Identification of an ethanol-soluble protein as beta-amylase and its purification from soybean seeds.
In the 60% ethanol extract of soybean seeds, a prominent protein band was visible after polyacrylamide gel electrophoresis, which had a molecular weight of about 55 x 10(3) M(r). This protein was purified to homogeneity by buffered ethanol extraction and preparatory gel electrophoresis. Since the N-terminus was apparently blocked, the protein was cleaved with cyanogen bromide and the largest fragment was isolated and a partial sequence determined. The sequence of the 27 N-terminal amino acid residues matched a published soybean beta-amylase peptide sequence. In addition, the purified protein had a high specific activity for beta-amylase and was not a glycoprotein. Furthermore, the partial sequence (106 nucleotides) of a cDNA clone, isolated from a soybean seed cDNA library by antibody screening, matched the cDNA sequence of soybean beta-amylase except for one base. Therefore, the ethanol-soluble protein was identified as beta-amylase. The enzyme was purified to homogeneity using a two-step purification procedure with a yield of over 50%.
Ren
H
H
Section of Plant Biology, Cornell University, Ithaca, NY 14853.
Madison
J T
JT
Thompson
J F
JF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D000431:Ethanol; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D013025:Glycine max / Q000737:chemistry*; D001614:beta-Amylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D000596:Amino Acids; D010940:Plant Proteins; D000431:Ethanol; D004247:DNA; D001614:beta-Amylase