Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

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 a good analytical response for the detection of antibiotics evaluated using electrochemistry studies. The linear response of the fabricated electrode was observed from 0.1 to 750 µM and the electrode limit of detection (LOD) was observed as 0.1 µM. The sensor confirms good accuracy, is highly selective, and sensitive for the target. While storing the modified electrode at 4 °C, the stability of biosensor was evaluated for 45 days, and activity loss of 30-40% was observed. The highly sensitive interface of Penicillinase@CHIT/PtNP-ZnO/ZnHCF/FTO electrode shows a promising future in forensic studies.

Modularized Field-Effect Transistor Biosensors.

Field-effect transistors (FETs), when functionalized with proper biorecognition elements (such as antibodies or enzymes), represent a unique platform for real-time, specific, label-free transduction of biochemical signals. However, direct immobilization of biorecognition molecules on FETs imposes limitations on reprogrammability, sensor regeneration, and robust device handling. Here we demonstrate a modularized design of FET biosensors with separate biorecognition and transducer modules, which are capable of reversible assembly and disassembly. In particular, hydrogel "stamps" immobilizing bioreceptors have been chosen to build biorecognition modules to reliably interface with FET transducers structurally and functionally. Successful detection of penicillin down to 0.25 mM has been achieved with a penicillinase-encoded hydrogel module, demonstrating effective signal transduction across the hybrid interface. Moreover, sequential integration of urease- and penicillinase-encoded modules on the same FET device allows us to reprogram the sensing modality without cross-contamination. In addition to independent bioreceptor encoding, the modular design also fosters sophisticated control of sensing kinetics by modulating the physiochemical microenvironment in the biorecognition modules. Specifically, the distinction in hydrogel porosity between polyethylene glycol and gelatin enables controlled access and detection of larger molecules, such as poly-l-lysine (MW 150-300 kDa), only through the gelatin module. Biorecognition modules with standardized interface designs have also been exploited to comply with additive mass fabrication by 3D printing, demonstrating potential for low cost, ease of storage, multiplexing, and great customizability for personalized biosensor production. This generic concept presents a unique integration strategy for modularized bioelectronics and could broadly impact hybrid device development.

Hydrogel Gate Graphene Field-Effect Transistors as Multiplexed Biosensors.

Nanoscale field-effect transistors (FETs) represent a unique platform for real time, label-free transduction of biochemical signals with unprecedented sensitivity and spatiotemporal resolution, yet their translation toward practical biomedical applications remains challenging. Herein, we demonstrate the potential to overcome several key limitations of traditional FET sensors by exploiting bioactive hydrogels as the gate material. Spatially defined photopolymerization is utilized to achieve selective patterning of polyethylene glycol on top of individual graphene FET devices, through which multiple biospecific receptors can be independently encapsulated into the hydrogel gate. The hydrogel-mediated integration of penicillinase was demonstrated to effectively catalyze enzymatic reaction in the confined microenvironment, enabling real time, label-free detection of penicillin down to 0.2 mM. Multiplexed functionalization with penicillinase and acetylcholinesterase has been demonstrated to achieve highly specific sensing. In addition, the microenvironment created by the hydrogel gate has been shown to significantly reduce the nonspecific binding of nontarget molecules to graphene channels as well as preserve the encapsulated enzyme activity for at least one week, in comparison to free enzymes showing significant signal loss within one day. This general approach presents a new biointegration strategy and facilitates multiplex detection of bioanalytes on the same platform, which could underwrite new advances in healthcare research.

Cephalosporin-NO-donor prodrug PYRRO-C3D shows ß-lactam-mediated activity against Streptococcus pneumoniae biofilms.

Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3'-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with ß-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following ß-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (ß-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks ß-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of ß-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the ß-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections.

Identification of peptide inhibitors of penicillinase using a phage display library.

There is a constant need to identify novel inhibitors to combat ß-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM(-1)) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV-vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 µM and a Ki' of 33.12 µM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of ß-lactamases and development of novel inhibitors of ß-lactamases.

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.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 µM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Dual-porosity hollow nanoparticles for the immunoprotection and delivery of nonhuman enzymes.

Although enzymes of nonhuman origin have been studied for a variety of therapeutic and diagnostic applications, their use has been limited by the immune responses generated against them. The described dual-porosity hollow nanoparticle platform obviates immune attack on nonhuman enzymes paving the way to in vivo applications including enzyme-prodrug therapies and enzymatic depletion of tumor nutrients. This platform is manufactured with a versatile, scalable, and robust fabrication method. It efficiently encapsulates macromolecular cargos filled through mesopores into a hollow interior, shielding them from antibodies and proteases once the mesopores are sealed with nanoporous material. The nanoporous shell allows small molecule diffusion allowing interaction with the large macromolecular payload in the hollow center. The approach has been validated in vivo using l-asparaginase to achieve l-asparagine depletion in the presence of neutralizing antibodies.

Effect of different combinations of antibodies and enzyme labels on ELISA of progesterone.

