International Biodeterioration & Biodegradation 52 (2003) 97–100

www.elsevier.com/locate/ibiod

Production of xylan-degrading enzymes from Amazon forest fungal

species

Roseli Garcia Medeiros

a

, Rog-erio Hanada

b

, Edivaldo Ximenes Ferreira Filho

a;∗

a

Departamento de Biologia Celular, Laboratorio de Enzimologia, Universidade de Brasilia, Brasilia, DF, CEP 70910–900, Brazil

b

Laboratorio de Patologia da Madeira, Instituto Nacional de Pesquisa da Amazˆonia, Manaus, Amazˆonia, Brazil

Received 1 June 2002; received in revised form 25 October 2002; accepted 19 November 2002

Abstract

Ten fungal species were isolated from decomposing wood in the Amazon forest. All produced xylan-degrading enzymes when cultivated

in liquid media containing oat spelt xylan. The best producing strains were identi9ed as Penicillium corylophilum, Aspergillus niger and

Trichoderma longibrachiatum. The best yields of -xylosidase and -arabinofuranosidase activities were Trichoderma harzianum and

Trichoderma sp. Xylanase activities from crude extract samples of P. corylophilum, A. niger and T. longibrachiatum were partially

characterized. They were most active at 40

◦

C (A. niger) or 45

◦

C (P. corylophilum and T. longibrachiatum) and pH 4.0–4.5. Reducing

agents (-mercaptoethanol and dithiothreitol),

L

-cysteine and

L

-tryptophan activated xylanase activity. In addition, dithiothreitol improved

the half-lives of these enzymes at 50

◦

C and 60

◦

C. By contrast, N-bromosuccinimide inhibited all the enzyme activities. Xylan and

dithiothreitol a>orded protection against xylan-degrading enzyme inactivation by N-bromosuccinimide, but failed to reverse it. The apparent

K

m

values on soluble and insoluble xylans from oat spelt showed that xylan-degrading enzymes from A. niger, T. longibrachiatum and

P. corylophilum were most active on the soluble form.

? 2003 Elsevier Ltd. All rights reserved.

Keywords: Xylanolytic fungi; Xylan; Xylan-degrading enzymes

1. Introduction

Xylans are branched polysaccharides found in the cell

walls of all land plants and composed of a backbone

containing -1, 4-linked-

D

-xylosyl residues and, depend-

ing on their source and extraction procedure, with side

chains of acetyl, glucuronic, methylglucuronic, arabino-

furanosyl, ferulic and coumaric groups (

Lee et al., 1993

;

Filho, 1998

). Xylans account for 20–35% of the total

dry weight of hardwoods and annual plants and repre-

sent a vast resource that can be used for production of

fermentable sugars and fuels (

Haltrich et al., 1996

;

Filho

1998

). The complete cleavage of xylan is carried out

by the synergistic action of -xylanase and its accessory

enzymes, including -xylosidase, -arabinofuranosidase,

-methylglucuronidase and acetyl xylan esterase (

Smith

et al., 1991

). Xylan-degrading enzymes have been reported

∗

Corresponding author. Fax: [phone].

E-mail address:

[e-mail]

(E.X.F. Filho).

to be produced by a wide variety of fungal species (

Kulkarni

et al., 1999

). These enzymes have potential application in

sacchari9cation of biomass and biobleaching of kraft pulps

(

Filho, 1998

;

Silveira et al., 1999

). The objectives of the

present work were to isolate and identify fungal species from

the Amazon forest and to evaluate their capacity to produce

xylan-degrading enzyme activity during growth in liquid

medium containing oat spelt xylan as the carbon source.

We also characterized the crude xylan-degrading enzyme

activities from Penicillium corylophilum, Aspergillus niger

and Trichoderma longibrachiatum.

2. Materials and methods
2.1. Chemicals

Oat

spelt

xylan,

carboxymethyl

cellulose,

p-nitrophenyl--

D

-xyloside,

p-nitrophenyl--

L

-arabino-

furanoside,

L

-cysteine, -mercaptoethanol, dithiothreitol

0964-8305/03/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0964-8305(02)00179-8

98

R. Garcia Medeiros et al. / International Biodeterioration & Biodegradation 52 (2003) 97–100

Table 1

Identity and source of isolates used for production of xylan-degrading enzymes

Fungus (species)

Isolate number

Source (wood)

Paecilomyces variotti Bainier

LPM 48

Virola sp.

