Purification of alpha-amylase from germinating little...
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Journal of Pharmacy Research Vol.4.Issue 5. May 2011 B.Usha et al. / Journal of Pharmacy Research 2011,4(5),1370-1371 1370-1371 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info *Corresponding author. K.P.J.Hemalatha Department of Biochemistry, Andhra University, Visakhapatnam-530 003, AndhraPradesh,India. INTRODUCTION Purification of a-amylase from germinating little millets (Panicum sumatrense) B. Usha and K. P. J. Hemalatha* Department of Biochemistry, Andhra University, Visakhapatnam-530 003, Andhra Pradesh, India. Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011 ABSTRACT The crude extract of α-amylase (alpha 1-4D-glucan glucanohydrolase EC 3.2.1.1.) was obtained from little millet (Panicum sumatrense L. Roth ex Roem. et Schult.) cotyledons excised from 3-day-old seedlings. The activity of α-amylase increased dramatically both in specific activity and total activity on day 3 when germination occurs in the dark. The enzyme was purified 10.15 fold with a five-step procedure including homogenization, acetone precipitation, ammonium sulfate fractionation, ion-exchange chromatography on DEAE cellulose, gel permeation chromatography on Sephadex G-150. The amylase purified was a monomer with molecular weight 46 kDa, which optimally hydrolyzes soluble starch at a temperature of 50°C and pH 5.0. The Michaelis constant for the enzyme catalyzed reaction ( K m ) is 1.6 mg soluble starch/ml. Key words: α-amylase, little millet, purification, properties, Panicum sumatrense Table. 1. Summary of the purification of a-amylase from 3-day old germinating little millets Step Volume Total Activity Total Protein Specific Activity Fold Percentage (ml) (Units)* (mg) (U /mg ) Purification Recovery Crude 250 431150 4375 98.54 1 100 Acetone 230 403300 4001 100.8 1.02 93.54 Precipitation Ammonium Sulfate 120 262000 1210 216.5 2.19 60.76 (30-65%) DEAE-Cellulose 80 177700 208.9 850.6 8.63 41.21 Sephadex G-150 40 125025 124.9 1001 10.15 29 *One unit is equivalent to 1 μmol of maltose released /min α-Amylases are starch-degrading enzymes that catalyze the hydrolysis of internal α-1, 4-O- glycosidic bonds in polysaccharides with retention of α-anomeric configuration in the products. Most of the α-amylases are metalloenzymes, which require calcium ions (Ca 2+ ) for their activity, structural integrity and stability. Amylases are widely distributed in microbes, plants and animals. The enzymic degradation of starch in plants has been attributed to α-amylase in starch storage tissues (Swain and Dekker, 1966a). They have been studied particularly in seeds, where they are responsible for the breakdown of polysaccharide reserves. Much of our knowledge of starch degradation during seed germination comes from studies with cereals (Kruger and Tkachuk, 1969; Murata et al. 1968; Nirmala and Muralikrishna, 2003b) where a number of amylase isozymes have been described. Starch is a high molecular weight α-D- glucan polymer consisting mainly a linear molecule namely amylose in α-1, 4 linkage and a branched polymer known as amylopectin having branches of α-1, 6 linkages (Banks and Greenwood, 1975). α-Amylase produces soluble oligosaccharides from starch and these are then hydrolysed by ß-amylase to liberate maltose. Finally, α-glucosidase breaks down maltose into glucose (Swain and Dekker, 1966b; Nomura et al. 1969). The purpose of this study is to isolate α-amylase from germinating little millet and exploit its properties for further use in view of the perspective that α-amylases have potential application in a wide number of industrial processes such as food, fermentation, textile, paper, detergent, and pharmaceutical industries. MATERIALS AND METHODS Determination of a-amylase activity and protein concentration Amylase was assayed according to the procedure of (Bernfeld, 1955). Gelatinized soluble starch (1%) in 0.05 M sodium acetate buffer (pH 5.0) was incubated with appropriately diluted enzyme at 45ºC for 20 min. The reaction was stopped by the addition of (1 ml) DNS reagent. One unit of enzyme activity was defined as μmol maltose equivalent released/min under the assay conditions. The specific activity was expressed as activity units/mg protein. Protein content was determined according to the method of (Lowry et al.1951) using BSA as standard. Purification of a-Amylase from germinating little millet