- Email: [email protected]
Hydrolysis of the potato glycoalkaloid α-chaconine by filamentous fungi
JOURNALOFBIOSCIENCE ANDBIOENGINEERING Vol. 94, No. 4,321-325. 2002
Hydrolysis of the Potato Glycoalkaloid a-Chaconine by Filamentous Fungi YUJI ODA,‘* KATSUICHI SAITO,’ AKIKO OHARA-TAKADA,’ AND MOTOYUKI MORI’ Department of Upland Agriculture Research, National Agricultural Research Center for Hokkaido Region, Memuro, Kasai, Hokkaido 082-0071, Japan’ Received 7 June 2002/Accepted 16 July 2002
Three strains of filamentous fungi have been isolated from potato sprouts to obtain an enzyme degrading the glycoalkaloids. All of the strains hydrolyzed a-chaconine and not a-solanine when grown on the sprouts. From strain HP341, identified as Plectosphaerella cucumerina, the enzyme hydrolyzing a-chaconine was purified on columns of DEAE-Toyopearl and Phenyl-Toyopearl. The partially purified enzyme hydrolyzed a-chaconine to PI-chaconine but not to &- or y-chaconine, suggesting that the enzyme is a rhamnosidase specific for the hydrolysis of the rhamnose (C&S glucose linkage in a-chaconine. Conversion of a-chaconine to &chaconine may be the first step of detoxification for filamentous fungi to grow on potato sprouts that accumulated antifungal a-chaconine. [Key words: glycoalkaloid,
a-chaconine,
rhamnosidase,
detoxification]
Potatoes contain secondary metabolites, steroidal glycoalkaloids, which are potential toxic substances. Human ingestion of 1 to 2 mg of potato glycoalkaloids per kg of body weight can cause poisoning with gastroenteric symptoms, coma, and even death (1). The toxicity may be due to disorders of the digestive system and general body metabolism, which are triggered by adverse effects on the central nervous system and disruption of cell membranes (1). The glycoalkaloid content in a potato tuber is 40 to 120 mg per kg of fresh weight (2) and increases more than ten-fold by light exposure, storage conditions, and processing damages (3). Once the glycoalkaloids are formed, typical food processing, which includes boiling, cooking, baking, frying, and microwaving, does not degrade them (4). For these reasons, potatoes that have turned green as a result of exposure to light are considered to be unsafe and usually rejected. The principal glycoalkaloids in potato tubers are IXchaconine and a-solanine (1). Both have a common chemical structure containing trisaccharide attached to the 3-OH position of the steroidal glycoalkaloid solanidine. In CLchaconine, the trisaccharide is composed of glucose and two residues of rhamnose, while a-solanine includes galactose, glucose, and rhamnose. The biological activity of the glycoalkaloids is governed by the type and number of sugar making up the trisaccharide chain (5). The difference in the nature of sugar explains the higher toxicity of a-chaconine than a-solanine (1). Stepwise removal of a sugar unit from the trisaccharide chain, producing p,- or P,-chaconine, ychaconine, and solanidine (Fig. l), gradually reduces the toxicity of a-chaconine (6). Sprouts, tubers, and blossoms of potato contained the enzymes liberating rhamnose, glu* Corresponding author. e-mail: [email protected] phone: +81-(0)155-62-9280 fax: +81-(0)155-62-9281 321
case, and galactose from the glycoalkaloids, and some of them were partially purified from the potato peel (7, 8). However, the hydrolyzing activity of glycoalkaloids in a potato plant may be insufficient for treating large quantities of discarded green potatoes. Chemical hydrolysis of potato glycoalkaloids requires the use of a strong acid and high temperature. The objective of our research is to develop an efficient method for the detoxification of glycoalkaloids accumulated in a potato crop. Here, we report on the isolation and characterization of the filamentous fungi hydrolyzing a-chaconine. MATERIALS
AND METHODS
Materials a-Chaconine and a-solanine were purchased from Sigma Chemical (St. Louis, MO, USA). Authentic PI-, &-, and ychaconines were prepared by partial hydrolysis of a-chaconine in 97.5% methanol-O.25N HCl at 60°C (9). Potato sprouts (dry matter, about 10%) collected from potatoes that had been stored for six months were treated at 105°C for 30 min, minced, and preserved at -40°C. Spores of fungi were formed as they were grown on Culture potato dextrose agar (Becton Dickinson, Sparks, MD, USA) for 5 to 7 d. After being suspended at 5 x IO6 spores per ml of 0.01% Tween 80, 0.2 ml of the suspension was inoculated to 10 g of the minced sprouts in a lOO-ml Erlemneyer flask and cultured for 5 d. All cultures were incubated at 25°C without shaking. Determination of the glycoalkaloids The cultured sprouts (10 g) as described above were mixed with 20 ml of 1.O% acetic acid and shaken at 120 rpm for 1 h. After centrifugation of the mixture at 10,000 xg for 20 min, the supernatant was passed through a commercial Cl8 cartridge to purify the glycoalkaloids by the method of Carman et al. (10). The glycoalkaloids were analyzed using a high-performance liquid chromatograph (HPLC, LC-1OAD system; Shimadzu, Kyoto) equipped with a reverse phase C8 column (Shim-pack HRC-C8; 25 cmx4.6 mm+) and a UV monitor.
322
ODA ET AL.
J. BIOSCI. BIOENG.,
a-Chacmine
f32-Chaconine
HO
FIG. 1. Chemical structures of a-chaconine and its hydrolysis products. The mobile phase was 0.58% ammonium phosphate in 30% acetonitrile at a flow rate of 0.8 ml/min. Enzyme assays The sprouts (10 g) grown with the fungi were mixed with 10 ml of distilled water and extracted by shaking at 4°C. The supernatant obtained by centrifugation of the mixture
was used as the crude enzyme. The substrate solution, including both 6.0 mg of cr-chaconine and 5.0 mg of cr-solanine per ml of 50% methanol, was prepared from the extract of sprouts, as described above. A standard assay mixture, which contained 0.025 ml of a 1 M acetate buffer (pH 5.0), 0.1 ml of the substrate solu-
HYDROLYSIS OF a-CHACONINE
VOL.94,2002 tion, and 0.05 ml of the crude enzyme in a total volume of 0.5 ml, was incubated at 30°C for 30 min unless otherwise stated. After boiling in water for 5 min stopped the reaction, and a-chaconine was determined by HPLC. One unit of a-chaconine-hydrolyzing activity was defined as the amount of emme that consumed 1 pmol of a-chaconine per min. The activity for hydrolyzingp-nitrophenyl a-L-rhamnoside was assayed as described elsewhere (11).
a-Solanine +
No inoculation
323
a-Chaconine +
fi
RESULTS In potato plants, the Isolation of filamentous fungi sprouts accumulate the highest level of glycoalkaloids, which inhibit fungal growth (1). The fungi, which can grow on the sprouts, are expected to convert the glycoalkaloids to less toxic substances; however, this phenomenon has rarely been observed. We have accidentally found molds on the surface of sprouts germinated from stored tubers and isolated several filamentous fungi on potato dextrose agar. Among them, three strains, which showed the distinct morphology of a colony, were selected and studied in detail. From the growth and morphological characteristics, strains HP339, HP340, and HP341 were estimated to be Cladosporium cladosporioides (12), Penicillium sp. (subgenus Furcatum section Furcatum sensu Pitt, 1980) (13), and Cephalosporium-like Hyphomycetes, respectively. Sequence analysis of 28s rDNA and 18S-28s rDNA spacer region revealed that strain HP34 1 was identified as Plectosphaerella cucumerina (anamorph: Plectosporium tabacinum) (14, 15). The glycoalkaloid conDegradation of a-chaconine tents were determined in the sprouts incubated after inoculation of the fungal spores. The three strains reduced the achaconine content (Fig. 2) but not the a-solanine content (data not shown), and R cucumerina HP341 lost a-chaconine in 24 h. In HPLC chromatograms, the decrease of achaconine was accompanied by the simultaneous increase of an unknown compound eluted at 13.3 min (Fig. 3). No other peak appeared in the chromatograms even when the sprouts were further incubated for 24 h. Properties of the enzyme hydrolyzing a-chaconine a-Chaconine-hydrolyzing activity was followed in the crude enzyme extracted from the sprouts grown with F!
- 6
Strain HP339
h
Strain HP340
b
I
16
14
12
10
8
Retention time (min) FIG. 3. HPLC chromatograms of the extract from the potato sprouts grown with the tilamentous fungi. The potato sprouts were incubated 25°C for 24 h after inoculation of fungal spores and used for the HPLC assay.
cucumerina HP341. The enzyme was gradually produced after inoculation as the pH increased, which may reflect vegetative growth (Fig. 4). The crude enzyme obtained from 100 g of the sprouts that had been incubated for 5 d was partially purified by the columns of DEAE-Toyopearl and Phenyl-Toyopearl and dialyzed against 20 mM TrisHCl buffer (pH 8.5) (Table 1). When the activity was assayed in the pH range of 4.0 to 5.5 at 30°C and in the range of 25°C to 60°C at pH 5.0, the enzyme gave maximal activities near pH 5.0 and 5O”C, respectively. Enzymatic reaction was conducted in the mixture containing authentic a-chaconine or a-solanine as substrates. The enzyme converted achaconine to P,-chaconine (Fig. 5) but did not affect asolanine (data not shown). Hydrolyzing activity of p-nitro-
254
9
- 6
0 0
10
20 C*e
30 period 0
40
50 0
I
2
3
4
5
6
FIG. 2. Degradation of a-chaconine in the potato sprouts by Mamentous fungi. The potato sprouts were incubated at 25°C after inoculation of fungal spores. Symbols: triangles, C. cludosporioides HP339; closed circles, Penicilhn sp. HP340; open circles, p cucumerina
cucumerina HP341. Symbols: open circles, a-chaconine-hydrolyzing
HP341.
activity; closed circles, pH.
Cuhureperiod (d)
FIG. 4. Production of an a-chaconine-hydrolyzing
enzyme by p
J. BIOSCI.BIOENG.,
ODA ET AL.
324
TABLE 1. Partial purification of an a-chaconine-degrading Step
enzyme from the extract of sprouts grown with p cucumerina HP341
Total activity (U)
Total protein
536 302 270
25.3 4.89 -1.29
Crude extract DEAE-Toyopearl650M Phenyl-Toyopearl650M
6d
Specific activitv
_
Ww) 21.2 61.8 209.0
_
Purification (fold)
Yield
1.0 2.9 9.9
100 56.3 50.4
(%I
The data are the mean values of three independent experiments.
PI P2
a
Y
Chaconine stana
Hydrolysis product
r\
I
12
14
16
Retention time FIG 5.
chromatogram e cucumerina
phenyl-a+rhamnoside was only detectable (x0.02 pmol/ min/mg protein) and corresponded to less than lOA of that for a-chaconine. It is unknown whether the activity toward p-nitrophenyl-a+rhamnoside is derived from a contaminant or the enzyme itself. However, the above observations indicate at least that the enzyme is a rhamnosidase highly specific for the rhamnose (C-C,) glucose linkage in a-chacanine. DISCUSSION Both a-chaconine and a-solanine are grouped as saponin that are found in various plant species. Saponins consist of triterpenoids, steroids, or steroidal glycoalkaloids as aglycones with one or more sugars, and they often repress the growth of fungi (16). The antifungal activity of these compounds may function in the chemical defense of the plants against attack from fungal pathogens (17). The major mechanism of toxicity involves the formation of complexes with membrane sterols, resulting in pore formation and loss of membrane integrity. Successful pathogens of the plants avoid antifungal saponins by changing their membrane composition or by removing their sugar chain for detoxification (18). a-Tomatine, the glycoalkaloids present in tomato, has tomatidine as aglycone and a tetrasaccharide moiety consisting of two molecules of glucose and one each of galactose and xylose (19). Some enzymes detoxifying a-tomatine have been characterized in the fungi infecting tomato, and the linkage of the sugar chain hydrolyzed by these enzymes is not the same (16). In the present experiments, three filamentous fungi degrading a-chaconine were isolated from the potato sprouts
