Xylan-Degrading enzymes from Aspergillus niger.pdf

Bioenerg. Res. (2012) 5:363

371

DOI 10.1007/s12155-011-9137-3

–

Isolation and Characterization of a Xylan-Degrading

Enzyme from Aspergillus niger van Tieghem LPM 93

with Potential for Industrial Applications

Natália von Gal Milanezi & Diana Paola Gómez Mendoza &

Félix Gonçalves de Siqueira & Luciano Paulino Silva & Carlos André Ornelas Ricart &

Edivaldo Ximenes Ferreira Filho

Published online: 13 July 2011

# Springer Science+Business Media, LLC. 2011

Abstract Aspergillus niger van Tieghem LPM 93 was

shown in an earlier study to produce the most thermo-

stable

3.02 IU/ml, respectively. Salts inhibited the activity of

XynI to different degrees. N-Bromosuccinimide caused

marked inhibition of XynI. On the other hand,

-xylanase, which was effective for improving

brightness and delignification of non-delignified and

oxygen-bleached samples of eucalyptus kraft pulp. Here,

we report the production, purification, and characteriza-

tion of a xylan-degrading enzyme (XynI) from this strain

grown in submerged liquid cultivation on medium

containing sugar cane bagasse as the carbon source.

XynI was isolated by ultrafiltration and gel-filtration

chromatography and characterized. The fungus displayed

high levels of xylanolytic activity after the second day of

cultivation, and this activity remained constant up to the

50th day. The molecular mass of XynI was in the range

of 32

mercaptoethanol and L -tryptophan were the best enzyme

activators.

Keywords Aspergillus niger . Sugar Cane Bagasse .

-Xylanase . Isoforms

Introduction

Lignocellulosic biomass is an important source of renew-

able energy. It consists primarily of the carbohydrate

polymers cellulose and hemicellulose and the phenolic

polymer lignin [ 21 , 44 ]. Hemicellulose refers to a large

group of heterogeneous polysaccharides. These polysac-

charides possess a great variety of substituents, including

sugars, in their side chains [ 44 ]. According to its structural

complexity, hemicellulose hydrolysis requires an enzymatic

pool composed of endo-1,4-

33 kDa as determined by mass spectrometry and

SDS-PAGE. The two-dimensional gel electrophoresis

analysis showed the existence of multiple forms of

–

xylanases in XynI. XynI showed the highest activity at

50°C and pH 4.5 and was stable in sodium acetate buffer

at pH 4.5. The K m and V max values were 47.08 mg/ml and

- D -xylanases (EC 3.2.1.8), 1,4-

- D -xylosidases (EC 3.2.1.37),

- L -arabinofuranosidases

: F. G. de Siqueira : E. X. F. Filho ( * )

Laboratory of Enzymology, Department of Cellular Biology,

University of Brasília,

Brasília, DF 70910-900, Brazil

e-mail: eximenes@unb.br

N. von Gal Milanezi

(EC 3.2.1.55),

- D -glucuronidases (EC 3.2.1.139), and

acetyl-xylan esterases (EC 3.1.1.72) [ 5 , 16 , 32 , 39 ].

Xylans are a major component of agroindustrial byproducts

and waste that represent rich carbon sources for the growth of

filamentous fungi and for the production of lignocellulolytic

enzymes [ 4 , 47 ]. Sugar cane (Saccharum officinarum)isan

important commodity for many developing countries

such as Brazil and India, the two biggest producers of

sugarcaneintheworld[ 17 ]. In this context, sugar cane

bagasse (SCB) is the largest Brazilian agroindustrial

waste, amounting to approximately 217

: C. A. O. Ricart

Laboratory of Biochemistry and Protein Chemistry,

Department of Cellular Biology, University of Brasília,

Brasília, DF 70910-900, Brazil

D. P. G. Mendoza

L. P. Silva

Laboratory of Mass Spectrometry,

Embrapa Genetic Resources and Biotechnology,

Brasília, DF 70770-917, Brazil

380×10 9 kg/year.

