Wilson Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Thermobifida fusca xyloglucan-specific endo-b-1,4-gluca-nase Xeg74 and the Xeg74 catalytic do
Trang 1Cloning, expression and characterization of a family-74
Diana C Irwin, Mark Cheng*, Bosong Xiang†, Jocelyn K C Rose‡ and David B Wilson
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA
Thermobifida fusca xyloglucan-specific
endo-b-1,4-gluca-nase (Xeg)74 and the Xeg74 catalytic domain (CD) were
cloned, expressed in Escherichia coli, purified and
charac-terized.This enzyme has a glycohydrolase family-74 CD that
is a specific xyloglucanase followed by a family-2
carbo-hydrate binding module at the C terminus.The Michaelis
constant (Km) and maximal rate (Vmax) values for hydrolysis
of tamarind seed xyloglucan (tamXG) are 2.4 lM and
966 lmol xyloglucan oligosaccharides (XGOs) min)1Ælmol
protein)1.More than 75% of the activity was retained after a
16-h incubation at temperatures up to 60C.The enzyme
was most active at pH 6.0–9.4 NMR analysis showed that
its catalytic mechanism is inverting.The oligosaccharide
products from hydrolysis of tamXG were determined by MS
analysis.Cel9B, an active carboxymethylcellulose (CMC)ase
from T fusca, was also found to have activity on xyloglucan
(XG) at 49 lmolÆmin)1Ælmol protein)1, but it could not hydrolyze XG units containing galactose.An XG/cellulose composite was prepared by growing Gluconacetobacter xylinuson glucose with tamXG in the medium.Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added T fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc.The function of this enzyme appears to be to break down the
XG surrounding cellulose fibrils found in biomass so that
T fuscacan utilize the cellulose as a carbon source Keywords: xyloglucanase; cellulase; inverting; regulation; plant cell walls
Converting plant biomass into ethanol for use as fuel has
been a long-term goal of scientists studying cellulases and
related glycosyl hydrolases.Much progress has been made
in identifying, cloning, expressing and characterizing
cellu-lases from both aerobic and anerobic bacteria and from
fungi.Hundreds of such enzymes have been identified and a
list of glycohydrolase families can be found at http://
afmb.cnrs-mrs.fr/CAZY/ Lynd et al.[1] have written a
comprehensive review of the current information on
pos-sible future strategies for biomass hydrolysis.A natural
biomass substrate, such as corn fiber [2], is structurally
complex and many other enzymes besides cellulases are
needed for efficient degradation of the polysaccharides to monosaccharides
The load-bearing structure of primary plant cell walls comprises a network of cellulose fibrils complexed through noncovalent associations with hemicelluloses such as xylo-glucan (XG) and arabinoxylan (AXG) [3].An additional network consists of pectic polysaccharides as well as other, less abundant, wall components, including structural pro-teins, proteoglycans and hydrophobic compounds [4] Cellulose microfibrils are composed of noncovalently, but tightly associated, linear chains of b-1,4-linked D -gluco-pyranosyl residues.XG is the predominant hemicellulose
in dicotyledon type I cell walls and has a b-1,4-linked glucopyranosyl backbone of repeating cellotetraose units with a-D-xylosyl residues attached to C6 of two or more of the first three residues.In addition, some of the xylosyl residues are substituted to form oligomeric side-chains containing galactosyl, arabinose or fucosyl residues [3].The nomenclature for XG subunits is given in Fig.1 [5].XG
is proposed to form a monolayer coating the surface of cellulose microfibrils and to penetrate the cellulose in amorphous areas [6].Experiments using differential extrac-tion of etiolated pea stems with a family-12 xyloglucanase, KOH and a cellulase, suggested that some of the XG is entrapped within or between cellulose microfibrils [3] Individual XG polymers are also thought to cross-link adjacent microfibrils, forming a complex three-dimensional lattice [4,6], underscoring the potentially important struc-tural role of XG
Thermobifida fusca YX is a thermophilic actinomycete that was originally isolated by Dexter Bellamy [7].It grows well at 50C in minimal medium with carbohydrate
Correspondence to D.B.Wilson, 458 Biotechnology Building,
Cornell University, Ithaca, New York, USA.
Fax: 1607 255 6249, Tel.: 1607 255 5706,
E-mail: dbw3@cornell.edu
Abbreviations: AXG, arabinoxylan; BMCC, bacterial microcrystalline
cellulose (CellulonTM); CBM, cellulose-binding module; CD, catalytic
domain; CMC, carboxymethylcellulose; GBG, bacterial cellulose;
GBX, bacterial cellulose/xyloglucan composite; PAHBAH,
p-hydroxybenzoic acid hydrazide; SC, phosphoric acid swollen
cellu-lose; tamXG, tamarind seed xyloglucan; TFSF, Thermobifida fusca
crude supernatant enzymes; Xeg, xyloglucan-specific
endo-b-1,4-glucanase; XG, xyloglucan; XGO, xyloglucan oligosaccharide.
Present addresses: *Beth Israel Hospital, New York City, NY, USA.
US Plant, Soil and Nutrition Laboratory, Tower Road., Ithaca,
NY 14853, USA.
Department of Plant Biology, Cornell University, Ithaca, New York,
USA.
(Received 3 April 2003, revised 19 May 2003, accepted 30 May 2003)
Trang 2polymers such as cellulose, starch, xylan, or mannan as the
sole carbon source and it is thought to be an important
organism in the degradation of biomass.Six T fusca
cellulase genes and a xylanase gene have been cloned,
expressed and characterized [8–11].The genome of this
organism has been sequenced (http://genome.ornl.gov/
microbial/tfus/) and includes many genes coding for a
variety of glycosyl hydrolases.One such gene codes for a
glycosyl family-74 hydrolase with a typical bacterial
family-2 cellulose-binding module (CBM) at the C terminus
In the present study we show that this enzyme is a
xyloglucanase and investigate its properties and its possible
role in the degradation of plant biomass
Materials and methods
Protein production and purification
Clone KPR17269, containing the gene for T fusca
xylo-glucan-specific endo-b-1,4-glucanase (Xeg)74, was obtained
from Stephanie Stilwagen Malfatti (DOE Genome Institute,
Walnut Creek, CA, USA).A PCR product of the gene was
made using the forward primer, 5¢-CGTCCACTCCGC
GGCCGCCCCCGCCTC, which creates a NotI site
(under-lined) near the predicted mature N-terminal codon (in
italics), and the reverse primer, 5¢-CCCTCGTGCGCTCG
AGGTACCAGGGCTTTGC, which has an XhoI site
after the C-terminal codon.Plasmid pD1164 was created
by ligating gel-purified fragments of pET26B+ (Novagen)
digested with NdeI and XhoI, a NdeI–NotI fragment
containing the T fusca Cel6A signal peptide and the above
PCR fragment digested with NotI and XhoI.This ligation
mixture was transformed into Escherichia coli DH5 alpha,
and plasmid minipreps (Qiagen) were used to identify a
transformant containing the desired plasmid which was
then transformed into E coli BL21-DE3 (Novagen) and
also into BL21 (DE3)-RP codon plus (Stratagene).An
Xeg74 catalytic domain (CD) expression plasmid was
created by ligating the 1.2-kb NotI–SphI fragment of
pD1164, the 5.4-kb SpeI–NotI fragment of pD1164 and a
1.0-kb SphI–SpeI PCR fragment, which created a stop
codon after the amino acid sequence GDLDG (mature
amino acid 736).The ligation mix was transformed into BL21 gold (DE3) (Stratagene) and the correct strain (pMC1, D1212) was identified, as described above.The portion of each plasmid created by PCR was sequenced and
no unwanted mutations were detected
D1170 (Xeg74) or D1212 (Xeg74CD) were cultured, from frozen stocks, overnight at 30C in Luria–Bertani (LB) broth containing 0.5% glucose and 60 lgÆmL)1 kanamycin.Thirty milliliters of culture was used to ino-culate 1 L of M9 containing 0.5% glucose and 60 lgÆmL)1 kanamycin.These cultures were grown to a D600 of 0.9, isopropyl thio-b-D-galactoside was added to 0.8 mM, and the cultures were allowed to grow overnight at 30C.The supernatant was clarified by centrifugation, NH2SO4was added to 1 2M, and the supernatant was further clarified by depth microfiltration using a CUNO Beta Pure polyolefin
#46368-02L cartridge.This material was loaded onto a phenyl sepharose CL-4B (Sigma) column (10 mLÆL)1 of supernatant) and the column was washed with two volumes
of 0.8M NH2SO4+ 10 mM NaCl + 5 mM Kpi, pH 6, followed by three volumes of 0.4M NH2SO4+ 5 mM NaCl + 5 mM Kpi, pH 6, and eluted with 5 mM Kpi,
pH 6.The purest fractions, as determined by SDS/PAGE, were combined and loaded on a Q-Sepharose Fast Flow (Pharmacia Biotech) column (2 mg of proteinÆmL)1 of column).The protein was eluted with a gradient (20· column volume) of 0–0.7M NaCl in 10 mM Bis/Tris,
pH 5.6, + 10% glycerol The purest fractions were com-bined and concentrated in a stirred cell using a PTGC
10 000 MWCO ultrafiltration membrane (Millipore).The proteins were stored at)70 C in 5 mMNaOAc, pH 5.5, containing 10% glycerol.The final yield of purified protein was 22 mgÆL)1Xeg74 and 14 mgÆL)1Xeg74CD T fusca crude cellulase was prepared by concentration of T fusca culture supernatant after growth on Solka Floc powdered cellulose (James River Corporation, Berlin, New Hamp-shire, USA), as previously described [2]
Cel9B was expressed in the supernatant of a Strepto-myces lividans clone, pSHE1, and purified as previously described [12].The molecular masses of the purified proteins were determined using a Bruker Biflex III MALDI-TOF spectrometer instrument at the Cornell University Bio-resources Center
