A bifunctional

␤-xylosidase-xylose isomerase from

Streptomyces sp. EC 10

Najia Belfaquih, Michel J. Penninckx*

Laboratoire de Physiologie et Ecologie Microbienne, Section Interfacultaire d’Agronomie, Universite´ Libre de Bruxelles, c/o Institut Pasteur,

642 rue Engeland, B-1180 Brussels, Belgium

Received 24 June 1999; received in revised form 24 Jan 2000; accepted 15 February 2000

Abstract

␤-Xylosidase (1,4-␤-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by

Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form
of carbon catabolite repression by glycerol.

␤-Xylosidase and xylose isomerase copurified strictly, and the preparation was found

homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp.
produced apparently a bifunctional

␤-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be

163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The
Streptomyces sp.

␤-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5

and 45°C. The xylose isomerase optimal temperature was 70°C and maximal activity was observed in a broad range pH (5– 8). Enhanced
saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35
amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced
by other microorganisms but not with other published N-terminal sequences of

␤-xylosidases. © 2000 Elsevier Science Inc. All rights

reserved.

1. Introduction

Xylans are major hemicellulose components of lignocel-

lulose biomass [1]. Several enzymes are required for com-
plete hydrolysis and assimilation of xylans, including
␤-endoxylanase, ␤-xylosidase, and xylose isomerase.

␤-Xylanase received the broader attention among these en-
zymes [2].

␤-Xylosidase (1,4-␤-D-xylan xylohydrolase, EC

3.2.1.37) hydrolyzes xylooligosaccharides, is essential for
complete saccharification of xylan, and has been purified
from fungi [3–5] and some bacteria [6 –9].

␤-Xylosidase

was detected in actinomycetes but deserved little attention,
except in the case of Thermomonospora fusca [10].

Xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5)

catalyzes the reversible isomerization of D-xylose to D-
xylulose in the first step of xylose metabolism following the
pentose phosphate cycle. It also catalyzes the isomerization
of glucose to fructose. Therefore, it is used industrially in

the production of high fructose syrup under the name glu-
cose isomerase [11]. The enzyme has been isolated from
many microorganisms and has been well studied [12].
Genes from Escherichia coli [13], Bacillus subtilis [14],
Clostridium species [15,16], Streptomyces species [17,18],
Ampullariella species [19] and Actinoplanes missouriensis
[20] have been sequenced.

Actinomycetes are recognized as dominant xylanolytic

species during several processes of biomass transformation
[21], and their enzymes may find new applications in the
pulp and paper industry [22,23] and in the recovery of
fermentable sugars from hemicelluloses [24].

The present paper reports on the regulation of production

and on the characterization of purified Streptomyces sp.
␤-xylosidase-xylose isomerase enzyme. The strain chosen
for purification was previously selected as an efficient de-
grader of the hemicellulosic fraction of straw [25] and
produces also three typical non-debranching endo-

␤-xy-

lanases [26]. Present data concluded that

␤-xylosidase

and xylose isomerase are produced under the form of
a single bifunctional protein enzyme in Streptomyces
sp. EC 10.

* Corresponding author. Tel.:

⫹0032-2-373-3301; fax: ⫹0032-2-373-

3309.

E-Mail address: [e-mail] (M.J. Penninckx).

www.elsevier.com/locate/enzmictec

Enzyme and Microbial Technology 27 (2000) 114 –121

0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
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2. Materials and Methods

2.1. Cultures and media

Streptomyces sp. EC 10 was described in [26].
Before use, the Streptomyces sp. strains were maintained

either frozen at

⫺80°C in 80% (w/v) glycerol or at 4°C as

1-week-old slant cultures on Difco (West Mosley, Surrey,
UK) actinomycetes isolation agar.

Spore suspensions prepared from the slants were resus-

pended in 0.9% NaCl and used for inoculation of the culture
medium. The basal medium without carbon source was
described in [26].

In induction experiments, 20 ml of spores suspension (10

mg dry weight) were first inoculated into 200 ml of the
culture medium containing 0.2% glycerol as the carbon
source. After 48 h of growth, the cells were harvested by
filtration on a Millipore 0.45

␮m membrane under sterile

conditions, washed with 100 ml of water, and inoculated
into 400 ml of the tested induction medium [27].

