Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina).

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices.

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Rational Design Strategy as a Novel Immobilization Methodology Applied to Lipases and Phospholipases.

Immobilization of lipases and phospholipases, mainly on water-insoluble carriers, helps in their economic reusing and in the development of continuous bioprocesses. Design of efficient lipase and phospholipase-immobilized systems is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop efficient immobilized systems. We conceived a new strategy for the rational design of immobilized derivatives (RDID) in favor of the successful synthesis of optimal lipase and phospholipase-immobilized derivatives, aiming the prediction of the immobilized derivative's functionality and the optimization of load studies. The RDID strategy begins with the knowledge of structural and functional features of synthesis components (protein and carrier) and the practical goal of the immobilized product. The RDID strategy was implemented in a software named RDID1.0. The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.

Characterization of a Novel Thermostable Enzyme from Thermus sp. 2.9 with Phospholipase and Acyltransferase Activities.

Phospholipases are classified in different enzyme families according to the ester bond they cleave within phospholipids. The use of phospholipases in industrial processes has prompted the search for new enzymes with differential properties. A gene encoding a novel phospholipase (PLP_2.9) was identified in the genome of the thermophilic strain Thermus sp. 2.9. The analysis of the primary sequence unveiled a patatin-like domain. The alignment of the amino acid sequence of PLP_2.9 to other bacterial patatin-related proteins showed that the four blocks characteristic of this type of phospholipases and the amino acids representing the catalytic dyad are conserved in this protein. PLP_2.9 was overexpressed in Escherichia coli and the purified enzyme was characterized biochemically. PLP_2.9 hydrolyzed p-nitrophenyl palmitate at alkaline pH over a wide range of temperatures (55-80°C), showing high thermostability. PLP_2.9 displayed phospholipase A and acyltransferase activities on egg yolk phosphatidylcholine. Due to its high thermostability, PLP_2.9 has potential applications as a catalyst in several industrial processes.

Comparative analysis of proteinase, phospholipase, hydrophobicity and biofilm forming ability in Candida species isolated from clinical specimens.

Candida species are the commensal organisms of human and animal mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to investigate the ability of clinical isolates of various Candida species to produce proteinase and phospholipase, hydrophobicity and biofilm forming ability that assumed to have a vital role in Candida pathogenicity. Eighty-four Candida strains belonged to Candida albicans (44.1%), C. glabrata (5.9%), C. guilliermondii (5.9%), C. krusei (10.8%), C. parapsilosis (26.2%), and C. tropicalis (7.1%) were examined for proteinase and phospholipase production, cell surface hydrophobicity and biofilm forming ability. The production of proteinase and phospholipase was detected in 81 (96.4%) and 79 (94.1%) of the strains, respectively. C. albicans showed the highest proteinase and phospholipase activity (mean Pz values of 0.42±0.25 and 0.72±0.28) and biofilm formation ability (0.66±0.22). C. parapsilosis had the highest hydrophobicity (42.97±16.1), which showed a good correlation with biofilm formation ability. A considerable percentage of non-albicans Candida strains produced significant amounts of proteinase and phospholipase with a good ability of biofilm formation in vitro. Taken together, our results further substantiated that enzymatic activity, hydrophobicity and the ability for biofilm formation are important virulence factors which may be account for pathogenicity of various Candida species distributed in albicans and non-albicans groups.

Structures of Azemiops feae venom phospholipases and cys-rich-secretory protein and implications for taxonomy and toxinology.

The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA2) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA2s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a1, Af-N49a2, and Af-N49b, respectively. The first three isoforms, comprising 3-4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has >80% sequence-similarity to other Glu(6)-PLA2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a1 are rather unique and loosely linked with crotalid PLA2s, while Af-N49b is related to the viperid PLA2s with Ser(1) substitution. Notably, the Asn(49)-substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential maps similar to each other and to those of antiplatelet PLA2s, while the Af-N49b model is similar to basic and myotoxic sPLA2 molecules. From Azemiops feae and four other Viperidae, we cloned five novel Cys-rich secretory proteins (CRISPs). Azemiops CRISP and natriuretic-peptide precursors share more sequence similarities with those of crotalid venoms than with viperid venoms, further supporting the theory that Azemiops are sister taxons to pit vipers, especially Tropedolaemus.

