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Biosci. Biotech. Biochem., 59 (12), 2223--2227, 1995

Purification and Partial Characterization of a fJ-l,3-Glucanase Secreted by theMycoparasite Stachyhotrys elegans

Russell J. TWEDDELL,* Suha H. JABAJI-HARE,**,t Mireille GOETGHEBEUR,*** Pierre M. CHAREST,*

and Selim KERMASHA* * *

Faculty ofAgricultural and Environmental Sciences, Macdonald Campus, McGill University, Ste-Anne-de-Bellevue, Quebec,Canada*Departement de phytologie, Pavillon c.-£. Marchand, Universite Laval, Ste-Foy, Quebec, Canada, GIK 7P4**Department of Plant Science, Macdonald Campus, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9*** Department ofFood Science and Agricultural Chemistry, Macdonald Campus, McGill University, Ste-Anne-de-Bellevue,Quebec, Canada, H9X 3 V9Received April 26, 1995

A /I-l,3-glucanase secreted by Stachybotrys elegans, when grown on minimal synthetic mediumcontaining Rhizoctonia solani cell wall fragments, was purified to homogeneity. The purification methodinvolved ammonium sulfate precipitation and ion-exchange and size-exclusion chromatographies. Themolecular mass of the enzyme was estimated under denaturing conditions to be about 94 kDa. The enzymehad an optimum pH of 5.0 and was most active between 40 and 50°C. Except for Mn2 +, the enzymeactivity was not sensitive to the metal ions tested and Km of 0.18 mg/ml was estimated for laminarin as asubstrate. Cell wall lytic activity of purified /I-l,3-glucanase was tested on actively growing R. solani hyphae./I-l,3-Glucanase induced morphological changes such as hyphal tip swelling, bursting, leakage of cytoplasm,and formation of numerous septae.

Lytic enzymes, particularly chitinases and f3-1,3­glucanases, are essential in the life cycles of higher fungi.They have been implicated in apical growth, 1) wall softeningduring hyphal branching, germination, hyphal fusion,2,3)and conidia formation. 4

) Recent studies indicate that duringmycoparasitism of plant pathogenic fungi, several of thesehydrolytic enzymes are produced by the mycoparasites andare involved in the degradation of fungal host cell walls. 5)

Among these enzymes, chitinases and f3-1,3-g1ucanases areconsidered key cell-wall degrading enzymes of higherfungi. 6, 7) In addition, these enzymes have been shown tobe produced by several potential biocontrol agents, andmay be an important factor in biological control. 8 - 11)

Recently Stachybotrys elegans (Pidopl.) W. Gams, a soilfungus, was shown to have a strong mycoparasitic activityagainst the soilborne pathogen Rhizoctonia solani Kuhn inin vitro studies. Ultrastructural studies of the interactionbetween the mycoparasite and R. solani,12) as well asbiochemical studies on the production ofhydrolytic enzymesby S. elegans 13

) strongly suggest the implication of theseenzymes, particularly f3-1,3-g1ucanases, in the degradationof R. solani cell walls. As far as we can tell, nothing wasreported about the f3-1 ,3-glucanases of this newly discoveredmycoparasite. The objectives of this study were to purifyand to partially characterize a f3-1,3-g1ucanase secreted byS. elegans, as well as to study its biological effects on themorphology of R. so/ani hyphae.

Materials and MethodsOrganism and culture conditions. The mycoparasite S. elegans was

isolated from soil in Izmir, Turkey. S. elegans was grown on sterile oatseeds for 10 days as described by Escande and Echandi. 14) The seeds werestored at 4°C and served as stock cultures. Inoculum for the productionof fi-I ,3-glucanases in batch cultures consisted of potato dextrose agar

t Corresponding author.

