Inhibitory mechanism of Penicillin V on mushroom tyrosinase.

Penicillin V is a bacteriolytic ß-lactam antibiotic drug. In the present work, we investigated the inhibitory effect of Penicillin V on the activity of mushroom tyrosinase for the first time. The molecular mechanism for the inhibition of tyrosinase by Penicillin V was investigated by means of kinetics analysis, fluorescence quenching and molecular docking techniques. The results showed that Penicillin V could inhibit both monophenolase and diphenolase activities with IC50 of 16.6 ± 0.5 and 11.0 ± 0.2 mmol/L, respectively. The inhibitory type of Penicillin V on mushroom was mixed type, and the values of KI and KIS were 13.46 and 17.26 mmol/L, respectively. The fluorescence quenching and molecular docking showed that Penicillin V could form static interaction near the catalytic pocket of the enzyme to hinder the transportation of substrate to the active site, as well as reduce the copper plasticity for catalysis. Our results contributed to the usage of Penicillin V as a novel tyrosinase inhibitor with dual effect in field of antimicrobial and food preservation and could also provide guidance for the design of novel tyrosinase inhibitors.

Molecular recognition and binding of beta-lactamase II from Bacillus cereus with penicillin V and sulbactam by spectroscopic analysis in combination with docking simulation.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 106 and 3.98 × 105 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics.

An improved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family.

Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Gram-positive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria. Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms.

Research on degradation of penicillins in milk by ß-lactamase using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

The degradation of penicillin G, penicillin V, and ampicillin in milk in the presence of ß-lactamase was investigated by ultra-performance liquid chromatography coupled with electrospray ionization-time-of-flight mass spectrometry. Degradation products of the 3 penicillins in milk were identified based on the fact that the metabolites or degradation products contain a substructure of penicillin, and their degradation pathways in acidic milk in presence of ß-lactamase were developed. The influence of factors on the degradation was investigated, including ß-lactamase dosage, temperature, time, and acidity. The ratio of the 2 degradation products (penicilloic acid and penilloic acid) is different at different temperatures and pH. Penicilloic acid was the dominant species obtained at pH 6 under 40°C, but, being unstable, it could not be used as a standard for accurate analysis of penicilloic acid, and also could not be used as target for detection of penicillins in milk. Penilloic acid was the dominant species obtained at pH 2 above 40°C; it was stable and could be used as a standard for quantitative analysis and as target for detecting whether penicillins were used in milk.

Among developmental regulators, StuA but not BrlA is essential for penicillin V production in Penicillium chrysogenum.

In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the ß-lactam antibiotic penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA. Inactivation of either brlA or stuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy, but did not affect biomass production during liquid-submerged growth. Genome-wide transcriptional profiling identified a complex StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary metabolism. Remarkably, inactivation of stuA, but not brlA, drastically downregulated expression of the penicillin biosynthetic gene cluster during solid and liquid-submerged growth. In agreement, penicillin V production was wild-type-like in brlA-deficient strains but 99% decreased in stuA-deficient strains during liquid-submerged growth, as shown by high-performance liquid chromatography (HPLC) analysis. Thus, among identified regulators of penicillin V production StuA has the most severe influence. Overexpression of stuA increased the transcript levels of brlA and abaA (another developmental regulator) and derepressed conidiation during liquid-submerged growth but did not affect penicillin V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development and secondary metabolism in P. chrysogenum.

Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.

Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the ß-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

Isolation and Characterization of Ochrobactrum tritici for Penicillin V Potassium Degradation.

Substantial concentrations of penicillin V potassium (PVK) have been found in livestock manure, soil, and wastewater effluents, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, bacterial strains capable of degrading PVK were isolated from sludge and characterized. Strain X-2 was selected for biodegradation of PVK. Based on morphological observations and 16S rRNA gene sequencing, strain X-2 was identified as an Ochrobactrum tritici strain. To enhance the PVK degradation ability of PVK, a whole-cell biodegradation process of Ochrobactrum tritici X-2 was established and optimized. In the whole-cell biodegradation process, the optimal temperature and pH were 30°C and 7.0, respectively. Under the optimized conditions, the degradation rate using 0.5 mg/ml PVK reached 100% within 3 h. During biodegradation, two major metabolites were detected: penicilloic acid and phenolic acid. The present study provides a novel method for the biodegradation of PVK using Ochrobactrum tritici strains, which represent promising candidates for the industrial biodegradation of PVK.IMPORTANCE Substantial concentrations of penicillin V potassium (PVK) have been found in the environment, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, antibiotic-degrading bacterial strains for PVK were isolated from sludge and characterized. Ochrobactrum tritici was selected for the biodegradation of PVK with high efficiency. To enhance its PVK degradation ability, a whole-cell biodegradation process was established and optimized using Ochrobactrum tritici The degradation rate with 0.5 mg/ml PVK reached 100% within 3 h. The potential biodegradation pathway was also investigated. To the best of our knowledge, the present study provides new insights into the biodegradation of PVK using an Ochrobactrum tritici strain, a promising candidate strain for the industrial biodegradation of ß-lactam antibiotics.

Isolation, Screening, and Characterization of Antibiotic-Degrading Bacteria for Penicillin V Potassium (PVK) from Soil on a Pig Farm.

(1) Background: Antibiotics are frequently used on farm animals, making animal husbandry a relatively large source of antibiotic pollution of the environment. The present study aims to isolate and acclimatize antibiotic-degrading bacterial strains for penicillin V potassium (PVK) from the contaminated soil of a pig farm. (2) Methods: Bacterial strains were isolated and acclimatized by continuous enrichment of cultures with PVK as the sole carbon source. The antibiotic susceptibility test, thiol mercury salt ultraviolet spectrophotometry (TMSUS), morphological observations, and 16S rDNA sequence analysis were used to identify and characterize the isolated strains. (3) Results: Four bacterial isolates (denoted as LM-1, LM-2, LM-3, LM-4) were obtained, and two of them (LM-1, LM-2) with the highest degradation rates were identified to belong to the same genera as Bacillus. These two isolates were found to be resistant to PVK antibiotic in an antibiotic sensitivity test. The TMSUS indicated that the strains LM-1 and LM-2 had good performance in PVK degradation (68% for LM-1, 66% for LM-2 in 48 h) when the initial PVK concentration was about 100 µg/mL. (4) Conclusions: Two bacterial strains isolated from the soil on a pig farm are effective in degrading PVK and can be potentially used for bioremediation of PVK antibiotic-contaminated soils.

Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and sulbactam to beta-lactamase II from Bacillus cereus.

The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding process were studied through spectroscopy analysis in combination with molecular dynamics (MD) simulation. The results show that in the binding process, a new coordination bond is observed between the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A weak conformational transformation from ß-sheets to random coils is observed in the loop2 of ligand-bound Bc II. The conformational transformation may depend on the functional group and binding pose of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of ligand-bound Bc II may lead to the opening of the binding pocket of Bc II. Therefore, loop2 can be considered a gate for control of ligand access in Bc II, hence its dynamic response should be considered in new drug design and development.

Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring.

The performance of a fiber optic and an optical conduit in-line attenuated total reflection mid-infrared (IR) probe during in situ monitoring of Penicillium chrysogenum fermentation were compared. The fiber optic probe was connected to a sealed, portable, Fourier transform infrared (FT-IR) process spectrometer via a plug-and-play interface. The optical conduit, on the other hand, was connected to a FT-IR process spectrometer via a knuckled probe with mirrors that had to be adjusted prior to each fermentation, which were purged with dry air. Penicillin V (PenV) and its precursor phenoxyacetic acid (POX) concentrations were determined by online high-performance liquid chromatography and the obtained concentrations were used as reference to build partial least squares regression models. Cross-validated root-mean-square errors of prediction were found to be 0.2 g L-1 (POX) and 0.19 g L-1 (PenV) for the fiber optic setup and 0.17 g L-1 (both POX and PenV) for the conduit setup. Higher noise-levels and spectrum-to-spectrum variations of the fiber optic setup lead to higher noise of estimated (i.e., unknown) POX and PenV concentrations than was found for the conduit setup. It seems that trade-off has to be made between ease of handling (fiber optic setup) and measurement accuracy (optical conduit setup) when choosing one of these systems for bioprocess monitoring.

Optimization of culture medium and conditions for penicillin acylase production by Streptomyces lavendulae ATCC 13664.

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylase. This extracellular enzyme recently has been reported to be a penicillin K acylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 microg/mL of CuSO4 x 5H2O was found to be best for acylase production. In such optimized culture medium, fermentation of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.

Phenoxymethylpenicillin amidohydrolases from Penicillium chrysogenum.

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6-aminopenicillanic acid (6-APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6-APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A:6-APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.

[Identification of the beta lactamase R-TEM of Pseudomonas aeruginosa]. / Identification de la beta lactamase R-TEM chez Pseudomonas aeruginosa

This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.

Coupling products of amino acids to penicillin V and cephalothin: synthesis and susceptibility to carboxypeptidases and lysosomal enzymes.

