Báo cáo khoa học: A thermoacidophilic endoglucanase (CelB) fromAlicyclobacillus acidocaldariusdisplays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases ppt

A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases Kelvin Eckert and Erwin Schneider Humboldt Universita ¨ t zu Berlin, Institut fu ¨ r Biologie/Bakterienphysiologie, Berlin, Germany A 100-kDa protein with endoglucanase activity was purified from Triton X-100 extract of cells of the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius. The enzyme exhibited activity towards carboxy methyl cel- lulose and oat spelt xylan with pH and temperature optima of 4 and 80 °C, respectively. Cloning and nucleotide sequence analysis of the corresponding gene (celB) revealed an ORF encoding a preprotein of 959 amino acids which is consistent with an extracellular localization. Purified recombinant CelB and a variant lacking the C-terminal 203 amino acid residues (CelB trunc ) displayed similar enzymatic properties as the wild-type protein. Analysis of product formation suggested an endo mode of action. Remarkable stability was observed at pH values between 1 and 7 and 60% of activity were retained after incubation for 1 h at 80 °C. CelB displayed homology to members of glycoside hydrolase family 51, being only the second entry with activity typical of an endoglucanase but lacking activity on p-nitro- phenyl-a- L -arabinofuranoside (pNPAraf). Highest sequence similarity was found towards the other endoglucanase F from Fibrobacter succinogenes (EGF), forming a distinct group in the phylogenetic tree of this family. Analysis of the amino acid composition of the catalytic domains demon- strated that CelB contains fewer charged amino acids than its neutrophilic counterparts, which is in line with adaptation to low pH. Wild-type and full-length recombinant CelB were soluble only in Triton X-100. In contrast, CelB trunc was completely water soluble, suggesting a role of the C-terminal region in cell association. This C-terminal hydrophobic region displayed local sequence similarities to an a-amylase fromthesameorganism. Keywords: endoglucanase; EC 3.2.1.4; enzyme 1,4-b- D -glu- can glucanohydrolase; glycoside hydrolase family 51; acidophile; Alicyclobacillus. Cellulose and hemicellulose (e.g. xylan), the major compo- nents of the plant cell wall, constitute complex substrates as variation can occur in the nature of the monomers, the linkages, chain length and degree of substitution. The complexity and variety of these substrates are mirrored by the numerous enzymes employed by microorganisms to degrade them. Thus, conversion of cellulose and xylan to soluble products requires endoglucanases (1,4-b- D -glucan- 4-glucanohydrolase; EC 3.2.1.4), exoglucanases, including cellodextrinases (1,4-b- D -glucan glucanohydrolase; EC 3.2.1.74) and cellobiohydrolases (1,4-b- D -glucan cello- biohydrolase; EC 3.2.1.91), b-glucosidases (b-glucoside glucohydrolase; EC 3.2.1.21), xylanases (1,4-b- D -xylan xylanohydrolase; EC 3.2.1.8) and b-xylosidases (1,4-b- D - xylan xylohydrolase; EC 3.2.1.37) [1]. To reflect structural features and to reveal the evolutionary relationships between these enzymes, glycoside hydrolases have been grouped into families on the basis of sequence similarity [2]. Some families contain enzymes with different substrate specificities while, on the other hand, enzymes with the same activity are found in different families [3]. Thus, cellulases are found in families 5–10, 12, 44, 45, 48, 51, 61 and 74, while xylanases have been assigned to families 10, 11, and 43. Cellulolytic and xylanolytic activities are also widespread in thermophilic microorganisms. Their occurrence is testi- mony to the presence of these substrates in thermophilic environments, either as plant litter in natural hot springs or in environments such as composite piles. Remarkably however, with a few exceptions, degradation of cellulose and hemicellulose among thermophiles is mostly due to anaerobic species and is absent in archaea [4]. Endoglucan- ases from aerobic thermophilic bacteria, displaying tem- perature optima between 55 and 70 °C and pH optima between 5 and 7 have been described so far for Acidother- mus cellulolyticus [5], Rhodothermus marinus [6], Thermobi- fida fusca [7], and Caldibacillus cellulovorans [4]. Based on 16 S-rRNA gene sequence, the latter is a close relative of members of the genus Alicyclobacillus that is characterized by the presence of alicyclic fatty acids as major components Correspondence to E. Schneider, Humboldt Universita ¨ t zu Berlin, Institut fu ¨ r Biologie/Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany. Fax: + 49 (0)30 20938126, Tel.: + 49 (0)30 20938121, E-mail: erwin.schneider@rz.hu-berlin.de Abbreviations: CelB trunc , C-terminally truncated CelB protein; CMC, carboxy methyl cellulose; EGF, endoglucanase F; GH, glycoside hydrolase; pNPAraf, p-nitrophenyl-a- L -arabinofuranoside. Note: Nucleotide sequence data are available in the EMBL database under the accession number AJ551527. (Received 20 June 2003, accepted 8 July 2003) Eur. J. Biochem. 270, 3593–3602 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03744.x of their membrane lipids. Members of this genus are acidophilic, strictly aerobic and have been described as noncellulolytic [4]. Alicyclobacillus acidocaldarius (ATCC 27009) was first isolated from an acidic creek in Yellowstone National Park, USA [8] and displays pH and temperature optima of 3–4 and 60 °C, respectively. Recently, we succeeded in the cloning, purification and crystallization of a cytoplasmic family 9 endoglucanase (CelA) from A. acidocaldarius [9,10]. The enzyme was active against cellobiosides, suggesting a role in degradation of imported oligosaccharides. Here, we report the gene cloning, sequen- cing and characterization of an extracellular endoglucanase (CelB) from the same organism that hydrolyses carboxy methyl cellulose (CMC), acid-swollen cellulose and oat spelt xylan. The enzyme displays a high degree of sequence similarity with members of GH family 51 of arabinofur- anosidases, but completely lacks this activity. Moreover, CelB is the first acidophilic addition to the family, exhibiting maximal activity at pH 4 and a remarkable tolerance to pH values ranging from 1 to 7. Experimental procedures Bacterial strain and culture conditions A. acidocaldarius ATCC 27009 was grown in minimal salt medium as described [11]. Carbon sources (at 0.2% each) were oat spelt xylan, birchwood xylan (Roth, Germany), starch (Sigma, Germany), sugar beet arabinan (Megazyme, Ireland), CMC (Serva Feinbiochemica, Germany) or glycerol. Maltose (Roth, Germany), cellobiose, glucose or xylose (Merck, Germany) were added to a final concentra- tion of 10 m M . Cloning procedures and plasmid constructions Restriction mapping, subcloning and Southern hybridiza- tion were carried out using standard molecular biology techniques according to [12]. Plasmid and phagemid DNA was purified with Qiagen’s plasmid kit. DNA sequencing was carried out commercially by Agowa (Berlin, Germany) on both strands according to the method of [13]. Chromosomal DNA from A. acidocaldarius was isolated as described in [11]. After partial digestion of DNA with SauIIIA, DNA fragments ranging from 8 to 12 kb were ligated into the Zap Express vector (Stratagene, Heidelberg, Germany), packaged using the Gigapack cloning kit (Stratagene) and plated using Escherichia coli xl1 MRF¢ (Stratagene) as host strain according to the manufacturer’s instructions. Screening took place by overlaying replica plates with top agar containing 10 m M isopropyl thio-b- D -galactoside,1%CMC,250m M b-alanine, pH 3.5, 1 m M MgSO 4 ,1.25m M CaCl 2 , 0.55% Gelrite (Merck) and incubating overnight at 57 °C. The relatively high concen- tration of b-alanine buffer should ensure a low pH of the top agar in order to select for acidophilic enzymes. Lysis zones around positive plaques were identified by flooding the plates with 0.1 M Tris, pH 8, and staining with Congo red according to [14]. Phagemids were derived and plated from positive plaques according to Stratagene using the ExAssist helper phage and the E. coli XLORL strain (Stratagene). The resulting plasmid harboring a 6.4-kb fragment was designated pKE25 (Fig. 1A). Plasmid pKE2201 was constructed by ligating a PstI- EcoRI fragment of pKE25 (Fig. 1A,B) into the expres- sion vector pBAD/HisB (Invitrogen). The resulting ORF (celB trunc ) translates into a protein with six histidine residues fused to Gly35 of the precursor. As the 3¢ region of the truncated ORF lacked a termination codon, a stop codon provided by the pBAD/HisB vector is used. This resulted in an extension of the protein by the sequence PKNSKLGCFFG C-terminal of Asp-757. Plasmid pKE25a5 was obtained by ligating a 5.7-kb KpnI fragment that was identified by Southern hybridization of digested chromosomal DNA with a digoxygenin-labeled (Boehringer) XhoI-NcoIfragmentofpKE25intoplasmid pUC18 [15] (Fig. 1A). Plasmid pKE101, harboring the complete celB gene was constructed by fusing the inserts from pKE25a5 and pKE2201 via a unique KpnIsiteinpBAD/HisB.Thus, Fig. 1. Overview of the celB region and cloning strategy, and sequence analysis of the 5¢ region of the celB gene. (A) Overview of the celB region and cloning strategy. Shown is the celB region of the A. aci- docaldarius chromosome (top line). Numbers indicate nucleotide positions relative to the 5¢-SauIIIA site of the original clone (pKE25). ORFs are represented by arrows in the direction of transcription. Dashed arrows show ORFs neighboring celB with putative assign- ments. The crooked arrow