Latex Proteases Research Articles

Effect of purified Calotropis gigantea latex protease on the cheese quality

Cheesemaking has been relying solely on chymosin for so long. But factors like increased cheese production, diminishing supply of chymosin as well as religious and dietary limitations have been hindering the demand-production balance of cheese in the world. Hence, the primary objective of this study was to utilize the three-phase partitioning (TPP) purified Calotropis gigantea latex protease in cheesemaking. The cheese prepared using purified protease was compared with chymosin cheese for physicochemical, sensorial, textural, and microbiological evaluation. The optimum conditions for purified protease from response surface methodology (RSM) analysis were 6.25 milk pH and 45℃ milk temperature. The physicochemical parameters (moisture content, protein, ash, calcium, salt content, and pH) of cheeses prepared from latex protease and chymosin were significantly different (p<0.05). The yield of latex protease cheese was significantly higher (p<0.05) than chymosin cheese. In terms of texture and aftertaste, the cheese made with latex protease had significantly lower (p<0.05) mean scores than chymosin cheese. Compared to chymosin cheese, the latex protease cheese had a generally inferior textural character, with significantly (p<0.05) lower values for hardness, chewiness, gumminess, cohesiveness, and resilience. The total viable count and Lactobacilli count of the cheeses produced with chymosin and latex protease showed a significant (p<0.05) difference. Hence, this study highlighted that the TPP purified C. gigantea latex protease could be used as a plant coagulant for cheesemaking.

Biotechnological application prospects of latex proteases from Apocynaceae family

Plant latex proteases from different species find applications in diverse industries ranging from food to medicine. Family Apocynaceae consists of species producing large quantities of milky latex. Latex is known to be a rich source of proteolytic enzymes or proteases. Proteases, mainly of the cysteine and occasionally serine type, have been characterized from the latices of different species of Apocynaceae. Many of these proteases have also demonstrated stability under a wide range of parameters including temperature, pH and presence of metal ions. The current review is an attempt to elucidate on latex proteases belonging to Apocynaceae family, highlighting those properties that offer potential for their large-scale applications in different industrial sectors.

Thermostable ficin from jelly fig (Ficus pumila var. awkeotsang) latex: purification, identification and characterization

The achenes/seeds of endemic jelly fig (Ficus pumila var. awkeotsang) fruit have been applied to prepare a traditional beverage in Taiwan. Upon fruit harvest, jelly fig latex exuded from stalks was discarded. Protease activity was monitored in its latex. Proteases capable of hydrolyzing proteins have many application aspects based on diverse characteristics. Commercial plant proteases are frequently from latex. The latex protease of jelly fig, termed FaFicin, was purified to homogeneity with a molecular mass of ~32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to liquid chromatographic-tandem mass spectrometric analyses, the expected protein band of protease was matched to ficin A, ficin B or chymopapain from common fig or papaya. Iodoacetamide, an inhibitor of cysteine protease, inhibited its protease activity completely. Hence FaFicin was identified as a papain-like cysteine protease (PLCP), exhibiting more than 80% and 70% activity as assayed at pH5-8 and 40-70 °C, respectively. It maintained ~89% of initial activity after 120 min at 55 °C and pH7. Moreover, FaFicin could degrade the myosin and actin of meat, and clot milk. The ficin FaFicin was obtained, purified and identified as a PLCP member from agricultural waste: jelly fig latex. It possessed activity under a wide range of pH values and temperature, and exhibited excellent thermostability. Based on its initial evaluation as a meat tenderizer and milk clotting reagent, the application of FaFicin was possible, which may extend utilization of jelly fig. © 2022 Society of Chemical Industry.

Autoproteolysis of Procerain and Procerain B mediated by structural changes

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Latex peptidases produce peptides capable of delaying fungal growth in bread

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60–90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

Euphorbia trigona latex nematicidal activity on the root-knot nematode Meloidogyne incognita

The aim of this study was to test nematicidal activity of the Euphorbia trigona latex on Meloidogyne incognita juveniles and to partially purify and characterize three proteases present in this latex. Three distinct proteases were partially purificated from E. trigona latex. They were named here trigonin 1, 2 and 3. Their molecular weights were estimated at: 36, 31 and 29 kDa, for trigonin 1, 2 and 3, respectively. The pH and temperature that provided highest protease activity were pH values of 4.0, 6.0 and 9.0, and 70 °C. The crude extract containing the three proteases present in E. trigona latex reduced significantly (p < 0.01) the number of live M. incognita juveniles in 96% after 24 hours treatment. The present study is the first report of E. trigona latex with nematicidal activity, therefore more studies regarding latex proteases action on nematodes are needed.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from theFicus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases andthe nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2 leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Purification, characterization and fibrino(geno)lytic activity of cysteine protease from Tabernaemontana divaricata latex.

Protease was isolated and purified from Tabernaemontana divaricata latex and its hemostatic potential was analyzed. Crude latex enzyme was purified through ion exchange and gel filtration chromatography. Purified protease was characterized and its thrombin-like (coagulant assay, fibrinogen polymerizing, and fibrinogenolytic activity) and plasmin-like (blood and plasma clot lysis) activities were assessed accordingly. The homogeneous nature of protease was confirmed with the identification of a single band approximately at 25-kDa molecular weight position. The purified enzyme showed an enhancement of 77.32% in clot inducing ability and 89.86% improvement in blood clot lysis in comparison to that by the crude enzyme. All three subunits (Aα, Bβ and γ chains) of human fibrinogen were hydrolyzed by the purified enzyme. PAGE results of the fibrinolytic activity and blood clot lytic effect by the purified enzyme indicated the plasmin-like activity. The study lays a foundation for the development of enzyme-based approaches for pharmaceutical innovations, in which plant latex proteases can be utilized as a potential natural agent for wound healing applications.

