Milk-Gelling Properties of Proteases Extracted from the Fruits of Solanum Elaeagnifolium Cavanilles.

There is little information on the milk coagulation process by plant proteases combined with chymosins. This work is aimed at studying the capability of protease enclosed in the ripe fruits of Solanum elaeagnifolium (commonly named trompillo) to form milk gels by itself and in combination with chymosin. For this purpose, proteases were partially purified from trompillo fruits. These proteases had a molecular weight of ~60 kDa, and results suggest cucumisin-like serine proteases, though further studies are needed to confirm this observation. Unlike chymosins, trompillo proteases had high proteolytic activity (PA = 50.23 UTyr mg protein-1) and low milk-clotting activity (MCA = 3658.86 SU mL-1). Consequently, the ratio of MCA/PA was lower in trompillo proteases (6.83) than in chymosins (187 to 223). Our result also showed that milk gels formed with trompillo proteases were softer (7.03 mPa s) and had a higher release of whey (31.08%) than the milk gels clotted with chymosin (~10 mPa s and ~4% of syneresis). However, the combination of trompillo proteases with chymosin sped up the gelling process (21 min), improved the firmness of milk gels (12 mPa s), and decreased the whey release from milk curds (3.41%). Therefore, trompillo proteases could be combined with chymosin to improve the cheese yield and change certain cheese features.

The aflatoxin inhibitors capsaicin and piperine from Capsicum chinense and Piper nigrum fruits modulate the antioxidant system in Aspergillus parasiticus.

Several aflatoxin inhibitors can modulate the antioxidant system in fungi. In this work, the effect of the ethanolic extract of Capsicum chinense and Piper nigrum fruits, capsaicin, and piperine on the expression of the aflE, aflG, aflH, aflI, aflK, aflL, aflO, aflP, and aflQ genes involved in the aflatoxin biosynthetic pathway in Aspergillus parasiticus were studied by qRT-PCR analysis. As well as, the effect on the expression of fungal antioxidant genes (sod1, catA, and cat2) and enzymatic activity of catalase (CAT) and superoxide dismutase (SOD). Results reveal that the highest (p < 0.05) radial growth inhibition (68 and 86%) and aflatoxins production inhibition (73 and 80%) was observed with capsaicin and piperine respectively, at 300 µg/mL, instead of the ethanolic extract at the same concentration. The qRT-PCR analysis showed that compounds and extracts at 300 µg/mL induced a down-regulation of aflatoxin genes and an up-regulation on the fungal antioxidant genes. CAT activity increased by 23.15, 36.65, 51.40, and 65.50%, in the presence of C. chinense and P. nigrum extract, capsaicin, and piperine exposure, respectively. While SOD activity was not significantly impacted (p > 0.05). In conclusion, the capsaicin and piperine, two antifungal and anti-aflatoxigenic compounds produce an up-regulation of antioxidant defense genes accompanied by an enhancement of catalase enzymatic activity in A. parasiticus.

cDNA Characterization and Expression of Selenium-Dependent CqGPx3 Isoforms in the Crayfish Cherax quadricarinatus under High Temperature and Hypoxia.

Glutathione peroxidase 3 (GPx3) is the only extracellular selenoprotein (Sel) that enzymatically reduces H2O2 to H2O and O2. Two GPx3 (CqGPx3) cDNAs were characterized from crayfish Cherax quadricarinatus. The nerve cord CqGPx3a isoform encodes for a preprotein containing an N-terminal signal peptide of 32 amino acid residues, with the mature Sel region of 192 residues and a dispensable phosphorylation domain of 36 residues. In contrast, the pereiopods CqGPx3b codes for a precursor protein with 19 residues in the N-terminal signal peptide, then the mature 184 amino acid residues protein and finally a Pro-rich peptide of 42 residues. CqGPx3 are expressed in cerebral ganglia, pereiopods and nerve cord. CqGPx3a is expressed mainly in cerebral ganglia, antennulae and nerve cord, while CqGPx3b was detected mainly in pereiopods. CqGPx3a expression increases with high temperature and hypoxia; meanwhile, CqGPx3b is not affected. We report the presence and differential expression of GPx3 isoforms in crustacean tissues in normal conditions and under stress for high temperature and hypoxia. The two isoforms are tissue specific and condition specific, which could indicate an important role of CqGPx3a in the central nervous system and CqGPx3b in exposed tissues, both involved in different responses to environmental stressors.

