ELSEVIER

Hydrolysis of wheat straw by a

thermostable endoxylanase:
Adsorption and kinetic studies
Caroline Zilliox and Philippe Deheire
Unite de Physicochimie et de Biotechnologie des PolymPres, Institut National de la Recherche
Agronomique, Villeneuve d Ascq Ckdex, France
The adsorption of a purified 20.7 kDa thermostable endo-1-4-P-xylanase (EC 3.2.1.8) from a Bacillus sp. on
wheat straw at 4C was studied. Adsorption data fitted the Langmuir-type adsorption isotherm with the maximum
amount of adsorbed xylanase being 521 kg protein g- straw. Adsorption of the xylanase on straw, h&tin, and
insoluble xylans was irreversible at 4C. The extent of hydrolysis was quantified by the measurement of total
neutral sugars liberatedfrom wheat straw-xylanase complexes at 60C. Maximum hydrolysis was observed using
350 pg enzyme g- straw and reached 11% of the xylans in the straw after 5 h of reaction. No proportionality
could be found between the level of xylanase adsorption on straw and the extent of hydrolysis at 60C. Adsorption
and hydrolysis experiments indicated that all the bound xylanase was not hydrolytically active. This suggested
that nonspec$c adsorption occurred on lignin. Analysis of the end products of the reaction indicated that xylose
and neutral and uranic acid-containing xylo-oligosaccharides were the major compounds. 0 1998 Elsevier
Science Inc.

Keywords:

Endoxylanase;

Bacillus sp.; wheat-straw

xylans; enzyme adsorption

Introduction
Agricultural
residues such as wheat straw represent large
renewable
resources for lignocellulosic
bioconversion.
Wheat straw is a widely available substrate. Its disposal
presents an environmental
problem. Transformation
of
this agricultural
by-product
is therefore desirable. Bioconversion of wheat straw is favored by its relatively low
lignin content (<20%, w/w),* and high content (up to
20%, w/w) of xylans.*T3 Equally, wheat straw could serve
as a source of pulp for papermaking
with the pretreatment
of wheat straw by ligninolytic
enzymes and endoxylanases lowering the energy requirement
for pulp preparation.4
The cell walls of forage crops are distinguished by two
notable characteristics.
Xylans are mainly in the form of
arabinoglucuronoxylans
and hydroxycinnamic
acids are
present. They are associated with the lignin via ester and
ether bondings. 5-7 For the entire degradation of xylans,

Address reprint requests to Dr. P. Debeire, INRA, Fractionnement

Enzymatique BP 1039, CPCB, Moulin de la House, 51687 Reims cedex 2,
France
Received 11 December; revised 19 March 1997; accepted 3 June 1997

Enzyme and Microbial Technology 22:58-63,

1998
@ 1998 Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010

many enzymes are implicated:

endoxylanases,
B-xylosidases [EC 3.2.1.371, o-arabinofuranosidase
[EC 3.2.1.551,
o-glucuronosidase
[EC 3.2.11, acetyl-esterase [EC 3.1.1.61,
feruloyl, and p-coumaryl esterases [EC 3.1.1.21.
Optimization
of lignocellulosic
bioconversion
by polysaccharide hydrolases requires a good knowledge of the
kinetic parameters of the reaction. The complexity of the
enzymatic hydrolysis of lignocellulosic
wastes stems from
the fact that they are insoluble substrates and thus their
enzymatic hydrolysis is always limited. Adsorption and
kinetic studies of enzymatic hydrolysis of lignocellulosic
materials have been widely investigated
with cellulasecellulose systems, *-lo whereas only a few studies have dealt
with the mechanisms of adsorption and hydrolysis of such
substrates by xylanases. l*
The objective of this work was to better understand the
mechanisms of hydrolysis of wheat straw using a purified
20.7 kDa endo-B-1,4-xylanase
produced by a new strain
of Bacillus sp. l3 Binding experi ments were performed at
4C in order to avoid enzymatic hydrolysis which could
modify the structure of straw and consequently,
the
parameters of the adsorption of the enzyme. Hydrolysis
experiments
were performed at 60C using straw-xylanase complexes
which had been previously
formed at
4C. The extent and specificity
of the binding of this

0141-0229/98/$19.00
PII so141-0229(97)00105-1

Wheat straw-xylan
enzyme
to straw were studied and the relationships
between
adsorption
and hydrolytic
efficiency
of the
enzyme were investigated.

