Pectin lyase from Fusarium oxysporum f. sp. radicis lycopersici: purification and characterization

J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.

747

Pectin lyase from Fusarium oxysporum f. sp. radicis lycopersici" purification and characterization M.A. Guevara a, M.T. Gonz~ilez-Ja6n b and P. Est6vez a aDepartamento de Biologia Vegetal, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia. bDepartamento de Gen6tica, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia.

Abstract A pectin lyase has been purified from Fusanum oxysporum f. sp. radicislycopersici. Proteins from cultures of 4 days on pectin, were precipitated with ammonium sulphate and separated with a Superdex 75HR1030 column and by preparative isoelectric focusing (LKB column of 110 ml capacity). A single band, with isoelectric point of 9.20, was detected by silver staining on analytical isoelectric focusing. The molecular mass, calculated from its partition coefficient on the Superdex column, was 18 kDa. The highest activity of this enzyme was attained at pH 9.5 and 50 ~ C. The pectin lyase showed high specificity for pectin, an "endo" mode of action and calcium dependence.

1. INTRODUCTION Fusarium oxysporum f.sp. radicis-lycopersici Jarvis and Shoemaker (FORL) (Jarvis and Shoemaker, 1978) [1] is a pathogen of tomato which, with the arrival of intensive tomato culture under glass, has developed to serious proportions [2]. This forma specialis of F. oxysporum affects largely the root and crown tissues of tomato and the symptoms occur as foot and root rot. FORL isolates are pathogenic on tomato plants with genes for resistance to races 1 and 2 of Fusarium oxysporum Schlecht. f.sp. lycopersici (Sacc.) Snyd. & Hans (FOL), that cause the common Fusarium wilt of the tomato. However, although resistance to FORL has been found and incorporated into commercial cultivars, the disease is a severe problem in wide areas of the North Hemisphere [3-9]. Colonization of tomato root tissues by FORL is associated with striking modifications of host cell walls, as it has been shown by ultrastructural studies which have been carried out describing the penetration of the fungus through

748

infected root tissues [4, 10-19]. The pattern of penetration, with disruption or even loss of middle lamella matrices [16], implicates production of pectic enzymes by FORL [11]. In fact, pectic enzymes have been considered to play a critical role in parasitism involving pathogens of dicotyledons, in which rhamnogalacturonan has a key role in wall structure [20]. The r2 isolate of Fusanum oxysporum f. sp. radicis-lycopersici (FORL) produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We have detected three forms that have lyase activity, an absolute requirement for calcium, and pIs of 9.20, 9.00 and 8.65. The two most alkaline forms had a weak preference for pectin whereas the other was more active on pectate. The three lyases were produced when the fungus grew on pectin and on restricted galacturonic acid (data presented in the "XV Congreso National de Microbiologia" [21] and sent for publication). The objective of this work has been the purification of the pectin lyase with pI 9.20, which is the most abundant.

2. MATERIALS AND METHODS

Fungus The isolate of F. oxysporum f.sp. radicis-lycopersici used in this study was strain r2, supplied by Dr. J. Tello (Instituto Naeional de Investigaciones Agrarias, Madrid). It was isolated as a single-spore culture, from an infected tomato plant (Lycopersicon esculentum Mill.) and grown on potato sucrose agar (PSA) at 22 ~ C. The ability of the isolate to the infect tomato was periodically checked as described by S~nehez et al. [22]. Stock cultures were maintained in Petri plates on potato sucrose agar at 5~ (transferred every month) and in soil for long time preservation.

