ORIGINAL PAPER

Purification and characterization of endoxylanase Xln-1from Aspergillus niger B03

Georgi Dobrev Æ Boriana Zhekova Æ Ginka Delcheva ÆLidia Koleva Æ Nicola Tziporkov Æ Ivan Pishtiyski

Received: 8 May 2009 / Accepted: 30 June 2009 / Published online: 10 July 2009

� Springer Science+Business Media B.V. 2009

Abstract An extracellular endoxylanase was isolated

from the xylanolytic complex of Aspergillus niger B03.

The enzyme was purified to a homogenous form using

consecutive ultrafiltration and anion exchange chromatog-

raphy. The endoxylanase was a monomer protein with a

molecular weight of 33,000 Da determined by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis, and

34,000 Da determined by gel filtration. The optimal pH

and temperature values for the enzyme action were 6.0 and

60�C, respectively. Endoxylanase was stable at 40�C, pH

7.0 for 210 min. The thermal stability of the enzyme was

significantly increased in the presence of glycerol and

sorbitol. The enzyme activity was inhibited by Cu2?, Fe2?,

Fe3?, and Ag1?, and it was activated by Mn2?. The sub-

strate specificity and kinetic parameters of the enzyme

were determined with different types of xylans. Endoxy-

lanase displayed maximum activity in the case of oat spelt

xylan, with an apparent Km value of 8.19 mg/ml. The

substrate specificity and the product profile of the enzyme

suggested it to be an endoxylanase.

Keywords Endoxylanase � Purification �Characterization � Aspergillus niger � Xylan

Introduction

Xylan is an abundant biopolymer found in plant tissues as a

major component of the cell walls. It is a complex

molecule composed of b-1,4-linked xylose chains with

branches containing arabinose and 4-O-methylglucuronic

acid. Due to its heterogeneity and complexity, the com-

plete hydrolysis of xylan requires a large variety of coop-

eratively acting enzymes. Endo-1,4-b-D-xylanases (EC 3.2.1.8)

randomly cleave the xylan backbone, b-D-xylosidases (EC

3.2.1.37) cleave xylose monomers from the non-reducing

end of xylooligosaccharides. The removal of side groups

is catalysed by a-L-arabinofuranosidases (EC 3.2.1.55),

a-D-glucuronidases (EC 3.2.1.139), acetylxylan esterases

(EC 3.1.1.72), ferulic acid esterases (EC 3.1.1.73) and

p-coumaric acid esterases (EC 3.1.1.-) (Collins et al. 2005).

Xylanolytic enzymes are produced by various groups of

microorganisms, including moulds, bacteria, and yeasts.

Filamentous fungi have been widely studied for their

ability to produce xylanases, and the genus Aspergillus has

displayed a great capacity for biosynthesis of the enzymes

(Kulkarni et al. 1999; Subramaniyan and Prema 2002; de

Vries and Visser 2001).

Endoxylanase plays a key role in xylan hydrolysis

(Collins et al. 2005). Endoxylanases from mould species

are usually found as a part of a multicomponent system

composed of enzymes with specialized functions. The

biosynthesis of a multienzyme complex is supposed to be a

strategy for superior hydrolysis, used by the microorgan-

ism. The xylosidic linkages in xylan molecules are not all

equivalent and equally accessible to xylanolytic enzymes.

The heterogeneous nature of xylan may be one of the

reasons for biosynthesis of multiple endoxylanases. The

divergent specificity of these enzymes could play a sig-

nificant role in their synergistic action towards the complex

substrate. Multiple endoxylanases have been reported in

numerous microorganisms (Latif et al. 2006; Murty and

Chandra 1992; Ryan et al. 2003; Silva et al. 1999; Wong

et al. 1988).

