Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest

Background Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration. Results A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 C for 20min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme 500 L, respectively. Conclusions This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203

NEAMTHM18

European-Union Civil Protection Mechanism, DG-ECHO, Agreement Number: ECHO/SUB/2015/718568/PREV26Published6T. Studi di pericolosità sismica e da maremoto1SR TERREMOTI - Sorveglianza Sismica e Allerta Tsunami2SR TERREMOTI - Gestione delle emergenze sismiche e da maremoto4IT. Banche dat

The making of the NEAM Tsunami Hazard Model 2018 (NEAMTHM18)

The NEAM Tsunami Hazard Model 2018 (NEAMTHM18) is a probabilistic hazard model for tsunamis generated by earthquakes. It covers the coastlines of the North-eastern Atlantic, the Mediterranean, and connected seas (NEAM). NEAMTHM18 was designed as a three-phase project. The first two phases were dedicated to the model development and hazard calculations, following a formalized decision-making process based on a multiple-expert protocol. The third phase was dedicated to documentation and dissemination. The hazard assessment workflow was structured in Steps and Levels. There are four Steps: Step-1) probabilistic earthquake model; Step-2) tsunami generation and modeling in deep water; Step-3) shoaling and inundation; Step-4) hazard aggregation and uncertainty quantification. Each Step includes a different number of Levels. Level-0 always describes the input data; the other Levels describe the intermediate results needed to proceed from one Step to another. Alternative datasets and models were considered in the implementation. The epistemic hazard uncertainty was quantified through an ensemble modeling technique accounting for alternative models' weights and yielding a distribution of hazard curves represented by the mean and various percentiles. Hazard curves were calculated at 2,343 Points of Interest (POI) distributed at an average spacing of ∼20 km. Precalculated probability maps for five maximum inundation heights (MIH) and hazard intensity maps for five average return periods (ARP) were produced from hazard curves. In the entire NEAM Region, MIHs of several meters are rare but not impossible. Considering a 2% probability of exceedance in 50 years (ARP≈2,475 years), the POIs with MIH >5 m are fewer than 1% and are all in the Mediterranean on Libya, Egypt, Cyprus, and Greece coasts. In the North-East Atlantic, POIs with MIH >3 m are on the coasts of Mauritania and Gulf of Cadiz. Overall, 30% of the POIs have MIH >1 m. NEAMTHM18 results and documentation are available through the TSUMAPS-NEAM project website (http://www.tsumaps-neam.eu/), featuring an interactive web mapper. Although the NEAMTHM18 cannot substitute in-depth analyses at local scales, it represents the first action to start local and more detailed hazard and risk assessments and contributes to designing evacuation maps for tsunami early warning

The overexpression of the SAPB of Bacillus pumilus CBS and mutated sapB-L31I/T33S/N99Y alkaline proteases in Bacillus subtilis DB430: New attractive properties for the mutant enzyme

International audienc

Inhibitory effect of fenugreek galactomannan on digestive enzymes related to diabetes, hyperlipidemia, and liver-kidney dysfunctions

International audienceThe present study was undertaken to investigate the effect of fenugreek galactomannan on intestinal glucose uptake in surviving diabetic rats. It explored their potential action with respect to lowering maltase, lactase, and sucrase activities in the small intestine of galactomannan-treated diabetic group compared to the diabetic control group. The findings indicate that the increase of blood glucose levels was significantly suppressed in the galactomannan-treated group than those in the diabetic rats. Moreover, the galactomannan isolated from fenugreek exhibited a prominent selective inhibitory effect against intestinal lipase activity. It was found to significantly delay the absorption of LDL-cholesterol and triglycerides and the increase in HDL-cholesterol. In addition, fenugreek galactomannan efficiently protect the hepatic function observed by the considerable decrease of aspartate and alanine transaminases (AST and ALT) and lactate deshydrogenase (LDH) contents in the serum of diabetic rats. The beneficial effects of fenugreek galactomannan were also evidenc-ed by their capacity to inhibit diabetes-induced kidney injury through lowering the urea and creatinine content in plasma. Overall, the conclusion of the present study indicate that fenugreek galactomannan displays a number of promising properties and attributes for future applications as therapeutic agents in biotechnological and bioprocess-based technologies, particularly those interested in the development of anti-diabetic and hypolipidemic drugs

Modulatory effect of fenugreek saponins on the activities of intestinal and hepatic disaccharidase and glycogen and liver function of diabetic rats

International audienceDiabetes mellitus is a serious health concern throughout the world and is often associated with a variety of bodily disorders such as liver toxicity and dysfunction. This study elucidates the effect of fenugreek saponin administration on disaccharidase and glycogen activities in the intestine and liver of surviving diabetic rats. It also evaluates the effect of saponin feeding using a number of liver toxicity indices, namely stress oxidant, liver dysfunction markers and metabolism. Our findings indicate that the fenugreek saponin fraction significantly modulated the disaccharidase and glycogen enzyme activities in the intestine and liver of rats, increased the hepatic glycogen content, suppressed the increase of blood glucose level and improved results in the oral glucose tolerance test (OGTT). The fenugreek saponin extract also efficiently protected the hepatic function, which was evidenced by the significant increases of superoxide dismutase (SOD), catalase (CAT), gluthation peroxidase (GPX), aspartate transaminase (AST), alanine transaminase (ALT) and lactate deshydrogenase (LDH) enzyme activities. Fenugreek saponin also induced a notable delay in the absorption of LDL-cholesterol and triglycerides and a remarkable increase in levels of HDL-cholesterol. A histological analysis of the hepatic tissues further established the positive effect of fenugreek saponin. Overall, the findings of the current study indicate that fenugreek saponins exhibit attractive properties and can be considered as promising candidates for future application as therapeutic agents in biotechnological and bioprocess-based technologies, particularly those related to the development of anti-diabetic, hepatoprotective and hypolipidemic drugs