hydrolysis of ws kinetic studies (zilliox, c. and debeire, p. 2000)
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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
d t
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 i n the straw aft er 5 h of reaction. N o proporti onali ty
could be ound betw een t he evel of xyl anase adsorpti on on straw and t he ext ent of hydrol ysis at 60. A dsorpti on
and hydrol ysis experi ments indi cated that all the bound xyl anase w as not hydrol yt icall y acti ve. This suggested
that nonspec c adsorpti on occurred on igni n. Anal ysis of the end products of the reacti on indi cated tha t yl ose
and neutr al and urani c acid-cont ai ni ng xyl o-ol igosacchari des w ere the maj or compounds.
0 1998 Elsevi er
Science I nc.
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. Bio-
conversion of wheat straw is favored by its relatively low
lignin content (
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Wheat straw xylan hydrolysis: C. Zilliox and P. Debeire
enzyme to straw were studied and the relationships
between adsorption and hydrolytic efficiency of the
enzyme were investigated.
Materials and methods
Enzyme purification
Xylanase was produced from a thermophilic Bacil lus strain D3.
This strain was obtained by chemical mutagenesis with ethyl
methanesulfonate of a thermophilic Bacil lus strain XE.14 Wild-
type and mutant strains of these Bacil lus 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 homoge-
neity by ion-exchange (Q Sepharose fast flow) and hydropho-
bicity 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 de-
scribed.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, respec-
tively. 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-
charides had been first hydrolyzed for 2 h at 100C with 2
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.
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 com-
plexes 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
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Table 3 Monosaccharide content of straw, straw xylan ex-
tracted and enzymatic products obtained by long-term hydroly-
sis of straw by xylanase
Carbohydrate ( )
Monosaccharide
Wheat
straw
Xylan
extracted
Enzymatic
products
Glucose
60.5
1.7
3.9
Xylose
30.9 76.7 71.7
Arabinose
4.1 8.8 10.3
Galactose
2.8
1.7
3.5
Mannose
1.7 0.2 1
Uranic acids
ND 10.9
9.6
ND, not determined
In order to know if the preadsorbed xylanase at 4C
could desorb during the reaction at 60C enzymatic activ-
ities 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 hydro-
lysis of straw were analyzed by GLC for neutral sugars and
by a calorimetric method for uranic acids. The monosac-
charide 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 indepen-
dantly 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 ob-
tained using wheat straw from another harvest. When the
straw was first heated to 100C for 10 min before xylano-
lytic hydrolysis, the end products of the reaction were
xylose-containing oligosaccharides of DP 2-4 with only a
small amount of xylose.
Wheat straw xylan hydro lysis: C. Zil l iox and P. Debeire
Discussion
Several studies have investigated the adsorption of cellu-
lases onto cellulose and cellulosic substrates. Ooshima et
a1.2
stated that the equilibrium of adsorption of Tri-
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 endo-
and 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 concentra-
tions. The xylanase was tightly bound on straw since
subsequent washings with water did not induce any signif-
icant desorption of the xylanase at 4C. In order to inves-
tigate 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 interac-
tions 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
S y f ; y4h
:w
y w S
24
___.__-I---...._____..--.-
h
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,
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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 xylanase-
straw 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 Tri choderma 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 xy-
lose. When this xylanase was incubated with birchwood
glucuronoxylan5 or straw arabino-glucuronoxylan (this
paper), only a very small amount of xylose with xylo-
oligosaccharides of DP 2-4 was produced. Furthermore,
pretreatment of the straw at 100C before enzymatic hydro-
lysis 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 xy-
lose. 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 ac-
counted 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.
cknowledgments
This work was supported by a grant from the Europol Agro
of Reims held by C. Zilliox.
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