Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK1
Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK2
Author for correspondence: Ruth Y. Eberhardt. Tel: +44 1223 496478. Fax: +44 1223 496023. e-mail: ruth.eberhardt{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: anaerobic fungi, cellulase, glycosyl hydrolases (families 5 and 45), endoglucanases, Piromyces equi
Abbreviations: CMC, carboxymethylcellulose; CMCase, carboxymethylcellulase; DNSA, dinitrosalicylic acid reagent; pNPC, p-nitrophenyl ß-D-cellobiopyranoside
The EMBL accession numbers for the sequences reported in this paper are AJ277482 and AJ277483.
a Present address: Finnfeeds International, PO Box 777, Marlborough SN8 1XN, UK.
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INTRODUCTION |
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The sequences of over 1500 glycosyl hydrolases are known and have been assigned to at least 77 families on the basis of hydrophobic cluster analysis and amino acid sequence similarities. Twelve of these families contain endoglucanases (Henrissat, 1991 , 1998
; Henrissat & Bairoch, 1993
, 1996
). Many glycosyl hydrolases are modular enzymes consisting of one or more catalytic domains joined to non-catalytic domains via linker sequences rich in proline and hydroxy amino acids (Gilkes et al., 1991
; Tomme et al., 1995
; Warren, 1996
).
Herbivores do not produce polysaccharide hydrolases, so rely on symbiotic relationships with micro-organisms (mostly bacteria and fungi), which inhabit their gastrointestinal tracts, to digest plant material. Anaerobic fungi were first isolated by Orpin (1975)
, from the rumen of a sheep. Since then they have been recovered from the digestive tracts of many different species of herbivores, including both ruminants and non-ruminants, where they are believed to be responsible for the digestion of 4070% of the ingested plant material (Li & Heath, 1993
; Trinci et al., 1994
). At least five genera of anaerobic fungi have been described to date including Neocallimastix, Orpinomyces, Piromyces, Caecomyces and Anaeromyces. The polysaccharide hydrolases produced by anaerobic fungi are amongst the most active that have been described to date; they are capable of degrading a wide range of polysaccharides and can completely solubilize both amorphous and highly crystalline cellulose (Li & Heath, 1993
; Selinger et al., 1996
; Wubah et al., 1993
). Unlike aerobic fungi, anaerobic fungi produce large, multienzyme cellulasehemicellulase complexes, similar to the cellulosome of Clostridium thermocellum (Ali et al., 1995
; Dijkerman et al., 1997
; Fanutti et al., 1995
; Hazlewood & Gilbert, 1998
; Teunissen et al., 1993
; Wilson & Wood, 1992
).
The anaerobic fungus Piromyces equi was isolated from the caecum of a pony (Munn, 1994 ; Orpin, 1981
). The cellulosehemicellulose degrading system of P. equi consists of a large multienzyme complex, which accounts for up to 90% of the cellulase, mannanase and xylanase activities produced by the fungus. This complex consists of at least ten polypeptides ranging from 50 to 190 kDa, including a 97 kDa putative scaffolding protein (Ali et al., 1995
; Fanutti et al., 1995
; Hazlewood & Gilbert, 1998
). Catalytic components of the complex are all modular enzymes and contain one, two or three copies of a highly conserved 40-residue non-catalytic sequence that is believed to function as a dockerin domain (Fanutti et al., 1995
). These putative dockerin domains are located at either the N terminus, the C terminus or between catalytic domains of P. equi enzymes (Hazlewood & Gilbert, 1998
).
Although genes encoding xylanases and mannanases from P. equi have been reported (Fanutti et al., 1995 ; Millward-Sadler et al., 1996
), no endoglucanase genes have been described to date. In this study we aim to characterize the endoglucanase components of the P. equi multienzyme complex. Here we describe the elucidation of the primary structures and enzymic properties of two endoglucanases from P. equi, Cel5A and Cel45A. Cel5A has four similar family-5 catalytic domains and shows cellulase, xylanase and mannanase activities. Cel5A is the first anaerobic fungal enzyme to be reported which has both N-terminal and the C-terminal dockerin domains within the same polypeptide. Cel45A is the first family-45 endoglucanase to be isolated from an anaerobic organism.
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METHODS |
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General recombinant DNA methodology.
