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Latex lipase of Euphorbia characias L.: An aspecific acylhydrolase with several isoforms
Abstract
The objective of the present work was to contribute to the understanding of the physiological role of latex lipolytic activity in Euphorbia characias. To this end, the acid and basic lipolytic activity of E. characias latex, as well as the substrate specificity on various triacylglycerols, were measured during the plant's vegetative and reproductive stages. Both activities appeared to increase during the reproductive stage and to peak at the beginning of the vegetative stage, when new leaves and branches are formed. For the first time, the phospholipolytic and esterase activity of E. characias latex is also reported. An extraction method in aqueous medium with the zwitterionic detergent CHAPS was successfully used to extract lipolytic activity from latex. Extraction permitted the selective recovery of a single protein spot, with a molecular weight of 37kDa, and presumably made of several acid isoforms which retained both lipolytic and phospholipolytic activity. The biochemical results suggest that lipolytic and phospholipolytic activity could depend on a single hydrolytic enzyme with several isoforms, equally expressed throughout the biological cycle of the plant. On the basis of the obtained results, we hypothesise that the E. characias latex lipase should be considered as an aspecific acylhydrolase with a combined lipase/phospholipase A activity.
... The extraction of lipases from the latex polymer matrix has not been easy. Classically, the fi rst step for the enrichment of the lipase activity present in the latex is the removal of soluble proteins in the mixture or a mix of water and a solvent in order to eliminate lipids present in the sample and achieve the extraction (24)(25)(26) . Other strategy that has been employed is the use of some nonionic and zwitterionic detergents (CHAPS, Triton TX-100, etc.). ...
... Other strategy that has been employed is the use of some nonionic and zwitterionic detergents (CHAPS, Triton TX-100, etc.). In the case of Euphorbia characias lipase, Fiorillo et al. ( 25 ) studied various detergents for extraction and combined them with various physical and chemical processes, including the use of a zwitterionic detergent (CHAPS) which improved the extraction yield. ...
Lipases from plants have very interesting features for application in different fields. This chapter provides an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from C. papaya is given.
... Lectin (inhibited by lactose and D-galactose), Euphorbia lactea, Euphorbia hermentiana, etc. (Euphorbiaceae) (Lynn and Clevette-Radford, 1986c) Chitin-binding protein, (Hevein-like) Hevea brasiliensis (Euphorbiaceae), Morus alba (Moraceae) (Gidrol et al., 1994;Broekaert et al., 1990;Wasano et al., 2009) GlcNAc-binding (Chitin-binding) protein (non-hevein like), Cucurbita maxima (Cucurbitaceae, phloem sap) (Read and Northcote, 1983;Walz et al., 2004;Kehr, 2006;Van Damme et al., 1998) Chitinase (also chitin-binding), Calotropis procera (Apocynaceae), Morus alba (Moraceae) Kitajima et al., 2010) Others Lipase, Euphorbia characias (Euphorbiaceae), Asclepias curassavica (Apocynaceae), Carica papaya (Caricaceae) (Giordani et al., 1991;Fiorillo et al., 2007;Gandhi and Mukherjee, 2000) Glutamyl cyclase, Carica papaya (Caricaceae) (Azarkan et al., 2004) Gum arabic glycoprotein, Acacia senegal (Fabaceae) (Goodrum et al., 2000) Phenyl alanine ammonia lyase (PAL), Lactuca sativa (Asteraceae) (Sethi et al., 2009) Phosphatase, Euphorbia esula, Euphorbia splendens (euphorbiaceae) (Lynn and Clevette-Radford, 1987b) Linamarase (b-glucosidase), Manihot esculenta (Euphorbiaceae) (Nambisan, 1999) in this section. Mulberry leaves exude latex ( Fig. 2A). ...
... In addition to the proteins described above that are reported in many plant families, there are still more latex proteins with a limited distribution among plant taxa that may potentially be involved in plant defenses against herbivores. These include lipases in the latex of Caricaceae, Euphorbiaceae, Apocynaceae (Fiorillo et al., 2007;Gandhi and Mukherjee, 2000;Giordani et al., 1991); glutamyl cyclase in Caricaceae (papaya) (Azarkan et al., 2004;Zerhouni et al., 1998); gum Arabic glycoprotein found from exudates of Acacia senegal (Fabaceae) (Goodrum et al., 2000); phenylalanine ammonia lyase (PAL) in the latex of romaine lettuce, L. sativa (Asteraceae), which is induced after insect herbivory in an insect-resistant line (Sethi et al., 2009); and phosphatase in the latex of Euphorbiaceae (Lynn and Clevette-Radford, 1987b). Phosphatase activities of VSPs (vegetative storage proteins), which are widely distributed in plants such as soybean and Arabidopsis thaliana, are associated with the toxic effects of VSPs against coleopteran and dipteran insects, suggesting that phosphatases could potentially function as defense proteins (Liu et al., 2005). ...
Plant latex and other exudates are saps that are exuded from the points of plant damage caused either mechanically or by insect herbivory. Although many (ca. 10%) of plant species exude latex or exudates, and although the defensive roles of plant latex against herbivorous insects have long been suggested by several studies, the detailed roles and functions of various latex ingredients, proteins and chemicals, in anti-herbivore plant defenses have not been well documented despite the wide occurrence of latex in the plant kingdom. Recently, however, substantial progress has been made. Several latex proteins, including cysteine proteases and chitin-related proteins, have been shown to play important defensive roles against insect herbivory. In the mulberry (Morus spp.)-silkworm (Bombyx mori) interaction, an old and well-known model system of plant-insect interaction, plant latex and its ingredients--sugar-mimic alkaloids and defense protein MLX56--are found to play key roles. Complicated molecular interactions between Apocynaceae species and its specialist herbivores, in which cardenolides and defense proteins in latex play key roles, are becoming more and more evident. Emerging observations suggested that plant latex, analogous to animal venom, is a treasury of useful defense proteins and chemicals that has evolved through interspecific interactions. On the other hand, specialist herbivores developed sophisticated adaptations, either molecular, physiological, or behavioral, against latex-borne defenses. The existence of various adaptations in specialist herbivores itself is evidence that latex and its ingredients function as defenses at least against generalists. Here, we review molecular and structural mechanisms, ecological roles, and evolutionary aspects of plant latex as a general defense against insect herbivory and we discuss, from recent studies, the unique characteristics of latex-borne defense systems as transport systems of defense substances are discussed based on recent studies.
