ORIGINAL PAPER

Purification and characterization of a novel extracellularb-1,3-glucanase complex (GluGgt) secretedby Gaeumannomyces graminis var. tritici

Yong-ting Yu Æ Zhen-sheng Kang ÆHenrich Buchenauer Æ Li-li Huang

Received: 11 May 2009 / Accepted: 9 July 2009 / Published online: 22 July 2009

� Springer Science+Business Media B.V. 2009

Abstract b-1,3-Glucanases produced by the take-all dis-

ease pathogen Gaeumannomyces graminis var. tritici (Ggt)

have been suggested to be implicated in infection and colo-

nization process of the pathogen in wheat. For further

studying the role of these enzymes in pathogenesis by

cytochemical technique, an extracellular b-1,3-glucanase

complex (GluGgt), was purified from culture filtrate of Ggt

by (NH4)2SO4 precipitation, hydrophobic-interaction,

anion-exchange and size-exclusion chromatography. The

complex consisted of two interconvertible isoforms (Ia and

Ib), both active proteins Ia and Ib appeared to be glycosylated

in native polyacrylamide gel electrophoresis (PAGE). The pI

of the active proteins Ia and Ib determined by IEF-PAGE

were 6.3 and 6.4, respectively. GluGgt yielded two subunits

with molecular masses of 66.2 and 56.0 kDa, respectively, in

15% SDS–PAGE. The complex had a pH optimum of 5.0 and

the optimal temperature was 50�C. The enzyme complex

proved to be stable at a temperature up to 60�C and retained

an optimal high activity in the pH range from 4.0 to 7.0.

Enzyme activity of GluGgt was obviously stimulated by

Mn2? ions and moderately inhibited in the presence of Hg2?

and Co2? ions and KMnO4. The Km of GluGgt was estimated

to be 1.08 mg ml-1 for laminarin as substrate and the Vmax

0.20 lmol min-1.

Keywords Gaeumannomyces graminis var. tritici �Exo-1 � 3-b-Glucanase � Glycoprotein �Interconvertible isoforms

Introduction

b-1,3-Glucanases, generally classified into endo-1,3-b-

glucanases (laminarinase, EC 3.2.1.6 and 1,3-b-glucan

glucanohydrolase, EC 3.2.1.39) and exo-1,3-b-glucanases

(1,3-b-glucan glucanohydrolase, EC 3. 2.1.58) according to

their hydrolytic action on specific substrates, are widely

distributed among viruses (Sun et al. 2000), bacteria

(Nagata et al. 1990), filamentous fungi (Rapp 1989; Stah-

mann et al. 1993), actinomycetes, yeasts, molluscs and

higher plants (Pitson et al. 1993). They have been reported

to play a variety of physiological and ecological roles such

as carbon source supplying and transferring, infection and

defense factors in parasitism or pathogenesis (Pitson et al.

1993; Sun et al. 2006; Kemp et al. 1999).

In recent years, there has been increasing interest in 1,3-

b-glucanases which are implicated in plant-pathogen

interaction (Tenberge et al. 1999; Kang and Buchenauer

2002; Tonon et al. 2002; Zhen and Li 2004), but there are

very few reports with regard to the role of the 1,3-b-glu-

canases of pathogen origin. The filamentous fungus

Gaeumannomyces graminis (Sacc.) von Arx & Oliver var.