In steroid immunoassays, selection of right combination of antibody and enzyme-labeled antigen determine the sensitivity and specificity of ELISA. Antibodies raised against different positions of progesterone adopting heterologous systems were reported to provide better assays for progesterone. Four different antibodies developed against progesterone-11α-hemiglutarate-BSA (P-11-HG-BSA), progesterone-11α-hemisuccinate-BSA (P-11-HS-BSA), progesterone-3-O-carboxymethyloxime-BSA (P-3-CMO-BSA), and progesterone-3-O-carboxymethyloxime-ovalbumin (P-3-CMO-ova) were tested in combination with enzyme-labeled P-11-HG, P-11-HS, progesterone-11α-carboxymethyl ether (P-11-CME), P-3-CMO, 17-hydroxyprogesterone-3-O-carboxymethyl oxime (17-P-3-CMO), and progesterone-4-carboxymethyl thioether (P-4-CMTE). These were variously labeled with penicillinase, alkaline phosphatase (ALP), and horseradish peroxidase (HRP). When antibody developed against P-11-HS-BSA was tested with P-3-CMO labeled separately with penicillinase, ALP, and HRP, the type of enzyme used had no effect on the performance of the assay. It was found that a homologous assay using P-3-CMO-ova as immunogen and P-3-CMO-HRP as label, as well as a heterologous ELISA with antibody raised against P-11-HS-BSA in combination with P-3-CMO-HRP, provided sensitive assays for progesterone. The use of 17α-hydroxy progesterone-3-O-carboxymethyl oxime-HRP with the same antibodies against P-3-CMO-BSA and P-11-HS-BSA also proved to be better than P-3-CMO-HRP. These findings implied that the sensitivity and specificity of ELISA to a great extent depended on the nature of the antibody produced, while the choice of enzyme labels could be manipulated.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1-3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Inhibition of the class C beta-lactamase from Acinetobacter spp.: insights into effective inhibitor design.

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.

Galvanostatic entrapment of penicillinase into polytyramine films and its utilization for the potentiometric determination of penicillin.

A sensitive and reliable potentiometric biosensor for determination of penicillin has been developed by exploiting the self-limiting growth of the non-conducting polymer, polytyramine. Optimum polytyramine-penicillinase (PTy-PNCnase) films for potentiometric detection of penicillin were accomplished with monomer solutions which contained 0.03 M tyramine, 37 U/mL penicillinase, 0.01 M KNO3, and 3 mM penicillin with an applied current density of 0.8 mA/cm2 and an electropolymerisation time of 40 seconds. The potentiometric biosensor gave a linear concentration range of 3-283 µM for penicillin and achieved a minimum detectable concentration of 0.3 µM. The biosensor was successfully utilized for the detection of Amoxycillin and gave an average percentage recovery of 102±6%. Satisfactory recoveries of penicillin G were also achieved in milk samples with the potentiometric biosensor when concentrations are ≥20 ppm.

Identification of beta-lactamase in antibiotic-resistant Bacillus cereus spores.

Beta-lactamase type I is reported for the first time to occur in the sporulated form in a penicillin-resistant Bacillus species. The enzyme was readily characterized from the B. cereus 5/B line (ATCC 13061) by mass spectrometry and two-dimensional gel electrophoresis.

Field-effect biosensor using virus particles as scaffolds for enzyme immobilization.

A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system. The developed field-effect penicillin biosensor consists of an Al-p-Si-SiO2-Ta2O5-TMV structure and has been electrochemically characterized in buffer solutions containing different concentrations of penicillin G. In addition, the morphology of the biosensor surface with virus particles was characterized by scanning electron microscopy and atomic force microscopy methods. The sensors possessed a high penicillin sensitivity of ~ 92 mV/dec in a nearly-linear range from 0.1 mM to 10 mM, and a low detection limit of about 50 µM. The long-term stability of the penicillin biosensor was periodically tested over a time period of about one year without any significant loss of sensitivity. The biosensor has also been successfully applied for penicillin detection in bovine milk samples.

Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase.

Rapid, low-cost and efficient assays for penicillinase activity and inhibition are of vital importance for therapeutics and diagnostics of bacterial resistance to antibiotics. Herein we report a novel approach for on-line enzyme assays for penicillinase utilizing capillary electrophoresis-integrated immobilized enzyme reactors (CE-IMERs). The CE-IMERs are fabricated based on penicillinase-mediated alginate hydrogelation, allowing single-step in-situ encapsulation of enzymes without any additional manipulation process. We show that the fabricated CE-IMERs have high enzyme loading capacity with approximately 61.8% of the original penicillinase in the sol mixture being encapsulated in the "egg-box" hydrogel matrix. Excellent intraday and interday stability and batch-to-batch reproducibility are proved, indicating the reliability of our method for accurate on-line enzyme assays for penicillinase. Enzymatic activities and inhibition of immobilized penicillinase are analyzed, the results of which are in good agreement with those using free enzymes. The proposed method is successfully used for determination of penicillin in pork samples, indicating the potential applications for analysis of complicated real samples.

Characteristics of beta-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii.

BACKGROUND: The presence of beta-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the beta-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India. RESULTS: The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126-1024 mg/L) and MIC90 (256-1024 mg/L) of beta-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87-96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77-79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5-7.1) enzymes. CONCLUSION: Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two beta-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region.

Silica-encapsulated nanomagnetic particle as a new recoverable biocatalyst carrier.

Enzymes are versatile biocatalysts with major advantages of ultrahigh reaction selectivity and specificity under mild conditions, which currently find increasing applications. However, their applications are often hampered by difficulties in recovery and recycling. As a result, we carried out detailed investigations on the synthesis and characterization of silica-encapsulated iron oxide magnetic nanoparticles of controlled dimension as an enzyme carrier. It is shown that the relatively smaller sized silica-coated magnetic nanoparticle prepared by the microemlusion technique can a carry bulky enzyme, beta-lactamase, via chemical linkages on the silica overlayer without severely blocking the enzymatic active center (which is commonly encountered in conventional solid supports). An activity study by Michalis-Menten kinetics reflects that this new type of immobilization allows enzyme isolation with accessibility as good as free enzyme. The recovery and reusability of the nanoparticle-supported enzyme upon application of magnetic separation are also demonstrated.

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.