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/ß-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

Enzymatic and hemolytic activity in different Candida species.

BACKGROUND: Candida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. AIMS: This study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. METHODS: Fifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. RESULTS: Forty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p<0.0001) and proteinase (p<0.05) production. CONCLUSIONS: It is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis.

Fatores de virulência de Candida spp. isoladas da cavidade bucal de crianças infectadas pelo HIV / Virulence factors of Candida spp. isolated from the oral cavity of HIV-infected children

Candida spp. são responsáveis por infecções graves em indivíduos imunossuprimidos. Diversos fatores de virulência contribuem para sua transição de comensais a patógenos, como a formação de biofilme, produção de fosfolipase, produção de protease e a resistência a antifúngicos. O objetivo deste estudo in vitro foi avaliar a formação de biofilme, produção de fosfolipase e protease e a susceptibilidade ao Fluconazol de isolados Candida spp., provenientes da saliva de crianças infectadas pelo HIV (Grupo HIV) e crianças saudáveis (Grupo controle). Também estes fatores foram correlacionados com o uso do HAART, estado imunológico, presença de AIDS e carga viral do grupo HIV. Foi analisado um total de 79 isolados, sendo 48 isolados de C. albicans (33/15) e 20 isolados do complexo C. parapsilosis lato sensu (12/8) dos grupos HIV e controle, respectivamente, e isolados de C. krusei (8), C. tropicalis (1), C. dubliniensis (1) e C. guilliermondii (1) do grupo HIV. Dados médicos e laboratoriais (CD4%, carga viral) foram coletados dos respectivos prontuários médicos. A formação de biofilme foi avaliada pela redução do XTT. Isolados de cada espécie com a habilidade de formar maior quantidade de biofilme maduro (grupo HIV) foram submetidos à microscopia confocal de fluorescência para a visualização da morfologia e estrutura do biofilme. As análises da produção de fosfolipase e protease se deram por meio da metodologia de placas de àgar de gema de ovo e Albumina de Soro Bovino, respectivamente. A susceptibilidade ao Fluconazol foi determinada por meio da técnica de microdiluição. Todos os isolados formaram biofilme (n= 79) e quantitativamente, esta formação foi semelhante em ambos os grupos (p>0.05). A formação de biofilme dos isolados de C. albicans foi maior do que a dos isolados de Candida não-albicans (p<0.05).A atividade de fosfolipase foi detectada em 40,5% (32/79) de todos os isolados e foi significativamente maior no grupo HIV (p=0.006) e nos isolados de C. albicans deste grupo (p=0,007). A atividade de protease foi detectada em 66 isolados (84,8%) e em ambos os grupos a maioria era produtor relativamente forte ou muito forte. Trinta e três (33/41, 7%) isolados eram resistentes ao Fluconazol, sendo 42,9% do grupo HIV e 39,1% do grupo controle. Não foi observada correlação entre a expressão dos fatores de virulência e os dados médicos relativos ao grupo HIV. No entanto, a expressão dos fatores de virulência dos isolados orais de Candida spp. de crianças infectadas pelo HIV se mostrou acentuada. Este achado pode destacar o papel da imunossupressão na regulação da expressão dos fatores de virulência de Candida spp (AU)

Candida species are responsible for life threatening infections in severely immunocompromised individuals. Several virulence attributes support their transition from commensal to parasite, as the biofilm formation, secretion of phospholipase, protease and the resistance to antifungals. The aims of this study were to assess the in vitro biofilm formation, phospholipase production, protease production and the susceptibility to Fluconazole of Candida spp. recovered from the saliva of HIV infected children and controls. Also, to correlate these features with the use of HAART, immunological status, presence of AIDS and viral load of HIV group. A total of 79 isolates were analyzed: 48 C. albicans isolates (33/15) and 20 C. parapsilosis sensu lato complex isolates (12/8) from HIV and control patients, respectively, while C. krusei (8), C. tropicalis (1), C. dubliniensis (1) and C. guilliermondii (1) from HIV patients only. Medical data was collected, considering antiretroviral therapy, CD4 count and viral load. The biofilm forming ability was analyzed through the XTT reduction assay technique. Representative isolates of each specie with the ability to form more quantity of mature biofilm (HIV group) underwent confocal laser scanning microscopy for the visualization of biofilm morphology and structure. Phospholipase and protease assays were performed through the egg yolk and Bovine Serum Albumin agar plate methods, respectively. Fluconazole susceptibility was determined through the microdilution assay. All isolates were able to form biofilm (n= 79). Quantitatively, Candida isolates from HIV-positive and negative children presented similar ability to form biofilm (p>0.05). In C. albicans isolates from both, HIV and control groups, the biofilm formation was higher than in non-albicans Candida isolates (p<0.05).Phospholipase activity was detected in 40.5% (32/79) of all isolates. Its activity was significantly higher in HIV group (p=0.006) and C. albicans isolates from HIV group (p=0.007). Protease activity was detected in 66 isolates (84.8%) and most of them from both groups were relatively/very strong producers. Thirty three (33/41.7%) of all isolates were resistant to Fluconazole. Most non-albicans Candida isolates from HIV (87.0%) and control (87.5%) groups were susceptible to this drug. No significant difference was detected between groups in biofilm, protease and Fluconazole susceptibility assays. Correlation with medical data in HIV group was not found. Candida spp. isolates of HIV-positive children present increased virulence expression due to significant higher in vitro phospholipase production. This finding may enlighten the relevant role played by immunosuppression in the modulation of Candida virulence attributes (AU)

Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus.

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4-5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases. By analyzing both esterase and phospholipase A2 activity, we determined the thermostability and the optimal pH, temperature dependence and substrates of these enzymes. We demonstrated that EfPLP shows the characteristics of a psychrophilic phospholipase. Furthermore, we analyzed the enzymatic activity of three engineered versions of the EfPLP, in which unique residues of EfPLP, Gly80, Ala201 and Val204, were substituted through site-directed mutagenesis with residues found in the E. crassus homolog (Glu, Pro and Ile, respectively). Additionally, three corresponding mutants of EcPLP were also generated and characterized. These analyses showed that the substitution of amino acids with rigid and bulky charged/hydrophobic side chain in the psychrophilic EfPLP confers enzymatic properties similar to those of the mesophilic patatin-like phospholipase, and vice versa. This is the first report on the isolation and characterization of a cold-adapted patatin-like phospholipase from eukaryotes. The results reported in this paper support the idea that enzyme thermal-adaptation is based mainly on some amino acid residues that influence the structural flexibility of polypeptides and that EfPLP is an attractive biocatalyst for industrial processes at low temperatures.

A grey mullet enzyme displaying both lipase and phospholipase activities: purification and characterization.

A lipase from the golden grey mullet viscera was purified to homogeneity by ammonium sulphate precipitation, gel filtration, anionic and cation exchange chromatographies. The pure enzyme tentatively named grey mullet digestive lipase (GmDL) is a monomer having a molecular mass of about 35 kDa, as determined by SDS-PAGE analysis. No similarity was found between the NH2-terminal amino acid residues of GmDL and those of other known digestive lipases. GmDL is a serine enzyme, like all known lipases from different origins. Interestingly, GmDL has not only lipase activity but also a phospholipase activity which requires the presence of Ca(2+) and bile salts. Specific activities of 64 U/mg, 55 U/mg and 63 U/mg were measured using tributyrin, olive oil emulsion or phosphatidylcholine as substrate, respectively at pH 8 and at 50°C. GmDL is therefore a thermo-active enzyme as compared to other fish lipases studied so far. It is worth to notice that grey mullet lipase was active in the presence of salt concentrations as high as 0.8M.

Purification and biochemical characterization of a novel alkaline (phospho)lipase from a newly isolated Fusarium solani strain.

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH(2)-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca(2+) and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5-10 and at temperatures below 45 °C.

Bacillus anthracis factors for phagosomal escape.

The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

Phospholipases in food industry: a review.

