Showing papers on "Penicillin amidase published in 1984"

Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin.

Abstract: Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.

Novel approaches to the purification of penicillin acylase.

Abstract: Penicillin acylase ofE. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0–7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purifiedf enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.

Penicillin acylase from the hybrid strains Escherichia coli 5K(pHM12): enzyme formation and hydrolysis of beta-lactam antibiotics with whole cells.

Abstract: Penicillin acylase formation by the hybrid strain Escherichia coli 5K(pHM12) was studied under different culture conditions and reached 200 to 250 mumol of 6-aminopenicillanic acid per min per g of bacteria (wet weight) for penicillin G. The Km of whole-cell acylase was determined with 9 to 11 mM for penicillin G at a pH optimum of 7.8 at 45 degrees C. A competitive product inhibition for phenylacetic acid of Ki = 130 mM was found. 6-Aminopenicillanic acid acts as a noncompetitive inhibitor, with a Ki of 131. The temperature optimum of the reaction lies at 54 degrees C. Penicillin G inhibits the reaction at Ki(S) = 1,565 to 1,570 mM. Whole-cell acylase reacts on a wide spectrum of penicillins and cephalosporins, but those substrates with a delta-aminoadipyl rest are not hydrolized. beta-Lactamase activity of less than 1% relative to the acylase activity was found at reaction temperatures between 28 and 45 degrees C. After a comparison of different methods for the estimation of beta-lactamase activity, we found that high-pressure liquid chromatography is to be preferred. During batch fermentation of E. coli 5K(pHM12), problems of plasmid stability in the host strain arose which were overcome by the addition of 4 mg of tetracycline per liter to the medium as a selective marker.

Phenylalkylsulfonyl derivatives as covalent inhibitors of penicillin amidase.

Abstract: It was demonstrated that phenylmethanesulfonyl fluoride-a very potent inhibitor of penicillin amidase from Escherichia coli-binds covalently to the enzyme in molar ratio 1:1. The chloride, the azide and the N-hydroxysuccinimide ester of phenylmethanesulfonic acid are also very strong inactivators of the amidase. Weaker inhibition was noted with para-substituted phenylmethanesulfonyl chlorides and with phenylethanesulfonyl and alkylsulfonyl chlorides. The inactivated amidase could be reactivated by incubation either with 6-amino-penicillanic acid or with proteins from E. coli extract. Benzyl isocyanate is also a potent covalent inhibitor of the amidase but inactivated amidase could be not reactivated in this way. It was demonstrated that representatives of all inactivator types bind to one active site of the amidase. Interdependence between inactivation rate and stability of some sulfonyl inhibitors was observed. No inhibition was noted the amide, the hydrazide and the methyl ester of phenylmethanesulfonic acid.

Kinetically Controlled Semisynthesis of β‐Lactam Antibiotics and Peptidesa

Abstract: where an activated substrate must be used, and the enzyme carries out a group transfer reaction. The yield of the desired product in this case is often much larger than the yield for the equilibrium-controlled process.'-' This requires that the catalyst be removed when the maximum is reached, and this is easily performed with immobilized enzymes. The detailed reaction mechanism for the kinetically controlled process' must be known for' a rational analysis of the factors controlling yield. Two different mechanisms have been proposed here. They are given in TABLE I . The mechanism without binding of the nucleophile N (6-APA i n Eq. 2, or an amino-acid derivative in peptide semisynthesis), before the deacylation of the acylenzyme E-A, is the most frequently used mechanism in the literature in this field. The other mechanism where the nucleophile N is bound to the acylenzyme before the deacylation may be inferred from the sequence specificity of the hydrolases and the principle of microscopic reversibility. The two mechanisms can be distinguished by measuring the initial rates of formation of AN (oaN) and A (uA) as functions of nucleophile concentration. The expressions for this and the yield at the kinetically controlled maximum are given in TABLE I.' Once the mechanism has been established, the yield-controlling factors can be rationally analyzed to evaluate the biotechnological potential of such processes. Some data pertinent to the kinetically controlled, enzyme-catalyzed synthesis of semisynthetic penicillins and peptides are presented here. Free and immobilized penicillin amidase (EC 3.5.1.1 1) from E. coli, the substrates and 6-APA used to prepare semisynthetic penicillins were gifts from Dr. Sauber (Hoechst AG) and Dr. Kramer (Rohm), respectively. Phenyl-acetyl-glycine was

A sensitive procedure for screening microorganisms for the presence of penicillin amidase.

Abstract: A procedure is described for screening bacteria for the presence of penicillin amidase. Cells, grown in the presence of phenylacetic acid, are incubated with phenoxymethylpenicillin (type I), benzylpenicillin (type II) or ampicillin and the 6-aminopenicillanic acid formed is detected and quantitatively estimated by its strong reaction with fluorescamine at pH 4. There is no requirement for separation of the penicillin substrate from the product but when a-aminobenzylpenicillin derivatives are used as enzyme substrates the amount of 6-aminopenicillanic acid formed must be determined by calculation. The procedure allowed positive and reliable identification of penicillin amidases in six organisms known to produce the enzyme and indicated that some of these enzymes had different properties in reactivity towards a-aminobenzylpenicillin derivatives.

A support-spacer-ligand system for specific affinity adsorption of penicillin acylase

Abstract: An affinity system for specific adsorption of penicillin acylase is reported in the present work. When all the active enzyme was adsorbed by an appropriate amount of carrier, 50% of the total protein containing only inactive portion remained in solution exhibiting strong and preferential adsorption of enzyme.