G. Dobrev � B. Zhekova (&) � G. Delcheva � L. Koleva �N. Tziporkov � I. Pishtiyski

Department of Biochemistry and Molecular Biology, University

of Food Technologies, 26 Maritza Blvd., 4000 Plovdiv, Bulgaria

e-mail: zhekova_b@yahoo.com

123

World J Microbiol Biotechnol (2009) 25:2095–2102

DOI 10.1007/s11274-009-0112-5

Production, isolation and characterization of xylan-

degrading enzymes have attached much attention for their

important practical applications in various industrial pro-

cesses, including modification of cereal-based foodstuffs,

improvement of animal feedstock digestibility, and delig-

nification of paper pulp.

The aim of the present research was to isolate and purify

an endoxylanase enzyme from the xylanolytic system of

Aspergillus niger B03 and to determine its main bio-

chemical properties.

Materials and methods

Strain cultivation

Aspergillus niger B03 was a gift from Biovet JSC (Pesh-

tera, Bulgaria). The strain was cultivated in 500-ml

Erlenmeyer flasks containing 50 ml of nutrient medium

with the following composition (g/l): corn cob 24.0, wheat

bran 14.6, malt sprouts 6.0, (NH4)2HPO4 2.6, urea 0.9. The

flasks were inoculated with 2.5 ml of 7-day-old culture,

containing 3 9 107 to 3 9 108 spore/ml and the strain was

grown at 28�C for 70 h with a 180 rev/min rotary shaking

(Dobrev et al. 2007). The mycelial biomass was removed

by filtration through Whatman No. 1 filter paper, and the

culture filtrate was used as a source of xylanolytic activity.

Purification procedure

The purification procedure consisted of ultrafiltration with

10,000 Da membrane and anion-exchange chromatography.

Anion-exchange chromatography was carried out in an

automated chromatographic system (Pharmacia Biotech)

using a DEAE-Sephadex A50 column (2.6 9 40 cm)

equilibrated with 10 mM sodium acetate buffer with pH

5.5. A sample, containing 51.25 mg of protein was loaded

onto the column and the unbound protein was washed with

the buffer solution. Elution was performed with gradient of

NaCl at a flow rate of 9.6 ml/h. Fractions of 11 ml were

collected, monitored at 280 nm, and assayed for xylano-

lytic and protein content.

Determination of Xln-1 molecular weight

The molecular weight of Xln-1 was determined by sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) and gel filtration.

SDS–PAGE was performed by the method of Laemli

(1970) with 10.0% polyacrylamide gel at a constant current

of 10 mA. The following protein calibration kit (Merck)

was used: ovotransferrin (78,000 Da), albumin (66,250 Da),

ovalbumin (45,000 Da), carbonic anhydrase (30,000 Da),

myoglobin (17,200 Da), cytochrome c (12,300 Da). The

staining procedure was done with Coomassie Blue R-350.

Gel filtration was performed on an automated chromato-

graphic system (Pharmacia biotech), equipped with a

Sephadex G-75 column (2.6 9 70.0 cm). The proteins were

eluted using 0.05 M solution of NaCl at a flow rate of

19.8 ml/h. Fractions of 7.0 ml were collected. The molecular

weight of the enzyme was estimated using the following

protein calibration kit: cytochrome c (12,300 Da), carbonic

anhydrase (30,000 Da), ovalbumin (45,000 Da), and bovine

albumin (67,000 Da).

Effect of pH and temperature on Xln-1 activity

and stability

The effect of pH and temperature on Xln-1 activity was

evaluated in the range of pH 2.5–8.0 and 30–90�C,

respectively. Citrate buffer solutions were used for pH

profile determination.

Thermal and pH stability was determined by pre-incu-

bating the enzyme at various temperatures, notably, 40 and

50�C, at pH 5.0 and 7.0 for different time intervals.

The influence of polyhydric alcohols was studied by pre-

incubating the enzyme in the presence of 10% (w/v)

glycerol and 10% (w/v) sorbitol at 60�C and pH 5.0.

Effect of metal ions on Xln-1 activity

The effect of metal ions on Xln-1 activity was assessed by

pre-incubating the enzyme in solutions containing 5, 10,

and 20 mM of different salts for 30 min at 30�C.