Materials and methods

hydrolysis:

C. Zilliox

and P. Debeire

charides had been first hydrolyzed for 2 h at 100C with 2 M

trifluoroacetic acid, reduced, and acetylated. For the determination
of neutral carbohydrate in wheat straw, hydrolysis was performed
by resuspending 20 mg of dry powder in 0.25 ml 72% (w/w)
sulfuric acid for 30 min at 25C followed by 2 h hydrolysis at
100C in 3 ml 6% (w/w) sulfuric acid.2 Water and lignin content
were determined by gravimetric methods.

Enzyme purification
Xylanase was produced from a thermophilic Bacillus strain D3.
This strain was obtained by chemical mutagenesis
with ethyl
methanesulfonate
of a thermophilic Bacillus strain XE.14 Wildtype and mutant strains of these Bacillus were deposited with
the Collection Nationale de Cultures des Microorganismes
de
1Institut Pasteur, Paris under the numbers I-1017 and I-1018.
The xylanase is an extracellular
enzyme which accounts for
50% of the total excreted proteins. It was purified to homogeneity by ion-exchange
(Q Sepharose fast flow) and hydrophobicity interaction (phenyl Sepharose CL4B) chromatography.15
The purified enzyme had an activity of 300 U ml- and a
specific activity of 2,000 U mg- protein at 60C a molecular
mass of 20.7 kDa, and a pI of 7.7. Maximal enzyme activity was
obtained with a temperature of 75C. No loss of activity was
observed after one day at 60C using a concentration
of 2 mg
xylanase ml - at pH 6.

Enzyme assays
Endo- 1,4+xylanase
activity was determined as previously described.16 Since this xylanase has an identical activity profile with
birchwood xylan when using either sodium acetate buffer (pH 5.8)
or distilled water, all enzymic incubations of this enzyme with
straw were performed in distilled water. Enzymic activity was
expressed in units (U) where 1 U is the amount of xylanase
required to release 1 pmol min- xylose reducing equivalent from
birchwood xylan (Sigma, St. Louis, MO). Xylanase activity on
straw was determined by the quantification of total neutral sugars
in the supernatant of a straw suspension after the removal of
insoluble material by centrifugation.

Substrates
Wheat straw was obtained from a farm in the north of France. The
leaves were removed and the internodes were ball-milled and
separated into fractions of different sizes using a sieve shaker. For
our studies, the 0.1-0.5 mm straw fraction was employed. Before
use, the straw was swollen at 60C for 16 h in water with
continuous stirring. In certain cases, thermal denaturation of straw
was subsequently performed by boiling 0.4 g of straw for 10 min
in 20 ml water. Hemicelluloses
from 2 g of wheat straw were
extracted for 3 h at 20C in 17 ml of 24% KOH solution containing
1% (w/v) NaBH,. After filtration on a sintered glass funnel
(medium porosity), the xylan was precipitated by the addition of
5 volumes of cold ethanol and 0.5 volume of acetic acid, filtered
on a glass microfiber filter (Whatmann GF/F), suspended in water,
dialyzed against distilled water, and freeze dried. Insoluble and
soluble wheat-straw xylans were separated by centrifugation of
suspensions of xylans in water at 60C. Lignin was isolated from
wheat straw by the dioxan extraction procedure.

General methods
Uranic acids and total neutral sugars were determined using the
m-phenylphenols
and phenol/sulfuric
acid methods,
respectively. Reducing sugars were quantified by the measurement of
ferricyanide reduction.
Monosaccharide
compositions of wheat
straw, xylans, and enzymic products were determined by GLC on
a SE-30 capillary column (Alltech, Deerfield, IL) after polysac-