Trichoderma longibrachiatum Rifai

LPM 139

Scleronema micranthum Ducke

Trichoderma harzianum Rifai

LPM 296

Couroupita guianensis Aubl.

Penicillium roquefortti Thom

LPM 303

Hura creaptans L.

Penicillium sp.

LPM 494

Simaruba amara Aubl.

Aspergillus niger van Tieghen

LPM 93

Cedrelinga cateniformis Ducke

Penicillium corylophilum Dierckx

LPM 119

Parkia multijuga Benth

Fusarium sp.

LPM 495

Simaruba amara Aubl.

Trichoderma sp. strain SM

LPM 496

Sche?era morototoni Decne and Planch

Trichoderma sp. strain SA

LPM 497

Simaruba amara Aubl.

(DTT), N-bromosuccinimide (NBS) and

L

-tryptophan were

purchased from Sigma Chemical Co. (St. Louis, MO).

2.2. Organism and enzyme production

All fungi species were isolated from decomposed wood

in the natural forest reserve of INPA (National Research

Institute of Amazonia, Brazil). The origins of isolates of

10 species of wood-inhabiting fungi are shown in Table

1

.

Pieces (10–20 mm

2

) of decomposed wood were placed in

a series of Petri plates containing 3% malt-agar and 250 mg

chloramphenicol, and incubated at 25

◦

C for 1 month. After

incubation, each puri9ed isolate was transferred to fresh 3%

malt-agar medium. The cultures may incubated at 25

◦

C until

the mycelium spread over most of the medium surface. They

were then stored at 5

◦

C. The identi9cation of fungi species

was done according to the protocols and have been previ-

ously described, the species and genus features described

in the following references:

Booth, 1971

;

Carmichael et al.,

1980

;

Klich and Pitt, 1988

;

Pitt, 1991

;

Rifai, 1969

;

Samson,

1974

. The puri9ed fungi were transferred to plates that con-

tained MYG (0.2% malt extract, 0.2% yeast extract, 2%

glucose and 2% agar) or BDA (2.0% potato broth, 2% glu-

cose and 1.5% agar) media. T. longibrachiatum and Tri-

choderma harzianum were maintained in MYG medium,

while Paecilomyces variotti, Trichoderma sp. strain SM,

Trichoderma sp. strain SA, Penicillium roquefortti, Peni-

cillium sp., P. corylophilum, Fusarium sp. and A. niger

were maintained in BDA medium at 28

◦

C. For produc-

tion of xylanase activity, the fungi were cultured in Er-

lenmeyer Lasks containing 0:5% (w=v) oat spelt xylan in

100 ml of minimal medium (0.7% KH

2

PO

4

, 0.2% K

2

HPO

4

,

0.05% MgSO

4

7H

2

O, 0.1% (NH

4

)

2

SO

4

) supplemented with

0.06% yeast extract. Flasks were inoculated with 2:9 ×

10

3

=ml of spore suspensions from routine subcultures. Cul-

tures were grown at pH 7.0 for 7 days at 28

◦

C with shak-

ing at 100 rpm. Subsequently the content of each Lask was

9ltered through Whatman 9lter paper number 1. The su-

pernatant solutions, hereafter called crude extracts, obtained

from 9ltration procedure were stored at 4

◦

C for subsequent

use as xylan-degrading enzyme preparations.

2.3. Enzyme assays

Xylan-degrading enzyme activity was determined, ex-

cept where noted otherwise, by mixing 50 l of enzyme

solution with 100 l of oat spelt xylan in 100 mM sodium

acetate bu>er, pH 5.0 at 50

◦

C for 30 min. The release

of reducing sugar was measured using the dinitrosalicylic

reagent method (

Silveira et al., 1999

). Xylan-degrading

enzyme activity was expressed as mol reducing sugar

formed min=ml enzyme solution, i.e., as IU/ml. The activity

of the enzyme preparations was also performed in the pres-

ence of amino acid modifying (-mercaptoethanol, NBS,

and DTT), and aminoacids (

L

-tryptophan and

L

-cysteine).

