Porcine Pancreatic α-Amylase and its Isoforms: Purification and Kinetic Studies

Abstract

Porcine pancreatic α-amylase was separated on DEAE-cellulose into two isoforms, i.e., PPA-I and PPA-II and their molecular weight was found to be ∼ 55 kDa by gel filtration. The pH and temperature optima of PPA and its isoforms were 6.9 and 45°C. Chlorides of metal ions Ca²⁺, Ba²⁺, Co²⁺, and Mg²⁺ enhanced the enzyme activity, whereas Al³⁺ and Hg²⁺ completely inactivated the enzyme. Oxalic and citric acids inhibition of PPA and its isoforms is concentration dependant. EDTA was found to be the most effective inhibitor. PPA and its isoforms released maltotriose and maltotetraose from starches.

Keywords:

• Cereal and millet starches
• ESI-MS
• Ion-exchange chromatography
• Malto-oligosaccharides
• Porcine pancreatic α-Amylase (PPA)
• PPA-I
• PPA-II

INTRODUCTION

Alpha amylases (α-1, 4 glucan-4-glucanohydrolase; EC 3.2.1.1) are a class of hydrolases widely distributed in microbes, plants and animals.[1] α-Amylase is an important industrial enzyme used in starch liquefaction to produce glucose, maltose, and also in brewing, baking, textile, paper, detergent and sugar industries.[2] Hithertoknown α-amylases are metalloenzymes containing at least one Ca²⁺ ion per enzyme molecule, which is essential for their activity and stability.[3] Calcium is required to maintain the structural integrity of amylases. Removal of calcium ions leads to either decreased thermostability or decreased enzymatic activity or both.[4]

The three-dimensional structure of porcine pancreatic α-amylase is extremely similar to human pancreatic α-amylase. Pancreatic α-amylase consists of three domains: a catalytic core domain (A) comprising a (β/α)₈ barrel, an extended loop inserted between the third β-strand and the third α-helix (B domain) and a C-terminal eight stranded β-sheet domain (C domain).[5] Molecular cloning and primary structure analysis of porcine pancreatic α-amylase showed highest homology to the human pancreatic α-amylase sequence (87.1%) among all the amylases known.[6,7]

Pancreatic α-amylase is mainly responsible for in vivo degradation of starch.[8] In humans starch digestion is initiated by the salivary amylase in the mouth, wherein starch is degraded into oligomers which are further degraded by pancreatic α-amylase into maltose, maltotriose and low molecular weight maltooligosaccharides in the small intestine.[9] Combination of soft ionization methods like Electron Spray Ion-Mass Spectrometry (ESI-MS) has been developed as a powerful technique to analyze the sequence and linkage positions of oligosaccharides.[10]

Porcine pancreatic amylase was separated into two components PPA-I and PPA-II and the measured molecular masses of PPA-I and PPA-II were 55400 ± 6Da and 55395 ± 6Da respectively.[11,12] Optimum pH values for amylases were neutral which is in tune with several mammalian amylases.[13]

Even though the sequencing of PPA and its isoforms has been elucidated in detail, the kinetic parameters of PPA in comparison with that of PPA-I and PPA-II was not studied. Hence, a detailed study was undertaken to (a) separate porcine pancreatic α-amylase isoforms; (b) compare the kinetic parameters of PPA with that of PPA-I and PPA-II; and (c) study the mode of action of PPA and its isoforms on cereal and millet starches by identifying the released products by ESI-MS.

MATERIALS AND METHODS

Materials

Finger millet, ragi (Eleusine coracana, variety Indaf-15), rice (Oryza sativa, variety Jaya), and maize (Zea mays, variety NAC - 6002) were procured from Zonal Agricultural Research Centre, Mandya of UAS, Bangalore, Karnataka, India. Wheat (Triticum aestivum, variety DPW- 225 was procured from the local market.

