Continuous penicillin G hydrolysis in an electro-membrane reactor with immobilized penicillin G acylase

Penicillin G (2%, w/v in phosphate buffer, pH 8) was hydrolysed in a flow-through, miniature electro-membrane reactor with the penicillin G acylase immobilized in 5% (w/v) polyacrylamide (diam. 10 mm, thickness 2.6 mm, enzyme activity 24 U ml^–1). The conversion of penicillin G increased from 0.15 to almost 0.5 when the electric current applied to the reactor was changed from –600 to +600 A/m² with a substrate residency of 1 h. Symbols and abbreviations c _j ^p & concentration of component j in product stream (M) c _j ^s & concentration of component j in substrate stream (M) c _s ^o & substrate concentration at reactor inlet (M) C _j ^p=c _j ^p/c _S ⁰ & scaled concentration of component j in product stream C _j ^s=c _j ^s/c _S ⁰ & scaled concentration of component j in substrate stream i & electric current density (A/m²) j & reaction component, j P, Q or S P & main reaction product (6-aminopenicillanic acid) PGA & penicillin G acylase Q & side reaction product (phenylacetic acid) S & substrate (penicillin G) Y _s=C _P ^s+C _P ^p & substrate conversion & mean residence time of substrate and product streams in reactor (h) =C _Q ^s+C _Q ^p+C _S ^s+C _S ^s & check-sum of scaled concentrations =C _P ^p/(C _P ^s+C _P ^p) & separation factor of 6-aminopenicillanic acid (0 1)     

Process of penicillin G hydrolysis catalyzed by penicillin acylase immobilized on acylic carrier

The usefulness of penicillin acylase immobilized onto butyl acrylate — ethyl glycol dimethacrylate (called in this paper acrylic carrier) in penicillin G hydrolysis performed in a stirred tank reactor is shown. The enzyme-acrylic carrier preparation does not deteriorate its own properties in the mixing condition of slurry reactor. The experiments were carried out in a batch and a continuous stirred tank reactor as well as continuous stirred tank reactors in series. It was found to be a satisfactory agreement between experimental and predicted results. It also indicated the optimal substrate concentration range which provides the most effective enzyme operation. A superiority of the three reactors in series over the batch reactor is shown.List of Symbols C_E g/m³ equivalent enzyme concentration - C_SO mol/m³ initial penicillin G concentration - K_A mol/m³ substrate affinity constant - K_iS mol/m³ substrate inhibitory constant - K_iP mol/m³ PhAA inhibitory constant - K_iQ mol/m³ 6-APA inhibitory constant - k₃ mol/g min constant rate of dissotiation of the active complex - r mol/m³ rate of reaction - t min. reaction time - t_j min. maintenance time - degree of conversion - _B, _F dimensionless time - min. residence time - PA penicillin acylase - PG penicillin G - PhAA phenylacetic acid - 6-APA 6-aminopenicillanic acid     

Model aided design of repeated fed-batch penicillin fermentation

Structured models of antibiotic fermentation that quantify maturation and aging of product forming biomass are fitted to experimental data. Conditions of superiority of repeated fed batch cultivation are characterized on the basis of a performance criterion that includes penicillin productivity and costs of operation. Emphasis is placed on the relevance of such research to the model aided design of optimal cyclic operation.List of Symbols c IU/mg cost factor - D s^–1 dilution rate - J IU · cm^–3 · h^–1 net productivity - k _p IU · mg^–11 · h^–1 specific product formation rate - k _pm IU · mg^–1 · h^–1 maximum specific product formation rate - p IU/cm³ concentration of penicillin - T s final time of fermentation - t s fermentation time - X kg/m³ concentration of biomass dry weight - X ₁kg/m³ concentration of young, immature biomass - X ₂ kg/m³ concentration of mature product forming biomass - X _c kg/m³ biomass concentration of the end of growth phase - X _mkg/m³ maximum biomass concentration Greek Letters s^–1 specific maturation rate - s^–1 specific aging rate - s^–1 specific growth rate - _m s^–1 maximum specific growth rate - _p s^–1 specific growth rate during the product formation phase - s cycle time - % volume fraction of draw-off Abbreviations CC chemostat culture - RFBC repeated fed batch culture - RBC repeated batch culture     

Kinetic behavior of penicillin acylase immobilized on acrylic carrier

The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols C _E g/m³ enzyme concentration - C _P,C _Q mol/m³ product concentrations - C _S mol/m³ substrate concentration - C _SO mol/m³ initial substrate concentration - K _A mol/m³ constant which defines the affinity of a substrate to the enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ mol/g/min constant rate of dissociation of the active complex - R(1) concentrational component of reaction resistance - R(2) resistance component derived from substrate affinity - R(3) resistance component due to the inhibition of the enzyme by substrate - R(4) resistance component due to the inhibition of the enzyme by PhAA - R(5) resistance component due to inhibition of the enzyme by 6-APA - r = dC_s/dt mol/m³ min rate of reaction - t min reaction time - (i) relative resistance of reaction     

Kinetic behaviour of penicillin acylase stabilized by poly (ethyleneimine)

The paper presents the kinetic evaluations of poly(ethyleneimine)-penicillin acylase preparations. The comparative studies show that the investigated system is much better than the native enzyme, and slightly worse than commercially available Boenringer preparation. Additionally, the high stability of PEI-enzyme system, very easy way of its preparation, high flexibility, and possibility to set the needed enzyme concentration are particularly favourable for use of the membrane bioreactor with PEI-enzyme system immobilized in its volume. Some advantages of the use of such bioreactor are also discussed.List of Symbols C _E IU/m³ activity concentration - C _S mol/m³ substrate concentration - C _P , C_Q mol/m³ products concentration - K _A mol/m³ constant which defines the affinity of a substrate to enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ , mol/IU min constant rate of dissociation of the active complex - mol/m³ min rate of reaction This work was supported by Government Committee of Science: Grant KBN # 3 0321 92 01     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Effect of penicillin on GABA-gated chloride ion influx

To investigate the mechanism of penicillin-induced convulsions, we have studied the effects of penicillin G (PC-G) on GABA-gated chloride ion influx in brain microsac preparations of mice. In the presence of 10^–4 M GABA, PC-G inhibited GABA-gated chloride ion influx in a dose-dependent manner. The dose-response curve for GABA in the presence of 10^–3 M PC-G was shifted rightward and there was a decrease in maximum response. The inhibitory effects of PC-G were not reversed by RO 15-1788, an antagonist of benzodiazepine (BZ) receptors, but were reversed by washing the microsac membranes. Therefore, PC-G probably exerts its proconvulsant effect by inhibiting GABA-gated chloride ion influx. However, it appears not to act through the BZ receptor of the GABA/BZ receptor complex.     

Quantitative physiology of Penicillium chrysogenum in penicillin fermentation

Using continuous and fed-batch penicillin fermentation systems some important metabolic parameters have been determined for the purpose of achieving process improvement and better process control. The specific uptake rates determined under the optimal conditions are: 0.33 mmol hexose/g cell/hr, 1.6 mmol oxygen/g cell/hr, 2mg NH₃-nitrogen/g cell/hr, 0.6 mg PO₄-phosphorus/g cell/hr, 2.8 mg SO₄-sulfur/g cell/hr, 1.8 mg phenyl acetic acid/g cell/hr. It was also found that during the production phase, or idiophase, the specific growth rate should be maintained at about 0.015 hr^?1 in order to support the maximum penicillin productivity of the given strain. Based on the results of this study a significant process improvement has been achieved through proper control of the supply and demand of the important nutrients and oxygen.     

Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

High risk of early sub-therapeutic penicillin concentrations after intramuscular benzathine penicillin G injections in Ethiopian children and adults with rheumatic heart disease

IntroductionIntramuscular benzathine penicillin G (BPG) injections are a cornerstone of secondary prophylaxis to prevent acute rheumatic fever (ARF) and rheumatic heart disease (RHD). Uncertainties regarding inter-ethnic and preparation variability, and target exposure profiles of BPG injection are key knowledge gaps for RHD control.MethodsTo evaluate BPG pharmacokinetics (PK) in patients receiving 4-weekly doses in Ethiopia, we conducted a prospective cohort study of ARF/RHD patients attending cardiology outpatient clinics. Serum samples were collected weekly for one month after injection and assayed with a liquid chromatography-mass spectroscopy assay. Concentration-time datasets for BPG were analyzed by nonlinear mixed effects modelling using NONMEM.ResultsA total of 190 penicillin concentration samples from 74 patients were included in the final PK model. The median age, weight, BMI was 21 years, 47 kg and 18 kg/m², respectively. When compared with estimates derived from Indigenous Australian patients, the estimate for median (95% confidence interval) volume of distribution (V/F) was lower (54.8 [43.9–66.3] l.70kg^-1) whilst the absorption half-life (t_1/2-abs2) was longer (12.0 [8.75–17.7] days). The median (IQR) percentage of time where the concentrations remained above 20 ng/mL and 10 ng/mL within the 28-day treatment cycle was 42.5% (27.5–60) and 73% (58.5–99), respectively.ConclusionsThe majority of Ethiopian patients receiving BPG as secondary prophylaxis to prevent RHD do not attain target concentrations for more than two weeks during each 4-weekly injection cycle, highlighting the limitations of current BPG strategies. Between-population variation, together with PK differences between different preparations may be important considerations for ARF/RHD control programs.     

A modified unstructured mathemathical model for the penicillin G fed-batch fermentation

Summary In this paper, an updated unstructured mathematical model for the penicillin G fed-batch fermentation is proposed, in order to correct some physical and biochemical shortcomings in the model of Heijnen et al. (1979,Biotechnol. Bioeng.,21, 2175–2201) and the model of Bajpai and Reuß (1980,J. Chem. Tech. Biotechnol.,30, 332–344). Its main features are the consistency for all values of the variables, and the ability to adequately describe different metabolic conditions of the mould. The model presented here can be considered as the translation of the latest advances in the biochemical knowledge of the penicillin biosynthesis.Nomenclature t time (h) - S amount of substrate in broth (g) - X amount of cell mass in broth (g) - P amount of product in broth (g) - V fermentor volume (L) - F input substrate feed rate (L/hr) - C _s S/V substrate concentration in broth (g/L) - C _x X/V cell mass concentration in broth (g/L) - C _P P/V product concentration in broth (g/L) - s _F substrate concentration in feed stream (g/L) - E _m parameter related to the endogenous fraction of maintenance (g/L) - E _p parameter related to the endogenous fraction of production (g/L) - K _x Contois saturation constant for substrate limitation of biomass production (g/g DM) - K _s Monod saturation constant for substrate limitation of biomss production (g/L) - K _p saturation constant for substrate limitation of product formation (g/L) - K _i substrate inhibition constant for product formation (g/L) - m _s maintenance constant (g/g DM hr) - k _h penicillin hydrolysis or degradation constant (hr^–1) - Y _x/s cell mass on substrate yield (g DM/g) - Y _p/s product on substrate yield (g/g) - specific substrate consumption rate (g/g DM hr) - specific growth rate (hr^–1) - _substr specific substrate to biomass conversion rate (hr^–1) - _x maximum specific substrate to biomass conversion rate (hr^–1) - specific production rate (g/g DM hr) - _p specific production constant (g/g DM hr)     

