Chen Chun-Chang, Cho Yen-Ching, Lai Chien-Chen, Hsu Wen-Hwei
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
J Agric Food Chem. 2009 Aug 12;57(15):6742-7. doi: 10.1021/jf8040337.
A secretory aspartic protease (also termed as rhizopuspepsin) was purified from Rhizopus oryzae NBRC 4749 by ion exchange chromatography with a yield of 45%. The enzyme was a nonglycoprotein with a molecular mass of 37 kDa as determined by SDS-PAGE analysis. N-terminal sequence and LC-MS/MS analyses revealed that this rhizopuspepsin corresponded to the hypothetical protein RO3G_12822.1 in the R. oryzae genome database. Comparison of genomic and cDNA genes demonstrated that the rhizopuspepsin contained two introns, whereas only one intron was reported in other rhizopuspepsin genes. Phylogenetic analysis also indicated that this rhizopuspepsin was distinct from other rhizopuspepsins. The temperature and pH optima for the purified rhizopuspepsin were 50 degrees C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 degrees C. The enzyme preferentially cleaved the peptides with hydrophobic and basic amino acids in the P1 site but had no activity for the Glu, Pro, Trp, and aliphatic amino acids containing the beta-branch side chain.
通过离子交换色谱法从米根霉NBRC 4749中纯化出一种分泌型天冬氨酸蛋白酶（也称为根霉胃蛋白酶），产率为45%。经SDS-PAGE分析测定，该酶为非糖蛋白，分子量为37 kDa。N端序列和LC-MS/MS分析表明，这种根霉胃蛋白酶与米根霉基因组数据库中的假定蛋白RO3G_12822.1相对应。基因组和cDNA基因的比较表明，根霉胃蛋白酶含有两个内含子，而其他根霉胃蛋白酶基因仅报道有一个内含子。系统发育分析还表明，这种根霉胃蛋白酶与其他根霉胃蛋白酶不同。纯化的根霉胃蛋白酶的最适温度和pH分别为50℃和pH 3.0，在40℃下观察到约3.5小时的半衰期。该酶优先切割P1位点含有疏水和碱性氨基酸的肽段，但对含有β-分支侧链的Glu、Pro、Trp和脂肪族氨基酸无活性。
