Standardized production of a homogeneous latex enzyme source overcoming seasonality and microenvironmental variables.

Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the enzymes is reported. The latex was mixed with distilled water and the mixture was cleaned through centrifugation. It was dialyzed with distilled water and centrifuged again to recover the soluble fraction [EP]. The dialyze is a key feature of the process. EP was characterized in terms of protein profile, chemical stability, among other criteria. Wild plants belonging to ten geographic regions and grown in different ecological conditions were used as latex source. Collections were carried out, spaced at three-month, according to the seasons at the site of the study. Proteolytic activity was measured as an internal marker and for determining stability of the samples. EP was also analyzed for metal content and microbiology. EP showed similar magnitude of proteolysis, chromatographic and electrophoretic profiles of proteins. Samples stored at 25 °C exhibited reduced solubility (11%) and proteolytic capacity (11%) after six months. Enzyme autolysis was negligible. Microbiological and metal analyses revealed standard quality of all the samples tested. EP induced milk clotting and hide dehairing after storage for up to six months.

Characterisation and standardisation of Wangashi cheese production steps.

BACKGROUND: ch aims to assess the effect of Calotropis procera plant stems as a coagulant treatment on the Wangashi cheese in order to characterize and standardize its production. Crude extract obtained from the Calotropis plant stems added to milk powder were used as a solution dissolved at various pH (4-8) and temperature (35-80°C) in order to examine the effect of pH and temperature on milk clotting and proteolytic activities. The pH 5.5 and temperature of 70°C were recorded as optimum pH and temperature. After that the concentration of the crude extract enzyme was assayed to purify it using ammonium sulfate precipitation at various percentage of saturation (20-80%) at determined optimum pH and temperature, whereby the saturation of 70% was detected to be the best because of its high specific activity, yield and purification fold. Two types of Wangashi cheese were produced in laboratory, one using directly the crude extract and the other the purified crude extract from Calotropis procera at optimum condition. Their chemical, textural and color properties were determined using standard methods. A significant difference between parameters tested was observed (p < 0.05). A decrease in moisture content, increase in protein content and also an improvement of color and textural parameters were recorded for the cheese obtained using purified crude extract Calotropis procer. METHODS: earch aims to assess the effect of Calotropis procera plant stems as a coagulant treatment on the Wangashi cheese in order to characterize and standardize its production. Crude extract obtained from the Calotropis plant stems added to milk powder were used as a solution dissolved at various pH (4-8) and temperature (35-80°C) in order to examine the effect of pH and temperature on milk clotting and proteolytic activities. The pH 5.5 and temperature of 70°C were recorded as optimum pH and temperature. After that the concentration of the crude extract enzyme was assayed to purify it using ammonium sulfate precipitation at various percentage of saturation (20-80%) at determined optimum pH and temperature, whereby the saturation of 70% was detected to be the best because of its high specific activity, yield and purification fold. Two types of Wangashi cheese were produced in laboratory, one using directly the crude extract and the other the purified crude extract from Calotropis procera at optimum condition. Their chemical, textural and color properties were determined using standard methods. A significant difference between parameters tested was observed (p < 0.05). A decrease in moisture content, increase in protein content and also an improvement of color and textural parameters were recorded for the cheese obtained using purified crude extract Calotropis procera s. RESULTS: earch aims to assess the effect of Calotropis procera plant stems as a coagulant treatment on the Wangashi cheese in order to characterize and standardize its production. Crude extract obtained from the Calotropis plant stems added to milk powder were used as a solution dissolved at various pH (4-8) and temperature (35-80°C) in order to examine the effect of pH and temperature on milk clotting and proteolytic activities. The pH 5.5 and temperature of 70°C were recorded as optimum pH and temperature. After that the concentration of the crude extract enzyme was assayed to purify it using ammonium sulfate precipitation at various percentage of saturation (20-80%) at determined optimum pH and temperature, whereby the saturation of 70% was detected to be the best because of its high specific activity, yield and purification fold. Two types of Wangashi cheese were produced in laboratory, one using directly the crude extract and the other the purified crude extract from Calotropis procera at optimum condition. Their chemical, textural and color properties were determined using standard methods. A significant difference between parameters tested was observed (p < 0.05). A decrease in moisture content, increase in protein content and also an improvement of color and textural parameters were recorded for the cheese obtained using purified crude extract Calotropis procera staems.

Biotechnological potential of a cysteine protease (CpCP3) from Calotropis procera latex for cheesemaking.

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.

Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex.

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest Vmax, Km, Kcat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC50 of around 50 µg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi.

Allergenicity reduction of cow's milk proteins using latex peptidases.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.

Latex peptidases of Calotropis procera for dehairing of leather as an alternative to environmentally toxic sodium sulfide treatment.