Radioactive penicillin G production by immobilized fungal vesicles

Summary Radioactive penicillin G production from l-[1-¹⁴C]-valine (1.75 GBq · mmol^-1) by native and by calcium alginate gel immobilized mycelium of Penicillium chrysogenum PQ-96 in a medium for antibiotic production as well as by vesicles isolated from the protoplasts of the same strain in a well-defined reaction mixture was investigated. Specific radioactivity of the penicillin G produced by the native vesicles was 1.45 GBq · mmol^-1 and that of the antibiotic synthesized by the calcium alginate gel immobilized vesicles was 1.48 GBq · mmol^-1. By comparison, the specific radioactivity of penicillin G produced by native mycelium was 0.42 GBq · mmol^-1 and of that synthesized by the immobilized mycelium was 0.96 GBq · mmol^-1. Production of radioactive penicillin G by native and immobilized vesicles in repeated use was also investigated. At the beginning of the production phase, the radioactive penicillin G synthesized by the immobilized vesicles was 25 nmol · mg protein^-1 · h^-1 and decreased after 8 days to a level of 11 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 7 days. The native vesicles showed a rapid decrease in radioactive antibiotic production. In comparison, the penicillin G production in a repeated use of immobilized vesicles decreased during 40 days from 140 nmol · mg protein^-1 · h^-1 to 60 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 35 days. The native vesicles showed after 4 days a lack of activity of penicillin G production. The stability of immobilized mycelium or vesicles in the process of radioactive penicillin G production is discussed.     

The effect of cultivation conditions on the penicillin production using a urethane foam-supported Penicillium chrysogenum

Urethane foam was applied to immobilize the fungus Penicillium chrysogenum in order to accelerate penicillin fermentation. Various operational conditions, such as cultivation temperature, initial pH value, composition of substrate and inoculum size of spores, which might influence penicillin fermentation significantly, were studied in a shaking flask culture system. The results are summarized as follows:

1. The maximum production amount of penicillin was achieved when cultivation temperature and initial pH were kept at 25±1°C and 4, respectively.
2. The maximum production yield of penicillin for substrate was obtainable when the concentration of lactose and that of cornsteep liquor in the basal medium were adjusted at 60 kg/m³ and 30 kg/m³, respectively.
3. It was found in this cultivation system that the optimum region concerning inoculum size was enlarged from 7.0·10⁵ to 1.0·10⁷ spores/dm³ while that of the traditional mehtod was very narrow around 1.0·10⁷ spores/dm³. Moreover, the production amount of penicillin produced by this new method was about twelve times as high as that produced by the traditional method.

     

Determination of glucose,urea and penicillin using enzyme-pH-electrodes

Conventional hydrogen ion glas electrodes have been used for the preparation of enzyme-pH-electrodes by either entrapping the enzymes within polyacrylamide gels around the electrode or as liquid layer trapped within a cellophane membrane. The enzymes were glucose oxidase, urase and penicillinase.The pH response to glucose concentration was about linear within 10^?1–10^?3 M glucose and for urea linear within 5·10^t—–5·10^?5M. The pH response to penicillin was about linear in the range from 10^?3–10^?2 M resulting in a pH shift of 1.4 units; reproduceable pH response was obtained down to concentrations of 3·10^?5 M.Studies as to the effect of buffer using an urease–pH-electrode showed a buffer concentration of 10^?2 M a substantial shift of about one pH-unit in the range of 10^?4 to 10^?2 M urea. Both urease- and penicillinase–pH-electrodes were tested as to stability showing no decrease in pH response except at high substrate concentration (1·10^?2 M) over a period of 2–3 weeks kept at room temperature.     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm³,100 dm³ and 1000 dm³ working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (q _pen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.List of Symbols C.E.R. mmol/(dm³h) Carbon dioxide evolution rate - D m Impeller diameter - D.O.T. % air saturation Dissolved oxygen tension - L _e m Mean effective length or main hyphal length - O.U.R. mmol/(dm³h) Oxygen uptake rate - P W Total power dissipation - q _pen units/(mg dry cell weight h) rate Specific penicillin production - R.Q. Respiratory quotient - 1/t _cs^–1 Circulation frequency     

Reactivation of immobilized penicillin G acylase: Influence of cosolvents and catalytic modulators