(PDA; Difco Laboratories, Detroit, Michigan) disks of IO-day oldmycelium of S. elegans grown from colonized oat kernels. Large-scaleproduction of fi-I ,3-glucanases was done in 2-liter and 3~liter Erlenmeyerflasks containing respectively 400 and 600 ml of minimal synthetic medium(MSM) amended with R. solani cell wall fragments 13) as a carbon source.The medium was composed (in g/Iiter) of: MgS04' 7H 20, 0.2; K 2HP04,0.9; KCI, 0.2; FeS04.7H 20, 0.002; MnS04, 0.002; ZnS04 , 0.002; NaN0 3,

1.0; yeast extract (Difco), 3.0; and R. solani cell wall fragments, 0.5. ThepH of the medium was adjusted to 5.2. The flasks were incubated withshaking (110 revolutions/min) at 24°C for 5 days. Crude culture filtrateswere collected by filtration through Whatman no. I filter paper and usedas a source of fi-I ,3-glucanases.

Enzyme purification. All procedures were done at 4°C. Crude culturefiltrate (13.7 liters) was concentrated to 1.7 liter by ultrafiltration(membrane type: PM I; effective area: 5 ft2; Romicon Inc., Woburn, MA).The concentrated crude culture filtrate hereafter referred to as fraction «FI), was freeze-dried and further purified.

(i) (NH4hS04 precipitation. F I was rehydrated with 70ml ofphosphate buffer (0.1 M, pH 7). (NH4hS04 was added to 40% saturationto the culture filtrate which was centrifuged (5000 x g, 20 min) and theprecipitate discarded. Ammonium sulfate was added to the supernatantto 60% saturation. The precipitate was removed by centrifugation(5000 x g, 20 min), dissolved in a small volume of phosphate buffer (0.1 M,pH 7.0), and dialyzed against citrate buffer (0.05 M, pH 4.7). The fraction(F II) was then freeze-dried and further purified.

(ii) Ion-exchange chromatography. The F II fraction obtained byammonium sulfate fractionation was rehydrated in Tris-HCl buffer (20 mM,pH 8.0; Buffer A) and chromatographed on a Resource Qanion-exchangercolumn (Pharmacia LKB Biotechnology, Uppsala, Sweden) using a FastProtein Liquid Chromatography system (FPLC system, Pharmacia LKBBiotechnology). The column was equilibrated with buffer A and samplesof 500 pi were put on the column. A linear gradient of buffer A and bufferB (Buffer A containing I M NaCI) was used for elution at a flow rate ofI ml/min. Fractions of 2 ml were collected. The fraction (tube 1f8) con­taining the majority of fi-I ,3-glucanase activity referred to as F III, wasdialyzed and freeze-dried.

(iii) Size-exclusion chromatography. F III obtained by ion-exchangechromatography was solubilized in phosphate buffer (0.05 M, pH 7.0), andseparated by size-exclusion chromatography on Superose 12 HR 10/30

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2224 R. J. TWEDDELL et al.

column (Pharmacia LKB Biotechnology) using the FPLC system. Thecolumn was equilibrated with the same phosphate buffer. Elution was doneat a flow rate of 0.5 ml/min, and I-ml fractions we're collected. The activefractions contained in tubes :117 and 8 were pooled, dialyzed and freeze-driedand hereafter are referred to as F IV.

Enzyme assays and protein measurement. [3-1,3-Glucanase (EC 3.2.1.39)activity was assayed by following the release offree glucose from laminarinusing a commercial glucose oxidase kit (Sigma Chemical Co., St. Louis,MO).9) The reaction mixture, which contained the enzyme dissolved in2 ml of citrate buffer (0.1 M, pH 4.7) and 1.6 mg of soluble laminarin, wasincubated at 40°C for 1h and the reaction was stopped by boiling. 13) Oneunit (U) of fl-l ,3-glucanase was defined as the amount of enzyme causingthe release of 1 jlmol of glucose per minute under these conditions. Specificactivity was expressed in units (U) per milligram of protein. Protein in theenzyme solutions was measured by the Lowry method as modified byHartree l5

