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R6sum6. Summary.
L'endoglucanase codde par le gone celB de The endoglucanase encoded by the celB gene
Clostridium thermocellum a dtd puri[ide ~ partir of Clostridium thermocellum was purified from
d'une souche d'Escherichia coli portant et expri- an E. coli strain carrying and expressing the
m a n t l e gone de C. thermocellum clond dans te C. thermocellum gene cloned in the plasmid
plasmide pBR322. La prdparation obtenue prd- pBR322. The preparation showed two active
sente deux bandes actives, d'une masse moldcu- bands, with Mr 55,000 and 53,000, presumably
laire apparente de 55.000 et 53.000, vraisembla- derived from the primary translation product by
blement ddrivdes du produit de traduction pri- proteolysis. Specific antiserum raised against these
maire par protdolyse. Un antisdrum spdcifique prd- bands was used to identify the corresponding anti-
pard contre ces bandes a permis d'identi[ier l'anti- gen in the culture supernatant of C. thermocellum :
gbne correspondant dans le surnageant de culture in a double immunodi[fusion test (Ouchterlony),
de C. thermocellum : dans un test de double a precipitin line was observed which [used comple-
immunodiJ[usion (Ouchterlony), on observe un arc tely with that formed by an E. coli extract contain-
de prdcipitation [usionnant complOtement avec ing endoglucanase B expressed from the cloned
celui [ormd par un extrait d'E. coli contenant gene. Proteins from C. thermocellum supernatant
l'endoglucanase B exprimde ~ partir du gkne clond. were further analyzed by SDS-polyacrylamide gel
Par ailleurs, les protdines du surnageant de C. ther- electrophoresis and transferred to a nitrocellulose
mocellum ont dtd sdpardes par dlectrophorbse sur sheet. After incubating the nitrocellulose blot with
gel de polyacryIamide en prdsence de SDS et trans- antiserum and subsequently with le5I-labeled pro-
[&des sur une feuille de nitrocellulose. AprOs avoir tein A, a band with Mr 66,000, corresponding to
incubd le ddcalque de nitrocellulose en prdsence the celB gene product expressed by C. thermocel-
d' antisdrum, puis de protdine A marquee lum, was detected by autoradiography.
l'iode 125, une bande de masse moIdculaire appa-
rente 66.000, correspondant au produit du gone
celB exprimd par C. thermocellum, a dtd repdrde
par autoradiographie.
Mots-cl~s : endoglueanase / g~ne / C. thermocellum. Key-words : endoglueanase gene / C. thermocellum.
Introduction.
Among the organisms that have been conside- which is thermostable and is not inhibited by cello-
red for the industrial fermentation of celluIose, biose or glucose, the major products of cellulose
Clostridium thermocellum, a thermophilic and degradation [I, 2] ; furthermore, it is able to fer-
cellulolytic anaerobe, has recently raised an in- ment mono- or oligosaccharides derived from
creassed interest. It has a high ceUulolytic activity, cellulose and hemicellulose into economically va-
fuged at 16,000 gmax for 15 min, redissolved in 15-20 ml ted subcutaneously at 3 week intervals to New Zealand
of 40 m M Tris-HC1, pH 7.7, and dialyzed 3 times against white rabbits. Serum was collected after the third injec-
500 ml of the same buffer. tion.