Agarose-gel electrophoresis, Southern hybridization, Northern hybridization and the transformation of E. coli were performed as described by Sambrook et al. (1989) . Plasmid DNA was prepared by an adaptation of the alkaline lysis method of Birnboim & Doly (1979)
or by using Qiagen resin columns (Qiagen), following the protocol recommended by the manufacturer. Restriction endonucleases and other DNA-modifying enzymes were used in accordance with the manufacturers instructions. Total RNA was extracted from a 2 l culture of P. equi by grinding fungal mycelia to a powder in liquid nitrogen, resuspending in GTC [4 M guanidine thiocyanate, 25 mM sodium citrate, 0·5 mM EDTA pH 7·5, 0·5% (w/v) sodium lauryl sarcosine] and homogenizing. The resulting solution was centrifuged at 2000 g for 10 min. CsCl was added to the supernatant to a final concentration of 0·1 g ml-1, transferred to tubes containing 5·7 M CsCl, 0·1 M EDTA and centrifuged at 80000 g for 24 h. The supernatant was discarded and the RNA pellet resuspended in TE (10 mM Tris, 1 mM EDTA) pH 7·6, 0·1% SDS. The RNA was further purified by chloroform/n-butanol extraction and ethanol precipitation, before resuspension in 1 mM EDTA pH 7·6. A cDNA library was constructed in
ZAPII from P. equi mRNA as described previously (Ali et al., 1995
) and screened for carboxymethylcellulase (CMCase) activity by plating onto NZY medium in a soft agar overlay containing 0·5 mM IPTG. After 16 h incubation at 37 °C, the plaques which had formed were overlaid with molten agar containing 0·5% carboxymethylcellulose (CMC), 50 mM K2HPO4 and 12 mM citric acid pH 6·5. After a further 4 h incubation at 37 °C, plates were stained with Congo red and destained with 1 M NaCl (Teather & Wood, 1982
) to identify positive clones. Clones expressing CMCase activity were excised and recircularized to form pBluescript phagemids.
Nucleotide sequencing.
Nucleotide sequencing was performed on plasmid DNA using a PRISM ready reaction dye-deoxy terminator cycle sequencing kit (Applied Biosystems). To sequence cel5A, nested deletions were generated with exonuclease III using the double-stranded nested deletion kit (Amersham-Pharmacia). cel45A was sequenced by chromosome walking: synthetic oligonucleotide primers were designed according to known sequence. Sequences were compiled and ordered using software written by the Genetics Computer Group at the University of Wisconsin.
Construction of pRE1 and pRE2.
The primers 5'-CGGGATCCGAACCAACTGGTAATATTCGTG-3' (primer A1) and 5 ' - A C T A C C G C T C G A G T T A T G G T T C TTCTTCAATAG-3' (primer A2) were used to amplify the region of cel5A encoding the catalytic domain nearest the C terminus (between nucleotides 3790 and 4884) to construct pRE1. To construct pRE2, the region of cel45A encoding the catalytic domain (between nucleotides 535 and 1260) was amplified by PCR using the primers 5'-CGGGATCCACTGGTTCGGGTGGTAACAGC-3' (primer B1) and 5'-GCCGCCGCTCGAGAGATAGGGATATACTGG-3' (primer B2). The reaction conditions for PCR were: denaturation 30 s at 94 °C, annealing 45 s at 50 °C, elongation 75 s at 72 °C, for 25 cycles. The amplified DNA sequences were digested with BamHI and XhoI and ligated into the thioredoxin fusion expression vector, pET32a, digested with the same restriction enzymes to form pRE1 and pRE2, encoding Trx-Cel5A' and Trx-Cel45A', respectively.
Purification of fusion proteins.
E. coli strains harbouring the plasmids pRE1 and pRE2 were grown to mid-exponential phase in 3 l (pRE1) or 6·7 l (pRE2) LB medium containing 50 µg ampicillin ml-1 at 37 °C. IPTG was added to each culture to a concentration of 0·5 mM and cultures were grown for a further 3 h at 37 °C (pRE1) or 30 °C (pRE2). Cells were pelleted by centrifugation at 10000 g for 10 min and then resuspended in 75 ml 100 mM NaCl, 20 mM Tris pH 8·0. The cells were then lysed by sonication. Triton X-100 was added to the pRE2 extract to a final concentration of 0·1% (v/v), followed by an incubation at 4 °C for 20 min. Cell debris was removed by centrifugation at 10000 g for 20 min and the resulting cell-free extract was incubated with 5 ml TALON metal affinity resin (Clontech) for 2 h at 4 °C. Unbound proteins were removed by 5 min centrifugation at 150 g, four washes with 100 mM NaCl, 20 mM Tris/HCl pH 8·0 and two washes with 10 mM imidazole, 100 mM NaCl, 20 mM Tris/HCl pH 8·0. To elute bound proteins, the TALON resin was applied to a column and 5 ml 50 mM imidazole, 100 mM NaCl, 20 mM Tris/HCl pH 8·0 was added. Five 1 ml eluates were collected; elution was repeated with 5 ml 100 mM imidazole, 100 mM NaCl, 20 mM Tris/HCl pH 8·0 to obtain a further five eluates. Eluates of Trx-Cel5A' and Trx-Cel45A' were checked by SDS-PAGE as described by Laemmli (1970) . The eluates containing fusion protein were pooled and dialysed twice against 10 mM Tris pH 8·0 to remove imidazole, Trx-Cel45A' was further purified by anion-exchange chromatography using a 2 ml BioQ column (Bio-Rad) on an automated Biologic workstation (Bio-Rad) and eluted in 10 mM Tris/HCl pH 8·0 with a gradient of 0500 mM NaCl.