Enzyme production FORL cultures were grown in 250 ml Edenmeyer flasks containing a carbon source in 100 ml salts medium shaken (in)on a rotary incubator (150 rev.min 1) at 22~ The medium contained easamino acids 0.46, KHzPO 4 0.1, MgSO4.7H20 0.05g/100ml, FeSO4.7H20 0.2, ZnSO4.7H20 1.0, NazMoO4.2H20 0.02, CuSO4.5H20 0.02, MnC12.4H20 0.02 ~tg m1-1. Flasks were inoculated with 1 ml of distilled water containing 1 x 10 6 conidia obtained by flooding fungal colonies on potato sucrose agar. The carbon source (pectin, galacturonic acid or glucose) to shake cultures, was added at 0.5% (w/v) or supplied from diffusion capsules for restricted supply [23, 24]. The capsules, containing D-galacturonic acid or glucose (30g/100ml), were provided with membrane layers allowing

749 linear rates of release of sugar over 20 to 24h, so that capsules needed to be changed only once daily. Capsules with membrane layers were sterilized at 120~ for 10 min and filled with the carbon source (sterilized by filtration) before placing in cultures. The pH of cultures was adjusted at 5.5 with NaOH before sterilization. Cultures with restricted supply of the carbon source were first grown on 0.5% (w/v) glucose for three days on a rotary incubator (150 rev.min ~) at 22~ Then, cells were removed from culture fluid by centrifugation (1800 g, 30 min), and grown for one day on 100 ml fresh medium supplied with the capsule containing glucose. After one day with restricted supply of glucose, cultures were grown for three more days with restricted supply of galacturonic acid or with restricted supply of glucose. For cultures on pectin, 1 ml of grown cells for three days on glucose, were transfered to fresh inorganic salts medium with 0.5% (w/v) pectin (apple pectin, Fluka) and grown for six days. Cultures from different times of growth were collected. Culture fluids were cleared by passing through glass fibre filter. After dialysis for 16-18 h against distilled water at 5~ filtrates were assayed for enzyme activities and proteins. Assay Method Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12, were incubated at 37~ for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms l~tmol of 4,5unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M-~cm-~ [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37~ Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HC1 buffer (pH 8.0) with 0.5 mM CaC12. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min -1 under the conditions of reaction. Specific viscosity (n~p) is (t/t0)-l, where t is the flow time (sec) of the reaction mixture and t o is the flow time of the buffer. The inverse~specific viscosity (n~p-~) is proportional to the incubation time and the amOunt of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. Protein determ ination. Protein was determined by Lowry's method [27], using bovine serum

750

albumin (Sigma) as a standar.

Purification of Pectin Lyase Preparation of enzyme. Culture fluids of three days on glucose 0.5% (w/v) and then four days on pectin 0.5% (w/v), cleared by passing through glass fibre filter, were used for the purification of PNL. A small quantity was remainder, dialyzed, and assayed for enzyme actitity and the remained was precipitated. Ammonium Sulfate Precipitation. The extract was made up to 40% saturation with the slow addition, with stimng, of ammonium sulfate at 4~ After several hours, the precipitate was removed by centrifugation at 30,000 g for 30 min and the supematant retained. It was brought to 100% saturation in similar conditions, the precipitate was collected by centrifugation, dissolved in the minimum of distilled water, dialyzed against water and then against 1% glycine, and lyophilized. Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 m NaC1; 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70: Albumin, 66,000 Da; Carbonic Anhidrase, 29,00; Cytochrome, 12,400; and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (Vo) were calculated from the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained from the relationship K ~ - (Vo-Vo)-(V~-Vo) where Vt represents the bed volume of column and Vo the void volume (which was calculated using blue dextran, Sigma). The molecular mass was determined using a standard curve of K,v vs the logarithm of the molecular masses of the standards [28, 29] Preparative Isoelectnc Focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB). The density gradient was formed with sorbitol [0-50% (w/v)]. Enzyme extract was distributed equally between the two gradient component solutions prior to the establishement of the gradient using a gradient former. The concentration of the career ampholytes, Servalyte 7-9 and 9-11 (Serva), was 1,2% (w/v) and the catode was placed at the botton of the column. The experiment was performed at 7~ with constant power (9.6 W) giving a maximum voltage of about 1600 V. After 48 h, fractions of 3 ml were removed from the bottom of the column. The pH values of the fraction were inmediately measured at 7~

