Enzyme Penicillinase Research Articles

Antibacterial and antioxidant activity of catechin, gallic acid, and epigallocatechin-3-gallate: focus on nanoformulations

Up to one million secondary metabolites are found in plant species, some of which may have desirable therapeutic activities. Among these secondary metabolites, catechin, gallic acid, and epigallocatechin-3-gallate are natural phenolic compounds with promising antioxidant and antibacterial activity. However, these compounds have disadvantages of poor solubility, low bioavailability in physiological conditions, and side effects in patients. Therefore new strategies could rely on formulations with other synthetic and natural materials. Nanoformulations of secondary metabolites could be new efficient strategies to treat many chronic bacterial infections. Combinations and conjugates of catechin, gallic acid, and epigallocatechin-3-gallate with various antibiotics could reduce the dose of these compounds, increase their antibacterial activity, and decrease cytotoxicity against healthy cells. For instance, a smart combination of two or more secondary metabolites may improve therapeutic applications in physiological conditions. In this regard, the growth of antibiotic-resistant bacteria, specifically multidrug-resistant bacteria with overexpression of efflux pumps and expression of the penicillinase enzyme, has been inhibited significantly. According to recent investigations, this review will discuss the advances and challenges of new micro and nanoformulations of these natural products.

Capacitive field-effect biosensor modified with a stacked bilayer of weak polyelectrolyte and plant virus particles as enzyme nanocarriers

This work presents a new approach for the development of field-effect biosensors based on an electrolyte-insulator-semiconductor capacitor (EISCAP) modified with a stacked bilayer of weak polyelectrolyte and tobacco mosaic virus (TMV) particles as enzyme nanocarriers. With the aim to increase the surface density of virus particles and thus, to achieve a dense immobilization of enzymes, the negatively charged TMV particles were loaded onto the EISCAP surface modified with a positively charged poly(allylamine hydrochloride) (PAH) layer. The PAH/TMV bilayer was prepared on the Ta2O5-gate surface by means of layer-by-layer technique. The bare and differently modified EISCAP surfaces were physically characterized by fluorescence microscopy, zeta-potential measurements, atomic force microscopy and scanning electron microscopy. Transmission electron microscopy was used to scrutinize the PAH effect on TMV adsorption in a second system. Finally, a highly sensitive TMV-assisted EISCAP antibiotics biosensor was realized by immobilizing the enzyme penicillinase onto the TMV surface. This PAH/TMV bilayer-modified EISCAP biosensor was electrochemically characterized in solutions with different penicillin concentrations via capacitance-voltage and constant-capacitance methods. The biosensor possessed a mean penicillin sensitivity of 113 mV/dec in a concentration range from 0.1 mM to 5 mM.

Optimal Conditions for Extraction and Purification of Penicillinase Enzyme

Optimal Conditions for Extraction and Purification of Penicillinase Enzyme

Enzyme-based sensing on nanohybrid film coated over FTO electrode for highly sensitive detection of antibiotics.

Cefepime and Meropenem are the new class of antibiotics, which are particularly used as last potent defender or the antibiotics of the last resort against multi-resistant bacterial species. In this paper, an impedance-based electrochemical biosensor was fabricated for identifying antibiotics of last resort in the forensic samples including gastric lavage and other body fluids. The sensor was developed using platinum nanoparticles (PtNPs) and electrodeposited zinc oxide- zinc hexacyanoferrate hybrid film (ZnO/ZnHCF) on the surface of a fluorine-doped glass electrode (FTO). Further, penicillinase was immobilized onto the modified electrode using penicillinase enzyme. The developed biosensor exhibits agood analytical response for the detection of antibiotics evaluated using electrochemistry studies. The linear response of thefabricated electrode was observed from 0.1 to 750µM and the electrode limit of detection (LOD) was observed as 0.1µM. The sensor confirms good accuracy, ishighly selective, and sensitive for the target. While storingthe modified electrode at 4°C, the stability of biosensor was evaluated for 45days, and activityloss of 30-40% was observed. Thehighly sensitive interface of Penicillinase@CHIT/PtNP-ZnO/ZnHCF/FTO electrode shows a promising future in forensic studies.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Rapid Determination of Benzylpenicillin Resistance in Staphylococcus aureus Bacteraemia Model.

