Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.     

基因工程在β-内酰胺药物研制上的应用

本文综合国内外有关文献介绍了青酶素Ｇ酰化酶的改造,异青霉素Ｎ合成酶和头孢菌素Ｃ水解酶的克隆,以及细菌血红蛋白基因克隆进丝状真菌。比较了化学法和两步酶法转化ｃｐｃ（头孢菌素Ｃ）成７－ＡＣＡ的优缺点。还讨论了基因工程β－内酰胺抗生素研制的发展方向。     

Cloning of a beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enterobacteriaceae

Abstract In this paper we describe the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that causes β-lactam resistance in both Escherichia coli HB101 and the parental strain E. cloacae 2249-1.
The increase in minimum inhibitory concentration (MIC) of the β-lactam antibiotics studied was not the result of enhanced β-lactamase production, but of a decrease in the concentration of the pore proteins OmpF and OmpC in E. coli and of a 37-kDa membrane protein in E. cloacae . The results obtained thus far indicate that we have cloned a gene encoding a 20 kDa polypeptide that is involved in the regulation of outer membrane protein synthesis.     

Deacetylation ofN-acetylthienamycin to thienamycin by a cell-free extract ofStreptomyces cattleya,the thienamycin producer

Summary A cell-free extract from the thienamycin producer,Streptomyces cattleya, has been found to deacetylate the co-product,N-acetylthienamycin. The pH optimum of the reaction is 7.5. Due to the lability ofN-acetylthienamycin, we used thed andl forms of the synthetic substrateN-chloroacetylvaline. We found that the enzyme is anl-deacetylase, has a molecular weight of 58 000, is stable up to 40°C, acts optimally at 45°C, is stable at pH 5–8, is not activated by divalent metal ions and is inhibited by Hg⁺⁺, Cu⁺⁺ andp-chloromercuribenzoate. This is the first report of an extract from a carbapenem producer which carries out the deacetylation ofN-acetylthienamycin, suggesting that the acetylated derivative is a precursor of thienamycin.Abbreviations THM thienamycin - N-AcTHM N-acetylthienamycin - CFE cell-free extract - N-Cl-Ac-l-Val N-chloroacetyl-l-valine - N-Cl-Ac-d-Val N-chloroacetyl-d-valine     

The isolation and characterization of antibiotic biosynthesis genes

Antibiotic biosynthesis pathways are found in a broad range of Gram positive prokaryotes, a smaller range of Gram negative prokaryotes and a limited range of eukaryotes. A variety of techniques can be used to identify the genes involved in the biosynthesis of these compounds ranging from genetic complementation and interspecific gene transfer to polymerase chain reaction amplification and transposon mutagenesis. The dissection of these cloned pathways and the understanding of their structure and regulation has led to insights into the structure and function of antibiotic biosynthesis genes. With new knowledge of the structural similarities and relationships between related antibiotic biosynthesis pathways, the possibility of directed manipulation of specific genes to allow synthesis of novel antibiotics is now possible.     

Microbial fuel cell-based diagnostic platform to reveal antibacterial effect of beta-lactam antibiotics

Beta-lactam antibiotics comprise the largest group of antibacterial agents. Due to their bactericidal properties and limited toxicity to humans they are preferred in antimicrobial therapy. In most cases, therapy is empiric since susceptibility testing in diagnostic laboratories takes a relatively long time. This paper presents a novel platform that is based on the microbial fuel cell (MFC) technology and focuses on the early antibiogram determination of isolates against a series of beta-lactam antibiotics. An advantage of the system is that it can be integrated into traditional microbiological diagnostic laboratory procedures. Tested bacterium suspensions are uploaded into the anodic chambers of each miniaturized MFC unit integrated into a panel system, containing different antibiotic solutions. Electronic signals gained in each MFC unit are continuously monitored and are proportional to the metabolic activity of the presenting test bacterium. Using this method, antibiotic susceptibility can be evaluated in 2–4 h after inoculation. Hereby we demonstrate the efficacy of the platform in antibiogram determination by testing the susceptibilities of Escherichia coli strain ATCC 25922 and Staphylococcus aureus strain ATCC 29213 against 10 beta-lactam antibiotics (penicillin, ampicillin, ticarcillin, cefazolin, cefuroxime, cefoperazone, cefepime, cefoxitin, cefaclor, imipenem). This paper also presents the construction of the background instrumentation and the panel system into which a printed circuit board (PCB) based electrode was integrated. Our results suggest that MFC based biosensors have the potential to be used in diagnostics for antibiogram determination.