Characterization of Thermostable α-Amylase from Germinating Cowitch (Mucuna pruriens (L.) DC.) Seeds
Abstract
Purification of α-amylase from germinating cowitch (Mucuna pruriens (L.) DC.) seeds (MpAmy) was carried out using two steps of 35-65% (w/v) Ammonium sulphate fractionation and β-cyclodextrin Sepharose 6B affinity chromatography. The enzyme was purified 216.4-fold with a final specific activity of 91.0 U/mg. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis exhibited a molecular weight of MpAmy at 58 kDa which corresponded with amylase activity by Zymographic method. Characterization of MpAmy showed high activity at the pH of 5.0-6.0 and the high temperature of 50.0-90.0 ºC. The optimum pH and temperature for the enzyme were at pH 5.0 and 60.0 ºC, respectively. Furthermore, MpAmy was found stable at a wide range of pH 5.0 to 7.0 pH and the temperature between 30 ºC to 90 ºC. The Michalis constant (Km) and Maximum velocity (Vmax) for hydrolysis of starch were 2.1 mg/mL and 0.2 µmoles/min/mL, respectively. The MpAmy showed the highest specific activity toward starch followed by amylopectin and glycogen as their natural substrates. The enzyme catalyzed Ethylidene-pNP-G7at 280.8 nmol/min/mL, indicating it was an alpha amylase. The results from enzyme characterization indicated that MpAmy is the Thermostable α-amylase. Therefore, MpAmy has the potential for applying in different industries, especially the starch industry. Keywords : α-amylase ; thermostable ; characterization ; purification ; Mucuna pruriens (L.) DC.References
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Tripathi, P., Lo Leggio, L., Mansfeld, J., Ulbrich-Hofmann, R., & Kayastha, A.M. (2007). α-Amylase from mung
beans (Vigna radiata-Correlation of biochemical properties and tertiary structure by homology modelling. Phytochemistry, 68, 1623–1631.
Vretblad, P. (1974). Immobilization of ligands for biospecific affinity chromatographic via their hydroxyl group, the
cyclohexa amylose-β-amylase system. FEBS Lett, 47, 86–89.
Wisessing, A., Engkagul, A., Wongpiyasatid, A., & Choowongkomon, K. (2010). Biochemical characterization of
the α-amylase inhibitor in mung beans and its application in inhibiting the growth of Callosobruchus
maculatus. J Agric Food Chem, 58, 2131–2137.
Wu, X., Wang, Y., Tong, B., Chen, X., & Chen, J. (2018). Purification and biochemical characterization of a
thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4–423. Int J Biol Macromol, 109, 329–337.
Xie, Z., Zhang, Z.L., Hanzlik, S., Cook E., & Shen, Q.J. (2007). Salicylic acid inhibits gibberellin – induced alpha-
amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol Biol, 64, 293–303.
Bradford, M.M. (1977). A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal Biochem, 72, 248–254.
Farooq, M.A., Ali, A., Hassan, A., Tahir, H.M., Mumtaz, S., Mumtaz, S. (2012). Biosynthesis and industrial
applications of α-Amylase: a review. Arch Microbiol, 203, 1281-1292.
Hamilton, L.M., Kelly, C.T., & Fogarty, W.M. (2000). Review: cyclodextrins and their interaction with amylolytic
enzymes. Enzyme Microb Technol, 26, 561–567.
Jegannathan, K.R., & Nielsen, P.H. (2013). Environmental assessment of enzyme use in industrial production – a
literature review. J Clean Prod, 42, 228–240.
Kumari, A., Singh, V.K., Fitter, J., Polen, T., & Kayastha, A.M. (2010). α-Amylase from germinating soybean
(Glycine max) seeds-purification, characterization and sequential similarity of conserved and catalytic amino acid residues. Phytochemistry, 71, 1657–1666.
Laemmli, U.K. (1970) Cleavage of structural protein during the assembly of head of bacteriophage T4. Nature, 227, 680–685.
Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 31, 426–428.
Muralikrishna, G., & Nirmala, M. (2005). Cereal α-amylases-an overview. Carbohydr Polym, 60, 163–173.
Noman, A.S.M., Hoque, M.A., Sen, P.K., & Karim, M.R. (2006). Purification and some properties of α-amylase from
post-harvest Pachyrhizus erosus L. tuber. Food Chem, 99, 444–449.
Paul, J.S., Gupta, N., Beliya, E., Tiwari, S., Jadhav, S.K. (2021). Aspects and recent trends in microbial α-amylase:
a review. Appl Biochem Biotechnol, 193, 2649-2698.
Posoongnoen, S., Ubonbal, R., Thummasirirak, S., Daduang, J., Minami, H., Yamamoto, K., & Daduang, S. (2015).
α-Amylase from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong)-nucleotide sequence analysis, cloning and expression. Plant Biotechnol, 32, 1–10.
Posoongnoen, S., & Thummavongsa, T. (2020). Purification and characterization of thermostable α-amylase from
germinating Sword bean (Canavalia gladiate (Jacq.) DC.) seed. Plant Biotechnol, 37, 31–38.
Posoongnoen, S., Ubonbal, R., Klaynongsruang, S., Daduang, J., Roytrakul, S., & Daduang, S. (2021).
Characterization and molecular cloning of secreted α-amylase with dominant activity from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong). Rev Bras Frutic, Jaboticabal, 43, 1–19.
Singh, K., & Kayastha, A.M. (2014). α-amylase from wheat (Triticum aestivum) seeds: its purification, biochemical
attributes and active site studies. Food Chem, 162, 1–9.
Singh, K., Ahmad, F., Singh, V.K., Kayastha, K., & Kayastha, A.M. (2017). Purification, biochemical
characterization and Insilico modelling of α-amylase from Vicia faba. J Mol Liq, 234, 133–141.
Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K.M., Soccol, C.R., & Pandey, A. (2006). α-amylases from
microbial sources – An overview on recent developments. Food Technol Biotechnol, 44, 173–184.
Sundarram, A., & Murthy, T.P.K. (2014). α-Amylase production and applications: A review. J Appl Environ
Microbiol, 2, 166–175.
Tripathi, P., Lo Leggio, L., Mansfeld, J., Ulbrich-Hofmann, R., & Kayastha, A.M. (2007). α-Amylase from mung
beans (Vigna radiata-Correlation of biochemical properties and tertiary structure by homology modelling. Phytochemistry, 68, 1623–1631.
Vretblad, P. (1974). Immobilization of ligands for biospecific affinity chromatographic via their hydroxyl group, the
cyclohexa amylose-β-amylase system. FEBS Lett, 47, 86–89.
Wisessing, A., Engkagul, A., Wongpiyasatid, A., & Choowongkomon, K. (2010). Biochemical characterization of
the α-amylase inhibitor in mung beans and its application in inhibiting the growth of Callosobruchus
maculatus. J Agric Food Chem, 58, 2131–2137.
Wu, X., Wang, Y., Tong, B., Chen, X., & Chen, J. (2018). Purification and biochemical characterization of a
thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4–423. Int J Biol Macromol, 109, 329–337.
Xie, Z., Zhang, Z.L., Hanzlik, S., Cook E., & Shen, Q.J. (2007). Salicylic acid inhibits gibberellin – induced alpha-
amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol Biol, 64, 293–303.
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2023-05-11
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