The following special chemicals were obtained from various agencies: Sugar standards (malto-oligosaccharides), diethyl amino ethyl (DEAE)-cellulose, porcine pancreatic α-amylase, ethylenediaminetetraacetic acid (EDTA), 3,5,d-nitro salicylic acid (DNS) were procured from Sigma Chemicals Company, St. Louis, MO, USA.

Column for HPLC analysis (μ Bondapak-NH₂ column) was obtained from Waters Associates, Milford, MA, USA. General chemicals used in this study were all of analytical grade and obtained from E-Merck, BDH, SRL and SD fine chemicals.

DEAE-Cellulose Chromatography

PPA (25 mg) was loaded onto a DEAE-cellulose column (φ 2.8 × 40 cm) pre-equilibrated with Tris-buffer (500 mL, 20mM, pH 8.0) at a flow rate of 30 mL/h and washed with the same buffer to remove the unbound proteins. A linear NaCl gradient (0–0.6 M) in equilibrating buffer was used to elute the bound protein which was collected (5 mL each) and monitored for protein (280 nm) as well as for amylase activity. In this present work, resolution of PPA-I and PPA-II components are obtained by DEAE-cellulose under slightly modified conditions.[14,15] The two activity peaks were designated as PPA-I and PPA-II in the order of their elution. PPA-I and PPA-II were concentrated and individually loaded on Sephadex G-100 glass column (φ 0.9 × 70 cm) pre-equilibrated with phosphate buffer (pH 6.9, 50 mM) and fractions (6 mL/h) were collected, monitored for protein and amylase activity.

Enzyme Assay

Amylases (PPA, PPA-I, and PPA-II) were assayed using gelatinized soluble starch (1g/100 mL, 1 mL) as substrate in sodium phosphate buffer (50 mM, pH 6.9), incubated with enzyme (50 μl) at 37°C for 60 min. The reaction was stopped by adding DNS reagent (1mL).[16] The tubes were heated in a boiling water bath for 10 min cooled to room temperature and absorbance was measured after appropriate dilution at 540 nm.[17] One unit of enzyme activity was defined as μmol of maltose equivalent released per min under the assay conditions.

Effect of Substrate Concentration (Kinetic Parameters)

Kinetics of PPA, PPA-I and PPA-II using starches isolated from different sources were studied at pH 6.9 and at 37°C.[18] The initial velocity was measured at various substrate concentrations [S], which were expressed as percentage for comparison. The values of Michaelis constants (K_m) and the maximum velocity (V_max) were determined from the Lineweaver-Burk plots.[19]

Effect of pH

Amylase activities were determined at various pH values using different buffers (50 nM concentration) such as sodium acetate (pH 4.0–5.5), sodium phosphate (pH 6.0–7.5) and Tris-HCl (pH 8.0–9.0) at 37°C. The maximum activity was taken as 100% and relative activities were plotted against different pH values.[20]

Temperature Optima

Amylase activities were determined at a temperature range of 30–70°C (with an interval of 5°C) with 1% soluble starch as substrate in 50 mM sodium phosphate buffer. The maximum activity was taken as 100% and relative activities were plotted against different pH values.[20]

pH Stability

Stability of amylases were determined using different buffers (ranging in their pH values from 3.0–9.0) such as phthalate-HCl (2.0–3.0), sodium acetate (4.0–5.0), sodium phosphate (6.0–8.0), Tris-HCl (8.0–9.0) at 50mM concentration. The residual activity was measured after pre-incubating the enzyme at pH 3.0–9.0 for 4 h (ranging from 15–240 min) at 37°C. The assay conditions were otherwise similar to that given above. The original activity was taken as 100%, and the relative activity was plotted against different time intervals.[21]

Thermal Stability

PPA and its isoforms in sodium phosphate buffer (50mM, pH 6.9) were incubated at a temperature range of 30–70°C for 15 min. The residual activity was estimated taking original activity as control (100%) and relative activity was plotted against different temperatures.[21]

