Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23 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) 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 Paenibacillus sp. 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 of Paenibacillus sp. BP-23 cellobiohydro- lase Cel48C, a unique type of cellulase 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 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 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 new cellulase; GHF48, family 48 catalytic domain of cellobiohydrolase 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 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 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 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 clone E. 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 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 Cellobiohydrolase Cel48C from Paenibacillus (Eur. J. Biochem. 270) 2915 Sequence 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, )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 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 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 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 low concentration 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 °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 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 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 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 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 Cellobiohydrolase Cel48C from Paenibacillus (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 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 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. 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(2002) Effect of cellulase-assisted refining on the proper- ties of dried and never-dried eucalyptus pulp. Cellulose 9, 115–125. Ó FEBS 2003 Cellobiohydrolase Cel48C from Paenibacillus (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 ´ 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