Although part of the bagasse is employed for internal

–

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Bioenerg. Res. (2012) 5:363

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371

energy generation in the sugar cane mills, some 20% of it

is not used [ 17 , 19 ]. The bagasse piles have low economic

value and represent an environmental problem due to the

risk of spontaneous combustion. A carbon source is an

essential component for fermentation by microorganisms,

influencing their metabolism and cellular growth [ 23 ].

SCB is an economically viable alternative carbon source

for the production of industrial enzymes from filamentous

fungi, bacteria, and yeasts. The enzyme described in this

study provides a potential to reduce the amount of

agroindustrial waste that is generated in many countries

as well as to develop essential green technologies.

Materials and Methods

Chemicals

All substrates, N-bromosuccinimide (NBS), dithiothreitol

(DTT), 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), 1-

ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC),

diethyl pyrocarbonate (DEPC) and 2,2

′

-dithiopyridine

(DTP), oat-spelt xylan, carboxymethyl cellulose (CMC),

polygalacturonic acid, galactomannan, microcrystalline cellu-

lose (avicel), p-nitrophenyl-

- D -xylopyranoside (pNPX), p-

nitrophenyl-

- D -glucopyranoside (pNPG) and p-nitrophenyl-

-Xylanases are glycosyl hydrolases (GH) known to

hydrolyze the polysaccharides from lignocellulosic biomass

[ 5 , 31 ]. Most of the fungal

- L -arabinofuranoside (pNPA) were purchased from Sigma

Aldrich Chemical Co. Chromatography resins and filter

paper (Whatman no 1) were from GE Healthcare. SCB

(S. officinarum L., variety Java) was from a local source.

-xylanases belong to the GH10

and GH11 families. The enzymes belonging to the GH10

family show some catalytic versatility and have higher

molecular masses and lower isoelectric points than those

from the GH11 family, which can efficiently hydrolyze

highly branched xylans and have lower molecular weights

and higher pI values [ 18 ].

Many microorganisms are capable of producing

Residue Pretreatment

SCB (S. officinarum L., variety Java) was ground in a

bench grinder, thoroughly washed with tap water and

autoclaved at 121°C for 2 h. After being autoclaved, it

was dried at 65°C for 48 h and ground to form a

homogeneous blend. A fine powder was obtained and used

as a substrate for the fungus.

xylanases [ 32 , 43 , 44 ]. Among these, filamentous fungi

are particularly promising for industry because they secrete

large amounts of

-xylanases into the environment,

eliminating the need for cell lysis [ 12 , 21 ]. Many industrial

processes can be developed using fungi or other micro-

organisms as enzyme sources and, in many cases, the

efficiency can be improved by using pure enzymes [ 4 ]. The

fungus Aspergillus niger is widely used in many biotech-

nological processes including biopulping, biorefineries,

food and pharmaceutical industries [ 30 ]. The most impor-

tant advantages associated with its use are its safety for

humans during enzyme production [ 35 ] and its versatile

metabolism, allowing its growth on many substrates and

under many environmental conditions [ 33 ].

In two previous publications [ 26 , 27 ], ten fungal

species were isolated from decomposed wood in the

natural forest reserve of National Research Institute of

Amazonia (Brazil), purified, and evaluated for their

capacity to produce xylan-degrading enzyme activity

during growth in liquid medium containing oat-spelt xylan

as the carbon source. A. niger vanTieghemLPM93was

the most efficient at producing thermostable

Enzyme Production

A. niger van Tieghem LPM 93 was obtained from the

fungus culture collection of the Enzymology Laboratory,

University of Brasília, Brazil and was maintained in PDA

medium (2.0% potato broth, 2.0% dextrose, and 2.0% agar) at

28°C and cultured on SCB. The basal culture medium

composition (g/l) was as follows: 7.0 g KH 2 PO 4 ,2.0g

K 2 HPO 4 ,0.1gMgSO 4 .7H 2 O, 1.0 g (NH 4 ) 2 SO 4 ,0.6gyeast

extract and 1% of SCB at pH 7.0. A portion (5.0 ml) of an A.