e) DEAE-Trisacryl chromatography : The dialyzed Immunological detection of endoglucanase B in crude
sample was loaded on a 2.6 × 27 cm column of D E A E - C. thermocellum culture supernatant : Double immuno-
Trisacryl (LKB) equilibrated in 40 m M Tris-HC1, pH 7.7. diffusion tests were performed according to Ouchter-
The resin was washed at 50 m l / h with the same buffer lony [11]. To detect the b a n d corresponding to endoglu-
containing 40 m M NaC1 until the A ~ returned to the canase B after polyacrylamide gel electrophoresis, the
baseline. The column was then eluted at 20 m l / h with separated proteins were transferred from the gel to a
a 360 ml gradient of 40 to 200 m M NaC1 in the same nitrocellulose sheet (Schleicher and Schtill B A 85) by
buffer. Salt concentration was followed by monitoring transverse electrophoresis for 3 h at 12 V / c m [12]. The
the conductivity of the fractions. nitrocellulose sheet was incubated for 30 m i n at 37°C
in 10 m M Tris-HC1, p H 7.4, containing 154 m M NaCI
f) Ultrogel AcA 44 chromatography : Active fractions and 5 per cent bovine serum albumin to saturate remai-
from the D E A E column were pooled and concentrated ning protein binding sites. After briefly rinsing in
by ultrafiltration on an Amicon P M 10 membrane. The 10 m M Tris-HCl, p H 7.4 + 154 m M NaC1, the blot
sample was loaded on a 1.5 × 90 cm column of Ultrogel was incubated overnight on a rotatory shaker at 4°C
A c A 44 (LKB) in 50 m M Tris-HC1, pH 7.0, and eluted with the same buffer containing 5 per cent bovine serum
with the same buffer a t 8 m l / h . Active fractions were albumin and 2 per cent of either preimmune or anti-celB
concentrated by ultrafiltration and either submitted to a antiserum. Excess immunoglobulins were washed by
second gel filtration on the same column or used as rinsing the blot 10 times with 10 m M Tris-HC1 +
such for preparative gel electrophoresis. 154 m M NaC1. The 5th and the 6th wash contained
Polyacrylamide gel electrophoresis : SDS-polyacryla- 0.05 per cent N P 40. After briefly equilibrating with
mide gel electrophoresis was performed according to Dulbecco's PBS buffer [1'9] containing 3 per cent bovine
Laemmli [17]. The same system was used for native gel serum albumin and 0.02 per cent NaNs, the sheet was
electrophoresis, except that SDS and mercaptoethanol incubated for 90 rain at room temperature o n a rotatory
were omitted and the samples were not heated. shaker in the same buffer containing 2-3 ~Ci of protein
A from S. aureus, labeled with 1:251 to a specific activity
Detection of bands having endoglucanase activity : of 50 m C i / m g [20]. Excess radioactivity was washed
After native gel electroporesis, the gel was laid on top as described above for immunoglobulins. The blot was
of a 0.75 m m sheet of 2 per cent agar containing 0.1 per briefy dried between paper towels, wrapped in plastic food
cent C M C in 50 m M Na~HPO4 + 12.5 m M citric acid, wrap and fluorographed for 3 h at - - 8 0 ° C with an
pH 6.3, and incubated for 30 min at 60°C. Zones of intensifying screen.
C M C hydrolysis were then detected by staining the agar
replica with Congo red as described [18].
Preparation of antiserum : 1-2 mg enzyme, purified
up to the first or second gel filtration step, were sub- R~sults.
mitted to native gel electrophoresis, using the whole
width of the slab gel. Zones containing endoglucanase
were located as described above, cut out and crushed Purification of endoglucanase B : Y i e l d s a n d
by forcing twice through an 18 gauge syringe needle. s p e c i f i c a c t i v i t i e s of t h e e n z y m e a t t h e v a r i o u s
Aliquots from the crushed gel containing 0.25 m g protein st~ps of p u r i f i c a t i o n a r e s u m m a r i z e d i n t a b l e I.
were mixed with Freund's complete adjuvant and injec- The relatively poor yield observed for the amino-
TABLE I.
Purification of endoglucanase B of E. coli HBIO1 (pCT207).
nium sulfate fractionation seems to be due to in- activity could be recovered from this fraction.
activation : analysis of the a m m o n i u m sulfate-con- However, attempts to omit the ammonium sulfate
taining supernatant after dialysis showed that little precipitation led to poor adsorption and resolu-
tion on the subsequent anion exchange column and
this step was thus kept in the purification proce-
I 2 3 4 5 dure.