Assay methods Tamarind seed xyloglucan (arabinose/galactose/xylose/glu-cose; 3 : 16 : 36 : 45) (tamXG) was obtained commercially (Megazyme, Ireland) or extracted as described previously [13]; XG-bean was isolated from the media of bean (Phaseolus vulgaris) suspension cell cultures, as described previously [14,15].Except where stated otherwise, xylo-glucanase activity was assayed in 500 lL microcentrifuge tubes by incubating 200 lL of samples containing 2–2.5 mgÆmL)1 tamXG, 0.05-M NaH2PO4/K2HPO4 (pH 7.5) buffer, and enzyme for 30 min at the desired temperature.Twenty-microliter samples (in triplicate) were removed and added to 1.5 mL of p-hydroxybenzoic acid hydrazide (PAHBAH) reagent [16] and boiled for 6 min according to the published procedure.The absorbance (A)
at 410 nm was read and a reference curve was prepared using glucose (0–25 lg)
Fig 1 Example of a xyloglucan (XG) oligosaccharide structure
(XXLG) and nomenclature [5] XXXG, XXLG, XLXG, and XLLG
are known subunits of tamarind seed xyloglucan (tamXG) [15].XGs
from other sources vary in composition and the XG from some
dicotyledonous plants has a- L -fucose (1 fi 2) added to some of the
galactose residues, while solanaceous plants produce more complex
arabinoxyloglucans (AXG) [23,26,28,34].In addition, residues may
contain either one or two O-acetyl groups on C-2, C-3 or C-6.
Trang 3The pH optimum assays used buffers prepared by mixing
30-mMsolutions of citric acid, NaH2PO4, boric acid, and
barbital to obtain the desired pH.The final concentration of
buffer in the assays was 15 mM.The temperature stability of
the enzyme was tested by incubation of 2.7 lMXeg74 for
16 h at 0–75C followed by performing the XG activity
assay at 50C
Kinetic constants were determined with substrate
con-centrations from 2 to 60 lgÆmL)1 and 0.375 pmolÆmL)1
enzyme.The bicinchoninate assay [17] was used to quantify
the reducing sugars produced.Michaelis constant (Km) and
maximal rate (Vmax) values were calculated from a plot of
the initial reaction rates vs.substrate concentration using
Kaleidagraph (SYNERGY Software) to fit the data to the
Michaelis-Menten equation
All reducing sugar assays included a glucose standard
curve.The average molecular mass of the XG
oligosaccha-rides (XGOs) was calculated from the manufacturer’s data
and was found to be 1293 Da.This value agreed with the
MS analysis of the tamXG digestion products.Exhaustive
digestion of tamXG by Xeg74 created a set of XGOs from
known amounts of substrate (0–1500 lg), and the
approxi-mate relationship between the glucose standard curve and
the nmoles of XGOs produced was determined for both the
PAHBAH and the bicinchoninic acid reducing sugar
methods.For the PAHBAH method, the nmol of
XGOs¼ nmol of glucose equivalents ·0.481; for the
bicinchoninic acid method, the nmol of XGOs¼ nmol of
glucose equivalents·0.686
NMR experiments were performed at the Chemistry and
Chemical Biology NMR facility, Cornell University, by the
method of Pauly et al.[18].TamXG (5 mgÆmL)1) was
added to 10 mM NaCl in D2O at 80C and stirred
overnight.Xeg74 was prepared for NMR experiments by
resuspending and concentrating it six times with 10-mM
NaCl in D2O using a Centricon 30
(Millipore).Three-hundred micrograms of Xeg74 was added to 0.7 mL of
tamXG in an NMR tube and spectra were taken over a
100-min time course
Samples were prepared for MS by reacting 0.4 nmol
Xeg74 or Cel9B with 5 mg of tamXG in 1 mL of 5 mM
NaOAc (pH 5.5) buffer containing 0.02% NaN3, at 50C
for 48 h on a rotator.Positive-mode electrospray MS was
performed on a Bruker Esquire LC ion-trap mass
spectro-meter
TLC of hydrolysis products was performed using
What-man LK5D 150-A silica gel thin-layer plates with two
ascents of the solvent, ethyl acetate/water/MeOH
(40 : 15 : 20).Plates were stained (100 mL of acetic acid,
1 mL of p-anisaldehyde, 2 mL of concentrated sulfuric
acid) and then heated for 1 h at 95C, as described by
Chirco & Brown [19] and Jung et al.[12].Glucose and
xylose oligomer standards were obtained from Seikagaku
America or Sigma
Preparation of GBG, GBX and tomato cell walls
Bacterial cellulose (GBG) and bacterial cellulose/xyloglucan
composite (GBX) were produced from
Gluconaceto-bacter xylinus(formerly Acetobacter xylinus) ATCC 53524
according to a procedure published previously [6].The
resultant gel-like material was harvested, rinsed six times on