indicates the celB promoter detailed in B. The thick vertical bar indicates the end of the ORF in celB trunc . Restriction sites relevant to the cloning strategy are given. At the bottom inserts of the constructed plasmids are drawn in relation to the celB region with the restriction sites used for excision of the insert prior to ligation in the host vector (in parentheses). The DNA fragment of pKE25 used for Southern hybridization is marked by a black box. (B) Sequence analysis of the 5¢ region of the celB gene. Shown are the nucleotide sequence and the corresponding amino acids. Indicated for the nucleotide sequence are the putative )10 and )35 promoter regions (underlined), the ribosome-binding site (double-underlined), the start codon (boldface) and the PstI site used for subcloning (dotted line). Indicated in the amino acid sequence are the putative cleavage site of the signal peptidase (arrow) and the amino acid sequence found in the N-terminus of the wild-type protein (identical positions underlined). 3594 K. Eckert and E. Schneider (Eur. J. Biochem. 270) Ó FEBS 2003 recombinant full-length CelB has an N-terminus identical with CelB trunc , but is derived from the full-length ORF with the wild-type termination codon (see also Fig. 1A). Computer-aided sequence analyses Sequences were analyzed using DNASIS (Hitachi). The hydropathy plot was obtained using the algorithm of Kyte and Doolittle [16] with a window size of 50. Database searches were conducted with BLASTP 2.2.5 and PSI - BLAST at NCBI [17]. Internal sequence similarities and local align- ments between two sequences were analyzed using PLALIGN 2.1 [18]. CLUSTALX [19] was used for alignments and construction of phylogenetic trees with the neighbor-joining method. Figures were drawn with GENEDOC [20] and TREE- VIEW [21]. Purification of wild-type CelB A. acidocaldarius cells were grown for three days on oat spelt xylan, reaching an OD at 650 nm of 2, harvested by centrifugation and resuspended in the same volume of minimal salt medium. Subsequently, cells were extracted with Triton X-100 (0.05% final) for 30 min at 57 °Cand recentrifuged for 15 min at 20 000 g. Routinely, 450 mL of Triton extract were adjusted to pH 6.5 by adding 10 m M BisTris buffer, and loaded onto a Q-Sepharose (Sigma) column (bed volume: 25 mL) equilibrated with 10 m M BisTris, pH 6.5, containing 0.94 m M CaCl 2 ,2m M MgSO 4 , and 0.005% Triton X-100 (buffer A). After washing with 150 mL buffer A, elution was performed with a NaCl gradient from 0 to 0.2 M in 500 mL of buffer A. CelB- containing fractions were collected, supplemented with b-alanine buffer, pH 3.5, to a final concentration of 40 m M andstoredat)80 °C. Purification of recombinant CelB and CelB trunc E. coli strain TOP10 (Invitrogen) hosting either the plasmid pKE101 for production of full-length CelB or pKE2201 for CelB trunc was grown in LB broth, contain- ing ampicillin (100 lgÆmL )1 ), to D 650 ¼ 0.5. Expression of celB and celB trunc was induced by addition of 0.2 and 0.02% arabinose, respectively, and growth continued for 4 h. Subsequently, cells were harvested, resuspended in buffer B (50 m M sodium phosphate, pH 7, 300 m M NaCl, 0.1 m M phenylmethylsulfonyl fluoride) to a D 650 of 25. Purification of full-length CelB proceeded with subsequent disintegration of the cells by sonication for 5 min (Sonifier II, Branson) followed by centrifugation at 130 000 g for 1 h at 4 °C. The resulting supernatant (2 mL) was mixed with 0.5 mL Ni-NTA agarose (Qia- gen) and Tween 20 was added to a final concentration of 0.1%. From hereon, Tween 20 and phenylmethylsulfonyl fluoride were present in all buffers. Binding took place for 30 min at 4 °C at an imidazole concentration of 10 m M after which the matrix was transferred to a column (diameter 0.5 cm) and washed with 5 mL of buffer) B containing 10 m M imidazole. Elution of bound protein was performed by raising the imidazole concen- tration stepwise from 25 to 200 m M . CelB-containing peak fractions were pooled and dialyzed overnight (dialysis tubing type 20, 12–16 kDa cut-off, Biomol, Germany) against buffer C (50 m M b-alanine, pH 3.5, 10 m M CaCl 2 ,10m M MgCl 2 ). CelB trunc was purified by disrupting the resuspended cells in a French press at 18 000 psi. After centrifugation 50 mL of the resulting supernatant were diluted 1 : 1 with buffer B and incubated with 5 mL Ni-NTA agarose for 30 min in the presence of 10 m M imidazole. Then, the resin was transferred to a column (diameter 1.5 cm), washed with 50 mL buffer B, containing 20 m M imidazole and protein was eluted with 65 mL buffer B, containing 50 m M imidazole. Peak fractions were pooled, concentrated 10-foldwithanAmiconconcentrator(YM30membrane) and dialyzed overnight against buffer C. Enzyme assays Under standard conditions enzyme activity was