B I O C H I M I E , 1983, 65, 495-500.
Identification of the endoglucanase encoded by the celB gene
of Clostridium thermocellum.
Pierre BEGUIN <>, Philippe CORNET *
and Jacqueline MILLET.
(Recu le 29-6-1983, acceptd le 28-7-1983).
R6sum6.
L'endoglucanase codde par le gone celB de
Clostridium thermocellum a dtd puri[ide ~ partir
d'une souche d'Escherichia coli portant et expri-
m a n t l e gone de C. thermocellum clond dans te
plasmide pBR322. La prdparation obtenue prd-
sente deux bandes actives, d'une masse moldcu-
laire apparente de 55.000 et 53.000, vraisembla-
blement ddrivdes du produit de traduction pri-
maire par protdolyse. Un antisdrum spdcifique prd-
pard contre ces bandes a permis d'identi[ier l'anti-
gbne correspondant dans le surnageant de culture
de C. thermocellum : dans un test de double
immunodiJ[usion (Ouchterlony), on observe un arc
de prdcipitation [usionnant complOtement avec
celui [ormd par un extrait d'E. coli contenant
l'endoglucanase B exprimde ~ partir du gkne clond.
Par ailleurs, les protdines du surnageant de C. ther-
mocellum ont dtd sdpardes par dlectrophorbse sur
gel de polyacryIamide en prdsence de SDS et trans-
[&des sur une feuille de nitrocellulose. AprOs avoir
incubd le ddcalque de nitrocellulose en prdsence
d' antisdrum, puis de protdine A marquee
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.
Introduction.
Among the organisms that have been conside-
red for the industrial fermentation of celluIose,
Clostridium thermocellum, a thermophilic and
cellulolytic anaerobe, has recently raised an in-
creassed interest. It has a high ceUulolytic activity,
<> To whom all correspondence should be addressed.
* On leave /tom Solvay and Cie, Brussels, Belgium.
Unitd de Physiologic Cellulaire,
Ddpartement de Biochimie
et Gdndtique Moldculaire, Institut Pasteur,
75724 Paris Cedex 15, France.
Summary.
The endoglucanase encoded by the celB gene
of Clostridium thermocellum was purified from
an E. coli strain carrying and expressing the
C. thermocellum gene cloned in the plasmid
pBR322. The preparation showed two active
bands, with Mr 55,000 and 53,000, presumably
derived from the primary translation product by
proteolysis. Specific antiserum raised against these
bands was used to identify the corresponding anti-
gen in the culture supernatant of C. thermocellum :
in a double immunodi[fusion test (Ouchterlony),
a precipitin line was observed which [used comple-
tely with that formed by an E. coli extract contain-
ing endoglucanase B expressed from the cloned
gene. Proteins from C. thermocellum supernatant
were further analyzed by SDS-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose
sheet. After incubating the nitrocellulose blot with
antiserum and subsequently with le5I-labeled pro-
tein A, a band with Mr 66,000, corresponding to
the celB gene product expressed by C. thermocel-
lum, was detected by autoradiography.
Key-words : endoglueanase gene / C. thermocellum.
which is thermostable and is not inhibited by cello-
biose or glucose, the major products of cellulose
degradation [I, 2] ; furthermore, it is able to fer-
ment mono- or oligosaccharides derived from
cellulose and hemicellulose into economically va-
Abbreviations :
C M C : carboxymethylcellulose ; Tris : tris(hydroxy-
methyl)aminomethane ; SDS : sodium dodecyl sulfate.
 496 P. Beguin and coll.
l u a b l e p r o d u c t s such as e t h a n o l a n d acetic a c i d
[3-6]. F i n a l l y , its t h e r m o p h i l i c c h a r a c t e r is of
a d v a n t a g e for i n d u s t r i a l processes, p a r t i c u l a r l y in
limiting p r o b l e m s of o v e r g r o w t h b y c o n t a m i n a t i n g
organisms. F o r these reasons, C. t h e r m o c e l l u m
a p p e a r s to be a g o o d c a n d i d a t e for the d e v e l o p -
m e n t of processes in w h i c h cellulose w o u l d be
h y d r o l y z e d a n d f e r m e n t e d in a single step.
Little is k n o w n as y e t a b o u t the enzymes requi-
r e d for cellulose d e g r a d a t i o n in C. t h e r m o c e l l u m
culture m e d i u m . U n t i l now, only two e n d o -
glucanases (1,4-~-D-glucan-4-glucanohydrolase
E.C.3.2.1.4) c o r r e s p o n d i n g to p o l y p e p t i d e s with
M r 5 6 , 0 0 0 a n d 8 3 - 9 4 , 0 0 0 h a v e b e e n purified a n d
c h a r a c t e r i z e d f r o m C. t h e r m o c e l l u m strains
N C I B 1 0 6 8 2 a n d L Q R I , respectively [7, 8]. A n o -
ther e n d o g l u c a n a s e , with M r 9 1 - 9 9 , 0 0 0 , has also
b e e n i s o l a t e d f r o m a n o t h e r t h e r m o p h i l i c Clostri-
d i u m species [9].
R e c e n t l y , however, the screening of a gene b a n k
has l e d to the i s o l a t i o n of two genes f r o m C. ther-
m o c e l l u m expressing e n d o g l u c a n a s e activity in
Escherichia coli [10]. O n e of these genes, t e r m e d