Adsorption

studies at 4C

Adsorption studies of the xylanase onto straw, avicel, lignin,

and insoluble xylans from straw were performed by separately
mixing each component
(0.4 g) with the purified xylanase
(7.5-1250 pg enzyme g- straw, 350 pg enzyme gg of each
other component)
in 20 ml of water. The mixtures
were
incubated at 4C with continuous
stirring and samples were
withdrawn periodically
and centrifuged
at 13,000 rpm for 5
min. No hydrolysis was detected at 4C. The amount of enzyme
in the supernatant
was determined
from the measurement
of
enzymic activity, since the lowest quantities of enzyme did not
permit protein quantification.
The quantity of adsorbed enzyme
was calculated by subtracting the amount of the enzyme in the
supernatants
from the amount of enzyme added initially. In
order to evaluate the reversibility
of the adsorption
of the
xylanase on straw, cellulose,
lignin, and xylan, desorption
experiments
were performed
at 4C. The enzyme-substrate
complexes
were centrifuged
at 8,000 pm for 15 min, the
supernatants
were removed, and equal volumes of cold water
were added to the pellets. The desorption of the enzyme was
checked by the measurement
of the enzyme activities in the
supernatants.
For the straw-xylanase
complex, three washings
were performed after 2. 4, and 24 h of incubation at 4C which
lasted 2, 2, and 17 h, respectively.
For the lignin-xylanase
and
xylan-xylanase
complexes, one washing took place after 24 h of
incubation and lasted 4 h.
All experiments
were performed
in triplicate and mean
values are reported. Statistical analyses (analysis of variance
and Fisher test) were computed
with SAS Software (SAS
Institute. Inc.. Cary. NC).

Hydrolysis of wheat straw at 60C by

preadsorbed xylanase
Adsorption at 4C of various amounts of xylanase on straw was
performed as described before. The wheat straw-xylanase complexes were subsequently incubated at 60C (0.4 g straw/20 ml
water). Samples were withdrawn periodically,
centrifuged
at
13.000 rpm for 5 min), and assayed for total neutral sugars to
measure the extent of hydrolysis.

Analysis of products from enzymic hydrolysis

The oligosaccharides
obtained by hydrolysis of straw (with an
enzymic concentration
of 350 pg gg straw) and xylans from
straw were analyzed by TLC on silica gel plates from Merck
(0.2 mm layer) using butan- 1-ol/acetic acid/water (2: 1: 1) as the
solvant system. The separated sugars were visualized using an
orcinol spray reagent (200 mg orcinol/lOO ml 20% sulfuric
acid).

Results
Adsorption

of xylanase on wheat straw at 4C

Different amounts of purified endoxylanase were incubated

with a fixed amount of wheat straw in order to measure the
extent of the adsorption. Pi is defined as the amount of
Enzyme Microb. Technol.,

1998, vol. 22, January

59

Papers
Table 1
xylanase

Kinetics of adsorption
on straw at 4C

of different

Pi (pg protein/g-
125

350

concentrations

of

Table 2 Adsorption of the xylanase (P, = 350 pg protein g -) on

lignin and insoluble xylans from straw at 4C

straw)
700

Lignin
1,250
Time (h)

Time (min)
5
10
15
30b
60b
120b

fads (pg protein/g-

89
94
107
113
113
114

2
2
k
k
-c
-c

2.5
1.4
2.9
4.7
3.5
5.3

244
273
278
290
300
285

i+
2
5
2
2

6.5
9
10.2
10.6
15.7
11.5

303
334
365
369
379
381

Pads (kg protein g-l)

strawP
+
+
2
k
2
?

12.7
9.3
10.6
12.4
11.8
12.2

403
505
573
558
552
563

-t
+
2
12
-c

15.2
19.9
10
16.5
10.8
10.6

Pi: amount of added enzyme in the reaction mixture; fads:

amount of adsorbed enzyme on straw
a Experiments were performed in triplicate. Mean values and
standard deviations are reported.
b Equality of Pads was tested for each Pi value by using a F-test
in a variance analysis procedure (u = 5%)

added enzyme in the reaction mixture (pg added protein

g - straw) and Pads as the amount of adsorbed enzyme
(adsorbed protein g- straw). No hydrolysis
of xylan
occurred at 4C. Whatever the enzymatic concentrations
(Pi), the adsorption reached a maximum within 30 min of
reaction (Table 1). The adsorption of the xylanase was
largely irreversible at 4C since extended washings of the
wheat straw-xylanase
complexes induced only slight desorption of the enzyme. Adsorption data of the complete
cellulase complex on cellulose are known to obey the
Langmuir-type
adsorption isotherm even for irreversible
adsorption:
Pads = [K,P,,,,J(l

Insoluble xylans

+ K,P)IP

where P is the free protein concentration (pg protein ml- ),

P ads,m is the maximal amount of adsorbed protein (pg-
protein g - straw), and K, is the adsorption equilibrium
constant (ml kg- protein). The data fit the Langmuir-type
adsorption isotherm (data not shown) with the values of
P ads,m at 521 pg protein g-l straw and Kp at 0.35 ml pg
protein. The maximum efficiency of adsorption of the
xylanase on straw (defined as P,d,IPi in percent) reached
80% for a Pi of 125 and 350 pg protein ml-. Similar
experiments
were performed using nonpretreated
wheat
straw with incubation at 60C for 16 h. The data also fit the
Langmuir-type
adsorption isotherm (data not shown) and
the values of P
and Kp were 78 pg protein g- straw
and 7.4 ml pgapsGotein,
respectively.

