Journal Article PreJuSER-10928

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

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2010
Elsevier Science New York, NY [u.a.]

Phytochemistry 71, () [10.1016/j.phytochem.2010.06.012]

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Abstract: 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.

Keyword(s): Amino Acid Sequence (MeSH) ; Amino Acids: analysis (MeSH) ; Germination: physiology (MeSH) ; Hydrogen-Ion Concentration (MeSH) ; Plants: enzymology (MeSH) ; Seeds: enzymology (MeSH) ; Seeds: metabolism (MeSH) ; Sequence Homology, Amino Acid (MeSH) ; Soybeans: enzymology (MeSH) ; Soybeans: metabolism (MeSH) ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization (MeSH) ; alpha-Amylases: metabolism (MeSH) ; Amino Acids ; alpha-Amylases ; J ; alpha-Amylase (auto) ; Purification (auto) ; Glycine max (auto) ; MALDI-TOF (auto) ; Sequence homology (auto)


Note: A.K. would like to thank University Grants Commission (UGC, India) for a fellowship and DAAD (Sandwich Model Fellowship) to carry out part of this work in Germany. A.K. also thanks Tobias Rosenkranz (ISB-2, Research Centre, Julich) for his skillful assistance and suggestions.

Contributing Institute(s):
  1. Biotechnologie 1 (IBT-1)
  2. Molekulare Biophysik (ISB-2)
Research Program(s):
  1. Biotechnologie (PBT)

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