title: Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2.
abstract: An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂ and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.
authors.affiliation: Graduate School of Biosphere Science Hiroshima University, Kagamiyama 1-4-4, Higashihiroshima, Hiroshima 739-8528, Japan.
authors.forename: Shengbin
authors.initials: S
authors.lastname: Rao
authors.forename: Osamu
authors.initials: O
authors.lastname: Mizutani
authors.forename: Takuya
authors.initials: T
authors.lastname: Hirano
authors.forename: Kazuo
authors.initials: K
authors.lastname: Masaki
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Iefuji
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine / Q000378:metabolism
chemical_list: D002364:Caseins; C026018:azocasein; D057055:Aspartic Acid Proteases; D010434:Pepsin A