Estudio de enzimas de la vía biosintética de cefamicina C en Nocardia Lactamdurans LC411 y en variantes AMY

Actualmente la tesis se encuentra en soporte de microficha nº 82 en la Universidad de León, Servicio de Publicaciones.-- 9 páginas.

[ES]: La biosíntesis de Cefamicina C por Nocardia Lactamdurans tiene lugar a través de una vía biosintética que se inicia con la formación del tripéptido L-Alfa-Aminoadipil) - L-Cisteinil-Dvalina (ACV) por la enzima ACV Sintetasa, seguida de la ciclación de este tripéptido a isopenicilina N por la isopenicilina N sintasa IPNS). A continuación, la isopenicilina N epimerasa (IPNE) convierte isopenicilina N a penicilina N. Esta última enzima es muy hábil. LA IPNS de N Lactamdurans es un monómero, ya que el peso molecular de la forma no desnaturalizada de la enzima, calculado por filtración en gel es idéntico al determinado por SDS-PAGE: 37500 DA. su punto isoeléctrico es 5.5. La IPNE de N Lactamdurans fue purificada 93.2 veces. Es estabilizaca por fosfato de piridoxal y tiene un peso molecular calculado por filtración en gel de 59000. Una banda proteíca de 59000 DA. y PI 5.1 - 5.26, se enriquece en fracciones activas analizadas en SDS-PAGE. Por otra parte, N Lactamdurans var. AMY-, una cepa que no produce cefamicina C, posee IPNS e IPNE antigénicamente similares a las de la cepa AMY+, proteínas que se visualizan en electroforesis bidimensional.

[EN]: The biosynthesis of cephalosporins by Acremonium chrysogenum (syn. Cephphalosporium acremonium) and cephamycins by Nocardia lactamdurans (syn. Streptomyces lactamdurans) takes place through similar biosynthetic pathways (reviewed by Martin & Liras, 1985). In both cases, the first step involves the formation of tripeptide δ-(L- α-aminoadipyl)-L-cysteinl-D-valine (ACV) by the enzyme ACV synthetase, followed by cyclization of ACV to isopenicillin N by isopenicillin N syntase (IPNS). Isopenicillin N epimerase (IPNE), which converts IPN into penicillin N by changing the L-α-aminoadipic acid side chain to the D-configuration was first detected in A. chrysogenum (Sawada et al., 1980). The enzyme was so labile that it could not be characterized further. The IPNS of N. Lactamdurans appears to be a monomer since the molecular weight to the natural (non-denatured) form calculated by gel filtration is identical to the molecular weight of SDS-denatured protein as estimated by PAGE: 37800 Da. Her pl is 5.5. The IPNE was extracted from N. Lactamdurans and purified 93.2-fold. The enzyme was unstable but could be partially stabilized by addition of pyridoxal phosphate. The purified enzyme did not require ATP fer activity in contrast to other amino acid racemases. The enzyme has a molecular weight of 59000 Da as determined by gel filtration. A protein band of 59000 Da and pl 5.1-5.26 was found to be enriched in active fractions which were obtained from N. lactamdurans and analized in SDS-PAGE, the band was also inmunodetected. In adition, N. Lactamdurans var. Amy, a non-producing cephamycin C strain has the inmunodetectables proteins IPNS and IPNE, and they can be visualized in two-dimensional electrophoresis system.

Peer reviewed