Rapid determination of antimicrobial susceptibility/resistance is an important factor in selecting an appropriate antimicrobial treatment and eradicating infections promptly. Conventional antimicrobial susceptibility tests (ASTs) are very time consuming. Thus, we developed a liquid chromatography-mass spectrometry (LC-MS/MS) method for rapidly determining the resistance of Staphylococcus aureus to penicillin-G in an animal-infection model. This technique will be able to detect those resistant strains whose resistance mechanism specifically controlled by penicillinase. The resistance status of S. aureus against penicillin-G was determined by conventional AST. Cultured S. aureus cells were inoculated to chicken for developing bacteraemia. The solution of penicillin-G was intravenously administered (10 mg/kg b.w.) to chickens just after infection detection. Blood samples were collected at different intervals after drug administration. The concentration of active penicillin-G and its metabolites were determined from the bacteria-free blood supernatant by utilizing the LC-MS/MS method. Evidence of infection in chicken was observed within 5 h of bacterial inoculation. The penicillinase enzyme generated by S. aureus transforms the active penicillin-G to an inactive metabolite by hydrolysis, which is evident by the mass shift from 335.10600 to 353.11579 Da as quantified using liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS). The signal intensity of inactive/hydrolysed penicillin-G is several-fold greater than that of the active penicillin-G in the blood sample of chicken infected with resistant strain and treated with penicillin-G. The antimicrobial resistance index (ARI) value of resistant S. aureus strain was more than 1, demonstrating the penicillin-G-resistance pattern of that strain. This method is able to determine the extent of β-lactam antimicrobial resistance within 1.5 h from the patient’s blood and is complementary with those existing AST methods which are usually practicing in the evaluation of β-lactam antibiotic resistance.

Penicillin Detection by Tobacco Mosaic Virus-Assisted Colorimetric Biosensors.

The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms. Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time. Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker. Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing. Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry. The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters. They extended the half-life of the sensors from 4 - 6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors. Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained. Initial experiments also indicate that the system may be transferred to label-free detection layouts.

Development of penicillin G biosensor based on penicillinase enzymes immobilized onto bio-chips.

Penicillin G (Pen-G) biosensor was developed by immobilizing penicillinase enzymes (Pen-X) onto tiny bio-chips using thioglycolic acid self-assembled monolayer (TGA-SAM). The selective pen-G biosensor was investigated by ferri/ferrocyanide couple using electrochemical method for catalytic hydrolysis of Pen-G/Pen-X in a very sensible approach. Pen-G was detected with modified bio-chip (Gold electrode of bio-chip/Thioglycolic acid/Penicillinase enzyme, in shortly AuE/TGA/Pen-X) by reliable cyclic voltammetric method at pH 7.1 in room conditions. The AuE/TGA/Pen-X modified bio-chip sensor demonstrates good linearity (50.0 nM to 5.0 mM; R = 0.9987), low-detection limit (~1.26 nM, SNR ~ 3), and higher sensitivity (~4.97 μA.μM(-1).cm(-2)), lowest-small sample volume (<70.0 μL), good stability, selectivity, and reproducibility. To the best of our knowledge, this is the first statement in which such a very high sensitivity, low-detection limit, and low-sample volume are required for Pen-G biosensor using AuE/TGA/Pen-X bio-chips assembly. The results of these studies have introduced for biomedical applications with interesting assembly (AuE/TGA/Pen-X) towards the development of selective Pen-G biosensors. The AuE/TGA-SAM system was implemented a facile approach to the integration of Pen-X/Pen-G fabricated bio-chips, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields.

Langmuir and Langmuir–Blodgett films of lipids and penicillinase: Studies on adsorption and enzymatic activity

Bioelectronic devices, such as biosensors, can be constructed with enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is fundamental for optimal performance. In this sense, nanostructured films in which molecular architectures can be controlled are of interest. In this present work, the adsorption of the enzyme penicillinase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid was investigated and characterized with surface pressure–area isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). The incorporation of the enzyme in the lipid monolayer not only caused the film to expand, but also could be identified through amide bands in the PM-IRRAS spectra, with the CN and CO dipole moments being identified, lying parallel to monolayer plane. Structuring of the enzyme into α-helices was identified in the mixed enzyme–phospholipid monolayer and preserved when transferred to solid as a Langmuir–Blodgett (LB) film. The enzyme–lipid LB films were then characterized with PM-IRRAS, atomic force microscopy and fluorescence spectroscopy. Measurements of the catalytic activity showed that the enzyme accommodated in the LB films preserved 76% of the enzyme activity in relation to the homogeneous medium. The method presented here not only allows for enhanced catalytic activity toward penicillin, but also can be useful to explain why certain film architectures exhibit better enzyme activity.