a filter with dH2O and stored at 4C in 0.04% NaN3 Culture reducing sugars were removed by rotating a solution of the coarsely chopped GBG or GBX in 0.05-M NaKPi, pH 7.4, for at least 2 h at 50C followed by further washing with 0.04% NaN3.Pieces of the gels were rinsed with water, blotted on filter paper and chopped finely with a razor blade.The dry mass of the material was approxi-mately 1% of the blotted gel and overdigestion of GBX with Xeg74 gave an XG content of 16%.In order to equalize the amount of cellulose to be digested, 42 mg of GBG or 50 mg
of GBX were weighed into 0.5-mL screw-cap microfuge tubes.Buffer (0.05-M NaKPi, pH 7.4) and enzymes were added to achive a final volume of 0.5 mL and the microfuge tubes were then incubated on a rotator for 16 h at 50C Reducing sugars in the supernatant were measured by the PAHBAH method, as described above, using glucose as a standard.Each data point represents the average of three separate digestions and the PAHBAH measurements for each digestion were run in triplicate
Tomato cell walls were isolated as follows [14,20]: 50 g of outer pericarp tissue from green tomatoes was finely diced, frozen in liquid nitrogen and ground into a fine powder This material was boiled in 500 mL of 95% EtOH for
40 min, filtered on MiraclothTM(Calbiochem), resuspended
in boiling EtOH, and refiltered.The solid material was resuspended in 500 mL of CHCl3/MeOH (1 : 1), filtered on
a glass frit and washed with 500 mL of acetone.These steps inactivate endogenous cell-wall enzymes and extract low-molecular-mass solutes.To remove starch, the solid mater-ial was resuspended in 15 mL of dimethylsulfoxide/H2O (9 : 1) and stirred for 1 h, centrifuged and washed three times with dimethylsulfoxide/H2O (9 : 1).The solid mater-ial was resuspended in trans-1,2-diaminocyclohexane-N,N,N¢,N¢-tetraacetic acid (CDTA)/bicarb buffer (50 mM CDTA, 50 mM Na2CO3) containing 20-mM NaBH4 (pH 6.5) to a final volume of 125 mL and stirred overnight (The CDTA chelates Ca2+, facilitating the release of pectin, and the NaBH4 removes reducing groups.) This material was filtered on MiraclothTM (Calbiochem), rinsed several times with CDTA/bicarb buffer, stirred overnight at room temperature, homogenized, filtered again, and washed with CDTA/bicarb buffer until the filtrate was clear.The solid material was resuspended and dialyzed against 0.01MTris (pH 7.0) containing 0.02% NaN3, at 4C.Assays were carried out, as for GBG and GBX, using 100 lL (6.8 mgÆmL)1dry mass) of the tomato cell wall preparation
as substrate in a total volume of 500 lL.The tomato XG was from Bree Urbanovicz and prepared by extraction of tomato cell walls with 1MKOH for 1 h and then with 4M KOH at room temperature with stirring overnight.The supernatants were filtered through nylon mesh and neut-ralized, on ice, to pH 7.0 with glacial acetic acid XG was precipitated from the 4Mextraction with two volumes of ethanol, cooled on ice, centrifuged, washed three times with cold ethanol, resuspended in water and then freeze dried Western blots
Culture supernatants were analyzed for the presence of Xeg74 by separation on SDS/polyacrylamide gels [21] followed by transfer to Immobilon-P poly(vinylidene diflu-oride) membranes (Millipore).Rabbit polyclonal antisera
Trang 4raised against recombinant Xeg74 was used as the first
antibody and goat anti-rabbit Ig alkaline phosphatase
conjugate (Bio-Rad) was used as the second antibody
Immunodetection was performed using nitro-blue
tetra-zolium and 5-bromo-4-chloro-3-indolyl phosphate,
accord-ing to the Bio-Rad protocol
The GenBank accession number for the T fusca genome
is NZ_AAAQ00000000.The file containing the Xeg74 gene
is NZ_AAAQ01000018 and the gene is Tfus0318, CDS
39974.42751 The protein identification is ZP_00056977
The accession numbers for T fusca cellulases are: Cel5A,
Q01786; Cel6A, P26222; Cel6B, Q60029; Cel9A, P26221;
Cel9B, Q08166; and Cel48A, AAD39947 T fusca YX
(BAA-629) and T fusca ER1 (BAA-630) have been
depo-sited in the American Type Culture Collection
Results
Cloning and purification
The gene for Xeg74 was cloned into pET26b using the
T fusca Cel6A signal sequence (MRMSPRPLRALL
GAAAAALVSAAALAFPSQAA) in place of its native
signal sequence.Cel6A, Cel6Acd, and Cel48A have been
expressed and secreted successfully by E coli [11,22] using
this signal sequence and it has a convenient NotI site at its
C terminus, which allows cloning in-frame with alanine as
the first amino acid of the mature protein.An alignment
of the amino acid sequences of 11 family-74 catalytic
