ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
176, 334-343 (1976)
lsolation and Characterization of a Cutinase culmorum and Its Immunological Comparison solani pisi’ C. L. SOLIDAY Department
AND
from Fusarium roseum with Cutinases from F.
P. E. KOLATI’UKUDY*
of Agricultural Chemistry and the Graduate Program in Biochemistry Washington State University, Pullman, Washington 99163
and Biophysics,
Received February 25, 1976 Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but notp-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition ofF. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the 13Hldiisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.
The cuticle of higher plants consists of a structural biopolymer, cutin, impregnated with wax (l-3). Cutin is a polyester composed of hydroxy fatty acids, containing 16 or 18 carbon atoms. It is thought to function as a barrier against the entry of pathogens into plants. Electron microscopic 1 Scientific Paper No. 4566, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99163. This work was supported in part by National Science Foundation Grant BMS74-09351 AOl. 2 Author to whom inquiries should be made.
studies indicate that enzymatic degradation of the cutin barrier may be the mode of fungal penetration into plants (4). The production of extracellular hydrolytic enzymes by plant pathogens has been suggested by the ability of some of these microorganisms to live on cutin as their sole source of carbon (5-7). The extracellular fluid of cutin-grown Fusarium solani f. pisi, a fungus pathogenic to pea roots, has been shown to catalyze depolymerization of cutin and hydrolysis of fatty acyl esters of p-nitrophenol (7). From the extracellular fluid, two iso-
334 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
CUTINASE
FROM F. ROSEUM
zymes of cutinase and a nonspecific esterase have been isolated and purified to homogeneity (81, and their properties have been described (9). This is the only pathogen from which such enzymes have been purified, and in order to assessthe possible role of such enzymes in pathogenesis, information concerning possible production of such enzymes by other pathogenic microorganisms is needed. In this communication, we describe the purification and characterization of a cutinase produced by a related pathogenic fungus Fusarium T-Oscum culmorum, whose host is wheat. The properties of the F. roseum cutinase are compared with those of the F. solani pisi cutinases, and the three cutinases thus far isolated are also compared immunologically. MATERIALS
AND METHODS
Cultures ofFusarium roseum were maintained on potato dextrose agar at 23”C, from which mycelia suspensions were taken for innoculation of cutin-containing media which consisted of 0.5 g of 60-mesh Golden Delicious apple cutin suspended in 100 ml of a basal mineral medium at pH 7.2 (7). F. roseum was cultured on the cutin-mineral medium for 12 days at 23°C. Purification of cutinase. The extracellular fluid from 60 cultures was filtered twice, yielding about 5 liters of fluid. Granular ammonium sulfate was slowly added to the stirring extracellular fluid until 50% saturation was achieved. The precipitate was sedimented by centrifugation at 20,OOOgfor 30 min, dissolved in 20 ml of 50 mM sodium phosphate buffer, pH 8.0, and dialyzed overnight against the same buffer at 4°C. The dialyzed solution was concentrated to 5-6 ml by ultrafiltration with an Amicon UMlO membrane and the solution was clarified by centrifugation. The concentrated enzyme solution was applied to a Sephadex G-100 column (2.2 x 34 cm) which had been equilibrated with 40 mM sodium phosphate buffer, pH 8.0, and the proteins were eluted with the same buffer. The column effluent was monitored for absorption at 280 nm, and the collected fractions (56 ml) were assayed forp-nitrophenyl butyrate andpnitrophenyl palmitate hydrolase activities by a spectrophotometric assay (7). The fractions possessing hydrolase activities were pooled and concentrated by ultrafiltration on a UMlO membrane. The resulting concentrate (6 ml) was dialyzed overnight against 100 mra Tris-HCl buffer at pH 9.0. The enzyme solution was then applied to a QAESephadex G-25 column (3 x 22 cm) which had been equilibrated in 100 mM Tris-HCl buffer, pH 9.0. The Culture culmorum
conditions.
