Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

JOURNAL OFBIOSCIENCE ANDBIOENGINEERING Vol. 93, No. 1,64-72.2002

Degradation of Cell Wall Materials from Sweetpotato, Cassava, aid Potato by a Bacterial Protopectinase and Terminal Sugar Analysis of the Resulting Solubilized Products LORENA

D. SALVADOR,’ YOSHIHITO

TOSHIHIKO SUGANUMA,‘* FUKUSHIGE,’ AND HAYAO

KANEFUMI TANOUE2

KITAHARA,’

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan’ and institute of Food Processing and Utilization, Kagoshima Prefectural Agricultural Experiment Station, Kamifukumoto-cho 5500, Kagoshima 891-0116, Japan’ Received 4 October 200UAccepted 6 November 2001

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Badus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAECPAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the gala&an as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction. [Key words: enzymatic degradation, protopectinase, terminal sugar analysis]

cell wall materials, sweetpotato

(Zpomoea batatas),

viscosity to produce a soluble-type dietary fiber. Protopectin is the water-insoluble precursor of pectin found in plant tissues. It is solubilized by restricted hydrolysis, resulting in the liberation of water-soluble pectin. The enzymes that catalyze this reaction are termed as protopectinases (PPases) (7). PPases liberate soluble pectin without simultaneous degradation of the solubilized pectin regardless of the kind of reaction catalyzed. PPase is not a single enzyme but rather a heterogeneous group of enzymes with different catalytic activities. Sakai et al. (8) have classified them into two types depending on their reaction sites. Atype PPases react with the homopolygalacturonan region (smooth region) of the protopectin and their action can either be by simple hydrolysis or by a transeliminative mechanism. B-type PPases on the other hand, react with the hairy region of protopectin consisting of rhamnogalacturonan and neutral sugar side-chains, and thus considered as glycan depolymerases. In order to determine which particular type of enzyme is responsible for pectin solubilization, analysis of the solubilized product is necessary. In this study, we focused on the use of terminal sugar analysis in determining the mode of action of the crude enzyme preparation. For example, a galactanase attacks the galactan moiety in the CWM to pro-

The manufacture of several food products from rootcrops such as.sweetpotato produce substantial amounts of waste materials. In Kagoshima, Japan for instance, about 60,000 tons of starch residue, the by-product of sweetpotato starch production is being generated yearly. Further utilization of this by-product will lead to improvement in the whole processing system. Our previous study (1) has shown that sweetpotato starch residue is a potential source of CWM and consequently dietary fiber. The importance of dietary fiber in the prevention of several diseases has been the subject of numerous studies (24). Among the plant dietary fiber components, pectin has received the most attention because of its usefulness in the food industry (5). It has also been reported that pectin exerts stronger effects on the improvement of defecation, reduction of blood cholesterol, and repression of hypertension, than the other cell wall polysaccharides (6). However, because of its high viscosity and poor solubility in water, pectin cannot be added to food products in an amount sufficient to give these effects. It is therefore necessary to reduce its * Corresponding author. e-mail: [email protected] phone.: +81-(0)99-285-8637 fax: +81-(0)99-285-8639 64

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duce an oligosaccharide, which has a galactose residue at the reducing end. Upon reduction, this terminal galactose is converted to its corresponding sugar alcohol, galactitol. This analysis illustrates the action pattern of the enzyme. We now report the degradation of CWMs from sweetpotato, cassava, and potato by a protopectinase from Bacillus sp. M4, isolated from soil in the field of our university. The solubilized products were fractionated by gel filtration chromatography and characterized in terms of their sugar composition. The mode of action of the enzyme was also determined by terminal sugar analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

