María Pilar Castillón

Variation of the levels of cyclic AMP and cyclic GMP during development of the insect Ceratitis capitata.

Abstract Changes in the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) during development were studied in the Dipterous Ceratitis capitata. The developmental patterns were different to each other. Cyclic AMP showed a sharp maximum in the larval stage to decrease afterwards during adult development. Changes of cyclic GMP exhibited an opposite pattern, although its levels were always higher than those of cyclic AMP.

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.

Optimization of 6-aminopenicillanic acid (6-APA) production by using a new immobilized penicillin acylase.

A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 23 factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5–10.5, and an optimal temperature of 60 °C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 °C.

Cyclic nucleotide cyclase variation during development of the insect Ceratitis capitata

Abstract Guanylate and adenylate cyclase activities were estimated in homogenates of the insect Ceratitis capitata at various stages of development. Guanylate cyclase activity was notably higher than adenylate cyclase activity in agreement with both cyclic nucleotide ratio and cyclic nucleotide-dependent protein kinase ratio reported in arthropod tissues. Variations in both enzyme activities during development were coincident in the adult development, while in other biological stages, as the larval development and puparium formation, the most significant changes affected to the activity of guanylate cyclase.

Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis

ABSTRACT Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM−1 s−1. Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5°C.

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log u P value of the solvent and the activation free energy for denaturation ( j G d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log u P h m 2.3 have protective effects, whereas solvents with log u P S m 1.8 are deleterious for penicillin acylase.

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.

Kinetic mechanism of β-glucosidase from Trichoderma reesei QM 9414.

Abstract β-Glucosidase is a key enzyme in the hydrolysis of cellulose to d -glucose. β-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl β-glucosides. The doulbe-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl β- d -glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 ± 0.2 mM and V = 2.09 ± 0.52 μmol · min−1 · mg−1 for salicin; K = 1.22 ± 0.3 mM and V = 1.14 ± 0.21 μmol · min−1 · mg−1 for cellobiose; K = 0.19 ± 0.02 mM and V = 29.67 ± 3.25 μmol · in−1 · mg−1 fro p-nitrophenyl β- d -glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by β-glucosidase on p-nitrophenyl β- d -glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl β- d -glucopyranoside hydrolysis.

Adenosine 3',5'-monophosphate phosphodiesterase activity during development of the insect Ceratitis capitata.

Summary Cyclic AMP phosphodiesterase activity was determined in homogenates of the insect Ceratitis capitata at several stages of development. These phosphodiesterase activities are interpreted on the bases of cyclic AMP concentrations and adenyl cyclase activities previously determined at the same stages of development. Variations of both enzyme activities are discussed in relation to the hormonal regulation of post-embryonic development in insects. The experimental results point to the possible existence of two sets of enzymes exhibiting different affinities for the naturally occurring cyclic nucleotides.

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

ABSTRACT The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (α-subunit) and 60.09 kDa (β-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Serβ1, Hisβ23, Valβ70, and Asnβ272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.