Reactivation of penicillin G acylase immobilized in glyoxyl-agarose after inactivation was studied with the purpose of increasing the lifespan of the biocatalyst by simple and reproducible strategies, considering unfolding–refolding and direct incubation in reactivation media. Reactivation yields were increased with respect to the control (fully aqueous medium) when cosolvents were added to the reactivation medium at concentrations below 50% (v/v). Best results were obtained with 30% (v/v) ethyleneglycol (EG) in both reactivation strategies. An increase in reactivation yield from 36.0 to 62.8% was obtained using the unfolding–refolding strategy, while an increase from 50.0 to 68.4% was obtained by direct incubation in aqueous media with respect to control. Catalytic modulators were also included in the reactivation medium: competitive inhibitors (phenylacetic acid and 2-thienylacetic acid) caused a reduction while non-competitive (7-ADCA and 6-APA) caused an increase in reactivation yield. Combining cosolvent and catalytic modulators, best results in both strategies were obtained with 30% (v/v) EG plus 100 mM 7-ADCA, where an increase in reactivation yield from 36.0 to 96.0% and from 50.0 to 98.0% was achieved with unfolding–refolding and direct incubation in reactivation media respectively. Apparent reactivation rate was higher in the case of direct incubation in reactivation media, best results being obtained when using 100 mM 7-ADCA and 30% (v/v) EG, with an increase with respect to the control (fully aqueous medium with no modulator) from 0.309 h^?1 to 1.129 h^?1, while for unfolding–refolding strategy increase was only from 0.124 h^?1 to 0.384 h^?1. Results indicate that direct incubation is a better strategy for penicillin G acylase reactivation and opens up the possibility of significantly increasing the operational lifespan of the biocatalyst by operating the reactor with repeated cycles of reaction and reactivation.     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Chemiluminescence assay for Listeria monocytogenes based on Cu/Co/Ni ternary nanocatalyst coupled with penicillin as generic capturing agent

Bacterial pathogen control is important in seafood production. In this study, a Cu/Co/Ni ternary nanoalloy (Cu/Co/Ni TNA) was synthesized using the oleylamine reducing method. It was found that Cu/Co/Ni TNA greatly enhanced the chemiluminescence (CL) signal of the hydroxylamine‐O‐sulfonic acid (HOSA)–luminol system. The CL properties of Cu/Co/Ni TNA were investigated systemically. The possible CL mechanism also was intensively investigated. Based on the enhanced CL phenomenon of Cu/Co/Ni TNA, a Cu/Co/Ni TNA, penicillin, and anti‐L. monocytogenes (Listeria monocytogenes) antibody‐based sandwich complex assay for detection of L. monocytogenes was established. In this sandwich CL assay, penicillin was employed to capture and enrich pathogenic bacteria with penicillin‐binding proteins (PBPs) while anti‐L. monocytogenes antibody was adopted as the specific recognition molecule to recognize L. monocytogenes. L. monocytogenes was detected sensitively based on this new Cu/Co/Ni TNA–HOSA–luminol CL system. The CL intensity was proportional to the L. monocytogenes concentration ranging from 2.0 × 10² CFU ml^?1 to 3.0 × 10⁷ CFU ml^?1 and the limit of detection wa 70 CFU ml^?1. The reliability and potential applications of our method was verified by comparison with official methods and recovery tests in environment and food samples.     

Immobilization of penicillin acylase on acrylic carriers

Penicillin acylase obtained from E. Coli (E. C. 3.5.1.11) was covalently bound via glutaric aldehyde to acrylic carriers crosslinked with divinylbenzene or ethylene glycol dimethacrylate. The best enzymatic preparation was obtained by using ethyl acrylate/ ethylene glycol dimethacrylate copolymer. 1 cm³ of the carrier bound 6.4 mg of protein, having 72% activity in relation to the native enzyme. The preparation lost only 10% of its initial activity after 100 d of storage at 4°C. A negligible effect of immobilization on the enzyme activity at different temperatures or pH as well as significant increase of the stability of the immobilized enzyme at elevated temperatures were observed.Abbreviations BA butyl acrylate - AE ethyl acrylate - PA penicillin acylase - 6-APA 6-aminopenicillanic acid - EGDMA ethylene glycol dimethacrylate - DVB divinylbenzene