I. de la Mata

Biotechnological applications of penicillin acylases: state-of-the-art.

Abstract. This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic β-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

New insights on nucleoside 2′-deoxyribosyltransferases: a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs

In recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2′-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

Abstract At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

Purification and characterization of penicillin V acylase from Streptomyces lavendulae

Penicillin V acylase was isolated and purified from culture supernatants of Streptomyces lavendulae. The enzyme that is largely extracellular was purified to homogeneity. Two substrates penicillin V and NIPOAB were used for inhibition studies. The kinetic constants were: KM(penV)=4.9mM, Vmax(penV)=0.47 μmol.min1.mg1; KM(NIPOAB)=11.9mM and Vmax(NIPOAB)=4.94×103 μmol.min1mg1. Penicillin G, phenoxyacetic acid, phenylacetic acid and 6-APA were competitive inhibitors but they inhibited slightly the enzyme. This results were interesting for the possible use of this penicillin V acylase in industrial biorreactors.

Chemical modification of tryptophan residues of d-amino acid oxidase from Rhodotorula gracilis

Abstract d -Amino acid oxidase was inactivated by N -bromosuccinimide (NBS) at 30°C and pH 8. The reaction followed pseudo-first order kinetics with second-order rate constants of 69.8 mM −1 min −1 for the apoenzyme and 0.63 mM −1 min −1 for the holoenzyme. The presence of substrates or benzoate protected the enzyme against inactivation. Difference absorption spectra at 280 nm, low consumption of NBS per mole of enzyme, the decrease in the fluorescence emission at 335 nm, integrity of the protein backbone and the absence of cysteine oxidation pointed to the modification of tryptophan residues. The statistical analysis of the residual fractional activity vs. the number of modified tryptophan residues led to the conclusion that one tryptophan residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme.