Extraction , Purification and Characterization of Endo-Acting Pullulanase Type I from White Edible Mushrooms

Article history: Received on: 09/10/2015 Revised on: 07/11/2015 Accepted on: 22/11/2015 Available online: 26/01/2016 Pullulanase (EC 3.2.1.41) has been isolated and purified from white edible mushrooms by ammonium sulphate precipitation (20-70%) followed by ion exchange chromatography (DEAE-cellulose) and gel filtration (Sephadex G 75-120), with final yield (20%) and purification fold (17.8). The molecular mass of pullulanase enzyme was 112 kDa as estimated by SDS-PAGE and the pI value was 6.2. The apparent Km and Vmax values for purified pullulanse on pulluan were 0.27 mM and 0.74 μM min -1 respectively. The activity was optimum at 40 ○ C and pH 6. Pullulanase showed moderate thermo-stability. A relative substrate specificity for hydrolysis of soluble starch, amylopectin and glycogen was 80, 60 and 30% respectively. Enzyme activity was highly activated by Fe +2 , Mn +2


INTRODUCTION
Pullulan is a polymer synthesized by the yeast-like fungus Aureobasidium pullulans.It is a linear α-D-glucan built of maltotriose subunits (Abdullah and French 1966).Pullulanase, It belongs to the α-amylase family, which is identified as glycoside hydrolase and break α-1,6 linkages in pullulan, starch, amylopectin and related oligosaccharides (Duan and Wu, 2015).Pullulanases are classified into five types based on the substrate specificity (Roy et al., 2003;Haki and Rakshit, 2003).(1) the glucoamylase type is an exo-acting carbohydrase (Reilly, 1979), which hydrolyzes pullulan from non-reducing ends to produce glucose; (2) the pullulanase type I specifically hydrolyses α-1,6 glycosidic linkages in pullulan or branched substrates such as amylopectin forming maltotriose (3) the isopullulanase type from Aspergillus niger, which hydrolyzes α-1,4 linkages of pullulan to produce isopanose; and (4) Pullulan hydrolase type I (neopullulanase) from Bacillus stearothermophilus (Ecel et al., 2015) which hydrolyzesα-1,4 linkages of pullulan to produce panose.(5) pullulanase type II (amylopullulanase) attacks α-1,6 linkages in pullulan and branched substrates in addition to α-1,4 links in polysaccharides other than pullulan (Bertoldo and . .Antranikian, 2002;Duffner et al., 2002).Pullulanases belong to group of glycosylhydrolases that are widely distributed in nature and are produced by an extremely wide variety of species such as animals, plants and microorganisms (Hyun and Zeikus, 1985;Zhang et al., 2013;Duan and Wu, 2015).Pulluanases have gained important in current saccharification processes as starchdebranching enzymes.In the starch processing industry for the production of maltose syrups and high purity glucose and fructose (Jensen and Norman 1984).This occurs when pullulanase is used in combination with glucoamylase or α-amylase, respectively in the saccharification process.Pullulanases that specifically attack the branching points of amylopectin are of special interest.The action of such enzymes would lead to the formation of linear oligosaccharides that can be attacked efficiently by other amylolytic enzymes, leading to high levels of glucose or maltose.Pullulanases are used in detergent industry as effective additives in dish washing and laundry detergents for the removal of starches under alkaline conditions (Van and Willem 1990) and in the manufacturing of low caloric beer (Olsen et al., 2000).It is also possible to use pullulanase as a dental plaque control agent (Marotta et al., 2002).Because of the various importance uses of pullulanase, it has been the subject of various applications so, the present study was conducted to extract, purify and characterize a highly active pullulanase from white edible mushrooms.

Preparation and extraction of crude enzyme
Mushrooms were extracted by distilled water at 9 ○ C with continuous shaking over a period of 2 h.This extract was then centrifuged at 3000 rpm for 15 min, and the supernatant was collected, dialyzed against distilled water and then used as the crude enzyme preparation.

Purification of pullulanase enzyme 1) Ammonium sulfate precipitation
The crude extract was brought to 20-70% saturation by gradually adding solid (NH 4 ) 2 SO 4 and stirred for 30 min at 4°C.The pellet was obtained by centrifugation at 12000 x g for 30 min and dissolved in 0.02M sodium phosphate buffer pH 6 and dialyzed extensively against the same buffer.

2) DEAE-cellulose column chromatography
The dialyzed sample was chromatographed on a DEAEcellulose column (12 x 2.4 cm i.d.) previously equilibrated with 0.02M sodium phosphate buffer pH 6.The adsorbed proteins were eluted firstly with sodium phosphate buffer (0.02M-pH 6) then with a stepwise NaCl gradient ranging from 0.05 to 0.5 M prepared in the equilibration buffer at a flow rate of 60 ml/hour.5 ml fractions were collected and the fractions containing pullulanase activity were pooled and lyophilized.