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

Gel electrophoresis and molecular mass measurement under denaturingconditions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) was done as described by Laemmli. 161 Protein standardsand samples were incubated in Laemmli buffer at 90°C for 10 min.SDS-PAGE was done in Phast Gel homogeneous 12.5% using thePharmacia Phast System (Pharmacia LKB Biotechnology). Electrophoresiswas done at lOrnA and was stopped when the dye front reached the anodicend. After electrophoresis, the gel was silver-stained according to the PhastSystem silver staining instruction manual (Pharmacia LKB Biotechnology).Phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin(43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor(20.1 kDa), and lactalbumin (14.4 kDa) were used as molecular massmarkers (Pharmacia LKB Biotechnology).

Calculation ofkinetic constants and pH and temperature optima. Kineticvalues were calculated by using various laminarin concentrations 0.05 to3 mg/ml in 1ml of citrate buffer (0.05 M, pH 4.7). A Lineweaver-Burkplot 1 7) was used to calculate Km and Vmax values. The effects of pH on[3-1 ,3-g1ucanase activity were measured using 0.1 M citrate phosphate bufferand 0.1 M phosphate buffer for pH ranges 3-5 and 6-8, respectively. 18)

The effects of temperature on enzyme activity were assayed in citrate buffer(0.05 M, pH 4.7) at temperatures of 30, 40, 50, 60, and 70°C.

Effects of metal ions. The effects of Fe2+ , Co 2 +, Ca2+, Mg2+, Mn2+,

Table I. Scheme of Purification of [3-1 ,3-Glucanase Secreted by S. elegans

and Zn2+ on [3-1,3-glucanase activity were measured by incubating theenzyme with each metal ion in citrate buffer (0.05 M, pH 4.7) at 40°C forI h. The activity assayed in the absence of metal ions was referred as 100%.

Bioassay. The effects of crude culture filtrate of S. elegans grown onR. solani cell walls, and of purified [3-1,3-glucanase on living hyphal tipsof R. solani were tested. PDA (Difco) disks of R. solani mycelium wereplaced on microscope slides that were previously coated with 2 ml of wateragar (1.5 %) containing 2 mg of crude culture filtrate with 0.01 U /mg ofglucanase activity. To test the effects of the purified enzyme, the sametechnique was used with slight modification due to the limited amountsof the purified enzyme available. Instead of incorporating the enzymeinto the water agar, 10 jll of purified [3-1 ,3-glucanase (0.0 I U /ml dissolvedin 0.05 M citrate buffer at pH 4.7) was applied directly to the activelygrowing R. solani hyphae 24 h after inoculation of microscope slides. Incontrol experiments, R. solani mycelia were exposed to citrate buffer(0.05 M, pH 4.7), boiled crude culture filtrate or boiled purified [3-1,3­glucanase. All slides were incubated at 24°C in a moist chamber and ex­amined at 40-min intervals with an Olympus BHS microscope with phasecontrast optics. Morphological anomalies were noted and photographed.

ResultsPurification of f3-1,3-g1ucanase

The purification scheme of f3-1,3-g1ucanase from S.elegans is summarized in Table 1. The results showed thatthe partially purified enzyme precipitated by (NH4hS04 at40-60% of saturation (F II) contained 59.7% of the totalactivity. The purification of F II by FPLC ion-exchangechromatography using Resource Qanion-exchanger column,demonstrated the presence of several peaks of protein (Fig.1); however, most of the 13-1 ,3-glucanase activity was locatedin tubes ~7-11 with tube ~8 showing the maximum specificactivity. Tube ~8 (F III) containing the majority off3-1,3-g1ucanase activity was further purified using FPLCsize-exclusion chromatography with Superose 12 HR 10/30column. This step yielded 2 peaks of protein (Fig. 2);however, only the peak contained in tubes ~7 and ~8 showedf3-1,3-glucanase activity (F IV). The enzyme was purified

