Potential Biological Activities of Peptides Generated during Casein Proteolysis by Curly Kale (Brassica oleracea L. var. sabellica L.) Leaf Extract: An In Silico Preliminary Study

This study is a brief report on the proteolytic activity of curly kale leaf extract against casein. Casein degradation products and an in silico analysis of the biological activity of the peptides obtained was performed. The efficiency of casein hydrolysis by curly kale extract was determined using SDS–PAGE and by peptide concentration determination. The pattern of the enzymatic activity was determined by MALDI–TOF MS analysis. The results showed that α- and β-casein were more resistant to curly kale extract hydrolysis, whereas κ-casein was absent in the protein profile after 8 h of proteolysis, and all casein fractions were completely hydrolyzed after 24 h of incubation. Based on sequence analysis, seven peptides were identified, with molecular mass in the range of 1151–3024 Da. All the peptides were products of β-casein hydrolysis. The identified amino acid sequences were analyzed in BIOPEP, MBPDB, and FeptideDB databases in order to detect the potential activities of the peptides. In silico analysis suggests that the β-casein-derived peptides possess sequences of peptides with ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibitory, antithrombotic, immunomodulatory, and antiamnesic bioactivity. Our study was first to evaluate the possibility of applying curly kale leaf extract to generate biopeptides through β-casein hydrolysis.

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.

Kinetic and Thermodynamic Characterization of the Protease from Bacillus licheniformis (ATCC 12759)

In this study, the kinetic of a thermo-stable extracellular protease produced by Bacillus licheniformis (ATCC 12759) cultured in skim latex serum fortified media was investigated. The enzyme was stable up to 65 oC after incubation for 60 min at pH 8. The Lineweaver-Burk exhibited vmax (maximum rate) of 37.037 U/mg min-1 and KM (Michaelis-Menten constant) of 8.519 mg/mL. The activation energy (Ea) of casein hydrolysis and temperature quotient (Q10) were found to be 4.098 kJ/mol and 1.038 - 1.034, respectively, at a temperature ranging from 35 oC to 65 oC. The results of the residual activity test allowed estimating activation energy for irreversible inactivation of the protease (denaturation) which was approximately Ea(d) = 62.097 kJ/mol. The thermodynamic parameters for the enzyme irreversible denaturation were as follow enthalpy (59.286 ≤ΔH*d≥ 59.535 kJ/mol), Gibbs free energy (97.375 ≤ ΔG*d≥ 93.774kJ/mol), and entropy (-122.797 ≤ ΔS*d≥ -101.992 kJ/mol). These thermodynamic parameters inferred that the thermo-stable proteases could be potentially important for industrial application, for example, in the detergent industries.Abstrak: Pada penelitian ini, kinetika protease ekstraseluler termo-stabil yang diproduksi oleh Bacillus licheniformis (ATCC 12759), yang dikultur dalam media yang diperkaya serum lateks skim diselidiki. Enzim stabil hingga 65 oC setelah diinkubasi selama 60 menit pada pH 8. Lineweaver-Burk menunjukkan vmax (laju maksimum) adalah 37.037 U/mg min-1 dan KM (konstanta Michaelis-Menten) 8.519 mg/mL. Energi aktivasi (Ea) dari hidrolisis kasein dan suhu quotient (Q10) ditemukan masing-masing sebesar 4.098 kJ/mol dan 1.038 - 1.034, pada suhu yang berkisar dari 35 oC hingga 65 oC. Hasil uji aktivitas residu memungkinkan estimasi energi aktivasi untuk inaktivasi ireversibel dari protease (denaturasi) yang kira-kira Ea (d) = 62.097 kJ/mol. Parameter termodinamika untuk denaturasi enzim ireversibel adalah sebagai berikut entalpi (59.286 ≤ΔH * d≥ 59.535 kJ / mol), energi bebas Gibbs (97.375 ≤ ΔG * d≥ 93.774kJ / mol) dan entropi (-122.797 ≤ ΔS * d≥ -101.992 kJ / mol). Parameter termodinamika pada penelitian ini menyimpulkan bahwa protease termo-stabil dapat berpotensi penting untuk aplikasi industry seperti dalam industri deterjen.

Immobilization of bromelain on cobalt-iron magnetic nanoparticles (CoFe2O4) for casein hydrolysis

By means of recycling an enzyme, bromelain was used in casein hydrolysis facilitated by a nanobiocatalyst consisting of bromelain, CoFe2O4 magnetic nanoparticles, chitosan, and glutaraldehyde. Bromelain was immobilized on the chitosan cobalt-magnetite nanoparticle surface via covalent bonds to form the nanobiocatalyst. Immobilized bromelain showed 77% immobilization binding, retaining 85 ± 2% of the initial catalytic activity. Nanoparticles and immobilized bromelain were characterized using UV-Vis and IR spectroscopies, X-ray, square wave voltammetry (SWV), cyclic voltammetry (CV), vibrating-sample magnetization (VSM), and transmission electron microscope (TEM). The Michaelis-Menten constant (KM) and VMAX of the free and immobilized enzyme were calculated: KM = 2.1 ± 0.18 mM and 1.8 mM, respectively and VMAX = 6.08 x 10-2 ± 2.1 x 10-2 U/min and 6.46 ± 0.91 U/min, respectively. The thermal stability of the free enzyme was higher than the immobilized enzyme: 95-98% and 83-87%, respectively. An optimum pH of 6 and a temperature of 20 °C were determined in both cases. Immobilized bromelain maintained 50% of the initial catalytic activity after the fifth use. The immobilized bromelain proved to be effective and reusable for casein hydrolysis. As novel contribution the characterization by VOC and CV was carried out.

Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source

Background: Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective: To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods: Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results: Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco®. However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco®. Conclusion: Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.