For enzyme purification, about 100 mg dry weight of

pregerminated spores of the Streptomyces sp. EC 10 strain,
in 30 ml were inoculated into 1 l of basal culture medium
supplemented with 0.2% (w/v) glycerol, contained in a 3-l
flask, and incubated for 36 h in an orbital shaker (154
strokes. min

⫺1

) at 30°C.

This preculture was added to 7 l of fresh medium in a

15-l Applikon bioreactor (Applitek, Deinze, BE). The cells
were cultivated for 48 h at 100% air saturation and agitation
rate of 250 rpm. After harvesting by sterile centrifugation at
10 420

⫻ g, the pellets were reinoculated into 2 l of basal

medium supplemented with 0.2% oat spelt arabinoxylan
(Sigma, St Louis, Mo, USA). Enzyme induction was per-
formed for 48 h in a 3-l Applikon bioreactor maintained at
100% air saturation and an agitation rate of 250 rpm. The
pellets were harvested by centrifugation and used as an
enzyme source for purification.

2.2. Enzyme purification

The pellets obtained after induction as described above

were resuspended in 40 ml of 10 mM K-phosphate pH 7.0
and sonicated with cooling at 4°C for 15 min with a Vibra
Cell VC 500 ultrasonic processor (Sonics Materials).

The suspension obtained was clarified by centrifugation

at 12 000

⫻ g for 10 min and the supernatant (i.e. cell

lysate) used for further operations.

2.2.1. Anion-exchange chromatography

The cell lysate was layed down on a 2.5

⫻ 45 cm column

packed with DEAE-Sephacel anion exchange resin (Phar-
macia, Belgium), previously equilibrated with 20 mM K-
phosphate buffer, pH 7.0, at 4°C. The column was first
washed with 500 ml of the equilibration buffer and the
proteins were eluted with a linear gradient of 0 –1 M NaCl
in 20 mM K-phosphate buffer, collecting 4-ml fractions.

Active fractions were pooled for further purification, dia-
lyzed overnight, and concentrated to about 5 ml in an
Amicon cell (Amicon, Beverly, MA, USA) using a PM 10
membrane.

2.2.2. Gel filtration

␤-Xylosidase and xylose isomerase were further chro-

matographed on a Biogel A (Bio-Rad Laboratories) column
(1.5

⫻ 45 cm) previously equilibrated with 50 mM K-

phosphate, pH 7.0. Filtration was done at the rate of 0.4 ml.
min

⫺1

. The active fractions were recovered, ultrafiltered to

about 10 ml as indicated above and kept at

20°C for prolonged storage. All purification steps were

performed at 4°C.

2.3. Enzyme activites and protein assays

2.3.1.

␤-Xylosidase assay

␤-Xylosidase assay was based on the release of p-nitro-

phenol from p-nitrophenyl-

␤-D-xylopyranoside (Sigma).

Appropriately diluted samples were incubated at 45°C with
20 mM substrate in the presence of 50 mM K-phosphate
buffer pH 7.0.

The reaction was stopped by addition of 100 mM NaOH-

glycine pH 10.9, and absorbance of the released p-nitrophe-
nol was immediately measured at 410 nm. Activity units (U)
are

␮mol p-nitrophenol. min

⫺1

.

2.3.2. Xylose-isomerase assay

The formation of D-xylulose from D-xylose was mea-

sured using the colorimetric assay as described in [28]. A
test mixture of 20

␮l, containing 0.1 M K-phosphate buffer

pH 7.0, 0.2 M D-xylose, and 0.4 M MnSO

4

, was incubated

together with 20

␮l of the enzyme solution for 10 min at

50°C. After this period the samples were put on ice to stop
the enzyme reaction and 40

␮l of 1.5% (w/v) cysteine

hydrochloride in water, 40

␮l of 0.12% (w/v) carbazole in

ethanol, and 1.2 ml of 70% (v/v) sulfuric acid were added.
After standing for 10 min the absorbance at 540 nm was
determined. The concentration of D-xylulose was deter-
mined from a standard curve of D-xylulose. One unit (U) of
enzyme activity is equal to the formation of one

␮mol

D-xylulose per min at 50°C.

In induction experiments, the culture medium to be an-

alyzed was first centrifuged as indicated above for enzyme
purification. The pellet was resuspended in 3 ml of 20 mM
K-phosphate pH 7.0 and lysed by sonication as described
above.

␤-Xylosidase and xylose-isomerase were assayed in

the cell lysate.

2.3.3. Protein

The protein contents were measured by the procedure

of Bradford [29] using BSA Cohn fraction V (Sigma) as
standard.

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N. Belfaquih, M.J. Penninckx / Enzyme and Microbial Technology 27 (2000) 114 –121

2.4. Electrophoresis procedures

PAGE-SDS was carried out with a Bio-Rad apparatus

(Mini Protean II) and a 10 –20% gradient polyacrylamide
gel, using Coomassie Blue staining for detecting protein
bands. Protein markers (Bio-Rad Laboratories) in the
14 000 –97 000 molecular weight range were used for char-
acterization of

␤-xylosidase-xylose isomerase enzyme.

Analytical isoelectric focusing (IEF) was carried out

using the Phast system and Phast Gel IEF 3–9 (Pharmacia).

2.5. End-product determination

The purified

␤-xylosidase (5 U 䡠 ml

⫺1

) was incubated

with different xylose oligomers solubilized at 10 mg

䡠 ml

⫺1

in 100 mM K-phosphate buffer pH 7.5, at 40°C. The sam-
ples prelevated during the course of the reaction were ana-
lyzed by HPLC using a Aminex Carbohydrate HPX-87 P
column (Waters Associates-Millipore-Benelux), equili-
brated and eluted with double distilled water. Peaks were
detected and quantified by differential refractometry [26].

2.6. Experiments on cooperative enzyme action

Purified enzyme preparations were assessed for their

contribution to xylan hydrolysis. Oat spelt arabinoxylan (50
mg) was placed in a conical flask with 3 U of purified
xylanases (XI, XII and XIII isoenzymes) and 3 U of

␤-xy-

losidase. Flasks were also set up with single enzymes as
controls. All the flasks were shaken at 50°C for 2 h and
samples of 250

␮l were removed at 15-min intervals and

assayed for the release of reducing sugars by the procedure
of Miller [30].

2.7. N-Terminal sequence

Purified

␤-xylosidase-xylose isomerase separated by

SDS-PAGE was blotted onto a PVDF-Membrane with a
Mini trans-Blot cell (Bio-Rad). The amino terminus was
sequenced with the Applied Biosystems 477 A gas-phase
protein sequencer.

2.8. Reproducibility of results

Only experiments are reported that were repeated at least

twice. Standard deviations were all within 10% of the mean
values presented.

3. Results and Discussion

3.1. Induction and repression pattern of

␤-xylosidase and

xylose isomerase

In preliminary experiments, it was found that

␤-xylosi-

dase and xylose isomerase from Streptomyces sp. EC 10

were exclusively associated with the cell pellet obtained
after centrifugation of the different culture medium tested.
This was also previously observed for

␤-xylosidase in other

Streptomyces sp. strains [28] and Thermomonospora fusca
[31], and in Thermoanaerobacter ethanolicus [32] and other
microorganisms for xylose isomerase.

Both activities were apparently strongly induced by xy-

lan and straw, but not by CMC (Table 1). The enzyme
activity peaked after 2 days of induction and declined there-
after (not illustrated). The decline was associated with a
cellular lysis shown by the fact that a large proportion of
3-day-old and more cells no longer retained the Gram stain.
␤-Xylosidase and xylose isomerase were also induced by
xylose, the component sugar of xylan. No induction oc-
curred when cells were grown on fructose, glucose or ga-
lactose, or aromatic residues found in lignocellulose
(vanillic and ferulic acids). When glycerol was present in
the media used for the induction experiments (Table 1), the
specific activity of both activities was much lower than in
media not supplemented with glycerol. No activity was
detected when growing the cells with glycerol alone. This
effect is consistent with a form of catabolite repression
mediated by glycerol.

3.2. Enzyme purification and estimation of purity

␤-Xylosidase and xylose isomerase were found to copu-

rify and eluted from DEAE-Sephacel column in the same
fractions (Fig. 1A). The peak fractions were combined and
subjected to a gel filtration after what the two enzymes
coeluted also in the same fractions (Fig. 1B).

The results of purification are summarized in Table 2.

The overall activity recovery was 15.07% for

␤-xylosidase

Table 1
␤-Xylosidase and xylose isomerase induction in Streptomyces sp EC10
and effect of glycerol. Enzyme activities was induced for 48 h with
0.2% substrate in the absence or (where shown) in the presence of 0.2%
glycerol as described in “Materials and methods”. The enzyme activity
was expressed as U.(mg protein)

⫺1

; bacterial protein was determined by

Kjeldahl analysis of total N in the culture solids. Experiments were done
in triplicate; standard deviations were all with 10% of the mean values
presented. ND: not detectable

Carbon sources

␤-Xylosidase
(U.mg

⫺1

protein)

Xylose isomerase
(U.mg

⫺1

protein)

Birchwood xylan

43.93

8.78

Oat-Spelt xylan

30.06

6.65

Straw

21

5.2

Xylose

16.42

5.2

Xylan

⫹ Glycerol

1.5

0.4

Glycerol

ND

ND

CMC

ND

ND

Fructose

ND

ND

Glucose

ND

ND

Galactose

ND

ND

Vanillic acid

ND

ND

Ferulic acid

ND

ND

116

N. Belfaquih, M.J. Penninckx / Enzyme and Microbial Technology 27 (2000) 114 –121

and 15.08% for xylose isomerase. Only one protein band
was confirmed by PAGE (not illustrated) and SDS-PAGE
(Fig. 2). The purified protein migrated also as one band of
pI 4.5 on IEF gel electrophoresis (not illustrated). These

data supported the hypothesis that

␤-xylosidase and xylose

isomerase activities are produced under the form of a single
bifunctional enzyme.

3.3. Molecular characteristics of

␤-xylosidase

and xylose isomerase

The pI of the purified

␤-xylosidase-xylose isomerase was

4.5 as deduced from IEF gel electrophoresis (not illustrat-
ed). The molecular weight of the native bifunctional en-
zyme, measured by gel filtration chromatography, on Biogel
A was 163 000. In the presence of SDS, the native enzyme
dissociated into subunits of 42 000 (Fig. 2), in accordance
with a plausible tetrameric structure. The estimated molec-
ular weight of the Streptomyces sp. native

␤-xylosidase is

within the range reported for other bacterial enzymes [10,
33,34]. Molecular weights ranging from 26 000 to 250 000
have been reported for fungal

␤-xylosidases [2], which

Fig. 1. Purification of intracellular xylosidase (‚) and xylose-isomerase (E)
activities from Streptomyces sp. EC 10. (

䡺) protein at 280 nm: A, DEAE-

Sephacel anion exchange chromatography; B, gel filtration chromatography.

Table 2
Summary of purification of

␤-xylosidase and xylose isomerase from Streptomyces sp. EC10

Purification steps

Crude extract

DEAE-Sephacel

Biogel A

Xylosidase

Xylose-isomerase

Xylosidase

Xylose-isomerase

Xylosidase

Xylose-isomerase

Total activity (U)

8287.5

1683.75

1614

336.75

1249

254

Protein (mg)

272.25

272.25

13.20

13.20

4.06

4.06

Specific activity (U/mg)

30.44

6.18

122.27

25.50

308

62.63

Yield (%)

100

100

19.50

20

15.07

15.08

Purification factor

1

1

4

4.1

10

10.1

Fig. 2. SDS-PAGE (Coomasie-stained) of the

␤-xylosidase-xylose isomer-

ase from Streptomyces sp. EC 10. Marker proteins, lane 1; purified xylo-
sidase-xylose isomerase, lanes 2 and 3 (7

␮g protein was applied in Lane

2 and 5

␮g in Lane 3). Molecular mass of marker proteins in kDa is shown

on the left.

117

N. Belfaquih, M.J. Penninckx / Enzyme and Microbial Technology 27 (2000) 114 –121

behaved as monomeric [5], dimeric [35], or tetrameric spe-
cies.

Bacterial

␤-xylosidases were reported either as an ho-

modimer of subunits having a molecular weight of 65 000 –
70 000 for Bacillus pumilus [36] or an heterotrimer, com-
prising one subunit of 63 000 and two of 85 000 in the case
of Clostridium acetobutylicum [7].

The T. fusca enzyme was assumed to be a trimer of

identical subunits [10]. Clostridium cellulolyticum produced
a

␤-xylosidase with a molecular weight of 43 000 as deter-

mined by SDS-PAGE electrophoresis [8].

3.4. Catalytic properties of the enzyme

The Streptomyces sp. EC 10

␤-xylosidase activity

showed a rather sharp pH optimum of 7.5 with practically
no activity below 6.0 and above 9.0 (Fig. 3A). It was also
found that this activity was stable at room temperature for at
least 48 h in the 5.0 to 8.0 pH range (Fig. 3A). Temperature
optimum was 45°C (Fig. 4A), the enzyme retaining only
20% of activity at 55°C. The enzyme retained 75% of its
activity when incubated at 45°C for 2 h, but only about 30%
survived at 60°C (Fig. 4A). The enzyme K

m

for pNPX was

calculated by using the substrate at concentration of 1 to 20
mM at 45°C and pH 7.5. Under these conditions, the en-
zyme obeyed strict Michaelian conditions with R

2

values of

more than 0.998 in Lineweaver-Burke double reciprocal
plots (not illustrated). The apparent K

m

of the purified

␤-xy-

losidase for pNPX was 13.5 mM (SD 0.01; N

⫽ 3).

The Streptomyces sp. EC10

␤-xylosidase exhibited com-

parable thermostability to other fungal and bacterial

␤-xy-

losidases but lower than the enzyme produced by the ther-
mophilic actinomycete T. fusca [10]. The T. fusca enzyme
exhibited also activity across a relatively broad pH range
(5.0 to 9.0), which was not the case for the Streptomyces sp.
and Bacillus pumilus [6]

␤-xylosidases, characterized by a

sharper pH optimum.

The T. fusca enzyme showed a higher affinity for the

pNPX substrate than the Streptomyces sp. EC10 enzyme,
with an apparent K

m

value of 0.89 mM [10].

The xylose isomerase activity showed maximum activity

at pH 7.0 and retained a high percentage of activity over a
wide pH range (Fig. 3B). It has also an optimum tempera-
ture at 70°C, is stable between 30 and 70°C and conserved
70% of its activity when incubated at 70°C during one hour
(Fig. 4B).

The size of Streptomyces sp. EC 10 purified protein is in

the range reported for the few bacterial

␤-xylosidases de-

scribed, but the acidic nature of the protein has more in
common with some of the monomeric

␤-xylosidases de-

scribed in fungi.

The purified enzyme hydrolyzed pNPX and had no de-

tectable endo-xylanase,

␣-arabinofuranosidase, endoglu-

canase or

␤-glucosidase activities, even after overnight in-

cubation.

Liberation of xylose (X) from xylobiose (X

2

) (Fig. 5A)

and xylotriose (X

3

) (Fig. 5B) was detected. Xylobiose ac-

cumulated transiently during xylotriose hydrolysis. This
identifies the

␤-xylosidase produced by Streptomyces sp.

EC 10 as a typical glycosidase which split xylobiose and
acted as an exoenzyme on xylotriose and presumably on
longer xylodextrins.

3.5. Synergy experiments

Purified

␤-xylosidase alone, exhibited a very limited

saccharifying activity on arabinoxylan, but enhanced sig-
nificatively the action of endoxylanases of the Streptomyces

Fig. 3. Effect of pH on stability (F) and activity (Œ) of purified

␤-

xylosidase and xylose isomerase. The pH stability was measured from the
residual activity after incubation in the buffers with indicated pHs at room
temperature for 2 h. The effect of pH on activity was examined after
incubation of the

␤-xylosidase with pNPX in the buffers with indicated

pHs at 45°C for 10 min and the xylose isomerase activity was examined
after incubation of xylose isomerase with xylose in buffers with indicated
pHs at 50°C for 10 min. A,

␤-xylosidase; B, xylose isomerase.

118

N. Belfaquih, M.J. Penninckx / Enzyme and Microbial Technology 27 (2000) 114 –121

sp. EC 10 strain, above the theoretical additive effect (Fig.
6). This observation agreed with a role for

␤-xylosidase in

hydrolyzing short oligosaccharides inhibitory to endoxyla-
nase activity.

