Purification and Characterization of a New Rhizopuspepsin from Rhizopus oryzae NBRC 4749 and Expression of Staphylococcus aureus BCRC 15205 Glutamyl Endopeptidase Gene in Bacillus subtilis

• Main Authors: Chun-Chang Chen, 陳俊彰
• Other Authors: 許文輝
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/23816018774557892632

博士 === 國立中興大學 === 分子生物學研究所 === 97 === 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 non-glycoprotein 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 gene 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 °C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 °C. This newly identified rhizopuspepsin preferentially cleaved the peptides with hydrophobic and positively charged amino acids in P1 site, but had no activity for the Glu, Pro, Trp, and aliphatic amino acids containing β-branch side chain. In contrast to P1 site, the P1’ site did not accommodate positively charged amino acids for the enzyme. But, it preferentially cleaved the peptides with aliphatic amino acids containing β-branch side chain in P1’ site. In this study, we also noticed that the rhizopuspepsin displayed aberrant mobility in SDS-PAGE analysis. Staphylococcus aureus glutamyl endopeptidase gene (sspA) was cloned into expressing vector, and expressed in Bacillus subtilis. Active glutamyl endopeptidase could be obtained from the wild-type B. subtilis DB2 expressing the sspA gene, whereas the enzyme remained in the precursor form from protease-deficient B. subtilis DB104 (nprE- aprE-). However, 6His tag was also deleted from the mature glutamyl endopeptidase produced by B. subtilis DB2, leading to difficulty in purification of recombinant glutamyl endopeptidase from culture supernatant. We also found that glutamyl endopeptidase variant with N68E mutation could form mature protein by autocatalysis.