domains shows that their amino acid identity to T fusca
Xeg74 ranges from 29 to 63% with the alignment starting
at GYTWR.Xeg74 was predicted by SignalP (http://
www.cbs.dtu.dk/services/SignalP-2.0/#submission) to have
the mature N-terminal sequence, APASATTGYTWR,
with the start of the conserved sequence at amino acid 8
The catalytic domain was produced from the same vector
after inserting a stop codon after amino acid 736 ending
with VGDLDG.This C-terminal sequence agrees well
with that of other family-74 catalytic domains and, in the
native protein, is followed by a linker region, (737)
PPPQPTEEP…, which is similar to the linker region in
T fuscaCel9A [12].Of the expressed protein, about 90%
was secreted to the culture supernatant and about 10%
was in the shock fluid, as determined by SDS gels (data
not shown).The molecular mass of purified Xeg74CD, as
determined by MS, was 79 443 Da (expected mass
79 480 Da).However, the mass spectrum of Xeg74
showed a broad set of peaks with mass values from
96 200 to 94 718 (expected mass 94 705 Da), indicating
that the signal peptide was being cut in different places,
resulting in the addition of up to 17 extra amino acids at
the N terminus of the protein.It is not clear why the
Xeg74CD signal peptide cuts cleanly and Xeg74 does not,
as both genes are cloned in the same way except for the
presence of the linker and the family-2 CBM at the C
terminus of XEG74.The molecular masses of Cel9A-68
(CD + family3c CBM) and Cel48ACD, both cloned with
the Cel6A signal peptide, were also determined by MS
and the peaks also had several small shoulders at higher
molecular masses although they were much sharper than
the peak for Xeg74.Possibly the more complicated
domain structure of Xeg74 (a catalytic domain, a linker,
and a CBM) results in nonspecific cleavage of the signal sequence
Characterization Xeg74 had very low activity on swollen cellulose (SC) or carboxymethyl cellulose, and assays using tamXG, barley b-glucan, Avicel, locust bean gum (galactomannan), soluble starch, xylan, pectin, or corn fiber as substrates showed that Xeg74 had significant activity only on tamXG.The products of Xeg74 hydrolysis of tamXG were analyzed by TLC (Fig.2A).Digestion of tamXG was complete within 1.5 h and produced three main bands with no carbohydrate remaining at the origin.In contrast, Xeg74 digestion of SC and carboxymethylcellulose (CMC), after overnight incu-bation, produced only very faint bands of cellotriose (G3), cellotetraose (G4) and cellopentaose (G5).Each of the six purified T fusca cellulases was tested for activity on tamXG and only Cel9B was active.However, it produced only two
of the three product bands and there was a large quantity of undigested material remaining at the origin (Fig.2B) Xeg74 had very limited activity on G4, G5 and G6 and only faint product bands were produced after overnight digestion (Fig.2C).Xeg74 was active on both bean and tomato XG and inactive on boiled barley b-glucan (Fig.2D,E)
The molecular masses of the digestion products of tamXG by Xeg74 and Cel9B were determined by MS (Fig.3).Using the known composition of tamXG [15,23], the possible products were determined, as shown in the figure.Xeg74 cleaved the backbone of XG to give products decorated with two or three xylose units and further decorated with up to two galactose molecules XXGG was not reported by York et al.[15] to be a component of tamXG, but there is a peak at mass 953.8, which appears to
be XXGG in Fig.3A.Cel9B only cleaved a portion of the tamXG (Fig.2B, lane 5) and did not produce any products containing galactose, XXGG appears to represent the major product of hydrolysis by this enzyme and this suggests that Cel9B prefers to cleave where there are two undecorated glucoses, although it can also cleave slowly to produce XXXG
The Kmwas determined to be 3 2 and 3 9 lgÆmL)1for Xeg74 and Xeg74CD, respectively.This corresponds to 2.4 and 3.0 lMusing a molecular mass of 1293 for the average XGO hydrolyzable unit.The Vmaxof Xeg74 was 966 lmol XGOÆmin)1Ælmol)1 protein, while that of Xeg74CD was somewhat higher, at 1257 XGOÆmin)1Ælmol)1protein.The specific activities were also determined using a similar assay
to that used for cellulases [8] with a substrate concentration