Cotyledons from 3-day old seedlings from germinating little millet were homogenized using mortar and pestle in 0.05 M sodium phosphate buffer (pH 6.9). Four milliliters of buffer was added per gram of fresh weight. α-Amylase was extracted for 1 h at 4°C. Extract was filtered through two layers of cheese cloth and centrifuged for 20 min at 4°C at 6,500×g. The supernatant was subjected to acetone precipitation 1:4 v/v. Acetone at -10°C was slowly added to crude extract while stirring. The mixture was allowed to stand for 30 min and then centrifuged at 6,500×g at 4°C for 20 min. The precipitate was collected and dissolved in 0.05 M sodium phosphate buffer (pH 6.9) and subjected to ammonium sulfate fractionation (30- 65%) and centrifuged. The precipitate redissolved and dialyzed overnight against the same buffer at 4°C was loaded onto a DEAE-cellulose column (1.5 cm × 30 cm) which was preequilibrated with 0.01 M phosphate buffer (pH 7.6). It was washed with the same buffer to remove unbound proteins at a flow rate of 30 ml/h. Bound components in the column were eluted with increasing gradient of 0-0.3 M NaCl in the eluting buffer, 3 ml fractions each were collected and monitored for protein as well as amylase activity. The fractions with high specific activity were pooled and subjected to gel permeation chromatography on Sephadex G-150 column (2.0 cm × 60 cm) which was preequilibrated with 0.01 M phosphate buffer (pH 7.6) and fractions (3 ml) each were collected, monitored for protein (280 nm) and amylase activity. Determination of molecular weight by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis Homogeneity and molecular weight of purified amylase was analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method according to (Laemmli, 1970). Gels were stained with Coomassie brilliant blue R250. In order to determine molecular weight, SDS-PAGE molecular weight standards in the molecular mass range from 14.3 to 97.4 kDa were used. Determination of Kinetic Constants The activity of the enzyme was assayed in 0.05 M sodium acetate buffer (pH 5.0) with varying substrate concentrations ranging from 0.2 to 2.0×10g/ml. K m and V max were calculated from LB plot (Lineweaver and Burk, 1934). pH optima α-Amylase activities were determined at various pH values using different buffers such as glycine-HCl (2.0-3.0), sodium acetate (4.0-5.0), sodium phosphate (6.0-7.0), Tris-HCl (8.0- 9.0) at 0.05 M concentration with 1% soluble starch as substrate. The relative activity at different pH values was calculated, taking the maximum activity obtained as 100%. Temperature optima α-Amylase activities were determined at a temperature range of 20-70°C (with an interval of 10°C) with 1% soluble starch as substrate. The relative activity at different temperatures was calculated, taking the maximum activity obtained as 100%. Influence of calcium ions on amylase thermostability The influence of calcium ions at various concentrations on α-amylase activity was studied. The samples were preincubated for 30 min in 50 mM acetate buffer (pH 5.0) at 45°C, supplemented with calcium ion and then enzyme assay was performed with soluble starch as substrate. RESULTS AND DISCUSSION Purification of Little millet a-Amylase The present study is an attempt towards purification of α-amylase from germinating little millet seeds. The activity of amylase increased from day 0 to day 3 where it exhibited its maximum activity. A summary of the purification procedure that was used for the little millet cotyledonary α-amylase is presented in (Table 1). The purification fold and percentage recovery were monitored by assay of enzyme against soluble starch. As a result of this procedure, a protein with specific activity of 1001 Units/mg was obtained. The purification fold was 10.15, whereas the recovery was 29%. α-Amylase from malted sorghum has been reported to be purified 24.7-fold, with a recovery of 17.1%, using 0-40% ammonium sulfate precipitation, ion exchange chromatography on DEAE cellulose and GPC on G-75 (Siva Sai Kumar et al. 2005).
Transcript of Purification of alpha-amylase from germinating little...
Journal of Pharmacy Research Vol.4.Issue 5. May 2011
B.Usha et al. / Journal of Pharmacy Research 2011,4(5),1370-1371
1370-1371
Research ArticleISSN: 0974-6943
Available online throughhttp://jprsolutions.info
*Corresponding author.K.P.J.HemalathaDepartment of Biochemistry,Andhra University,Visakhapatnam-530 003,AndhraPradesh,India.
INTRODUCTION
Purification of α-amylase from germinating little millets (Panicum sumatrense)B. Usha and K. P. J. Hemalatha*
Department of Biochemistry, Andhra University, Visakhapatnam-530 003, Andhra Pradesh, India.
Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011ABSTRACT
The crude extract of α-amylase (alpha 1-4D-glucan glucanohydrolase EC 3.2.1.1.) was obtained from little millet (Panicum sumatrense L. Roth ex Roem. et Schult.) cotyledons excised from3-day-old seedlings. The activity of α-amylase increased dramatically both in specific activity and total activity on day 3 when germination occurs in the dark. The enzyme was purified 10.15fold with a five-step procedure including homogenization, acetone precipitation, ammonium sulfate fractionation, ion-exchange chromatography on DEAE cellulose, gel permeationchromatography on Sephadex G-150. The amylase purified was a monomer with molecular weight 46 kDa, which optimally hydrolyzes soluble starch at a temperature of 50°C and pH 5.0.The Michaelis constant for the enzyme catalyzed reaction (Km) is 1.6 mg soluble starch/ml.
Key words: α-amylase, little millet, purification, properties, Panicum sumatrense
Table. 1. Summary of the purification of α-amylase from 3-day old germinating little millets
Step Volume Total Activity Total Protein Specific Activity Fold Percentage (ml) (Units)* (mg) (U /mg ) Purification Recovery
Crude 250 431150 4375 98.54 1 100Acetone 230 403300 4001 100.8 1.02 93.54PrecipitationAmmonium Sulfate 120 262000 1210 216.5 2.19 60.76(30-65%)DEAE-Cellulose 80 177700 208.9 850.6 8.63 41.21Sephadex G-150 40 125025 124.9 1001 10.15 29
*One unit is equivalent to 1 µmol of maltose released /min
α-Amylases are starch-degrading enzymes that catalyze the hydrolysis of internal α-1, 4-O-glycosidic bonds in polysaccharides with retention of α-anomeric configuration in the products.Most of the α-amylases are metalloenzymes, which require calcium ions (Ca2+) for theiractivity, structural integrity and stability. Amylases are widely distributed in microbes, plantsand animals. The enzymic degradation of starch in plants has been attributed to α-amylase instarch storage tissues (Swain and Dekker, 1966a). They have been studied particularly inseeds, where they are responsible for the breakdown of polysaccharide reserves. Much of ourknowledge of starch degradation during seed germination comes from studies with cereals(Kruger and Tkachuk, 1969; Murata et al. 1968; Nirmala and Muralikrishna, 2003b) where anumber of amylase isozymes have been described. Starch is a high molecular weight α-D-glucan polymer consisting mainly a linear molecule namely amylose in α-1, 4 linkage and abranched polymer known as amylopectin having branches of α-1, 6 linkages (Banks andGreenwood, 1975). α-Amylase produces soluble oligosaccharides from starch and these arethen hydrolysed by ß-amylase to liberate maltose. Finally, α-glucosidase breaks down maltoseinto glucose (Swain and Dekker, 1966b; Nomura et al. 1969). The purpose of this study is toisolate α-amylase from germinating little millet and exploit its properties for further use inview of the perspective that α-amylases have potential application in a wide number ofindustrial processes such as food, fermentation, textile, paper, detergent, and pharmaceuticalindustries.
MATERIALS AND METHODS
Determination of α-amylase activity and protein concentration Amylase was assayed according to the procedure of (Bernfeld, 1955). Gelatinized solublestarch (1%) in 0.05 M sodium acetate buffer (pH 5.0) was incubated with appropriately dilutedenzyme at 45ºC for 20 min. The reaction was stopped by the addition of (1 ml) DNS reagent.One unit of enzyme activity was defined as µmol maltose equivalent released/min under theassay conditions. The specific activity was expressed as activity units/mg protein. Proteincontent was determined according to the method of (Lowry et al.1951) using BSA as standard.