niger van Tieghem LPM 93 spore suspension (10 8 spores/

ml) was introduced into an Erlenmeyer flask (2 l) containing

500 ml of liquid medium with agroindustrial residue as the

carbon source. SLC was carried out at a substrate concen-

tration of 1.0% (w/v) for 6 days at 28°C with agitation at

120 rpm. After the culture had grown, the medium was

passed through filter paper (Whatman No. 1). The resulting

filtrate, hereafter called crude extract, was stored at 5°C and

used for further isolation and characterization of the

-xylanase

[ 27 ]. The crude xylanase preparation from A. niger van

Tieghem LPM 93 was effective for improving brightness

and delignification of non-delignified and oxygen-

bleached samples of eucalyptus kraft pulp [ 27 ]. The aim

of the present study was to isolate and characterize a

xylan-degrading enzyme (XynI) produced by the meso-

philic fungus A. niger vanTieghemLPM93whengrown

by submerged liquid cultivation (SLC) containing SCB as

carbon source.

xylanase samples. For

-xylanase induction, aliquots were

harvested every 24 h during 50 days and used to estimate the

enzyme activity and protein concentration.

Enzyme Purification

The crude extract was concentrated approximately 10-fold

by ultrafiltration using an Amicon System (Amicon Inc.,

Bioenerg. Res. (2012) 5:363

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371

365

Beverly, MA 01915, USA) with a membrane having a

cutoff point of 10 kDa (PM 10) at 10°C and 2.5 kgf/cm 2 .

Aliquots (500 ml) of the ultrafiltrate were precipitated

with 60% (w/v) saturation of ammonium sulfate and

allowed to settle for 15 h at 5°C. The precipitate was

obtained by centrifugation at 4,500×g for 20 min at 4°C

and dissolved in 50 ml of 50 mM sodium phosphate

buffer, pH 7.0. It was designated as UFPM10. Aliquots of

UFPM10 (18 ml) were fractionated by gel-filtration

chromatography on a Sephadex G-50 (2.7×60.0 cm)

column pre-equilibrated with 50-mM sodium phosphate

buffer,pH7.0,containing0.15MNaCl.Fractions(5ml)

were collected at flow rate of 20 ml/h, and those

corresponding to

acetate (pH 3.0

–

6.0), 50 mM sodium phosphate (pH 6.0

–

7.5), or 50 mM Tris

9.0), respectively, at 45°C

and 50°C. All buffers, regardless of pH, were adjusted to

thesameionicstrengthwithNaCl.Theeffectsofseveral

salts (MgCl 2 ,MgSO 4 .7H 2 O, AlCl 3 ,HgCl 2 ,NaCl,ZnSO 4 ,

CaCl 2 ,KC ,FeC 3 ,FeSO 4 ,CuSO 4 ,MnC 2 ,CuC 2 ,

AgNO 3 , and CoCl 2 ) and other agents (DTP, DTNB,

EDC, DEPC, L -tryptophan, L -cysteine,

–

HCl (pH 7.5

–

iodoacetamide,

DTT,

xylanase activity were tested after 30 min of incubation at

29°C in the presence of the individual reagents at final

concentrations in the range of 0.5

-mercaptoethanol, NBS, SDS, and EDTA) on

–

10 mM, followed by

the standard

-xylanase assay under the following

-xylanase activity, hereafter named

XynI, were pooled and stored at 5°C.

conditions: 25

l of XynI, 75

l of the reagent, and

l of xylan. For the kinetic experiments, soluble and

insoluble xylans from oat spelt were used as substrates in

concentration ranges of 4

Enzymatic Assays

6.0 mg/ml, respec-

tively. The substrates were saturating and the enzyme

activities were proportional to the amount of enzyme

added. Soluble and insoluble xylans were prepared as

described by Filho et al. [ 8 , 9 ]. K m and V max values were

estimated from the Michaelis

–

50 and 0.5

–

Endoglucanase,

-xylanase, polygalacturonase and manna-

nase activities were determined by mixing 50

l of enzyme

sample with 100

lof1%(w/v) substrate (CMC, oat-spelt

xylan or polygalacturonic acid, sodium salt) or 0.5% (w/v)

substrate (galactomannan) at 50°C for 30 min, respectively.

