Search

Penicillin G acylase (PGA) is an enzyme that hardly interacts with polycationic polymers (e.g., polyethyleneimine, PEI) and thus the enzyme cannot be stabilized against the action of organic solvents by its co-immobilization with the polymer in the same support, neither covalently attached to the support nor adsorbed on the already immobilized enzyme. However, a new mutant PGA bearing eight additional Glu residues homogenously distributed throughout the enzyme surface may interact with the polymer. The co-immobilization of the enzyme and PEI on glyoxyl-agarose allows one to fully take advantage of the stabilization produced by the multipoint covalent attachment and by the protective hydrophilic micro-environment generated by the polycationic polymer, enabling a significant stabilization of the immobilized PGA in the presence of organic solvents.

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions:? chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.

The production, under environmentally benign conditions, of efficient and more cost-effective anti-infective agents (available to the whole mankind) is one of most ambitious dreams of the industrial medicinal chemistry. Semi-synthetic βlactam antibiotics are very effective anti-infective agents. They are very stable and can be used via oral delivery. They exhibit a very wide spectrum of anti-bacterial activity and minimal side effects after being massively used for a very long time. In this way, we can assume that semi-synthetic β-lactam antibiotics are going to be one of the key anti-infective agents in the next years. The condensation of natural or modified antibiotic nuclei with different acyl donor chains is one of the key steps for the industrial synthesis of these anti-infective agents. Up to now, these condensations are mainly carried out through classical chemical methods and this implies a number of economical, ecological and technological drawbacks (high energy requirements, many protection and deprotection steps, utilization of toxic methylene chloride as solvent, etc). Enzyme biocatalysts may be very useful to catalyse these selective condensations under very mild experimental and environmental conditions. In fact, the possibility of using enzymes to carry out such biotransformations, at laboratory scale, has been discussed and demonstrated a long time ago. However, industrial synthesis of βlactamic antibiotics is still carried out via unfavorable chemical routes. In fact, enzymes have not been produced in the nature to act in industrial reactors; so they are usually very unstable, inhibited by substrates and products and they may not have ideal catalytic properties for industrial uses (high reaction rates, required selectivity, ability to reach quantitative synthetic yields, stable enough to run a number of reaction cycles, etc). These limitations of enzyme biocatalysis become even more significant mainly when the enzyme is going to be used with non-natural substrates, catalysing non-natural processes and working under non-conventional conditions. However, in the last ten years, a great number of papers have reported substantial improvement of these enzymatic synthetic approaches (new enzymes, better enzyme derivatives, improved reaction designs and so on) and it seems that a massive industrial implementation of enzymes in antibiotic synthesis is approaching. In this review, we would like to make a critical discussion of these very interesting advances in the application of enzyme biocatalysts for the industrial synthesis of semi-synthetic antibiotics.