Evaluation of inhibitory activity, purification and X-ray crystallography of Alpha-Amylase inhibitor from Phaseolus vulgaris cultivars of Uttarakhand, India.

The present work is based on analysis of inhibitory activity of alpha-amylase inhibitor in selected cultivars of Phaseolus vulgaris of Uttarakhand. Fifteen samples were assessed for inhibitory activity of alpha-amylase inhibitor. Significant variations were found in different cultivars. Crude extract of alpha-amylase inhibitor from sample PUR (Purola) have shown maximum inhibitory activity (70.2 ± 0.84). Crude extract of all the cultivars have shown considerable variations in inhibitory activity in the temperature ranging from 20ºC to 100ºC. Based on inhibitory activity and heat stability profile, the alpha amylase inhibitor was purified from PUR cultivar. The purified inhibitor was found to be stable even at 90ºC with an inhibitory activity of 97.20 ±0.09. The molecular weight of purified inhibitor on Native PAGE (Polyacrylamide gel electrophoresis) was found to be 31kd, consisting of two subunits of 17kd and 14kd on SDS-PAGE.


Introduction
Plant seeds produce a variety of proteinaceous inhibitors of proteases and amylases. These inhibitors are characterized on the basis of sequences similarity and class of enzyme they inhibited 1 . Seed of common bean (Phaseolus vulgaris L.) contain certain inhibitors for digestive enzyme α-amylase. The amylase inhibitor does not inhibit the activity of plant, fungal and bacterial α-amylases, but inhibits the activity in mammals and some insects 2 . The α-amylase inhibitor strongly inhibits the larval midgut α-amylase activities of adzuki bean weevil (Collasobruchus chinesis L.) and Cowpea weevil (Collasobruchus maculatus), non-pest species of common bean 3 . The amylase inhibitors can be classi ed according to their tertiary structure in six different classes, namely lectin like, knottin like, cereal type, kunitz like, γ-purothinin like and thaumatin like 4 . Phaseolus vulgaris α-amylase inhibitors are also known as starch blockers and has been developed into more effective control agents for diabetes and obesity 5 .
A starch blocker is a substance that interferes with the breakdown of starch leading to reduced digestibility such that energy derived from the starch is reduced or rate of body absorption of energy in the form of glucose is reduced 6 . Several amylase inhibitors drugs (acarbose, voglibose) are in use for diabetic patients, often in conjugation with insulin 7 . Although the biochemical properties of legume α-amylase inhibitor have been studied for over 20 years some discrepancies dealing with their physico-chemical and functional properties have been frequently reported. In addition, only a little is known on their structural features, and their inhibition mechanism remains to be studied in details. Amylase inhibitor has been shown to have nutraceutical properties as well 8 . Since Uttarakhand is a rich repository of beans as more than 50 cultivars have been found, therefore the present study was undertaken to evaluate the starch blocking activity i.e. amylase inhibitor activity, heat stability of alpha-amylase inhibitor in selected cultivars, puri cation and crystallographic analysis of alpha-amylase inhibitor.