Filter paper activity (FPase) [ 25 ] was determined using

150

Menten equation with a

nonlinear regression data analysis program [ 24 ]. Each

assay described above was repeated at least three times;

the standard deviation was less than 20% of the mean.

–

l of enzyme with filter paper as the substrate at 50°C

for 1 h. Avicelase activity was determined by mixing 50

of avicel suspension (1%, w/v) with 100

l of enzyme

sample at 50°C for 2 h. The amount of reducing sugar

released was measured using dinitrosalicylic reagent [ 28 ].

Activity was expressed as micromoles of reducing sugar

formed per minute per milliliter of enzyme solution, i.e., as

IU/ml. Glucose, xylose, mannose, and galacturonic acid

were used as standards.

Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) of

-xylanase samples were carried out as

described by Laemmli [ 22 ] using 12% gels. After electro-

phoresis, the protein bands were silver stained by the

method of Blum et al. [ 2 ]. For the detection of

-Xylosidase,

-glucosidase, and

-xylanase

activity, zymograms were carried out as described by Wang

et al. [ 43 ]. Replicate denaturing electrophoretic gel,

containing 0.1% oat-spelt xylan, was submitted to zymo-

gram analysis. It was stained for

-arabinofuranosidase activities were determined with the

substrates pNPX, pNPG, and pNPA, respectively [ 45 , 46 ].

Protein concentration was determined by the Bradford

assay [ 3 ] using bovine serum albumin as a standard.

-xylanase activity in a

Congo red solution (0.5 mg/ml) for 15 min at room

temperature and washed with 1 M NaCl to remove excess

dye and fixed with 1 M HCl. The molecular mass of XynI

was estimated by SDS-PAGE using low molecular mass

markers (GE Healthcare). For two-dimensional gel electro-

phoresis the samples were previously treated with the 2D-

Clean-Up Kit (GE Healthcare) and resuspended in 350

Enzyme Characterization

The influence of the temperature on

-xylanase activity

was measured by performing the standard activity assay at

temperatures ranging from 30°C to 70°C. The temperature

stability of

-xylanase was determined by pre-incubating

the enzyme samples at 45°C, 50°C, and 55°C and removing

samples at intervals to measure the activity as described

before. The enzyme stability was also measured using

50 mM sodium acetate buffer, pH 4.5 at 45°C and 50°C,

and in the presence of L- tryptophan or

lof

solution containing DTT (85 mM), Triton X-100 (2.5%, w/v),

IPG buffer at pH 3

10 (GE® 0.5%, w/v), urea (7 M),

thiourea (2 M), and isopropanol (10%). The samples were

applied to 18 cm pH 3

–

10 linear immobilized pH gradient

-mercaptoethanol

at a final concentration of 10 mM at 45°C. The influence

of pH on

strips (Immobiline

Dry Strips, GE Healthcare) by in gel

rehydration and analyzed by isoelectric focusing on an Ettan

IPGphor III apparatus (GE Healthcare). The second dimen-

sion (8

™

-xylanase activity was assessed by incubating

l of enzyme solution, 50

lofxylan(1%,w/v), and

15% polyacrylamide gradient, SDS-PAGE) was

carried out in BioRad Protean® II xi Cells.