that contain high amounts of the glycoalkaloids. Strain HP34 1 produced an a-chaconine-hydrolyzing enzyme that was shown to be rhamnosidase-specific for the rhamnose (C-C,) glucose linkage but not for the rhamnose (C-C,) glucose linkage in a-chaconine. The substrate specificity of the enzymes from the three strains may be common because only &chaconine was formed from a-chaconine in the sprouts grown with each fungus (Fig. 3). The species names of the three strains, Cladosporium cladosporioides, Penicillum sp., and Plectosphaerella cucumerina, have not been reported to be pathogenic to potato plants in Japan (20). Detoxification of antifungal saponins is involved in the resistance of pathogenic fungi but may not be sufficient to infect saponin-containing plants (21). The tomato pathogen Gibberella pulicaris was reported to metabolize a-chaconine to P,-chaconine by the enzyme specific for the rhamnose (C,-C,) glucose linkage (22). The steroidal avenacosides A and B, which are located in the leaves and shoots of oats, include sugar chains composed of the rhamnose (C-C,) glucose linkage such as achaconine (16). Both avenacosides are inactive and convert antifungal 26-desglucoavenacosides in response to attack or wounding ( 16). The phytopatbogenic fungus Stagonospora avenae reduces the antifungal activity of 26-desglucoavenacosides by removing the sugar chain (23). Morrissey et al. (24) stated that the sugar chain is sequentially hydrolyzed by the action of one a-rhamnosidase and two p-glucosidases and that a-rhamnosidase plays a crucial role in saponin resistance. Alternatively, the product lacking the rhamnose residue attached to the C, position of glucose in the side chain is less toxic to pathogenic fungi infecting oats. It may be possible that the three strains isolated in the present experiments reduce the toxicity of a-chaconine by a rhamnosidase toward rhamnose (C,-C,) glucose linkage. The hydrolysis product P,-chaconine seems to be less toxic for fungi, while it is still harmful to animal cells. The developmental toxicity of P,-chaconine in the frog embryo was higher than that of I$- and y-chaconine (6). Further degradation by microbial enzymes to solanidine, which has a low level of biological activity, is required to accomplish our research project. ACKNOWLEDGMENTS This work was supported in part by a grants-in-aid from Japan Food Industry Center (Tokyo) and Special Coordination Funds for Promoting Science and Technology (Leading Research Utilizing Potential of Regional Science and Technology) of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
VOL. 94,2002
HYDROLYSIS OF a-CHACONINE
REFERENCES
203-208 (1996). 12. Domsch, K. H., Gams, W., and Anderson, T. II.: Compen-
1. Friedman, 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
M. and McDonald, G. M.: Potato glycoalkaloids: chemistry, analysis, safety, and plant physiology. Crit. Rev. Plant Sci., 16, 55-l 32 (1997). Friedman, M. and Dao, L.: Distribution of glycoalkaloids in potato plants and commercial potato products. J. Agric. Food Chem., 40,419-423 (1992). Kozukue, N. and Mixuno, S.: Effects of light exposure and storage temperature on greening and glycoalkaloid content in potato tubers. J. Jpn. Sot. Hortic. Sci., 59, 673677 (1990). (in Japanese) Bushway, R J. and Ponnampalam, R: a-Chaconine and asolanine content of potato products and their stability during several modes of cooking. J. Agric. Food Chem., 29, 814-817 (1981). Friedman, M., Rayburn, J. R, and Bantle, J. A.: Structural relationships and developmental toxicity of Solanum alkaloids in the frog embryo teratogenesis assay-Xenopus. J. Agric. Food Chem., 40,1617-1624 (1992). Bayburn, J. R, Bantle, J. A., and Friedman, M.: Role of carbohydrate side chains of potato glycoalkaloids in developmental toxicity. J. Agric. Food Chem., 42, 15 1l-1 5 15 ( 1994). Busbway, A. A., Bushway, R J., and Kim, C. II.: Isolation, partial purification and characterization of a potato peel glycoalkaloid glycosidase. Am. Potato J., 65, 62163 1 (1988). Bushway, A. A., Bushway, R. J., and Kim, C. II.: Isolation, partial purification and characterization of a potato peel asolanine cleaving glycosidase. Am. Potato J., 67, 233-238 (1990). Friedman, M. and McDonald, G. M.: Acid-catalyzed partial hydrolysis of carbohydrate groups of the potato glycoalkaloid a-chaconine in alcoholic solutions. J. Agric. Food Chem., 43, 1501-1506 (1995). Carman, A. S., Jr., Kuan, S. S., Ware, G. M., Francis, 0. J., Jr., and Kirschenheuter, G. P.: Rapid high-performance liquid chromatographic determination of the potato glycoalkaloids a-solanine and a-chaconine. J. Agric. Food Chem., 34,279-282 (1986). Oda, Y. and Tonomura, K.: a-Galactosidase from the yeast Torulaspora delbrueckii IF0 1255. J. Appl. Bacterial., 80,
325
13. 14.
