Showing papers on "Penicillin amidase published in 2009"
TL;DR: The microstructure and the catalytic properties of crosslinked enzyme aggregates of penicillin acylase (PA) obtained under different conditions were investigated in this article, where the period of time left between enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation.
Abstract: The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 16 μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA
48 citations
TL;DR: Two proteolytic enzymes were chosen: penicillin amidase and caspase-3, and the corresponding self-cleavable chemiluminescent substrates were synthesised, and their evaluation using an in vitro assay has enabled us to prove the decomposition of the linker under physiological conditions and the selectivity for the targeted enzyme.
Abstract: A new generation of dioxetane-based chemiluminescent substrates suitable for detecting protease activities is described. Our strategy involves the use of a self-cleavable spacer as the key molecular component of these protease-sensitive chemiluminescent probes. Among the assayed strategies, the PABA (para-aminobenzylic alcohol) linker associated with an ether linkage enables the release of the light-emitting phenolic 1,2-dioxetane moiety through an enzyme-initiated domino reaction. To validate this strategy, two proteolytic enzymes were chosen: penicillin amidase and caspase-3, and the corresponding self-cleavable chemiluminescent substrates were synthesised. Their evaluation using an in vitro assay has enabled us to prove the decomposition of the linker under physiological conditions and the selectivity for the targeted enzyme.
41 citations
TL;DR: A self‐renaturing enzyme‐chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous‐organic mixtures is designed to broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions.
Abstract: Molecular chaperones in aqueous-organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self-renaturing enzyme-chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous-organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme-chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous-methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems.
34 citations
TL;DR: Experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium that implies significant savings in terms of costs and environmental protection.
Abstract: The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.
23 citations
TL;DR: The potential of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process, was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation.
Abstract: Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross-linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re-incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472–479. © 2009 Wiley Periodicals, Inc.
21 citations
TL;DR: In this article, the 3G3K mutant of the Penicillin G acylase (PGA) from E. coli obtained by site-directed mutagenesis was investigated.
21 citations
TL;DR: Combining macromolecular crowding with glycine quenching was one of the efficient strategies adopted to improve microwave-assisted covalent PA immobilization.
20 citations
TL;DR: It was observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 degrees C or lower temperatures, and the elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
17 citations
TL;DR: Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, and the optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier−1 in the hydrolysis of penicillin V at pH 8.0 and 40°C.
Abstract: Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier−1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t1/2=625 h vs. t1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.
12 citations
TL;DR: The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.
Abstract: We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol–water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol–water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the β-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37°C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol–water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis. Biotechnol. Bioeng. 2009;102: 417–424. © 2008 Wiley Periodicals, Inc.
12 citations
TL;DR: In this article, a two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved.
Abstract: A two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved. Ethylene glycol (EG) was chosen as the organic co-solvent. Factors including co-solvent content, enzyme loading, reaction temperature and substrate concentration were investigated. The optimum conditions were as follow: pH 8.0 phosphate buffer solution, 50% EG (v/v), 25 °C, 100 mM PGK and 300 mM d -phenylglycine methyl ester (D-PGM), 43.2 IU/ml IPA-750. The maximum yield was 57.3% after a reaction time of 17 h. It is the first report about the synthesis of ampicillin from penicillin G potassium salt in one-pot combining the enzymatic hydrolysis and the subsequent enzymatic condensation, and the novel methodology will have important application in the β-lactam antibiotics industry.
TL;DR: It was found that solvents with high hydrophobicity and lower dielectric constant were relatively effective in increasing PVA activity, and systematic selection of suitable solvent for best performance was allowed.
Abstract: Erwinia aroideae (DSMZ 30186) is a potential microbial culture to produce intracellular penicillin V acylase (PVA). The whole cell PVA activity was improved by permeabilization with various organic solvents. The cell-bound PVA activity showed an eightfold increase upon treatment with chloroform (5 μL/mg dry biomass ) for 10min and diethyl ether (10 μL/mg dry biomass ) for 45min. Hexane, toluene, ethyl acetate and dichloromethane enhanced the enzyme activity up to two-, six-, four- and two-fold, respectively; whereas, PVA activity declined drastically on permeabilization with acetone, pyridine and alcohols. The physicochemical properties of the organic solvents used for permeabilization were correlated with the change in activity. It was found that solvents with high hydrophobicity (log P>0.68) and lower dielectric constant (<9) were relatively effective in increasing PVA activity. These results allow systematic selection of suitable solvent for best performance.
TL;DR: Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.
Abstract: Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.
TL;DR: In this article, the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation was investigated, and the factorial design indicated that the main factors that positively affect enzyme production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H 2O, Na2SO4, and casein carbonate and magnesium chloride.
Abstract: The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.