–

l of each the following buffers: 50 mM sodium

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Bioenerg. Res. (2012) 5:363

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371

Mass Spectrometry

production and gradual release of hydrolytic enzymes for

the consumption of the substrate. The presence of soluble

sugars in the culture medium apparently did not signifi-

cantly inhibit the production of

Protein spots detected on the two-dimensional gel electro-

phoresis of the XynI purified fraction were excised, reduced

with DTT, alkylated with acrylamide, and digested with

trypsin (Promega, Madison, USA) as previously described

[ 29 ]. Protein digests were analyzed by peptide mass

fingerprinting (PMF) and peptide fragment fingerprinting

by matrix-assisted laser desorption/ionization time of flight

(MALDI-TOF) mass spectrometry using an Autoflex II

MALDI-TOF-TOF mass spectrometer (Bruker Daltonics,

Bremen, Germany). For analysis, 2

-xylanases, but it may

have been responsible for maintaining the enzyme activity

without significant variation from the second day of growth

of the fungus. It is also possible that the sugar released into

the environment was used by the fungus as an energy

source because there was no nutrient addition during the

period studied. The medium collected on the sixth day of

growth contained a protein peak that coincided with a peak

of high xylanolytic activity. The amount of total protein

varied during the growth period studied. This protein

profile probably includes other proteins, in addition to

lofeachdigestwas

mixedw th1

-cyano-4-

hydroxycinnamic acid in 70% (v/v) acetronitrile, 0.1%

(w/v) TFA) on the surface of an AnchorChip

lofma ix(10

xylanases, which are simultaneously produced and may be

involved in the complex process of SCB degradation.

Therefore, based on the growth curve of the fungus and in

order to obtain large amounts and a high diversity of

xylanolytic enzymes, we established six days for fungal

growth in liquid medium containing SCB.

The influence of SCB on the synthesis of

plate

(Bruker). External calibration was performed using a

peptide standard kit (Bruker Daltonics). Known trypsin

autolysis and keratin peaks were used for internal

calibration. Peptide masses (MH + ) were recorded in 750

to 3,000 Da range. The peptide mass spectra were

generated using the software FlexControl v. 2.4 (Bruker

Daltonics). The same software was used to acquire and

process the peak lists that was employed for database

searches using BioTools v. 2.0 (Bruker Daltonics) linked

to Mascot ( http://www.matrixscience.com/ ) against the

National Center for Biotechnology Information protein

database (NCBI; Bethesda, USA). Monoisotopic masses of

tryptic peptides were used to identify the proteins by PMF.

Error tolerance for peptide mass was lower than 100 ppm and

no restrictions were imposed on protein molecular mass.

Further search parameters were: one missed cleavage site for

trypsin, methionine oxidation as a variable modification and

propionamide cysteine (acrylamide alkylation) as a fixed

modification. Hits were considered significant if the protein

score exceeded the threshold score calculated by Mascot

software assuming a p value of <0.05.

™

-xylanase

was examined by electrophoresis under denaturing con-

ditions (data not shown). The SDS-PAGE of the crude

extract samples from the inducing medium revealed protein

bands with molecular weights ranging from 14 to 90 kDa.

A pronounced protein band of approximately 30 kDa was

detected between 2 and 16 days of incubation. It was

coincident with bands that stained for

-xylanase activity

after zymogram analysis (Fig. 1c ). After the 1st day of

incubation, a protein band with high molecular weight

(above 66 kDa) could be seen. Protein bands with low

molecular weight (less than 14 kDa) were only detected

after the 6th day of incubation.

Enzyme Purification

The pool of xylanolytic enzymes obtained from the SLC

containing SCB as the carbon source was isolated by a

combination of ultrafiltration, ammonium sulfate precipita-

tion and chromatographic procedures. The crude extract

was concentrated 10-fold by ultrafiltration.

Results and Discussion

Induction Profile

-Xylanase

activity was found in the retentate and ultrafiltrate. The

amount of protein of ultrafiltrate (0.5 mg) was much lower

than the retentate (25.5 mg). The xylanase activity of the

ultrafiltrate and concentrate were 0.5 and 1.20 IU/ml. For

further purification, the ultrafiltrate was precipitated with

60% of ammonium sulfate saturation. The

The induction profile during growth of A. niger van

Tieghem LPM 93 on SCB showed that

-xylanase activity

increased steadily without a lag and reached a plateau that

lasted from the second day to the end of the cultivation

period. The growth profile was accompanied by several

peaks of protein. A multiplicity of forms is commonly

described for

-xylanase

activity was only found in the precipitate, which, in turn,

was fractionated by gel-filtration chromatography on

Sephadex G-50 column (Fig. 1a ). A single peak of

-xylanases from fungi and bacteria as result

of differential mRNA processing and posttranslational

modifications [ 44 ]. This profile of induction suggests a

progressive access to the hemicellulose structures that

permeate the cellulose fibers of SCB, stimulating the

xylanase activity was eluted before a major peak of protein.

