Modulación de las propiedades catalíticas de la penicilina G acilasa mediante la inmovilización dirigida de la enzima nativa y modificada genéticamente

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-03-2006Penicillin G acylase (PGA) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6- aminopenicillanic acid, the starting material for the synthesis of several semi-synthetic antibiotics. This enzyme can also be used to catalyze the inverse reaction of semi-synthesis of plactam antibiotics. PGA from Escherichia coli is the most widely used hydrolytic and synthetic enzyme. This acylase is used as immobilized derivative on solid supports in order to ensure good activity-stability properties under operational conditions. However, it has been reported that different immobilized derivatives of PGA may exhibit different catalytic properties. The main objective of this Ph.D. Thesis is the preparation of immobilized derivatives of native and recombinant PGA with good synthetic properties for the kinetically controlled synthesis of a precursor of Cephamandole. This main objective has been focussed via three main sub-objectives: a.- Evaluation of synthetic properties of different derivatives of native PGA. Different derivatives has been prepared: a.- with different orientation of the enzyme on the support, b.- by using supports with different physical properties, c.- by blocking the support (after immobilization of the enzyme) with different small ligands. These derivatives have been evaluated regarding to a critical parameter in enzymatic synthesis: the ratio between synthetic and hydrolytic activity of the immobilized enzyme. b.- By using a recombinant PGA (with a number of Lys groups on the opposite face to the active centre) we have been able to immobilized the enzyme (through this region) on glyoxyl-agarose. The synthetic properties of these new derivatives were also evaluated. C.- A new mutant of PGA was designed in order to achieve the best immobilization and the best synthetic properties. The pac gene from E.coli ATCC 11105 was mutagenized by PCR at its 3' end, corresponding to the end of the 13 chain. The overproduction of the recombinant enzyme was obtained by cloning the mutagenized pac gene finto the pET101 /D-TOPO expression vector. After purification, the mutant was immobilized and the catalytic properties of the free and immobilized enzyme were compared with those of the native enzyme, in the kinetically controlled synthesis of an intermediate of Cefamandol. The introduction of this new tag improves the immobilization efficiency as well as the catalytic properties of the immobilized enzyme. In particular, the immobilized mutated enzyme shown much better synthetic properties than the native enzyme and maintained its catalytic efficiency also after immobilization