15. 16. 17.
dium of soil fungi, ~01s. 1 and 2. Academic Press, New York (1980). Pitt, J. I.: A laboratory guide to common Penicillum species. CSIRO Food Research Laboratory, North Ryde (1985). Palm, M. E., Gams, W., and Nirenberg, H. I.: Plectosporium, a new genus for Fusarium tabacinum, anamorph of PZectosphaereZZacucumerina. Mycologia, 87,397406 (1995). Uecker, F. A.: Development and cytology of PlectosphaereZZacucumerina. Mycologia, 85,47&479 (1993). Osbourn, A. E.: Saponins and plant defence - a soap history. Trend Plant Sci., 1,4-9 (1996). Osbourn, A. E.: Antimicrobial phytoprotectants and fungal pathogens: a commentary. Fungal Genet. Biol., 26, 163-168
(1999). 18. Osbourn, A. E.: Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell, 8, 1821-183 1 (1996). 19. Friedman, M., Kozukue, N., and Harden, L. A.: Preparation and characterization of acid hydrolysis products of the tomato glycoalkaloid a-tomatine. J. Agric. Food Chem., 46, 2096-2101 (1998). 20. The Phytopathological Society of Japan: Common name of plant diseases in Japan, 1st ed. Japan Plant Protection Association, Tokyo (2000). (in Japanese) 21. Martin-Hernandez, A.M., Dufresne, M., Hugouvieux, Y., Melton, R., and Osbourn, A. E.: Effects of targeted replacement of the tomatinase gene on the interaction of Septoria Zycopersici with tomato plants. Mol. Plant-Microbe Interact., 13, 1301-1311 (2000). 22. Becker, P. and Weltring, K-M.: Purification and characterization of a-chaconinase of Gibberella pulicaris. FEMS Microbial. Lett., 167, 197-202 (1998). 23. Wubben, J. P., Price, K. R, Daniels, M. J., and Osbourn, A. E.: Detoxification of oat leaf saponins by Septoria avenae. Phytopathology, 86,986992 (1996). 24. Morrissey, J. P., Wubben, J. P., and Osbourn, A. E.: Stagonospora avenae secretes multiple enzymes that hydrolyze oat leaf saponins. Mol. Plant-Microbe Interact., 13, 10411052 (2000).
Hydrolysis of the Potato Glycoalkaloid a-Chaconine by Filamentous Fungi YUJI ODA,‘* KATSUICHI SAITO,’ AKIKO OHARA-TAKADA,’ AND MOTOYUKI MORI’ Department of Upland Agriculture Research, National Agricultural Research Center for Hokkaido Region, Memuro, Kasai, Hokkaido 082-0071, Japan’ Received 7 June 2002/Accepted 16 July 2002
Three strains of filamentous fungi have been isolated from potato sprouts to obtain an enzyme degrading the glycoalkaloids. All of the strains hydrolyzed a-chaconine and not a-solanine when grown on the sprouts. From strain HP341, identified as Plectosphaerella cucumerina, the enzyme hydrolyzing a-chaconine was purified on columns of DEAE-Toyopearl and Phenyl-Toyopearl. The partially purified enzyme hydrolyzed a-chaconine to PI-chaconine but not to &- or y-chaconine, suggesting that the enzyme is a rhamnosidase specific for the hydrolysis of the rhamnose (C&S glucose linkage in a-chaconine. Conversion of a-chaconine to &chaconine may be the first step of detoxification for filamentous fungi to grow on potato sprouts that accumulated antifungal a-chaconine. [Key words: glycoalkaloid,
a-chaconine,
rhamnosidase,
detoxification]
Potatoes contain secondary metabolites, steroidal glycoalkaloids, which are potential toxic substances. Human ingestion of 1 to 2 mg of potato glycoalkaloids per kg of body weight can cause poisoning with gastroenteric symptoms, coma, and even death (1). The toxicity may be due to disorders of the digestive system and general body metabolism, which are triggered by adverse effects on the central nervous system and disruption of cell membranes (1). The glycoalkaloid content in a potato tuber is 40 to 120 mg per kg of fresh weight (2) and increases more than ten-fold by light exposure, storage conditions, and processing damages (3). Once the glycoalkaloids are formed, typical food processing, which includes boiling, cooking, baking, frying, and microwaving, does not degrade them (4). For these reasons, potatoes that have turned green as a result of exposure to light are considered to be unsafe and usually rejected. The principal glycoalkaloids in potato tubers are IXchaconine and a-solanine (1). Both have a common chemical structure containing trisaccharide attached to the 3-OH position of the steroidal glycoalkaloid solanidine. In CLchaconine, the trisaccharide is composed of glucose and two residues of rhamnose, while a-solanine includes galactose, glucose, and rhamnose. The biological activity of the glycoalkaloids is governed by the type and number of sugar making up the trisaccharide chain (5). The difference in the nature of sugar explains the higher toxicity of a-chaconine than a-solanine (1). Stepwise removal of a sugar unit from the trisaccharide chain, producing p,- or P,-chaconine, ychaconine, and solanidine (Fig. l), gradually reduces the toxicity of a-chaconine (6). Sprouts, tubers, and blossoms of potato contained the enzymes liberating rhamnose, glu* Corresponding author. e-mail: [email protected] phone: +81-(0)155-62-9280 fax: +81-(0)155-62-9281 321
case, and galactose from the glycoalkaloids, and some of them were partially purified from the potato peel (7, 8). However, the hydrolyzing activity of glycoalkaloids in a potato plant may be insufficient for treating large quantities of discarded green potatoes. Chemical hydrolysis of potato glycoalkaloids requires the use of a strong acid and high temperature. The objective of our research is to develop an efficient method for the detoxification of glycoalkaloids accumulated in a potato crop. Here, we report on the isolation and characterization of the filamentous fungi hydrolyzing a-chaconine. MATERIALS
AND METHODS
Materials a-Chaconine and a-solanine were purchased from Sigma Chemical (St. Louis, MO, USA). Authentic PI-, &-, and ychaconines were prepared by partial hydrolysis of a-chaconine in 97.5% methanol-O.25N HCl at 60°C (9). Potato sprouts (dry matter, about 10%) collected from potatoes that had been stored for six months were treated at 105°C for 30 min, minced, and preserved at -40°C. Spores of fungi were formed as they were grown on Culture potato dextrose agar (Becton Dickinson, Sparks, MD, USA) for 5 to 7 d. After being suspended at 5 x IO6 spores per ml of 0.01% Tween 80, 0.2 ml of the suspension was inoculated to 10 g of the minced sprouts in a lOO-ml Erlemneyer flask and cultured for 5 d. All cultures were incubated at 25°C without shaking. Determination of the glycoalkaloids The cultured sprouts (10 g) as described above were mixed with 20 ml of 1.O% acetic acid and shaken at 120 rpm for 1 h. After centrifugation of the mixture at 10,000 xg for 20 min, the supernatant was passed through a commercial Cl8 cartridge to purify the glycoalkaloids by the method of Carman et al. (10). The glycoalkaloids were analyzed using a high-performance liquid chromatograph (HPLC, LC-1OAD system; Shimadzu, Kyoto) equipped with a reverse phase C8 column (Shim-pack HRC-C8; 25 cmx4.6 mm+) and a UV monitor.
322
ODA ET AL.
J. BIOSCI. BIOENG.,
a-Chacmine
f32-Chaconine
HO
FIG. 1. Chemical structures of a-chaconine and its hydrolysis products. The mobile phase was 0.58% ammonium phosphate in 30% acetonitrile at a flow rate of 0.8 ml/min. Enzyme assays The sprouts (10 g) grown with the fungi were mixed with 10 ml of distilled water and extracted by shaking at 4°C. The supernatant obtained by centrifugation of the mixture
was used as the crude enzyme. The substrate solution, including both 6.0 mg of cr-chaconine and 5.0 mg of cr-solanine per ml of 50% methanol, was prepared from the extract of sprouts, as described above. A standard assay mixture, which contained 0.025 ml of a 1 M acetate buffer (pH 5.0), 0.1 ml of the substrate solu-
HYDROLYSIS OF a-CHACONINE
VOL.94,2002 tion, and 0.05 ml of the crude enzyme in a total volume of 0.5 ml, was incubated at 30°C for 30 min unless otherwise stated. After boiling in water for 5 min stopped the reaction, and a-chaconine was determined by HPLC. One unit of a-chaconine-hydrolyzing activity was defined as the amount of emme that consumed 1 pmol of a-chaconine per min. The activity for hydrolyzingp-nitrophenyl a-L-rhamnoside was assayed as described elsewhere (11).