Purification of α-Amylase from germinating little milletCotyledons from 3-day old seedlings from germinating little millet were homogenized usingmortar and pestle in 0.05 M sodium phosphate buffer (pH 6.9). Four milliliters of buffer wasadded per gram of fresh weight. α-Amylase was extracted for 1 h at 4°C. Extract was filteredthrough two layers of cheese cloth and centrifuged for 20 min at 4°C at 6,500×g. Thesupernatant was subjected to acetone precipitation 1:4 v/v. Acetone at -10°C was slowly addedto crude extract while stirring. The mixture was allowed to stand for 30 min and thencentrifuged at 6,500×g at 4°C for 20 min. The precipitate was collected and dissolved in 0.05M sodium phosphate buffer (pH 6.9) and subjected to ammonium sulfate fractionation (30-65%) and centrifuged. The precipitate redissolved and dialyzed overnight against the samebuffer at 4°C was loaded onto a DEAE-cellulose column (1.5 cm × 30 cm) which waspreequilibrated with 0.01 M phosphate buffer (pH 7.6). It was washed with the same buffer toremove unbound proteins at a flow rate of 30 ml/h. Bound components in the column wereeluted with increasing gradient of 0-0.3 M NaCl in the eluting buffer, 3 ml fractions each werecollected and monitored for protein as well as amylase activity. The fractions with high specificactivity were pooled and subjected to gel permeation chromatography on Sephadex G-150column (2.0 cm × 60 cm) which was preequilibrated with 0.01 M phosphate buffer (pH 7.6)
and fractions (3 ml) each were collected, monitored for protein (280 nm) and amylase activity.
Determination of molecular weight by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresisHomogeneity and molecular weight of purified amylase was analyzed by denaturing sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method according to (Laemmli,1970). Gels were stained with Coomassie brilliant blue R250. In order to determine molecularweight, SDS-PAGE molecular weight standards in the molecular mass range from 14.3 to 97.4kDa were used.
Determination of Kinetic ConstantsThe activity of the enzyme was assayed in 0.05 M sodium acetate buffer (pH 5.0) with varyingsubstrate concentrations ranging from 0.2 to 2.0×10g/ml. Km and Vmax were calculated fromLB plot (Lineweaver and Burk, 1934).
pH optimaα-Amylase activities were determined at various pH values using different buffers such asglycine-HCl (2.0-3.0), sodium acetate (4.0-5.0), sodium phosphate (6.0-7.0), Tris-HCl (8.0-9.0) at 0.05 M concentration with 1% soluble starch as substrate. The relative activity atdifferent pH values was calculated, taking the maximum activity obtained as 100%.
Temperature optimaα-Amylase activities were determined at a temperature range of 20-70°C (with an interval of10°C) with 1% soluble starch as substrate. The relative activity at different temperatures wascalculated, taking the maximum activity obtained as 100%.
Influence of calcium ions on amylase thermostabilityThe influence of calcium ions at various concentrations on α-amylase activity was studied.The samples were preincubated for 30 min in 50 mM acetate buffer (pH 5.0) at 45°C,supplemented with calcium ion and then enzyme assay was performed with soluble starch assubstrate.
RESULTS AND DISCUSSION
Purification of Little millet α-AmylaseThe present study is an attempt towards purification of α-amylase from germinating littlemillet seeds. The activity of amylase increased from day 0 to day 3 where it exhibited itsmaximum activity. A summary of the purification procedure that was used for the little milletcotyledonary α-amylase is presented in (Table 1). The purification fold and percentage recoverywere monitored by assay of enzyme against soluble starch. As a result of this procedure, aprotein with specific activity of 1001 Units/mg was obtained. The purification fold was 10.15,whereas the recovery was 29%. α-Amylase from malted sorghum has been reported to bepurified 24.7-fold, with a recovery of 17.1%, using 0-40% ammonium sulfate precipitation,ion exchange chromatography on DEAE cellulose and GPC on G-75 (Siva Sai Kumar et al.2005).
Journal of Pharmacy Research Vol.4.Issue 5. May 2011
B.Usha et al. / Journal of Pharmacy Research 2011,4(5),1370-1371
1370-1371
Determination of molecular weightIn this study little millet amylase was purified tohomogeneity, with a molecular mass of 46 kDa asdetermined by the SDS-PAGE (Fig.1). In general, themolecular weight of malt cereal enzymes ranged from42-46 kDa (Greenwood and Milne, 1968).
