RESUMO
The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.
Assuntos
Escherichia coli/enzimologia , Penicilina Amidase/metabolismo , Providencia/enzimologia , Saccharomyces cerevisiae/metabolismo , DNA Recombinante/genética , DNA Recombinante/metabolismo , Dimerização , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Bacterianos/genética , Glicosilação , Penicilina Amidase/química , Penicilina Amidase/genética , Saccharomyces cerevisiae/genética , Análise de Sequência , Transformação GenéticaRESUMO
The nucleotide (nt) sequence of the gene encoding penicillin G amidase (PA) of Arthrobacter viscosus strain ATCC 15,294 was determined. The sequence contained an open reading frame of 2406 nt with a G+C content of 37%. The deduced amino-acid sequence shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.
Assuntos
Arthrobacter/enzimologia , Proteínas de Bactérias/genética , Genes Bacterianos/genética , Penicilina Amidase/genética , Sequência de Aminoácidos , Arthrobacter/genética , Proteínas de Bactérias/química , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Dados de Sequência Molecular , Penicilina Amidase/metabolismo , Filogenia , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
The nucleotide sequence of Penicillin G amidase (PA,E.C.3.5.1.11) of Providencia rettgeri was determined. We aligned our P. rettgeri PA with other known Gram negative periplasmically located beta-lactam amidases. The analysis revealed a high homology with other Enterobacteric amidases (60%-65%), while with similar Pseudomonas sp. amidases the homology exceeded 25%. These homologies indicate their common ancestry.