assayed at a protein concentration of 1.3 lgÆmL )1 in buffer C with 0.25% CMC for 1 h at 70 °C. Subsequently, reducing sugar content was determined according to [22]. One unit (U) is defined as the amount of enzyme releasing 1 lmol of reducing equivalents per minute. Xylanase activity was measured accordingly using oat spelt xylan solubilized as described previously [9]. In addition to the substrates used for cultivation, linear arabinan from beet arabinan (Mega- zyme, Ireland), avicel PH101 (Fluka, Germany), phosphoric acid-swollen cellulose, prepared according to Wang et al. [23] (0.25% each), and pNPAraf (Sigma, Germany) (10 m M ) were employed. pH stability was determined by incubating concentrated CelB trunc (25 lgÆmL )1 )in75 m M of the indicated buffers, supplemented with 10 m M CaCl 2 and 10 m M MgCl 2. After incubation overnight at 4 °C, the sample was diluted 40-fold in buffer C and activity was assayed under standard conditions. Thin-layer chromatography After substrate hydrolysis in buffer C analysis of released products was performed as described previously [9,24]. N-Terminal amino acid sequence analysis Protein samples (40-fold concentrated Triton extract or purified wild-type CelB) were subjected to SDS/PAGE and stained with Serva Blue R, after which CelB was exci- sed. Cyan bromide treatment and sequencing were kindly performed by R. Schmid (University of Osnabru ¨ ck, Germany) as described [25,26]. Analytical methods SDS/PAGE and staining with Serva Blue R (Serva) was carried out as described in [11] using 10% acrylamide. Silver staining was performed according to [27]. For activity staining SDS gels containing either 0.2% CMC or 0.2% oat spelt xylan were used and treated with 50 m M b-alanine, pH 3.5, 0.94 m M CaCl 2 ,2m M MgSO 4 according to [28]. The number of washing steps was reduced to three. Subsequent incubation took place for 1 h at 57 °C. Immunoblot analyses were performed by transferring proteins from SDS gels onto nitrocellulose membranes Ó FEBS 2003 A thermoacidophilic cellulase from Alicyclobacillus (Eur. J. Biochem. 270) 3595 using a ÔTrans-Blot semidryÕ apparatus (Bio-Rad) [29]. Subsequently, the membranes were incubated with a polyclonal antiserum raised against purified wild-type CelB (Biogenes, Germany). Antigen–antibody complexes were visualized with peroxidase-conjugated donkey anti-rabbit immunoglobulins using enhanced chemiluminescence (Luminol, NEN, USA) and exposure to Hyperfilm (Amer- sham-Buchler, Germany). Protein concentration was determined with the Micro BCA Protein Assay Reagent Kit (Pierce). Results Purification of a xylan-degrading enzyme from A. acidocaldarius In the initial stage of this work, we screened A. acidocalda- rius for extracellular thermoacidophilic enzymes with poly- saccharide-degrading activities. The organism was found to utilize a variety of polysaccharides including xylan as sole source of carbon and energy. However, we failed to detect xylanase activity in the culture supernatant. Thus, assuming a cell-associated enzyme, we succeeded in extracting xylan- degrading activity from intact cells with Triton X-100. The xylanase activity remained cell-bound, even after the culture reached the stationary phase of growth. SDS/PAGE of Triton-extracted proteins followed by silver staining revealed about 10 major proteins with molecular masses ranging from 30 to 100 kDa (Fig. 2A, lane 1). Zymogram analysis demonstrated that at least five of these proteins displayed activity towards oat spelt xylan (not shown) and CMC (Fig. 2A, lane 2). To begin with, we concentrated our further efforts on the 100-kDa protein. Purification of the protein was achieved by ion exchange chromatography using Q-Sepharose in the presence of 0.05% Triton X-100 (Fig. 3, lane 1). From a 1-L culture of A. acidocaldarius  2.0 mg of the 100-kDa protein exhibiting, on average, a CMCase activity of 10.3 UÆmg )1 and a xylanase activity of 0.9 UÆmg )1 were obtained routinely. N-Terminal sequence analysis of the protein revealed the peptide sequence ADV(T?)STPI(A?)MEXQV, while ana- lysis of a peptide fragment generated by cyanogen bromide gave rise to the sequence (M)VAEL(G?)REINAY. No homology to an entry in the database was found using BLASTP. The purified 100-kDa protein was injected into rabbits to raise polyclonal antibodies. Subsequent immunoblot ana- lysis of the Triton extract revealed that, in addition to the 100-kDa protein, two other protein bands strongly cross- reacted with the antiserum (Fig. 2A, lane 3). This result may imply that these proteins represent degradation products of the 100-kDa protein. Thus, the additional bands observed in the zymogram (Fig. 2A) are likely to represent other enzymes that participate in the complete degradation of CMC or xylan. Furthermore, Western blot analysis of Triton extracts