Determination of Kinetic ConstantsEffect of different substrate concentrations on the ini-tial velocity was calculated and the Km and Vmax forlittle millet α-amylase determined were found to be1.6 mg starch/ml and Vmax 1388 units /min/ mg ofprotein, respectively (Fig. 2). The Km value reportedfor the α-amylases from several other sources are verysimilar (Beers and Duke, 1990 ; Tripathi et al. 2007)and 0.57 mg and 1.3 mg starch/ ml were reported fromwheat (Fahmy et al. 2000).
Fig.1 SDS-PAGE for homogeneity oflittle millet α-amylase. (A) Molecularmass markers; a. 97.4 kDa pyrophosphorylase b; b. 66 kDa bovine serum albu-min; c. 43.0 kDa ovalbumin; d. 29.0 kDacarbonic anhydrase;e. 14.3 kDalysozyme. (B) Purified α-amylase.
pH optimumThe little millet amylase has an acidic pH optimum of 5.0 as do most plant amylases.Increasing or decreasing the pH resulted in decrease in the activity of the enzyme in the pHrange studied (Fig. 3). Similar acidic pH range 4.5 to 6.5 for activity of amylase has beenreported for amylases from finger millet (Nirmala and Muralikrishna, 2003b), shoots andcotyledons of pea (Pisum sativum L.) seedlings (Beers and Duke, 1990) and wheat (Kruger andTkachuk, 1974).
Fig.2 Lineweaver-Burk plot of little millet α-amylase reaction velocities versus starch as sub-strate concentration. One milliliter of the reaction mixture containing 0.05 M sodium acetatebuffer pH 5.0, suitable aliquot of enzyme and concentrations of starch ranging from 0.2 to2.0×10-3 g/ml. Enzyme activity was assayed as per materials and methods.
0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 - o . 6 -0 . 4 - 0 . 2
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Temperature optimumα-Amylase exhibited maximum activity after 20 min incubation at 50°C. An increase intemperature of 10°C compared to the optimum resulted in a 21% decrease of activity in thepresence of substrate soluble starch but further increasing of the temperature to 70°C resultedin further loss of enzyme activity (Fig. 4). This value is comparable to the one reported for ragi
Fig.3 pH optimum of little millet α-amylase. One milliliter of the reaction mixture containing1% starch and suitable aliquot of enzyme and buffers at varying pH 0.05 M glycine-HCl (pH2.0-3.0), sodium acetate (pH 4.0-5.0), sodium phosphate (pH 6.0-7.0), and Tris-HCl (8.0-9.0).Enzyme activity was assayed as per materials and methods.
Fig.4 Temperature optimum of little millet α-amylase. One milliliter of the reaction mixturecontaining 1% starch, 0.05 M sodium acetate buffer pH 5.0, and suitable aliquot of enzyme. Theenzyme activity was measured at varying temperatures ranging from 20-70°C.
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Source of support: University Grants Commission (UGC), New Delhi, India., Conflict of interest: None Declared
α-2 amylase (Nirmala and Muralikrishna, 2003b). A temperature optimum of 60°C wasreported for sorghum α- amylase (Siva Sai Kumar et al. 2005).
REFERENCES 1. Banks, W. and Greenwood, C.T. 1975. Starch and its components. Edinburg University Press:
Edinburg 67-110.2. Beers, E. and Duke, S.1990. Characterization of α-amylase from shoots and cotyledons of pea
(Pisum sativum L.) seedlings. Plant Physiol., 92: 1154-1163.3. Bernfeld, P. 1955. Amylases a and ß. In: Methods in Enzymology, ed. by Colowick S.P. and Kalpan
N. O., Academic Press: New York, 1: 149-158.4. Fahmy, A.S., Mohamed, M.A., Mohamed, T.M. and Mohamed, S.A. 2000. Distribution of α-
amylase in the Gramineae. Partial purification and characterization of α-amylase from Egyptiancultivar of wheat Triticum aestivium. Bull. NRC, Egypt, 25: 61-80.
5. Greenwood, C.T. and Milne, E.A. 1968. Studies on starch degrading enzymes. Part VIII.A com-parison of α-amylase from different sources: Their properties and action pattern. Die Starke, 20:139-150.
