Sunday, August 10, 2008
P15
Purification and characterization of a novel rhizopuspepsin from Rhizopus oryzae
Chun-Chang Chen, Yen-Ching Cho, Chien-Chen Lai, and Wen-Hwei Hsu. Institute of Molecular Biology, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung, Taiwan
A novel aspartic protease, designated as rhizopuspepsin 6, was purified from culture broth of the R. oryzae NBRC 4749 by HiTrap Q FF column with a recovery of 44%. SDS-PAGE analysis showed that this protein had an apparent molecular mass of approximately 37 kDa. LC-MS/MS and automated Edman degradation analyses indicated that rhizopuspepsin 6 is corresponding to the hypothetical protein (RO3G _12822.1) in the R. oryzae genome. Sequence comparison between cDNA and genomic DNA revealed two introns in rhizopuspepsin 6 gene, while other reported rhizopuspepsin genes contained only one intron. The amino acid sequence of rhizopuspepsin 6 shared 66-72 % identity with other existing rhizopuspepsins. Phylogenetic tree analysis indicated that the rhizopuspepsin 6 is a new member of rhizopuspepsin, which diverges into a separate clade from already existing rhizopuspepsins. The optimal pH and temperature of the enzyme were pH 3.0 and 50°C , respectively. Kinetic assay using chromogenic peptides, Lys-Pro-Ala-Lys-Xaa-p-nitrophenylalanine-Arg-Leu, as substrates indicated that rhizopuspepsin 6 had similar cleavage specificity to other rhizopuspepsins. This enzyme preferentially cleaved at hydrophobic and basic amino acid in P1 position, but had reduced activity for hydrophobic amino acid containing β-branched side chain.
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