The purification procedure provided a yield of 9.5% and a

14.9-fold purification. Since other forms of

-xylanase

Bioenerg. Res. (2012) 5:363

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Fig. 1 a Chromatographic

profile of UFPM10 in a

Sephadex G-50 column. Total

protein (solid line) and xylano-

litic activity (dashed line).

b SDS-PAGE (12%) of the

purification steps of the crude

extract from A. niger van.

Tieghem LPM 93 grown on

liquid medium containing SCB.

Line 1, markers; line 2, crude

extract; line 3, UFPM10; line 4,

XynI. c Zymogram: line 1,

crude extract; line 2, UFPM10;

line 3, XynI

0.50

0.40

0.30

0.20

0.10

0.00

Fraction Number

kDa

1 2 3 4 1 2 3

97 –

66 –

45 –

30 –

20.1 –

14.4 –

were detected in the retentate, and these enzymes may act

synergistically to effect xylan breakdown, the fold purifi-

cation, and recovery values were underestimated [ 46 ]. This

phenomenon is often described during purification of

geous, especially for filamentous fungi [ 11 ], and this

property could be explored for biotechnological applica-

tions.

In support of

the SDS-PAGE result, zymogram

xylanases produced by fungi. Teixeira et al. [ 42 ] reported

yield and fold purification of 4.58 and 16.88, respectively

for

analysis revealed one

-xylanase activity band coincident

with that staining for protein. A clear hydrolysis activity

zone was formed against a dark background (Fig. 1c ).

-xylanase of Aspergillus awamori. The ultrafiltration

procedure retained most of the the

-xylanase activity in

the retentate. Moreover, comparison of these values with

those reported for the

Enzyme Characterization

-xylanases from other sources is not

very meaningful because of the high interlaboratory

variability in assays, and because

The substrate specificity of XynI was restricted to xylan. It

was devoid of measurable pectinase, mannanase, cellulase,

-xylanases differ from

one another with respect to their mechanism of action. The

apparent purity of the enzyme was demonstrated by SDS-

PAGE and zymogram analysis (Figs. 1b, c ). The gel under

denaturing conditions showed a single band. The molecular

mass of XynI was found to be in the range of 32

-glucosidase

activities. The specificity of XynI for xylan as substrate is

an important parameter for its use in pulp bleaching,

whereas in this process the enzyme has to be cellulase free.

The rate dependence of the

-xylosidase,

-arabinofuranosidase and

-xylanase reaction on soluble

and insoluble xylans followed Michaelis

33 kDa,

as estimated by SDS-PAGE. This is in agreement with the

range determined more accurately for the native enzyme by

using mass spectrometry, a value range that compares well

to previously reported data on A. awamori xylanase [ 42 ]. A

single peak was detected on the mass spectrum (Fig. 2 ).

Those results revealed the ability of

–

Menten kinetics.

Nonlinear regression data analysis determination of kinetic

parameters of XynI acting on soluble and insoluble oat-

spelt xylans showed that the enzyme had affinity only for

soluble xylans, with K m and V max values of 47.08 mg/ml

and 3.02 IU/ml, respectively, suggesting that the presence

of a particular type of substituent (arabinofuranosyl group)

in the vicinity would be a requirement for the action of

XynI. In this case, the substituent (arabinofuranosyl

residue) may be required for the proper orientation of xylan

in the catalytic site. Consistent with this possibility is the

–

-xylanases to change

their conformation and pass through membranes with a

cutoff of 10 kDa [ 9 , 36 , 40 ]. The ability to pass through the

small pores in the wood and thus to penetrate the

hemicellulose-lignin-cellulose matrix could be advanta-

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