6. Janeck, S. and Belaz, S. 1992.α-amylases and their approaches leading to their enhancedstability.FEBS letter, 304: 1-3.
7. Kruger, J.E. and Tkachuk, R. 1969. Wheat α-amylases. I. Isolation. Cereal Chem., 46: 219-235.8. Kruger, J.E. and Tkachuk, R. 1974 Wheat α-amylases. II. Physical characterization. Cereal Chem.
51: 508-529.9. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacte-
riophage T4. Nature, 277: 680-685.10. Lineweaver, H. and Burk, D. 1934. The determination of enzyme dissociation constants. J.Amer.
Chem. Soc., 56: 658-666.11. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the
Folin phenol reagent. J. Biol. Chem., 193: 265-275.12. Murata, T., Akazawa, T. and Furuchi, S. 1968. Enzymic mechanism of starch breakdown in germi-
nating rice seeds. I. An analytical study. Plant Physiol,. 43: 1899-2005.13. Nirmala, M. and Muralikrishna, G. 2003a. Properties of three purified α-amylases from malted
finger millet (Ragi, Eleusine coracana, Indaf-15). Carbohydrate Polymers, 54: 43-50.14. Nirmala, M. and Muralikrishna, G. 2003b. Three α-amylases from malted finger millet (ragi,
Eleusine coracana, Indaf-15)-Purification and partial characterization. Phytochemistry, 62: 21-30.15. Nomura, T., Yasuhiro, K. and Akazawa, T. 1969. Enzymic Mechanism of Starch Breakdown in
Germinating rice Seeds. II. Scutellum as the Site of Sucrose Synthesis. Plant Physiol., 44: 765-769.16. Siva Sai Kumar, R., Sridevi Annapurna, S. and Appu Rao, A.G. 2005. Thermal stability of a-
amylases from malted jowar (Sorghum bicolor) J.agric.Food Chem., 53: 6883-6888.17. Sprinz, C., In Belitz, H.D. 1999. Gross W. (Eds.) Food Chemistry, Springer Verlag, Berlin, Heidel-
berg, New York 92-151.18. Swain, R.R. and Dekker, E.E. 1966a. Seed germination studies.I. Purification and properties of an
α-amylase from the cotyledons of germinating peas. Biochem. Biophys. Acta, 122: 75-86.19. Swain, R.R.and Dekker, E.E. 1966b. Seed germination studies. II. Pathways for starch degradation
in germinating pea seedlings. Biochem. Biophys. Acta, 122: 87-100.20. Tripathi, P., Leggio,L.L., Johanna, J., Mansfeld, R., Ulbrich, H., and Kayastha, A.M.2007. α-
Amylase from mung beans (Vigna radiata) – Correlation of biochemical properties and tertiarystructure by homology modeling, Phytochem., 68: 1623-1631.
21. Vihinen, M. and Mantsilla, P. 1989. Microbial amylolytic enzymes. Critical review in biochemistryand molecular biology, 24: 329-348.
Influence of calcium ions on the activity of amylaseStudies on the effect of calcium ions on α-amylase activity revealed that the presence of calciumenhance the enzyme activity by 19.0%. Cereal amylases are known to be metallo enzymescontaining at least one Ca2+ per molecule (Janeck and Belaz, 1992) and its number may go uptoten (Vihinen and Mantsilla, 1989). Enhancement of amylase activity by Ca2+ ions is based onits ability to interact with negatively charged amino acid residues such as aspartic and glutamicacids, which resulted in stabilization as well as maintenance of enzyme conformation. Inaddition, calcium is known to have a role in substrate binding (Sprinz, 1999).
CONCLUSIONMore recently, many authors have presented good results in developing α-amylase purificationtechniques, which enable applications in pharmaceutical and clinical sectors which requirehigh purity amylases. The use of α-amylase in starch based industries has been prevalent formany decades for commercial production. The search for amylase production from new sourcesis a continuous process. In this study an α-amylase was purified to homogeneity fromgerminating little millets following conventional methods of protein purification.
ACKNOWLEDGEMENTSThis work was supported by a grant from the University Grants Commission (UGC), NewDelhi, India.
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