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Proteolysis and Rheological Properties of Cream Cheese Made with a Plant-Derived Coagulant from Solanum elaeagnifolium.

Cream cheese is a fresh acid-curd cheese with pH values of 4.5⁻4.8. Some manufacturers add a small volume of rennet at the beginning of milk fermentation to improve the texture of the cream cheese. However, there is no information about the effect that proteases other than chymosin-like plant-derived proteases may have on cream cheese manufacture. This work aimed to describe some proteolytic features of the protease extracted from fruits of Solanum elaeagnifolium Cavanilles and to assess the impact that this plant coagulant has on the viscoelastic properties of cream cheeses. Results showed that caseins were not hydrolyzed extensively by this plant-derived coagulant. In consequence, the ratio of milk clotting units (U) to proteolytic activity (U-Tyr) was higher (1184.4 U/U-Tyr) than reported for other plant proteases. The plant coagulant modified neither yield nor composition of cream cheeses, but viscoelastic properties did. Cream cheeses made with chymosin had a loss tangent value (tan δ = 0.257) higher than observed in cheeses made with 0.8 mL of plant-derived coagulant per liter (tan δ = 0.239). It is likely that casein fragments released by the plant-derived coagulant improve the interaction of protein during the formation of acid curds, leading to an increase in the viscoelastic properties of cream cheese.

Selenoprotein M gene expression, peroxidases activity and hydrogen peroxide concentration are differentially regulated in gill and hepatopancreas of the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation.

In many organisms, episodes of low O2 concentration (hypoxia) and the subsequent rise of O2 concentration (reoxygenation) result in the accumulation of reactive oxygen species and oxidative stress. Selenoprotein M (SelM), is a selenocysteine containing protein with redox activity involved in the antioxidant response. It was previously shown that in the white shrimp Litopenaeus vannamei, the silencing of SelM by RNAi decreased peroxidase activity in gill. In this work, we report the structure of the SelM gene (LvSelM) and its relative expression in hepatopancreas and gill after 24h of hypoxia followed by 1h of reoxygenation. The gene is composed by four exons interrupted by tree introns. In gills and hepatopancreas, SelM expression increased after 24h of hypoxia followed by 1h of reoxygenation, while peroxidases activity diminished in hepatopancreas but increased in gills. Hydrogen peroxide (H2O2) concentration was higher in hepatopancreas in response to hypoxia for 6h and did not change after 24 of hypoxia followed by reoxygenation; conversely, no change was detected in gill. SelM appears to be a key enzyme in gill oxidative stress regulation, since the higher expression is associated with an increase in peroxidases activity while maintaining H2O2 concentration. In contrast, in hepatopancreas there is a higher expression after hypoxia and reoxygenation for 24h, but peroxidases activity was lower and the change in H2O2 occurred after 6h of hypoxia and this level was maintained during reoxygenation.

White shrimp Litopenaeus vannamei catalase: gene structure, expression and activity under hypoxia and reoxygenation.

Catalase (EC 1.11.1.6) is an antioxidant enzyme involved in redox equilibrium, regulating hydrogen peroxide (H(2)O(2)) concentration, a harmful reactive oxygen species (ROS) that is produced during hypoxia. Hypoxia occurs commonly in aquatic environments and in shrimp farms. We studied the catalase gene of the shrimp Litopenaeus vannamei and tested its expression and enzyme activity during hypoxia (1.5mg/L O(2); 6 and 24h) and reoxygenation (1h after hypoxia). The complete gene is 2974bp long and has four introns of 821, 223, 114 and 298bp, respectively. The first intron has tree microsatellites, with GT and (T)AT(GT) repeated sequences. L. vannamei catalase is part of an invertebrate clade including crustaceans and rotifers. Catalase expression and activity is different in gills and hepatopancreas. Expression in gills increased 3.2 and 3-fold in response to hypoxia and reoxygenation (6 and 24h hypoxia, followed by 1h reoxygenation) compared to normoxia, while no differences were detected in the expression and activity in hepatopancreas. Catalase activity in gills had a contrary response to expression in hypoxia and reoxygenation.

