title: Purification and characterization of a new Rhizopuspepsin from Rhizopus oryzae NBRC 4749.
abstract: 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.
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Chun-Chang
authors.initials: CC
authors.lastname: Chen
authors.forename: Yen-Ching
authors.initials: YC
authors.lastname: Cho
authors.forename: Chien-Chen
authors.initials: CC
authors.lastname: Lai
authors.forename: Wen-Hwei
authors.initials: WH
authors.lastname: Hsu
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007438:Introns; D008969:Molecular Sequence Data; D010802:Phylogeny; D012233:Rhizopus / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D016282:Aspartic Acid Endopeptidases; C035794:rhizopuspepsin