Estimation of Xln-1 substrate specificity

The substrate specificity of Xln-1 was tested with 10 mg/ml

oat spelt xylan (Sigma), birchwood xylan (Sigma), beech-

wood xylan (Sigma), carboxymethyl cellulose (Sigma),

starch (Fluka) and pectin (Fluka) by determination of the

reducing sugars by the dinitrosalicylic acid method (Miller

1959). The enzyme activity against remazol brilliant vio-

let birchwood xylan (RBV-xylan) was determined as

described by Ronen et al. (1991). When 1.0 mM p-nitro-

phenyl-b-D-xylopyranoside, and p-nitrophenyl-b-D-gluco-

pyranoside (Sigma) were used as substrates, the activity

was determined as described by Ponpium et al. (2000).

Kinetic parameters determination

Kinetic parameters were determined using oat spelt,

birchwood and beechwood xylan as substrates in the range

2096 World J Microbiol Biotechnol (2009) 25:2095–2102

123

of 3.0–9.0 mg/ml initial concentration by applying the

Lineweaver-Burk transformation.

Investigation of xylan hydrolysis

Xylan hydrolysis was studied by determination of the rel-

ative viscosity of xylan solution and the xylooligosaccha-

rides formed by Xln-1.

Xylan hydrolysis was performed with 40.0 mg/ml

birchwood xylan, 12 U of Xln-1 at pH 5.0 and 40�C. The

relative viscosity of xylan solution was measured with an

Oswald viscometer (Delcheva et al. 2007) and the reducing

sugars content in the reaction mixture was determined by

the dinitrosalicylic acid method (Miller 1959).

The composition of the hydrolysates was analysed by

thin layer chromatography (TLC) on a silica gel plate

(Merck 20 9 20 cm). The hydrolysis was carried out with

birchwood xylan 10.0 mg/ml, 12 U of Xln-1 at pH 5.0 and

40�C for 19 h. Samples of 2.0 ll from 2.0 mg/ml solutions

of xylooligosacharides mixture (Wako), 1.0 mg/ml xylose,

and 1.0 ll from the reaction mixtures were applied to the

start line. The mobile phase consisted of propanol, ethyl-

acetate, and water in a ratio of 7:1:2. The chromatogram

was developed once until the mobile phase reached 15 cm

from the start line. After being air-dried in a horizontal

position it was sprayed with a mixture, containing 0.5 g

carbazol, 5.0 ml sulphuric acid and 95.0 ml ethanol and

heated for 10 min in an oven at 120�C. The carbohydrates

appeared as purple spots on a blue background (Adachi

1964).

For HPLC analysis of xylose, the reaction was stopped

by adding a twofold greater volume of ethanol, and the

unhydrolysed xylan was removed by centrifugation. A

2 ml sample of the supernatant was evaporated to a con-

stant dry weight, and the dried sample was dissolved in

0.5 ml of distilled water. Xylose concentration was ana-

lysed using HPLC Waters system with refractive index

detector R401 on a carbohydrate column (waters). The

mobile phase was a mixture of acetonitrile and water in a

ratio of 87:13 with a flow rate of 0.6 ml/min, and the

column temperature was 30�C.

The activation energy of enzymatic and acidic hydro-

lysis of birchwood xylan was determined by using the

Arrhenius plot in the range of 30–70�C.

Determination of xylanolytic activity

Xylanolytic activity was determined by mixing 0.2 ml

enzyme solution with 1.8 ml 10.0 mg/ml solution of oat

spelt xylan in 100 mM sodium acetate buffer with pH 5.0,

at 50�C for 10 min (Bailey et al. 1992). One unit of

xylanolytic activity is defined as the amount of enzyme

releasing 1 lmol of xylose equivalent per minute under the

assay conditions.

Determination of carbohydrate content of Xln-1

and deglycosylation treatment

The carbohydrate content of Xln-1 was determined accord-

ing to the method of Dubois et al. (1956). Deglycosylation of

the enzyme was performed by treatment with PNGase F

(BioLabs) at pH 7.5 and 37�C for 4 h according to the

instructions of the manufacturer.