Mammal, plant, and mainly microbial phospholipases are continuously being studied, experimented, and some of them are even commercially available at industrial scale for food industry. This is because the use of phospholipases in the production of specific foods leads to attractive advantages, such as yield improvement, energy saving, higher efficiency, improved properties, or better quality of the final product. Furthermore, biocatalysis approaches in the food industry are of current interest as non-pollutant and cleaner technologies. The present chapter reviews the most representative examples of the use of phospholipases in food industry, namely edible oils, dairy, and baking products, emulsifying agents, as well as the current trend to the development of novel molecular species of phospholipids with added-value characteristics.

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Actividad fosfolipasa extracelular de aislamientos de Malassezia de individuos con y sin dermatosis / Extracellular phospholipase activity of Malassezia strains isolated from individuals with and without dermatological disease

Antecedentes. El género Malassezia incluye numerosas levaduras lipofílicas que pertenecen a la microbiota cutánea del hombre y de otros mamíferos. Algunas especies se han asociado con diversas dermatosis. Apenas se conocen los factores que permiten la transformación de las levaduras del género Malassezia de un microorganismo comensal a uno patógeno, pero la producción de diversas enzimas, como lipasas, fosfolipasas y lipooxigenasa, podría contribuir a la actividad patogénica de estas levaduras. Objetivos. En la presente investigación hemos determinado y comparado la actividad de la fosfolipasa extracelular a partir de 60 aislamientos de Malassezia en seres humanos para relacionar esta característica con las especies del género y con el origen (dermatosis o no) de las cepas examinadas. Métodos. La producción de fosfolipasa se determinó utilizando el método semicuantitativo de la placa de yema de huevo. Resultados y conclusiones. Malassezia obtusa, Malassezia slooffiae, Malassezia globosa y Malassezia restricta crecieron mal en el medio de cultivo seleccionado, por lo que no fue posible determinar la actividad fosfolipasa. Malassezia pachydermatis mostró la mayor actividad enzimática. Produjeron fosfolipasa 29 cepas de Malassezia sympodialis; los aislamientos de pacientes con pitiriasis versicolor se caracterizaron por una actividad fosfolipasa significativamente mayor que los aislamientos de individuos sanos. Esta observación sugiere que la actividad fosfolipasa de Malassezia podría desempeñar un papel en el inicio de las lesiones cutáneas, en particular en el caso de la mencionada micosis(AU)

Background. The Malassezia genus includes mainly lipophilic yeasts belonging to the cutaneous microbiota of man and other mammals. Some Malassezia species have been associated with various dermatological diseases. The factors permitting the transformation of yeasts of the Malassezia genus from a commensal organism to a pathogenic agent are still little known but the production of various enzymes such as lipase, phospholipase and lipoxygenase could contribute to the pathogenic activity of these yeasts. Aims. Here we have determined and compared the extracellular phospholipase activity of sixty human isolates of Malassezia so as to relate this feature to the species of Malassezia and to the origin (from dermatological diseases or not) of the strains examined. Methods. Phospholipase production was determined using the semi-quantitative egg-yolk plate method. Results and conclusions. Malassezia obtusa, Malassezia slooffiae, Malassezia globosa, Malassezia restricta had difficulty developing in the chosen culture medium so that it was not possible to measure phospholipasic activity. Malassezia pachydermatis showed the highest phospholipase activity. Twenty-nine Malassezia sympodialis strains produced phospholipase; the isolates from patients with pityriasis versicolor had significantly higher phospholipasic activity than those isolated from healthy individuals. This observation suggests that the phospholipasic activity of Malassezia may play a role in the onset of skin lesions, especially in the case of pityriasis versicolor(AU)

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom exerts its anticoagulant activity by enzymatic hydrolysis of plasma phospholipids and by non-enzymatic inhibition of factor Xa in a phospholipids/Ca(2+) independent manner.

