Showing papers on "Penicillin amidase published in 1987"

Penicillin Amidase from E. coli

Abstract: Penicillin amidase (EC 3.5.1.1 1) is an enzyme that can catalyze the hydrolysis and synthesis of amide bonds between phenylacetic or phenoxyacetic acid and their derivatives and 6-aminopenicillanic acid (6-APA) or 7-aminocephalosporanic acid (7-ACA) and its derivatives.' Penicillin amidases with different molecular weights (60-260 kDa), substrate specificities and pH optima have been found in more than 100 microorganisms. The biological function of these enzymes is still not known. The enzymes from E . coli (penicillin G amidase) and P. ostreatus (penicillin V amidase) are currently used on an industrial scale for the hydrolysis of penicillin G and V2 respectively. The main product-6-APA-is used for the synthesis of semisynthetic penicillins and cephalosporins. These enzymes can also be used as biocatalysts for the synthesis of the semisynthetic 8-lactam antibiotics. Two different processes can be applied here.3 In the equilibrium controlled process the enzyme is used as a hydrolase in the reverse direction that accelerates the condensation reaction to its thermodynamic equilibrium. The product yield cannot be influenced by the properties of the enzyme. In the second process-kinetically controlled-activated substrates must be used. The enzyme is here used as a transferase that catalyzes the transfer of the side-chain to the P-lactam nucleophile (6-APA, 7-ACA etc.). The maximum product yield in this process is a function of the properties of the enzyme.' Thus, for the kinetically controlled process the enzyme properties that influence the yield must be known. To obtain this information studies using pure enzyme are required. Some results on the E . coli penicillin amidase pertinent in this context are presented here.

Hydrophobic interaction chromatography of penicillin amidase

Abstract: For hydrophobic binding of penicillin amidase to modified Sepharose, a phenyl group or a hydrophobic aliphatic moiety (leucyl, octyl) is necessary. Concentration and purification of the enzyme can then be achieved in a single step.

Penicillin Acylase Activity and Stability in PEG Solutions

Abstract: Penicillin acylase (EC 3.5.1 . I I)(PA) is a commercial enzyme used for the deacylation of benzylpenicillin to 6-aminopenicillanic acid (6-APA), which is used for the production of semisynthetic penicillins. The recirculation of the enzyme has been studied in an aqueous two-phase system.' The most efficient phase system, in which PA was partitioned almost totally to the bottom phase, contained high concentrations of polyethylene glycol (PEG) and potassium phosphate. High concentrations of PEG have been reported to influence the activity of PA.' The influence of PEG on the stability of invertase has recently been reported.' The activity of PA was investigated in solutions of PEG with differcnt molecular weight and different concentrations in relation to the water activity of the solutions (FIG. I ) . The water activity of the PEG solutions was calculated according to a formula found by Norrish4

Two molecular forms of penicillin amidase synthesized by Escherichia coli.

Abstract: The Swatek's method was further simplified for the assay of penicillin amidase activity. The absorbance of colour obtained during determination of 6-aminopenicillanic acid was dependent on concentration of 4-dimethylaminobenzaldehyde and on temperature. Antiodies induced in rabbits with one molecular form of penicillin amidase from E. coli PCM 271 (PA-1 or PA-2) did not cross-react with the other amidase form. No differences in substrate specificity on inactivation with SDS and in alkaline medium between the two amidase forms were observed. Concentrated urea inactivated PA-2 irreversibly and PA-1 reversibly. N-Bromosuccinimide inactivated almost completely only PA-1. Two E. coli PCM 271 strain variants were separated by microbial selection. Each of them produced only one amidase form. Also two amidase forms were found in cells of E. coli ATCC 11105, whereas E. coli ATCC 9636 and ATCC 9637 synthesize only PA-1.

Enzyme-linked immunosorbent assay (ELISA) of penicillin amidases.

Abstract: Non-competitive, sandwich enzyme immunoassay for both penicillin amidases from Escherichia coli is described. The assay involves the use of monospecific antibodies and their conjugates. The amidases inactivated by heating and by acid- or alkali-treatment cannot be assayed. Reproducible results for each amidase were achieved within 5-6 hours in the range of 3-500 ng/ml (0.25-40 mU/ml) and coefficient of variation was 13%.