Immunochemical detection of penicillins by using biohybrid magnetic particles

An Enzyme Linked Immunosorbent Assay (ELISA) with the ability to detect some of the most widely used penicillins such as penicillin G, amoxicillin and ampicillin has been developed and validated in milk samples to ensure food safety. The assay can achieve limits of detection (0.1 μg L−1) much lower than the MRLs (Maximum Residue Levels) established by the EU (4 μg L−1). The procedure allows the analysis of the samples with a simple and fast treatment of the matrix with biohybrid magnetic particles to hydrolyze the β-lactam ring. A new immunizing hapten has been described and the polyclonal antibodies produced showed high specificity for open ring penicillins and no cross-reactivity with other antibiotic families. The assay has been designed to detect simultaneously both penicillin species in milk, the antibiotics and their hydrolyzed forms. Concerning the open-ringed forms their detection in food samples will be especially important in the next few years because of the illegal use of penicillinase enzymes by farmers to mask the real concentrations of β-lactams in milk.

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67±13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality.

Fast determination of antibiotics in whole blood

There is a need for analytical methods capable of monitoring blood antibiotic levels in real time. Here we present a method for quantifying antibiotic levels in whole blood that does not require any sample pretreatment. The tests employ the enzyme penicillinase to assay for penicillin G, penicillin V and ampicillin using a flow-injected biosensor, the Enzyme Thermistor. Optimal flow rates, sample volumes and pH were determined to be 0.5 mL/min, 100 μL and 7.0, respectively. Analysis of the antibiotics diluted in buffer gave a linear range of 0.17–5.0 mM. Calibration curves prepared using blood spiked with the antibiotics gave a linear range of 0.17–2.0 mM. Linear regression values for all of the calibration curves were 0.998 or higher. Assay cycle time was 5 min. The relative standard deviation value for 100 determinations of a mock blood sample spiked with penicillin G was 6.71%. Despite the elimination of sample pretreatment, no detectable clogging or signal drift was observed. The assay provides a fast, simple, reliable analytical method for determining antibiotic concentrations in blood without the need for any sample pretreatment. This is an important first step towards developing a device capable of real-time monitoring of antibiotic levels in whole blood. The technology has the potential to significantly improve the outcomes of patients undergoing critical care.

Antibacterial, Antialgal and β-lactamase Inhibition Activity of Penicillium Purpurogenum var Rubrisclerotium

One of the biggest challenges encountered by the medical world is antibiotic resistance posed by the superbugs. Similarly, algae pose a threat by growing in swimming pools, aquariums, causing toxicity in potable waters etc. In this study, a fungal strain was isolated from algal source and studied for its antibiotic activity. The strain was identified as Penicillium purpurogenum var rubrisclerotium and was tested for its antibacterial activity against S.aureus, B.subtillis, E.coli and P.aeruginosa; antialgal activity against Chlorella vulgaris and β-lactamase inhibitor activity against Penicillinase enzyme (B. cereus) using sulbactum as a control for β-lactamase inhibition, and Penicillin as a substrate. The fermented broth exhibited antibacterial, antialgal activity against test cultures along with a significant β-lactamase inhibitory activity, measured by iodometric and agar gel diffusion bioassay methods. The broth was further concentrated by treating it with butyl acetate and tried against test organisms which resulted in many fold increase in the activities.

ZnO Nanorods Based Enzymatic Biosensor for Selective Determination of Penicillin.

In this study, we have successfully demonstrated the fabrication of a biosensor based on well aligned single-crystal zinc oxide (ZnO) nanorods which were grown on gold coated glass substrate using a low temperature aqueous chemical growth (ACG) method. The ZnO nanorods were immobilized with penicillinase enzyme using the physical adsorption approach in combination with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) as cross linking molecules. The potentiometric response of the sensor configuration revealed good linearity over a large logarithmic concentration range from 100 µM to 100 mM. During the investigations, the proposed sensor showed a good stability with high sensitivity of ~121 mV/decade for sensing of penicillin. A quick electrochemical response of less than 5 s with a good selectivity, repeatability, reproducibility and a negligible response to common interferents such as Na1+, K1+, d-glucose, l-glucose, ascorbic acid, uric acid, urea, sucrose, lactose, glycine, penicilloic acid and cephalosporins, was observed.

An investigation on antibiotic resistance of Neisseria gonorrhoeae isolated from gonorrheal patients in Zahedan, Iran from 2007 to 2010

Present study was aimed to determine antibiotic susceptibility and penicillinase production by Neisseria gonorrhoeae strains isolated from gonorrheal patients in Zahedan (south-east Iran). In a descriptive study from 2007 to 2010, 400 suspected patients were studied by history review, medical examination, Gram staining and culture in Thayer-Martin medium. Antibiotic susceptibility test of isolated strains was done by disk diffusion method and penicillinase test in penicillin resistant isolates by aciodometric method. The culture of 77(19.2%) of gonorrheal patients were positive. The resistance rate against applied antibiotics was as follow: penicillin (79.2%), ciprofloxacin (53.2%), ceftriaxone (3.8%), spectinomycin (2.5%), cefixim (12.9%), co-trimoxazole (93.5%), tetracycline (88.3%) and gentamicin (29.8%). In the meantime, 83.1% of penicillin resistant isolates produced penicillinase enzyme. Ceftriaxone, spectinomycin and cefixime are the sole antibiotics that could be considered as selective drugs. Quinolones which were regarded as an effective group of antibiotics until recently, haven lost their efficacy. Resistance against other antibiotics is rapidly growing, thus, conducting experimental tests and determination of minimum inhibitory concentration and clinical trial studies at fixed intervals can contribute to diagnosis of resistance of gonococci and rapid and successful treatment of their infections. Key words: Neisseria gonorrhoeae, gonorrhea, penicillinase (β-lactamase).

Use of Information Visualization Methods Eliminating Cross Talk in Multiple Sensing Units Investigated for a Light-Addressable Potentiometric Sensor

The integration of nanostructured films containing biomolecules and silicon-based technologies is a promising direction for reaching miniaturized biosensors that exhibit high sensitivity and selectivity. A challenge, however, is to avoid cross talk among sensing units in an array with multiple sensors located on a small area. In this letter, we describe an array of 16 sensing units of a light-addressable potentiometric sensor (LAPS), which was made with layer-by-layer (LbL) films of a poly(amidomine) dendrimer (PAMAM) and single-walled carbon nanotubes (SWNTs), coated with a layer of the enzyme penicillinase. A visual inspection of the data from constant-current measurements with liquid samples containing distinct concentrations of penicillin, glucose, or a buffer indicated a possible cross talk between units that contained penicillinase and those that did not. With the use of multidimensional data projection techniques, normally employed in information visualization methods, we managed to distinguish the results from the modified LAPS, even in cases where the units were adjacent to each other. Furthermore, the plots generated with the interactive document map (IDMAP) projection technique enabled the distinction of the different concentrations of penicillin, from 5 mmol L(-1) down to 0.5 mmol L(-1). Data visualization also confirmed the enhanced performance of the sensing units containing carbon nanotubes, consistent with the analysis of results for LAPS sensors. The use of visual analytics, as with projection methods, may be essential to handle a large amount of data generated in multiple sensor arrays to achieve high performance in miniaturized systems.

Layer-by-Layer Assembly of Carbon Nanotubes Incorporated in Light-Addressable Potentiometric Sensors

The integration of carbon nanotubes in conjunction with a chemical or biological recognition element into a semiconductor field-effect device (FED) may lead to new (bio)chemical sensors. In this study, we present a new concept to develop field-effect-based sensors, using a light-addressable potentiometric sensor (LAPS) platform modified with layer-by-layer (LbL) films of single-walled carbon nanotubes (SWNTs) and polyamidoamine (PAMAM) dendrimers. Film growth was monitored for each layer adsorbed on the LAPS chip by measuring current−voltage (I/V) curves. The morphology of the films was analyzed via atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM), revealing the formation of a highly interconnected nanostructure of SWNTs-network into the dendrimer layers. Constant current (CC) measurements showed that the incorporation of the PAMAM/SWNT LbL film containing up to 6 bilayers onto the LAPS structure has a high pH sensitivity of ca. 58 mV/pH. The biosensing ability of the devices was tested for penicillin G via adsorptive immobilization of the enzyme penicillinase atop the LbL film. LAPS architectures modified with the LbL film exhibited higher sensitivity, ca. 100 mV/decade, in comparison to ca. 79 mV/decade for an unmodified LAPS, which demonstrates the potential application of the CNT-LbL structure in field-effect-based (bio)chemical sensors.