Purification step

---~-. --_..._-- -_..._---

Culture filtrate (concentrated) (F I)(NH4)zS04 fractionation (40-60%) (F II)Resource Q (F III)Superose 12 HR 10/30 (F IV)

Total protein(mg)

641.256.6

1.370.19

Total activityU(U)

55.8133.303.971.44

Specific activityb(U/mg)

0.0870.592.907.58

Purification fold

16.8

33.387.1

Recovery(%)

10059.77.12.6

U One unit of [3-1 ,3-glucanase (U) is defined as the amount of enzyme causing the release of 1jlmol of glucose per minute under the conditionsdescribed.

b Specific activity was expressed in units (U) per mg of protein.

0.20 3.0 1.0

E0.15 0.75

l:~'20co ;; ·iii !:~

0.10u-

Gl 1.5 ~ [ 0.50u Ul: :Em IIIIII Z.c u E(;

Gl_Q.~

CIl 0.05C/l-

.c 0.25c(

0 0.0 0.04 8 12 16 20 24

Tube Number

Fig. 1. Profile ofChromatographic Purification ofFraction II (F II) on Ion-exchange Resource QColumn Chromatography, Using FPLC System.

(e) absorbance at 280nm; (0) P-l,3-glucanase specific activity.

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f3-1,3-Glucanase Secreted by S. elegans 2225

100

~80

~ 60.s;tiIIIQl 40.~mGi 20a:

o+-~--'--,--,,......,~........,.--r--'---;

20 40 60 80Temperature (0C)

~ 80~.s;ti 60III

~ 40iGia: 20

Morphological effects of p-J,3-glucanaseA number of morphological changes in hyphae of R.

solani were induced by the crude culture filtrate and by thepurified enzyme. In both cases, vacuolization, swelling, lysis,and bursting of the tips were observed (Figs. 6B and D;

100

A Lineweaver-Burk plot was used to demonstrate theeffect of substrate concentration on initial velocities ofrelease of glucose from laminarin catalyzed by 13-1,3­glucanase. The Km and Vmax of the enzyme were estimatedto be 0.18 mg/ml and 41.5/lmol glucose/pure enzymemg/min, respectively (Fig. 5).

.005 8.0

E .004r::

::-~0

~ .003 :~.~0'0

GI 4.0 <0..ur::

~~III .002,g

0 g:s111 ~--,g< .001

0 04 8 12 16

Tube Number

87.l-fold with a recovery of 2.6% (Table I). The SDSelectropherogram of purified fraction (F IV) showed thepresence of homogeneous 13-1 ,3-glucanase (Fig. 3).

Characterization of p-J,3-glucanaseThe molecular mass of the purified P-l,3-glucanase was

found to be about 94 kDa by an SDS-electropherogram(Fig. 3). The effect of temperature was studied by incubatingthe enzyme at temperatures between 30 and 70°C. Theenzyme was most active between 40 and 50°C (Fig. 4a).The effect of pH on P-l,3-glucanase activity showed thatthe optimum pH is 5.0 (Fig. 4b). The effect of various metalions was tested on P-l,3-g1ucanase activity. The results(Table II) indicate that Mn2 + had a stimulated effect onthe enzyme activity, but Fe2 +, C02 +, Ca2 +, Mg2 +, andZn2 + had no marked effect on enzyme activity.

Fig. 2. Profile of Chromatographic Purification of Fraction III (F III),Obtained from Ion-exchange Chromatography, on Size-exclusion Superose12 HR 10/30 Column Chromatography, Using FPLC System.

(.) absorbance at 280 nm; (0) {3-1,3-glucanase specific activity.

kDa

94.0-

67.0- t_43.0-

2

o~...,......,....,,...,...,.......-.........--,...,.......,..l

2 3 4 5 6 7 8 9pH

Fig. 4. Effects of Temperature and pH on f3-1,3-Glucanase Activity.

a: {3-1,3-Glucanase activity of 0.62 flg of the enzyme was measured at variousincubation temperatures under the following assay conditions: the enzyme wasdissolved in I ml citrate buffer (0.05 M, pH 4.7) containing 0.8 mg of laminarin andthe reaction was incubated for I h. b: Effects of pH on {3-1,3-glucanase activity. Aquantitative analysis of the enzyme activity as a function of pH was done by incubatingthe enzyme in the following 0.1 M buffers: citrate phosphate (pH 3,4,5) and phosphate(pH 6, 7, 8). The reactions were performed by incubating 1.6 flg of purified enzymewith 0.8 mg of laminarin for I h at 40°C. The total volume of the reaction mixturewas I ml. Each value represents the mean of 2 separate measurements.

Table II. Effects of Metal Ions on f3-1,3-Glucanase Activity

30.0-Metal ions (l mM) Relative activity"

(%)

" In the presence of various metal compounds (final concentration,1mM), the f3-I,3-glucanase activity of 0.37 f..l.g of the enzyme wasmeasured under the following assay conditions: the enzyme wasdissolved in 1ml citrate buffer (0.05 M, pH 4.7) containing 0.8 mg oflaminarin and the reaction was incubated at 40°C for 1h. Each valuerepresents the mean of 2 separate measurements.

20.1-

14.4- ."11;

Fig. 3. SDS-PAGE of the Purified f3-1,3-Glucanase.

SDS PAGE was done in Phast Gel homogeneous 12.5% by using the PharmaciaPhast System. After electrophoresis, the gel was silver stained according to thedescription of the Phast System silver staining instruction manual (Pharmacia LKB).Lane I: molecular mass markers, phosphorylase b (94kDa), bovine serum albumin(67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsininhibitor (20.1 kDa), and lactalbumin (14.4kDa). Lane 2: purified {3-I,3-glucanase(well was filled with 7 J.lg of protein).

NoneFeCl 3 '6H zOCoClz '6HzOCaClz ·2HzOMgClz '6HzOMnClz '4HzO

ZnClz

100100104104102119103

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2226 R. J. TWEDDELL et al.

•

1/5 (mglml)-1

Fig. 5. Lineweaver-Burk Plot of P-I ,3-Glucanase.

The enzyme (0.82 /lg) was assayed with varying amounts of substrate (Iaminarin) inI ml of citrate buffer (0.05 M, pH 4.7). The reaction was done at 40°C for I h. Glucosereleased was assayed by the glucose oxidase method (Sigma). The rates of productformation (/lmol glucose/min) at different substrate concentrations were set out.

arrowhead). In addition, numerous septae were observedin hyphae that were exposed to pure glucanase only (Fig.6D, arrow). All of these changes except for septal formation,were discernible within 2 hours following exposure of R.solani mycelia to a solution of purified glucanase. Bycontrast, R. solani hyphae grown under all controlconditions showed no signs of damage (Figs. 6A and C).

exclusion chromatographies. The rate of recovery was 2.6%.This is comparable to those of fJ-l,3-glucanases purifiedfrom Bacillus circulans (2%),24) Penicillium italicum (2%),21)and from infected bean seedlings (2.2%).25)

The molecular mass of the enzyme was estimated underdenaturing conditions to be about 94 kDa. Of fungalfJ-l,3-g1ucanases, the molecular mass of fJ-l ,3-g1ucanase ofS. elegans is higher than that of Penicillium italicum(68 kDa),21) Rhizopus arrhizus (32.1 kDa),26) R. solani(29 kDa), 27) and Trichoderma longibrachiatum (70 kDa). 22)In a previous study, we detected two isoforms of fJ-l,3­glucanase when crude culture filtrate of S. elegans waselectrophoresed on a native gel. 13) However in this study,one single band was detected by the SDS-electropherogramof purified fJ-l ,3-g1ucanase suggesting that only one isoformwas purified.

The optimal pH (5.0) and temperature (40-50°C) forenzyme activity were comparable to values reported forfJ-l,3-glucanases from other microorganisms (Streptomycessp., pH 5.5 and 55°C20); T. longibrachiatum, pH 4.8 and55°C22 ); Schizophyllum commune, pH 5.5 and 50°C).28)fJ-l ,3-Glucanase was found to be insensitive to most of themetal ions tested, suggesting that metals do not markedlyaffect enzyme activity. The K m of the fJ-l,3-glucanase forlaminarin (0.18 mg/ml) was higher than that for fJ-l,3­glucanase secreted by the fungi T. Longibrachiatum(0.0016%)22) and P. italicum (0.04 mg/ml), 21) but lower thanthat of fJ-l ,3-g1ucanase secreted by Rhizopus chinensis (3.4mg/ml)23) and Ruminococcus flavefaciens (0.37 mg/ml). 19)

In a previous study, 13) we demonstrated that crude culturefiltrates of S. elegan'i containing chitinase and fJ-l,3­glucanase activities had the ability to release glucose fromR. solani cell wall fragments. These findings strongly implythat fJ-l ,3-glucanases are capable of degrading R. solani cell

252015105o

:5120~.E~ 80

~

-5

DiscussionThis work reports for the first time on the purification

and characterization of a fJ-l,3-glucanase secreted by themycoparasite S. elegans, and on its effects on R. solanimycelia. The enzyme was purified to homogeneity from aculture filtrate of S. elegans grown on MSM containing R.solani cell wall fragments. The purification method wassimpler than other procedures used for the purification offJ-l ,3-glucanases from various organisms. 19 - 23) It involvedammonium sulfate precipitation and ion-exchange and size-

Fig. 6. Morphological Changes Induced in R. solani Hyphae by S. elegam' P-I ,3-Glucanase.

A: R. so/ani hyphae growing on water agar containing boiled culture filtrates at 0.01 V/ml (control). B: Lysed hyphal tips of R. so/ani (arrowhead) when grown on wateragar containing active culture filtrate at 0.01 V/m!. C: Hyphae of R. so/ani showing normal growth when exposed to boiled purified {j-I,3-glucanase at 0.01 Vlml (control).D: Lysed hyphal tip of R. so/ani when exposed to purified {j-I,3-glucanase at 0.01 V/ml (arrowhead). Notice the formation of numerous septae (arrow).

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fJ-l,3-Glucanase Secreted by S. elegans 2227

walls. In this study, the application of either crude culturefiltrate of S. elegans or purified f3-1,3-glucanase to activelygrowing R. solani hyphae had similar effects, i.e., hyphaltip swelling, bursting and leakage of cytoplasm resultingfrom its lytic activity. These results indicate that the purifiedf3-1,3-glucanase of S. elegans has the ability to hydrolyzethe R. solani cell walls without the participation ofchitinases. Hence f3-1,3-glucanase may be considered as akey enzyme in the ability of S. elegans in lysing R. solanicell walls. These results may be related to the fact that R.solani cell wall contains mostly f3-1 ,3-glucan polymers withsignificantly lower amounts of chitin. 29)

The findings reported in this study highly suggest theimportant role of f3-1,3-glucanase in the mycoparasitismprocess between S. elegans and R. solani. Purification ofthe chitinases secreted by S. elegans will be undertaken inorder to verify their lytic activity against R. solani cell wall,their possible interaction with f3-1,3-glucanase, and theirimportance in the mycoparasitism process.

Acknowledgments. The authors acknowledge the technical assistanceof B. Beliveau, D. Chou, and A. Goulet. This work was supported by theNatural Sciences and Engineering Research Council to S. H. Jabaji-Hareand partly by the Conseil des Recherches en Peche et en Agro-Alimentairedu Quebec to S. H. Jabaji-Hare and P. M. Charest.

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