3) Sephadex G 75-120 column Chromatography
The concentrated solution containing the pullulanase activity was applied onto a Sephadex G75-120 column (40 cm x 1.8 cm i.d.).The column was equilibrated and developed with 0.02 M sodium phosphate buffer pH 6 at a flow rate of 30 ml/hour and 2 ml fractions were collected.

Enzyme assay
Pullulanase activity was assayed by measuring the reducing sugar released from pullulan.The reaction mixture (1ml) containing pullulan (1% w/v) in sodium phosphate buffer 0.02 M, pH 6 and enzyme was incubated at 40 °C for 30 min.The reducing sugar was measured by the dinitrosalicylic acid method (Millar, 1959).One unit of pullulanase activity is defined as the amount of enzyme which produces 1µmol of reducing sugar with glucose as the standard per min under the optimum conditions.

Electrophoretic analysis and isoelectric point
Native gel electrophoresis was carried out with 7% PAGE (Smith, 1969).In order to examine the subunit composition of the pullulanase, protein samples were also analyzed by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli, 1970 after the samples had been heated at 100 °C for 5 min.Low-molecular weight marker proteins were used as standards.Following native PAGE and SDS-PAGE the proteins were stained with Coomassie blue (Weber and Osborn, 1969).
The isoelectric point of the pullulanase was determined by isoelectric focusing under native conditions.Electro-focusing was performed according to O 'Farrell, (1975) and the isoelectric point (pI) value was calculated from a calibration curve (Ubuka et al., 1987).The proteins were stained with 0.25% Coomassie brilliant blue R-250.

Protein determination
Protein was determined by the dye binding assay method of Bradford (1976) using Bovine serum albumin (BSA) as a standard protein.

Effect of temperature on enzymatic activity and thermal stability
To determine the temperature effect on the enzyme activity the reaction mixture containing pullulan and purified pullulanase were incubated at different temperatures for 30 min and the activity was assayed.Thermal stability was determined by incubating the enzyme samples in different temperatures at different time intervals.

Effect of pH on pullulanase activity
To 0.5 ml of 1% (w/v) of pullulan in various pH's (4-8) at 0.02 M concentration, 0.5 ml of the purified pullulanase was added for 30 min at 40 ○ C.

Substrate specificity
The ability of the purified enzyme to hydrolyze various carbohydrates was examined at 40 ○ C and pH 6 in 0.02 sodium phosphate buffer.The carbohydrates tested were pullulan, amylopectin, soluble starch, glycogen and dextrin at a concentration 1% (w/v).

Effect of metal ions and chelating agents on the activity of pullulanase
The purified pullulanase was preincubated with various metal ions at 0.1 M concentration and chelating agents at 1M concentration.
The purified treated enzyme was incubated at 40 ○ C for 30 min in 0.02 M sodium phosphate buffer, pH 6.The enzyme sample without any additives was considered as control.

Kinetic determination
Michaelis-Menten constant (K m ) and maximum velocity (V max ) of pullulanase were estimated according to Lineweaver and Bark, 1934 using different concentration of pullulan.

End product analysis
Purified pullulanase samples were added to pullulan at a concentration of 1% (w/v) in 0.02 M sodium phosphate buffer, at pH 6.The reaction mixtures were incubated at 40 ○ C for 1, 2 and 24 hours.The products were analyzed by HPTLC plate with malto-oligosaccharide (G 3 -G 6 ), maltose and glucose used as standards.Chromatography was carried out using the solvent system, Butanol: acetic acid: water (40:10:50 v/v/v).Carbohydrates were detected by staining with anilinediphenylamine phosphoric acid reagent.The enzymatic products was visualized as blue spots after incubating the plate at 70 ○ C for 5 min (Asha et al., 2013).

Purification of pullulanase enzyme from mushroom.
A typical purification scheme of pullulanase from the mushroom is presented in Table (1).The purification procedure was carried out by ammonium sulfate precipitation (20-70%) followed by ion exchange chromatography on DEAE-cellulose column and gel filtration chromatography on Sephadex G75-120 column.The specific activity of the pullulanase of mushrooms crude extract was found to be 10 units/mg protein.Similar purification procedure of pullulanase was reported from the fungus Hypocrea jecorina (Orhan et al., 2014).The DEAE-cellulose elution profile (Figure 1) revealed the presence of one peak containing pullulanase activity eluted with 0.02 M sodium phosphate buffer pH 6.The DEAE-cellulose fraction of this peak were pooled, concentrated by lyophilization and applied onto a Sephadex G75-120 column (Figure 2) which revealed the presence of one peak of pullulanase enzyme activity.The pullulanase was purified with a specific activity of 97 units/ mg protein and 17.8 purification fold with 20% yield (Table 1).