Penicillin biosensor based on a capacitive field-effect structure functionalized with a dendrimer/carbon nanotube multilayer

Silicon-based sensors incorporating biomolecules are advantageous for processing and possible biological recognition in a small, reliable and rugged manufactured device. In this study, we report on the functionalization of field-effect (bio-)chemical sensors with layer-by-layer (LbL) films containing single-walled carbon nanotubes (SWNTs) and polyamidoamine (PAMAM) dendrimers. A capacitive electrolyte-insulator-semiconductor (EIS) structure modified with carbon nanotubes (EIS-NT) was built, which could be used as a penicillin biosensor. From atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM) images, the LbL films were shown to be highly porous due to interpenetration of SWNTs into the dendrimer layers. Capacitance–voltage (C/V) measurements pointed to a high pH sensitivity of ca. 55mV/pH for the EIS-NT structures. The biosensing ability towards penicillin of an EIS-NT-penicillinase biosensor was also observed as the flat-band voltage shifted to lower potentials at different penicillin concentrations. A dynamic response of penicillin concentrations, ranging from 5.0μM to 25mM, was evaluated for an EIS-NT with the penicillinase enzyme immobilized onto the surfaces, via constant–capacitance (ConCap) measurements, achieving a sensitivity of ca. 116mV/decade. The presence of the nanostructured PAMAM/SWNT LbL film led to sensors with higher sensitivity and better performance.

Penicillin detection with nanocrystalline‐diamond field‐effect sensor

AbstractNanocrystalline‐diamond (NCD) films have been utilised for the detection of penicillin G for the first time. The developed penicillin‐sensitive biosensor consists of a field‐effect capacitive electrolyte‐diamond‐insulator‐semiconductor (EDIS) structure with an immobilised enzyme layer that covers the gate region of the sensor. Undoped NCD thin films of about 100 nm thickness were grown on a p‐Si‐SiO2 (50 nm thermally grown SiO2) structure by microwave plasma‐enhanced chemical vapour deposition. The enzyme penicillinase has been adsorptively immobilised directly onto the O‐terminated NCD surface. The EDIS biosensors have been characterised in buffer solutions with different content of penicillin G by means of capacitance–voltage and constant‐capacitance method. The developed penicillin biosensor possesses a low detection limit of 5 µM and a high sensitivity of 60–70 mV/decade in a wide linear range of 0.005–2.5 mM penicillin G concentration. (© 2008 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Characterisation of capacitive field-effect sensors with a nanocrystalline-diamond film as transducer material for multi-parameter sensing

The feasibility of a capacitive field-effect EDIS (electrolyte-diamond-insulator-semiconductor) platform for multi-parameter sensing is demonstrated by realising EDIS sensors with an O-terminated nanocrystalline-diamond (NCD) film as transducer material for the detection of pH and penicillin concentration as well as for the label-free electrical monitoring of adsorption and binding of charged macromolecules, like polyelectrolytes. The NCD films were grown on p-Si-SiO 2 substrates by microwave plasma-enhanced chemical vapour deposition. To obtain O-terminated surfaces, the NCD films were treated in an oxidising medium. The NCD-based field-effect sensors have been characterised by means of constant-capacitance method. The average pH sensitivity of the O-terminated NCD film was 40 mV/pH. A low detection limit of 5 μM and a high penicillin G sensitivity of 65–70 mV/decade has been obtained for an EDIS penicillin biosensor with the adsorptively immobilised enzyme penicillinase. Alternating potential changes, having tendency to decrease with increasing the number of adsorbed polyelectrolyte layers, have been observed after the layer-by-layer deposition of polyelectrolyte multilayers, using positively charged PAH (poly (allylamine hydrochloride)) and a negatively charged PSS (poly (sodium 4-styrene sulfonate)) as a model system. The response mechanism of the developed EDIS sensors is discussed.