Kinetic study of penicillin acylase from Alcaligenes faecalis

Abstract

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m -0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, K m = 0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (A\ = 0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (As' = 54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6-nitro-3-phenylacetamidobenzoic acid (k cat = 95 s _1 and k cat IK m = 2.1 X10 7 M _1 s _1 ) and benzylpenicillin (A cat = 54 s _1 and k cat IK m -1.3 X10 -7 M 1 s 1 ) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

“…Although NIPAB and analogous spectroscopic substrates have been used in screening procedures for acylase activities in microorganisms (13, 16 -18) and in kinetic studies (4,19,20), their use for determination of kinetic parameters for the conversion of invisible substrates has not been described. With the method reported here, steady-state kinetic constants for invisible substrates, including inhibition constants of the product, can be obtained by measuring the effect on the time course of hydrolysis of a chromogenic reference substrate.…”

“…The 6-APA produced during hydrolysis of penicillin G can be detected directly using HPLC methods, by fluorescence measurements after reaction with fluorescamine (2), or by colorimetric measurements after derivatization with 4-(dimethylamino)benzaldehyde (3). The released phenylacetic acid can also be monitored by acid/base titration (4).…”

Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents

“…[11] In our continued investigations of the enzymatic synthesis of ampicillin in organic media at low water activity we turned our attention to the relatively unknown penicillin acylase from Alcaligenes faecalis [12,13] on account of its stability [13] and pH tolerance. [14] We now report the effects of the nature of the solvent and the water content on the course of the enzymatic coupling of d-PGA and 6-APA using a CLEA of A. faecalis penicillin acylase. Our goal was to increase the synthesis/hydrolysis ratio by reducing the amount of water in the system, thereby providing a more economically viable process.…”

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

“…[4][5][6][7] Moreover, it may be employed in many other reactions, like the synthesis of antibiotics, resolution of racemic mixtures, synthesis of amides and selective deprotections. [8][9][10][11][12][13][14][15] Although PGA has a moderate thermostability which has permitted its rapid implementation in hydrolytic reactions, it is rapidly inactivated by most of the currently used organic cosolvents or in anhydrous media, [16][17][18][19][20] reaction conditions which are required for antibiotic synthesis to shift thermodynamic equilibrium in the synthetic direction or to increase the solubility of hydrophobic compounds. [14,[21][22][23][24][25][26] One strategy to improve the enzyme stability in organic media may be to generate hydrophilic environments around the enzyme, promoting a certain partition of the organic solvent molecules and a drop in the organic solvent concentration in the enzyme environment.…”