Perspectives for the use of latex peptidases from Calotropis procera for control of inflammation derived from Salmonella infections

BackgroundAnti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera. PurposeA mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection. MethodsLPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium. ResultsLPp2 was not toxic at dosages lower than 62.2 μg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2. ConclusionWe conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella.

Thrombin-like serine protease, antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA2, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.

Tabernaemontana divaricata Stem and Latex Proteases as Haemostatic Agent with Temporally Spaced Intense Fibrinogenolytic and Mild Fibrinolytic Activity

Background: Proteases play a crucial role in the pharmacological properties of latex producing plants. Some of them exhibited intervention with fibrinogenolysis and/or fibrinolysis, two crucial wound healing events. Objective: To evaluate wound healing potential of crude and partially purified enzyme from Tabernaemontana divaricata (stem and latex). Materials and Methods: Proteolytic activity, clot inducing/dissolving potential, fibrinogen polymerization, recalcification time, blood clot lysis and Tricine-SDS PAGE for enzyme treated fibrinogen and human plasma clot were performed. Results: Latex PPE exhibited significant proteolytic activity (115.8 ± 0.3 U/ml) compared to that of the stem (28.78 ± 0.2 U/ml). Enzyme preparations exhibited temporally spaced clot inducing and subsequent dissolving properties favoring hemostatic effect, procoagulant effect being dominant and the first event. Significant reduction in fibrinogen absorbance at 540 nm with time, recalcification time and human fibrinogenolytic product analysis on Tricine PAGE substantiated procoagulant effect. Disappearance of Aα and Bβ fibrinopeptides by both stem and latex PPEs in the PAGE was observed. γ subunits were completely hydrolysed by latex PPE, however, it showed comparative resistance to stem PPE. Reduction in blood clot weight and fibrin subunit intensity supported thrombolytic property. Conclusion: The study provides evidence of the procoagulant and thrombolytic activity associated with T. divaricata proteases.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.

Crystal structure and specific location of a germin-like protein with proteolytic activity from Thevetia peruviana

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical β-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.

Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization. Application to the synthesis of an antihypertensive peptide

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Ya) and protein yield (Yp).Asclepain-OGS showed the highest Ya (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Yp was 75 ± 0.4 %. Ya for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.

Serine protease from Artocarpus altilis (breadfruit) latex

Breadfruit is recognized as a plant with the potential to be cultivated as an alternative staple food due to its high starch content and envisioned as a solution to mitigate hunger. The plant bears abundant fruits throughout the year with its productivity is comparable to other staple crops and it thrives well in countries where the poor population is high. Breadfruit however, exudes copious amount of latex upon harvesting and the stained epicarp often perceived as low in quality. Standardized methods of harvesting in plantations include a stage of latex draining by inverting the fruit for several hours. The latex will be drained to the ground and considered as an agricultural waste with no current commercial application. Despite being considered as a nuisance, plant latexes is a rich source of proteases functioned as a defensive mechanism against pathogenic attacks. In the effort to identify its potential, the breadfruit latex protease was purified and its characteristics were determined. The stability of the protease was investigated and its kinetics of inactivation was estimated in this research. Based on the analyses, breadfruit latex was discovered to consist of a serine protease with highly stable properties, potentially developed as an alternative commercial protease.

Hemostatically potent small molecular weight serine protease from Maclura spinosa (Roxb. ex Willd.) accelerates healing of subcutaneous dermal wounds in Swiss albino mice

Latex of Maclura spinosa has been used by Kollamalayali tribal community of Western Ghats for curdling of milk. In our previous study we have reported the presence of multiple proteases in the latex, among which the serine protease possesses potent role in hemostasis. With a view to further characterize these proteases the current study was taken up. Maclura spinosa latex contains a serine protease which is resolved using molecular size exclusion column chromatography and an anion exchanger resin in a consecutive manner. The specific activity of the enzyme Maclura spinosa latex protease (MSLP) was found to be 56.15 units/mg and recovery to be 2.68% with a fold purity of 0.41. Being a typical serine protease, MSLP is significantly inhibited by PMSF up to 72.97%. The optimum temperature and pH for the enzyme were found to be 50 °C and 8 respectively. Excision wound healing assay in Swiss albino mice using MSLP, showed accelerated wound closure up to 89.35 ± 1.209% compared to 91.938 ± 1.649% shown by positive control. Further PMSF treated MSLP sample did not show considerable wound healing which confirms exclusive involvement of serine proteases. Examination of biochemical markers viz, hydroxy proline content in healing tissue and catalase activity of fatty tissue also ascertain the potential of MSLP in wound healing. Histopathological studies of healing tissue provide confirmatory evidence with dense collagenation of tissue and fibroblast proliferation rendered by MSLP in the respective treated subjects.

Laticifers, Latex, and Their Role in Plant Defense.

Latex, a sap produced by cells called laticifers, occurs in plants of wide taxonomic diversity. Plants exude latex sap in response to physical damage. Questions about the function of latex or the underlying mechanisms persist, but a role in defense is likely. The presence of constitutive peptidases in latex sap in addition to inducible and de novo synthesized pathogenesis-related proteins (PR-proteins), raises the question about the role that each sap component plays to protect plants and how synergism occurs among sap proteins in the course of herbivory or infection. Here we discuss a variety of functions for laticifer and latex in plant defense. We propose that latex peptidases build the front line of defense against herbivores or pathogens.

Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model.

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.

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.