Synergy between enzyme components has been exten-

sively studied for fungal cellulases [37], but few reports
have appeared concerning xylanolytic enzymes. Biely and
coworkers suggested a synergy between fungal acetyl es-

terase and endo-

␤-xylanases [38]. Bachmann and McCarthy

[31] reported some cooperative action between xylanolytic
components of T. fusca. A much clearer understanding of
synergy between xylanolytic enzymes has still to emerge,
and will probably appear considerably more elaborate than
in the case of cellulases due to the more complex nature of
xylan substrate as compared to cellulose.

3.6. N-Terminal sequence

The amino-terminal amino acid sequence of the purified

enzyme was determined and only one sequence was ob-
served:

Fig. 4. A, effect of temperature on stability (F) and activity (Œ) of purified
␤-xylosidase. For stability, the enzyme solution in K- phosphate buffer (50
mM, pH 7.0) was incubated for 1 h at various temperatures, and then the
residual activity was assayed. For activity, the enzyme activity was assayed
at various temperatures by the standard assay method. B, effect of tem-
perature on stability (F) and activity (Œ) of purified xylose-isomerase. For
stability, the enzyme solution in K-phosphate buffer (100 mM, pH 7.0) was
incubated for 1 h at various temperatures, and then the residual activity was
assayed. For activity, the enzyme activity was assayed at various temper-
atures by the standard assay method.

Fig. 5. Pattern of hydrolysis of xylobiose and xylotriose by Streptomyces
sp. EC 10

␤-xylosidase. A, xylobiose; B, xylotriose. The xylodextrins were

incubated in the presence of

␤-xylosidase as indicated in Section 2. X,

xylose; X

2

, xylobiose; X

3

, xylotriose.

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N. Belfaquih, M.J. Penninckx / Enzyme and Microbial Technology 27 (2000) 114 –121

Ser-Tyr-Gln-Pro-Thr-Pro-Glu-Asp-Lys-Phe-Thr-Phe-

Gly Leu-Try-Thr-Val-Gly Try-Gln-Gly Arg-Asp-Pro-Phe-
Gly Asp-Ala-Thr-Arg-Gly Ala-Leu-Asp-Pro.

It is worthy of note that this is the only

␤-xylosidase

sequence determined in Streptomyces species. Moreover,
this sequence shows no homology with

␤-xylosidase from

other microorganisms, but the similarity is found toward
some xylose isomerase sequences. The Streptomyces sp. EC
10 amino acid sequence shows 91% identity with a xylose
isomerase produced by Streptomyces sp. SK strain, 80%
identity with a xylose isomerase from Actinoplanes missou-
riensis, 73% identity with a D-xylose ketol-isomerase from
T. thermophilus. Other similarities were found with Strep-
tomyces rubiginosus, Streptomyces diastaticus, Streptomy-
ces violaceoniger, and Arthrobacter sp. [39]. No

␤-xylosi-

dase activity was apparently investigated for these purified
xylose isomerase enzymes.

In Trichoderma reesei [40], Butyrivibrio fibrisolvens

[41] and Thermoanaerobacter ethanolicus [34] the purified
␤-xylosidase showed ␣-arabinofuranosidase activity and
both activities copurified strictly as shown here for

␤-xylo-

sidase-xylose isomerase. A molecular genetic study in Bac-
teroides ovatus [42] reported that

␤-xylosidase and ␣-ar-

abinofuranosidase were encoded by the same DNA region
within a cluster of genes involved in hemicellulose degra-
dation. A bifunctional xylanase-

␤-xylosidase has also been

cloned in Caldocellum saccharolyticum [43].

Our data conclude that at least in the Streptomyces genus,

␤-xylosidase and xylose isomerase are produced under the
form of a single bifunctional protein enzyme. It is plausible
to conclude that bifunctional enzymes are more common in
microbial hemicellulose degradation than previously ob-
served. This would be underlied by the fact that biomass
degrading enzymes act often in a synergy relationship.

Moreover,

the

Streptomyces

sp.

␤-xylosidase-xylose

isomerase could be used for producing xylulose, the first
metabolic intermediate in the xylose assimilatory pathway.

Acknowledgments

This work was supported by the EEC Non Nuclear En-

ergy, and by a personnal grant of the Belgian National Fund
for Scientific Research (FNRS) to M.P.N.B. was supported
by studentship from Marocco and the Van Buren fondation.

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