of 2.5 mgÆmL)1and increasing amounts of enzyme.The data shows a linear relationship up to 50% digestion and the specific activities were 578 and 875 lmol XGOÆmin)1Æ lmol)1 enzyme for Xeg74 and Xeg74CD, respectively The extra amino acids left on the N terminus from the signal peptide may have caused the whole protein to have lower specific activity than the CD, or the smaller size of the CD may enable it to fit into the xyloglucan/cellulose matrix more easily, increasing the availability of the insoluble xyloglucan substrate to the enzyme.The specific activity of Cel9B was also measured in this way and was found to be
46 lmol XGOÆmin)1Ælmol enzyme at 15% digestion
Trang 5However, the maximum digestion obtained was about
30% of the total substrate and the curve was not linear
In contrast, Cel9B has activities of 5410 and 363 lmol
cellobioseÆmin)1Ælmol)1, respectively, on CMC and SC
while Xeg74 activity on CMC and SC was 1.1 and 1.9 lmol
cellobioseÆmin)1Ælmol)1, respectively
Xeg74 retained more than 60% activity at pH 6.0–9.4
and 90% activity at pH 6.9–8.6 (Fig 4A) When assayed
with 0.025M (pH 7.5) NaH2PO4/K2HPO4, Hepes, Tris/
HCl, or NaOAc buffers, the enzyme had 100%, 83%, 73%,
and 92% activity, respectively.Temperature stability tests
showed that the enzyme retained full activity after 16 h of
incubation at 55C and 76% activity after incubation at
60C (Fig.4B).Thirty-minute activity assays showed that
the activity increased with temperature up to 76C
(Fig.4B)
Enzymatic hydrolysis of b-glycosidic bonds occurs by
two general mechanisms giving rise to either retention or
inversion of the anomeric configuration [24].Figure 5
shows the [1H]-NMR spectra of tamXG after reaction with
Xeg74 for 0, 5, 20 and 100 min The a-anomeric proton
appears at 5 min, indicating an inverting enzyme, and after
20 min the b-anomeric proton appears as the result of
mutarotation.The NMR assignments relied on the work of
Pauly et al.[18] for the family-12 Aspergillus aculeatus
xyloglucanase, a retaining enzyme, in which the b-anomeric
proton appeared first followed by the subsequent
appear-ance of the a-anomeric proton
Concentrated supernatants from T fusca cultures
grown on Solka Floc, xylan, or corn fiber, all had activity
on tamXG and revealed the same product pattern on
TLC as observed for Xeg74 (Fig.2B) The xylan-grown
supernatant produced four additional brown-colored bands of lower molecular mass; although the composition
of these bands is not known, the brown color indicates that they contain xylose.A Western blot with polyclonal rabbit antiserum prepared against purified Xeg74 showed that the level of this protein was highest in the super-natants of the Solka Floc-, xylan-, or corn fiber-grown cultures and was present at low levels when T fusca was grown on glucose, cellobiose, xylose, or bacterial micro-crystalline cellulose (BMCC) (Fig.6) T fusca was not able to grow with tamarind, tomato, or bean XG as the sole carbon source.When glucose was added to the culture, growth resumed, ruling out the presence of an inhibitor.Although attempts to culture T fusca on tamXG produced little or no growth, high levels of Xeg74 were induced (Fig.6, lane 11) and TLC analysis of the culture supernatant showed that the XG had been degraded to the expected products (data not shown).The Xeg74 antiserum also reacted with T fusca Cel6A, but not with the Cel6A catalytic domain (Fig.6, lanes 9 and 10), which indicates that the CBM of Cel6A cross-reacts with the Xeg74 antiserum.The family-2 CBMs of all six cellulases show 33–52% amino acid identity with the Xeg74 CBM (43% identical to Cel6A CBM).Presumably these two CBMs have antigenic epitopes in common while the others do not.This cross-reaction is useful for comparing the differences in induction of the two enzymes.Cel6A is induced by growth on cellobiose, Solka Floc, BMCC, and corn fiber, while Xeg74 is present
in all of the supernatants but is found at a higher level in cultures grown on XG, xylan, Solka Floc, and corn fiber (Fig.6).The low level of Xeg74 found in BMCC cultures
Fig 2 TLC analyses of reaction products (A–E) All reactions were run at 50 C for 16 h except for lane 2, which was incubated for 1.5 h The enzymes and substrates used are noted on the figure under each lane using the following abbreviations: 74, xyloglucan-specific endo-b-1,4-glucanase (Xeg)74; and 9B, Cel9B.CF, XY, and SF indicate hydrolysis by enzymes in concentrated crude supernatants from cultures of Thermobifida fusca ER1 grown on corn fiber, xylan, or Solka Floc, respectively.Abbreviations: tom, tomato; tam, tamarind; and G6, cellohexose.Standards for each TLC analysis were glucose, cellobiose, cellotriose, cellotetraose, cellopentaose (G1–G5) and xylose and xylobiose (X1–X2).