c e l A , has b e e n s h o w n to code for a p r o t e i n which
cross-reacts with an a n t i s e r u m raised against the
M r 5 6 , 0 0 0 e n d o g l u c a n a s e , h e n c e f o r t h called e n d o -
g l u c a n a s e A.
T h e a i m of this w o r k was to detect a n d identify
the e n z y m e expressed b y the s e c o n d gene, t e r m e d
celB, a m o n g the m a n y different p r o t e i n s p r e s e n t
in C. t h e r m o c e l l u m c u l t u r e supernatant.
I n o r d e r to o b t a i n an i m m u n o l o g i c a l p r o b e ,
e n d o g l u c a n a s e B was p u r i f i e d f r o m an E. coli
strain c a r r y i n g the celB gene a n d used to p r e p a r e
a specific antiserum. T h e presence of m a t e r i a l
i m m u n o l o g i c a l l y i d e n t i c a l to E. coli-made endo-
g l u c a n a s e B was d e t e c t e d in c o n c e n t r a t e d culture
s u p e r n a t a n t of C. t h e r m o c e l l u m using O u c h t e r l o n y
d o u b l e i m m u n o d i f f u s i o n tests [11]. T o further
identify this c o m p o n e n t , crude C. t h e r m o c e l l u m
cellulase was s u b m i t t e d to p o l y a c r y l a m i d e gel
e l e c t r o p h o r e s i s f o l l o w e d by e l e c t r o p h o r e t i c trans-
fer to a nitrocellulose sheet. A b a n d r e a c t i n g with
a n t i - e n d o g l u c a n a s e B a n t i b o d i e s was then detected
b y i n c u b a t i n g the n i t r o c e l l u l o s e blot with anti-
s e r u m a n d s u b s e q u e n t l y with a25I-labeled p r o -
tein A f r o m S t a p h y l o c o c c u s aureus [12].
Materials and Methods.
Bacterial strains : C. thermocellum strain was
NCIB10682. E. coli strains were HB101 (pro leu thi
hsdR recA ~rt) harbouring plasmids pCT105 or pCT207,
BIOCHIMIE, 1983, 65, n ° 8-9.
which were derived from pBR322 [13] by insertion of
C. thermocellum DNA containing either the celA gene
(pCT105) or the celB gene (pCT207).
Growth conditions : E. coli was grown aerobically in L
broth [14] supplemented with 100 rag/1 ampicillin at
37°C. Ceils were harvested by centrifugation at the end
of the exponential growth phase. S. thermocellum was
grown anaerobically at 60°C in complete medium [7]
containing 10 g/1 MN 300 cellulose (Macherey, Nagel,
Diiren, W. Germany) and the culture supernatant har-
vested at the end of the growth phase.
Endoglucanase assay: Endo~[3-1,4-glucanase was assay-
ed by incubating the enzyme at 60°C in a 1.5 per cent
(w/v) solution of CMC (Sigma, medium viscosity) in
50 mM K~HPO~ -t- 12.5 mM citric acid, pH 6.3. At
defined times, aliquots were withdrawn from the incuba-
tion mixture and the appearance of reducing sugars was
assayed by the Somogyi-Nelson method [15]. One unit
of activity corresponds to the release of one micromole
glucose equivalent per hour.
Protein assay : The protein concentration was deter-
mined either by following the A ~ continuously or by the
Coomassie blue G-250 binding assay [16], using bovine
serum albumin as a standard.
Preparation of crude C. thermocellum cellulase : The
supernatent from a C. thermocellum culture grown in the
presence of cellulose was precipitated with ethanol and
cellulase was redissolved and dialyzed against 50 mM
K~HPO~ -t- 12.5 mM citric acid, p i t 6.3, as described
previously [7]. The preparation was further concentrated
15-fold by ultrafiltration on an Amicon PM 10 membrane.
Purification of endoglucanase B : Unless otherwise sta-
ted, all operations were performed at 0-4°C.
a) Preparation o/ crude extract : 50-100 g frozen bac-
terial cells of E. coli HB101 (pCT207) were resuspended
in 2.1 volumes of 40 mM Tris-HC1, pH 7.7, containing
15 jxg/ml DNase I (Worthington). Cells were disrupted
in an Aminco French pressure cell at 16,000 psi (110
MPa). The extract was diluted 5-fold in the same buffer
and cleared by centrifuging at 16,000 gmax for 15 min.
b) Streptomycin sulfate precipitation : 10 ml/g protein
of a 100 mg/ml streptomycin sulfate solution in 40 raM
Tris-HCl, pH 7.7 were slowly added while stirring.
After stirring for 45 min, the precipitate was removed
by centrifugation at 16,000 gmax for 15 min.
c) Heat precipitation : 500 ml samples of the strepto-
mycin-treated extract were transferred to 1 1 conical
flasks and heated with gentle swirling in a 60°C water
bath. 10 min after the temperature of the extract had
reached 60°C, the flasks were chilled in an ice bath and
the precipitated proteins were centrifuged at 16,000 gmax
for 15 min. Traces of contaminating pellet material were
removed from the supernatant by filtration through a
Whatman G F / C filter.
d) Ammonium sulfate precipitation : The enzyme was
precipitated by slowly adding 295 g powdered ammonium
sulfate per 1 of heat-treated extract while stirring. After
stirring for 45 rain, the precipitated protein was centri-
 C e l B gene-encoded endoglucanase of C. t h e r m o c e l l u m . 497

