JOURNAL OF BIOSCIENCE AND BIOENGINEERING 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-chaco- nine, 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 anti- fungal a-chaconine.

[Key words: glycoalkaloid, a-chaconine, rhamnosidase, detoxification]

Potatoes contain secondary metabolites, steroidal glyco- alkaloids, which are potential toxic substances. Human in- gestion 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 dis- orders of the digestive system and general body metabo- lism, 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 proc- essing, 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 IX- chaconine and a-solanine (1). Both have a common chemi- cal structure containing trisaccharide attached to the 3-OH position of the steroidal glycoalkaloid solanidine. In CL- chaconine, the trisaccharide is composed of glucose and two residues of rhamnose, while a-solanine includes galactose, glucose, and rhamnose. The biological activity of the glyco- alkaloids is governed by the type and number of sugar mak- ing up the trisaccharide chain (5). The difference in the na- ture 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, y- chaconine, 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: yujioda@affrc.go.jp 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 ac- cumulated 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 y- chaconines were prepared by partial hydrolysis of a-chaconine in 97.5% methanol-O.25N HCl at 60°C (9). Potato sprouts (dry mat- ter, 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.

Culture Spores of fungi were formed as they were grown on 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 mix- ture 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 col- umn (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% aceto- nitrile at a flow rate of 0.8 ml/min.

was used as the crude enzyme. The substrate solution, including

Enzyme assays The sprouts (10 g) grown with the fungi both 6.0 mg of cr-chaconine and 5.0 mg of cr-solanine per ml of

were mixed with 10 ml of distilled water and extracted by shaking 50% methanol, was prepared from the extract of sprouts, as de-

at 4°C. The supernatant obtained by centrifugation of the mixture scribed 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-

VOL. 94,2002 HYDROLYSIS OF a-CHACONINE 323

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-nitro- phenyl a-L-rhamnoside was assayed as described elsewhere (11).

RESULTS

Isolation of filamentous fungi In potato plants, the 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 sev- eral 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 Cephalo- sporium-like Hyphomycetes, respectively. Sequence analy- sis of 28s rDNA and 18S-28s rDNA spacer region revealed that strain HP34 1 was identified as Plectosphaerella cucum- erina (anamorph: Plectosporium tabacinum) (14, 15).

Degradation of a-chaconine The glycoalkaloid con- tents were determined in the sprouts incubated after inocu- lation of the fungal spores. The three strains reduced the a- chaconine content (Fig. 2) but not the a-solanine content (data not shown), and R cucumerina HP341 lost a-chaco- nine in 24 h. In HPLC chromatograms, the decrease of a- chaconine 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

0 0 10 20 30 40 50

C*e period 0

FIG. 2. Degradation of a-chaconine in the potato sprouts by Ma- mentous fungi. The potato sprouts were incubated at 25°C after inocu- lation of fungal spores. Symbols: triangles, C. cludosporioides HP339; closed circles, Penicilhn sp. HP340; open circles, p cucumerina HP341.

a-Solanine a-Chaconine

+ +

fi No inoculation

Strain HP339 h

Strain HP340 b

I

8 10 12 14 16

Retention time (min)

FIG. 3. HPLC chromatograms of the extract from the potato sprouts grown with the tilamentous fungi. The potato sprouts were in- cubated 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 Tris- HCl buffer (pH 8.5) (Table 1). When the activity was as- sayed 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 activ- ities near pH 5.0 and 5O”C, respectively. Enzymatic reaction was conducted in the mixture containing authentic a-chaco- nine or a-solanine as substrates. The enzyme converted a- chaconine to P,-chaconine (Fig. 5) but did not affect a- solanine (data not shown). Hydrolyzing activity of p-nitro-

25 4 9

- 6

0 I 2 3 4 5 6

Cuhure period (d)

FIG. 4. Production of an a-chaconine-hydrolyzing enzyme by p cucumerina HP341. Symbols: open circles, a-chaconine-hydrolyzing activity; closed circles, pH.

324 ODA ET AL. J. BIOSCI. BIOENG.,

TABLE 1. Partial purification of an a-chaconine-degrading enzyme from the extract of sprouts grown with p cucumerina HP341

Step Total activity Total protein Specific activitv Purification Yield (U)

Crude extract 536 DEAE-Toyopearl650M 302 Phenyl-Toyopearl650M 270

The data are the mean values of three independent experiments.

6d 25.3

_ Ww) _ 21.2

(fold)

1.0 (%I

100

a PI P2 Y

stana Chaconine

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 contami- nant 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-cha- canine.

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 agly- cones with one or more sugars, and they often repress the growth of fungi (16). The antifungal activity of these com- pounds may function in the chemical defense of the plants against attack from fungal pathogens (17). The major mech- anism 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 com- position or by removing their sugar chain for detoxification (18). a-Tomatine, the glycoalkaloids present in tomato, has tomatidine as aglycone and a tetrasaccharide moiety con- sisting of two molecules of glucose and one each of galac- tose 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 de- grading a-chaconine were isolated from the potato sprouts

4.89 61.8 2.9 56.3 1.29 209.0 9.9 50.4 -

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, Peni- cillum 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 re- sistance of pathogenic fungi but may not be sufficient to infect saponin-containing plants (21). The tomato pathogen Gibberella pulicaris was reported to metabolize a-chaco- nine to P,-chaconine by the enzyme specific for the rham- nose (C,-C,) glucose linkage (22).

The steroidal avenacosides A and B, which are located in the leaves and shoots of oats, include sugar chains com- posed of the rhamnose (C-C,) glucose linkage such as a- chaconine (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-desglucoavena- cosides 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-glucosi- dases and that a-rhamnosidase plays a crucial role in sapo- nin resistance. Alternatively, the product lacking the rham- nose 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 rham- nosidase toward rhamnose (C,-C,) glucose linkage. The hy- drolysis product P,-chaconine seems to be less toxic for fungi, while it is still harmful to animal cells. The develop- mental toxicity of P,-chaconine in the frog embryo was higher than that of I$- and y-chaconine (6). Further degrada- tion by microbial enzymes to solanidine, which has a low level of biological activity, is required to accomplish our re- search 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 Japa- nese Government.

VOL. 94,2002 HYDROLYSIS OF a-CHACONINE 325

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