from A. acidocaldarius cells grown on different substrates demonstrated that, in addition to oat spelt xylan, synthesis of the 100-kDa protein was induced by birchwood xylan, CMC, and cellobiose, but not by glycerol, glucose, xylose, maltose, starch or arabinan (Fig. 2B). Cloning and sequence analysis of the 100-kDa protein The cloning procedure with the Zap Express vector (see Experimental procedures for details) yielded a gene bank with 2 · 10 6 plaque-forming units (p.f.u.) with insert sizes ranging from  3–10 kb. Screening of 45 000 plaques for Fig. 2. Identification of CelB in Triton extract from A. acidocaldarius. (A) Triton extract (25 lL per lane) from cells grown on oat spelt xylan after SDS/PAGE and silver staining (lane 1), zymogram analysis with CMC (lane 2) and Western blotting (lane 3) with antibodies raised against wild-type CelB. (B) Western blots of Triton extracts (25 lL) from A. acidocaldarius grown on different substrates. Lanes 1, cello- biose; 2, starch; 3, arabinan; 4, birchwood xylan; 5, xylose; 6, CMC; 7, glycerin;8,glucose;9,maltose. Fig. 3. SDS/PAGE of purified wild-type and recombinant forms of CelB. Lane 1, wild-type CelB (0.2 lg), silver stained; 2, full-length recombinant CelB (3 lg),stainedwithServaBlueR;3,recombinant CelB trunc (3 lg), stained with Serva Blue R. 3596 K. Eckert and E. Schneider (Eur. J. Biochem. 270) Ó FEBS 2003 CMC activity with the substrate overlay method and subsequent excision resulted in five phagemids. One clone harbored a previously described intracellular cellulase CelA [9] as identified by Western blotting, but a second clone reacted with antibodies raised against the wild-type 100- kDa protein. Nucleotide sequencing revealed an incomplete ORF which coded for a protein that displayed high sequence similarities with endoglucanases and arabinofur- anosidases. Eventually, screening digested chromosomal DNA by Southern hybridization with a fragment from the 3¢ end of the incomplete ORF gave rise to an overlapping clone that contained the rest of the ORF. The complete ORF encoded a preprotein of 959 amino acids with a molecular mass of 100 849 kDa. A possible start codon (TTG) with a putative ribosome-binding site was found together with possible )10(TATAAC) and )35(TTGACA) regions (Fig. 1B). SignalP [30] detected a possible signal peptide whose cleavage site was located C-terminal of amino acid Ala25 of the preprotein (Fig. 1B). Nineteen amino acids situated C-terminally of the cleavage site with a sequence with 79% identity to the sequence obtained from the N-terminus of the purified wild-type 100-kDa protein were found (Fig. 1B). More- over, residues 485–496 of the translated ORF showed only one substitution when compared with the internal sequence of the 100-kDa protein (see above). In both cases, the observed mismatches concerned only those residues that could not unequivocally be identified by amino acid analysis. Taken together, we conclude that the ORF is likely to encode the 100-kDa protein that was purified from A. acidocaldarius cells. The ORF was designated celB. The celB gene is flanked by two divergently transcribed putative ORFs, encoding a LacI/GalR type transcription regulator (152 nucleotides downstream of celB)andaLysE type exporter (176 nucleotides upstream of celB), respect- ively (Fig. 1A). A database search using BLASTP revealed highest sequence similarity of CelB (28% identity, 45% similarity over a length of 410 amino acids) with endoglucanase F (EGF) from Fibrobacter succinogenes S85 ([31], GenBank accession number U39070) which belongs to family 51 of glycoside hydrolases (GH51). Among the 32 other mat- ches found, 19 were arabinofuranosidases. After three iterations PSI - BLAST showed only three proteins not classified as arabinofuranosidases among the top 30 matches. Sequence comparison of CelB with all members of GH51 revealed a central catalytic domain ranging from amino acids Thr223 to Pro702. Catalytic residues have been inferred from sequence alignments in this family [32] and have been experimentally confirmed [33–35]. The conserved motif Gly Asn Glu is also present in CelB identifying Glu366 as the acid/base catalyst. Furthermore, Glu510 is a possible candidate for the catalytic nucleophile residue. A phylogenetic tree constructed from the align- ment of the catalytic domains showed that CelB forms a distinct cluster with EGF (Fig. 4). These two are the only enzymes characterized as endoglucanases in GH family 51. Adjacent to the catalytic region, a stretch of 20 amino acids (residues Ser720–Asp739) was found with 60% of the residues being proline, aspartate, serine or threonine, which are typical of linker sequences [36]. This was the highest occurrence of these amino acids in the whole sequence. Thus, this region may function as a linker between the