fuged at 16,000 gmax for 15 min, redissolved in 15-20 ml
of 40 m M Tris-HC1, pH 7.7, and dialyzed 3 times against
500 ml of the same buffer.
e) DEAE-Trisacryl chromatography : The dialyzed
sample was loaded on a 2.6 × 27 cm column of D E A E -
Trisacryl (LKB) equilibrated in 40 m M Tris-HC1, pH 7.7.
The resin was washed at 50 m l / h with the same buffer
containing 40 m M NaC1 until the A ~ returned to the
baseline. The column was then eluted at 20 m l / h with
a 360 ml gradient of 40 to 200 m M NaC1 in the same
buffer. Salt concentration was followed by monitoring
the conductivity of the fractions.
f) Ultrogel AcA 44 chromatography : Active fractions
from the D E A E column were pooled and concentrated
by ultrafiltration on an Amicon P M 10 membrane. The
sample was loaded on a 1.5 × 90 cm column of Ultrogel
A c A 44 (LKB) in 50 m M Tris-HC1, pH 7.0, and eluted
with the same buffer a t 8 m l / h . Active fractions were
concentrated by ultrafiltration and either submitted to a
second gel filtration on the same column or used as
such for preparative gel electrophoresis.
Polyacrylamide gel electrophoresis : SDS-polyacryla-
mide gel electrophoresis was performed according to
Laemmli [17]. The same system was used for native gel
electrophoresis, except that SDS and mercaptoethanol
were omitted and the samples were not heated.
Detection of bands having endoglucanase activity :
After native gel electroporesis, the gel was laid on top
of a 0.75 m m sheet of 2 per cent agar containing 0.1 per
cent C M C in 50 m M Na~HPO4 + 12.5 m M citric acid,
pH 6.3, and incubated for 30 min at 60°C. Zones of
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-
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
by forcing twice through an 18 gauge syringe needle.
Aliquots from the crushed gel containing 0.25 m g protein
were mixed with Freund's complete adjuvant and injec-
ted subcutaneously at 3 week intervals to New Zealand
white rabbits. Serum was collected after the third injec-
tion.
Immunological detection of endoglucanase B in crude
C. thermocellum culture supernatant : Double immuno-
diffusion tests were performed according to Ouchter-
lony [11]. To detect the b a n d corresponding to endoglu-
canase B after polyacrylamide gel electrophoresis, the
separated proteins were transferred from the gel to a
nitrocellulose sheet (Schleicher and Schtill B A 85) by
transverse electrophoresis for 3 h at 12 V / c m [12]. The
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
and 5 per cent bovine serum albumin to saturate remai-
ning protein binding sites. After briefly rinsing in
10 m M Tris-HCl, p H 7.4 + 154 m M NaC1, the blot
was incubated overnight on a rotatory shaker at 4°C
with the same buffer containing 5 per cent bovine serum
albumin and 2 per cent of either preimmune or anti-celB
antiserum. Excess immunoglobulins were washed by
rinsing the blot 10 times with 10 m M Tris-HC1 +
154 m M NaC1. The 5th and the 6th wash contained
0.05 per cent N P 40. After briefly equilibrating with
Dulbecco's PBS buffer [1'9] containing 3 per cent bovine
serum albumin and 0.02 per cent NaNs, the sheet was
incubated for 90 rain at room temperature o n a rotatory
shaker in the same buffer containing 2-3 ~Ci of protein
A from S. aureus, labeled with 1:251 to a specific activity
of 50 m C i / m g [20]. Excess radioactivity was washed
as described above for immunoglobulins. The blot was
briefy dried between paper towels, wrapped in plastic food
wrap and fluorographed for 3 h at - - 8 0 ° C with an
intensifying screen.
R~sults.
Purification of endoglucanase B : Y i e l d s a n d
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
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.
The relatively poor yield observed for the amino-