Dehairing of crude leather is a critical stage performed at the beginning of its processing to obtain industrially useful pieces. Tanneries traditionally apply a chemical process based on sodium sulfide. Since this chemical reactive is environmentally toxic and inefficiently recycled, innovative protocols for reducing or eliminating its use in leather depilation are welcomed. Therefore, latex peptidases from Calotropis procera (CpLP) and Cryptostegia grandiflora (CgLP) were assayed for this purpose. Enzyme activity on substrates representative of skin such as hide powder azure (UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK), and epidermis (UEP) was determined, while depilation activity was assayed on cow hide. Only CpLP was active against keratin (13.4 UK) and only CgLP was active against elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3 UEP) showed higher activity against the other substrates than CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays, CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator) released hairs from cow hide pieces. Macroscopic and microscopic analyses of the hide revealed that the dehairing process was complete and the leather structure was preserved. The proteolytic system of C. procera is a suitable bioresources to be exploited by tanneries.

First insights into the diversity and functional properties of chitinases of the latex of Calotropis procera.

Chitinases (EC 3.2.1.14) found in the latex of Calotropis procera (Ait) R. Br. were studied. The proteins were homogeneously obtained after two ion exchange chromatography steps. Most proteins were identified individually in 15 spots on 2-D gel electrophoresis with isoelectric points ranging from 4.6 to 6.0 and molecular masses extending from 27 to 30 kDa. Additionally, 66 kDa proteins were identified as chitinases in SDS-PAGE. Their identities were further confirmed by mass spectrometry (MS) analysis of the tryptic digests of each spot and MS analysis of the non-digested proteins. Positive reaction for Schiff's reagent suggested the proteins are glycosylated. The chitinases exhibited high catalytic activity toward to colloidal chitin at pH 5.0, and this activity underwent decay in the presence of increasing amounts of reducing agent dithiothreitol. Spore germination and hyphae growth of two phytopathogenic fungi were inhibited only marginally by the chitinases but were affected differently. This suggested a complex relationship might exist between the specificity of the proteins toward the fungal species. The chitinases showed potent insecticidal activity against the Bruchidae Callosobruchus maculatus, drastically reducing survival, larval weight and adult emergence. It is concluded that closely related chitinases are present in the latex of C. procera, and the first experimental evidence suggests these proteins are involved more efficiently in defence strategies against insects rather than fungi.

Aislamiento y caracterizacion parcial de fracciones con actividad ribonucleasa a partir del latex de calotropis procera (Aiton) W.T. Aiton y pedilanthus tithymaloides (L.) poit / Isolation and partial characterization of fractions with ribonuclease activity from latex of calotropis procera (Aiton) W.T. Aiton and pedilanthus tithymaloides (L.) poit

Con el objetivo de aislar y caracterizar parcialmente las enzimas ribonucleasas (RNasas) contenidas en el látex de Calotropis procera y Pedilanthus tithymaloides, se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con acetato de sodio y centrifugación a 16.000 x g durante 15 min y fraccionadas por cromatografía de intercambio iónico. Se estimó la masa molecular a través de ecuaciones de regresión lineal. Se realizaron pruebas de glicosilación. En ambas especies, las proteínas con actividad RNasa presentaron una masa molecular entre 28 y 30 kDa. No existe evidencia de proteínas glicosiladas en el látex de C. procera. En P. tithymaloides la RNasa es una proteína glicosilada.

In order to isolate and characterize partially ribonucleases (RNases) enzymes contained in the latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from mature plants. Soluble proteins were extracted with sodium acetate and centrifugation at 16,000 xg for 15 min and fractionated by ion exchange chromatography. Molecular mass was estimated by linear regression equations. Glycosylation tests were conducted. In both species, proteins with RNase activity showed a molecular mass between 28 and 30 kDa. No evidence of glycosylated proteins in latex from C. procera. In P. tithymaloides, RNase may be a glycosylated protein.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.

We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Escherichiacoli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5' pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5' cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C.procera.

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

cDNA cloning and molecular modeling of procerain B, a novel cysteine endopeptidase isolated from Calotropis procera.

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed.

Physicochemical properties and tenderness of meat samples using proteolytic extract from Calotropis procera latex.

This study was conducted in order to tenderise muscle foods (pork, beef and chicken) by using crude enzyme extract from Calotropis procera latex. Chunks of knuckle muscle from pork and beef as well as of breast muscle from chicken were marinated with distiled water (control) and 0.05%, 0.1%, 0.2%, 0.3% and 0.5% (w/w) of crude enzyme extract powder for 60 min at 4°C. The marinated samples were then subjected to various physical and chemical property determinations. A decrease in moisture content was observed when the crude enzyme extract was added. Firmness and toughness of the muscle samples significantly decreased with the increased addition of crude enzyme extract (p<0.05). The water holding capacity and cooking yield of the treated samples showed no significant difference throughout the crude enzyme extract addition (p>0.05). Crude enzyme extract had no effect on the pH of the pork sample, but it slightly increased the pH in the beef and chicken. An increase in protein solubility and TCA-soluble peptides content was observed in all of the treated samples. The electrophoresis pattern of the muscle treated samples also revealed extensive proteolysis occurring in each muscle type. From the results, it is determined that latex from Calotropis procera can be used as an alternative source of proteolytic enzymes for the effective tenderising of meat.

Glutaraldehyde-activated chitosan matrix for immobilization of a novel cysteine protease, procerain B.

Proteases have several applications in the food industry. We report the immobilization of procerain B, a novel cysteine protease, on glutaraldehyde-activated chitosan beads through covalent attachment. Glutaraldehyde not only serves as a cross-linking agent but also links the procerain B on the surface of bead through primary amine group (either lysine side chain or N-terminal) by Schiff base linkage. Immobilized procerain B was characterized for optimum functional range and stability with respect to pH and temperature. The chitosan-immobilized procerain B has broad pH and thermal optima. The effects of substrate concentration and reusability of immobilized beads were also studied. It showed nearly 50% activity until the 10th use.

Exploring applications of procerain b, a novel protease from Calotropis procera, and characterization by N-terminal sequencing as well as peptide mass fingerprinting.

Procerain B is a novel cysteine protease isolated from Calotropis procera by our group and published recently. We have further characterized the enzyme by N-terminal sequencing and peptide mass fingerprinting. Procerain B showed maximum sequence similarity (80%) with Asclepain. Moreover, the characteristic VDWR motif of cysteine proteases is present in procerain B. The N-terminal and peptide mass fingerprinting analysis showed a distinct nature of the enzyme. Various applications of the enzyme were also evaluated. Procerain B is very effective in milk-clotting and may be a potential candidate for this process in the cheese industry. Additionally, the enzyme has potential application as dietary supplement to aid digestion. Effects of various metal ions on milk-clotting activity were also studied. The milk-clotting activity was increased in case of few metals while others have a negative effect. It is worth mentioning that the easy availability of plant material and simple purification method makes industrial production of the enzyme feasible. A protease with easy purification and suitable properties for application is always desired.

Characterization and optimization of carboxylesterase-catalyzed esterification between capric acid and glycerol for the production of 1-monocaprin in reversed micellar system.

Calotropis procera R. Br. carboxylesterase (EC 3.1.1.1) solubilized in reversed micellar glycerol droplets containing a very small amount of water (less than 5ppm) and stabilized by a surfactant effectively catalyzed the esterification between glycerol and capric acid to produce 1-monocaprin. Reaction variables including surfactant types, organic solvent media, reaction time, G-value ([glycerol]/[capric acid]), R-value ([water]/[surfactant]), pH, temperature, and types of metal ion inhibitors on the carboxylesterase-catalyzed esterification were characterized and optimized to efficiently produce 1-monocaprin. Bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and isooctane were the most effective surfactant and organic solvent medium, respectively, for 1-monocaprin formation in reversed micelles. The optimum G- and R-values were 3.0 and 0.05, respectively, and the optimum pH and temperature were determined to be 10.0 and 60 degrees C, respectively. K(m,app.) and V(max,app.) were calculated from a Hanes-Woolf plot, and the values were 9.64 mM and 2.45 microM/min mg protein, respectively. Among various metal ions, Cu(2+) and Fe(2+) severely inhibited carboxylesterase-catalyzed esterification activity (less than 6.0% of relative activity).

UNBS1450 from Calotropis procera as a regulator of signaling pathways involved in proliferation and cell death.

Despite significant progress in oncology therapeutics in the last decades, the urge to discover and to develop new, alternative or synergistic anti-cancer agents still remains. For centuries it has been known that the coarse shrub Calotropis procera is a very promising source of ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insecticidal, anti-inflammatory, anti-diarrhoeal, larvicidal and cytotoxic chemicals. Different compounds like norditerpenic esters, organic carbonates, the cysteine protease procerain, alkaloids, flavonoids, sterols as well as numerous types of cardenolides have provided this plant for centuries with scientists' interest. The chemical class of cardenolides and their related bioactivity evaluation and structure-activity relationship (SAR) studies pointed out their therapeutic utility and led to the discovery of promising drug candidates. Recently the cardiotonic steroid UNBS1450 01 (derived from 2-oxovoruscharin 02) from C. procera was shown to additionally exert an anti-cancer activity. UNBS1450 01 has been proven to be a potent sodium pump inhibitor, showing anti-proliferative and cell death-inducing activities. This anti-cancer potential of UNBS1450 01 is achieved by disorganization of the actin cytoskeleton after binding to the sodium pump at the cellular membrane, by inducing autophagy-related cell death, by repressing NF-kappaB activation as well as by down-regulating c-Myc in cancer cells. We aim to review pharmacologically important chemical extracts from C. procera and focus more specifically on the anti-cancer activities of UNBS1450 01.

Procerain, a stable cysteine protease from the latex of Calotropis procera.

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.