Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2

• Published in: Journal of bioscience and bioengineering 2011-11, Vol.112 (5), p.441-446
• Main Authors: Rao, Shengbin, Mizutani, Osamu, Hirano, Takuya, Masaki, Kazuo, Iefuji, Haruyuki
• Format: Article
• Language: English
• Subjects: alpha-casein; Amino Acid Sequence; amino acid sequences; amino acids; Animals; Aspartic Acid Proteases - chemistry; Aspartic Acid Proteases - isolation & purification; Aspartic Acid Proteases - metabolism; Basidiomycetous; beta-casein; Biological and medical sciences; Biotechnology; Caseins - metabolism; chromatography; Cloning; Cloning, Molecular; complementary DNA; Cryptococcus; Cryptococcus (Tremellomycetes); Cryptococcus - enzymology; Cryptococcus - metabolism; Electrophoresis, Polyacrylamide Gel; enzyme activity; Extracellular; Fundamental and applied biological sciences. Psychology; genes; hemoglobin; Humans; hydrolysis; insulin; kappa-casein; milk clotting; Molecular Sequence Data; molecular weight; open reading frames; pepsin; Pepsin A - metabolism; polyacrylamide gel electrophoresis; Protease; protein hydrolysates; proteolysis; Purification; Saccharomyces cerevisiae - metabolism; sequence analysis; Substrate Specificity; swine; Swine - metabolism; yeasts
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2011.07.013

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-NH 2 and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH 2. 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.