Metal Ion Effect

PPA and its isoforms were incubated with 5 mM solution of salts of metal ions (chlorides of Ca²⁺, Ba²⁺, Co²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Al³⁺, Hg²⁺) and their activities were determined. The enzyme activities without metal ions were taken as 100% and relative activities determined in the presence of metal ions were calculated.[20]

Effect of Inhibitors

The inhibitors such as citric acid (1–15 mM), oxalic acid (1–15 mM), and EDTA (2.5–150 μm) were incubated with PPA and its isoforms at 37°C for 15 min and the residual activity was measured. The enzyme activities without inhibitors were taken as 100% and relative activities were plotted against different inhibitor concentrations.[21]

Digestibility

ESI-MS of maltooligosaccharides

Starches were isolated according to the procedure adopted by Mohan, et.al.[22] Isolation of starches were similar to that of starch isolation from legumes.[23] Starches were gelatinized and subjected to digestion by PPA, PPA-I and PPA-II for 60 min and solutions were precipitated with three volumes of absolute ethanol and kept overnight at 4°C. The undigested residual starches were separated by centrifugation and the supernatants consisting of oligosaccharides were resolved and identified by HPLC (LC-8 A CR4A, Shimadzu) on μ-Bonda pack amino column (4.1 mm × 30 cm) using acetonitrile : water (70:30) as eluent with a flow rate of 1 mL / min.[24,25,26]

Structural characterization of maltooligosaccharides by Electron Spray Ion-Mass Spectrometry (ESI-MS)

The fragmentation pattern of the maltooligosaccharides produced by PPA and its isoforms are described using ESI-MS.[27,28] Mass spectra of the oligosaccharides obtained by pancreatic α-amylase digestion of the starches were measured by Alliance, Waters 2695 mass spectrometer using positive mode electrospray ionization. Capillary voltage was 3.5 kV, core voltage 100 V, source 80°C, desolvation temperature 150°C, core gas (Argon) 35 L h⁻¹ and desolvation gas (Nitrogen) 500 L h⁻¹.[29]

RESULTS AND DISCUSSION

DEAE-Cellulose Chromatography

Porcine pancreatic α-amylase was separated into two activity peaks on DEAE cellulose column and designated as PPA-I and PPA-II (Fig. 1) with a linear gradient of NaCl (0–0.6 M). PPA-I and PPA-II were eluted at concentrations of 0.25 and 0.36 M NaCl respectively. Porcine pancreatic α-amylase (PPA) was resolved to homogeneity from tissue extracts into two forms PPA-I and PPA-II as reported by Granger, et. al.[30] These two forms have the same kinetic properties and molecular masses with distinct pI of 7.5 and 6.4, respectively.[31,12]

Figure 1 Elution profile of porcine pancreatic alpha amylase on DEAE—cellulose column.

Effect of Substrate Concentration (Kinetic Parameters)

The effect of different concentrations of cereal and millet starches on the initial velocity was calculated. K_m and V_max for PPA, PPA-I, and PPA-II were found out from the Line-Weaver double reciprocal plots.[19,32] The K_m values of PPA for ragi, rice, wheat and maize starches varied between 0.8–3.3% and the V_max was between 2841–6212 U/mg protein, respectively. For PPA-I, K_m values for ragi, rice, wheat, and maize ranged between 1.3–2.5% whereas V_max was found to be between 2740–4201 U/mg protein. K_m values of PPA-II for ragi, rice, wheat and maize were in the range of 1.0–2.96% and the V_max values ranged between 2755–5102 U/mg protein (Table 1). Maize starch was the most difficult to digest followed by ragi, wheat and rice starches as indicated by the relatively high K_m value.