TABLE I.
Purification of endoglucanase B of E. coli HBIO1 (pCT207).

Volume Total protein Total activity Specific Yield

Steps (ml) (mg) (units) activity
(units/mg) %

Crude extract 1,070 9,920 74,400 7.5 100

Streptomycin sulfate supernatant 1,050 7,080 61,200 8.6 82
Heat-treated extract 980 1,510 40,800 27 54
A m m o n i u m sulfate precipitate (dialyzed) 26 227 13,200 58 18
Pool from D E A E column 23 12.3 8520 690 11.4
Pool from 1st Aca 44 column (concentrated) 2 2.45 6120 2,480 8.2
Pool from 2rid Aca 44 column (concentra-
ted) 2 1.25 4440 3,570 6

75 g frozen cells were processed as described in Materials and Methods.

BIOCHIMIE, 1983, 65, n ° 8-9. 36
498 P. Beguin and coll.
nium sulfate fractionation seems to be due to in-
activation : analysis of the a m m o n i u m sulfate-con-
taining supernatant after dialysis showed that little
I 2 3 4 5 dure.
J92.5
~~ , 69
~46
W
H
~ m 3 0
: ~ 21.5
~14.3
FIG. 1. - - Gel electrophoresis o] purified endogluca-
nase B under native and denaturing conditions.
Lane I : 16 p~g enzyme (purified through the 2nd gel
filtration step), electrophoresed on a 10 per cent poly-
acrylamide gel without SDS and mercaptoethanol and
stained with Coomassie brilliant blue R-250.
Lane 2: endoglucanase stain performed on a CMC-
containing replica of lane 1 [18].
Lane 3 : 8 ~xg enzyme, heated for 3 min in sample
buffer containing 2 per cent SDS and 5 per cent mercapto-
ethanol and electrophoresed on an SDS gel containing
10 per cent polyacrylamide.
Lane 4 : a crushed piece of non-denaturing gel contain-
ing the active bands shown in lanes i and 2 was heated
in denaturing buffer for 45 rain at 60°C and 3 min at
100°C. The sample was then electrophoresed on the same
gel as in lane 3.
Lane 5: molecular weight markers run on the same
gel as samples of lanes 3 and 4 ; 92.5 : phosphorylase B ;
69 : bovine serum albumin ; 46 : ovalbumin ; 30 : carbonic
anhydrase; 21.5: soybean trypsin inhibitor, 14.3: lyso-
zyme.
BIOCHIMIE, 1983, 65, n ° 8-9.
activity could be recovered from this fraction.
However, attempts to omit the ammonium sulfate
precipitation led to poor adsorption and resolu-
tion on the subsequent anion exchange column and
this step was thus kept in the purification proce-
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-
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-
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-
naturing conditions shows an identical doublet
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
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-
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
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.
Since even after two cycles of gel filtration, the
preparation was found to contain some impurities,
antisera were prepared by injecting crushed gel
slices containing only the active bands.
Identification o[ the celB gene product in
C. thermocellum : With concentrated C. thermo-
cellum culture supernatant, antiserum raised
against endoglucanase B purified from E. coli gave
a precipitin line which fused with the line formed
against an extract from an E. coli strain bearing
the celB gene. No reaction was observed with
extracts of E. coli bearing the celA gene (fig. 2).
This result shows that endoglucanase B is indeed
expressed by C. thermocellum and present in the
culture medium.
To identify the ceIB gene product made by
C. thermocellum, crude culture supernatant was
 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),

FIG. 3. - - D e t e c t i o n oJ the CeIB gene p r o d u c t oJ C.

analyzed by SDS-polyacrylamide gel eIectropho-
resis and the proteins transferred to nitrocellulose.
Material cross-reacting with endoglucanase B was
detected by incubating the blot with anti-celB pro-
tein antiserum followed by lz~I-labeled protein A
from S. aureus. A strong radioactive band with
Mr 66~000 was detected with immune serum
(fig. 3). For unknown reasons, a slight band of
similar molecular weight was also seen with con-
trol serum ; its intensity was however much redu-
ced as Compared to the band observed with
immune serum.
thermocellum.
5 ~1 (18 units) concentrated C. t h e r m o e e l l u m culture
supernatant were denatured for 5 rain at 100°C in sample
buffer containing 2 per cent SDS and 5 per cent mercapto-
ethanol, electrophoresed on an SDS gel containing
10 per cent polyacry!amide and submitted to the immuno-
lagical detection procedure described in Materials and
Methods.
L a n e 1 : blot treated with preimmune serum.
Lane 2 : blot incubated with anti-endoglucanase B anti-
serum. Molecular weight markers were the same as in
figure 1.

nism, and in coming back subsequently to its ori-

ginal source to identify it by immunological 'tech-
Discussion. niques. This was motivated by the fact that cloning
and spontaneous expression of the ceIB gene in
E. coli were achieved before any antiserum was
A somewhat unusual approach was followed in available to correlate it with a known enzyme of
this work, which consisted in purifying an enzyme C. thermocellum, as had been the case for the celA
after cloning and expressing it in a foreign orga- gene [10]. An E. coli strain bearing the cloned
BIOCHIMIE, 1983, 65, n ° 8-9.
500 P. Beguin and coil.
gene was chosen as a source to purify the antigen
because it was the only starting material for which
endoglucanase activity corresponded exclusively
to the ceIB gene product. Whether endogluca-
nase B was present in C. thermocellum culture
supernatant was not known and, at any rate, the
presence of a variety of endoglucanases [18]
would have made difficult to guess which of them
was encoded by the celB gene. Furthermore, the
purification was greatly facilitated by the absence
of high molecular weight aggregates like those
formed by C. thermocellum extracellular proteins
[1, 7, 22].
Endoglucanase B appears to be a new enzyme,
not related to endoglucanase A nor to the endo-
glucanase purified by Ng and Zeikus from C. ther-
mocellum L Q R I [7, 8], which has a higher mole-
cular weight. Previous studies of extracts of E. coli
strains expressing the celB gene demonstrated that
the enzyme strongly decreases the viscosity of
C M C solutions, with little release of reducing
sugars, a feature typical of endoglucanases [10].
It shows a reduced activity toward trinitrophenyl-
ated CMC, as compared to endoglucanase A [10].
Very little degradation of native cellulose could
be detected, even when extracts containing endo-
glucanases A and B were mixed together (unpu-
blished data). Additional components, possibly
including an exoglucanase, are thus required for
the hydrolysis of native cellulose. This conclusion
parallels results of Johnson and Demain [23. 24],
showing that. while degradation of CMC by
C. thermocellum supernatant was achieved by
endoglucanases not affected by aerobic conditions,
full activity toward microcrystalline cellulose
(Avicel) required the activity of another compo-
nent bearing oxidation-sensitive SH groups.
The availability of a specific antiserum should
be helpful for the purification of the C. thermo-
cellum enzyme, since it provides a means to iden-
tify it unambiguously and possibly to isolate it by
immunoaffinity chromatography. It would then be
possible to determine its N-terminal sequence and
to compare it with the D N A sequence of the celB
gene, in order to study the processing of extra-
cellular proteins in C. t h e r m o c e l l u m ; further-
more, catalytic properties would be best studied
in detail with an intact enzyme and not with
E. coil-synthesized endoglucanase, which lacks
part of the original molecule.
BIOCHIMIE, 1983, 65, n ° 8-9.
Acknowledgments.
The authors w&h to thank Mrs. M. Rocancourt ]or
her expert technical help, Mr, G. Ldp£e [or growing
the strains in ]ermentors, Dr. M. Kaminsk ]or preparing
the antiserum, Dr. N. Guiso-Maclou[ for gifts o/
l~51-labeled protein A, Dr. J.-P. Aubert ]or helpJul dis-
cussions and Drs. Johnson and Demain for communica-
ting results prior to publication. This work was supported
by research ]unds ]rom University Paris 7 and by a
research contract from SoIvay and Cie, Brussels, Belgium,
and Rh6ne-Poulenc Recherches, Paris, France.
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