Characterization of novel cysteine proteases from germinating cotyledons of soybean [Glycine max (L.) Merrill]

The enzymatic properties of novel cysteine proteases D3-alpha and beta whic h were purified from germinating soybean cotyledons were investigated. The enzyme activities were exhibited in the presence of a thiol reagent, such a s 2-mercaptoethanol, and apparently inhibited by E-64, a cysteine protease inhibitor. Hydrolytic activities toward carbobenzoxy-Phe-Arg-MCA were detec ted at a pH above 4.0, The optimum temperature for activities was about 40 degrees C. The isoelectric point of D3-alpha and beta was 4.4 and 4.7, resp ectively. The molecular mass of D3-alpha and beta, measured by MALDI/TOF ma ss spectrometry, was 26,178 and 26,429 Da, respectively. The substrate spec ificities of the enzymes were examined using peptide-MCAs and peptides, and cathepsin L-like broad specificity was observed at pH 4.0, These results d emonstrated that these enzymes are cysteine endopeptidases [EC 3.4.22.-] li ke papain [EC 3.4.22.2].