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

Enzyme Microb. Technol., 1998, vol. 22, January 59


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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,

Enzyme Microb. Technol., 1998, vol. 22, January

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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.

References

1. Aman, P. Composition and structure of cell wall polysaccharides in

forages In:

Forage Cell Wal l Structure and Di gestibi l i t y

(Jung,

2.

3.

4.

5.

6.

7.

8.

9.

10.

II.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

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: Str aw Decay and Its Eficr on

Disposal and Ut i l izat ion

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 convention-

nal pulping of wheat straw.

Tupp i 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.

Phy to chemi str ?, 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

vi ri de. Bi ot echnol . Bioeng. 1987, 30, 25 -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 M icmb. Technol . 1988,

10,

503-507

Moloney, A. and Coughlan, M. P. Sorption of Talaromyces emer-

sonii cellulase on cellulosic substrates. Biot echnol. Bi oeng. 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. Bi oresource Technol . 1993. 44, 25-32

Viikari. L.. Kantelinen, A., Buchert, J.. and Pub, J. Enzymatic

accessibility of xylans in lignocellulosic materials.

Appl. Microbial.

Bi ot echnol . 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:

Xyl ans and X ylanase

(Visser, J., Beldman, G., Kusters-van Som-

eren, 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 thermo-

philic

Bacil lus

xylanase. J.

Mol . Bi ol . 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 M icrob.

Technol. 199 1, 13, 6 17-62 1

Monties, B. Preparation of dioxane lignin fractions by acidolysis. In:

Methods i n Enzymology

Vol 161

Biom ass Part B, Li gnin. Pecti n,

und Chit in. Academic Press, London, 1988, 31-35

Blumenkrantz, N. and Asboe-Hansen, G. New method for quanti-

tative determination of uranic acids.

Ana l. 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. Bio-

them. 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 quan-

titative 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 vir ide

in

cellulose.

Bi ot echnol . Bioeng. 1983, 25,

3103-3114

62 Enzyme Microb. Technol., 1998, vol. 22, January


6/6

Wheat straw xylan hydrolysis: C. Zilliox and P. Debeire

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

25. Das. N. N.. Das, S. C.. Dutt. A. S., and Roy, A. Lignin-xylan ester

walls in flowering plants: Consistency of molecular structure with me

linkage in jute fiber (Corchnru.s cup.~~lur~s~.

Cw bohydr. Res. 198

1,

physical properties of the walls during growth.

Plant J.

1993,3, I-30

94, 73-82

24. Eriksson, 0. and Goring, A. I. Structural studies on the chemical 26. Das, N. N.. Das, S. C.. Sarkar. A. K., and Mukherjee, A. K.

bonds between lignins and carbohydrates in spruce wood.

Wood Sci.

Lignin-xylan ester linkage in

Mestu ji hrr (Hi biscus ccmnabin usJ.

Tech&. 1980, 14,261-279

Carbohydr. Res. 1984. 129, 197-207

Enzyme Microb. Technol., 1998, vol. 22, January 63

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Hydrolysis of WS Kinetic Studies (Zilliox, C. and Debeire, P. 2000)