... The latex of Carica papaya is already known as a rich source of cysteine endopeptidases, including papain, chymopapain and caricain [10]. These proteinases can be extracted in the form of water-soluble proteins from the latex [11,12]. The presence of lipase activity was first reported by Giordani et al. [13], but this activity was found to be associated with the insoluble pellet obtained after latex centrifugation [6,[11][12][13]. ...
... These proteinases can be extracted in the form of water-soluble proteins from the latex [11,12]. The presence of lipase activity was first reported by Giordani et al. [13], but this activity was found to be associated with the insoluble pellet obtained after latex centrifugation [6,[11][12][13]. Until recently, all attempts to solubilise the enzymatic activity from this latex fraction were unsuccessful [14]. ...
Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.
... The mesocarp of the fruit of oil palm contains the highest level of lipase activity recorded for a plant tissue [21]. The lipase from Euphorbia latex has been studied by a few groups [22][23][24][25]. It was found to be soluble in organic solvents, and a solvent-based procedure has been used to purify this enzyme [24,25]. ...
... The antimicrobial function of PLA2s is well documented [44]. Recently, a PLA activity was also shown in the latex of Euphorbia [22]. No obvious PLA2 candidate has been detected by MS analysis of latex major proteins. ...
Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.
... Plant latex lipases have in common optimal pH activities at neutral to alkaline pH values, preference for carboxylic acids of short and medium chain lengths, and sn-1,3 regioselectivity. These enzymes proved to be very interesting biocatalyst for diverse monoesters synthesis, hydrolysis of triglycerides, alcoholysis of sunflower oil, resolution of naproxen, lipids modification and asymmetric resolutions [21,[24][25][26][27][28][29][30]. Within this context, ASL has an optimal pH and temperature of 8.5 and 60 • C respectively, and showed a marked preference towards the hydrolysis of butyrate esters [31]. ...
... Plant latex lipases have in common optimal pH activities at neutral to alkaline pH values, preference for carboxylic acids of short and medium chain lengths, and sn-1,3 regioselectivity. These enzymes proved to be very interesting biocatalyst for diverse monoesters synthesis, hydrolysis of triglycerides, alcoholysis of sunflower oil, resolution of naproxen, lipids modification and asymmetric resolutions [21,[24][25][26][27][28][29][30]. Within this context, ASL has an optimal pH and temperature of 8.5 and 60 • C respectively, and showed a marked preference towards the hydrolysis of butyrate esters [31]. ...
The present contribution investigates the biocatalytic performance of the lipase obtained from the latex of the native plant known as Araujia sericifera (ASL) in the hydrolysis followed by the esterification of released free fatty acids and transesterification of glycerides of sunflower waste cooking oil WCO. A specific enzymatic activity of 719.05 μmol mg⁻¹ h⁻¹ (60 % conversion of triglycerides towards free fatty acids) was obtained in the hydrolysis of WCO with 0.05 % of biocatalyst (typically, 10.0 g WCO and 5.0 mg of enzyme) and 50 % w/w of water added in 30 min of reaction at 25 °C in homogeneous type of reaction system. The lipase has the capacity to catalyze the hydrolysis of the triglycerides mainly towards monoglycerides and diglycerides in a lesser extent. The released FFAs and the remaining glycerides reacted in tandem by addition of short chain alcohols immediately after the hydrolysis (without addition of more enzyme to the reaction media). Above 90 % conversion of the FFAs was obtained with methanol, ethanol, n-propanol and n-butanol at contents as low as 1:0.2 oil: alcohol molar ratio at 25 °C. The transesterification of the remaining monoglycerides (about 20 %) was also observed although no further reaction of the triglycerides was detected even under a great excess of alcohol.
... Aside from microorganism, plant seeds and plant latex are potential sources of lipase due to their high availability, low production cost and ease of handling (Avelar et al., 2013). Plant-based lipases that have been reported are castor seeds (Avelar et al., 2013), physic nuts (Barros et al., 2010), oatseeds (Piazza dan Haas, 1999), sunflower seeds (Barros et al., 2010), ricebran (Loeb et al., 1949), papaya latex (Domínguez de María et al., 2006), frangipani latex (Cambon et al., 2006), Euphorbia characias latex (Fiorillo et al., 2007), etc. In other work, we reported frangipani latex as the most active lipase sources compared to other plant lipase sources such as castor seeds, rice bran, and papaya latex. ...
