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

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption. Copyright 2000 John Wiley & Sons, Inc.

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, Km = 0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, Km = 0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (Ki = 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 (K(S)' = 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)/Km = 2.1 x 10(-7) M(-1) s(-1)) and benzylpenicillin (k(cat) = 54 s(-1) and k(cat)/Km = 1.3 x 10(-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.