Determination of protein concentration

Protein concentration was determined by the Lowry

method with bovine serum albumin (Sigma) as a standard.

Results and discussion

Purification of endoxylanase (Xln-1)

The culture liquid of Aspergillus niger B03, containing

xylanolytic activity was subjected to a purification proce-

dure, consisting of two consecutive steps—ultrafiltration

and column chromatography (Table 1). After the ultrafil-

tration, xylanolytic activity was present in the retentate

with a yield of 85.06%. For further purification, the

retentate was subjected to anion-exchange chromatography

on DEAE-Sephadex A50 (Fig. 1). Two peaks showing

xylanolytic activity were detected. The first peak (Xln-1)

was eluted together with the unabsorbed protein. This

indicated that the enzyme was characterized with a pI value

higher than 5.5. Xln-1 was purified with a yield of 4.16%

and a purification factor of 3.95. The low yield and puri-

fication fold values may be explained by the presence of

other xylan-degrading enzymes in the crude retentate. The

Table 1 Procedures for Xln-1

purificationStep Total

activity (U)

Total

protein (mg)

Specific

activity (U/mg)

Purification

(fold)

Yield (%)

Crude supernatant 10,194.00 76.00 134.13 1.00 100.00

Ultrafiltration 8,671.05 51.25 169.19 1.26 85.06

DEAE-Sephadex A50 423.90 0.8 529.88 3.95 4.16

World J Microbiol Biotechnol (2009) 25:2095–2102 2097

123

analysis by anion-exchange chromatography revealed two

xylanolytic enzymes, and in a previous study a b-xylosi-

dase was isolated from the culture liquid after gel filtration

(Delcheva et al. 2008). These enzymes may act synergis-

tically for the attainment of complete xylan hydrolysis.

Similar results have been reported by other authors (Franco

et al. 2004; Silva et al. 1999).

The purity of Xln-1 was tested by SDS–PAGE. The

protein was detected as a single band on the electrophe-

rogram, indicating a homogeneous enzyme. The homoge-

neity of Xln-1 was confirmed by gel filtration on Sephadex

G50. The analysis revealed a single protein peak, corre-

sponding to all xylanolytic active fractions.

Molecular weight of Xln-1

The results from SDS–PAGE and gel filtration were used

for estimation of the molecular weight of Xln-1. It was

determined to be 33,000 Da by SDS–PAGE and 34,000 Da

by gel filtration. The close values achieved by both meth-

ods suggested Xln-1 to be a monomer protein. The analysis

of the carbohydrate content showed that the enzyme had a

high degree of glycosylation, estimated to be 74.1%.

Similar results were found for other xylanases (Mansour

et al. 2003).

Effect of pH and temperature on Xln-1 activity

The effect of pH on the enzyme activity is presented on

Fig. 2. For comparison, data about the crude enzyme is

included. Xln-1 showed a maximum activity at pH 6.0,

whereas the optimal pH for the crude enzyme was 4.0.

The optimal temperature value for maximal xylanolytic

activity was 60�C for Xln-1, and 50–55�C for the crude

enzyme (Fig. 3).

Effect of pH and temperature on Xln-1 stability

An important feature of industrially applied enzymes is

their stability. The stability of Xln-1 was studied at 40�C,

and 50�C, at pH 5.0, and 7.0 (Figs. 4, 5). The stabilities of

Xln-1 and the crude enzyme differed significantly at pH 7.0

(Figs. 4b, 5b). The purified Xln-1 retained 80% of its

activity for 120 min, when incubated at 40�C and pH 7.0

(Fig. 4b). The crude enzyme showed only 40% residual

activity at the same conditions.

A more significant difference was observed at 50�C and

pH 7.0 (Fig. 5b). Xln-1 showed 86% residual activity after

45 min of incubation, and the crude enzyme retained only

6% of the initial activity. When the enzymes were incubated

at pH 5.0, they showed a similar behavior (Figs. 4a, 5a).

These results may be explained by the complex action of

the xylanolytic enzymes contained in the crude enzyme

preparation. Probably, some of the xylanolytic enzymes

Fig. 1 Xln-1 purification by anion exchange chromatography on

DEAE-Sephadex A50: the symbols (u) represent xylanolytic activity,

solid lines represent A280, and broken lines represent NaCl concen-

tration

0

20

40

60

80

100

2 3 4 5 6 7 8

pH

Rel

ativ

eac

tivity

(%)

Fig. 2 Effect of pH on Xln-1 activity: (u) homogenous enzyme, (e)

crude enzyme

0

20

40

60

80

100

25 30 35 40 45 50 55 60 65 70 75

Temperature ( C)°

Rel

ativ

e ac

tivity

(%

)

Fig. 3 Effect of temperature on Xln-1 activity: (u) homogenous

enzyme, (e) crude enzyme

2098 World J Microbiol Biotechnol (2009) 25:2095–2102

123

were inactivated at pH 7.0, which led to a decrease in the

total activity in the crude enzyme preparation.

In order to explain the high stability of Xln-1 at 50�C,

the thermal stability of the enzyme after a deglycosylation

treatment with PNGase F was also studied (data not

shown). Deglycosylated enzyme was incubated at 50�C, at

pH 5.0 for 30 min. In this case a change in the residual

activity was noticed. The residual activity of the deglyco-

sylated enzyme was 42%, and the untreated Xln-1 showed

75% of its activity. This result confirmed the hypothesis

that Xln-1 is a glycoprotein. Glycosylation of Xln-1

probably improves its stability.

Since protein stabilization by low molecular weight

solutes is a widely used strategy (Lemos et al. 2000;

Sa-Pereira et al. 2004), the stabilizing effect of glycerol and

sorbitol on Xln-1 was studied (Fig. 6). Glycerol and sor-

bitol showed a good stabilizing effect. The enzyme was

totally inactivated, when incubated for 150 min at the

conditions used, whereas in the presence of glycerol and

sorbitol 50% of residual activity was detected at the same

conditions. Probably the polyhydric alcohols stabilize the

enzyme by decreasing the water activity by formation of

hydrogen bonds with water. This results in a low water

environment and a decrease in the degree of protein

unfolding (Lemos et al. 2000).

(a)

0

20

40

60

80

100

0 30 60 90 120 150 180 210

0 30 60 90 120 150 180 210

Time (min)

Time (min)

Res

idua

l act

ivity

(%

)R

esid

ual a

ctiv

ity (

%)

(b)

0

20

40

60

80

100

Fig. 4 Stability of Xln-1 at 40�C, (a) pH 5.0 and (b) pH 7.0: (u)

homogenous enzyme, (e) crude enzyme

0

20

40

60

80

100(a)

(b)

0 30 60 90 120

Time (min)

Time (min)

Res

idua

l act

ivity

(%

)R

esid

ual a

ctiv

ity (

%)

0

20

40

60

80

100

0 30 60 90 120

Fig. 5 Stability of Xln-1 at 50�C, (a) pH 5.0 and (b) pH 7.0: (u)

homogenous enzyme, (e) crude enzyme

0

20

40

60

80

100

0 25 50 75 100 125 150

Time (min)

Res

idua

l act

ivity

(%

)

Fig. 6 Effect of polyhydric alcohols on Xln-1 stability: (h) glycerol

(4) sorbitol, (u) reference

World J Microbiol Biotechnol (2009) 25:2095–2102 2099

123

Effect of metal ions on Xln-1 activity

The effect of different concentrations of metal ions on Xln-

1 activity is summarized in Table 2. Xln-1 was totally

inactivated, when incubated in the presence of Cu2?, even

at the lowest tested concentration. Fe2? and Fe3? also

showed a negative effect. The presence of heavy metals

resulted in a decrease in enzyme activity. Xln-1 was sig-

nificantly activated by Mn2?. The relative activity reached

160% at 10 mM concentration of Mn2?.