Assays.
Protein concentrations of Trx-Cel5A' and Trx-Cel45A' were determined by dye binding (Bradford, 1976 ). Enzyme activity was assayed in 50 mM K2HPO4, 12 mM citric acid (PC buffer) pH 6·5 in the presence of 1 mg BSA at 37 °C (Trx-Cel5A') or 60 °C (Trx-Cel45A'), unless otherwise stated. Reducing sugar release was measured with the dinitrosalicylic acid reagent (DNSA) (Miller, 1959
). All assays were performed for 5, 10, 15, 30 and 60 min to determine the initial rate of activity. Substrates tested were CMC (BDH), acid-swollen cellulose (Sigmacell, treated according to Wood, 1971
), bacterial microcrystalline cellulose [harvested from Acetobacter hansenii, washed with 4% (v/v) NaOH, neutralized with HCl and washed in distilled H2O], Avicel (Merck), soluble birchwood xylan, barley ß-glucan (Megazyme), lichenin (Cetraria islandica; Sigma), laminarin (Laminaria digitata; Sigma), carob galactomannan (Megazyme), debranched arabinan (Megazyme) and potato galactan (Megazyme). Concentrations of substrate used ranged from 0·1% to 1·125% w/v. Cellobiohydrolase activity was measured using 100 µl 1 mM p-nitrophenyl ß-D-cellobiopyranoside (pNPC) (Sigma) in a 1 ml reaction volume, stopping the reaction with 500 µl 1 M Na2CO3 and measuring p-nitrophenyl release spectrophotometrically at 400 nm. Assays to determine pH optima were performed in citric acid, Na2HPO4 buffer (pH 4·07·6) or sodium barbitone, HCl buffer (pH 7·98·1). Temperature inactivation studies were carried out by preincubating enzyme and buffer at a given temperature for 0, 2, 5, 10, 15, 20, 30, 45 and 60 min, before assaying for activity against CMC as described above. One unit of enzyme activity was defined as the quantity of enzyme required to liberate 1 µmol product in 1 min.
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RESULTS AND DISCUSSION |
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Molecular architecture
The deduced primary structures of Cel5A and Cel45A, encoded by cel5A and cel45A, respectively, revealed that the N-terminal regions conformed to typical signal peptides, which is consistent with the observation that endoglucanases are components of the extracellular cellulase system of P. equi. Cel5A has three copies of a highly conserved approximately 40-residue sequence, one between residues 23 and 57, and a further two between residues 1631 and 1708. Cel45A has three repeats of this 40-residue sequence between residues 22 and 152. This highly conserved 40-residue sequence is present in many of the polysaccharide hydrolases isolated from anaerobic fungi and is believed to function as a dockerin domain, which mediates binding of the catalytic subunits of the fungal multienzyme cellulase complex to a scaffolding protein (Dalrymple et al., 1997 ; Fanutti et al., 1995
; Hazlewood & Gilbert, 1998
; Li et al., 1997b
). Fungal dockerins have been previously found in one, two or three copies at either the N terminus, the C terminus or between two catalytic domains (Fig. 2
). Cel5A is the first reported example of an enzyme with dockerins at two separate locations within the molecule. This may have arisen via the fusion of two endoglucanase genes encoding proteins with dockerin domains. Alternatively, the presence of dockerins at both ends of the enzyme could provide a means for linking complexes together to form clusters analogous to the polycellulosome of C. thermocellum (Mayer et al., 1987
; Shoham et al., 1999
).
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ACKNOWLEDGEMENTS |
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Received 17 February 2000;
revised 11 May 2000;
accepted 17 May 2000.