Results And Discussion
Assessment of starch blocking activity of α-amylase inhibitor: Alpha-amylase inhibitor protein inhibits the α-amylase enzyme and interferes in digestion of starch. Therefore, inhibitory activity of α-amylase inhibitor in selected cultivars can be used as a measure of starch blocking activity. Inhibition of pancreatic amylase was observed in all the seed samples thus showing the presence of α-amylase inhibitor ( Table 2). The cultivars were found to differ signi cantly in inhibitory activity. The maximum inhibitory activity was found to occur in sample PUR (70.2 ± 0.84 %) and minimum in sample DUN (39.43 ± 0.47 %). Sample PUR and MUN-2 have not shown a signi cant difference in inhibitory activity, similarly sample MAJ and RUD have nearly similar inhibitory activity, whereas all other cultivars are found to differ signi cantly in inhibitory activity of amylase inhibitor. Research into the mechanism of the Phaseolus vulgaris α-amylase inhibitor action shows that the inhibitor is effective in preventing starch digestion by restricting access to active site of the enzyme. The molecular-level binding of the action of the amylase inhibitor on human pancreatic amylase and PPA was reviewed in detail 9 . The study reveals that during inhibition, the components of inhibitor molecule play an important role in this mechanism. The main components that participate in the mechanism include two loops of the inhibitor made up of residues 20-45 and 172-19010, the amylase domains A/B and the active site non-loop residues (Asp197, Glu233, Asp300 and Arg74) in human pancreatic amylase 11 Effect of temperature on α-amylase inhibitor: Signi cant variations in inhibitory activity were found between all the cultivars. The inhibitory activity of cultivars increases upto 60 or 70 º C, afterwards there is decrease in the values of inhibitory activity. Sample DWA, MAJ, ALM, RAM and MUN-2 has shown maximum inhibitory activity at 50 º C and above this temperature the inhibitory activity decreases. Similarly, sample CHM, DUN, DHA, JOSH, TAP, MUN-1, MAJ and CHK have shown an increase in activity upto 60 º C and then there is decrease in activity with an increase in temperature ( Table 3). The minimum inhibitory activity at 20 º C was shown by sample DUN (36.45 ± 0.65 %) and maximum by sample PUR (71.34 ± 0.42 %) than other cultivars. On the other hand, at 100 º C the minimum inhibitory activity was shown by sample DWA (25.22 ± 0.40 %) and maximum by sample PUR (70.23 ± 0.28 %). Out of the fteen cultivars, sample PUR has shown a consistent stability in amylase inhibitor activity even at 100 º C (Table 5). Heat stability of α-amylase inhibitor has been shown by many studies. The inhibitor has been found to be stable at a temperature range of 40-90 º C 12,13 . The inhibitor is completely inactivated at 100 º C by boiling for 10 min 8 .
Puri cation and Molecular weight determination of α-amylase inhibitor: Based upon the inhibitor activity and effect of temperature on crude extract of amylase inhibitor in selected cultivars, puri cation of amylase inhibitor was done from sample PUR. Puri cation was done by ammonium salt precipitation (80-90%) followed by dialysis and gel-ltration chromatography using Sephadex G-50 column. The fractions were collected at constant ow rate and were assayed for protein estimation (%) and speci c activity (%). The speci c activity was found to increase after each puri cation procedure ( Table 4). The fractions eluted from sephadex column were analysed for inhibitory activity against PPA (Fig. 1) The puri ed fraction of sample PUR on SDS-PAGE was found to resolve into two bands of molecular weight of 14 and 17kd (Fig. 2). These bands may be due to denaturation of pure amylase inhibitor into two subunits.
Native PAGE of puri ed inhibitor from sample-PUR has shown a single band corresponding to molecular weight of 31kd (Fig. 3). The results obtained in present study were similar with literature references 8 , where the molecular weight of α-amylase inhibitor from Vigna sublobata was found to be 14kd on SDS-PAGE.
X-ray crystallography of puri ed α-amylase inhibitor: Crystals of puri ed enzymes from PUR cultivars were obtained by hanging drop methods and was found to differ in shape, wavelength, and space groups and in solvent content. X-ray analysis of sample-PUR was puri ed to homogeneity and crystallized at 293 K (Fig. 4). The crystals diffracted beyond 1.0 Å resolution using synchrotron single beam x-ray crystallography. The crystal belongs to the monoclinic space group P2 1 2 1 2, with Unit-cell parameters (A°) a = 74.56, b = 60.45, c = 64.40. Percent solvent content was 42.05%. Similar type of crystallography has been reported by literature references 14 in bifunctional amylase/subtilisin inhibitor puri ed from rice in which the crystal was found to be monoclinic with unit cell parameters of a = 79.99, b = 62.95, c = 66.70 Å.
As conclusion, the present work describes the comparative analysis of alpha amylase inhibitor activity in selected cultivars of kidney beans and its puri cation from sample PUR. The present study revealed that the inhibitory activity of plant alpha-amylase inhibitor against mammalian amylases could cause a marked decrease in the availability of digested starch. This could suggest a potential in the prevention and treatment of diabetes and nutritional problems, which result in obesity. Based on the results of this study, the α-amylase inhibitors from Phaseolus vulgaris may have potential in the prevention and therapy of obesity and diabetes.

Materials And Methods
Seed samples: Seeds of Phaseolus vulgaris have been collected from different geographical locations of Uttarakhand. Fifteen cultivars showing variation in seed coat colour, size and shape were selected. The seeds were authenticated and deposited in National Bureau of Plant and Genetic Research, New Delhi, India (Table 1).
Chemicals and Reagents: Sephadex G-50, PPA (Porcine pancreatic amylase) were purchased from Sigma (India). Protein markers were purchased from Genei (India) and others required chemicals from Himedia (India).
Crude extract Preparation: The extraction of seed proteins from seed our was done according to the method described by literature 3 . 100 mg of nely grounded seed our was taken, homogenised in extraction buffer and was incubated at 4 º C for 1hr. The homogenate was then centrifuged at 15000 rpm for 15 min at 4 º C. The supernatant was collected and stored in aliquots at -20°C for further analysis. The protein content was measured by method described by Bradford 15 Amylase inhibitory activity: The amylase inhibitory activity was determined according to literature descriptions 8 with some modi cations. A soluble starch solution (0.4 ml,1 % w/v) was made in 80mM phosphate buffer (pH = 6.9) and a solution of PPA (0.2 ml, 0.001 % w/v) in 20mM acetate buffer (pH-4.5, containing 20mM CaCl 2 and 10 mM NaCl) was added into it and then incubated for 15 min at 37 º C. The reaction was stopped by addition of 0.8ml of Dinitrosalicylic acid reagent (1gm DNS, 200 mg crystalline phenol and 50 mg of sodium sulphite dissolved in 1% NaOH). The contents were heated in a boiling water bath for 5 min, and after cooling it was diluted with 4ml of water. Absorbance of the mixture was read at 540 nm against blank prepared without using PPA. Amount of maltose produced was calculated from standard curve of maltose. The above method was also used to describe α-amylase inhibitor activity but PPA solution and puri ed inhibitor solutions (0.2ml) were pre-incubated for 15 min before addition of soluble starch solution. Alpha-amylase inhibitory activity was calculated according to equation shown below: