Isabel de la Mata

Identification of an essential glutamate residue in the active site of endoglucanase III from Trichoderma reesei

n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.

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.

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.

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.

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.