the surface ofa glass slide. A small volume
(15 lL) of cell extracts from E. coli/pUCel48C (Cel48C), E. coli/
pBRC7 (Cel9B) and E. coli/pUC19 (C-), plus concentrated super-
natant from parental strain Paenibacillussp.BP-23 (BP-23) were
applied onto the gel and incubated for 18 h at 37 °Cpriortodetection
of activity by Congo red staining. (B,C) In both gels specific molecular
mass markers are shown (M).
2916 M. M. Sa
´
nchez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
SDS/PAGE analysis of cell extracts from E. coli/pU-
Cel48C showed the presence of two bands of 122 and
114 kDa, not found in cell extracts of control E. coli/
pUC19 (Fig. 2). According to the predicted molecular mass
of Cel48C, the upper 122-kDa band would correspond to
the complete protein, while the lower 114-kDa band would
be a product of enzyme proteolysis, an effect frequently
observed in multidomain glycosyl hydrolases, and described
to occur mostly at the join points between modules [21].
The ability of cloned Cel48C to bind cellulose was tested
and used for enzyme purification. Following the procedure
described at the Materials and methods section, Cel48C
strongly bound to ASC, Avicel and BMCC (not shown),
although elution of the enzyme from ASC could not be
achieved. Among the different elutants used for protein
separation after binding, water provided the highest
efficiency. The ability ofCel48C to strongly bind Avicel
allowed the development ofa simple batch-affinity chro-
matography system for purification of the cloned enzyme,
using Avicel as the ligand substrate. SDS/PAGE of the
eluted samples indicated that the enzyme had been purified
essentially to homogeneity (Fig. 2), showing a molecular
mass consistent with that calculated from SDS/PAGE. The
average yield of purification was estimated to be 40–65%
recovery of the desired protein. The purified cellulase was
subsequently concentrated, lyophilysed and stored at room
temperature. Activity ofCel48C was essentially the same
after storage.
Mode ofactionof Cel48C
The hydrolytic profile ofCel48C on polymeric or oligomeric
substrates was determined by measuring the reducing sugar
equivalent release and by TLC analysis. In general, the
activities shown by Cel48C on all substrates assayed were
extremely low, as happens for most family 48 cellobio-
hydrolases [6,11]. The enzyme displayed maximum activity
at 45 °C and pH 6.0, being active after 48 h incubation
under these conditions. The highest rate of hydrolysis was
found on ASC (4.88 mUÆmg protein
)1
), reaching half of the
maximum reaction velocity at a concentration of 0.21%
ASC. Activity was also found on BMCC (1.88 mUÆmg
prot
)1
), whereas activity on Avicel was much lower
(0.48 mUÆmg protein
)1
). No reducing sugars were released
from CMC, starch, birchwood xylan, polygalacturonic acid,
or laminarin, and no methylumbeliferone was released from
4-methylumbeliferyl a-
D
-glucoside. Based on these results,
Cel48C appears to be an exocellulase with a narrow
substrate specificity [4,9]. The major product of ASC,
BMCC and Avicel hydrolysis detected by TLC after 18 h
digestion was cellobiose (Fig. 3), as is typical for cellobio-
hydrolases, including those acting on crystalline cellulose
[4,11]. However, analysis of the kinetics of ASC digestion
with Cel48C showed the additional production of minor
amounts of cellotriose and cellotetraose (not shown),
suggesting that the enzyme could bear some minor endo-
glucanase activity, as described for certain exocellulases [10].
Hydrolysis of cellodextrins was also assayed by TLC
(Fig. 3). Cellobiose and cellotriose were not hydrolysed by
Cel48C, as happens for other cellobiohydrolases [10,11].
Cellotetraose was mostly hydrolysed to cellobiose, and
degradation of cellopentaose produced both cellobiose and
cellotriose. As in the case of ASC, analysis of the kinetics of
cellotetraose digestion showed the presence of minor
amounts of cellotriose only after 168 h incubation (not
shown), indicating that the hypothetical endoglucanase
activity ofCel48C on this substrate is very low, acting
mostly in an exo- mode as a processive enzyme (EC 3.2.1.91)
[9,12]. Further evidence for the processivity ofCel48C was
obtained after a 48-h digestion of ASC and Avicel with
Cel48C. The products of digestion were analysed by TLC
by loading both the supernatants of the reaction and the
insoluble fractions of the digested samples. As shown in
Fig. 3, no soluble sugars appeared at the insoluble fraction
of either digested sample, indicating that the main activity of
Cel48C is that ofa cellobiohydrolase, acting processively on
these substrates.
To analyse the exo-modeofactionof Cel48C,
p-nitrophenol liberation fom p-nitrophenyl (pNP)-glyco-
sides was assayed by spectroscopy. Interestingly, hydrolysis
of pNP-cellobioside, a substrate readily hydrolysed by exo-
glucanases and used as an indicator for cellobiohydrolase
activity [11] did not release pNP. In addition, no chromo-
genic activity was found on pNP-cellotrioside, pNP-
cellotetraoside or pNP-cellopentaoside, indicating that
p-nitrophenol was not released from these substrates either.
However, analysis by TLC of the products released after
hydrolysis of pNP-glycosides showed that Cel48C caused
indeed the degradation of pNP-cellotetraoside and pNP-
cellopentaoside, with liberation of pNP-cellobioside and
cellobiose or cellotriose, respectively (Fig. 3). The results
obtained indicate that, although very low, the enzyme bears
activity on these substrates and suggest that Cel48C cannot
proceed from the free nonreducing end of the sugar chain.