catalytic domain and the C-terminal portion of the enzyme. A database search with the N-terminal region of CelB (residues 1–222) revealed no significant similarities to other proteins. Fig. 4. Phylogenetic tree of catalytic domains belonging to GH family 51. CelB (doubly underlined) forms a distinct group with EGF (under- lined). Also underlined is AbjA (CAA76421) from the thermophile T. xylanilyticus.Barlength,extentofexchangeof0.1perresidue. GenBank/GenPept accession codes are given (Agrobacterium tume- faciens C58: AAL43920, ORF Atu3104; Arabidopsis thaliana: AAD40132, ORF At5g26120/T1N24.13; AAF19575, ORF At3g10740/T7M13–18; Aspergillus niger: AAC41644, arabinofurano- sidase A; A. niger var. awamorii: IFO4033,BAB21568, ArfA; Bacil- lus halodurans C-125: BAB05580, ORF AbfA (BH1861); BAB05593, ORF Xsa (BH1874); Bacillus subtilis ssp. subtilis str. 168: CAA61937, arabinofuranosidase 1; CAA99576, arabinofuranosidase 2; Bactero- ides ovatus: AAA50391, arabinosidase 1; AAA50393, arabinosidase 2; Bifidobacterium longum NCC2705: AAN24035, BL0181; AAN24368, AbfA1; AAN24945, BL1138; AAN24971, AbfA2; AAN25400, AbfA3; Caulobacter crescentus CB15: AAK23403, ORF CC1422; Cellvibrio japonicus: AAK84947, arabinofuranosidase; Clostridium acetobutyl- icum ATCC 824: AAK81366, ORF CAC3436; Clostridium sterco- rarium: AAC28125, arabinofuranosidase; Cytophaga xylanolytica: AAC38456, arabinofuranosidase I; AAC38457, arabinofuranosidase II; F. succinogenes S85: AAC45377, EGF; G. stearothermophilus T-6: AAD45520, abfA; Hordeum vulgare: AAK21879, AXAH-I; AAK21880, AXAH-II; Mesorhizobium loti MAFF303099: NP 104667, Mll3591; Oryza sativa: BAC10349, OJ1200 °C08.20; CAD39869, OSJNBb0058J09.6; Sinorhizobium meliloti 1021: CAC49446, AbfA; Streptomyces chartreusis: BAA90771, arabinofuranosidase I; Strepto- myces coelicolor A3(2): CAA20794, ORF SCI35.05c; CAB86096, AbfA; Streptomyces lividans 66: AAA61708, AbfA; T. xylanilyticus D3: CAA76421, AbjA; Thermotoga maritima: AAD35369, ORF TM0281). Ó FEBS 2003 A thermoacidophilic cellulase from Alicyclobacillus (Eur. J. Biochem. 270) 3597 Purification of recombinant CelB and CelB trunc Recombinant full-length CelB could be purified by Ni-NTA affinity chromatography provided that 0.1% Tween 20 or 0.5% Triton X-100 were present throughout the purification procedure to keep the protein in solution. Routinely, 18 mg of purified CelB exhibiting a CMCase activity of 11.4 UÆmg )1 (average of two independent preparations) were obtained from a 1-L culture of E. coli TOP 10 (pKE101) (Fig. 3, lane 2). CelB trunc , lacking the C-terminal 203 amino acids was purified from E. coli strain TOP 10 (pKE2201) by essen- tially the same procedure but in the absence of detergent (Fig. 3, lane 3). The protein displayed a similar activity towards CMC of 10.1 UÆmg )1 (average of two independent preparations), strongly suggesting that the C-terminal portion is dispensable for activity. Thus, further character- ization of the enzymatic properties was carried out predominantly with CelB trunc . pH and temperature dependence CelB trunc was most active at pH 4 but still displayed 50% of its activity at pH 3 and 5, respectively, while no activity was recorded at pH values below 2 or above 6.5. Wild-type CelB behaved similarly (Fig. 5A). Likewise, CelB trunc and the wild-type protein basically exhibited the same temperature dependence with an optimum at 80 °C. No activity was found at 100 °Corbelow40°C. The optimum curve of the recombinant protein is broader than that of the wild-type for unknown reasons (Fig. 5B). Together, the pH and temperature dependence of the enzyme reflect the environ- mental conditions to which A. acidocaldarius is exposed. pH and temperature stability CelB trunc also displayed remarkable tolerance to acidic pH, being stable (80% residual activity) overnight at pH values ranging from 1.5 to 7. Increasing alkaline conditions irreversibly inactivated the enzyme (Fig. 6A). CelB trunc was considerably stable at 80 °C, still exhibiting 60% of the control activity after 1 h. Preincubation at 70 °Cforthat amount of time even stimulated the activity. In contrast, a 10-min incubation at 90 °C prior to assaying the residual activity resulted in complete inactivation of the enzyme (Fig. 6B). Enzymatic properties and substrate specificity Determination of kinetic parameters of CelB trunc using CMC as substrate yielded a K m of 0.35 mgÆmL )1 and a V max of 10.8 UÆmg )1 , resulting in a k cat of 0.881 min )1 . Under standard conditions, optimal activity was obtained in the presence of Ca 2+ and Mg 2+ ions (10 m M each). Omitting the bivalent cations decreased the activity by 50%. A small stimulating effect in the presence of these cations was also described for AbfA from GH family 51 [37]. In contrast, 10 m M Zn 2+ caused a 78% inhibition of CelB trunc