Fatty acids are intermediate substances in synthesis of oleochemical products. Enzymatic technology of fatty acids production (also known as lipolysis) is now developing as potential substitution for the conventional production of fatty acid, i.e. thermal hydrolysis of triglyceride. It offers more economical process condition, low energy consumption, and minimal product degradation compared to the conventional process. This research aims to evaluate performance of various organic solvents as reaction media in lipolysis with plant latex lipase. Organic solvents observed were chloroform, n-hexane, diethyl ether, benzene, acetone, ethanol, methanol, n-heptane, and isooctane. Analysis of each organic solvent effect on lipolysis was described based on solvents properties. Conversion of lipolysis with organic solvents is 0,10-1,25 times fold compared to conversion of non-solvent lipolysis. We suggest that dielectric constant and viscosity are the two main organic solvent properties affecting lipase performance in lipolysis. Overall, n-hexane, n-heptane, and isooctane are recommended to be used as reaction media in lipolysis with plant lipase because their effects to degree of lipolysis are positive. Keywords: lipolysis; lipase; organic solvent; frangipani
... The results obtained were in agreement with Tsuchiya et al. (1999) who suggest that the vegetable lipases intervene in the metabolism, rearrangement and degradation of chlorophyll during the growth process and the senescence of leaves, thus in the fruits ripening process. In addition, Fiorillo et al. (2007) reported that the lipolytic activity of crude latex of Euphorbia characias L. progressively increased from January to May corresponding to the reproductive stage; the activity peaked in June and decreased from July to November (over the course of the vegetative stage). However, the correlation between lipase and protease activities was showed by Caro et al. (2000) Table 1. ...
In this study, enzyme activities and latex production were assessed on twenty fig cultivars of San Pedro, Common, Smyrna and Caprifig-types, throughout different maturity stage (unripe, mid-ripe and ripe) for three times a day (morning, mid-day and late afternoon). Comparison of latex yield revealed negative correlation between the developmental changes of enzymes in the target fig types. The highest latex yield was 1% recorded in the Common-type cultivar Kahli and both San Pedro-type cultivars Bither Abiadh and Bither Akhal, while this yield declined 10-fold in the Smyrna-type cultivar Njali at mid-maturity stage. The protease activity exhibited the highest level of 1,312,652 U/ml in cultivar Bidh Beghal (Smyrna-type) at the ripening stage that was correlated with the increase of lipase level by 286.33 U/ml at the mid-maturity stage, while its inhibition was recorded by 100% at the unripe stage. The synergic effect between lipase and protease activities was recorded during the receptivity period, which means that enzyme expression of the fig latex depends upon the emitted volatile compounds. The apparent reliance on constituents as attractants and few and specific pollinators may indicate co-evolution.
... An interesting alternative is represented by plant lipases, which are relatively easy to obtain [9]. In this sense, some plant latex-producing families such as Caricaceae, Euphorbiaceae, and Apocynaceae showed lipase activity [10]. From these lipases, Carica papaya latex lipase (CPL) has been, by far, the most studied in different industrial applications due to the abundance of this resource, its easy production and versatility [11]. ...
The present contribution screens the specific activity of various inorganic and enzymatic based materials in the esterification of oleic acid that is typically used as a test reaction for the production of biodiesel from high free fatty acid feedstocks. The inorganic materials investigated in this contribution are bulk fosfotungstic heteropoly acid of the Wells Dawson structure H6P2W18O62.nH2O (HPA), as well as dispersed on titanium dioxide 18 % w/w H6P2W18O62/TiO2 and the insoluble cesium salt of the Wells Dawson heteropoly anion Cs2H4P2W18O62. Additionally, the commercial biocatalyst Novozym® 435 (immobilized lipase B of Candida antarctica) and a self-supported lipase of vegetable origin obtained from the latex Araujia sericifera (ASL) were studied among the materials of enzymatic nature. The density and accessibility of Brønsted acid sites have a key role in the specific activity of the fosfotungstic based heteropoly compounds. The HPA dispersed over an oxide support catalyzed the esterification of the fatty acid in a heterogeneous fashion with the highest activity (6.4 µmol mg⁻¹ h⁻¹ at 85 °C) among the inorganic materials. In contrast, the enzymatic materials are more active at lower temperature than the inorganic ones. Particularly, ASL catalyzed the homogenous methanolysis with the highest specific activity (30.7 µmol mg⁻¹ h⁻¹ at 40 °C) at the lowest temperature among the materials assayed.
Graphical Abstract
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... Some family plants known as laticifers are Apocynaceae, Asclepiadaceae, Euphorbiaceae, Moraceae and Sapotaceae (Moulin et al., 1994;Palocci et al., 2003;Giordani et al., 1991;Villeneuve et al., 2005;Dhuique-Mayer et al., 2001, 2003. The laticifers plants show intense metabolic activity, with the latex containing lipids, rubbers, resins, and sugars, as well as several proteins and many different enzymes (that is, peroxidases, proteases, esterases, and phosphatases) (Fiorillo et al., 2007;El Moussaoui et al., 2001;Lynn and Clevette-Radford, 1987). The laticifers plants also show secondary metabolic activity directed toward the production of defence-related molecules, which accumulate in appreciable amounts in the latex. ...
Industrial-scale processes currently developed make use of chemical catalysis processes that are highly efficient but require very complex product purification steps. Enzymatic catalysis through plant lipases as biocatalysts is an alternative which, in contrast to chemical catalysis processes, appeared simple to perform, and can be done at low investment cost. Although microbial lipases have been extensively studied, little research has been focused on the use of plant lipases namely plant latex lipases. The present article outlines the most advanced knowledge concerning plant latex characterization in order to show how plant latex can be a promising alternative to catalyze transesterification for biodiesel production. This paper provides an overview regarding the main aspects of latex, such as the reactions catalyzed, physiological functions, specificities, sources and their industrial applications.
... In addition, some latex proteins are confined to specific plant taxa and have been suggested to be involved in plant defense. These compounds include phosphatase in Euphorbiaceae [5]; lipase in Caricaceae, Euphorbiaceae, Apocynaceae [6][7][8]; and glutaminyl cyclase in Caricaceae (papaya) [9,10]. Carica papaya Linn. ...
