Penicillin Substrate Research Articles

Vector Machines Regression Applied in Penicillin Fermentation Process Control

According to the characteristics of biochemical processes, this paper studied the small samples of the data process. Small amount of data can be quickly measured variable modeling. Introduces the support vector machine (SVM) modeling as small sample theory, the least squares support vector machine (LS-SVM) algorithm based on improved SVM applied to the typical fermented penicillin biochemical processes in the past. Online prediction model only off-line testing of important process variables based on the simulation results simulation platform show that the method is only by learning the few batches of sample data. Establish a penicillin product concentration, cell concentration and substrate concentration. This paper analyzes the characteristics of the penicillin fermentation process, and the use of SVM method for modeling, give potency relationship between the factors and its influence. Through experiments, the influence of SVM model parameters to adjust performance. Through a variety of models from the established field data can be found, SVM is better than ANN modeling.

Soil nematode community changes associated with compost amendments

Abstract Five composts (C1: fresh olive pomace, straw, chicken manure, urea; C2: fresh olive pomace, lettuce residues, cow manure, straw, sawdust; C3: sewage sludge, municipal green residues; C4: grass, leaves, tree branches, soil; C5: by-product from penicillin production (mycelium), straw and sawdust) were tested in a pot experiment to investigate their short-term effect on the nematode community of a grassland soil. Composts were mixed with soil at the rates of 10, 25, 50 and 100 g (kg soil)–1 and barley was sown in each potting mixture after a 2-month decomposition period. Nematodes were extracted from each pot 5 months after barley sowing, identified at genus level and grouped into bacterial, fungal and root fungal feeders, predators, omnivores and plant parasites. Bacterial feeders, predators and omnivores were favoured by amendments with composted penicillin substrate and partly by compost from olive pomace, whereas composts from green wastes and sewage sludge suppressed these trophic groups except for predators. All compost treatments suppressed fungal feeders as well as significantly reducing the density of plant-parasitic nematodes compared with non-amended soil. The highest suppressiveness on plant-parasitic nematodes was found for the composts from urban green residues, penicillin substrate and olive pomace plus cow manure. Suppressive effect was hypothesised to be related to ammoniacal nitrogen content of the composts. Composts from fungal mycelium or olive pomace seem to be the most suitable for application in nematode management strategies due to their low impact on beneficial trophic groups.

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

Study of the oxidative half‐reaction catalyzed by a non‐heme ferrous catalytic center by means of structural and computational methodologies

AbstractDeacetoxycephalosporin C synthase (DAOCS) is a mononuclear ferrous enzyme that catalyzes the expansion of the five‐membered thiazolidine ring of the penicillin nucleus into the six‐membered dihydrothiazine ring of the cephalosporins. In the first half‐reaction with dioxygen and 2‐oxoglutarate, a reactive iron–oxygen species is produced that can subsequently react with the penicillin substrate to yield the cephalosporin. We describe quantum mechanical calculations of the first part of the reaction based on the high‐resolution structures of the active site of DAOCS and its complexes with ligands. These studies are aimed at understanding how the reactive species can be produced and contained in the active site of the enzyme. The results demonstrate the priming of the active site by the co‐substrate for oxygen binding and hint to the presence of a stable iron–peroxo intermediate in equilibrium with a more reactive ferryl species and the formation of CO2 as a leaving group by decarboxylation of 2‐oxoglutarate. A conclusion from these studies is that substitution of CO2 by the penicillin substrate triggers the oxidation reaction in a booby‐trap‐like mechanism. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

Conformational Flexibility of the C Terminus with Implications for Substrate Binding and Catalysis Revealed in a New Crystal Form of Deacetoxycephalosporin C Synthase

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268-299) is rotated by about 16 degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.

Using Steric Hindrance to Design New Inhibitors of Class C β-Lactamases

β-lactamases confer resistance to β-lactam antibiotics such as penicillins and cephalosporins. However, β-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of β-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R 1 side chain of β-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective β-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of the penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.

Contrasting fates for 6-α-methylpenicillin N upon oxidation by deacetoxycephalosporin C synthase (DAOCS) and deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS)

6-α-Methylpenicillin N was synthesised via known routes from 6-aminopenicillanic acid, and tested as a substrate for recombinant DAOCS and DAOC/DACS. Incubation with DAOCS resulted in conversion of 2-oxoglutarate without oxidation of the penicillin substrate (‘uncoupled turnover’). Incubation with DAOC/DACS resulted in oxidation to the cephem aldehyde. This is the first example of substrate-induced ‘uncoupled turnover’, which has been proposed to be an editing mechanism for these enzymes.

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the ΔR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 Å) and the ΔR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 Å). The latter may mimic a product complex, and supports proposals for a metal-bound CO 2 intermediate during catalysis.

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.

Overexpression and Biosynthetic Deuterium Enrichment of TEM-1 β-Lactamase for Structural Characterization by Magnetic Resonance Methods

An expression system has been developed that allows high levels of production of TEM-1 β-lactamase with ease of biosynthetic incorporation of nuclear isotopes. The gene for mature TEM-1 β-lactamase fused to the leader sequence of the ompA protein was subcloned into the pET-24a(+) vector by introduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induction at 25°C supported by LB medium supplemented with osmolytes (300 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-type TEM-1 β-lactamase was recovered at a level of 140 mg/L. The production level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the efficiency of biosynthetic incorporation of covalent deuterium showed maximal (90%) incorporation with minimal medium containing 99% 2H2O and sodium d3-acetate (99 atom% 2H). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced. The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labeled penicillin substrate and hydrogens on the penicillin moiety in a cryokinetically isolated acylenzyme reaction intermediate because of the decrease in overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies requiring large quantities of protein in isotopically enriched forms.

Perspectives of oral cephalosporins in upper respiratory tract infections

Perspectives of oral cephalosporins in upper respiratory tract infections

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans.

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.

Reversible deactivation of beta-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex.

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].

A sensitive procedure for screening microorganisms for the presence of penicillin amidase.

A procedure is described for screening bacteria for the presence of penicillin amidase. Cells, grown in the presence of phenylacetic acid, are incubated with phenoxymethylpenicillin (type I), benzylpenicillin (type II) or ampicillin and the 6-aminopenicillanic acid formed is detected and quantitatively estimated by its strong reaction with fluorescamine at pH 4. There is no requirement for separation of the penicillin substrate from the product but when alpha-aminobenzylpenicillin derivatives are used as enzyme substrates the amount of 6-aminopenicillanic acid formed must be determined by calculation. The procedure allowed positive and reliable identification of penicillin amidases in six organisms known to produce the enzyme and indicated that some of these enzymes had different properties in reactivity towards alpha-aminobenzylpenicillin derivatives.

54a] Penicillin acylase (assay)

This chapter presents the assay procedures of penicillin acylase. The most specific method of assaying these enzymes involves the determination of the rate of formation of 6-APA from a readily available penicillin, such as benzylpenicillin or phenoxymethylpenicillin. The method of choice involves the hydroxylamine assay of 6-APA after removal of residual penicillin substrate by solvent extraction at pH 2. Alternatively, the 6-APA may be assayed in the presence of residual substrate by the p-dimethylaminobenzaldehyde method. The method lends itself to automation, but the β-lactam ring opened form of 6-APA also reacts. A non-automated version has also been described. A less specific but readily performed method involves the pH-stat titration of carboxylic acid side chain liberated from the substrate penicillin. This method is suitable for following large-scale reactions or obtaining kinetic data if no β-lactamase enzyme is present to liberate penicilloic acid.