Indian Journal of Biotechnology Vol 9, April 2010, pp 160-165

Degradation of flatulence-causing oligosaccharides in soymilk by α-galactosidase—A novel thermotolerant from

Penicillium purpurogenum Ramalingam1*, Rudra2, N Saraswathy1 and S Sadasivam1

1Department of Biotechnology, Kuamaraguru College of Technology, Coimbatore, India 2Department of Biotechnology, Majhighariani Institute of Science and Technology, Rayagada, Orissa

Received 21 November 2008; revised 1 June 2009; accepted 10 August 2009

α-Galactosidase from Penicillium purpurogenum was induced to a greater extent in the culture medium by arabinose, galactose, lactose and guar galactomannan. The enzyme was purified by acetone precipitation and DEAE-Sephacel column chromatography. The purified enzyme showed a single band in both native-PAGE and SDS-PAGE. The molecular mass of enzyme was found to be between 107 and 110 kDa. The enzyme exhibited the optimum pH and temperature at 5.0 and 55ºC, respectively. α-Galactosidase was strongly inhibited by Ag+ and Hg2+, while Fe2+ enhanced the activity. PMSF did not inhibit α-galactosidase activity, whereas N-bromosuccinimide completely inhibited the enzyme activity. Further, galactose and melibiose moderately inhibited the enzyme activity. The Km values for PNPG, melibiose, raffinose and stachyose were found to be 0.20, 5.26, 10 and 9.1, respectively. α-Galactosidase completely hydrolyzed flatulence-causing raffinose and stachyose of soymilk in 1.45 h.

Keywords: Characterization, α-galactosidase, purification, raffinose, soymilk, stachyose

Introduction α-Galactosidase (α-D-galactoside galactohydrolase;

EC 3.2.1.22) catalyzes the hydrolysis of simple sugars and complex polysaccharides including blood group type-B1-3. α-Galactosidase has various industrial applications. It is extensively used in beet sugar industry for the hydrolysis of raffinose, which is an obstacle for the normal crystallization of sucrose. During processing, raffinose concentration increases from 0.15% to more than 5%. α-Galactosidase from Mortierella vinacea is used in beet sugar industry to hydrolyze the raffinose into sucrose and galactose, thereby reducing the inhibitory effect of raffinose on sucrose crystallization4. In the food industry, α-galactosidase is used to remove flatulence-causing oligosaccharides in legumes. Raffinose, stachyose, and verbascose are the major galacto-oligosaccharides in various legume seeds including soybeans. Soymilk is prepared from soybean seeds and is nutritionally comparable to cows’ milk except the presence of flatulence-causing raffinose family sugars and beany flavour5. α-Galactosidase is not found in human

digestive juice. As a result, raffinose-family of oligosaccharides in legume-based diets escapes digestion in human and these oligosaccharides promote the growth of the probiotic bacteria including Bifidobacterium sp.6. Enzymatic treatment was more effective than other methods for the removal of raffinose-family oligosaccharides in soymilk7. α-Galactosidase from guar has the ability to act on

galactomannans at low water activities. This property is exploited biotechnologically to produce modified galactomannan for various food applications8. α-Galactosidase is used in pulp and paper industry to increase the bleaching effect on pulp9. Thermostable α-galactosidase also finds its application in hydraulic fracturing of oil and gas wells10. Blood groups (A, B, AB & O) are determined by virtue of the type of polysaccharides that appears at their surfaces. α-Galactosidase from coffee bean is used to covert the blood group type B to type O by removing the terminal α-1,3-linked galactose moiety3. α-Galactosidase is widely distributed in bacteria,

fungi, plants and animals1,2,11. It has extensively been purified and fully characterized from various fungal sources1,2. α-Galactosidase from Penicillium purpurogenum was purified to homogeneity12,13, but the purified enzyme was not characterized with

_____________________ *Author for correspondence: Tel: 91-422-2669401; Fax: 91-422-2669406 E-mail: gobiram@rediffmail.com

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respect to the effect of sugars, reagents, kinetic properties and also its application in the hydrolysis of raffinose and stachyose of soymilk. Isolation of fungi from various sources for a novel α-galactosidase is a continuous process in our laboratory. Penicillium purpurogenum was isolated from soil samples that had the capacity to grow extensively on guar galactomannan as a carbon source and also degrades raffinose and stachyose. This paper reports the purification, characterization and applications of a novel extracellular, thermotolerant α-galactosidase from the fungal isolate, P. purpurogenum. Materials and Methods Fungus

