No abstract

Proteolysis of soy protein isolates (SPI) was investigated by using pepsin with a pH of 1.5 to 4.0 at 37°C and papain at a temperature of 37°C to 80°C with pH 7.0. The glycinin fraction in native SPI was selectively hydrolyzed by pepsin in the pH 1.5 to 2.5 range. On the other hand, the ␤ ␤ ␤ ␤ ␤-conglycinin fraction in native SPI was selectively hydrolyzed by papain at 70°C. This selective proteolysis would be significantly correlated with the denaturation of glycinin and ␤ ␤ ␤ ␤ ␤-conglycinin in SPI. A protocol for preparing hydrolysates selectively enriched with glycinin or ␤ ␤ ␤ ␤ ␤-conglycinin was proposed.

The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34,000 (B-chain)]. Lipase II has a molecular weight of 30,000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6.0-6.5 and a temperature optimum of 35 degrees C, while, for Lipase II these values were pH 6.0 and 40 degrees C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.

The crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 A. The structure was refined to an R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical alpha/beta hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an alpha-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface.