RESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Rhizopus/enzimologia , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Íntrons , Dados de Sequência Molecular , Filogenia , Rhizopus/química , Rhizopus/classificação , Rhizopus/genética , Especificidade por SubstratoRESUMO
A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6beta position of compactin was developed. Agar plates containing different carbon sources and 500 microg compactin mL(-1) were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program.
Assuntos
Bactérias/metabolismo , Lovastatina/análogos & derivados , Testes de Sensibilidade Microbiana , Pravastatina/biossíntese , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Hidroxilação , Lovastatina/metabolismo , Lovastatina/farmacologiaRESUMO
Genes involved in fatty acid degradation (fad) were isolated from Pseudonocardia autotrophica BBRC12444. Six open reading frames and a bi-directional promoter region were identified by DNA sequence analyses and primer extension. The fad gene cluster included five ORFs, designated fadA, fadB, fadR, fadC, and fadD. Base on their amino acid sequence identity, the gene products were identified as acyl-CoA ligase (FadA), enoyl-CoA hydratase (FadB), transcriptional regulator (FadR), cytochrome P450 monooxygenase (FadC), and ferredoxin (FadD). Regulatory protein, FadR, could bind to an operator sequence located in the divergent promoter region between fadR and fadC genes, implicating the control of fatty acid degradation. The real-time quantitative PCR assays revealed that the expression of the fadA, fadB, fadR, and fadC genes was induced by long chain fatty acids and repressed by glucose. All results demonstrated that the fad gene cluster participated in the pathway of the fatty acid catabolism. This is the first bacterial fad gene cluster to be reported.
