Purification and Characterization of a CM-Glycinin Digesting Protease from Soybean Seeds

A glycinin-digesting protease was purified from 4-h-imbibed soybean seeds to apparent homogeneity by a combination of DEAE-cellulose, gel filtration and Shodex IEC QA-824 chromatography. The purified soybean protease showed a single band on SDS-PAGE and degraded the carboxymethylated glycinin molecule at neutral or alkaline pH. The glycinin-digesting protease has an apparent molecular mass of 98 kilodaltons as estimated by SDS-PAGE under both reduced and non-reduced conditions. A peptidic substrate for the soybean protease was detected and purified from a tryptic digest of glycinin A₅A₄B₃ complex ; it corresponded to Ala(375)-Lys(381) of the B₃ subunit. It was found that the protease could hydrolyze the peptide bond between Tyr(378) and Asn(389) of that peptide. The soybean protease was significantly inhibited by PMSF, indicating the endopeptidase is a serine protease. Moreover, the glycinin-digesting activity is also susceptible to EDTA and the inactivated enzyme could be restored by the addition of MnCl₂, suggesting that Mn²⁺ is an important factor for the endopeptidase activity. To our knowledge, this is the first report of purification and characterization of a glycinin-digesting protease from soybean seeds.