The reaction mixtures contained individual reagents at a

9nal concentration of 0.1 or 4:6 mM (NBS) and 9:3 mM

(-mercaptoethanol, DTT,

L

-tryptophan and

L

-cysteine).

Appropriate controls were included in all cases. Xylan,

DTT and

L

-tryptophan were also pre-incubated for 10 min

with crude enzyme sample just before addition of NBS

and the assay was carried out as above. The crude enzyme

sample was also pre-incubated with NBS for 10 min prior

addition of DTT. Carboxymethyl-cellulase assay was per-

formed in the conditions as described above. The activity

against 9lter paper was measured as described by

Mandels

et al. (1976)

. -xylosidase and -arabinofuranosidase ac-

tivities were determined as reported elsewhere (

Ximenes

et al., 1996

). For the kinetic experiments, soluble and

insoluble samples from oat spelt xylan were used as substra-

tes in concentration ranges of 0.15–0:9 mg=ml (soluble xy-

lan) and 0.15–2:0 mg=ml (insoluble xylan). The substrates

were prepared as described by

Filho et al. (1993)

. K

m

val-

ues were estimated from Michaelis–Menten equation with a

non-linear regression data analysis program (

Leatherbarrow,

1987

). The determination of optimum temperature of

xylan-degrading enzyme samples was carried out in the tem-

perature range of 30–80

◦

C at pH 7.0. Optimum pH values

were determined by measuring the enzyme activity at 50

◦

C

at pH values from 3.0 to 8.0. The following bu>ers were

used: 50 mM sodium acetate bu>er (3.0–5.5), and sodium

phosphate bu>er (6.0–8.0). The bu>ers, regardless of pH,

were adjusted to the same ionic strength with NaCl. The

R. Garcia Medeiros et al. / International Biodeterioration & Biodegradation 52 (2003) 97–100

99

temperature stability of xylan-degrading enzyme samples

was determined by pre-incubating the enzyme at 50

◦

C and

60

◦

C. At various times, aliquots were withdrawn and the

residual activity was measured under standard conditions.

2.4. Protein concentration

Protein concentration was measured by the method of

Bradford (1976)

, using bovine serum albumin as standard.

3. Results and discussion

3.1. Screening of xylan-degrading enzyme activities

All fungal species were able to produce reducing sugars

from xylan (results not shown). Most of the crude extract

samples showed weak but measurable -xylosidase and

-arabinofuranosidase activities. The enzymes were most

active in T. harzianum and Trichoderma sp strain SM.

Fusarium sp. did not show xylosidase activity. The best

producers of xylan-degrading enzyme activity were P. cory-

lophilum, A. niger and T. longibrachiatum, respectively.

Most of the enzyme samples did not contain cellulase

activity (9lter paper-degrading activity or carboxymethyl

cellulase). Only A. niger, Trichoderma sp. strain SM and

P. corylophilum were able to degrade, at a low level, 9lter

paper and carboxymethyl cellulose (results not shown).

3.2. Characterization of xylan-degrading enzyme activity

from crude extracts of P. corylophilum, A. niger and

T. longibrachiatum

Xylan-degrading enzyme activities from crude extract

samples of P. corylophilum, A. niger and T. longibrachia-

tum were partially characterized. The pH and temperature

were typical of xylan-degrading enzymes from mesophilic

fungi (

Silveira et al., 1999

). Those from P. corylophilum

and T. longibrachiatum were most active at 45

◦

C, while A.

niger had a maximum enzyme activity at 40

◦

C. The enzyme

samples displayed highest activity at pH range of 4.5–5.5.

The e>ect of pH and temperature on xylan-degrading en-

zyme activity from P. corylophilum is presented in Figs.

1

and

2

, respectively. All enzyme samples showed more aNn-

ity against soluble xylan (Table

2

). The xylan-degrading

enzyme activity from A. niger showed the lowest K

m

value.

The involvement of some modifying agents on xylan-

degrading enzyme activities from crude extract samples

of P. corylophilum, A. niger and T. longibrachiatum

was investigated. Crude enzyme samples from T. longi-

brachiatum and A. niger were activated by

L

-cysteine,

L

-tryptophan, DTT and -mercaptoethanol (Table

3

).

DTT and -mercaptoethand did not improve the xylan-

degrading enzyme activity from P. corylophilum. The crude

xylan-degrading enzymes were inhibited by NBS at 4:6 mM.

At 0:1 mM NBS, the xylan-degrading enzyme activities of

P. corylophilum, A. niger and T. longibrachiatum were not

Fig. 1. E>ect of pH on xylan-degrading enzyme activity from P.

coryophilum.

Fig. 2. E>ect of temperature on xylan-degrading enzyme activity from P.

coryophilum.

Table 2

K

m

Determination of xylan-degrading enzyme activities from fungal

species on soluble and insoluble xylans

Fungus

Soluble xylan

Insoluble xylan

(mg/ml)

(mg/ml)

Trichoderma longibrachiatum

0.7

1.2

Penicillium corylophilum

0.6

0.8

Aspergillus niger

0.5

1.0

a>ected (results not shown).

L

-Tryptophan is believed to

be involved in the substrate-binding of the xylan-degrading

enzymes (

Coughlan, 1992

). One must be cautious in deter-

mining the mode of inhibition since NBS is a strong oxi-

dizing agent and is capable of eliciting a variety of e>ects

on proteins (

Clarke, 1987

). Evidence for involvement of the

L

-tryptophan residue at the active site is given by the com-

plete protection of the crude xylan-degrading enzyme ac-

tivity from P. corylophilum by 1% xylan and DTT against

NBS. On the other hand, xylan and DTT failed to reverse

the xylan-degrading enzyme of P. corylophilum from in-

activation by NBS (results not shown).

The industrial application of xylan-degrading enzymes

in bio-bleaching process for paper pulp requires ther-

mostable enzymes (

Filho, 1998

). The e>ect of temperature

on the stability of the xylan-degrading enzyme activities

was therefore determined. After 6 h incubation at 45

◦

C

and pH 5.5, all enzyme samples lost 50% of their initial

activity. Xylan-degrading enzymes from P. corylophilum,

T. longibrachiatum and A. niger retained 40%, 47% and

100

R. Garcia Medeiros et al. / International Biodeterioration & Biodegradation 52 (2003) 97–100

Table 3

E>ect of reducing agents,

L

-tryptophan and

L

-cysteine on xylan-degrading enzyme activities from fungal species

Fungus

L

-Cysteine

-Mercaptoetanol

DTT

L

-Tryptophan

(% control)s

(% control)

(% control)

(% control)

Trichoderma longibrachiatum

160

380

180

260

Penicillium corylophilum

141

100

100

125

Aspergillus niger

145

145

109

181

Fig. 3. Thermal stability of xylan-degrading enzymes from P. cory-

lophilum (), T. longibrachiatum (

4

) and A. niger ().

65% of their activity, respectively, after incubation at 50

◦

C,

pH 5.5 for 60 min. The presence of DTT in the incu-

bation mixture markedly increased the thermostability of

xylan-degrading enzyme samples. Xylan-degrading enzyme

activity from A. niger was the most thermostable of the

three enzyme samples, with a half-life of greater than 5 h

at 60

◦

C and pH 5.5 while those from P. corylophilum and

T. longibrachiatum showed half-lives of 1 h. At 50

◦

C and

pH 5.5, all enzyme samples showed half-lives greater than

6 h (Fig.

3

).

This present research work was done to select fungal

strains for application in kraft pulp pre-bleaching process.

P. corylophilum produced the highest xylan-degrading en-

zyme activity, followed by A. niger and T. longibrachiatum

when grown in medium containing oat spelt xylan as the car-

bon source. To our knowledge, this is the 9rst report on the

production and characterization of a crude xylan-degrading

enzyme sample from P. corylophilum. Further studies will

be carried out to purify and determine the role of the puri-

9ed enzyme systems on the hydrolysis of xylan, their use in

biobleaching and the involvement of

L

-tryptophan, cysteine

and other amino acid residues in enzymatic activity.

Acknowledgements

This work was supported by research grants from Bio-

amazˆonia/BASA (Brazil) and PADCT III/CNPq (Brazil).

EX.F.F. and R.G.M. are recipient of research fellowship and

postgraduate maintenance scholarship, respectively, from

CNPq (Brazil).

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