The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel.. Primer FWC48A, designed from the known s
Trang 1Exo-mode of action of cellobiohydrolase Cel48C
A unique type of cellulase among Bacillales
Marta M Sa´nchez, F I Javier Pastor and Pilar Diaz
Department of Microbiology, Faculty of Biology, University of Barcelona, Spain
Sequence analysis of a Paenibacillus sp 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 a cellobiohydrolase in Paenibacillus sp BP-23 would contribute to widen up its range of action 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 from a variety of
groups [1,2] Breakage of cellulose seldom occurs as an
isolated process, but is instead part of a 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 action of 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 of action 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)
Trang 2linker 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), has been
estab-lished [18]
Strain Paenibacillus sp 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 of Paenibacillus sp BP-23
cellobiohydro-lase Cel48C, a unique type of cellucellobiohydro-lase among 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 FASTA3 (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 throughSIGNALP 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, PROSITE
and SWISS-MODEL 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 of a 2.2-kb DNA fragment, using a cell suspension from Paenibacillus 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 Paenibacillus sp BP-23 chromosome containing the truncated ORF found in plasmid pBRC7 (A) and complete ORF and multidomain structure of Cel48C (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 newcellulase; GHF48, family 48 catalytic domain of cellobiohydrolase Cel48C Transcription orientation is indicated by thick black arrows The small arrows indicate the position of the primers used for sequencing by gene walking FW48A corresponds to the first primer used, starting at the known region of the truncated ORF.
Trang 3consecutive use of primers FWC48B to FWC48G, until the
complete nucleotide sequence of the newORF was obtained
(Fig 1) The known DNA coding sequence was used to
design a newset 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 coliXl1-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 mM)
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 mM 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), and the
corresponding pellets washed for three times with the
same buffer For analysis of bound proteins, the last
pellets were eluted using 0.2M glucose, 1M NaCl, 1.5M
urea or H2O before loading onto SDS–polyacrylamide
gels (10% acrylamide) for protein analysis and binding
determination
Enzyme purification
The ability of Cel48C 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 of a 5% suspension of Avicel
in water Binding to Avicel was performed in batch for
1 h at 4°C in 50 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 mM
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 F254, 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 of Paenibacillus sp BP-23 (cel48C_ PAE23) cellobiohydrolase coding gene was submitted to the EMBL under accession number AJ488933 (Q8KKF7) Results and discussion
Isolation of recombinant cloneE coli/pUCel48C Sequence analysis of Paenibacillus sp 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 of a gene cluster
The complete DNA sequence of cel48C 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 cells and w ould help
to explain why family 48 cellulases are more difficult to clone, with only 12 family members identified up to now [11]
Trang 4Sequence analysis
Analysis of the complete nucleotide sequence of both
strands of cel48C showed the presence of a
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 of a cluster
constituted by the two contiguous genes coding for
cellu-lases Cel9B and Cel48C A palindromic 18 nucleotide
(GTGCAG)3 repeat with the appearance of a
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 of Cel48C under certain growth
conditions, as described for several Avicel-inducible
cellu-lases [32] An additional palindromic sequence with the
appearance of a terminator was found after the stop codon
of cel48C, acting as a signal structure for protein synthesis
termination
The protein deduced from cel48C 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 of a 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, Paenibacillus sp BP-23 [3,7]
The deduced amino acid sequence of Cel48C 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 of Cel48C 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,
)3 and )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)3 and )5 of the tunnel structure could account for differences in substrate speci-ficity as a result of a differential recognition capacity Purification and properties of Cel48C
For qualitative detection of Cel48C 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 of Cel48C to hydrolyse other insoluble cellulosic substrates, a newmethod 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 of a 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 Paenibacillus sp BP-23 supernatants, probably due to the lowconcentration of Cel48C 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 of a 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 Paenibacillus sp BP-23 (BP-23) were applied onto the gel and incubated for 18 h at 37 °C prior to detection
of activity by Congo red staining (B,C) In both gels specific molecular mass markers are shown (M).
Trang 5SDS/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 of Cel48C to strongly bind Avicel
allowed the development of a 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 of Cel48C was essentially the same
after storage
Mode of action of Cel48C
The hydrolytic profile of Cel48C 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 of Cel48C 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 of Cel48C 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 of a cellobiohydrolase, acting processively on these substrates
To analyse the exo-mode of action of 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 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 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)5 and )3
of the tunnel structure of Cel48C [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 showvery lowand 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
Trang 6of 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 Paenibacillus sp 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 of Paenibacillus sp 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 of action on naturally occurring cellulosic substrates,
as happens in cellulosome-containing Clostridium species [12] This system would confer strain Paenibacillus sp 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 Centre de Refere`ncia en Biotecnologia (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.
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