Crude latex of Carica papaya L. has been known to offer a lot of benefits and potentials
especially in the agricultural industry and human health. This study focuses on
the latex coming from its fruits of Papaya CX variety. Seven to eight
longitudinal incisions were made in order to allow latex to appear and drain in
the collecting devices. 439.5 g dried latex was stored in plastic
containers and freezed. Results showed that dried latex contained higher amount
of crude protein (57.24 ± 0.69%), followed by moisture (17.76 ± 0.09%),
ash (7.00 ± 0.01%), crude fat (5.21 ± 0.13%) and crude fiber (0.67 ± 0.09%)
based on the complete proximate analysis. In the enzyme analysis, papain had
protease activity of 2655 units·g-1 at pH 5.5 and 285 units·g-1 at pH 9.0. These results provided evidence that papain as a protease enzyme is
found in the crude latex of papaya which is a major constituent in
various proteolytic activities. Crude latex from C. papaya L. can be utilized to address the issues in agricultural farms to
accelerate production and reduce environmental hazards.
... Lipases obtained from the latex of other plant families have been characterized in terms of diverse biocatalytic features, but they have been less studied in terms of their potential biotechnological applications. 18,19 On the other hand, oilseed plant lipases are usually present in very low concentration levels, which represents a drawback for their use in large scale or pilot scale and justifies why, until now, their current applications are mainly on a laboratory scale. Plant lipases from sources other than oil seeds can be considered as promising catalysts for higher production levels. ...
Lipase activity found in the insoluble fraction of Araujia sericifera Brot. (Apocynaceae) (ASL) latex, a native South American milkweed, was characterized by use of different test reactions and under different reaction conditions. In this context, hydrolytic lipase activity towards both natural and synthetic substrates and towards fatty acid esterifications was assayed at different temperatures, pH values, and biocatalyst loads. In the case of natural substrates (cottonseed oil), highest lipase activity was found at pH 8.5 and 60 °C. In the hydrolysis of synthetic substrates (p-nitrophenyl esters) the lipase showed preference for the lowest molecular-weight p-nitrophenyl ester assayed (butyrate). Results of the direct esterification of fatty acids of different chain length in organic media showed that esterification levels of up to 45% could be obtained in one hour of reaction. Activity results were compared with the activity shown by the commercial immobilized lipase Novozym 435 under defined reaction conditions. The high activity exhibited by ASL in the hydrolysis of natural substrates and particularly in the direct esterification of different fatty acids in organic medium, together with its high storage stability, suggests that this plant lipase is a promising biocatalyst for various biotechnological applications.
... Activity of phenylalanine ammonia lyase, polyphenol oxidase and many other defense-related enzymes is much higher in the laticifers than in the leaves of rubber tree (Hevea brasiliensis H.B.K.) (Broekaert et al. 1990, Martin 1991, Gidrol et al. 1994, Pujade-Renaud et al. 1994, Wititsuwannakul et al. 2002. Wounding of laticifers is also known to induce other defense-related enzymes in latex of papayas (Azarkana et al. 2004, Kydt et al. 2007), fig tree (Ficus carica L.) (Kim et al. 2003, Taira et al 2005, rooster tree (Calotropis procera Ait.) , and Albanian spurge (Euphorbia characias L.) (Mura et al. 2005(Mura et al. , 2007Fiorillo et al. 2007). Thus, plant latex acts as a chemical defense due to alteration in its constituents upon insect damage. ...
... Similarly, class II chitinases and papain occurring in Carica papaya latex also play a defensive role against herbivorous insects [61] and deter them from feeding by making the food unpalatable to insects [60]. Thus, proteins, enzymes and allergens protect plants from herbivorous insect attack [64,65], but it is still unclear that repellent activity in plant latexes is due to presence of proteins or volatile substances occur in latex fluid [66] . As literature reveals, plant latexes contain different chemical components which show high anti-feedant, effects on herbivorous insects, when treated with different doses [67] in artificial diets [68]. ...
In the present investigation various bioassays were conducted to evaluate the anti-termite ef-ficacy of plant latex based formulations to con-trol population of Indian white termite in sub-tropical soil. Results reveal that crude latex, its fractions and combinatorial fractions have shown very high toxicity against O. obesus. The LD 50 values for different latex fractions of 24 h were in a range of 5.0 -17.613 μg/mg while com-bined mixtures of Calotropis procera have shown synergistic activity against termites and caused comparably high mortality with LD 50 1.987 -6.016 μg/mg. The mortality rate was found dose and time dependent as it was found to be increased with an increase in dose and ex-posure period. In olfactometry tests, C. procera latex solvent fractions have shown significant repellency at a very low dose 0.010 -0.320 μg/mg. Interestingly, solvent fractions have sig-nificantly repelled large numbers of worker ter-mites due to volatile action of active compo-nents of latex and different additives. ED 50 val-ues obtained in crude latex were 0.121 μg/mg body weights while combinatorial formulations have shown ED 50 in between 0.015 -0.036 μg/mg. Statistical analysis of repelled and un-repelled termites gave a low Chi-square value (X 2 value = 0.890) which is an indicator of independence of repellent action in randomly selected termite groups. In field experiments pre-soaked cotton threads impregnated with Calotropis procera crude latex were tagged around tree trunks of Tectona grandis provided a wider protection against O. obesus. By employing these pre-coa-ted threads, termite infestation and tunneling activity were significantly decreased (p < 0.05 and 0.01). When germinating crop plants were sprayed with various plant latex formulations, these have caused very high protective efficacy against termite infestation. It has significantly reduced crop losses up to 6.45%. There was a significant difference in infestation obtained in control and treatment groups (P < 0.05 and 0.01) which shows that Calotropis procera possesses enough anti-termite potential against Indian white termite, O. obesus population. If used these, formulations may also provide wide a range of control against other kinds of pests including house hold, medical and veterinary. However, Calotropis procera latex based for-mulations can be recommended for effective control of termites in high infestation areas by applying spray, or in form of poison baits or as fumigant in pure form.