One of the fungal isolates from soil samples showed luxuriant growth on agar plates containing guar galactomannan as sole source of carbon. It was selected and identified as P. purpurogenum by the staff of the Department of Plant Pathology, Indian Agricultural Research Institute (IARI), New Delhi. The organism was maintained on Czapek-Dox agar at 4ºC and was sub-cultured periodically. Induction of α-Galactosidase

Different carbon sources at 2% (w/v) were sterilized separately and added aseptically to the Czapek-Dox broth (pH 5.5). The flasks were inoculated with 48 h grown mycelial suspension. Batch submerged fermentation was carried out by placing the inoculated flasks on an orbital shaker at 120 rpm and 28±2ºC for 6 d. At the end of the 6th d the contents of the flasks were filtered through Whatman filter paper no. 1 to remove mycelia and the filtrate was tested for α-galactosidase activity. Purification of α-Galactosidase

Chilled acetone was added to the cell-free spent medium in a ratio of 1:1.5 (%, v/v) with gentle agitation and kept aside at 4ºC. After 12 h, the upper clear supernatant was decanted and discarded. The bottom mixture was centrifuged and the pellet, thus, obtained was dissolved in a small volume of 25 mM acetate buffer (pH 5.0). The enzyme solution was dialyzed for 18 h at 4ºC with an intermittent change in buffer and centrifuged at 9,000× g for 15 min. The resultant supernatant dialyzed against phosphate buffer (25 mM, pH 6.0) for 24 h and centrifuged to remove the precipitate. The supernatant was loaded on to a DEAE-Sephacel column (2 × 10 cm) equilibrated with phosphate buffer (25 mM, pH 6.0). The column was washed thoroughly with

equilibration buffer till the effluent reaches zero absorbance at 280 nm. The bound enzyme was eluted with a linear gradient of 0.5 M NaCl with a flow rate of 25 mL h-1. Each fraction collected was analyzed for α-galactosidase enzyme and protein concentration at 280 nm. The fractions (35 to 41) showing maximum α-galactosidase activity were pooled and dialyzed to remove the salt. The dialyzate was centrifuged to remove the precipitates and the supernatant was used in subsequent experiments. Enzyme Assay and Protein Estimation α-Galactosidase activity was measured using

p-nitrophenyl-α-D-galactopyranoside (PNPG) as substrate. The reaction mixture contained 0.1 mL of appropriately diluted enzyme solution, 0.8 mL of 0.1 M acetate buffer (pH 5.0) and 0.1 mL of 2 mM PNPG solution in distilled water. The mixture was incubated for 15 min at 55ºC and then the reaction was arrested by the addition of 3 mL of 0.2 M sodium carbonate solution. The quantity of p-nitrophenol liberated was measured by the absorbance at 405 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 µmol of p-nitrophenol per min per mL of enzyme under assay conditions. Protein concentration was estimated by the method of Lowry et al14 using bovine serum albumin as standard. SDS-PAGE for the purified enzyme was carried out by the method of Laemmli15.

Characterization of α-Galactosidase The optimum pH for enzyme activity was

determined by incubating the enzyme with PNPG in buffers ranging from pH 3.0 to 8.0 at 50ºC. The pH stability of an enzyme was conducted by pre-incubating the enzyme up to 2 h in phosphate buffer, pH 6.0 and 7.0. After incubation the residual enzyme activity was determined at optimum pH and 50ºC. The optimum temperature for enzyme was carried out at the optimum pH by performing the enzyme assay at different temperatures ranging from 10º to 70ºC. Thermal stability studies were carried out pre-incubating the enzyme at the optimum pH 5.0 at 60ºC for 2 h and the residual activity was determined at optimum pH 5.0 and temperature 55ºC.

The effect of different metal ions (5 mM), sugars (10 mM) and reagents (5 mM) on enzyme activity was carried out by pre-incubating the enzyme in acetate buffer (0.1 M, pH 5.0) at the indicated concentration for 30 min. Then, the residual enzyme activity was carried out under optimal conditions by the adding 100 µL of 2 mM PNPG to the reaction mixture.