Substrate specificity of Xln-1

Substrate specificity of the homogenous Xln-1 is presented

in Table 3. The enzyme showed a higher activity against

oat spelt xylan in comparison to birchwood, and beech-

wood xylans. This fact may be explained by the different

composition and structure of the substrates. Birchwood and

beechwood xylans are obtained from hard wood, and they

are found to be in the form of 4-O-methylglucuronoxylan.

4-O-methyl-glucuronic acid is linked to every tenth xylose

unit by a-1,2-glycoside bond. Furthermore, about 60–70%

of sugar C-2 and/or C-3 hydroxyl groups are esterified with

acetic acid (Beg et al. 2001; Coughlan and Hazlewood

1993). Xylan acetylation limits severely the points for

enzyme hydrolysis (Kulkarni et al. 1999; Subramaniyan

and Prema 2002). This is a probable explanation for the

lower activity against these xylan types.

Xln-1 showed activity against coloured RBV-birchwood

xylan, and no activity was detected when p-nitrophenyl-

b-D-xylopyranoside was used as a substrate. These results

indicated Xln-1 to be an endoxylanase.

Kinetic parameters of Xln-1

The kinetic parameters of the enzyme in the case of dif-

ferent substrates showed that the highest apparent Km value

was in the case of oat spelt xylan (8.19 mg/ml). Signifi-

cantly lower values were determined for birchwood

(3.01 mg/ml) and beechwood (1.57 mg/ml) xylans, indi-

cating a greater affinity of the enzyme. This could be

explained by the lower degree of branching in these

xylan types (Sarbu et al. 2003). On the other hand, the

high acetylation degree of the substrates may result

in lower Vmax values, which were respectively oat spelt

xylan 9.5, birchwood xylan 6.64 and beechwood xylan

5.69 mg/ml min.

Xylan hydrolysis by Xln-1

Xylan hydrolysis was studied by assaying the change in the

relative viscosity and the reducing sugars content (Fig. 7).

A sharp decrease of xylan viscosity and a corresponding

increase in reducing sugars content was detected during the

first 10 min of the reaction. The rates of viscosity decrease

and reducing sugars increase were reduced after 15 min of

reaction. These results confirmed the hypothesis that the

enzyme is an endoxylanase.

The composition of xylan hydrolysates was analysed by

TLC. At the reaction conditions used Xln-1 formed xylo-

biose, xylotriose and xylooligosacharides with a higher

degree of polymerization. Xylose was not detected on the

chromatogram. Xylose concentration was determined to be

0.11 mg/ml by HPLC. This value represented only 6.1% of

Table 2 Effect of metal ions on Xln-1 activity

Metal ions Residual activity (%)

Concentration (mM)

5 10 20

None 100.00 100.00 100.00

Pb(NO3)2 36.89 33.77 24.75

(CH3COO)2Pb 57.82 56.79 57.03

(CH3COO)2Cd 72.51 69.49 71.48

AgNO3 78.79 0 0

NaCl 87.19 87.21 86.97

KCl 90.63 91.91 92.35

CaCl2 98.75 95.43 95.87

CuCl2 0 0 0

CuSO4 0 0 0

CoCl2 108.12 98.65 93.52

FeCl3 60.63 35.23 23.76

FeSO4 39.70 39.76 38.25

MgSO4 94.38 94.72 93.91

MnCl2 136.56 155.50 117.50

MnSO4 140.62 163.90 131.30

Li2SO4 89.38 88.46 87.21

ZnSO4 70.32 66.27 67.43

Table 3 Substrate specificity of Xln-1

Substrate Specific

activity (U/mg)

Oat spelt xylan 649.25

Birchwood xylan 497.17

Beechwood xylan 475.09

RBV-birchwood xylan 240.57

Carboxymethyl cellulose 0

Starch 0

Pectin 0

p-Nitrophenyl-b-D-xylopyranoside 0

p-Nitrophenyl-b-D-glucopyranoside 0

2100 World J Microbiol Biotechnol (2009) 25:2095–2102

123

the reducing sugars, formed by the enzyme. These results

supported the hypothesis for the endo mechanism of

Xln-1. A similar product profile was reported for other

endoxylanases (Lv et al. 2008; Maalej et al. 2008; Martınez-

Trujillo et al. 2003).