A homodimeric acidic PLA(2) (RVVA-PLA(2)-I) of 58.0 kDa molecular weight purified from Russell's viper (Daboia russelli) venom demonstrated dose-dependent catalytic, strong anticoagulant and indirect hemolytic activities and inhibited blood coagulation cascade in both enzymatic and non-enzymatic mechanisms. In in vitro condition, RVVA-PLA(2)-I showed preferential hydrolysis of phosphatidylcholine with a K(m) and V(max) values of 0.65 mM and 28.9 µmol min(-1), respectively. Biochemical study and GC-analysis of plasma phospholipids hydrolysis by PLA(2) revealed that anticoagulant activity of RVVA-PLA(2)-I was partly attributed by the enzymatic hydrolysis of pro-coagulant phospholipids PC, followed by PS. The spectrofluorometric and gel-filtration analyses documented binding of RVVA-PLA(2)-I with activated factor X and PC; however, it does not bind with factor Va, prothrombin and thrombin. Therefore, this anticoagulant PLA(2) inhibits the blood coagulation cascade non-enzymatically by binding with coagulation factor Xa, even in the absence of phospholipids and Ca(2+) and thus slows down the blood coagulation by partially inhibiting the prothrombin activation. Chemical modification of essential amino acids present in the active site, neutralization with Azadirachta indica leaves extract (AIPLAI) and heat-inactivation study reinforce the association of catalytic and anticoagulant activity of RVVA-PLA(2)-I and also throw a light on its non-enzymatic mechanism of anticoagulant action.

Recent progress on phospholipases: different sources, assay methods, industrial potential and pathogenicity.

Significant studies on phospholipases optimization, characterization, physiological role and industrial potential have been conducted worldwide. Some of them have been directed for biotechnological advances such as gene discovery and functional enhancement by protein engineering. Others reported phospholipases as virulence factor and major cause of pathophysiological effects. A general overview on phospholipase is needed for the identification of new reliable and efficient phospholipase, which would be potentially used in number of industrial and medical applications. Phospholipases catalyse the hydrolysis of one or more ester and phosphodiester bonds of glycerophospholipids. They vary in site of action on phospholipid which can be used industrially for modification/production of new phospholipids. Catalytically active phospholipase mainly use phosphatidylcholine as major substrate, but they can also show specificity with other phospholipids. Several accurate phospholipase assay methods are known, but a rapid and reliable method for high-throughput screening is still a challenge for efficient supply of superior phospholipases and their practical applications. Major application of phospholipase is in industries like oil refinery, health food manufacturing, dairy, cosmetics etc. All types of phospholipases can be involved as virulence factor. They can also be used as diagnostic markers for microbial infection. The importance of phospholipase in virulence is proven and inhibitors of the enzyme can be used as candidate for preventing the associated disease.

Epidemiology and phospholipase activity of oral Candida spp. among patients with central nervous system diseases before and after dental cleaning procedure

Patients suffering of diseases that affect central nervous system may be considered more susceptible to the infectious diseases of mouth. Sixty-nine patients suffering of cerebral palsy, Down's syndrome and metal retardation were submitted to saliva examination for the presence of Candida spp. before and after a procedure of dental cleaning. The isolates were submitted to assay for verifying phospholipase production. 55.10 percent of the patients provided isolation of Candida spp. The frequency of isolation obtained before dental procedure was: C. albicans (83.33 percent), C. krusei (8.33 percent) and C. kefyr, C. parapsilosis and C. glabrata (2.78 percent each). The frequency after the procedure was: C. albicans (68.57 percent), C. parapsilosis (11.43 percent), C. krusei and C. kefyr (8.57 percent each) and Candida glabrata (2.86 percent). We verified significantly difference (p < 0.01) between populations obtained at the two examinations. Phospholipase production was verified only among C. albicans strains and the proportion of producers was higher when testing isolates obtained after dental cleaning procedure. Studies focused on Candida spp. isolation are useful for better comprehension of the role of these yeasts on the oral flora from patients with cerebral palsy, Down's syndrome and metal retardation.

Crab digestive phospholipase: a new invertebrate member.

Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14 kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA(2) (PPPL), CDPL displayed its maximal activity at 50 degrees C and not at 37 degrees C. A specific activity of 40 U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50 degrees C) in the presence of 8 mM sodium deoxycholate (NaDC) and 10 mM CaCl(2). In contrast to PPPL, purified CDPL was completely inactivated at 60 degrees C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.