0.5
1
4
6
24
After washing

fadJfi (%)

(4 h)

323
332
306
313
324
316

2
-+
2
2
2
t

9.2
10
12
11.2
9
10.5

90

225
1
1602
8
204 2 8.1
258 2 12.3
324 2 12
317 2 11.6

90.5

5: amount of added enzyme in the reaction mixture; fads:

amount of adsorbed enzyme on straw components
a Experiments were performed in triplicate. Mean values and
standard deviations are reported ((Y = 5%)

lignin or xylan, 90% of the xylanase remained bound on the

two substrates.

Hydrolysis of wheat straw at 60C by the

adsorbed xylanase
Various amounts of xylanase were adsorbed on straw at 4C
and the enzyme-straw complexes were incubated at 60C.
Figure I represents the extent of hydrolysis of straw for
different quantities of adsorbed xylanase. The different
curves relate to different incubation times. The production
of sugars was not proportional to the quantity of adsorbed
enzyme whatever the incubation time. Theoretically,
the
initial rates of hydrolysis might be directly related with the
initial amount of adsorbed enzyme. In our experiments,
initial velocity measurements could be measured using short
incubation times (15 min). Proportionality
between initial
velocities and Pads was observed only for very small
amounts of Pads (< 10 pg protein g- straw).
With Pads of 300 pg protein g- straw and higher, the
extent of hydrolysis reached a maximum at 11% hydrolysis
of the straw xylan after 5 h. Using the same incubation time,
even small amounts of adsorbed xylanase (Pads of 27 p.g
g-) led to the 7% hydrolysis of the straw xylan.

Adsorption of xylanase on cellulose and lignin and

xylan from straw at 4C
The results from the adsorption experiments on lignin and
insoluble xylans from straw are given in Table 2. The
xylanase did not bind microcrystalline
cellulose (Avicel,
data not shown). The adsorption of the xylanase on lignin
was fast whereas the adsorption of the xylanase on insoluble
xylans was slow. Both reactions were irreversible since
extended washings at 4C did not induce significant desorption of the xylanase. With a Pi of 350 p.g protein g-

60

Enzyme Microb. Technol.,

1998, vol. 22, January

Figure 1 Hydrolysis of wheat straw at 60C with different

amounts of preadsorbed xylanase if,,,) at different incubation
times: 0.5 h, W; 1 h, 0; 2 h, 0; 3 h, 0; and 5 h, A

Wheat straw-xylan
Table 3 Monosaccharide
content of straw, straw xylan extracted and enzymatic products obtained by long-term hydrolysis of straw by xylanase
Carbohydrate

Monosaccharide
Glucose
Xylose
Arabinose
Galactose
Mannose
Uranic acids

(%)

Wheat
straw

Xylan
extracted

Enzymatic
products

60.5
30.9
4.1
2.8
1.7
ND

1.7
76.7
8.8
1.7
0.2
10.9

3.9
71.7
10.3
3.5
1
9.6

ND, not determined

In order to know if the preadsorbed xylanase at 4C

could desorb during the reaction at 60C enzymatic activities were measured at different times in the supernatants of
the reaction mixture with a Pads of 470 kg g-l. A slight
desorption was observed after 1 h at 60C which accounted
for 90 p,g of enzyme g- straw. After 2 h at 6OC, the
amount of free enzyme decreased to a value of 30 pg free
xylanase g- straw which remained constant for the rest of
the incubation period.