Table 1 Kinetic constants of PPA, PPA-I, and PPA-II for cereal and millet starches. (K_m and V_max)

Starches	PPA		PPA-I		PPA-II
	Km	Vmax	Km	Vmax	Km	Vmax
Ragi	2.3	4149	2.5	4201	1.4	4149
Rice	0.8	2841	1.3	2740	1.0	2755
Wheat	1.0	3571	1.8	3125	1.5	3571
Maize	3.3	6212	2.5	3571	2.9	5102
Km (%) ; Vmax (U/mg protein). Mean values of duplicate analysis.

pH and Temperature Optima

The optimum pH for the hydrolytic activity of PPA under physiological conditions has long been believed to be around neutrality on the basis of the study on soluble starch.[33] Effect of pH on the activity of amylase is influenced by the kind of substrate and the assay conditions.[34] In the present study soluble starch was taken as the substrate at 1% concentration. pH optima of PPA, PPA-I and PPA-II were 6.9 (Fig. 2a). The activities of PPA and its isoforms are maximum at pH 7.0 compared to the activities observed in the acidic pH. The activities of PPA, PPA-I and PPA-II in sodium acetate buffer at pH 5.0 were 80, 85, and 65% respectively. PPA and its isoforms showed maximum activity in sodium phosphate buffer at pH 6.9. Tris-HCl buffer had a stabilizing effect on the activities of PPA, PPA-I and PPA-II. PPA retained about 80% activity at pH 8.5. PPA-I and PPA-II had maximum of 80 and 60% activities at pH 8.0. The results obtained in the present investigation are in accordance with the published literature.[33]

Figure 2 (a) pH optima of PPA, PPA-I and PPA-II; (b) Temperature optima of PPA, PPA-I and PPA-II.

PPA, PPA-I, and PPA-II showed temperature optima at 45°C. All the three enzymes retained up to 50% activity at 50°C. The activity increased sharply with gradual increase in temperature up to 45°C, whereas it showed decrease with further increase in temperature, indicating loss in the active conformation of the enzyme. PPA, PPA-I and PPA-II showed only 20–30% activity at 70°C (Fig. 2b).

pH and Temperature Stability

PPA was found to be stable in the pH range of 7.0–8.0 with 80% activity after 4 h incubation whereas PPA-I and PPA-II were stable in the pH range of 6.0–8.0 with almost 80% activities after 4 h incubation. However, the activity of these amylases decreased drastically at pH 2.0–3.0 after pre-incubation for 30 min (Fig. 3 a,b,c).

Figure 3 pH stability of PPA and its isoforms. (a) PPA; (b) PPA-I; (c) PPA-II.

α-Amylases are generally stable in the pH range of 5.5–8.0. Exceptions exist on both sides of the pH scale as reported for bacterial amylases.[35,36] An acidophilic α-amylase from a Bacillus sp. has a pH optima of 2.0[35] and an alkalophilic α-amylase from another Bacillus sp. has pH optima of 10.5.[36] This broad range of optimum pH values for activity of individual α-amylases indicated their evolutionary adaptability to environmental circumstances.[37]

More than 95% activity was retained by PPA, PPA-I and II up to 45°C (Fig. 4). At 50°C the activities of PPA, PPA-I and PPA-II dropped to 50, 63, and 65% respectively. However, at 70°C, PPA and its isoforms have completely lost their activity.

Figure 4 Effect of temperature on stability of PPA, PPA-I and PPA-II.

The lowest temperature optimum was reported as 25°C for Fusarium oxysporum α-amylase,[3,38] whereas the highest for Bacillus licheformis α-amylase (90°C).[39] The temperature optima of α-amylases, are observed between 50 and 60 °C. The extra thermostability of thermophilic Bacillus licheniformis α-amylase has been found to be mainly due the additional salt bridges involving a few specific lysine residues.[40] Bacillus stearothermophilus α-amylase,[41] as well as a mutant α-amylase from Bacillus amyloliquefaciens[42] have been suggested to be stable against thermal denaturation through ionic interaction.

