Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Structural Analysis Revealed the Interaction of Cardenolides from Calotropis procera with Na+/K+ ATPases from Herbivores.

BACKGROUND: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera. OBJECTIVE: We aimed to evaluate the interaction between cardenolides and Na+/K+ ATPases from herbivores. METHODS: The Na+/K+ ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera. RESULTS: The replacement of serine in sensitive Na+/K+ ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides. CONCLUSION: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera.

Antidermatophytic activity of synthetic peptides: Action mechanisms and clinical application as adjuvants to enhance the activity and decrease the toxicity of Griseofulvin.

BACKGROUND: Dermatophytes belonging to the Trichophyton genus are important human pathogens, but they have developed resistance to griseofulvin, the most common antifungal drug used to treat dermatophytosis. OBJECTIVE: This study was aimed to evaluate the antidermatophytic activity of synthetic peptides, as well as mechanisms of action and synergistic effect with griseofulvin. METHODS: Scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FM) were employed to understand the activity and the mechanism of action of peptides. RESULTS: Here we report that synthetic peptides at 50 µg/mL, a concentration 20-fold lower than griseofulvin, reduced the microconidia viability of T. mentagrophytes and T. rubrum by 100%, whereas griseofulvin decreased their viability by only 50% and 0%, respectively. The action mechanism of peptides involved cell wall damage, membrane pore formation and loss of cytoplasmic content. Peptides also induced overproduction of reactive oxygen species (ROS) and enhanced the activity of griseofulvin 10-fold against both fungi, suggesting synergistic effects, and eliminated the toxicity of this drug to human erythrocytes. Docking analysis revealed ionic and hydrophobic interactions between peptides and griseofulvin, which may explain the decline of griseofulvin toxicity when mixed with peptides. CONCLUSION: Therefore, our results strongly suggest six peptides with high potential to be employed alone as new drugs or as adjuvants to enhance the activity and decrease the toxicity of griseofulvin.