tritici Walker (Ggt) causes wheat take-all disease, a

destructive root disease of wheat worldwide. The pathogen

infects wheat from the roots by infectious hyphae, which

develop from mycelium surviving saprophytically in the

dead roots and stem bases in the soil. The disease starts as

Y. Yu � Z. Kang � H. Buchenauer � L. Huang (&)

College of Plant Protection and Shaanxi Key Laboratory

of Molecular Biology for Agriculture, Northwest A&F

University, 712100 Yangling, Shaanxi,

People’s Republic of China

e-mail: huanglili@nwsuaf.edu.cn

Y. Yu

e-mail: yytxn@nwsuaf.edu.cn

H. Buchenauer

Insitute of Phytomedicine (360), University Hohenheim,

70593 Stuttgart, Germany

123

World J Microbiol Biotechnol (2009) 25:2179–2186

DOI 10.1007/s11274-009-0123-2

root rot and progresses upward into the bases of the stems,

consequently disrupting the flow of water to the tops and

cause premature death of the infected plant. During

infection and colonization by Ggt, one of the typical host

reactions is the formation of lignitubers in the infected

wheat root cells, which like papillae, may act as physical

barriers to retard further spread of the pathogen (Skou

1975). Cytochemical tests of lignitubers demonstrated the

presence of callose, apart from lignin, xylan and cellulose

(Kang et al. 2000). This suggests that Ggt may secrete

corresponding cell wall-degrading enzymes such as cellu-

lases, xylanases and b-1,3-glucanases to disintegrate these

morphological barriers during infection of wheat roots. The

biochemical study of Dori et al. (1995) has confirmed that

Ggt can produce cellulases and xylanases in vitro. In our

previous work the presence of b-1,3-glucanase activities in

axenic culture filtrates from Ggt has been reported (Wang

et al. 2003). The objective of this study was to purify and

partially characterize the b-1,3-glucanases secreted by G.

graminis var. tritici, which is a prerequisite to study the

biological role of these enzymes in pathogenesis at the

host-parasite interface in vivo.

Materials and methods

Organism and culture conditions

Fungal isolate 9826 of Ggt was used in this study (Wang

et al. 2003). The fungus was cultivated on potato dextrose

agar (PDA) in Petri dishes. For production of 1,3-b-glu-

canases, 150 ml of minimal synthetic medium (MSM, pH

5.5) (Van Hoof et al. 1991) in 0.25 l Erlenmeyer flasks

amended with wheat bran (10 g l-1) as a carbon source and

beef extract (10 g l-1) as the nitrogen source were used.

The nutrient medium was inoculated with 5 PDA disks

grown with Ggt and the flasks were incubated on a rotary

shaker (150 rev min-1) at 25�C for 15 days. Culture fil-

trate was collected by filtration through Whatman no. 1

filter paper and used as source of b-1,3-glucanase.

Enzyme and protein assay

The b-1,3-glucanase activity was measured by mixing

100 ll of sample with 100 ll 50 mM sodium acetate (pH

5.5) containing 200 lg laminarin (Sigma). The mixture

was incubated at 37�C for 30 min and the reducing sugar

produced was determined by the method described by

Rapp (1989). Glucose was used as the standard. One unit of

activity of b-1,3-glucanase is defined as the amount of

enzyme that catalyses the release of reducing groups

equivalent to 1 lmol glucose per min at 37�C. Protein in

the enzyme solution was measured by the Bradford method

using bovine serum albumin (BSA, Sigma) as the standard.

Specific activity was expressed in units (U) per milligram

of protein.

Purification of b-1,3-glucanase secreted by Ggt

All procedures of the b-1,3-glucanase purification were

carried out at 20�C unless otherwise stated. Ammonium

sulphate was added to the culture filtrates (2,240 ml) to

60% saturation. After 48 h at 4�C, the filtrate was centri-

fuged at 22,0009g for 15 min. The precipitate was dis-

solved in 30 ml 50 mM sodium acetate buffer (pH 5.5) and

the insoluble material was removed by centrifugation at

8,0009g for 10 min. The supernatant was dialysed in

cellulose dialysis tubes (Bioshorp, nominal MW cut-off

8,000–14,000) at 4�C against 4 l of the same buffer and

freeze-dried. Most of the freeze-dried enzyme was dis-

solved in 20 mM Tris–HCl (pH 7.5, containing 2 M NaCl)

and applied to a phenyl-Sepharose-6 fast flow (high sub)

pre-packed column (0.7 9 2.5 cm, Amersham Biosci-

ences, Uppsala, Sweden) pre-equilibrated with the same

buffer, using a High Protein Liquid Chromatography sys-

tem (AKTA Purifier 10 system, Amersham Biosciences).

The column was eluted with 2 M NaCl in 20 mM Tris–

HCl (pH 7.5) followed by elution with a linear gradient of

2.0 to 0 M NaCl in Tris–HCl buffer at a flow rate of

0.5 ml min-1. Fractions of 1.5 ml were collected and

monitored for protein (A280) and b-1,3-glucanase activity.

Active fractions were pooled, desalted by dialysed against

de-ionized water (4�C) and loaded onto a DEAE-Sephar-

ose fast flow pre-packed column (0.7 9 2.5 cm, Amer-

sham Biosciences) which had been equilibrated with

20 mM borate-NaOH buffer (pH 9.0). The bound protein

was eluted by a linear gradient of 0–1 M NaCl after

washing the column with 6 ml of 20 mM borate-NaOH

buffer (pH 9.0). The flow rate was 0.5 ml min-1 and

1.5 ml-fractions were collected. Active fractions of bound

parts were pooled, desalted and freeze-dried. This active

fraction was dissolved in 25 mM phosphate buffer (pH 6.5,

containing 0.15 M NaCl) and separated by size-exclusion

chromatography on a Superdex 200 10/300 GL

(1.0 9 30 cm, Amersham Biosciences) pre-packed column

that was equilibrated with the same buffer. The column

was eluted with the same buffer at a flow rate of

0.5 ml min-1 and fractions of 1 ml were collected. The

b-1,3-glucanase positive fractions were pooled, desalted

and freeze-dried.

Native Polyacrylamide gel electrophoresis (PAGE),

activity staining and electro-elution

Native polyacrylamide resolving gels (7.5%) and stacking

gels (5%) prepared in a Mini-Protean� 3 system (Bio-Rad

2180 World J Microbiol Biotechnol (2009) 25:2179–2186

123

Laboratories, Hercules, CA, USA) were applied to analyse

the enzyme activity in the crude culture supernatant and to

determine the homogeneity of b-1,3-glucanase after puri-

fication (Laemmli 1970). After electrophoresis, protein

bands were visualized by staining with Coomassie Brilliant

blue R-250 (CBB R-250, Fluka). For activity staining,

another prepared gel was washed three times with distilled

water and b-1,3-glucanase activity staining was performed

according to the method described by Stahmann et al.

(1993). For recovering b-1,3-glucanase in the gel after

native PAGE, mini-gel pieces containing the proteins of

interest were excised and electroeluted. The eluent was

dialysed against de-ionized water at 4�C and freeze-dried

(Sun et al. 2006).

Sodium dodecyl sulfate (SDS)–PAGE

The subunit of the enzymes were analysed by SDS–

PAGE (Laemmli 1970). The mini-gel pieces containing

b-1,3-glucanase activity was excised from the native

PAGE gel described above; equilibrated first in equili-

bration buffer I [6 M urea, 2% (w/v) SDS, 20% (v/v)

glycerin, 0.375 M Tris and 2% (w/v) dithiothreitol (DTT),

pH 6.8] at room temperature for 20 min; and then in

equilibration buffer II [6 M urea, 2% (w/v) SDS, 20% (v/v)

glycerin, 0.375 M Tris and 2.5% (w/v) iodoacetamide, pH

6.8] at room temperature for 15 min to eliminate the

redundant DTT. After removing the remaining buffer II

from the gel strip, the strip was loaded to a 15% dena-

tured SDS–PAGE gel for electrophoresis at 40 V for

60 min and continued at 80 V for 120 min. The gel was

stained with CBB R-250.

Enzyme characterization

Native molecular mass of b-1,3-glucanase

The molecular weights of native state of the proteins was

determined by Matrix Assisted Laser Desorption ionisation

time-of-flight mass spectrometry (MALDI-TOF-MS) with a

REFLEXTM III system (Bruker, Germany) in National

Center of Biomedical Analysis (NCBA, China). The protein

molecular weight of the native state was also analysed by

gel filtration (Superdex 200 10/300 GL column, normal

separation range of 10–600 kDa or a self-prepared column

followed the instruction filled with Sephacryl S-300HR,

normal separation range of 10–1,500 kDa) and by non-

reducing PAGE using the a Pharmacia HMW calibration kit

(consisting of thyroglobulin (669 kDa), ferritin (440 kDa),

catalase (232 kDa), lactate dehydrogenase (140 kDa)) as

the standard. Non-reducing PAGE was performed using the

method as described by Angulo-Valadez et al. (2007).

Isoelectric focusing (IEF) and Glycosylation assay

Isoelectric focusing (IEF) was performed in a 8 9 7 9

0.75 cm PAGE gel (Mini-Protein� 3 system, Bio-Rad)

using the method described by Stahmann et al. (1993). The

pH gradient was generated with a 0.8% Ampholine (3.5–

10), 1.2% Ampholine (4–6) and 1.2% Ampholine (5–7) (all

from LKB, Bromma, Sweden) solution, using a broad

range of pI marker standards (pH 3–10, Pharmacia).

Glycosylation assay was performed on native PAGE gel

staining with the periodic acid/Schiff reagent according to

Gerard (1990).