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Kinetic studies were carried out at optimum pH 5.0 and temperature 55ºC. The Michaelis-Menten constants (Km) were determined using melibiose, raffinose and stachyose at concentrations ranging from 1 to 10 mM. The Km for PNPG was also determined at concentrations ranging from 0.01 to 0.1 mM. Degradation of Raffinose and Stachyose in Soymilk

Soymilk used for the enzymatic degradation was prepared by the method of Mulimani and Ramalingam16. To 100 mL of soymilk, 10 mL of partially purified enzyme (24 U) was added, stirred well and incubated for 2 h. Every 30 min time intervals, 10 mL of the reaction mixture were withdrawn, to which 10 mL of absolute ethanol was added to arrest the reaction by precipitating proteins. The contents were filtered through Whatman filter paper no. 1 and the filtrate was concentrated to 1 mL syrup in a thermostat water bath maintained at 75ºC. The oligosaccharides in the concentrated syrup (both enzyme treated and untreated) were separated and estimated by the method as described by Tanaka et al17. Results and Discussion Effect of Sugars on Enzyme Induction

Different sugars induced α-galactosidase to a varied extent from P. purpurogenum. Among the sugars tested, galactose, lactose and guar gum showed maximum enzyme induction (Table 1). On the contrary, a minimum α-galactosidase induction was seen with glucose, sucrose and starch as carbon source in the medium. The substrates melibiose and raffinose had an appreciable α-galactosidase enzyme induction; whereas, the enzyme induction was slight with arabinose, xylose and maltose. L-Arabinose was reported to induced α-galactosidase from Saccharomyces carlsbergensis and the contamination of galactose in commercial samples of L-arabinose was actually responsible for the enzyme induction18. Further, induction of α-galactosidase was reported by arabinose, galactose and guar gum from white-rot fungus, Pleurotus florida2. On the other hand, galactose, melibiose, raffinose, stachyose, D-fucose, glucose, D-arabinose and D-xylose failed to induce the

enzyme from Talaromyces flavus. Instead, quinovose, a specific inducer, strongly induced the enzyme19. The coinduction of β-L-arabinofuranosidase and α-D-galactosidase enzymes was observed in fungus, Trichoderma reesei when it was cultivated in presence of L-arabinose, L-arabitol, D-galactose or dulcitol20. Melibiose, a natural substrate/inducer of α-galactosidase was failed to induce the enzyme in filamentous fungi21. Purification and Characterization of α-Galactosidase

Table 2 represents summary of the purification steps. The total enzyme activity of the protein precipitate produced by acetone was 2,290 U. The α-galactosidase was further purified by DEAE-Sepharose column chromatography. The active fractions from DEAE-Sepharose were pooled, dialyzed and lyophilized. The enzyme was purified to 9.44-fold and the yield was 2%. The final enzyme preparation from DEAE-Sepharose column showed a single band in SDS-PAGE as shown in Fig. 1. The molecular mass of the final protein on SDS-PAGE was found to be between 107 and 110 kDa. The molecular mass of α-galactosidase from the present study differs with that of earlier purified α-galactosidase from other fungi, such as, P. purpurogenum12 (67 kDa), Ganoderma lucidum22 (56 kDa), Aspergillus oryzae23 (65 kDa), Pleurotus florida2 (99 kDa) and P. ochrochloron24 (60 kDa).

Table 2—Purification of extracellular α-galactosidase from P. purpurogenum

Step Total volume (mL)

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Purification fold

Cell-free filtrate 1200 2640 372 7.1 100 1 Acetone Precipitation 67 2290 91 25 4.84 3.52 Ion-exchange Chromatography 4 826 3.5 236 2.00 9.44

Table 1—Induction of α-galactosidase from P. purpurogenum on different carbohydrates

Carbon source (2%, w/v)

Enzyme activity (U)

Arabinose 0.19 Xylose 0.29 Glucose 0.01 Galactose 3.10 Melibiose 0.56 Maltose 0.16 Lactose 1.11 Sucrose 0.09 Raffinose 0.17 Starch 0.08 Guar gum3.52

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The purified α-galactosidase was optimally active at pH 5.0. The enzyme retained more than 90% of its original activity at pH 6.0 and 7.0. Further, the enzyme maintained 67 and 60% of its maximal activity at pH 6.0 and 7.0, respectively for 2 h. Most fungal α-galactosidases showed their optimal pH in acidic range between 4.5 and 5.52,12,13,24. While α-galactosidases from bacterial sources were found active near neutral pH1,11. The pH stability of α-galactosidase from the present study can be exploited for the hydrolysis of raffinose and stachyose in soymilk without lowering its pH. The optimum temperature for the enzyme was 55ºC. Further, the enzyme retained 75% of its initial activity at 60ºC for 2 h. Usually, fungal α-galactosidases showed optimum temperature above 50ºC and retained more than 50% of their activity at 60ºC in 1 h2,12,13,23. Enzymes which are active above 50ºC are preferred in food industry because it checks the growth of contaminant mesophilic microbes during processing.