... to the above-described proteins that were reported from many plant groups, some latex proteins are confined to specific plant taxa and have been suggested to be involved in plant defense. These compounds include phosphatase in Euphorbiaceae (Lynn & Clevette-Radford 1987); lipase in Caricaceae, Euphorbiaceae, Apocynaceae (Fiorillo et al. 2007, Gandhi & Mukherjee 2000, Giordani et al. 1991); glutaminyl cyclase in Caricaceae (papaya) (Azarkan et al. 2004, Zerhouni et al. 1998); and gum arabic glycoprotein, a high-molecular-weight, hydroxyproline-rich arabinogalactan-protein found from exudates of Acacia senegal (Fabaceae) (Goodrum et al. 2000). Finally, linamarase in cassava leaves and latex is a β-glucosidase that specifically degrades linamarine, also present in the leaves and roots of the same plant, and results in the production of hydrogen cyanide that is toxic to most organisms. ...
Latex is a sticky emulsion that exudes upon damage from specialized canals in about 10% of flowering plant species. Latex has no known primary metabolic function and has been strongly implicated in defense against herbivorous in-sects. Here we review historical hypotheses about the function of latex, evi-dence that it serves as a potent defense, and the chemistry and mode of action of the major constituent defense chemicals and proteins across a diversity of plant species. We further attempt to synthesize the characteristics of latex as a coordinated plant defense system. Herbivores that feed on latex-bearing plants typically evade contact with latex by severing the laticifers or feeding intercellularly, or may possess physiological adaptations. Convergent evolu-tion appears to be rampant both in plants with latex and insects that exploit latex-bearing plants. Because latex shows phenotypic plasticity, heritability, and macoevolutionary lability, it is an ideal system to study plant-herbivore interactions using evolutionary approaches.
... There has been a growing interest in plant lipases in recent years since they are cheap, very versatile, and stable in organic media [11][12][13]. Lipases from plant families like Asclepiadaceae [14], Euphorbiaceae [15][16][17], Caricaceae [18][19][20][21], or Rosaceae [22] have been described as useful biocatalysts for several applications. Very little work on 'unusual' or tropical seeds has been reported [21]. ...
Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive
oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70°C, respectively. The lipase was
stable between 30 and 80°C for 5min. K
m and V
max values were determined as 48mM and 23.06×10−3U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu2+, Ca2+, Hg2+, Mn2+, and Ni2+ ions, while Mg2+ and Zn2+ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80
and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50mM urea and thioacetamide.
Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents,
the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1year at −20°C.
KeywordsPurification-Characterization-Walnut seed-Lipase-Enzymatic activity-Stability
... Lipolytic latexes are also known from latex of some Apocynaceae and Euphorbiaceae (Palocci et al. 2005). With the exception of the study on Euphorbia characias L., by Fiorillo et al. (2007), research on plant latex lipases in the Euphorbiaceae is poorly studied compared to research on the Caricaceae. However, unlike most Euphorbia species, Acalypha, Macaranga, and Neoscortechnia do not produce milky latex. ...
Hanging by a Thread: Natural Metallic Mordant Processes in Traditional Indonesian Textiles. Despite the availability of synthetic dyes and the impact of significant religious, social, and economic change, textile weavers
in more remote areas of Indonesia continue naturally dyed textile production as a living tradition. This paper documents mordant
plants in Sulawesi, West Kalimantan, and nine islands in eastern Indonesia (Bali, Flores, Java, Lembata, Nusa Penida, Rai
Jua, Savu, Sumba, and West Timor). These plants, such as various Symplocos species, are hyperaccumulators of aluminum compounds. Other plants used as sources of alkaline ash, of saponifiable oils
and fats and for ritual purposes in the dyeing process, are also recorded.
Di Ambang Kepunahan: proses mordan dengan menggunakan logam dari tumbuhan dalam pembuatan kain tradisional di Indonesia. Ditengah maraknya pemakaian warna sintetis serta terjadinya perubahan dalam keyakinan, keadaan sosial dan ekonomi, penenun
di beberapa daerah terpencil tetap memproduksi kain warna alam sebagai sebuah tradisi. Jurnal ini membahas tumbuhan mordant
atau perekat warna serta tantangan yang dihadapi dalam pemakaiannya di daerah Sulawesi dan Kalimantan serta di sembilan pulau
lain di Indonesia mencakup Bali, Flores, Jawa, Lembata, Nusa Penida, Rai Jua, Sabu, Sumba dan Timor Barat. Tumbuhan mordant
yang dibahas, seperti Symplocos, menganndung zataluminum yang tinggi. Tumbuhan lain yang dipergunakan sebagai sumber abu alkali, minyak dan lemak saponifiable,
serta yang dipakai dalam ritual proses mordant juga dibahas dalam artikel ini.
Key WordsNatural mordants–oil seeds–
Symplocos
... Recently, a vast number of cysteine proteases have been described as occurring in many latex-producing plants (Liggieri et al. 2004;Morcelle et al. 2004a, b). Chitinases, lectins, lipases, oxidative and other hydrolytic enzymes have also been described as occurring in latex fluids (Stirpe et al. 1993;Azarkan et al. 1997;Giordani et al. 1992;Amani et al. 2007;Fiorillo et al. 2007). These findings on the biochemical composition of laticifer fluids support the hypothesis of their defensive role (Taira et al. 2005). ...
