A Novel Lipase from Streptomyces exfoliatus DSMZ 41693 for Biotechnological Applications.

Genome mining of Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step hydrophobic interaction chromatography. The enzyme is a monomeric protein of 27.6 kDa, which belongs to subfamily I.7 of lipolytic enzymes according to its phylogenetic analysis and biochemical characterization. The purified enzyme shows the highest activity at 60 °C and an optimum pH of 8.5, whereas thermal stability is significantly improved when protein concentration is increased, as confirmed by thermal deactivation kinetics, circular dichroism, and differential scanning calorimetry. Enzyme hydrolytic activity using p-nitrophenyl palmitate (pNPP) as substrate can be modulated by different water-miscible organic cosolvents, detergents, and metal ions. Likewise, kinetic parameters for pNPP are: KM = 49.6 µM, kcat = 57 s-1, and kcat/KM = 1.15 × 106 s-1·M-1. SeLipC is also able to hydrolyze olive oil and degrade several polyester-type polymers such as poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), and poly(ε-caprolactone) (PCL). Moreover, SeLipC can catalyze the synthesis of different sugar fatty acid esters by transesterification using vinyl laurate as an acyl donor, demonstrating its interest in different biotechnological applications.

Overexpression of penicillin V acylase from Streptomyces lavendulae and elucidation of its catalytic residues.

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 (alpha-subunit) and 60.09 kDA (beta-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobicpocket involved in catalytic activity, including Serbeta1, Hisbeta23, Valbeta70, and Asnbeta272, 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.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % ß-sheet, 19.4 % ß-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/ß hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

Nucleoside 2'-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus--preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides.

Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5.

Magnetic chitosan beads for covalent immobilization of nucleoside 2'-deoxyribosyltransferase: application in nucleoside analogues synthesis.

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe(3)O(4)) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2'-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2'-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2'-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2'-deoxyriboside and 5-trifluorothymidine.

Characterization of a novel subgroup of extracellular medium-chain-length polyhydroxyalkanoate depolymerases from actinobacteria.

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the α/ß-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ(Bd) has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ(Bd) is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions.

The phaZ ( Sex ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% ß-sheet, 17.1% ß-turns, and 35.2% random coil, with a midpoint transition temperature (T (m)) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-ß-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.

Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters.

Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date.

Pandemics and Other Health Crises: A Special Report from a European Parliament Workshop.

Once an emergency has passed, general attention typically returns to dealing with day-to-day system management, and the opportunity to learn from the crisis and improve is missed. Lessons from the coronavirus disease 2019 (COVID-19) crisis must be learned, and the necessary changes made at all levels, both in terms of improving collaboration and strengthening health systems. This special report provides the conclusion of a workshop held in the European Parliament (EP) in Brussels, Belgium. The event explored the modalities of response and preparation to the COVID-19 pandemic, and to health crises in general. The workshop considered actions at different levels: international organizations (global level), European Union (EU) Member States ([MS] national level), and health services (local level). It provided an opportunity to look back at several initiatives taken during the pandemic, and to draw inspiration from them.

Enzymatic synthesis of nucleoside analogues using immobilized 2'-deoxyribosyltransferase from Lactobacillus reuteri.

Covalent attachment of recombinant Lactobacillus reuteri 2'-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2'-deoxyadenosine synthesis from 2'-deoxyuridine and adenine at 40°C and pH 6.5. Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2'-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of 2,6-diaminopurine-2'-deoxyriboside with negligible loss of catalytic activity.

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies.

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni(2+)-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.

Novel Bifunctional Acylase from Actinoplanes utahensis: A Versatile Enzyme to Synthesize Antimicrobial Compounds and Use in Quorum Quenching Processes.

Many intercellular communication processes, known as quorum sensing (QS), are regulated by the autoinducers N-acyl-l-homoserine lactones (AHLs) in Gram-negative bacteria. The inactivation of these QS processes using different quorum quenching (QQ) strategies, such as enzymatic degradation of the autoinducers or the receptor blocking with non-active analogs, could be the basis for the development of new antimicrobials. This study details the heterologous expression, purification, and characterization of a novel N-acylhomoserine lactone acylase from Actinoplanes utahensis NRRL 12052 (AuAHLA), which can hydrolyze different natural penicillins and N-acyl-homoserine lactones (with or without 3-oxo substitution), as well as synthesize them. Kinetic parameters for the hydrolysis of a broad range of substrates have shown that AuAHLA prefers penicillin V, followed by C12-HSL. In addition, AuAHLA inhibits the production of violacein by Chromobacterium violaceum CV026, confirming its potential use as a QQ agent. Noteworthy, AuAHLA is also able to efficiently synthesize penicillin V, besides natural AHLs and phenoxyacetyl-homoserine lactone (POHL), a non-natural analog of AHLs that could be used to block QS receptors and inhibit signal of autoinducers, being the first reported AHL acylase capable of synthesizing AHLs.

Biochemical and structural studies of two tetrameric nucleoside 2'-deoxyribosyltransferases from psychrophilic and mesophilic bacteria: Insights into cold-adaptation.

Nucleoside 2'-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2'-deoxynucleosides and the following transfer of the 2'-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures.

Lactobacillus reuteri 2'-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides.

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% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees 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 degrees 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.

Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics.

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.

Immobilized enzyme reactors based on nucleoside phosphorylases and 2'-deoxyribosyltransferase for the in-flow synthesis of pharmaceutically relevant nucleoside analogues.

In this work, a mono- and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the in-flow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2'-deoxy, 2',3'-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in 65-59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield). Both IMERs proved to be promising alternatives to chemical routes for the synthesis of nucleoside analogues. The developed in-flow system represents a powerful tool for the fast production on analytical scale of nucleosides for preliminary biological tests.

The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged D-amino acid oxidase from Trigonopsis variabilis.

d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K(d)=1.5+/-0.1x10(-7) M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed alpha/beta protein with 8 alpha-helices and 14 beta-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% alpha-helix content, 20% beta-structure content (14% antiparallel and 6% parallel beta-sheet), 15% beta-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures (T(m)) of 37.9 degrees C and 41.4 degrees C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney (pkDAAO) and Rhodotorula gracilis (RgDAAO) d-amino acid oxidases, a "head-to-head" interaction between subunits in the TvDAAO dimer might be expected.

Resource consumption and costs of palliative care services in Spain: a multicenter prospective study.

Patients (n=395) with terminal-stage cancer receiving attention from palliative care services (PCSs) were recruited over a period of 15 consecutive days from 171 participating PCS units. Resource consumption and costs were evaluated for 16 weeks of follow-up, and the findings were compared with a study conducted in 1992 so as to assess change over time. The most frequent health care interventions were homecare visits, hospital admissions, and patient-consultant phone calls. PCS provided 67% of all services and consultation interventions in 91% of patients. Compared with the historical data, there was a significant shift from the use of conventional hospital beds toward palliative care beds, a reduced hospital stay (25.5-19.2 days; P=0.002), an increase in the death-at-home option (31%-42%), a lower use of hospital emergency rooms (52%-30.6%; P=0.001), and an increase in programmed care. Compared to the previous resource consumption and expenditure study in 1992, the current PCS policy implies a cost saving of 61%, with greater efficiency and no compromise of patient care.