a-Solanine +
No inoculation
323
a-Chaconine +
fi
RESULTS In potato plants, the Isolation of filamentous fungi sprouts accumulate the highest level of glycoalkaloids, which inhibit fungal growth (1). The fungi, which can grow on the sprouts, are expected to convert the glycoalkaloids to less toxic substances; however, this phenomenon has rarely been observed. We have accidentally found molds on the surface of sprouts germinated from stored tubers and isolated several filamentous fungi on potato dextrose agar. Among them, three strains, which showed the distinct morphology of a colony, were selected and studied in detail. From the growth and morphological characteristics, strains HP339, HP340, and HP341 were estimated to be Cladosporium cladosporioides (12), Penicillium sp. (subgenus Furcatum section Furcatum sensu Pitt, 1980) (13), and Cephalosporium-like Hyphomycetes, respectively. Sequence analysis of 28s rDNA and 18S-28s rDNA spacer region revealed that strain HP34 1 was identified as Plectosphaerella cucumerina (anamorph: Plectosporium tabacinum) (14, 15). The glycoalkaloid conDegradation of a-chaconine tents were determined in the sprouts incubated after inoculation of the fungal spores. The three strains reduced the achaconine content (Fig. 2) but not the a-solanine content (data not shown), and R cucumerina HP341 lost a-chaconine in 24 h. In HPLC chromatograms, the decrease of achaconine was accompanied by the simultaneous increase of an unknown compound eluted at 13.3 min (Fig. 3). No other peak appeared in the chromatograms even when the sprouts were further incubated for 24 h. Properties of the enzyme hydrolyzing a-chaconine a-Chaconine-hydrolyzing activity was followed in the crude enzyme extracted from the sprouts grown with F!
- 6
Strain HP339
h
Strain HP340
b
I
16
14
12
10
8
Retention time (min) FIG. 3. HPLC chromatograms of the extract from the potato sprouts grown with the tilamentous fungi. The potato sprouts were incubated 25°C for 24 h after inoculation of fungal spores and used for the HPLC assay.
cucumerina HP341. The enzyme was gradually produced after inoculation as the pH increased, which may reflect vegetative growth (Fig. 4). The crude enzyme obtained from 100 g of the sprouts that had been incubated for 5 d was partially purified by the columns of DEAE-Toyopearl and Phenyl-Toyopearl and dialyzed against 20 mM TrisHCl buffer (pH 8.5) (Table 1). When the activity was assayed in the pH range of 4.0 to 5.5 at 30°C and in the range of 25°C to 60°C at pH 5.0, the enzyme gave maximal activities near pH 5.0 and 5O”C, respectively. Enzymatic reaction was conducted in the mixture containing authentic a-chaconine or a-solanine as substrates. The enzyme converted achaconine to P,-chaconine (Fig. 5) but did not affect asolanine (data not shown). Hydrolyzing activity of p-nitro-
254
9
- 6
0 0
10
20 C*e
30 period 0
40
50 0
I
2
3
4
5
6
FIG. 2. Degradation of a-chaconine in the potato sprouts by Mamentous fungi. The potato sprouts were incubated at 25°C after inoculation of fungal spores. Symbols: triangles, C. cludosporioides HP339; closed circles, Penicilhn sp. HP340; open circles, p cucumerina
cucumerina HP341. Symbols: open circles, a-chaconine-hydrolyzing
HP341.
activity; closed circles, pH.
Cuhureperiod (d)
FIG. 4. Production of an a-chaconine-hydrolyzing
enzyme by p
J. BIOSCI.BIOENG.,
ODA ET AL.
324
TABLE 1. Partial purification of an a-chaconine-degrading Step
enzyme from the extract of sprouts grown with p cucumerina HP341
Total activity (U)
Total protein
536 302 270
25.3 4.89 -1.29
Crude extract DEAE-Toyopearl650M Phenyl-Toyopearl650M
6d
Specific activitv
_
Ww) 21.2 61.8 209.0
_
Purification (fold)
Yield
1.0 2.9 9.9
100 56.3 50.4
(%I
The data are the mean values of three independent experiments.
PI P2
a
Y
Chaconine stana
Hydrolysis product
r\
I
12
14
16
Retention time FIG 5.
chromatogram e cucumerina
phenyl-a+rhamnoside was only detectable (x0.02 pmol/ min/mg protein) and corresponded to less than lOA of that for a-chaconine. It is unknown whether the activity toward p-nitrophenyl-a+rhamnoside is derived from a contaminant or the enzyme itself. However, the above observations indicate at least that the enzyme is a rhamnosidase highly specific for the rhamnose (C-C,) glucose linkage in a-chacanine. DISCUSSION Both a-chaconine and a-solanine are grouped as saponin that are found in various plant species. Saponins consist of triterpenoids, steroids, or steroidal glycoalkaloids as aglycones with one or more sugars, and they often repress the growth of fungi (16). The antifungal activity of these compounds may function in the chemical defense of the plants against attack from fungal pathogens (17). The major mechanism of toxicity involves the formation of complexes with membrane sterols, resulting in pore formation and loss of membrane integrity. Successful pathogens of the plants avoid antifungal saponins by changing their membrane composition or by removing their sugar chain for detoxification (18). a-Tomatine, the glycoalkaloids present in tomato, has tomatidine as aglycone and a tetrasaccharide moiety consisting of two molecules of glucose and one each of galactose and xylose (19). Some enzymes detoxifying a-tomatine have been characterized in the fungi infecting tomato, and the linkage of the sugar chain hydrolyzed by these enzymes is not the same (16). In the present experiments, three filamentous fungi degrading a-chaconine were isolated from the potato sprouts
that contain high amounts of the glycoalkaloids. Strain HP34 1 produced an a-chaconine-hydrolyzing enzyme that was shown to be rhamnosidase-specific for the rhamnose (C-C,) glucose linkage but not for the rhamnose (C-C,) glucose linkage in a-chaconine. The substrate specificity of the enzymes from the three strains may be common because only &chaconine was formed from a-chaconine in the sprouts grown with each fungus (Fig. 3). The species names of the three strains, Cladosporium cladosporioides, Penicillum sp., and Plectosphaerella cucumerina, have not been reported to be pathogenic to potato plants in Japan (20). Detoxification of antifungal saponins is involved in the resistance of pathogenic fungi but may not be sufficient to infect saponin-containing plants (21). The tomato pathogen Gibberella pulicaris was reported to metabolize a-chaconine to P,-chaconine by the enzyme specific for the rhamnose (C,-C,) glucose linkage (22). The steroidal avenacosides A and B, which are located in the leaves and shoots of oats, include sugar chains composed of the rhamnose (C-C,) glucose linkage such as achaconine (16). Both avenacosides are inactive and convert antifungal 26-desglucoavenacosides in response to attack or wounding ( 16). The phytopatbogenic fungus Stagonospora avenae reduces the antifungal activity of 26-desglucoavenacosides by removing the sugar chain (23). Morrissey et al. (24) stated that the sugar chain is sequentially hydrolyzed by the action of one a-rhamnosidase and two p-glucosidases and that a-rhamnosidase plays a crucial role in saponin resistance. Alternatively, the product lacking the rhamnose residue attached to the C, position of glucose in the side chain is less toxic to pathogenic fungi infecting oats. It may be possible that the three strains isolated in the present experiments reduce the toxicity of a-chaconine by a rhamnosidase toward rhamnose (C,-C,) glucose linkage. The hydrolysis product P,-chaconine seems to be less toxic for fungi, while it is still harmful to animal cells. The developmental toxicity of P,-chaconine in the frog embryo was higher than that of I$- and y-chaconine (6). Further degradation by microbial enzymes to solanidine, which has a low level of biological activity, is required to accomplish our research project. ACKNOWLEDGMENTS This work was supported in part by a grants-in-aid from Japan Food Industry Center (Tokyo) and Special Coordination Funds for Promoting Science and Technology (Leading Research Utilizing Potential of Regional Science and Technology) of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
VOL. 94,2002
HYDROLYSIS OF a-CHACONINE
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