Alpha-amylase from germinating soybean (Glycine max) seeds--purification, characterization and sequential similarity of conserved and catalytic amino acid residues.
Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS-PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI-TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25-85 degrees C. Apparent Michaelis constant (K(m)((app))) for starch was 0.71 mg/mL and turnover number (k(cat)) was 280 s(-1) in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 degrees C showed first-order kinetics with rate constant (k) equal to 0.0063 min(-1). Soybean alpha-amylase showed high specificity for its primary substrate starch. High similarity of soybean alpha-amylase with known amylases suggests that this alpha-amylase belongs to glycosyl hydrolase family 13. Cereal alpha-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant alpha-amylase. Soybean can be used as commercially viable source of alpha-amylase for various industrial applications.
Kumari
Arpana
A
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India.
Singh
Vinay Kumar
VK
Fitter
Jörg
J
Polen
Tino
T
Kayastha
Arvind M
AM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D018525:Germination / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D010944:Plants / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000516:alpha-Amylases / Q000378:metabolism*
D000596:Amino Acids; D000516:alpha-Amylases