Temperature and pH optimum and stability

The optimum temperature was determined by carrying out

the enzyme assay reaction from 20 to 80�C. Likewise,

enzyme stability was determined at various temperatures

over 10 min with residual activity being measured as

described above.

Optimum pH was determined by performing the stan-

dard enzyme assay except the appropriate buffers con-

taining 50 mM each component within the pH range of

3.0–9.0: sodium acetate (pH 3.0–5.5), phosphate (pH 6.0–

8.0), and Tris–HCl (pH 8.0–9.0). The pH stability of the

purified enzyme was examined by measuring the residual

activity after incubation the enzyme mixture at each

desired buffers for 10 h at 4�C.

Effect of metal ions and other regents

The effects of metal ions Mn2?, Co2?, Hg2?, Mg2?, Fe3?,

Zn2?, Ca2?, Ba2? (all as chloride salts) and Cu2? (sul-

phate) ions as well as EDTA and KMnO4 on enzyme

activity were measured by incubating enzyme with each

metal ion in 50 mM sodium acetate buffer (pH 5.5) at 37�C

for 30 min.

Kinetic parameters

The apparent Km and Vmax values were calculated from

Lineweaver-Burk plots with data obtained by b-1,3-glu-

canase activity assay described above using laminarin of

various concentrations as substrate.

Hydrolysis product analysis

The purified enzyme solutions of 20 ll were incubated

with 20 ll of 2 mg ml-1 laminarin in 0.2 M sodium ace-

tate (pH 5.5) at 37�C. Samples were removed after 5, 20,

40 and 60 min, the reaction was stopped by transferring the

tubes in a bath of boiling water for 5 min. The reaction

World J Microbiol Biotechnol (2009) 25:2179–2186 2181

123

products were identified by thin-layer chromatography

(TLC) as described by Nagata et al. (1990).

Substrate specificity

Substrates were used at a concentration of 2 mg ml-1 for

laminarin (Sigma), pullulan (Sigma), lichenan (Sigma),

p-nitrophenyl-b-D-galactopyranoside (pNPG, Merck,

Germany), salicin (Merck), soluble starch (Merck) and

4 mg ml-1 for chitin (Sigma) and carboxymethylcellulose

(CMC, Sigma). The standard assay conditions, as described

above, were used for laminarin, salicin, CMC, soluble

starch and chitin. The amount of reducing sugars released

from chitin was detected by the DNS method using

N-acetylglucosamine (GlcNAc) as the standard. When

using pNPG as substrate, enzyme activity was determined

followed the method as described by Del Rey et al. (1979).

Results

Purification of b-1,3-glucanase excreted from Ggt

The crude enzyme supernatant was subjected to a native

PAGE to identify b-1,3-glucanase activity of the protein

content. The crude enzyme extract revealed two bands

(Fig. 1a) following staining either with CBB-R250 or with

laminarin for detecting b-1,3-glucanase activity (Fig. 1b).

After purification by ammonium sulfate fractionation,

phenyl-Sepharose 6 FF column chromatography, DEAE-

Sepharose FF column chromatography and Superdex 200

10/300 column chromatography, the b-1, 3-glucanase was

purified 9.37-fold with a yield of 9.03% (Table 1). The

purified enzyme extract analysed by native PAGE (Fig. 1c)

revealed two bands corresponding to the activity fractions

in crude enzyme extract, designated Ia and Ib.

Mini-gel pieces containing Ia and Ib after native PAGE

were also excised and subjected to electroelution, respec-

tively, and both eluent of Ia and of Ib represented b-1,

3-glucanase activity determined by enzyme activity assay

as described above (data not shown). Eluted protein frac-

tions of Ia and of Ib were also analysed by native PAGE,

both of them were also separated into two bands with

almost the same mobility manners as Ia and Ib in the

purified enzyme extract (Fig. 2b, lanes 1, 2). Another cycle

of excision, electroelution and subsequently native PAGE

were performed for the four protein fractions separated

from Ia and Ib following same method. Interestingly,

similar scenario represented again, four eluted protein

fractions each was separated into two bands with the same

mobility manners as Ia and Ib (Fig. 2c, lanes 1–4). These

results indicating that Ia and Ib in the purified enzyme

extract are two interconvertible concomitants.

Ia and Ib, separated by native PAGE, was excised from

mini-gels and subjected to SDS–PAGE, as described in

Materials and methods. After electrophoresis and staining

with CBB R-250, the gel exhibited both enzyme fractions

consists of two subunits with molecular masses of 66.2 and

56.0 kDa (Fig. 3), revealed the homogeneity of Ia and Ib.

The purified enzyme complex was thus designated b-1,

3-glucanase complex (GluGgt).

Characterization of GluGgt

The molecular weight of the native proteins (Ia and Ib)

could not be identified by MALDI-TOF-MS, possibly due

to the machine’s narrow testing range (10–400 kDa). Thus,

we attempted to assess the molecular weights of Ia and Ib

by gel filtration and non-reducing PAGE. The molecular

weights of Ia and Ib, more accurately the enzyme complex-

as there was only one activity peak in a single tailed protein

peak (A280) for the enzyme complex during elution course

of gel filtration, determined to be higher than that of thy-

roglobulin (669 kDa) by this technique, which fell beyond

the range of the markers used. While determined by non-

reducing PAGE, the molecular weight of Ia also showed

higher than that of thyroglobulin (669 kDa) and the

molecular weight of Ib was similar to that of ferritin

(440 kDa) (data not shown).

IEF-PAGE of the purified enzyme resulted in two bands

representing Ia and Ib on native PAGE. The isoelectric

points of Ia and Ib were 6.3 and 6.4, respectively. Glyco-

sylation of the enzyme complex (Ia and Ib) was demon-

strated by the periodic acid/Schiff stain, indicating that the

b-1,3-glucanases excreted by Ggt were glycoproteins.

IaIb

(a) (b) (c)

*

Fig. 1 Detection of 1,3-b-glucanase in the crude enzyme supernatant

and the purified extract on native PAGE gels (7.5%). a Crude enzyme

stained by CBB R-250, b Crude enzyme containing 1.3-b-glucanase

after activity staining using laminarin, c Purified 1,3-b-glucanase

stained with CBB R-250. * Shows the position of Ib in an activity

stained gel

2182 World J Microbiol Biotechnol (2009) 25:2179–2186

123

The optimal temperature of GluGgt was 50�C and it was

stable at B60�C (Fig. 4a). The enzyme was most active at

pH 5.0 and more than 90% of the maximal activity

remained following 10 h incubation within a pH range

from 4.0 to 7.0 (Fig. 4b).

The effect of various metal ions was tested on the b-1,

3-glucanase activity of GluGgt. The results presented in

Table 2 indicate that Mn2? ion had a obvious stimulating

effect on the enzyme activity towards laminarin; at a

concentration of 2 mM, the activity was increased by 16%.

Fe2? ion enhanced the enzyme activity slightly at a con-

centration of 2 mM. Hg2? and Co2? ions as well as

KMnO4 impaired the enzyme activity moderately at each

2 mM of the compounds by 40.2, 21.9, 51.2%, respec-

tively. EDTA, Mg2? and Cu2? ions presented slight

inhibiting effect on the enzyme activity.

The apparent Km and Vmax of GluGgt were calculated to

be 1.08 mg ml-1 and 0.20 lmol min-1 for laminarin,

respectively, (Fig. 5). TLC analysis of the laminarin-

hydrolysis products showed that laminarin was digested by

GluGgt after incubation for 5, 20, 40 and 60 min, glucose

was the only product identified. Thus, the enzymes

appeared to be exo-1,3-b-glucanase.

Substrate specificity of GluGgt was determined against

different glucans (Table 3). Among the substrates tested,

laminarin was hydrolysed by the enzyme at the highest

rate. Salicin was also degraded at a significant degree;

pNPG and CMC were degraded much more slowly. Almost

no activity was detected when chitin and soluble starch

were applied as substrate and no activity was detected

when the substrates pullulan and lichenan were used.

Discussion

This study reports for the first time on the purification and

characterization of a b-1,3-glucanase complex (GluGgt)

secreted by G. graminis var. tritici. The enzyme complex

contains two interconvertible isoforms (Ia and Ib), both Ia

and Ib are composed of two subunits of 66.2 and 56.0 kDa.

