A thermoacidophilic endoglucanase CelB from Alicyclobacillusarabinofuranosidases belonging to family 51 of glycoside hydrolases Kelvin Eckert and Erwin Schneider Humboldt Universita¨t zu
Trang 1A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus
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 80C, 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 (CelBtrunc) 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
80C 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, CelBtrunc 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 from the same organism
Keywords: endoglucanase; EC 3.2.1.4; enzyme 1,4-b-D -glu-can glu-glu-canohydrolase; 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 70C 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
16S-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)
Trang 2of 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 60C, 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 acidocaldariusATCC 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 mM
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 kbwere
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 mM isopropyl
thio-b-D-galactoside, 1% CMC, 250 mMb-alanine, pH 3.5, 1 mM
MgSO4, 1.25 mM CaCl2, 0.55% Gelrite (Merck) and
incubating overnight at 57C 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 0.1MTris, 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 (celBtrunc) 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-NcoI fragment of pKE25 into plasmid pUC18 [15] (Fig 1A)
Plasmid pKE101, harboring the complete celB gene was constructed by fusing the inserts from pKE25a5 and pKE2201 via a unique KpnI site in pBAD/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).
Trang 3recombinant full-length CelB has an N-terminus identical
with CelBtrunc, 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 withBLASTP2.2.5 andPSI-BLASTat
NCBI [17] Internal sequence similarities and local
align-ments between two sequences were analyzed usingPLALIGN
2.1 [18] CLUSTALX [19] was used for alignments and
construction of phylogenetic trees with the neighbor-joining
method Figures were drawn withGENEDOC[20] and
TREE-VIEW[21]
Purification of wild-type CelB
A acidocaldariuscells 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 57C and
recentrifuged for 15 min at 20 000 g Routinely, 450 mL of
Triton extract were adjusted to pH 6.5 by adding 10 mM
BisTris buffer, and loaded onto a Q-Sepharose (Sigma)
column (bed volume: 25 mL) equilibrated with 10 mM
BisTris, pH 6.5, containing 0.94 mMCaCl2, 2 mMMgSO4,
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 0.2M 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 mMand stored at)80 C
Purification of recombinant CelB and CelBtrunc
E coli strain TOP10 (Invitrogen) hosting either the
plasmid pKE101 for production of full-length CelB or
pKE2201 for CelBtruncwas grown in LB broth,
contain-ing ampicillin (100 lgÆmL)1), to D650¼ 0.5 Expression
of celB and celBtrunc 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 mM sodium phosphate, pH 7, 300 mM
NaCl, 0.1 mM phenylmethylsulfonyl fluoride) to a D650
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 4C 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 4C at an imidazole concentration of
10 mM after which the matrix was transferred to a
column (diameter 0.5 cm) and washed with 5 mL of
buffer) B containing 10 mM imidazole Elution of bound
protein was performed by raising the imidazole
concen-tration stepwise from 25 to 200 mM CelB-containing
peak fractions were pooled and dialyzed overnight
(dialysis tubing type 20, 12–16 kDa cut-off, Biomol, Germany) against buffer C (50 mM b-alanine, pH 3.5,
10 mM CaCl2, 10 mM MgCl2)
CelBtruncwas 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 mM imidazole Then, the resin was transferred to a column (diameter 1.5 cm), washed with
50 mL buffer B, containing 20 mMimidazole and protein was eluted with 65 mL buffer B, containing 50 mM
imidazole Peak fractions were pooled, concentrated 10-fold with an Amicon concentrator (YM30 memb rane) 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 70C 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 mM) were employed pH stability was determined by incubating concentrated CelBtrunc(25 lgÆmL)1) in 75 mMof the indicated buffers, supplemented with 10 mMCaCl2and
10 mM MgCl2. After incubation overnight at 4C, 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 mM b-alanine,
pH 3.5, 0.94 mMCaCl2, 2 mM MgSO4 according to [28] The number of washing steps was reduced to three Subsequent incubation took place for 1 h at 57C Immunoblot analyses were performed by transferring proteins from SDS gels onto nitrocellulose membranes
Trang 4using a Trans-Blot semidry 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-riusfor 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)1and a xylanase activity of 0.9 UÆmg)1were 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· 106plaque-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), stained with Serva Blue R; 3, recomb inant CelB trunc (3 lg), stained with Serva Blue R.
Trang 5CMC 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) and a LysE
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 Bar length, extent of exchange of 0.1 per residue 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).
Trang 6Purification of recombinant CelB and CelBtrunc
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)
CelBtrunc, 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 CelBtrunc
pH and temperature dependence
CelBtruncwas 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, CelBtrunc and the
wild-type protein basically exhibited the same temperature
dependence with an optimum at 80C No activity was
found at 100C or b elow 40 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
CelBtruncalso 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) CelBtrunc
was considerably stable at 80C, still exhibiting 60% of
the control activity after 1 h Preincubation at 70C for that
amount of time even stimulated the activity In contrast, a
10-min incubation at 90C 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 CelBtrunc using
CMC as substrate yielded a Kmof 0.35 mgÆmL)1and a Vmax
of 10.8 UÆmg)1, resulting in a kcatof 0.881 min)1
Under standard conditions, optimal activity was obtained
in the presence of Ca2+ and Mg2+ions (10 mM 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 mMZn2+caused a 78% inhibition of CelBtrunc
activity Inhibition by Zn2+is typical of many GHs [38,39],
but not of all members of family 51 [37,40]
Apart from CMC, CelBtruncwas 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 CelBtrunc 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 cellocello-biose 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 xylopentacellotetra-ose, 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.
Trang 7no 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 acidocaldariuscells 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
80C
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) or 90 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 TLCanalysis 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.
Trang 8low 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
40C, respectively [31] For AbjA, the respective values are
pH 5.9 and 75C [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 (CelBtrunc), 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, CelBtrunc 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 This is also in contrast to all other memb ers of 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], a very low activity on CMC (0.08% of the arabinofuranosidase activity) was reported This remarkable finding is further corroborated by the fact that, based on sequence comparison of their catalytic domains, CelB and EGF form a distinct cluster within family 51, although both enzymes differ in their pH- and temperature optima Thus, substrate specificity seems to put more constraints on the sequence than do pH and temperature adaptation EGF and CelB are examples of divergent evolution in a family of proteins which results in new substrate specificity
Acknowledgments
The authors thank Gabriele Brune for excellent technical assistance, Evert Bakker and Sylke Wilken (University of Osnabru¨ck) for contributions in the initial stage of this work, and Roland Schmid (University of Osnabru¨ck) for performing N-terminal sequence analyses This work was supported by the Fonds der Chemischen Industrie and by a fellowship (to K E.) of the Deutsche Bundesstiftung Umwelt (DBU).
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