MATERIALS

AND METHODS

Materials The CWMs from sweetpotato, cassava, and potato were prepared by treatment of starch residues with an a-amylase according to the method of Noda et al. (9) as previously described (1). The starch residue used as substrate in the determination of PPase activity was provided by the Kagoshima Prefectural Experiment Station (Kagoshima City). Soya fiber, S-DN SY-4037, was received from Fuji Oil Co. Ltd. (Osaka) while corn fiber was purchased from Wako Pure Chemicals Ind., Ltd. (Osaka). Skim milk and yeast extract were acquired from Difco Laboratories (Detroit, MI, USA) while fish meat extract, casein, casamino acid, and Polypepton were from Wako Pure Chemicals. Carboxymethyl cellulose (CMC), gum arabic, locust bean gum, tragacanth gum, and guar gum were obtained from Nacalai Tesque Inc. (Kyoto). Oat spelts xylan was from Sigma Chemical Co. (St. Louis, MO, USA) while arabinan was purchased from Megazyme International Ireland Ltd. (County Wicklow, Ireland). The endo-1,4-/%D-gakdanase was acquired also from Megazyme as well as the substrates used for the enzyme assays namely, AZCL-debranched arabinan, AZCL-p-glucan, AZCL oat spelts xylan, AZCL-wheat arabinoxyIan, AZCL-potato galactan, AZO-avicel, and AZ-rhamnogalacturonan. The standard amylose samples used in gel-filtration chromatography were obtained from Nakano Vinegar Co. Ltd. (Aichi) while blue dextran 2000 and amylose EX-I (DP= 17) were from Pharmacia Fine Chemicals Ltd. (Uppsala, Sweden) and Hayashibara Biochemical Lab., Inc. (Okayama), respectively. All chemicals and standard sugars and amino acids used were of analytical reagent quality and were purchased from either Wako Pure Chemicals or Nacalai Tesque. Microorganism and enzyme production Bacillus sp. M4 was isolated from soil in the field of Kagoshima University Korimoto campus (Kagoshima City). This strain was selected from many candidates isolated from soil because of its ability to disintegrate sweetpotato tissue (maceration activity). The organism was maintained on agar slants containing 0.5% fish meat extract, 1.O% Polypepton, and 0.5% NaCl, with the pH adjusted to 7.2. For the culture medium for enzyme production, several carbon and nitrogen sources were tested. The test culture medium for carbon source was composed of 0.5% carbon source, 0.1% NH,NO,, 0.1% KH,PO,, 0.05% MgSO,.7H,O, and 0.02% KCl. For the test for nitrogen source, the medium consisted of either 1% compound nitrogen, 0.5% amino acid, or 0.2% inorganic nitrogen, and 0.2% soya fiber and the same concentrations of KH,PO,, MgSO,.7H,O, and KCI. The final medium used for enzyme production was composed of soya fiber (5 g/l), skim milk (5 g/Z), KH,PO, (1 g/Z), MgSO,.7H,O (0.5 g/Z) and KC1 (0.2 g/Z), with a pH of 7.0 and this medium was termed as soya fiber-skim milk (S-S) medium. The organism was inoculated in 5 ml of the S-S medium in a test tube and precultivated on a shaker (144 rpm) at 37°C for 24 h. This cul-

65

ture broth was then used to inoculate 100 ml of the same medium in a 500 ml-Erlenmeyer flask, and cultivation was continued for 48 h at the same temperature and speed. The culture broth was centrifuged at 15,000 rpm for 20 min and the supernatant was dialyzed against 40 mM Tris-HCI buffer, pH 7.0 with 0.5 mM CaCl, for 48 h. The dialyzed enzyme solution was added with 0.1% thymol and was kept at 4°C. Enzyme activity assays Arabinanase activity was measured using AZCL-debranched arabinan as substrate. The reaction mixture contained 2 mg substrate, 950 pl of 40 mM Tris-HCl buffer, pH 7.0 with 0.5 mM CaCl, and 50 ul of appropriately diluted enzyme solution. The mixture was incubated at 30°C for 30 min and then centrifuged at 15,000 rpm for 10 min. The absorbance of the supematant at 660 mn was measured. The absorbance of a completely solubilized substrate (2 mg/ml) was measured and used for calculating the activity. One unit of arabinanase activity is defined as the amount of enzyme that solubilizes 1 ug of the substrate per minute under these assay conditions. Xylanase, galactanase, arabinoxylanase, S-glucanase, and cellulase activities were measured under the same conditions using the corresponding substrates. Rhamnogalacturonase activity was determined using AZ-rhamnogalacturonan as soluble substrate. Enzyme solution (0.1 ml) was added to 0.25 ml substrate solution (20 mg per 1 ml of 40 mM TrisHCl buffer, pH 7.0 with 0.5 mM CaCl,) and the mixture was incubated at 30°C for 30 min. The reaction was terminated by immersion in boiling water for 5 min, and high molecular weight substrate was precipitated by the addition of 1 ml ethanol. The reaction tubes were allowed to stand at room temperature for 5 min and were centrifuged at 4000 rpm for 10 min. The absorbance of the supematant was then measured at 590 nm. Polygalacturonic acid lyase (PGL) activity was measured spectrophotometrically at 235 nm using 0.1% polygalacturonic acid in 40 mM Tris-HCl buffer, pH 7.0 with 0.5 mM Ca*‘. A molar extinction coefficient of 4600 M-‘cm-’ was used (10). Cell wall polysaccharide-degrading activity (or PPase activity) was assayed according to the method of Ishii (11) using sweetpotato starch residue as substrate. The reaction mixture consisted of 36 mg substrate, 19.6 ml of 40 mM Tris-HCl buffer, pH 7.0 with 0.5 mM CaCI, and 0.4 ml of enzyme solution. The mixture was incubated at 37°C with constant mixing (32 rpm) for 6 h. After the reaction, 1 ml of the reaction mixture was taken and centrifuged at 15,000 rpm for 10 min. Degree of degradation was determined by measuring the total sugar or galacturonic acid content by the phenol-H,SO, (12) or the modified carbazole (13) methods, respectively. Enzymatic degradation of CWM and soya fiber CWMs from sweetpotato, cassava, and potato, and soya fiber were degraded with the M4 enzyme using the same conditions employed in the PPase activity determination. After 24 h incubation, the solubilized products were separated from the cell wall residues by filtration using a G3-glass filter, and the supematant was freezedried. For the treatment using commercial endo-galactanase, 20 mM acetate buffer, pH 4.0 was used instead of Tris-HCl buffer. Gel-ftitration chromatography Gel filtration chromatography of the solubilized products was performed on a Biologic HR chromatography system (Bio-Rad Laboratories, Hercules, CA, USA), equipped with a Superose 12 HR lo/30 column (30x 1 cm, Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England). The standard samples used were blue dextran 2000 (MW=2,000,000), amylose AS 110 (MW= 1lO,OOO),AS 30 (MW =30,000), AS 10 (MW=lO,OOO), amylose EX-I (DP=17) (MW= 2772), maltopentaose (MW=828), and glucose (MW=180). The solubilized products were eluted with 10 mM Tris-HCl buffer, pH 7.0, with 0.1 M NaCl and 0.02% NaN, at a flow rate of 0.5 ml/min. Fractions were assayed for total sugar and galacturonic acid by the phenol-H,SO, and modified carbazole methods, respectively. The

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fractions with considerable amounts of sugars were divided into four fractions, which were pooled separately. Salt was removed from each fraction by gel-filtration in a Sephadex G-25 column (34 x 1.6 cm, Pharmacia Fine Chemicals Ltd.). The desalted fractions were then freeze-dried. Reduction of the solubilized prodReduction with NaBH, ucts was done according to the method of Yamaguchi et al. (14) with some modifications. Two mg of the sample was dissolved in distilled water and then added with 4 mg of NaBH,. The solution was allowed to stand at room temperature for 3 h. After standing, Amberlite h-120 (I-I+) ion exchange resin (Pfaltz and Bauer Inc., Waterbury, CT, USA) was slowly added until the bubbling stops. After filtration, the solution was evaporated to dryness under reduced pressure, followed by addition and evaporation of methanol (5 x 2 ml) to remove the boric acid. Monosaccharide composition analysis Chromatography of the solubilized products was performed on a Dionex Bio-LC system using a CarboPac PA10 column (Dionex Corp., Sunnyvale, CA, USA) as described in a previous paper (1). Terminal sugar analysis Chromatography of the reduced products was also performed on the same system, after hydrolysis with 1 M H,SO, at 100°C for 3 h, using a column for sugar alcohols (CarboPac MA 1 column, 250 x 4 mm) and guard column. Detection was made by a pulsed amperometric detector using a quadruple-potential waveform (15). The neutral monosaccharides and their corresponding sugar alcohols were eluted isocratically using 380 mM NaOH. All determinations were carried out at room tem-

TABLE 1. Enzyme activities present in the culture filtrate of Bacillus sp. M4

Enzyme Arabinanase Arabinoxylanase g-Glucanase Galactanase Xylanase Cellulase Rhamnogalacturonanase Polygalacturonic acid lyase Cell wall polysaccharide-degrading

Activity

activity

1670 11 21 136 11 0.3 33 3b 42”

a Expressed as ugImin/ml. b Expressed as umol/min/ml. ’ Expressed as % degradation in terms of total sugar content. Activities were determined by incubating the corresponding substrates in 40 mM Tris-HCI buffer, pH 7.0 containing 0.5 mM CaCl, at 3O’C for 30 min (37°C for 6 h for cell wall polysaccharide-degrading activity).

perature using a flow rate of 0.4 ml/min. Response factors of the monosaccharides and sugar alcohols were calculated using galactose as reference sugar after drying to constant weight under vacuum at 50°C using P,O,. RESULTS From the soil Activities of crude enzyme preparation in our campus, we have isolated a rod-shaped, gram positive, aerobic, and spore-forming bacterium. It was also found to have a catalase activity and showed a positive response for VP reaction. Based on these characteristics, we have identified this bacterium as a member of the genus Bacillus and called it Bacillus sp. M4. In the cultivation of Bacillus sp. M4 for enzyme production, several substances were examined as carbon and nitrogen sources, to establish a suitable culture medium. For the carbon source, the use of a commercial soluble-type dietary fiber from soy bean (soya fiber) significantly increased most of the activities (data not shown). It was found to be a better carbon source than arabinan and pectic acid for the arabinanase and PGL activities, respectively, as well as for the PPase activity. It has been previously reported that the use of soybean flour extract promoted PPase production in Bacillus subtilis (16). As for the nitrogen source, skim milk was found to be the most effective in inducing all the activities among the substances tested (data not shown). Based on these results, soya fiber and skim milk were selected as the carbon and nitrogen source, respectively in the culture medium for enzyme production. The culture filtrate from the soya fiber-skim milk medium of Bacillus sp. M4 was used as the crude enzyme preparation. It contained PGL as well as several glycan depolymerase activities (Table 1). Arabinanase activity was particularly high and significant levels of galactanase and rhamnogalacturonanase activities were also found. How-

PB FIG. 1. Effect of pH on the PPase (open circle), PGL (cross), rhamnogalacturonase (closed circle), galactanase (open triangle), xylanase (open square), and arabinanase (closed square) activities of Bacillus sP.-M4 enzyme. The corresponding substrates were added with 0.1 M alvcine buffer of various DH (~H7.5-11.0) and the reaction mixtur&-were incubated at 30°C ‘for jb min (37°C for 6 h for PPase activity). Relative activity was expressed as the percentage of the activity at a specific pH relative to the optimum activity.

ever, cellulase activity was nearly absent. The various enzyme activities were found to be optimum at relatively high pH values (between pH 8.0-9.5) (Fig. 1). PPase activity was highest between pH 8.0-9.0. Rhamnogalacturonase activity was found to be optimum at pH 8.0, while PGL activity was highest at pH 9.5. These results indicate that the Bacillus sp. M4 produces alkaline type of enzymes. In a time course exDegradation patterns of CWMs periment, the rate and extent of degradation of the CWMs by Bacillus sp. M4 enzyme was monitored in terms of the amounts of total sugar and galacturonic acid released during the incubation period. Sweetpotato CWM exhibited a faster rate of degradation than the other two CWMs (Fig. 2). After 1 h of reaction, about 3 1% (0.56 mg/ml) of the CWM (as total sugar) has already been solubilized. The degradation continued to increase gradually until it reached a maximum (42%, 0.76 mg/ml) after 6 h of incubation. Among the CWMs, it was in this sample where the highest amount

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67

0 0

4

8

12

16

20

24

0

Incubation time (h)

8

4

12

16

20

24

0

4

8

12

16

20

24

Incubation time (h)

Incubation time 01)

FIG. 2. Time courses of the degradation of sweetpotato (A), cassava (B), and potato (C) CWMs by Bacillus sp. M4 enzyme. Degradation of CWMs was computed by dividing the amount of total sugar (open square) or galacturonic acid (open circle) released in the reaction mixture by the amount of CWM used in the determination. Total sugar content was corrected for the coloration contributed by galacturonic acid.

of galacturonic acid was released (0.43 mg/ml). Cassava CWM has a degradation pattern almost similar with that of sweetpotato, but the extent of the release of total sugar and especially galacturonic acid was only minimal. Maximum degradation was reached after 24 h wherein about 26% (0.46 mg/ml) of the CWM (as total sugar) has been solubilized. The amount of galacturonic acid released was also very small (0.14 mg/ml). Potato CWM was degraded by the enzyme in a different manner. The amount of total sugar and galacturonic acid initially released was relatively low, but it increased significantly as the incubation continued. After 24 h, degradation in terms of total sugar was very near (40%, 0.72mgIml) with that of sweetpotato CWM (42%, 0.76 mg/ml). However, the maximum amount of galacturonic acid released was still less than that measured in sweet-

potato (0.30 mg/ml). When the enzyme concentration was increased by 10 fold, potato CWM was degraded to about 66% (1.18 mg/ml) in terms of total sugar after 24 h incubation (data not shown). Characterization of the solubilized products The sugar composition of the solubilized products obtained after 24 h incubation with Bacillus sp. M4 enzyme was determined by HPAEC-PAD after hydrolysis with 1 M H,SO, (Table 2). Each of the solubilized product from the CWM was mainly composed of galacturonic acid and the neutral sugars galactose, arabinose, and rhamnose. Galactose was present in the highest amount among the neutral sugars (between 62 to 85%). As already mentioned above, the amount of galacturonic acid detected in sweetpotato was relatively higher (57%) than those of the other two samples (29 and

TABLE 2. Sugar composition of the solubilized products from the enzymatic degradation of sweetpotato, cassava, and potato cell wall materials by Bacillus sp. M4 enzyme Sample SP solubilized product Fr. I Fr. II Fr. Ill Fr. IV CA solubilized product Fr. I Fr. II Fr. Ill Fr. IV

Yield” (“/)

10.9 9.0 15.5 4.0

10.1 5.1 20.5 8.1

Neutral sugar composition (weight %)

Gal UA contentb (%) \ I

Fuc

Rha

Ara

XYl

Man

Glc

Gal

0.5

10.0

23.2

nd.

n.d.

4.2

62.2

57.0

3.3 n.d. n.d. n.d.

23.2 21.2 10.1 nd.

23.5 37.6 18.0 45.2

4.8 3.4 0.3 n.d.

n.d. nd. nd. n.d.

10.2 2.1 0.6 1.1

35.0 35.7 70.9 53.6

47.7 94.4 1.6 2.5

0.7

6.2

10.7

2.5

n.d.

5.1

74.8

29.0

11.1 0.6 n.d. n.d.

10.0 22.1 1.9 nd.

29.5 27.6 7.7 9.9

17.9 2.8 0.2 n.d.

n.d. n.d. n.d. n.d.

14.6 1.2 4.0 7.9

17.1 45.3 86.1 82.2

71.1 80.1 31.3 2.6

85.6 41.5 n.d. n.d. 1.4 2.9 9.7 0.5 PO solubilized product 35.1 77.5 n.d. 9.3 14.7 16.0 15.6 9.5 9.3 Fr. I 60.9 31.7 nd. 0.3 0.9 25.3 12.0 4.0 0.4 Fr. II 90.3 8.1 n.d. nd. n.d. 2.7 7.0 17.3 n.d. Fr. Ill 82.0 1.0 n.d. n.d. n.d. n.d. 18.0 8.2 n.d. Fr. IV * Yield of fraction (%). Calculated by dividing the weight of the fraction obtained by that of the solubilized product applied to Superose 12 column multiplied by 100. b Determined by the modified carbazole method and corrected for the effect of the presence of NaN,. Expressed as mg Gal UA/mg sample multiplied by 100. n.d., Not detected.

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41%). Based on the results of the previous study, the sugar compositions obtained suggest that the pectin fraction of the CWM is the most probable cell wall component that was solubilized by the Bacillus sp. M4 enzyme. This also implies that the M4 enzyme is a protopectinase since it was able to solubilize the protopectin in the CWMs, resulting in the liberation of a water-soluble pectin polymer. The solubilized products were further analyzed by gel filtration on a Superose 12 column. Four fractions were obtained and were numbered in the order they were eluted (Fig. 3). Each of the four fractions was desalted on Sephadex G-25 column, freeze-dried and then subjected to sugar composition analysis. The solubilized product was separated into its higher molecular weight (higher MW, Fr. I and II) and lower molecular weight (lower MW, Fr. III and IV)

Fr. II

1

0.5

FIG 3. Elution profiles of the solubilized products from the degradation of sweetpotato (A), cassava (B), and potato (C) CWMs by Bacillus sp. M4 enzyme on Superose 12 column using 10 mM TrisHCl buffer, pH 7.0 containing 0.1 M NaCl and 0.02% NaN,. Total sugar (open square) and galacturonic acid (open circle) contents of the fractions. The values for galacturonic acid content were not corrected for the effect of NaN,. The fractions were grouped as follows: for SP, Fr. I (Fr. 20-32), Fr. II (Fr. 33-35), Fr. III (Fr. 36-37), Fr. IV (Fr. 3840); for CA, Fr. I (Fr. 2431), Fr. II (Fr. 32-34), Fr. III (Fr. 35-38) Fr. IV (Fr. 39-41); and for PO, Fr. I (Fr. 18-33), Fr. II (Fr. 34-36), Fr. III (Fr. 37-t0), Fr. IV (Fr. 4143). The standard samples used were blue dextran 2000 (a), amylose AS 110 (b), AS 30 (c), AS 10 (d), amylose EX-I, DP= 17 (e), maltopentaose (f), and glucose (g).