Amino acids have been coupled to the carboxyl group of penicillin V and cephalothin by methods that keep the beta-lactam ring intact. Derivatives were successfully obtained with both neutral (Leu, Val, Ala, Ile, Trp, Tyr, Gly) and one acidic (Glu) amino acids. The new compounds were inactive in vitro against Staphylococcus aureus or Micrococcus luteus. Incubation in the presence of purified carboxypeptidases (A, B), soluble lysosomal fractions from liver, or cellular homogenates from liver, kidney, fibroblasts, and macrophages did not allow recovery of the antibacterial activity. Injection in mice also failed to cause liberation of microbiologically active compounds. HPLC studies confirmed that the amide linkage between the antibiotic and the amino acid was not hydrolyzed in the presence of soluble lysosomal fractions from liver. However, conversion of cephalothin and cephalothin-leucine to desacetyl derivatives was observed in the presence of soluble lysosomal fractions and extracts from liver and semipurified orange peel acetylesterase(s). It is concluded that amino acid derivatives of beta-lactam antibiotics do not offer potential chemotherapeutic use as prodrugs.

Uptake of benzylpenicillin, cefpiramide and cefazolin by freshly prepared rat hepatocytes. Evidence for a carrier-mediated transport system.

The kinetics and mechanism of the hepatic uptake of beta-lactam antibiotics were studied by using freshly prepared rat hepatocytes. The initial uptake rates of benzylpenicillin and cefpiramide represented both saturable and nonsaturable transport processes, whereas that of cefazolin showed an apparently nonsaturable uptake process within the concentration range below 4 mM. The apparent nonsaturable uptake rate constants for benzylpenicillin, cefpiramide and cefazolin were 0.580, 0.047 and 0.289 nmoles/min/mg protein/mM respectively. The apparent values of Kt and Vmax describing the saturable transport were 0.473 +/- 0.158 mM and 2.02 +/- 0.48 nmoles/min/mg protein for benzylpenicillin and 0.847 +/- 0.254 mM and 0.70 +/- 0.18 nmoles/min/mg protein for cefpiramide respectively. The Arrhenius plot of benzylpenicillin uptake of 200 microM presented a single straight line in the range of 22 degrees-37 degrees with an activation energy of 16.8 kcal/mole. An energy requirement was also demonstrated for benzylpenicillin uptake as metabolic inhibitors (antimycin A, NaCN, rotenone and 2,4-dinitrophenol) significantly reduced the initial uptake rate of benzylpenicillin (P less than 0.05). Uptake of benzylpenicillin (200 microM) was not inhibited by ouabain (1 mM). Benzylpenicillin uptake was inhibited competitively by phenoxymethylpenicillin, cefpiramide and cefazolin with the inhibition constants, Ki, of 0.680, 0.583 and 11.7 mM respectively. Benzylpenicillin also inhibited competitively the uptake of cefpiramide with a Ki of 0.655 mM. From these results it was considered that a carrier-mediated uptake system participates in the hepatic uptake of at least four of the beta-lactam antibiotics examined in this study.

L-lysine repression of penicillin biosynthesis and the expression of penicillin biosynthesis genes acvA and ipnA in Aspergillus nidulans.

The addition of 0.1 M L-lysine to the fermentation medium reduced the production of penicillin by about 50% in Aspergillus nidulans. To analyse this effect at the molecular level, the expression of the penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, was studied by using translational fusions with different reporter genes (strain AXB4A, acvA-uidA, ipnA-lacZ fusions; AXB4B, acvA-lacZ, ipnA-uidA fusions) integrated in single copy at the chromosomal argB locus of Aspergillus nidulans. Irrespective of the reporter genes used the expression of acvA and ipnA fusion genes was repressed in L-lysine grown cultures. The expression of a fusion gene of an A. nidulans primary metabolism gene (oliC-lacZ) was not affected by L-lysine.

Influence of medium composition on penicillin V production in a stirred tank reactor.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.

Strain improvement of Penicillium chrysogenum by recombinant DNA techniques.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.

Penicillin amidase from Proteus rettgeri.

1. Penicillin amidase from Proteus rettgeri was purified 580-fold by a four-step chromatographic procedure. Titration with phenylmethanesulphonyl fluoride showed that the purified preparation contains 53% of the enzyme. 2. The molecular weight of the amidase was found to be 65.000. The enzyme is strongly inhibited by N-bromosuccinimide and zinc ions. It hydrolyses penicillins, cephalosporins and some synthetic substrates, and in addition it catalyses synthesis of ampicillin from methyl ester of phenylglycine and 6-aminopenicillanic acid. 3. The immobilized amidase obtained by copolymerization of the chemically modified enzyme with acrylamide was applied for preparative hydrolysis of benzylpenicillin.