Composition of wheat straw and the

hydrolysis products
Glucose and xylose were quantified by GLC and amounted
to 33% and 17%, respectively, of the total weight of wheat
straw. Xylans (23%) were recovered by alkaline extraction
from straw by gravimetric determination.
The centesimal
carbohydrate composition of wheat straw and xylans are
given in Table 3. The oligosaccharides
obtained by hydrolysis of straw were analyzed by GLC for neutral sugars and
by a calorimetric method for uranic acids. The monosaccharide content of isolated xylans and oligosaccharides was
similar (Table 3).
The hydrolysis products of wheat straw and the xylan
preparation
from straw by the purified xylanase were
examinated by TLC (Figure 2). For the hydrolysis of the
xylan preparation,
xylose-containing
oligosaccharides
of
DP 2-5 were detected as the major compounds.
Small
amounts
of xylose-containing
oligosaccharides
of DP
higher than 5 were released with a trace of xylose independantly of incubation time. In contrast, the TLC pattern of
xylose-containing
oligosaccharides
obtained from enzymic
hydrolysis of straw changed during the incubation time.
After 1 h of incubation, the major compounds were of DP
2-4 whereas after a prolonged incubation time (24 h), the
major compound was xylose. The same results were obtained using wheat straw from another harvest. When the
straw was first heated to 100C for 10 min before xylanolytic hydrolysis, the end products of the reaction were
xylose-containing
oligosaccharides
of DP 2-4 with only a
small amount of xylose.

hydrolysis:

C. Zilliox and P. Debeire

Discussion
Several studies have investigated the adsorption of cellulases onto cellulose and cellulosic substrates. Ooshima et
of adsorption of Tria1.2 stated that the equilibrium
choderma viride cellulase on Avicel was reached within 30
min between 5-50C. Beldman ef a1.s used a contact time of
1 h to establish an equilibrium of the adsorption of endoand exoglucanases from T. viride on crystalline cellulose. In
our studies, adsorption of xylanase on straw was reached
within 30 min at 4C for all initial enzymatic concentrations. The xylanase was tightly bound on straw since
subsequent washings with water did not induce any significant desorption of the xylanase at 4C. In order to investigate the specificity of the binding of the xylanase on straw.
adsorption studies of the xylanase on the major components
of straw (cellulose, xylan, and lignin) were performed at
4C. The xylanase did not bind microcrystalline
cellulose
showing the lack of a cellulose binding domain in this
protein. Nonspecific adsorption of the xylanase on lignin
was reached within 30 min whereas the specific adsorption
of the xylanase on insoluble xylans was surprisingly slow
(24 h of incubation). Both adsorptions were irreversible.
This xylanase is hydrophobic in character,13 and strongly
adsorbed on phenyl Sepharose. It requires the use of 25%
ethylene glycol for elution; therefore, hydrophobic interactions could be involved in the nonspecific adsorption of this
enzyme on lignin.
With Pi = 350 kg protein g- substrate,
similar
amounts of enzyme bound straw (280 pg protein g-
straw) and lignin or insoluble xylans (3 15 kg protein g-
lignin or xylans). In the cell wall, xylans and lignin are
embedded in a matrix of cellulose.23 The slight difference
between these adsorption values could indicate that the
enzyme molecules
diffuse into the straw and bind the
xylans and lignin which are located inside the straw.
Using nonpretreated
straw at 60C for 16 h, binding of
the xylanase was weak (Pads.,, at 70 kg protein g-
straw) compared to binding on pretreated straw (Pads.,, at

$f

y4h

___.__-I---...._____..--.-

:w

Xy Xw S
24

Figure 2 TLC of standard xylose-containing

oligosaccharides
(S) and those obtained by time course hydrolysis of wheat straw
(Wat 1,5, and 24 h incubation), preheated wheat straw at 100C
(W/I at 24 h incubation), and wheat straw xylans (Xw at 1, 5, and
24 h incubation) by xylanase. X, = Xyl; X, =. Xyl,; X, = Xyl,;
x, = XVI,

Enzyme Microb. Technol.,

1998, vol. 22, January

61

Papers
521 pg protein gg straw). These results showed that
thermal treatment of straw leads to an increase in the
accessibility
of the fixation sites of the enzyme.
Hydrolysis experiments at 60C with bound xylanasestraw complexes showed that there is no direct relationship
between the amount of bound enzyme and the extent of
hydrolysis; moreover, small amounts of bound xylanase
(Pads at 27 kg protein g- straw) were sufficient to
hydrolyze 7% of the xylans in the straw after 5 h of
incubation. These results suggest that the low quantity of
xylanase hydrolyzes the easily hydrolyzable xylans which
are located, for example, at the periphery of the particules of
straw. When higher quantities of enzyme were used, the
efficiency of the enzyme decreased. One explanation is that
the excess of xylanase could be adsorbed nonspecifically
on
lignin as shown by Chernoglazov et aL9 who observed that
the purified glucanases of Trichoderma reesei could be
adsorbed onto lignin and become inactive. Other possible
explanations are that the excess xylanase was bound onto
highly substituted, hydrolysis-resistant
xylans or associated
with less accessible sites where the enzyme was less mobile
and less active. The major question which remains to be
elucidated arises from the kinetic studies of adsorption
which showed that the xylanase binds, in an irreversible
manner, to the lignin faster than to the xylans. If this
phenomenon
occurs when the xylanase adsorbs on the
straw, how can the enzyme, which is tightly bound to the
lignin, hydrolyze the xylans?
Analysis of the hydrolytic products of the xylanase-straw
reaction showed that after a prolonged incubation time,
xylo-oligosaccharides
of DP 2-4 were converted into xylose. When this xylanase was incubated with birchwood
glucuronoxylan5
or straw arabino-glucuronoxylan
(this
paper), only a very small amount of xylose with xylooligosaccharides
of DP 2-4 was produced. Furthermore,
pretreatment of the straw at 100C before enzymatic hydrolysis did not lead to increased xylose production. These
results suggest that a P-xylosidasic activity is present in the
straw which hydrolyzes the xylo-oligosaccharides
into xylose. Covalent linkages between uranic acids from xylans
and other compounds such as lignin have already been
demonstrated in spruce wood,24 Hibiscus cannabinus, and
Corchorus capsularis. 2x*6 In this study, uranic acids accounted for 11% of the xylans isolated from the straw and
the xylose-containing
oligosaccharides
produced by the
xylanase contained 9.6% of uranic acids. These results
show that a part of glucuronic acids are not involved in
covalent linkages between xylans and lignin and thus do not
prevent the liberation of the oligosaccharides
from the
straw.

2.

3.

4.

5.

6.

7.

8.

9.

10.

II.

12.

13.

14.

15.

16.

17.

18.

19.

Acknowledgments

20.

This work was supported by a grant from the Europol Agro

of Reims held by C. Zilliox.

References
1.

62

22.

Aman, P. Composition and structure of cell wall polysaccharides

in
forages In: Forage Cell Wall Structure and Digestibility (Jung,

Enzyme Microb. Technol.,

21.