activity. Inhibition by Zn 2+ is typical of many GHs [38,39], but not of all members of family 51 [37,40]. Apart from CMC, CelB trunc was also found to hydrolyze phosphoric acid-swollen cellulose (0.81 UÆmg )1 ) and, like the wild-type protein, showed activity towards oat spelt xylan (0.6 UÆmg )1 ). In contrast, no activity was found with crystalline cellulose (Avicel PH101), birchwood xylan, starch and, most strikingly, with arabinan, linear arabinan or pNPAraf, in spite of the described sequence similarity to other arabinofuranosidases. In order to discriminate an endo or exo mode of action degradation of CMC and oat spelt xylan by CelB trunc was analyzed by TLC. A time course showed that in the initial reaction only high molecular mass products were released from oat spelt xylan (Fig. 7A). The appearance of disaccharides (xylobiose and cello- biose in the case of oat spelt xylan and cellobiose in the case of CMC) as final degradation products was only observed at a high enzyme concentration (80 lgÆmL )1 ) (Fig. 7B,C). To confirm the endowise action of the enzyme, its hydro- lytic properties were investigated using linear cello- and xylooligomers as substrates. Cellobioside and xylobioside, respectively, were the final products (Fig. 7D). Interestingly, a G3 and an X4 intermediate were formed from cellotetra- ose and xylopentaose, respectively, although in both cases Fig. 5. pH and temperature optima of wild-type CelB (d) and recom- binant CelB trunc (j). (A) pH optimum. Cellulase activity was assayed under standard conditions at 70 °C at the indicated pH values. Glycine (pH 1–3) and citrate phosphate (pH 3–7) were used as buffers. Control activities (100%) for wild-type CelB and CelB trunc were 12.2 and 10.8 UÆmg )1 , respectively. (B) Temperature optimum. Cellulase activity was assayed at pH 3.5 for 30 min. Control activities (100%) for wild-type CelB and CelB trunc were 8.8 and 8.1 UÆmg )1 , respectively. 3598 K. Eckert and E. Schneider (Eur. J. Biochem. 270) Ó FEBS 2003 no glucose could be detected (Fig. 7D). This might be due to a possible transglycosidase activity of the enzyme under the experimental conditions used. Such an activity is not uncommon to glycosidases that, like those grouped in family 51, hydrolyze their substrates by a retaining cleavage mechanism [41–43]. The appearance of weak spots repre- senting larger products than the starting substrates cello- or xylopentaose might be taken as further indication for the above notion (Fig. 7D). Together, we conclude that CelB has the hallmark qualities of an endoglucanase which acts mainly on CMC and noncrystalline cellulose but is also capable of hydrolyzing xylan. Discussion A cell-associated 100-kDa protein (CelB) with xylan-degra- ding activity could be extracted with Triton X-100 from A. acidocaldarius cells grown on oat spelt xylan. Purification and characterization of both wild-type and recombinant forms of the protein demonstrated it to be a thermoacido- philic endoglucanase, with activities against CMC, acid- swollen cellulase and oat spelt xylan. The protein is remarkably stable at acidic pH and temperatures up to 80 °C. These thermoacidophilic properties are in line with the growth characteristics of the organism which reflect its natural habitat. How tolerance to acidic pH is achieved in proteins is poorly understood. Schwermann et al.[44] observed that acidophilic proteins possess a reduced density of both positively and negatively charged residues at their surface and proposed that this phenomenon might contri- bute to acidostability by preventing electrostatic repulsion at Fig. 6. pH and temperature stability of CelB trunc . (A) pH stability. Enzyme was incubated overnight at 4 °C in the indicated buffers after which residual acitivity was assayed under standard conditions. Con- trol activity (100%) was 8.7 UÆmg )1 . See Experimental procedures for details. (B) Temperature stability. Enzyme was incubated at 70 (j), 80 (m)or90°C(·) for the indicated times and residual activity was measured under standard conditions. Control activity prior to heat treatment (100%) was 11.7 UÆmg )1 . Fig. 7. TLC analysis of degradation products of various substrates after incubation with CelB trunc . (A) Time course of degradation products of 0.25% xylan at an enzyme concentration of 16 lgÆmL )1 (B) Hydro- lysis of xylan at high enzyme concentrations (80 lgÆmL )1 , underlined). (C) Extensive hydrolysis of 0.5% CMC. (D) Hydrolysis (1 h) of oligosaccharides (10 m M ) at an enzyme concentration of 16 lLÆmL )1 . Arrows indicate G3 and X4 intermediates that might have arosen from degradation of larger transglycosylation products. Possible transgly- cosylation products larger than the starting substrates are marked by asterisks. Substrate blanks (–) were incubated along with the samples. Numbers give incubation time in minutes. M, marker; G, glucose; C, cellobiose; G4, cellotetraose; G5, cellopentaose; X, xylose; X2 xylo- biose; X5, xylopentaose. Ó FEBS 2003 A thermoacidophilic cellulase from Alicyclobacillus (Eur. J. Biochem. 