components (Fig. 3). It can be noticed that the measured values of galacturonic acid in the eluted fractions were relatively low. This was found to be due to the presence of NaN, in the elution buffer, which may have interfered in the development of color in the carbazole reaction resulting in a decrease in absorbance at 530 nm (data not shown). The neutral sugar composition of each fraction is also given in Table 2. The galacturonic acid contents were corrected for the effect of the presence of NaN,. Also shown are the % yield values (by wt.) of the fractions. It can be seen that in all three samples, Fr. III has the highest % yield, and thus is the major fraction. Galacturonic acid was mostly detected in the higher MW fractions, Fr. I and II. As for the neutral sugars, galactose, arabinose, and rhamnose, are the major neutral sugars in the higher MW fractions while it was mostly galactose in the lower MW fractions. Small but significant amounts of fucose were also detected in Fr. I of sweetpotato and potato (3.3 and 9.3%, respectively), aside from cassava wherein the amount detected was higher (11.1%). This has also been observed in our previous study (1). Terminal sugar analysis of the solubilized products In order to determine which of the enzyme activities is primarily responsible for the degradation of the pectin fraction of the CWMs, the mode of action of the crude enzyme preparation must be ascertained. For this purpose, we determined the sugars present in the reducing terminal side of the solubilized product obtained after enzymatic treatment. As a model system, soya fiber was treated with a commercial endo- 1,4-P-D-galactanase from Aspergillus niger. Aside from the galactanase activity (564 U/ml), this enzyme preparation has also been found to contain a minor arabinanase activity (66 U/ml) (data not shown). After 24 h incubation, the reaction mixture was centrifuged and the filtrate was freeze-dried and then subjected to terminal sugar analysis. A control sample (i.e. without enzyme treatment) was also subjected to the same analyses. The compositions obtained are given in Table 3. Galactose and arabinose are the major neutral sugars present in soya fiber comprising about 57 and 25%, respectively. This is similar with the sugar composition of soy bean cell walls previously reported (17, 18). As for the terminal sugar composition, it TABLE 3. Sugar composition analysis of the reduced products solubilized from soya fiber by an endo-1,4-S-Dgalactanase or Bacillus sp. M4 enzyme Sugar or sugar alcohol composition (weight %) Fuc+Rha Ara Xyl+Glc Gal Gal-01 Glc-01 Rha-01 Ara-01

Sample Control (no enzyme)

After galactanase treatment

After Bacillus sp. M4 treatment

3.8 24.6 14.1 56.5 0.4 0.6 n.d. nd.

6.8 30.0 12.5 22.5 30.7 nd. nd. n.d.

5.4 28.8 15.6 39.2 8.0 n.d. 1.2 1.1

Mannose, fucitol. xvlitol, and mannitol were also determined but were not detected. _ nd., Not detected.

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TABLE 4. Terminal sugar composition of the solubilized products from the enzymatic degradation of sweetpotato, cassava, and potato cell wall materials by Bacillus sp. M4 enzyme Sample SP solubilized product Fr. I Fr. II Fr. III Fr. IV CA solubilized product Fr. I Fr. II Fr. III Fr. IV PO solubilized product Fr. I Fr. II Fr. III Fr. IV

Sugar/Sugar alcohol composition (weight %) Fuc+Rha

Ara

1.5 (n.d.) 20.0 18.4 0.4 0.3

18.5 (n.d.) 27.7 31.3 13.2 27.7

4.4 (4.5) 5.3 3.8 0.8 1.4

51.2 (51.2) 41.0 30.2 59.5 43.1

i!$

(Z) 22.9 26.1 9.2 8.9

(Z) 34.9 4.8 8.4 13.3

5.8 (4.7) 10.8 11.6 4.2 11.3

3.1 (1.2) 23.8 3.0 0.2 n.d.

18:5 3.3 n.d. (Z.) 19.8 20.8 3.0 nd.

Xyl+Glc

Gal

Rha-ol

Ata-

Glc-ol

Gal-01

n.d. (;i:) n.d. n.d. nd.

15.5 (23.3) 1.0 2.2 24.2 21.9

(& n.d. n.d. 0.2 0.8

n.d. (n.d.) 1.2 0.3 n.d. nd.

25.1 (37.0) 1.6 0.2 19.2 21.5

2.0 (2.3) n.d. n.d. 0.8 1.0

n.d. (n.d.) 0.8 0.5 n.d. nd.

23.0 (33.2) 1.5 0.9 22.8 26.5

(n?) 4.5 13.3 0.8 2.2

1.6 (2.5) 0.3 0.7 1.1 3.4

45.3 (48.5) 16.5 37.9 53.6 54.8

8.3 (0.7) 1.4 12.2 6.1 0.7

55.3 (58.6) 41.5 50.0 65.6 60.8

(n?) 1.8 13.2 3.3 0.3

BValues in parentheses are the sugar and sugar alcohol composition of the solubilized products determined after passing through SepPak cartridge before reduction with NaBH,. Mannose, fucitol, xylitol and mannitol were also determined but were not detected. n.d., Not detected.

can be seen that only galactitol was present in the sample treated with the enzyme while there were no significant amounts of sugar alcohols detected in the control sample (Table 3). This suggests that the commercial enzyme specifically acted on the galactan moiety of the sample and thus confirmed its mode of action as an endo-galactanase. Then, soya fiber was also subjected to enzymatic degradation using Bacillus sp. M4 enzyme (Table 3). Galactitol was the major sugar alcohol detected with small amounts of rhamnitol and arabitol also present, suggesting that a galactanase may be the primary activity responsible for the solubilization of soya fiber. TLC analysis of the solubilized products showed that the treatment with Bacillus sp. M4 enzyme resulted in the release of a larger block from soya fiber than the endo-galactanase (data not shown). The solubilized products from the CWMs were subjected to terminal sugar analysis and the compositions obtained are shown in Table 4. Galactitol is the major sugar alcohol present in the three samples, implying that the M4 enzyme attacked the CWMs to release products having galactose at the reducing end. This also suggests that a galactanase is the primary activity responsible for the solubilization of pectin in the CWMs. In addition, small amounts of rhamnitol and arabitol were also detected. The solubilized products were also passed into the SepPak QMA cartridge before reduction to remove galacturonic acid. The removal of galacturonic acid by the cartridge also resulted in the removal of rhamnose and subsequently rhamnitol (Table 4). This indicates that it is associated with galacturonic acid, probably as a rhamnogalacturonan molecule. The fractions obtained after gel filtration of the solubilized products were also subjected to the same analysis. The terminal sugar compositions of the fractions of the three samples were very similar (Table 4). Rhamnitol was the

main sugar alcohol present in the higher MW fractions, specifically in Fr. II, although it was galactitol in Fr. 111, the major fraction. This suggests that a rhamnogalacturonase is also responsible for pectin solubilization, especially in producing larger products. DISCUSSION The culture filtrate from Bacillus sp. M4 was found to degrade the CWMs from sweetpotato, cassava, and potato (Fig. 2). A relatively higher amount of galacturonic acid was solubilized from sweetpotato CWM. In our previous study (1) we have reported that among the three CWMs, sweetpotato contained the highest amount of galacturonic acid (3 1%) as compared to 17 and 24% of cassava and potato, respectively. The neutral sugar compositions of the solubilized products from the three CWMs were quite similar (Table 2). They are mainly composed of galactose, arabinose, and rhamnose. These monosaccharides are the major sugar components of pectic polysaccharides (1, 19, 20). This implies that among the cell wall polysaccharide components, the protopectin moiety is the one most preferably attacked by the Bacillus sp. M4 enzyme. Fucose was also detected in the solubilized products of the three rootcrops. In our previous study, mcose was not detected in sweetpotato and potato CWMs (1). This suggests that the mcose-containing moiety in the CWMs of sweetpotato and potato was not extracted by ammonium oxalate during the chemical fractionation of the CWMs. MC Neil et al. (21) have in fact mentioned that minor amounts of fucose may also be present in rhamnogalacturon I and II. The ability of the crude enzyme preparation to attack CWMs and consequently liberate water soluble pectic polysaccharides indicates that it has a protopectin-solubiliz-

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ing activity, otherwise known as PPase activity. Sakai and Okushima (22) have pioneered the study of this group of enzymes. Since then, several PPases from various origins have been isolated (16, 23-36). PPases are classified into two main types depending on their reaction mechanisms. Atype PPases such as PGL, react with the homopolygalacturonan region of protopectin while the B-type like arabinanase reacts on the neutral sugar side chains that may connect the rhamnogalacturonan chain and cell wall constituents (8). We have found several enzyme activities in the culture filtrate of Bacillus sp. M4 (Table 1). Aside from PGL, several glycan depolymerase activities were also present, such as galactanase, rhamnogalacturonanase, and especially arabinanase. PGL is known to be the major pectic-depolymerizing enzyme detected. It is more commonly produced by bacteria in contrast to polygalacturonase (PG) which is formed mostly by fungi. We have determined the effect of EDTA on the PPase and PGL activities. The results showed that 2 mM EDTA completely inhibited the PGL activity as no increase in the absorbance at 235 run was observed, even when the supernatant from the PPase activity determination was incorporated into the PGL assay reaction mixture (data not shown). PPase activity on the other hand, was only slightly inhibited as the activity was almost retained even in the presence of the same concentration of EDTA. This suggests that PGL is not the primary activity responsible for the solubilization of the protopectin in the CWMs, since even without the PGL activity, the PPase is still operative. These results also indicate that the PPase activity present in the culture filtrate of Bacillus sp. M4 is contributed by B-type rather than the A-type at least at pH 7.0. In order to ascribe the pectin releasing activity to a particular kind of B-type PPase, we have determined what sugar residue is present in the reducing terminal of the solubilized product. This analysis would give an indication of the cleavage site by the enzyme and thereby identify the mode of action of the enzyme, which attacked the heteropolysaccharide of the CWM. In this study, we used a simple method of doing this by reduction of the solubilized products, hydrolysis of the reduced products and subsequent chromatography using an HPAEC-PAD system. To test the applicability of this method, we have used a model system by treating soya fiber with a commercial endo-galactanase. The results have shown that the only sugar alcohol detected in the sample treated with the enzyme was galactitol (Table 3). In the reaction of Bacillus sp. M4 enzyme with the three CWMs, galactitol was found to be the major sugar alcohol present in the solubilized products of all three CWMs (Table 4). Minor amounts of rhamnitol and arabitol were also detected. Similar results were obtained in the treatment of soya fiber with the Bacillus sp. M4 enzyme (Table 3). Further analysis after fractionation was performed and it was found that significant amounts of rhamnitol were present in the higher MW fractions while galactitol was present in the lower MW fractions, especially in Fr. III, the major fraction. This demonstrates that the Bacillus sp. M4 enzyme attacked the galactan moiety in the protopectin, thus implying that a galactanase is the primary enzyme activity responsible for pectin solubilization. Several galactanases

have been previously isolated from various strains of B. However, none of these galactanases have been reported to contain a PPase activity. The presence of rhamnose in the reducing terminal side of the higher MW fractions suggests that a rhamnogalacturonase may also be operative in the solubilization of the galacturonic acid moiety of the CWMs. Since the pioneering work of Schols et al. (41) on rhamnogalacturonase (RGase), now known as RG-hydrolase, several other types of RGase have been reported. These are RG-acetylesterase (42), RG-rhamnohydrolase (43), RG-lyase (44), and RG-galacturonohydrolase (45). We have found that the absorbance at 235 nm of a reaction mixture of 0.1% rhamnogalacturonan and M4 enzyme increased with time, although the reaction was quite slow (data not shown). Based on these results, it is possible that the RGase present in the Bacillus sp. M4 crude enzyme preparation is of the RG-lyase type. Sakai et al. have reported a PPase from B. subtilis which has an arabinase as the primary activity responsible for the degradation of arabinan in sugar beet pulp (28, 29). However, in the case of the crude enzyme of Bacillus sp. M4, it appears that arabinanase is not the primary activity responsible for the release of the pectic polysaccharides from the three CWMs, although the arabinanase activity measured in the culture filtrate was remarkably high (Table 1). The arabinanase activity attacks a debranched substrate of a-1,5arabinan but not a branched one. Thus it is possible that this particular substrate is not present in the CWMs. The data presented here suggest that a B-type PPase is present in the culture filtrate of Bacillus sp. M4, which may have caused the solubilization of the protopectin in the CWMs and the subsequent release of a soluble pectin fraction at neutral pH. The primary activity responsible for pectin solubilization was found to be a galactanase, although arabinanase activity was highest among the enzyme activities detected. A rhamnogalacturonase also acted as a secondary activity, whose action resulted in the release of a galacturonic acid-rich, larger molecular weight product. Studies on the physical properties and physiological functions of the isolated soluble-type pectin fraction are necessary to fully maximize its potential. Further investigations on the purification as well as characterization of the enzyme are also important for a better understanding of its mode of action and to allow its comparison with other related enzymes.

subtilis (37-40).

ACKNOWLEDGMENTS This study was supported in part by the Regional Collaborative Research Program for the Promotion and Development of Advanced Technology for 1996 from the Ministry of Agriculture, Forestry, and Fisheries, as well as a grant from the Ministry of Education, Culture, Science, and Technology (No. 12839015).

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