The results of the effect of metal ions on α-galactosidase activity are shown in Table 3. Metal

ions, such as, Cu2+, Mg2+, Zn2+, Ca2+ and K+ had no inhibiting effect on enzyme. Ag+ and Hg2+ ions completely inhibited the enzyme activity, whereas Fe2+ ions enhanced the enzyme activity. Earlier, α-galactosidase from P. purpurogenum and G. lucidum was also reported to be inhibited by mercuric chloride and silver nitrate ions13. Further, EDTA and 1,10-phenanthroline did not inhibit α-galactosidase, indicating that metal ion is not required for the enzyme activity. PMSF also did not inhibit α-galactosidase activity. While, N-bromosuccinimide completely inhibited enzyme activity, suggesting that tryptophan is required for enzyme activity. N-Bromosuccinimide was earlier reported to inhibit α-galactosidase from A. oryzae2,23. Arabinose, xylose, glucose, lactose, maltose, sucrose and raffinose (at 10 mM level) didn’t inhibit α-galactosidase activity (Table 4). Fructose slightly enhanced the enzyme activity, while galactose and melibiose moderately

Fig. 1—SDS-PAGE of α-galactosidase from P. purpurogenum (20 µg of protein was applied onto 12.5% polyacrylamide gel and stained with Coomassie brilliant blue R-25): Lane 1, Purified enzyme; & Lane 2, Mol wt marker.

Table 3—Effect metal ions and inhibitors on relative activity of α-galactosidase*

Metal ion/Reagents Concentration

(mM) Relative

activity (%)

Control NIL 100 Fe2+ 5 119 Cu2+ 5 75 Mg2+ 103 Zn2+ 5 94 Ca2+ 5 105 Ag+ 5 0 Hg2+ 5 0 EDTA 5 100 1,10-Phenanthroline 5 100 PMSF 5 103 N-Bromosuccinimde 5 0

*Each value was an average of triplicate determinations

Table 4—Effect of sugars on relative activity of α-galactosidase*

Sugar Concentration (mM)

Relative activity (%)

Control Nil 100 Arabinose 10 100 Xylose 10 98 Fructose 10 100 Galactose 10 38 Glucose 10 100 Lactose 10 104 Maltose 10 112 Melibiose 10 65 Sucrose 10 110 Raffinose 10 103

*Each value was an average of triplicate determinations

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inhibited the activity. In earlier reports, galactose strongly inhibited α-galactosidase from A. oryzae and P. florida1,2,23, while the enzyme from Bacillus stearothermophilus was slightly inhibited by galactose25.

Kinetic parameters (Km and Vmax) for PNPG, melibiose, raffinose and stachyose were also determined. From the reciprocal plots, Km and Vmax values for PNPG were determined as 0.20 mM and 0.02 µM min-1 mg-1, respectively. The Km value for PNPG was found to be lower than that for the natural substrates, such as melibiose, raffinose and stachyose. The Km values for melibiose, raffinose and stachyose were found to be 5.26, 10 and 9.1 mM, respectively. The Km values determined for PNPG and raffinose were lower than those quoted in the literature for B. stearothermophilus (0.625 mM)25 and Thermomyces lanuginosus (16.4 mM)26.

The partially purified α-galactosidase completely hydrolyzed flatulence-causing raffinose and stachyose of soymilk in 1.45 h as evident by the disappearance of these oligosaccharides (Table 5). The percent degradation of flatulence-causing oligosaccharides (stachyose and raffinose) was proportional to an increase in the duration of enzymatic treatment. The results indicate that α-galactosidase from P. purpurogenum has a potential in soymilk processing and other food applications. α-Galactosidases from plant and microbs have earlier been reported for the hydrolysis of raffinose and stachyose of soymilk2,5,7,25. References

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Table 5—Stachyose and raffinose content (% dry wt) of soymilk treated with α-galactosidase enzyme from P. purpurogenum *

Time (min) 0 30 60 90 120 Stachyose 5.8 3.9 1.6 0.4 0 Raffinose 2.1 1.3 0.5 0.1 0

*Each value was an average of triplicate determinations

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α-galactosidase in Penicillium ochrocloron by guar (Cyamopsis tetragonolobus) gum, Biotechnol Appl Biochem, 17 (1993) 361-371.

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