RESUMO
Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.
Assuntos
Carboxiliases , Genética , Clonagem Molecular , Escherichia coli , Metabolismo , Vetores Genéticos , Huperzia , Genética , Lisina , Metabolismo , Proteínas de Plantas , Genética , Proteínas Recombinantes , GenéticaRESUMO
A gas chromatographic analysis method was employed to determine the cellular fatty acids (CFAs)profiles of the spores of some aerobic endospore4orming bacilli. Purified spore cultures of 51 experimentas strains were processed to acquire whole cell fatty acids methyl esters for the subsequent gas chromatographic analysis,and the corresponding vegetative cells were set as control. The reproducibility study of spore fatty acids revealed that,the fatty acids components of spores were stable enough for research purpose,provided under standardized experimentas procedure. The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains. The fatty acids analysis of spores seemed to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.
RESUMO
A gas chromatographic analysis method was employed to determine the cellular fatty acids (CFAs)profiles of the spores of some aerobic endospore forming bacilli.Purified spore cultures of 51 experimentas strains were processed to acquire whole cell fatty acids methyl esters for the subsequent gas chromatographic analysis,and the corresponding vegetative cells were set as control.The reproducibility study of spore fatty acids revealed that,the fatty acids components of spores were stable enough for research purpose,provided under standardized experimentas procedure.The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains.The fatty acids analysis of spores seemed to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.