Some fungal species, such as Penicillium italicum (San-

chez et al. 1982), Phytophthora infestans (Bodenmann

et al. 1985), Sclerotium glucanicum (Rapp 1989), Botrytis

cinerea (Stahmann et al. 1993) and Acremonium persici-

num (Pitson et al. 1995) also produced more than one

b-1,3-glucanases. Different b-1,3-glucanases originating

from the same organism, among these reported, were either

produced under different culture conditions (e.g. carbon

Table 1 Purification of b-1,3-

glucanase from

Gaeumannomyces graminis var.

tritici

a,b Values are the mean and

standard deviation of triplicate

determination

Steps Total activity

(U)aTotal protein

(mg)bSpecific activity

(U/mg)

Purification

fold

Yield

(%)

Culture filtrate 85.98 ± 0.11 803.11 ± 2.31 0.11 1 100

(NH4)2SO4 60% 24.94 ± 0.06 105.42 ± 1.63 0.24 2.18 29.00

Phenyl-Sepharose 16.01 ± 0.03 39.79 ± 0.86 0.40 3.64 18.62

DEAE-Sepharose 11.01 ± 0.01 21.63 ± 0.19 0.51 4.63 12.80

Superdex 200 10/300 GL 7.77 ± 0.01 7.23 ± 0.01 1.07 9.73 9.03

1 1 2 1 2 3 4

IaIb

(a) (b) (c)

Fig. 2 Products of electro-elution of Ia and Ib on native PAGE gels

(7.5%). a Lane 1—purified 1,3-b-glucanases; b lane 1—electro-

elution product of Ia, lane 2—electro-elution product of Ib; c lane 1—

electro-elution product of Ia of lane 1 in (b), lane 2—electro-elution

product of Ib Lane 1 in (b), lane 3—electro-elution product of Ia of

lane 2 in (b), lane 4—electro-elution product of Ib of lane 2 in (b).

All stained with CBB R-250

1 2 3 kDa

97.2

66.4

44.3

30.1

21.0

14.4

Fig. 3 SDS–PAGE of Ia and Ib on SDS PAGE gel (15%). Lane 1—

Ia, lane 2—Ib, lane 3—Lower molecular protein markers, top-to-

bottom: rabbit phosphorylase b (97.4 kDa), bovine serum albumin

(66.2 kDa), rabbit actin (43.0 kDa), bovine carbonic anhydrase

(31.0 kDa), trypsin inhibitor (20.1 kDa) and hen egg white lysozyme

(14.4 kDa).The gel was stained with CBB R-250

World J Microbiol Biotechnol (2009) 25:2179–2186 2183

123

source) or could each be separated from their counterparts,

and there seemed no obvious relevance between them. The

bacterial species Flavobacterium dormitator secreted four

types of endo-b-1,3-glucanases (I, II, III and IV) in culture

broth, b-1,3-glucanase I was produced first, followed by

production of II and IV and was suggested to be a prote-

olysis product of b-1,3-glucanase II (Nagata et al. 1990). In

another case, Forster and Rasched (1985) reported that

P. infestans produced a pectinesterase complex (PE I)

which also consisted of two separate enzymes, which was

confirmed by appearance of two activity peaks during

separation by chromatofocusing and two bands in SDS–

PAGE, but they could not be separated by a number of

different techniques.

GluGgt secreted by G. graminis var. tritici is a novel

b-1,3-glucanase complex for the interconvertible relation-

ship of Ia and Ib. After recovery Ia (or Ib) from the native

PAGE gel by electroelution, Ia (or Ib) would again turn

into Ia and Ib. There should be a certain equilibrium

between Ia and Ib in the aqueous solution, more bio-

chemical tests should be carried out to explore the internal

equilibrium mechanism and external effect factors. Ib, with

a molecular weight slightly higher than 440 kDa, is pos-

sibly a tetramer of which the monomer (*120 kDa) con-

sisted of the 66.2 and 56.0 kDa subunits. The molecular

weight of Ia was not easily evaluated, since there are no

suitable molecular standards bigger than 669 kDa that

could be used in gel filtration and gel electrophoresis. The

absence of available devices to analyse the molecular

weight of so big a protein makes us unable to determine the

molecular weight of Ia at present. Ia and Ib seemed too big

compared to b-1,3-glucanases produced by other fungi, the

biggest one reported to date being a b-1,3-glucanase pro-

duced by P. infestans (160–230 kDa, Bodenmann et al.