The crustacean selenoproteome similarity to other arthropods homologs: a mini review

Selenoproteins (Sels) are involved in oxidative stress regulation. Glutathione peroxidase (GPx) and thioredoxin reductase are among the most studied Sels in crustaceans. Since their expressions and activities are affected by pathogens, environmental and metabolic factors, their functions might be key factors to orchestrate the redox cellular balance. The most studied invertebrate selenoproteome is from Drosophila. In this fly, SelD and SelB are involved in selenoproteins synthesis, whereas SelBthD, SelH and SelK are associated with embryogenesis and animal viability. None of the Sels found in Drosophila have been identified in marine crustaceans yet, and their discovery and function identification is an interesting research challenge. SelM has been identified in crustaceans and is differentially expressed in tissues, while its function remains to be clarified. SelW and G-rich Sel were recently discovered in marine crustaceans and their functions are yet to be clearly defined. To fully understand the crustacean selenoproteome, it is still necessary to identify important Sels such as the SelD, SelBthD and SelB homologs. This knowledge can also be useful for marine crustacean industry to propose better culture strategies, enhanced health and improved profits.

Hypoxia, reoxygenation and cytosolic manganese superoxide dismutase (cMnSOD) silencing in Litopenaeus vannamei: effects on cMnSOD transcripts, superoxide dismutase activity and superoxide anion production capacity.

The effects of silencing the mRNA of cytosolic manganese superoxide dismutase (cMnSOD), an enzyme involved in the antioxidant defense, were analyzed in Whiteleg shrimp, Litopenaeus vannamei adults. Shrimp were intramuscularly injected with long dsRNAs corresponding to the N-terminal portion of the cMnSOD and held under normoxic conditions for 24h. Another group of shrimp was exposed to hypoxia for 6h followed by reoxygenation for 1h. Shrimp injected with long dsRNAs had lower cMnSOD transcripts in gills and hepatopancreas. In the cMnSOD silenced shrimp, superoxide dismutase (SOD) activity decreased in gills but not in hepatopancreas. Shrimp subjected to hypoxia had lower cMnSOD transcripts and SOD activity in gills and hepatopancreas; the production of superoxide anion (O2*-) by hemocytes was also lower in this group. Reoxygenation reverted the effect of hypoxia increasing the levels of cMnSOD transcripts, SOD activity and the production of O2*-. These results suggest that cMnSOD contributes significantly to the SOD activity in gills and hepatopancreas and indicate its importance in the redox system regulation for L. vannamei.

Expression and silencing of Selenoprotein M (SelM) from the white shrimp Litopenaeus vannamei: effect on peroxidase activity and hydrogen peroxide concentration in gills and hepatopancreas.

Selenoprotein M (SelM), is a selenocysteine containing protein with redox activity involved in the antioxidant response. In the white shrimp Litopenaeus vannamei, SelM expression in gills is induced transiently during viral infection by the White Spot Syndrome Virus (WSSV). We report that SelM expression was detected in healthy shrimp L. vannamei in gills, muscle, hepatopancreas and pleopods, with more abundance in the hepatopancreas and gills. SelM transcripts were silenced by intramuscular injection with double-stranded RNAs (dsRNAs). In gills and hepatopancreas, all shrimp injected with long dsRNAs had lower SelM transcripts levels compared with controls. Peroxidase activity and hydrogen peroxide concentration were measured to detect effects on antioxidants. Peroxidase activity decreased upon silencing of SelM in gills, but no significant effect was detected in hepatopancreas. In contrast, total cell hydrogen peroxide concentration did not change in gills and hepatopancreas of silenced shrimp. Non-heme peroxidases are new players in the oxidative stress system that need to be addressed in detail, as well as selenium as a critical micronutrient for the antioxidant and innate immune systems in crustaceans.