751

Ultrathin-layer analytical isoelectric focusing Proteins were separated according their pI by isoelectric focusing (IEF) at 7~ on a LKB 2117 Multiphor II apparatus. Ultrathin layers (0.4 mm) of polyacrylamide gels with ampholytes pH 2-11 were cast for isoelectric focusing as recommended by the manufacturer. Polyacrylamide solutions containing 5.2 % acrylamide (Pharmacia), 0.17 % N,N'-methylenebisacrylamide (Pharmacia), 1.1 ml of Servalyte career ampholytes (Serva), 0.6 ml of 1 % ammonium persulphate (Pharmacia) and 20 lal TEMED (Pharmacia), per 12.72 ml of the total volume, were cast on a glass as support using an Ultro Thin Layer Casting Tray (Bio Rad). Electrode wicks for the anode and catode were soaked in 1 M H3PO4 and 0.5 M NaOH respectively. Gels were preelectrofocused for 30 minutes at a constant 5.0 W. Samples of 10 ~g of protein in 10 lal were applied onto the gel via a small tab of glass fibre paper. Subsequent electrofocusing was earned out for 60 min at a constant 15 W with a maximum of 1400 V; sample application tabs were removed 30 min after focusing began. Broad pI Calibration Kit standards (Pharmacia) were used for pI estimation. Agarose overlays (2 mm thickness) for enzymes detection, containing pectin, were cast by capillary action between two glass plates separated by spacers. On one of these glass plates, a GelBond support film (LKB) was affixed by a thin film of water. The agarose solution was heated to 95~ and the gel mold was heated to 50~ before casting. The agarose solutions contained 1 % agarose, 0 . 1 % of pectin in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12 [25]. After focusing, gels were incubated in the appropriate buffer for 5 min. Then agarose overlays were placed on the surface of the isoelectricfocusing gels, incubated at 37 ~ for 20 min and the overlays stained with 0.05% ruthenium red. Afterwards, isoelectricfocusing gels were stained with silver (Bio-Rad kit) for protein detection.

Substrate specificity and mode of action Mode of action and substrate specificity of the purified enzyme were determined by following the decrease in viscosity and the increase in absorbance at 235 nm in reaction mixtures in the presence of 0.187 % substrate (pectin or pectate) at pH 8.0.

Optimum pH Optimum pH was determined by following the decrease in viscosity of the reaction mixture using 0.187 % pectin as a substrate in 0.05 M tris-HC1 buffer (pH 7.0-9.0) or glycine buffer (pH 9-10). Controls were run without enzyme preparation.

752

Effect of temperature Optimum temperature was determined at pH 8.0 by following the decrease in viscosity of reaction mixtures containing 0.187 % pectin, at temperatures between 30 ~ and 55~ Controls were run without enzyme preparation. Requirem ent of calcium The effect of C a 2+ w a s assayed by viscosimetry in reaction mixtures containing 0.187 % pectin, buffered at pH 8.0, and ethylene diaminetetraacetic acid (EDTA) (0.005 M) or GaG12 (0-0.02 M).

3. RESULTS

Production of pectin enzymes on restricted galacturonic acid and on pectin FORL was grown on restricted galacturonic acid and on pectin in order to ascertain the production of lyases by FORL and if different forms were produced. Figure 1 shows the time course of PNL activity during growth of the fungus in the two culture conditions experienced: pectin lyase was produced both (1)

4 1

E ctO ('O Cq

< (D o

200 o 3

0.02 restricted restricted galacturonic/ /' glucose acid ? // /

< .-- 0.01

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t~ e-

6

1

2

3

4

5

6

7

Days of culture

,'7"

(2) 0.09

40 ~-

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._= it) GI

p tin

0.03

7

r

i

1

2

3

20 '~

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5

6

Days of culture

7

i

i

8

9

Figure 1. Time-course of pectin lyase activity in cultures of F. oxysporum f. sp. radici s - l y c o p e rsici. Fungus was first grown on unrestricted glucose for three days, then the biomass was: or shifted to restricted glucose and, after one day, to restricted galacturonic acid (1); or shifted to pectin (2). Enzyme activity was determined as increase in A235 nm (-*-) and by viscosimetry (-o-) and determined for 10 min.