The defensive role of the latex of Calotropis procera has recently been reported. In this study, latex proteins involved in detrimental effects on insects were evaluated on another
important crop pest. The latex was fractionated to obtain its major protein fraction, which was then used to evaluate its
insecticidal properties against Callosobruchus maculatus (Coleoptera: Bruchidae) in artificial bioassays. Laticifer proteins (LP) were investigated to characterize their action in
such an activity. LP was highly insecticidal at doses as low as 0.1% (W/W). This effect was slightly augmented in F1 generation reared in artificial seeds containing LP at similar proportions of F0, but was fully reversed when F1 developed in LP-free seeds. The insecticidal proteins were not retained in a chitin column, and did not lose their insecticidal
activity, even after heat treatment or pronase digestion. However, these samples inhibited papain (EC 3.4.22.2) activity and
gut proteases of C. maculatus larvae, and a reverse zymogram showed the presence of protein bands resistant to papain digestion. These activities were
not observed in unheated LP as they were probably masked by abundant endogenous cysteine protease (EC 3.4.22.16) activity
present in unheated LP. LP was resistant to proteolysis when assayed with C. maculatus gut extract. However, gut proteins of C. maculatus were digested when incubated with LP. These observations and the deleterious effects of LP upon C. maculatus, reinforce the hypothesis that laticifer fluids are involved in plant defense against insects and indicate C. procera latex to be a source of promising insecticidal proteins. The inhibitor of proteolysis present in the latex seems to be resistant
to heat and proteolysis and is certainly involved in the detrimental effects observed.
KeywordsApocynaceae-Bruchidae-
Calotropis procera
-Plant defense-Proteases-Protease inhibitor
... During the last few decades, considerable attention has therefore focused on these enzymes (Lee, Xia, & Zhang, 2008). Various plant sources of lipases and suitable techniques for isolating and purifying them have been well documented (Fiorillo, Palocci, Soro, & Pasqua, 2007;Giordani, Moulin, & Verger, 1991;Palocci et al., 2007;Suzuki, Honda, & Mukasa, 2004). Lipases belonging to plant families such as Euphorbiaceae (Giordani et al., 1991;Moulin, Teissere, & Bernard, 1994), Asclepiadaceae (Giordani et al., 1991), Brassicaceae (Hills & Mukherjee, 1990) and Caricaceae (Giordani et al., 1991) have been described as providing useful biocatalysts for several applications. ...
High levels of lipase activity are known to occur in Carica papaya latex, and this activity is being used in some biotechnological applications. The lipolytic activity of C. papaya lipase (CPL) on dietary triacylglycerols (TAG) has not yet been studied. Hence, the aim of this study was to characterise the specific activity of CPL on dietary TAG present in a crude preparation. Also, we have determined its stability during the lipolysis of a test meal at various pH values mimicking those occurring in the gastro-intestinal tract, with or without bile, and have compared these properties with those of porcine pancreatic extract (PPE) and human pancreatic lipase (HPL). CPL showed maximum stability at pH 6.0, both with and without bile. Some residual activity was still observed at pH 2 (20%), whereas the pancreatic lipases tested were immediately completely inactivated at this pH. In the absence of bile, the highest specific activities were measured at pH 6 in the case of CPL, PPE and HPL. Adding bile slightly decreased the CPL activity in the 4–6 pH range, thus shifting the optimum CPL activity to pH 7, where the presence of bile had no effect. Lipolysis levels decreased with the pH, but CPL was still more active than PPE at pH 5 on a relative basis. These results suggest that CPL might be a promising candidate for use as a therapeutic tool on patients with pancreatic exocrine insufficiency.
Over 350 million years have passed since the documentation of the first interaction between plants and insects. Numerous plant defense qualities and associated counter-adaptive features have developed as a result of these interactions between insects and plants. These characteristics might be either morphological or biological in nature. One of the most significant and useful biochemical characteristics in plants is latex. Latex has a sticky property due to presence of secondary metabolites in it, which aids in entangling or sealing the mouthparts of small insects. These metabolites also chemically interact with the insects interfering with crucial bodily processes. Plant latex has amazing properties that help protect plants from insects and inhibit them in general. It may be possible to control insect pests in a natural, secure, and long-lasting manner by correctly identifying plant latex with strong insecti-cidal properties and developing formulations of plant latex.
Lipases from vegetable sources have been the focus of intense and growing research. The use of enzymes from plants has the advantage of employing industrial waste products. The lipase activity of Euphorbia peplus L. (Euphorbiaceae) was investigated for the first time. The Euphorbia peplus latex lipase (EpLL) was purified after ammonium sulfate fractionation and anion exchange chromatography on a DEAE-Cellulose column leading to 12.57-fold purification. The EpLL displayed a probable molecular weight of about 40 kDa. The lipase activity was optimum at a temperature of 40 °C and pH 8, the specific activities of EpLL were found to be 249 ± 12.45 and 161.4 ± 8.07 U/mg when tributyrin (TC4) and olive oil were used as substrate respectively. The enzyme retained 80 % of its activity when incubated for 1 h at 50 °C. The EpLL was strongly destabilised by divalent metal ions (Fe2+, Mg2+, Zn2+ and Cu2+). Lipase was slightly stimulated by Triton X-100 and Tween-80, while strongly inhibited by sodium dodecyl sulfate. A good stability of the enzyme in the presence of organic solvents was reveled suggesting its industrial utility.
Interest in the production of biodiesel—a clean renewable fuel—is increasing worldwide. Industrial-scale processes currently being developed use homogenous and heterogeneous chemical catalysis processes that are highly efficient but require very pure reagents and complex product purification steps, or high temperature and pressure processing conditions. Enzymatic catalysis using plant lipases as biocatalysts is an alternative which, contrary to chemical catalysis processes, is simple to perform, at low investment cost, and therefore potentially easier to disseminate, especially in developing countries. Although microbial lipases have been extensively studied, little research has been focused on the use of plant lipases in biodiesel production. These latter lipases can, however, be readily extracted from the plant organs that contain them (seeds, bran or latex) and they are less expensive to use than microbial lipases. The aim of the present article was thus to review published research findings and outline the most advanced knowledge concerning alkyl ester production catalyzed by plant lipases. This work focuses mainly on the conditions for using plant lipases in certain synthesis reactions (biomass selection, preparation and purification of lipase extracts) and current knowledge on reaction parameters that affect the catalytic activity. Moreover, biodiesel production using plant lipases and ethanol instead of methanol certainly seems to be in line with the development of innovative environment-friendly technologies.
Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.
The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.
The latex of Euphorbia lathyris can utilize acetate, pyruvate and mevalonate for triterpene synthesis in vitro. Acetyl-CoA, hydroxymethylglutarate, hydroxymethylglutaryl-CoA and isopentenyl pyrophosphate were not effective as precursors for triterpene biosynthesis. Acetate is utilized only by the terpenoid pathway and by the tricarboxylic acid cycle; it is not used for fatty acid synthesis in this system. However, phospholipids were found to be efficient acyl donors for triterpene ester synthesis. The observed selectivity of precursor utilization as well as the observed rates for product formation indicate separate sites for triterpenol and triterpene ester synthesis and that one is not precursor for the other.
The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.
A lipase from the latex of Euphorbia characias was purified using a method involving extraction with apolar solvent and adsorption chromatography on silica gel. The lipase (specific activity, 1500 international units/mg of protein) was eluted from silica gel complexes with a lipid. The main protein fraction, which had a molecular mass of 38 kDa, was inactive when dissociated from the lipid fraction. When the lipid and protein fractions were reassociated, 72% of the lipolytic activity was recovered. This lipolytic activity was inhibited by diethyl p-nitrophenyl phosphate, which was shown to bind the lipase with a molar ratio of 0.75. High specific activities (1000 international units/mg) were measured for the lipase of E. characias on lipid extracts rich in galactosyl diacylglycerols. The apolipase was sequenced up to residue 23. The B chain of ricin has a strong homology (43.5%) with that sequence and cross-reacted with antibodies raised against the purified lipase from E. characias. The activity of the B chain of ricin was comparable (54 international units/mg) to that of the apolipase of E. characias (100 international units/mg) mixed with the same lipid cofactor complex. The primary structure (residues 68-72) of the B chain of ricin contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly. Its reactivity with diethyl p-nitrophenyl phosphate indicates the presence of an activated serine that, in addition to its well-documented lectin activity for galactosides, suggests that the B chain of ricin may be a galactosyl diacylglycerol lipase, closely analogous to the lipase from E. characias.
In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.
The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC(4)) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent K(m) (K(m,app)) of 2.4 mM, a k(cat) of 200 min(-1) and a specific activity of 1.0 unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC(10)), with a K(m,app) of 3.5 mM, a k(cat) of 173 min(-1) and a specific activity of 3.5 units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH 7.0 in the presence of 0.2 M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20 mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30 mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC(10) and BAL-TC(4) hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC(4) and pNPC(10), and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins.
Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to
identify this enzyme, proteomic analysis was performed on purified oil bodies. A ∼60-kDa protein was identified (RcOBL1),
which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an α/β-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima ∼4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not
active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that
the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence
for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like
proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.
The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The qualitative and quantitative variations in the acetone extracts of Euphorbia characias growing in Sardinia were studied throughout one year (May 1986 to April 1987) by capillary column GC and GC-MS. The composition of the triterpene alcohols as well as the sterol fraction was investigated.
Purified particles isolated from the latex of Euphorbia species catalyse the synthesis of triterpene esters from endogenous triterpenol and free fatty acid as the acyl donor. Exogenous ATP and CoA did not stimulate the enzyme activity. The enzyme is probably located in the protein layer that surrounds the particle. The experimental results indicate that the triterpenol esterification occurred via a triterpenol esterase reaction. The esterase exhibits fatty acid, and to a lesser extent, triterpenol specificity. Short-chain and polyunsaturated fatty acids are preferred to longer chain and less unsaturated acids. Among the various free triterpenols present in Euphorbia latex particles, cycloartenol was the favoured substrate for the esterase.In vitro incubations with whole latex and labelled acetate showed that newly synthesized labelled triterpenols are absorbed by the particles as esters of almost unlabelled fatty acids. The possible physiological significance of the latex particle triterpenol esterase is discussed.
Lipase fatty acid typoselectivities of Euphorbia characias latex and commercially available crude preparation of bromelain were determined in the hydrolysis of homogeneous triacylglycerols (TAG) and natural TAG mixtures. Their activities were compared to a commercially available crude preparation of papain. Under optimal lipolysis conditions at pH 8.0 and 10 min of incubation time, maximal activities were observed at 45, 55, and 50°C, respectively, for E. characias latex, crude bromelain, and crude papain. Commercially available crude preparations of bromelain exhibited very poor hydrolysis activity. Latex from E. characias, which contained 340 mg of dried material per milliliter of fresh latex, exhibited a high lipase activity and a short-chain fatty acid preference in the hydrolysis of homogeneous TAG. For all substrates, it showed a better activity than crude papain. Lipase fatty acid typoselectivities of crude bromelain and crude papain also were studied in interesterification reactions of tributyrin with a series of homogeneous TAG. Experiments showed that crude bromelain [water activity (A
w
)∶ 0.21] had no activity in interesterification. Regarding reactions with crude papain (A
w
∶ 0.55), yields of newly formed TAG decreased with increasing chain length of TAG, except for the reaction with trimargarin. For interesterification of tributyrin with unsaturated TAG, triolein reacted faster than polyunsaturated TAG. During these interesterification reactions, the proportion of new TAG with two butyroyl residues was higher than new TAG with only one butyroyl residue. This phenomenon was more pronounced for reactions with long-chain TAG.