Effect of Metal Ions

Effect of di- and trivalent ions on the enzyme activities of PPA, PPA-I, and PPA-II are given in Table 2. Activity of PPA has increased in the presence of divalent ions such as Ca²⁺, Co²⁺, Mg²⁺ and Ba²⁺. Hg²⁺ and Al³⁺ completely inhibited the activities of all the three amylases, i.e. PPA, PPA-I and PPA-II. Cu²⁺ ions also inhibited the activities of the all the three enzymes to a larger extent.

Table 2 Effect of metal ions on the activities of PPA, PPA-I and PPA-II

Salts (5mM)	PPA	PPA-I	PPA-II
Control	100	100	100
CaCl2	114.9	58.6	48.9
BaCl2	108.4	14.1	0.8
CoCl2	105.9	21.2	2.6
MgCl2	111.6	18.8	25.5
ZnCl2	15.5	12.6	5.2
Cucl2	0.7	0.3	0.4
AlCl3	0	0	0
HgCl2	0	0	0
Mean values of triplicate analysis.

The inactivation is attributed to the binding of this metal ion either to the catalytic residue or by replacing the Ca²⁺ from the substrate-binding site of the enzyme. Ca²⁺ had stabilizing effect on PPA-I and PPA-II. Enhancement of amylase activity by Ca²⁺ ions is based on its ability to interact with negatively charged amino acid residues such as aspartic acid and glutamic acids, which resulted in stabilization, as well as maintenance of enzyme conformation. In addition, calcium is known to have a role in substrate binding.[43] The presence of Ca²⁺ is needed for the optimum activity and stability of the enzyme. The inhibition by other metal ions might be due to the competition for calcium binding site. With respect to the other metal ions, Al³⁺ completely inactivated PPA, PPA-I and PPA-II.

Alpha-amylases have long been known to require at least one Ca²⁺ ion per enzyme molecule for maintaining their tertiary structure and catalytic activity.[44,45] PPA was reported to have a calcium ion-binding site in which Ca²⁺ creates an ionic bridge between two β-structures. The essential Ca²⁺ ion appears to stabilize the active site cleft by inducing an ionic bridge between domains A and B. Role of Mg²⁺ in maintaining the stability and structure of the α-amylase is well documented.[34] Significant increase in the activity of α-amylase from B. amyloliquifaciens from a commercial source was reported by the exogenous addition of Co²⁺.

Effect of Citric and Oxalic Acids

Carboxylic acids such as citric and oxalic acids inhibit α-amylases by selective removal of Ca²⁺ present in the enzyme. The effect of citric and oxalic acids on the activities of PPA, PPA-I, and PPA-II were tested in different concentrations (0–15.0 mM). Citric acid (10 mM) completely inhibited the enzyme activities of PPA and PPA-II, whereas 7.5 mM concentration completely inhibited PPA-I activities (Fig. 5A). Oxalic acid (10mM) completely inhibited PPA activity whereas, PPA-I and PPA-II were inhibited at 5.0 mM and 7.5mM respectively (Fig. 5B). The differential inhibition effect of citric/oxalic acid on the activities of PPA, PPA-I, and PPA-II may be attributed to relative binding of Ca²⁺ to the enzymes.

Figure 5 (a) Effect of citric acid on the activities of PPA and its isoforms; (b) Effect of oxalic acid on the activities of PPA and its isoforms.

Effect of EDTA

EDTA at 125 μm concentration completely inhibited the activity of PPA and its isoforms (Fig. 6). The inhibition by EDTA is due to its chelating of bound calcium present in PPA and its isoforms.[46,43] Calcium ions appear to stabilize the active site cleft by inducing an ionic bridge between the domains both in the native enzyme and its isoforms. However, EDTA inhibited isoforms I and II much more strongly than PPA. This may be perhaps due to the presence of more amount of bound calcium in the native enzyme than its isoforms, which may require higher concentration of EDTA to chelate the bound calcium present in the native enzyme.

Figure 6 Effect of EDTA on PPA and its isoforms.