Trang 6shows that it is not the cellulose in Solka Floc which is
inducing the higher level of Xeg74 but some minor
component
Role of Xeg74
G xylinussynthesizes cellulose I chains that extrude parallel
to the bacterial wall and which coalesce into bundles of
highly crystalline microfibrils [25].When G xylinus was
grown in the presence of glucose and tamXG, 38% of the
added XG was incorporated into the cellulose pellicle and
microscopic analysis showed the formation of cross-linkages
between the ribbons [6].The level of incorporated XG was
similar to that found in primary cell walls and much of the
XG was thought to be intimately bound to the surface or
woven into the cellulose fibers [6].The role of Xeg74 in plant
biomass degradation was studied using the GBX composite
produced by G xylinus when grown on glucose plus
tamXG and, as a control, GBG was prepared by growth
on glucose.A mixture of purified T fusca cellulases (cel
mix) consisted of Cel5A (an endocellulase), Cel6A (a
nonreducing end-directed exocellulase), Cel9A (a processive
endocellulase), and Cel48A (a reducing end-directed exocellulase).Under the assay conditions, Xeg74 enzyme alone (0.05 nmol, 4.7 lg) produced 58 lg of reducing sugar from GBX and 6.9 lg from GBG.The cel mix alone was not able to degrade GBX to any appreciable extent; however, when Xeg74 was added, the activity was very similar to that
of concentrated T fusca crude supernatant enzymes (TFSF) (Fig.7A) The reactions of the cel mix and the cel mix + Xeg74 on GBG were very similar to that of TFSF
on GBG (data not shown).XG protects the cellulose microfibrils from degradation by cellulases.Xeg74CD was found to have 86% of the activity of whole Xeg74 when combined with the cel mix on GBX, implying that the CBM contributes to the degradation, but is not essential Tomato cell walls that had been processed to inactivate endogenous enzyme activity and remove low-molecular-mass solutes and starch, were used as a substrate in a similar assay.Figure 7B shows that the amount of reducing sugar produced by the cel mix, plus and minus Xeg74, was about the same.However, TFSF was able to produce four times
as much reducing sugar as the cel mix.Analysis of the hydrolysate by TLC showed glucose and cellobiose to be the
Fig 3 MS analysis of the products of tamarind seed xyloglucan
(tam-XG) hydrolysis by xyloglucan-specific endo-b-1,4-glucanase (Xeg)74
(A) and Cel9B (B) The theoretical mass values are given in parentheses
for possible products.
Fig 4 Temperature and pH optima for Xeg74 activity (A) Percentage xyloglucanase activity at various pH values.(B) Effect of temperature
on the activity and stability of xyloglucan-specific endo-b-1,4-gluca-nase (Xeg)74.Stability was tested by incubating xyloglucan-specific endo-b-1,4-glucanase (Xeg)74 at the indicated temperatures for 16 h followed by dilution and a 30-min assay at 50 C.
Trang 7dominant products, with only faint bands at the higher
molecular massess expected for XGOs (Fig.2D, lane 19)
The products from Xeg74 hydrolysis of the 4M
KOH-extracted tomato XG are shown in Fig.2E, lane 22.XGO
bands are produced, but much of the substrate remains
undegraded, at the origin.An activity assay measuring
reducing sugars showed that a maximum of 26% of this
tomato XG extract could be degraded by Xeg74.This may
reflect the presence of small amounts of polysaccharides
other than XG in the extract and/or variability in the
structure of tomato XG.For example, the AXG of
solanaceous plants, such as tomato, is characterized by
the presence of arabinose-containing side-chains, such as
b-ara-(1fi 3)-a-L-ara-(1fi 2)-a-D-xyl [26].It is possible that
these residues do not fit in the active site of Xeg74
Discussion
In this study, results were obtained which showed that XG
protects the cellulose in the cellulose/xyloglucan composite
and that the activity of Xeg74 with a synergistic mixture of
cellulases can easily degrade this material.However, tomato
cell walls have a much more complex structure, and
additional enzymes in the TFSF crude mixture are essential
for tomato cell-wall digestion.Similar results were seen in studies by Vincken et al.[27,28], which showed good synergistic activity on water-unextractable solids from apples (about 24% xyloglucan and 33% cellulose) when Trichoderma virideEndo IV (a family-12 CMCase/xyloglu-canase) was added to a mixture of EXOIII and EndoI.The amount of cellobiose released was twice as high as the amounts released by EXOIII and EndoI alone; however, even the most effective combination was not able to solubilize all of the cellulose
The results of the NMR experiment show that Xeg74 and presumably all family-74 enzymes catalyze hydrolysis with inversion of the anomeric configuration.This is in contrast
to the family-12 A aculeatus xyloglucanase, which is a retaining enzyme [18].The Xeg74 gene does not have an upstream 14-bp DNA-binding site for the regulatory protein, CelR, which is found in the six T fusca cellulase genes.This is consistant with our observation that Xeg74 and Cel6A are regulated differently.It is interesting that
T fuscaproduces Xeg74 during incubation with tamXG, even though it cannot grow on the XGOs released from XG hydrolysis.The amount of enzyme produced in a nongrow-ing culture appears to be equal to or even a little higher than the amount produced in growing cultures, using either Solka Floc or corn fiber as the sole carbon source.Another example of an enzyme produced in a nongrowing culture is
a polyester-degrading extracellular hydrolase from T fusca DSM43793 [29], which is induced in cultures containing EcoflexTM(BASF AG, Germany), a random co-polyester of 1,4-butanediol, terephthalic acid and adipic acid, as the sole carbon source.It is curious that T fusca grows on xylose and yet does not metabolize the a-D-xylose in the XGOs produced by Xeg74.There are open-reading frames in the
T fuscagenome which are homologous to a-xylosidases; however, neither extracts nor concentrated supernatants
Fig 6 Western blot of an SDS/polyacrylamide gel using rabbit polyclonal antiserum against xyloglucan-specific endo-b-1,4-glucanase (Xeg)74 Supernatants (10 lL) from cultures of Thermobifida fusca grown on glucose (lane 1), cellobiose (lane 2), xylan (lane 3), Solka Floc (lane 4), and corn fiber (lane 5); lane 6, Benchmark molecular mass standards; lane 7, Xeg74 (0.05 lg); lane 8, Xeg74CD (0.05 lg); lane 9, Cel6ACD (0.5 lg); lane 10, Cel6A (0.05 lg); supernatants (10 lL) from cultures of T fusca grown on tamXG, no growth was apparent (lane 11), bacterial microcrystalline cellulose (BMCC) (lane 12), Solka Floc (lane 13).
Fig 5 [1H] NMR spectra of tamarind seed xyloglucan (tamXG)
reac-ted with xyloglucan-specific endo-b-1,4-glucanase (Xeg)74 at 50 °C The
a-anomeric proton appears rapidly, indicating an inverting enzyme,
while the b-anomeric proton appears later as a result of mutarotation.
Trang 8from T fusca cultures grown on Solka Floc, xylan, or corn
fiber had measurable activity on p-nitro-phenol a-D-xylose
[2]
The first family-74 enzyme to be reported was an
A aculeatusenzyme (FIII avicelase), which produces
cello-triose and cellobiitol from reduced cellopentaose, clearly
showing that it releases cellobiose from the reducing end
[30].However, the enzyme was not tested on XG and the
degree of hydrolysis was only about 0.7% Thermotoga
maritimaCel74 is most active on barley b-glucan, which has
a mixed-linkage b-1,3/4 glucose backbone and only 23% as
much activity on tamXG [31] A niger EglC [32] resembles
Xeg74 in having the highest activity on tamXG with about
5% as much activity on CMC or b-glucan and it has a
C-terminal family-1 fungal CBM.EglC is regulated by
XlnR, a transcriptional activator which binds to the DNA sequence, GGCTAA.We did not find a protein homolog-ous to XlnR in the T fusca genome, nor did we find GGCTAA upstream of the Xeg74 start codon.The Geotrichumsp M128 family-74 enzyme is an exoglucanase, which attacks the reducing end of XG, releasing GG, XG,
or LG [33].This protein is unique in having four regions of amino acids (235–251; 310–318; 361–372; 398–413) that are not found in the other family-74 proteins.These may form loops that make the active site a tunnel rather than an open cleft, leading to exoglucanase rather than endoglucanase activity.The nature and presence of a CBM varies among the family-74 proteins: four of 13 family-74 genes code for a family-2 CBM; three have a family-1 fungal CBM; one has
a family-3 CBM; and five have no CBM.The low Kmof
T fuscaXeg74 shows that the binding of the catalytic site to the substrate is very tight and perhaps this makes the CBM useful but not essential for hydrolytic activity.Overall, the characterized family-74 enzymes show a variety of substrate specificities with most having a strong preference for a cellulosic backbone substituted with xylose (and further decorated) side-chains.The structure of the Xeg74 active cleft should provide interesting insights into its mechanism
of action and efforts are underway to solve the structure of Xeg74CD
In summary, Xeg74 is produced at low levels on all substrates, is induced at higher levels by growth on Solka Floc, corn fiber and xylan, and is also highly induced by XG
in the media, although additional growth does not take place.The purpose of the enzyme seems to be to degrade
XG surrounding cellulose microfibrils to allow the organism access to a substrate which it can hydrolyze and metabolize efficiently.This function is consistent with the presence of a family-2 CBM on the enzyme.Xeg74 may also function to help T fusca grow on other plant cell-wall polymers, such
as xylan or mannan, which may be protected by xyloglucan
Acknowledgements This work was supported by DE-FG02-01ER63150, from the US Department of Energy.
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