CULMORUM
335
proteins were eluted with two bed volumes of the same buffer, followed by two bed volumes each of pH 8.3 and 7.0 buffers (100 mM Tris-HCl). The column effluent was monitored for absorption at 280 nm, and the fractions (5-6 ml) were checked for hydrolase activity by the spectrophotometric assay (7). The fractions which eluted at pH 9.0 and contained hydrolase activity were pooled and concentrated by ultrafiltration. The concentrate was dialyzed against citrate-phosphate buffer, pH 4.8 (5 mM citrate and 10 mM phosphate). Final purification was achieved by cation exchange chromatography of the enzyme solution applied to a SP-Sephadex G-25 column (1.8 x 15 cm) equilibrated in citrate-phosphate buffer, pH 4.8. The column was washed with two bed volumes of buffer before a linear gradient of O-300 mM sodium chloride was applied. The hydrolase activity of the eluant was monitored by the spectrophotometric assay. Electrophoresis. Cationic polyacrylamide disc gel electrophoresis was performed as described by Gabriel (10). The separation gels were prepared by tilling 65 x 5 mm glass tubes to 45 mm with 7.5% polyacrylamide in acetate buffer, pH 3.8. After polymerization, 10 mm of a stacking gel solution (2.5% polyacrylamide in acetate buffer, pH 5.0) was added to each tube. The running buffer consisted of a palanine solution (3.12 g/liter), adjusted to pH 4.5 with acetic acid. Electrophoresis was performed with 2 n-A/tube until the methylene blue tracking dye approached the bottom of the gel. The gels were stained with 1% amido black in 7% acetic acid for 2 h and then destained by an Ames Model 1801quick gel destainer or by soaking in 7% acetic acid. Amino acid analysis. The amino acids derived from the enzymes were analyzed with a Beckman automatic amino acid analyzer, Model 121C, using the method of Spackman et al. (11). Approximately 0.5-mg samples of the cutinase were hydrolyzed with 6 M HCl at 100°C for 24 h. Identical samples were used to measure the quantities of cysteine and methionine (12) and tryptophan (13). SDS3 electrophoresis. Dodecyl sulfate polyacrylamide gel electrophoresis was performed with 15% polyacrylamide gels as described by Weber et al. (14). The standards were alcohol dehydrogenase (40,0001, pepsin (35,000), a-chymotrypsinogen (25,700), trypsin (23,300), and ribonuclease (13,700). The proteins were heated with 2% SDS and 100 mM dithioerythritol in a boiling water bath for 3 min before electrophoresis. After electrophoresis at 7 n-&tube, the gels were stained with Coomassie blue
a Abbreviations used: SDS, sodium dodecyl sulfate; DTE, dithioerythritol; Dip-F, diisopropylfluorophosphate; PNP, p-nitrophenyl palmitate; PNB, p-nitrophenyl butyrate.
336
SOLIDAY
AND
overnight and then destained with an Ames quick gel destainer. Cutinase assays. Appropriate aliquots of the enzyme were incubated in a total volume of 1 ml of 50 mM glycine-NaOH buffer (pH 10.0) with 4.2 mg of tritiated cutin in a gyrating water bath shaker for 10 min at 30°C. The suspension of tritiated cutin, used as substrate, was prepared with great care as previously described (8). The reaction was terminated with the addition of 0.2 ml of 1 M HCl, and the reaction mixture was centrifuged at 5000 rpm for 15 min. Aliquots (0.2 ml) of the supernatant were assayed for radioactivity in 15 ml of Aquasol with a Packard 3003 liquid scintillation spectrometer. All measurements of radioactivity were done with a 14% counting eftlciency and a standard deviation of
OF
BIOCHEMISTRY
AND
BIOPHYSICS
176, 334-343 (1976)
lsolation and Characterization of a Cutinase culmorum and Its Immunological Comparison solani pisi’ C. L. SOLIDAY Department
AND
from Fusarium roseum with Cutinases from F.
P. E. KOLATI’UKUDY*
of Agricultural Chemistry and the Graduate Program in Biochemistry Washington State University, Pullman, Washington 99163
and Biophysics,
Received February 25, 1976 Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but notp-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition ofF. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the 13Hldiisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.
The cuticle of higher plants consists of a structural biopolymer, cutin, impregnated with wax (l-3). Cutin is a polyester composed of hydroxy fatty acids, containing 16 or 18 carbon atoms. It is thought to function as a barrier against the entry of pathogens into plants. Electron microscopic 1 Scientific Paper No. 4566, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99163. This work was supported in part by National Science Foundation Grant BMS74-09351 AOl. 2 Author to whom inquiries should be made.
studies indicate that enzymatic degradation of the cutin barrier may be the mode of fungal penetration into plants (4). The production of extracellular hydrolytic enzymes by plant pathogens has been suggested by the ability of some of these microorganisms to live on cutin as their sole source of carbon (5-7). The extracellular fluid of cutin-grown Fusarium solani f. pisi, a fungus pathogenic to pea roots, has been shown to catalyze depolymerization of cutin and hydrolysis of fatty acyl esters of p-nitrophenol (7). From the extracellular fluid, two iso-
334 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
CUTINASE
FROM F. ROSEUM
zymes of cutinase and a nonspecific esterase have been isolated and purified to homogeneity (81, and their properties have been described (9). This is the only pathogen from which such enzymes have been purified, and in order to assessthe possible role of such enzymes in pathogenesis, information concerning possible production of such enzymes by other pathogenic microorganisms is needed. In this communication, we describe the purification and characterization of a cutinase produced by a related pathogenic fungus Fusarium T-Oscum culmorum, whose host is wheat. The properties of the F. roseum cutinase are compared with those of the F. solani pisi cutinases, and the three cutinases thus far isolated are also compared immunologically. MATERIALS
AND METHODS
Cultures ofFusarium roseum were maintained on potato dextrose agar at 23”C, from which mycelia suspensions were taken for innoculation of cutin-containing media which consisted of 0.5 g of 60-mesh Golden Delicious apple cutin suspended in 100 ml of a basal mineral medium at pH 7.2 (7). F. roseum was cultured on the cutin-mineral medium for 12 days at 23°C. Purification of cutinase. The extracellular fluid from 60 cultures was filtered twice, yielding about 5 liters of fluid. Granular ammonium sulfate was slowly added to the stirring extracellular fluid until 50% saturation was achieved. The precipitate was sedimented by centrifugation at 20,OOOgfor 30 min, dissolved in 20 ml of 50 mM sodium phosphate buffer, pH 8.0, and dialyzed overnight against the same buffer at 4°C. The dialyzed solution was concentrated to 5-6 ml by ultrafiltration with an Amicon UMlO membrane and the solution was clarified by centrifugation. The concentrated enzyme solution was applied to a Sephadex G-100 column (2.2 x 34 cm) which had been equilibrated with 40 mM sodium phosphate buffer, pH 8.0, and the proteins were eluted with the same buffer. The column effluent was monitored for absorption at 280 nm, and the collected fractions (56 ml) were assayed forp-nitrophenyl butyrate andpnitrophenyl palmitate hydrolase activities by a spectrophotometric assay (7). The fractions possessing hydrolase activities were pooled and concentrated by ultrafiltration on a UMlO membrane. The resulting concentrate (6 ml) was dialyzed overnight against 100 mra Tris-HCl buffer at pH 9.0. The enzyme solution was then applied to a QAESephadex G-25 column (3 x 22 cm) which had been equilibrated in 100 mM Tris-HCl buffer, pH 9.0. The Culture culmorum
conditions.