Analysis of the purified enzyme on native poly-
acrylamide gels showed two protein bands coin-
ciding with two bands of endoglucanase activity
(fig. 1, lanes 1 and 2). Denaturing SDS gels show-
J92.5 ed the presence of two major bands with Mr
55,000 and 53,000 (fig. 1, lane 3), accounting for
about 80 per cent of the total protein by densito-
~~ , 69 meter scanning. The correspondence between
these bands and those seen on native gels is seen
in figure 1, lane 4 : after extraction of the active
bands from a native gel, electrophoresis under de-
~46 naturing conditions shows an identical doublet
W
H
with Mr 55,000 and 53,000.
It has been shown elsewhere [13] that E. coli
minicells expressing the celB gene synthesize only
one immunoreactive polypeptide, which is specific
of the C. thermocellum D N A insert ans has a
~ m 3 0 molecular mass of 66,000 daltons. Furthermore.
the size of the insert (2.7 kilobases) is too short to
account for the separate coding of two proteins
larger than 50,000 daltons. It is therefore likely
that the two bands observed derive from a single
gene product, presumably by a proteolytic mecha-
: ~ 21.5 nism which is not detected in minicells. A similar
phenomenon has been reported concerning plas-
mids expressing Staphylococcus aureus protein A
[21] : normal E. coli cells contained two maior
polypeptides related to protein A with Mr 43,000
~14.3 and 41,000, whereas the corresponding gene pro-
duct synthesized in minicells appeared as a set of
polypeptides having sizes between 45,000 and
59,000 daltons.
FIG. 1. - - Gel electrophoresis o] purified endogluca-
nase B under native and denaturing conditions. Since even after two cycles of gel filtration, the
Lane I : 16 p~g enzyme (purified through the 2nd gel preparation was found to contain some impurities,
filtration step), electrophoresed on a 10 per cent poly- antisera were prepared by injecting crushed gel
acrylamide gel without SDS and mercaptoethanol and slices containing only the active bands.
stained with Coomassie brilliant blue R-250.
Lane 2: endoglucanase stain performed on a CMC- Identification o[ the celB gene product in
containing replica of lane 1 [18]. C. thermocellum : With concentrated C. thermo-
Lane 3 : 8 ~xg enzyme, heated for 3 min in sample cellum culture supernatant, antiserum raised
buffer containing 2 per cent SDS and 5 per cent mercapto- against endoglucanase B purified from E. coli gave
ethanol and electrophoresed on an SDS gel containing
10 per cent polyacrylamide. a precipitin line which fused with the line formed
Lane 4 : a crushed piece of non-denaturing gel contain- against an extract from an E. coli strain bearing
ing the active bands shown in lanes i and 2 was heated the celB gene. No reaction was observed with
in denaturing buffer for 45 rain at 60°C and 3 min at extracts of E. coli bearing the celA gene (fig. 2).
100°C. The sample was then electrophoresed on the same
gel as in lane 3. This result shows that endoglucanase B is indeed
Lane 5: molecular weight markers run on the same expressed by C. thermocellum and present in the
gel as samples of lanes 3 and 4 ; 92.5 : phosphorylase B ; culture medium.
69 : bovine serum albumin ; 46 : ovalbumin ; 30 : carbonic
anhydrase; 21.5: soybean trypsin inhibitor, 14.3: lyso- To identify the ceIB gene product made by
zyme. C. thermocellum, crude culture supernatant was
BIOCHIMIE, 1983, 65, n ° 8-9.
CelB gene-encoded endoglucanase of C. thermocellum. 499
9 2.5
~m69
;--4 6
30
~i ~ :~i
-::::
:::::
H,~H
21.5
Fro. 2. - - D o u b l e immunodi[Jusion tests.
Wells contained: A b : 100 ,~t anti-endoglucanase B
antiserum; 1 : 5 0 pxl (75 units) ammonium sulfate frac-
tion from E. coli H B I 0 1 (pCT207) ;, 2 : 1 5 0 ~xl (540 units) -- 14.3
concentrated C. t h e r m o c e l l u m culture supernatant; 3 :
100 ~1 (360 units) concentrated C, thermocellum culture :::2;:::::;
.... ?i :)i:
supernatant ; 4 : 50 ~1 (75 units) ammonium sulfate frac-
tion from E, coli HB101 (pCT105),