1998, vol. 22, January

H. G., Buxton, D. R., Hatfield, R. D., and Ralph, J., Eds.). A.S.A.,
C.S.S.A.. S.S.S.A., Madison, WI, 1993, 183-199
Harper, S. H. T. and Lynch, J. M. The chemical components and
decomposition of wheat straw leaves, internodes, and nodes. J. Sci.
Food Agric. 1981, 32, 1057-1062
Lynch, J. M. Straw residues as substrates for growth and product
formation by soil microorganisms.
In: Straw Decay and Its Eficr on
Disposal and Utilization (Grossbard, E., Ed.). Hatfield Polytechnic,
John Wiley & Sons, Chichester, 1979. 47-56
Sermanni, G. G., Dhannibale, A., Perani, C.. Porri, A., Pastina. F.,
Minelli, V.. Vitale. N., and Gelsomino, A. Characteristics of paper
handsheets after combined biological pretreatments and conventionnal pulping of wheat straw. Tuppi J. 1994, 77, 151-157
Lam, T. B. T., Iiayama, K., and Stone. B. Distribution of free and
combinated
phenolic acids in wheat internodes. Phytochemisrv
1990. 29,429-433
Lam. B. T. B., Iiyama, K., and Stone, B. Cinnamic acid bridges
between cell well polymers in wheat and phalaris
internodes.
Phytochemistr?, 1992, 31, 1179-l I83
Scalbert. A., Monties, B., Lallemand, J. Y., Guittet, E., and Roland,
C. Ether linkages between phenolic acids and lignin fractions from
wheat straw. Phytochemistry 1985, 24, 1359-1362
Beldman, G.. Voragen, A. G. J.. Rombouts, F. M., Searle-van
Leeuwen, M. F., and Pilnik, W. Adsorption and kinetic behavior and
purified endoglucanases
and exoglucanases
from Trichoderma
viride. Biotechnol. Bioeng. 1987, 30, 25 I-257
Chemoglazov,
V. M., Ermolova, 0. V.. and Klyosov, A. A.
Adsorption of high-purity endo-1,4-B-glucanases
from Trichodema
reesei on components of lignocellulosic materials: cellulose, lignin,
and xylan. Enzyme Micmb. Technol. 1988, 10, 503-507
Moloney, A. and Coughlan, M. P. Sorption of Talaromyces emersonii cellulase on cellulosic substrates. Biotechnol. Bioeng. 1983,
25, 27 l-280
Nidetzky, B.. Steiner, W., Hayn. M.. and Esterbauer, H. Enzymatic
hydrolysis of wheat straw after steam pretreatment: Experimental
data and kinetic modelling. Bioresource Technol. 1993. 44, 25-32
Viikari. L.. Kantelinen, A., Buchert, J.. and Pub, J. Enzymatic
accessibility of xylans in lignocellulosic materials. Appl. Microbial.
Biotechnol. 1994, 41, 124-129
Samain, E., Touzel, J. P., Brodel, B.. and Debeire, P. Isolation of a
thermophilic
bacterium producing high levels of xylanase. In:
Xylans and Xylanase (Visser, J., Beldman, G., Kusters-van Someren, M. A.. and Voragen, A. G. J., Eds.). Elsevier Science
Publishers B. V., Amsterdam. 1992, 467-470
Pickersgill, R. W., Debeire, P., Debeire-Gosselin,
M., and Jenkins,
J. A. Crystallization
and preliminary X-ray analysis of a thermophilic Bacillus xylanase. J. Mol. Biol. 1993. 230, 664-666
Debeire-Gosselin,
M., Loonis. M., Samain, E., and Debeire, P.
Purification and properties of a 22 kDa endoxylanase excreted by a
new strain of thermophilic
Bucillus. In: Xylans and Xylanases
(Visser. J., Beldman, G., Kusters-van Someren, M. A., and Voragen,
A. G. J., Eds.). Elsevier Science Publishers, 1992, 463-466
Pellerin, P., Gosselin, M., Lepoutre. J. P., Samain, E., and Debeire,
P. Enzymatic production of oligosaccharides
from corncob xylan.
Enzyme Microb. Technol. 199 1, 13, 6 17-62 1
Monties, B. Preparation of dioxane lignin fractions by acidolysis. In:
Methods in Enzymology Vol 161 Biomass Part B, Lignin. Pectin,
und Chitin. Academic Press, London, 1988, 31-35
Blumenkrantz, N. and Asboe-Hansen,
G. New method for quantitative determination
of uranic acids. Anal. Biochem. 1973, 54,
484-489
Dubois, M., Gilles, K, A., Hamilton, J. K., Rebers, P. A., and Smith.
F. Calorimetric
method for determination
of sugars and related
substances. Anal. Chem. 1956, 28, 350-356
Kidby. D. K. and Davidson, D. J. A convenient ferricyanide
estimation of reducing sugars in the nanomole range. Anal. Biothem. 1973, 55, 321-325
Hoebler. C.. Barry, J. L., David, A., and Delort-Lava], J. Rapid acid
hydrolysis of plant cell wall polysaccharides
and simplified quantitative determination of their neutral monosaccharides by gas-liquid
chromatography.
J. Agric. Food Chem. 1989. 37, 360-367
Ooshima, H.. Sakata, M., and Harano, Y. Adsorption of cellulase
from Trichoderma viride in cellulose. Biotechnol. Bioeng. 1983, 25,
3103-3114

Wheat straw-xylan
23.

24.

Carpita, N. C. and Gibeaut, D. M. Structural models of primary cell

walls in flowering plants: Consistency of molecular structure with me
physical properties of the walls during growth. Plant J. 1993,3, I-30
Eriksson, 0. and Goring, A. I. Structural studies on the chemical
bonds between lignins and carbohydrates in spruce wood. Wood Sci.
Tech&.
1980, 14,261-279

25.

26.

hydrolysis:

C. Zilliox

and P. Debeire

Das. N. N.. Das, S. C.. Dutt. A. S., and Roy, A. Lignin-xylan ester
linkage in jute fiber (Corchnru.s cup.~~lur~s~. Cwbohydr. Res. 198 1,
94, 73-82
Das, N. N.. Das, S. C.. Sarkar. A. K., and Mukherjee, A. K.
Lignin-xylan
ester linkage in Mestu jihrr (Hibiscus ccmnabinusJ.
Carbohydr. Res. 1984. 129, 197-207

Enzyme Microb. Technol.,

1998, vol. 22, January

63