Carmen Acebal

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.

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

SummaryTrichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.

Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition.

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.

Interaction of penicillin V acylase with organic solvents:: Catalytic activity modulation on the hydrolysis of penicillin v

Abstract The modulation of hydrolytic activity of penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae by organic solvents is reported. On one hand, the addition of water-soluble cosolvents increases the catalytic activity up to a critical concentration of the non-aqueous solvents, yet further increase of the latter leads to protein denaturation. For alcohols and aprotic polar solvents, there are linear correlations between the critical concentration of water miscible cosolvent (at which enzyme deactivation does not begin to take place) and the dielectric constant of the cosolvents added. On the other hand, water-immiscible solvents can show activating or inhibitory effects that may be related to interactions between the structure of the solvent and the enzyme.

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

ABSTRACT A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (Tm) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.

Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of recombinant poly-3-hydroxybutyrate depolymerase from Streptomyces exfoliatus

Cross-linked enzyme aggregates of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces exfoliatus (PhaZ(Sex)-CLEAs) have been prepared. Acetone was used as the precipitating agent, while addition of bovine serum albumin (BSA) facilitated CLEAs formation. Conditions for enzyme precipitation and cross-linking have been optimized, and confocal scanning microscopy showed a homogeneous enzyme distribution in the biocatalyst. Obtained PhaZ(Sex)-CLEAs presented an average size of 50-300 μm, showing a high PHB depolymerase activity of 255 U/g wet biocatalyst at 40°C and pH 7.0. Temperature-activity profile of PhaZ(Sex)-CLEAs at pH 8.0 showed that the highest activity for pNPB hydrolysis was achieved at 60°C, whereas pH-activity profile at 40°C indicated that highest activity for PHB hydrolysis was achieved at pH 7.0. Additionally, immobilized biocatalyst could be recycled at least for 20 consecutive batch reactions without loss of catalytic activity, and showed higher pH and temperature stability, and better tolerance to several organic solvents than its soluble counterpart.

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.

Lysolecithin:Lysolecithin acyltransferase from rabbit lung: Enzymatic properties and kinetic study

Abstract The enzyme lysolecithin:lysolecithin acyltransferase has been isolated from the soluble fraction of rabbit lung and it has been found responsible for two different reactions: (a) transacylation between two molecules of lysolecithin to give dipalmitoylphospha-tidylcholine and glycerophosphorylcholine and (b) hydrolysis of the substrate yielding free fatty acid and glycerophosphorylcholine. Both activities copurify 80-fold with a hydrolysis/transacylation ratio about 2 in all purification steps. The enzyme shows a strong tendency to aggregate with other proteins without loss of activity. The hydrolysis/transacylation ratio is strongly dependent on experimental conditions and these must be carefully controlled if reproducibility is desired. The dependence of this ratio on several parameters is discussed. Based on identical behavior against temperature, β-mercaptoethanol, and iodoacetate and on the appearance of a unique band on electrophoresis, it can be concluded that only one protein is responsible for both activities. The molecular weight ranges between 58,000 and 60,000 D and the amino acid composition shows its acidic character, as described for the enzyme from other sources. The kinetic pattern of both reactions is different and depends on the physical state of the substrate; when the enzyme binds to monomers, the substrate is hydrolyzed to free fatty acid, whereas the binding to micelles favors the transacylation giving phosphatidylcholine. The kinetic constants have been evaluated and a model is proposed for both reactions. The activation energy has been measured and the proximity of the values for the two reactions suggests that the limiting step is the same for hydrolysis and transacylation. The implication of this enzyme in surfactant synthesis is discussed.

Activation and Stabilization of Penicillin V Acylase from Streptomyces lavendulae in the Presence of Glycerol and Glycols

Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis.

D-amino acid oxidase from Rhodotorula gracilis is a FAD-containing enzyme that belongs to the oxidase class that is characterized by the ability of the reduced flavin to react quickly with oxygen, yielding hydrogen peroxide and the oxidized cofactor. Hydrogen peroxide, necessary for the production of glutaryl-7-ACA from cephalosporin C had a deleterious effect on the enzyme. H(2)O(2) induced the oxidation of tryptophan and cysteine residues of the protein that could be involved in the dimerization process, required for the attainment of a fully competent enzyme. H(2)O(2) had also a kinetic effect on the reaction catalyzed by D-amino acid oxidase. It was a pure noncompetitive inhibitor; the corresponding inhibition constants were K(is) = 0.52 mM and K(ii) = 0.70 mM.