Showing papers on "Penicillin amidase published in 1997"

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis

Abstract: Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers

Abstract: The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 by cross-linking with dextran dialdehyde molecules, at a molecular mass of 11 500, 37 700 and 71 000 Da, was studied. The thermal inactivation mechanisms of the native and modified PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active but temperature-sensitive transient states. The highest enhancement to the thermostability of PGA was obtained using dextran-71000-dialdehyde modification, as a␣nearly ninefold increase at temperatures above 50 °C. The modification of PGA by dextran-11500-dialdehyde resulted in a considerable reduction of the Vm and Km parameters of the enzyme. However, other dextran dialdehyde derivatives used for modification did not cause a meaningful change in either Vm and Km. Modification by dextran dialdehyde derivatives did not result in significant change to either the optimal temperature or the activation energy of PGA. All modified PGA preparations showed lower inactivation rate constants but higher half-lives for inactivation than those of the native PGA at all temperatures studied. As indicated by the half-life times and ki values, dextran 71000-dialdehyde was found to be more effective at cross-linking in the thermo-stabilization of PGA than any other agent studied in this work.

Stabilization of Escherichia coli penicillin G acylase by polyethylene glycols against thermal inactivation.

Abstract: The effects of five polyethylene glycol (PEG) compounds of different molecular weight on the thermal stability of penicillin G acylase (PGA) obtained from a mutant ofEscherichia coli ATCC 11105 have been investigated. The molecular weights of PEG compounds were 400, 4000, 6000, 10,000, and 15,000. The thermal inactivation mechanisms of both native and PEG-containing PGA were considered to obey first order inactivation kinetics during prolonged heat treatments. Optimal concentrations of PEGs at molecular weights of 400,4000, 6000,10,000, and 15,000 were found to be 250,150,150,100, and 50 mM, respectively. The greatest enhancement of thermostability was observed with PEG 4000 and PEG 6000, as a nearly 20-fold increase above 50°C. PGA showed almost the same temperature activity profile and optimal temperature values both in the presence and absence of PEG. The addition of PEGs did not cause any change in the optimal temperature value of PGA, but the parametersV m ,K m , the activation energy, and thek cat values of enzyme were markedly decreased because of the mixed inhibition by PEG compounds. The type of inhibition was found to be hyperbolic uncompetitive.

Bacillus sphaericus Penicillin V Acylase: Purification, Substrate Specificity, and Active-Site Characterization

Abstract: Penicillin V acylase from Bacillus sphaericus was purified to homogeneity with an overall yield of 15%. The enzyme exhibited comparatively high specificity for penicillin V, penicillin G, and other related compounds being hydrolyzed at less than 10% of the rate of penicillin V. Moreover, the high rate of hydrolysis was observed when the side chain of the substrate molecule was unsubstituted. Lysine-modifying reagents inactivated the enzyme rapidly. Kinetics and titration studies indicated the involvement of lysine in the catalytic activity of the enzyme.

Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads.

Abstract: Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide andN,N’- methylenebisacrylamide. A solution of monomer that was made up from methacrylamide andN,N’-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013–0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45‡C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.

Cross-linked stabilization of Escherichia coli penicillin G acylase against pH by dextran-dialdehyde polymers

Abstract: The inactivation kinetics of Escherichia coli penicillin G acylase (PGA), and cross-linked stabilization of the enzyme by dextran-dialdehyde derivatives of molecular weights of 11500, 37000 and 71000, were similar from pH 2 to pH 10. Inactivation of the native and modiÞed PGA obeyed Þrst order kinetics. The lowest inactivation rate constants for native and dextran-11500-dialdehyde modiÞed PGA were 9.0 3 10 Ð4 and 1.5 3 10 Ð4 min Ð1 respectively at pH 7.0. The highest pH stabilization (nearly ten-fold) was obtained at pH 7.0.

Modified fluorimetric assay for estimating ampicilloate concentrations and its use for detecting β-lactamase and penicillin acylase activity in bacteria

Abstract: Sodium ampicilloate concentrations were estimated fluorimetrically by heating solutions with ascorbic acid, EDTA and a modified Lowry A reagent which was prepared by including copper sulfate and potassium sodium tartrate in 0.5 mol dm - 3 acetate buffer at pH 4. A concentration range of 0.5–50 µmol dm - 3 was used for the estimations. The reaction was used to estimate β-lactamase activity on ampicillin but the substrate also showed some fluorescence and a calculation was required to determine the amount of ampicilloate formed when both substances were present in the one reaction mixture. The β-lactamase was inhibited by treatment with trichloroacetic acid so the procedure could be used to assay the enzyme activity after a fixed time. 6-Aminopenicillanic acid did not fluoresce on treatment with the modified reagent and organisms which contained penicillin acylase lowered the amount of ampicillin which could be converted to ampicilloate. When penicillin acylase and β-lactamase co-existed in the one organism, the respective activities were determined by use of the copper–ascorbate–EDTA fluorescence assay for ampicilloate coupled with a fluorescamine assay for 6-aminopenicillanic acid determinations. On prolonged incubation, some organisms containing penicillin acylases lowered the amount of ampicilloate which formed a fluorescent product. This effect was attributed to deacylation of ampicilloate by the penicillin acylases.

Purification of Penicillin G Amidase using Quaternary Ammonium Salts and effect on the activity of the immobilised enzymes

Abstract: This paper describes the purification of Penicillin G Amidase (EC 3.5.1.11) using quaternary ammonium salts with the aim of increasing the activity of immobilised enzymes prepared from the purified solutions. Two different quaternary ammonium salts were tested with different solutions of the enzyme. It was concluded that the quaternary ammonium salts used selectively precipitated the non-enzymatic protein leaving in solution practically all the enzyme resulting in a high yield of purification. Optimal conditions for purification using the two types of quaternary ammonium salts were determined. Immobilisation studies were performed from various purified enzyme solutions, using different amounts of a quaternary ammonium salt. The immobilised enzymes so obtained showed a much higher activity than the immobilised enzyme obtained from non-purified enzyme solutions.

Biocatalysis in the Chiral Recognition of meso‐Diamides ‐ An Efficient Route from Cyclic Olefinic Hydrocarbons to Optically Pure Diamino‐ Polyols.

Abstract: meso-Diamino-di(tri,tetr)ols 1 – 8 were synthesized starting from cheap carbocyclic olefins and cis -diepoxy derivatives. Enantioselective hydrolysis of the corresponding bis(phenylacet)amides with penicillin amidase from E. coli (EC 3.5.1.11) was effected in good yields (73 – 90%) and with high optical purities (ee 91 − >97).

Chemoenzymic Synthesis of Pα-Methyl Deoxynucleoside Triphosphates.

Abstract: 5′-O-(methylphosphonyl)-N-(phenylacetyl)-2 ′-deoxycytidine, deoxyadenosine and deoxyguanosine were pyrophosphorylated and the resulting N-protected P α-methyl nucleoside triphosphates were deblocked by treatment with penicillin amidase at pH 7.8, 25°C to give P α-methyl nucleoside triphosphates.