alpha-Amylase from germinating soybean (Glycine max) seeds - Purification, characterization and sequential similarity of conserved and catalytic amino acid residues

作者：Kumari Arpana; Singh Vinay Kumar; Fitter Joerg^*; Polen Tino; Kayastha Arvind M

来源：Phytochemistry, 2010, 71(14-15): 1657-1666.

DOI：10.1016/j.phytochem.2010.06.012

• alpha-Amylase
• Purification
• Glycine max
• MALDI-TOF
• Sequence homology

Starch hydrolyzing 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 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.

• 出版日期2010-10