Electrophoretic analysis and isoelectric point of the purified pullulanase
The purity of pullulanase was investigated by electrophoretic analysis.Samples from crude extract, DEAE-cellulose and Sephadex G75-120 fractions of pullulanase were analyzed electrophoretically on 7% native PAGE (Figure 3).Single protein band of the purified pullulanase was obtained indicating the tentative purity of the preparation.The molecular mass of the purified pullulanase enzyme was determined by SDS-PAGE to be 112 kDa (Figure 4).Different molecular masses were reported; pullulanase from Bacillus sp. has a molecular mass of 106 kDa in consistent with our preparation (Kunamneni and Singh, 2006).Pullulanase from the fungus Hypocrea jecorina has a molecular mass of 136 kDa (Orhan et al., 2014).Pullulanase from bacterium Fervidobacterium pennavorans has a molecular mass of 240 kDa (Koch et al., 1997).The native protein exhibited pI of 6.2 (Figure 5).This is consistent with the isoelectric point (pI) value of pullulanase from Bacillus subtilis which was around pH 6 (Asoodeha and Lagziana, 2012).

Effect of temperature on the activity and thermal stability of purified pullulanase
The maximum activity was observed at 40 ○ C (Figure 6).Thermal stability was studied at different temperatures at different time intervals, purified pullulanase showed moderate stability as the residual activity after 30 min incubation at 60 ○ C was 50%, while the residual activity was 20% after incubation for 45 min at 70 ○ C (Figure7).Asha et al. (2013) showed that the maximum activity of pullulanase from Bacillus halodurans was observed at 50 ○ C. Thermal stability was studied at 50 ○ C for an extended range of incubation period.Up to 45 min.incubation had less effect on enzyme activity.At 1 st hour, 60% residual activity was observed, 2 nd hour 46% residual activity and at overnight incubation, 17% residual activity was observed.

Effect of pH on pullulanase activity
As shown in Figure ( 8) the pH value at which the pullulanase showed maximum activity was observed at pH 6.The same result was reported by Ling et al., 2009, while Ara et al., 1995 showed that maximum activity was observed at pH 9.5.
On contrary, Ling et al. (2009) reported that starch, amylopectin and dextran gave low relative enzyme activity 28, 20 and 0.2% respectively.Effect of metal ions and chelating agents on the activity of pullulanase Hg 2+ , Ag+ and Co 2+ showed strong inhibition on pullulanase activity, while Mg 2+ slightly inhibited the enzyme.Almost Ni 2+ , Na + and Cu 2+ have no effect on the activity.Fe 2+ , Mn 2+ and Ca 2+ ions have very strong effect on stimulating the activity of pullulanase (Table 3).
Ara et al., 1995 reported that pullulanase activity of the enzyme was strongly inhibited by Hg 2+ and Mn 2+ .While Co 2+ ions slightly stimulated the pullulanase activity.Table (4) Showed that EDTA and DTT stimulate the activity of pullulanase while iodoacetate and sodium floride slightly inhibited the enzyme activity, while mercaptoethanol has no effect.Ara et al. (1995) showed that monoiodoacetate had moderately inhibitory effect on the enzymatic activity, while Asha et al. (2013) reported that EDTA and DTT did not obviously inhibit the pullulanase activity.

Kinetic properties of pullulanase
The activity of pullulanase from mushrooms on pullulan as a substrate showed Michaelis-Menten Kinetics.The apparent Michaelis-Menten constant (K m ) value for pullulan was 0.27 mM, while the value of V max was 0.74 µmol min -1 (Fig. 9).The K m of pullulanase type II from Bacillus cereus H 1.5 was 1.1 mg mL -1 , while the value of V max was 0.275 µmol min -1 (ling et al., 2009).

End products of pullulanase action
Maltotriose was the only trimeric (degree of polymerization, DP3), product of pullulan hydrolysis after incubation at 1, 2 and 24 hours (Figure 10), indicating that the enzyme hydrolysis is specific for α-1-6 glucosidic linkage of pullulan and the enzyme is endoacting enzyme and belong to type I pullulanase.

CONCLUSION
Pullulanase type I produced by edible white mushrooms was capable to attack specifically α-1,6 linkages in pullulan to generate maltotriose as the major end product.Pullulanase is moderately stable at the high temperature range.Current research work is focused on the extraction, purification and characterization of an industrial enzyme such as pullulanase by very simple and inexpensive methods from white edible mushrooms which are considered very safe source in food industry.Pullulanases have wide scale application in pullulan processing industry on account of their thermo-stability and ability to degrade row pullulan.The high substrate specificity of pullulanase together with its thermal stability makes this enzyme a good selection in the starchprocessing and detergent industries and other biotechnological applications.

Fig. 2
Fig. 2 A typical elution profile for the chromatography of mushroom DEAEcellulose fraction on Sephadex G-75-120 column (40 cm x 1.8 cm i.d.) previously equilibrated with 0.02 M sodium phosphate buffer pH 6.0.

Fig. 8 :
Fig. 8: The effect of different pH's on pullulanase activity at 0.02M concentration of the tested buffers.

Table 2 :
Substrate specificity of pullulanae from edible mushrooms.

Table 3 :
Effect of metals on pullulanase activity.

Table 4 :
Effect of chelating agents on pullulanase activity.