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

Early postpartum hormonal therapy improves fertility of dairy cows

A study of 234 Holstein cows was conducted to determine if hormonal treatments of gonadotropin-releasing hormone (GnRH or Cystorelin®) and(or) prostaglandin F2a (PGF or Lutalyse®) given early after calving would improve subsequent fertility of dairy cows. Treatment of cows having abnormal conditions associated with calving (puerperal problems) reduced interval from calving to conception by 43 to 48 days when GnRH was given once between days 10 and 14 postpartum or when PGF was administered once between 20 and 24 days after calving compared with cows given only saline (controls). The reduction in days open was 27 to 29 days overall for all cows (normal and abnormal) treated with either hormone compared with controls. Cows (normal and abnormal) given either hormone required 26 to 41 % fewer inseminations per conception than controls. Reasons for improved fertility are discussed. We conclude that early postpartum treatments with GnRH or PGF improved fertility of dairy cows, especially those that experienced puerperal problems

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22 h of incubation at 45 °C was 3000 U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40–60%)-dialysis followed by heat-treatment (70 °C for 30 min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29 kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60 °C, respectively. Its half-life times at 70 and 80 °C were 8 and 4 h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5 L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis

Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R

In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO4) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100 U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680 U/mL). Additionally, a new extracellular 51 kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70 °C and pH 10. Its half-life times at 70 and 80 °C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5 L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5 L; which offers an interesting potential for its application in the laundry detergent industry