270) 3599 low pH. To test this hypothesis, we compared the amino acid composition of the catalytic domain of CelB with those from two other members of GH family 51, EGF from F. succinogenes and AbjA (Genpept accession number CAA76421) from the thermophile Thermobacillus xylani- lyticus. EGF has a pH and temperature optimum of 5.3 and 40 °C, respectively [31]. For AbjA, the respective values are pH 5.9 and 75 °C [40]. CelB displayed a lower percentage of charged amino acids, especially lysine and arginine, which were reduced by 10.1% together compared with EGF and 6.4% in comparison with AbjA. On the other hand, CelB contains a higher percentage of alanine and proline and of uncharged polar residues. Thus, these data are at least not in contradiction to the above notion. Wild-type and full-length recombinant CelB were found to be soluble only in the presence of detergent, while the truncated form of the protein (CelB trunc ), lacking the C-terminal 203 residues was readily soluble in buffer. A hydropathy plot of the protein is consistent with this observation, predicting a C-terminal hydrophobic region encompassing residues 700–900 (not shown). Further- more, CelB trunc exhibited the same pH and temperature dependence as the wild-type protein, suggesting that the C-terminal fragment is not essential for catalysis. Rather, these data point to a role in cell association, possibly by specific protein–protein interaction with the S-layer of the organism [45], although sequence analysis did not iden- tify a typical S-layer binding domain [46]. A possible approach to confirm this notion would be to determine the cellular localization of homologously expressed C-terminally truncated variants of CelB. Unfortunately, such experiments are not feasible due to the fact that A. acidocaldarius cannot yet be manipulated by genetic means. However, some evidence in support of the above notion is provided by a study on an a-amylase (AmyA) from the same organism. Like CelB, AmyA remains attached to the cells during exponential growth [44], and is only released into the medium as the culture approaches the stationary phase. Moreover, the cell-associated form of the enzyme is extractable by Triton X-100. Interest- ingly, a hydropathy plot of AmyA [47] revealed a hydrophobic N-terminal region ( residues 110–340) (not shown) to which a function has not yet been assigned. High activity of CelB against CMC and TLC analysis of the time course with initial production of high molecular mass products from CMC and oat spelt xylan characterize the enzyme as an endoglucanase. Except for birchwood xylan, these activities are in line with induction of celB gene expression when grown on these substrates. However, birchwood xylan is highly acetylated [48], which may cause steric hindrance resulting in low activity of CelB against this substrate even though it leads to gene expression. Together, these data suggest that the enzyme may play a role in the degradation of both cellulose and xylan in vivo. This is further underlined by the fact that hydrolysis of xylan and CMC by CelB leads to formation of disaccharides, albeit at high enzyme concentrations. Consequently, com- plete degradation of the substrates will likely require cooperative efforts of CelB with other glycoside hydrolases. Indeed, zymogram analysis demonstrated the presence of additional protein bands with CMCase and xylanase activity in the Triton X-100 extract (Fig. 2A). The resulting oligo- saccharides may then be transported into the cytoplasm and subsequently hydrolyzed by enzymes such as CelA [9]. In terms of substrate specificity CelB is similar only to one other member of GH family 51, an endoglucanase (EGF) from F. succinogenes. Like CelB, EGF has no activity on pNPAraf.Thisisalsoincontrasttoallothermembersof this family for which such an activity was tested [33,40, 49,50,51]. Only in the case of AbfA from Geobacillus ste- arothermophilus T-6[37],averylowactivityonCMC (0.08% of the arabinofuranosidase activity) was reported. 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Matsuo, N.K.S., Kuno, A., Kobayashi, H. & Kusakabe, I. (2000) Purification, characterization and gene cloning of two a- L -arabinofuranosidases from Streptomyces chartreusis GS901. Biochem. J. 346, 9–15. 3602 K. Eckert and E. Schneider (Eur. J. Biochem. 270) Ó FEBS 2003 . A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family. CAA99576, arabinofuranosidase 2; Bactero- ides ovatus: AAA50391, arabinosidase 1; AAA50393, arabinosidase 2; Bifidobacterium longum NCC2705: AAN24035, BL0181; AAN24368, AbfA1;