In fact, if pNP-cellopentaoside were hydrolysed from the
nonreducing end, the expected products would be cellobiose
and pNP-glucoside (or pNP-cellotrioside) [10], not found
after Cel48C hydrolysis. Although it cannot be ruled out
that the presence of the aromatic group may affect the
expected pattern of degradation, cellobiose should be
released after hydrolysis from the nonreducing end [9]. As
expected, no hydrolysis of pNP-cellobioside or pNP-cello-
trioside could be detected (Fig. 3), supporting the hypothe-
sis that cellobiose cannot be released from the nonreducing
ends of these substrates [9,10]. Such an exo- processive mode
of action suggests that Cel48C hydrolyses polysaccharides
and cellodextrins from the reducing end of the sugar chain
[9,10]. The presence a small additional amino acid loop
found close to the substrate recognition subsites )5and)3
of the tunnel structure ofCel48C [6,8] could account for a
differential substrate recognition and could be responsible
for the anomalous activity found on pNP-glycosides.
Reducing-end directed processive exocellulases have
already been described among family 48 glycosyl hydrolases
[8,9], with some members also having some endoglucanase
activity like Cel48C [10,11]. Nevertheless, the real function
of such class of enzymes has not been solved to date, as they
show very low and restricted activity on most common
cellulosic substrates. Production by bacteria of family 48
exocellulases with no apparent activity may indicate that
this kind of enzymes play a yet unknown role in the
breakdown of cellulosic substrates in nature, acting prob-
ably as key components of the cellulolytic system of certain
cellulase-producing bacteria [12]. Study of their mechanism
Ó FEBS 2003 CellobiohydrolaseCel48CfromPaenibacillus (Eur. J. Biochem. 270) 2917
of action and knowledge of their natural substrate may be of
great interest to understand the biological role of family 48
cellobiohydrolases. For this purpose, further synergism
assays [5,11,33] are being performed using combinations of
Cel48C and other endo- or exo-cellulases from either the
same or different strains.
The biochemical and structural properties shown by
Cel48C, the first cellobiohydrolase described among Bacil-
lales, and the general properties of the Paenibacillussp. BP-23
cellulolytic system that consists of two endoglucanases from
families 5 and 9 with homology to Clostridium species
cellulases [20,21], and a reducing-end processive cello-
biohydrolase (Cel48C), similar to those found in anaerobic
bacteria [34], seem to be closer to the cellulolytic systems of
anaerobic cellulosome-containing bacteria than to those
of Bacillus species [35,36], suggesting a higher degree of
proximity ofPaenibacillussp.BP-23 to glucan-hydrolysing
anaerobic bacteria [15]. However, like Cellulomonas fimi
cellobiohydrolase B [14], Cel48C bears its own CBM and two
fibronectin domains that would enable the cell to widen up its
range ofaction on naturally occurring cellulosic substrates,
as happens in cellulosome-containing Clostridium species
[12]. This system would confer strain Paenibacillussp. BP-23
the properties of an efficient system for biotechnological
applications such as pulp and paper manufacture [37].
Acknowledgements
We thank the Serveis Cientifico-Te
`
cnics of the University of Barcelona
for technical aid in sequencing. This work was partially financed by the
Scientific and Technological Research Council (CICYT, Spain), grants
QUI98-0413-CO2-02 and PPQ2001-2161-CO2-02, by the III Pla de
Recerca de Catalunya (Generalitat de Catalunya), grant 2001SGR-
00143, and by the Generalitat de Catalunya to the ÔCentre de
Refere
`
ncia en BiotecnologiaÕ (CeRBa). M. Sa
´
nchez is a recipient of a
fellowship from the Spanish Ministery of Education and Science.
Fig. 3. TLC analysis of the products of hydrolysis released by Cel48C. (A) Production of cellobiose and cellotriose from polysaccharides and
cellodextrins after 18 h incubation at 45 °C. (M): G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose. Lanes (1) and
(2): ACS incubated without (1) and with (2) Cel48C. Lanes (3) and (4): BMCC incubated without (3) and with (4) enzyme. Lanes (5) and (6): Avicel
incubated without (5) and with (6) enzyme. Lanes (7) and (8): cellobiose (G2) incubated without (7) and with (8) enzyme. Lanes (9) and (10):
cellotriose (G3) incubated without (9) and with (10) enzyme. Lanes (11) and (12): cellotetraose (G4) incubated without (11) and with (12) enzyme.
Lanes (13) and (14): cellopentaose (G5) incubated without (13) and with (14) enzyme. (B) 48-h incubation of ASC and Avicel without (1, 3), or with
enzyme (2, 4). Samples correspond to the supernatants of the incubated samples (1, 2) or to the insoluble fractions of the reaction mixtures (3, 4). (C)
Hydrolysis of pNP-glycosides by Cel48C. Lanes: (1) pNP-glucoside (pNPG) (2) pNP-cellobioside (4) pNP-cellotrioside (6) pNP-cellotetraoside (8)
pNP-cellopentaoside incubated without enzyme. Lanes: (3) pNP-cellobioside (5) pNP-cellotrioside (7) pNP-cellotetraoside (9) pNP-cellopentaoside
incubated with Cel48C as above. Lane (10) corresponds to the products of hydrolysis of cellopentaose (G5) and is shown as size marker.
2918 M. M. Sa
´
nchez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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Ó FEBS 2003 CellobiohydrolaseCel48CfromPaenibacillus (Eur. J. Biochem. 270) 2919
. Exo-mode of action of cellobiohydrolase Cel48C
from
Paenibacillus
sp. BP-23
A unique type of cellulase among Bacillales
Marta M. Sa
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nchez, F. I. Javier. and
characterization of Paenibacillus sp. BP-23 cellobiohydro-
lase Cel48C, a unique type of cellulase among Bacillales,
bearing a multidomain structure and