A commercial preparation of a lipase produced by Candida cylindracea catalysed the hydrolysis of both long- and short-chain esters of p-nitrophenol. Six major bands of hydrolytic activity to α-naphthyl acetate were detected on polyacrylamide gel electrophoresis and two on isoelectric focusing. The esterase activity fractionated into two major peaks of activity on ion-exchange chromatography and into several peaks of activity on hydrophobic interaction chromatography. These esterase activities showed different substrate specificities to p-nitrophenyl esters, tributyrin and cetyl palmitate.
Latex sera from 18 Euphorbia species were surveyed for protein, carbohydrate and total solid contents. Protease, esterase, alkaline and acid phosphatase, N-acetyl-β-glucosaminidase, α-mannosidase, α- and β-galactosidase, α- and β-glucosidase, α-amylase, lysozyme, leucine amino peptidase and cellulase activities were also measured. The effects of nine protease inhibitors were assayed as were haemagglutinating (lectin) contents. Two-dimensional (isoelectric focusing and SDS) PAGE maps of the sera were made. The results obtained show that the latices have widely varying biochemical properties.
The activity and substrate specificity of latex lipases from Euphorbia species (E. characias, E. wulfenii, E. pinea, E. myrsinites and E. dendroides) were investigated. High lipolytic activity was found only in E. characias and for the first time in E. wulfenii latex. For both species the lipolytic activity on various triglycerides, and under different temperature and pH conditions, in both crude latex and in partially purified enzymes was quantified. Optimised extraction and purification methods permitted the recovery of the enzymatic fraction with high lipolytic activity. This fraction is probably constituted by a pool of different lipolytic enzymes. Finally, lipolytic activity was also measured for E. characias and E. wulfenii during vegetative and reproductive stages.
An important aspect of lipolytic enzymes is the unique physicochemical character of the reactions they catalyse at lipid-water interfaces, involving interfacial adsorption and subsequent catalysis sensu stricto. Lipases are now used as catalysts in aqueous as well as low-water media and accept various molecules as substrates. They were previously defined in kinetic terms, based on ‘interfacial activation’. This phenomenon was not found among esterases. Recently determined 3D structures of some, but not all, upases show a ‘lid’ controlling access to the active site. Thus, the presence of a lid, and ‘interfacial activation’, are unsuitable criteria for classifying specific esterases. Consequently, lipases can be pragmatically redefined as carboxyl-esterases acting on long-chain acylglycerols: they are simply fat-splitting ‘ferments’.
Lipase activity (measured with tributyrin as substrate) was detected for the first time in fresh latex of branched non-articulated laticifers of Euphorbia characias, Asclepias curassavica and A. serilofera. Activity was also detected in spray-dried latex from anastomosing articulated laticifers of Carica papaya. The lipolytic activity of all the latices was associated with sedimentable particles. Attempts to solubilize the enzymatic activity from particulate fractions of Carica papya latex were unsuccessful. The esterase inhibitors phenylmethylsulphonyl fluoride or benzamidine had no effect on the lipase activity. The enzyme in Carica papya latex shows greatest activity with tributyrin and is stable in non-water-miscible organic solvents.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A lipase from the latex of Euphorbia characias was purified using a new method involving extraction by apolar solvent and adsorption chromatography on silica. The lipase (specific activity 1,500 IU/mg of protein) was eluted from silica complexed with a lipid. The main proteic fraction, showing a molecular weight of 38,000, was inactive when dissociated from the lipid fraction. It was necessary to reassociate the lipid and proteic fractions for 72% of the lipolytic activity to be recovered.
The present work documents, on a qualitative and quantitative basis, the lipolytic activity of ricin protein RCA60 on glycerophospholipids. RCA60 demonstrates a low level of hydrolysis towards radioactive dipalmitoyl-glycerophosphatidylcholine. This observation was confirmed on a better substrate, palmitoyl-oleoyl-glycerophosphatidylcholine, after analysis of the reaction products by thin-layer and gas chromatography. A comparable hydrolytic activity was observed when palmitoyl-oleoyl-glycerophosphatidylethanolamine was used as substrate. The nature of the hydrolysis products supports the conclusion that RCA60 demonstrates phospholipase A1 and A2 activities as well as a lysophospholipase activity of A1 and A2 type. The insensitivity of this lipolytic activity towards calcium ions and the presence of the already described consensus sequence of lipases, Gly-Xaa-Ser-Xaa-Gly, in the primary sequence of the B-chain of RCA60 support the idea that the lipolytic activity of RCA60 is more related to the lipase family than to the phospholipases A. We hypothesize that such activity contributes to the mechanism which underlies the expression of the cytotoxicity of RCA60.
Growing insights into the many roles of glycoconjugates in biorecognition as ligands for lectins indicates a need to compare plant and animal lectins. Furthermore, the popularity of plant lectins as laboratory tools for glycan detection and characterization is an incentive to start this review with a brief introduction to landmarks in the history of lectinology. Based on carbohydrate recognition by lectins, initially described for concanavalin A in 1936, the chemical nature of the ABH-blood group system was unraveled, which was a key factor in introducing the term lectin in 1954. How these versatile probes are produced in plants and how they are swiftly and efficiently purified are outlined, and insights into the diversity of plant lectin structures are also given. The current status of understanding their functions calls for dividing them into external activities, such as harmful effects on aggressors, and internal roles, for example in the transport and assembly of appropriate ligands, or in the targeting of enzymatic activities. As stated above, attention is given to intriguing parallels in structural/functional aspects of plant and animal lectins as well as to explaining caveats and concerns regarding their application in crop protection or in tumor therapy by immunomodulation. Integrating the research from these two lectin superfamilies, the concepts are discussed on the role of information-bearing glycan epitopes and functional consequences of lectin binding as translation of the sugar code (functional glycomics).
The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca(2+) and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo.
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