Inhibition of amylase activity by EDTA indicated that the enzyme is a metallo-protein. EDTA binds most divalent metal ions. Removal of Ca²⁺ from the enzyme exposes the 2-sulphdryl groups and thereby the enzyme loses its activity. The inhibition may be due to the preferential and rapid formation of enzyme-inhibitor complex over substrate-enzyme complex indicating this to be a competitive type of inhibition.[46]

Digestibility

PPA, PPA-I, and PPA-II released mainly oligosaccharides of varying degree of polymerization (G2 to G7) and negligible amounts of higher oligosaccharides (DP = >8) from cereal and millet starches. The pattern obtained from cereal starches differ especially with respect to amounts of G3 and G4 oligosaccharides (Data not shown). The in vitro digestibility pattern of Curcuma starch was carried out using α-amylase from Bacillus sp. The digestibility of tuber starch from Curcuma was high when compared to cereal starches.[47]

Structural Characterization of Maltooligosaccharides by Electron Spray Ion-Mass Spectrometry (ESI-MS)

The fragmentation pattern of the starches by pancreatic α-amylase was confirmed by ESI-MS (Fig. 7i, ii, iii). PPA digested starches of ragi showed the MS spectrum of the ion at 365.3 m/z (maltose: fragmentation, 180 × 2 = 360 − 18 = 342 + 23 = 365), 527.41 m/z (maltotriose: fragmentation, 180 × 3 = 540 − 36 = 504 + 23 = 527) and 689.4 m/z (maltotetraose: fragmentation, 180 × 4 = 720−54 = 666 + 23 = 689) and trace of maltohexaose at 1013 m/z (fragmentation, 180 × 6 = 1080−90 = 990 + 23 = 1013). PPA digested starches of rice showed the MS spectrum of the ion at 365.3, 527.41, and 689.4 m/z and small amount of maltopentaose at 851 m/z (fragmentation, 180 × 5 = 900 – 72 = 828 + 23 = 851). 23 is the sodium adducts [M+ Na]⁺ that is detected in all the samples. With respect to PPA digestion of wheat and maize starches, MS spectrum of the ion was observed at 365.3, 527.41 and 689.4 m/z, which correspond to maltose, maltotriose, and maltotetraose. Digestion of starches by PPA-I and PPA-II showed similar pattern of fragmentation at 527.41 (maltotriose: fragmentation, 180 × 3 = 540 − 36 = 504 + 23 = 527) and 689.4 (maltotetraose: fragmentation, 180 × 4 = 720 − 54 = 666 + 23 = 689) m/z.

Figure 7(i) ESI-MS spectra of maltooligosacharides obtained by PPA digestion of starches: (a) ragi; (b) rice; (c) wheat; (d) maize.

Figure 7(ii) ESI-MS spectra of maltooligosacharides obtained by PPA-I digestion of starches: (a) ragi; (b) rice; (c) wheat; (d) maize.

Figure 7(iii) ESI-MS spectra of maltooligosacharides obtained by PPA-II digestion of starches: (a) ragi; (b) rice; (c) wheat; (d) maize.

CONCLUSIONS

PPA was separated into two isoforms i.e. PPA-I and PPA-II on DEAE-cellulose and their molecular weight was found to be 55 kDa. They were inactivated below pH 4.0 and above 70°C. Ca²⁺ was enhancing the activities of PPA and its isoforms. EDTA, oxalic acid and citric acid inhibited PPA and its isoforms. Kinetic studies revealed a similar degradation pattern of starches by PPA, PPA-I, and PPA-II. PPA and its isoforms released preponderantly maltotriose and maltotetraose from the cereal and millet starches as identified by ESI-MS.

ACKNOWLEDGMENTS

The authors thank Dr V. Prakash F.R.Sc. Director C.F.T.R.I., for his kind encouragement and support. AG thanks the Council of Scientific Industrial Research (C.S.I.R) New Delhi for the Senior Research Fellowship.