1985). But the 66.2 and 56.0 kDa subunits make it

0

10

20

30

40

50

60

70

80

90

100

20 30 40 50 60 70 80

Temperature (°C)

Rel

ativ

e en

zym

e ac

tivity

(%

)0

10

20

30

40

50

60

70

80

90

100

3 4 5 6 7 9

pH

Rel

ativ

e en

zym

e ac

tivity

(%

)

(a) (b) Fig. 4 Effect of temperature (a)

and pH (b) on activity (d) and

stability (s) of 1,3-b-

glucanases from Ggt. Activity

was estimated as a percentage of

the maximum (defined as

100%). All values are the means

of three replicates; standard

deviations (SD) were \5% of

mean

Table 2 Effects of metal ions on 1.3-b-glucanase activity of GluGgt

Compounds (2 mM) Relative activitya (%)

None 100.0 ± 0.47

Fe3? 103.6 ± 0.20*,**

Co2? 78.1 ± 0.80*,**

Mn2? 116.4 ± 0.27*,**

Mg2? 94.9 ± 0.18*,**

Zn2? 100.5 ± 0.27

Hg2? 59.8 ± 0.27*,**

Ba2? 100.5 ± 0.37

Ca2? 102.1 ± 0.81*

Cu2? 96.1 ± 0.73*,**

EDTA 90.4 ± 0.88*,**

KMnO4 48.8 ± 0.98*,**

The results are expressed as the percentage (%) of relative enzyme

activitya Mean and standard deviation of triplicate determination

*,** Indicate activities differ significantly at P B 0.05 and P B 0.01

levels by Student’s t-test, respectively, compared with control (none)

-2

0

2

4

6

8

10

12

14

16

18

-1 0 1 2

1/[Laminarin] (ml/mg)

1/V

(min

/um

ol)

1/Vmax-1/Km

Fig. 5 Linewaver-Burk plot of GluGgt. The purified enzyme was

assayed with various laminarin concentrations (0.5–2.5 mg ml-1) in

50 mM sodium acetate buffer (pH 5.5) at 37�C for 30 min. The rates

of glucose (lmol min-1) formation at different substrate concentra-

tion were set out

2184 World J Microbiol Biotechnol (2009) 25:2179–2186

123

possible, for the molecular weight of monomer they made

up would be 120 kDa.

The pI value reported for fungal b-1,3-glucanases varied

in a range of 3.0–9.8 and mostly between 4.0 and 8.0, were

different not only between organisms but also within one

organism. The pI values of Ia was 6.3 and of Ib 6.4, which

were similar to those of b-1,3-glucanases from Cochlio-

bolus carbonum (Van Hoof et al. 1991), a pathogen of

maize. The pI values of Ia and Ib were so similar probably

because they were composed of the same two subunits. The

finding that Hg2? and Co2? ions markedly inhibited the

activity of the purified enzyme, may suggest that SH-

groups are involved as functional groups in the molecule of

the enzyme (Sanchez et al. 1982; Kulminskaya et al. 2001).

b-1,3-Glucanases of plant pathogen origin have been

generally reported to be implicated in nutrition of the

pathogen, induction of the host defense reaction and pen-

etration of the plant cell walls during the infection process

(Van Hoof et al. 1991). b-1,3-Glucanases secreted by Ggt

were supposed to be implicated in the last (Kang et al.

2000). Based on the prepared anti-GluGgt antibody,

immunolocalization of GluGgt in the Ggt-infected wheat

root indicated that there were 1,3-glucanase of Ggt in the

lignitubers surrounding the infection hyphae (Yu 2008).

This result indicated that 1,3-glucanase of Ggt may be

implicated in degrading callose in lignitubers. However,

the cell wall pore size of higher plant estimated to be 4 nm

and it is relatively small compared with the macro-1,3-

glucanase complex secreted by Ggt (Carpita and Gibeaut

1993; Tenberge et al. 1999). If GluGgt does participate in

degrading callose in papillae or lignitubers, how does this

1,3-glucanase complex get to the callose deposition sites?

Do other cell-degrading enzymes produced by Ggt, such as

pectinase, xylanase and cellulase (Dori et al. 1995) work

synergistically with GluGgt during cell wall penetration

process? Further studies on these enzymes need to be done

to answer these questions.

Acknowledgments This work was supported by the National Nat-

ural Science Foundation (No. 30270863), Program for Changjiang

Scholars and Innovative Team in University of Education Ministry of

China (No. 200558), and 111 Project from Education Ministry of

China (No. B07049). We gratefully acknowledge Dr. W. N. Song and

Dr. Y. Xiang for valuable advices in the paper preparation.

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