753

on galacturonic acid as well as on pectin. Maximum of activity was obtained at 60 h on galacturonic acid (1) and 106 h on pectin (2).

Purification of Pectin liase Culture fluids of four days on pectin were cleared by passing through glass fibre filter and fractionated by ammonium sulfate, gel filtration and preparative isoelectric focusing: Table 1 summarizes the purification steps. A peak with PNL activity was eluted from the Superdex 75HR1030 column (figure 2) and subjected to preparative isoelectric focusing (figure 3). Table 1 Step

Protein

Total activity

Specific activity

Purification

Yield

mg

U

U/mg

fold

%

Extract

35.96

995.92

27.69

-

100

(NH4)2SO4

2.50

62.76

25.10

0.91

6.30

Gel Filtration

0.24

36.99

154.12

5.56

3.71

IEF

0.03

23.59

899.35

32.48

2.36

"13 0

! ~0

0.06

5"

:r,. .

E o~ o

ro

m

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>

-

0

10

20

_

30

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.

40

50

60

70

80

i

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_

90

Fraction no.

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 M NaC1; 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content ( - - ) and PNL activity (-r

754

12

\

8"1" \

I

0

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r

A

0

5

10

_ _ _ I . . . .

i

20

25

15

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m

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e

30

Fraction no.

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity ( § and pH (- -).

Molecular Weight The PNL, eluted from the Superdex column, showed a molecular weight of around 18 kDa (figure 4). lsoelectric Point Figure 5 shows the pattem of lyase isoenzymes along the purification process: at first, three bands with lyase activity (pls 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1); in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pI 9.20 (B 2), but more proteins were detected by silver staining (A 2).

121

1

80

r

PNL

40 L.. m

~ 0

~

2010-

~ -

4

4 1

0

0.1

I

I

I

0.2

0.3

0.4

K av

Figure 4. Estimation of molecular weight by calibration of Superdex 75HR1030. Standard proteins: 1, Albumin (66,000 Da); 2, Carbonic Anhidrase (29,000 Da); 3, Cytochrome c (12,400 Da); 4, Aprotinin, (6,500 Da). The line has been drawn using the equation lg m = 5.01930882 2.757789171 * kay; r = - 0.9965.

755

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3).

A 9.30 8,65 8.45 8.15 7.35 6.85 6.55 --5.85

B

M 1

2

3

1

2

3

9 ~''~i

9.20 9.00 8.65

5.20 4.55 3.50

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 mm) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 lag of protein in 10 lal of 1% glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HC1 buffer at pH 8.0 with CaC12. M.- Broad pI Calibration Kit protein (Pharmacia), samples of 5 lag of protein were applied. 1.Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Properties of purified enzyme Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein: it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures.

756

Effect of pH on the activity of PNL. The enzyme exhibited maximum activity at pH 9.5 (figure 6). Effect of temperature. The optimum temperature for the PNL activity was 50~ (figure 7). Effect of Ca2+. The addition of 0.005 M EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Calcium concentrations of 0.001 M and lower were without effect on PNL activity, the optimum concentration being in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity.

~

100

9._> @ <

80 6o

(!;)

.>-

40

"~ nr'

20 0

I

I

I

I

I

I

I

7

7.5

8

8.5

9

9.5

10

pH

Figure 6. Effect of pH on the activity. Reaction mixtures, buffered at different pH values: 7-9 (tris/HC1), 9-10 (glycine), were incubated under standard conditions. Both buffers were 0.05M of final concentration in the reaction mixture.

/ ~" 100 ~-~ 9 80

~> 40 ~.

20

o/

~I~

~1 ~

30

40

45

50

55

Temperature (~

Figure 7. Effect of temperature on the PNL. The optimum temperature was determined using temperatures between 30 ~ and 55~

under standard conditions.

757

"r

100 80

60

~: eo 0 EDTA 0

0.1

0.5

1

5

10

15

20

CaCI 2Concentration (mM)

Figure 8. Effect of CaC12 and EDTA on the PNL. The addition of EDTA (0.005 M) and CaC12 (0-0.02 M) to the reaction mixtures were assayed under standard conditions.

4. DISCUSSION It has been generally believed that, among plant pathogens promoting pectolysis, bacteria produce predominantly pectate lyase while fungi usually secrete pectin lyase (Phoma medicaginis var. pinodella synthesizes a pectin lyase [25 ). However, both types of lyase activity are frequently present in an organism: Fusarium solani f. sp. phaseoli produces a calcium-dependent lyase that degrades both pectin and pectic acid under alkaline conditions [30]. Fusanum solam f. sp. pisi produces an endopectate lyase that seems to be involved in pathogenesis [31 ]. It has been suggested that both types of enzyme should be considered as pectin lyase, and that they be distinguished according to their preference for highly esterified and low-esterified pectin [32]. As other pectolytic microorganisms the r2 isolate of Fusarium oxysporum f. sp. radicis-lycopersici produces a battery of pectic enzymes differing in substrate preference, reaction mechanism, and action pattern. When separated by isoelectric focusing and stained for activities, we have detected three forms that have lyase activity and pIs of 9.20, 9.00 and 8.65 (figure 5). Phoma medicaginis var. pinodella synthesizes a pectin lyase that has a pI of 7.9 [25], the pectate lyase from Fusarium solani f. sp. pisi has a pI of 8.3 [31], the multiple endopectate lyases from Hypomyces solani f. sp. cucurbitae obtained from culture and from infected tissue possess isoelectric points in the range of 10.2-10.3 and 10.510.6 respectively [33].

758

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. The PNL exhibits an optimum pH of 9.5 (figure 6) and an optimum temperature of 55 ~ C (figure 7). Lyases catalyze the reaction in an alkaline or in a neutral medium at high temperatures [32]: pectin lyase from Phoma medicaginis var. pinodella showed an optimum pH of 7.5 [25], endopectate lyase from Fusarium solam f. sp. pisi showed an optimum pH of 9.4 [31 ], and pectate lyase from Rhizoctonia solani showed an optimum pH of 8.0 [34]. The molecular weight calculated by Superdex chromatography was 18 kDa (figure 2). Endopectate lyases from Hypomyces solani f. sp. cucurbitae from culture and infected tissue have molecular sizes between 32 and 42 kDa [33]. Fusarium solani f. sp. pisi possess an endopectate lyase of 26 kDa [31] and Erwinia aroideae possess one of 67 kDa [25]. Phoma medicaginis var. pinodella has two forms of the pectin lyase with molecular weight of 29.5 and 118 kDa which suggested the existence of monomeric and tetrameric components [25]. The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma m edicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM C a 2+ [25]. The lyase from Fusarium solani f. sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent: the pectate lyase from Rhizoctoma solam [34] and the endopectate lyase from Fusanum solam f. sp. pisi [31 ]. Two characteristics of the lyase that we have purified may be significant. First, the small molecular size of the protein may confer it a high mobility that could be helpful to its movement through host cell walls. In second place, it is an endo-type enzyme, fact that has been considered essential for maceration of plant tissues [35]. In this sense it is noteworthy that between the battery of pectic enzymes produced by FORL, this pectin lyase is the only protein that behaves as an endo-type.

759 REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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