The activation energies of enzymatic and acidic hydro-

lysis of birchwood xylan were determined to be 31.73, and

61.21 kJ/mol, respectively. The activation energy of the

reaction performed by Xln-1 was twofold lower than the

value obtained for the acidic hydrolysis. These results

confirmed the advantages of enzyme processes in respect to

the lower temperature needed.

A comparison of the properties of Xln-1 and endoxy-

lanases produced by other mould strains is presented in

Table 4. Most endoxylanases are reported to be monomer

proteins with a molecular weight in the range of 19.0–

52.0 kDa. The optimal value of pH for maximal endoxy-

lanase activity vary in the range 3.0–6.0, and the optimal

temperature value is 45–60�C (Table 4). The properties of

the isolated Xln-1 are similar to those of family 11 gly-

cosidehydrolases. Xln-1 is an endo acting enzyme, it does

not show activity against aryl-b-glycosides, and is char-

acterized by a low molecular weight.

Conclusion

A xylan-degrading enzyme (Xln-1) was isolated from

the xylanolytic enzyme complex of Aspergillus niger B03.

Xln-1 was highly active against different xylans, it

hydrolysed RBV-xylan and did not act on aryl-b-glycosides.

The enzyme caused a decrease in xylan viscosity and did not

release xylose. These results indicated that Xln-1 is an

endoxylanase. The specific properties of Xln-1 (optimal pH

value at pH 6.0, optimal temperature value at 60�C, signifi-

cant stability at 50�C, endo action mechanism) allow the

enzyme to be applied in bread production.

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30Time (min)

Rel

ativ

e vi

scos

ity

0

4

8

12

16

Red

ucin

g su

gars

(m

mol

/L)

Fig. 7 Xylan hydrolysis by Xln-1 (40.0 mg/ml birchwood xylan, 12

U Xln-1, pH 5.0, 40�C: (u) relative viscosity, (m)

Table 4 Comparative characterization of endoxylanases from Aspergillus sp.

Strain Enzyme Mm

(kDa)

pI Optimal

pH

Optimal

temperature (�C)

Km

(mg/ml)

Reference

Asp. niger B03 Xln-1 33a

34b

[5.5 6.0 60 8.19c

3.01d

This work

Asp. niger A-3 P-2

P-3

28.5

24.5

5.25

5.15

4.0

4.5

60

40

1.78d

2.20d

Bakalova et al. (1995)

Asp. oryzae F-III-II-I 38.5a 4.58 4.5 37 5.56d Bakalova et al. (1996)

Asp. oryzae Not cited 46.5 3.6 5.0 55 – de Vries and Visser (2001)

Asp. awamori Not cited 39

23

26

5.7–6.7

3.7

3.3–3.5

5.5

5.0

4.0

55

50

45–50

– Subramaniyan and Prema (2002)

Asp. sojae Not cited 32.7

35.5

3.5

3.8

5.0

5.5

60

50

– Subramaniyan and Prema (2002)

Asp. fumigatus Xylanase II 19 – 5.5 55 3.01c Silva et al. (1999)

Asp. aculeatus F III 52 3.8 5.0 70 – de Vries and Visser (2001)

Asp. carneus M34 Not cited 18.8a 7.7–7.9 6.0 50 – Fang et al. (2008)

Asp. ficuum AF-98 Not cited 35a – 5.0 45 3.747d Lu et al. (2008)

a Determined by SDS–PAGEb Determined by gel filtrationc Substrate oat spelt xyland Substrate birchwood xylan

World J Microbiol Biotechnol (2009) 25:2095–2102 2101

123

Acknowledgments The research was carried out with a strain of

Aspergillus niger, provided by Biovet JSC. The work was supported

by the Grant-in-Aid for Scientific Research of Priority area (Agri-

cultural science) from the Ministry of Education of Bulgaria.

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