CULMORUM
335
proteins were eluted with two bed volumes of the same buffer, followed by two bed volumes each of pH 8.3 and 7.0 buffers (100 mM Tris-HCl). The column effluent was monitored for absorption at 280 nm, and the fractions (5-6 ml) were checked for hydrolase activity by the spectrophotometric assay (7). The fractions which eluted at pH 9.0 and contained hydrolase activity were pooled and concentrated by ultrafiltration. The concentrate was dialyzed against citrate-phosphate buffer, pH 4.8 (5 mM citrate and 10 mM phosphate). Final purification was achieved by cation exchange chromatography of the enzyme solution applied to a SP-Sephadex G-25 column (1.8 x 15 cm) equilibrated in citrate-phosphate buffer, pH 4.8. The column was washed with two bed volumes of buffer before a linear gradient of O-300 mM sodium chloride was applied. The hydrolase activity of the eluant was monitored by the spectrophotometric assay. Electrophoresis. Cationic polyacrylamide disc gel electrophoresis was performed as described by Gabriel (10). The separation gels were prepared by tilling 65 x 5 mm glass tubes to 45 mm with 7.5% polyacrylamide in acetate buffer, pH 3.8. After polymerization, 10 mm of a stacking gel solution (2.5% polyacrylamide in acetate buffer, pH 5.0) was added to each tube. The running buffer consisted of a palanine solution (3.12 g/liter), adjusted to pH 4.5 with acetic acid. Electrophoresis was performed with 2 n-A/tube until the methylene blue tracking dye approached the bottom of the gel. The gels were stained with 1% amido black in 7% acetic acid for 2 h and then destained by an Ames Model 1801quick gel destainer or by soaking in 7% acetic acid. Amino acid analysis. The amino acids derived from the enzymes were analyzed with a Beckman automatic amino acid analyzer, Model 121C, using the method of Spackman et al. (11). Approximately 0.5-mg samples of the cutinase were hydrolyzed with 6 M HCl at 100°C for 24 h. Identical samples were used to measure the quantities of cysteine and methionine (12) and tryptophan (13). SDS3 electrophoresis. Dodecyl sulfate polyacrylamide gel electrophoresis was performed with 15% polyacrylamide gels as described by Weber et al. (14). The standards were alcohol dehydrogenase (40,0001, pepsin (35,000), a-chymotrypsinogen (25,700), trypsin (23,300), and ribonuclease (13,700). The proteins were heated with 2% SDS and 100 mM dithioerythritol in a boiling water bath for 3 min before electrophoresis. After electrophoresis at 7 n-&tube, the gels were stained with Coomassie blue
a Abbreviations used: SDS, sodium dodecyl sulfate; DTE, dithioerythritol; Dip-F, diisopropylfluorophosphate; PNP, p-nitrophenyl palmitate; PNB, p-nitrophenyl butyrate.
336
SOLIDAY
AND
overnight and then destained with an Ames quick gel destainer. Cutinase assays. Appropriate aliquots of the enzyme were incubated in a total volume of 1 ml of 50 mM glycine-NaOH buffer (pH 10.0) with 4.2 mg of tritiated cutin in a gyrating water bath shaker for 10 min at 30°C. The suspension of tritiated cutin, used as substrate, was prepared with great care as previously described (8). The reaction was terminated with the addition of 0.2 ml of 1 M HCl, and the reaction mixture was centrifuged at 5000 rpm for 15 min. Aliquots (0.2 ml) of the supernatant were assayed for radioactivity in 15 ml of Aquasol with a Packard 3003 liquid scintillation spectrometer. All measurements of radioactivity were done with a 14% counting eftlciency and a standard deviation of
