Expression Level of a Phenylalanine Ammonia-Lyase Gene in Poinsettia Is Negatively Correlated with Poinsettia Branch-Inducing Phytoplasma Titer.

Poinsettia is an important ornamental cultivated worldwide. Commercial poinsettias are almost universally infected with a pathogen known as the poinsettia branch-inducing phytoplasma (PoiBI), which can increase the level of branching in host plants and make the plants more desirable to consumers. Despite PoiBI's crucial role in poinsettia production, little is known about PoiBI-poinsettia interactions in regard to the pathogen's in planta population dynamics. The expression profiles of a phenylalanine ammonia-lyase gene (Euphorbia pulcherrima PAL [EpPAL]) and the PoiBI titers in poinsettia tissues were investigated. Differential gene expression analyses using quantitative PCR (qPCR) showed that EpPAL expression levels differed significantly across tissue types. The highest expression levels were detected in stems, followed by root. Lower EpPAL expression levels were detected in leaf tissues, particularly in source leaves closer to the base; the average expression level in these leaves was only one-seventh of that detected in stems. Phytoplasma concentrations in source leaves close to the base were significantly greater than the other tissue types; the average value was 7.6-fold of that detected in stem tissues, which had the lowest phytoplasma titers. A negative correlation between EpPAL expression level and PoiBI load was detected, suggesting that the products of EpPAL-associated pathways or other genes indirectly associated with EpPAL may interfere with PoiBI's growth. While additional studies are needed to validate these interpretations, the results from this work provide new insights into PoiBI-poinsettia interaction and showed that correlations between pathogen load and defense-related genes could be detected in phytoplasma-associated pathosystems. IMPORTANCE Phytoplasma-plant interactions are interesting subjects for fundamental and applicative research. Although many studies have characterized molecular interplays between these pathogens and hosts, knowledge on relationships between phytoplasmas' in planta population dynamics and host gene expression remains scarce. By using the poinsettia branch-inducing phytoplasma (PoiBI) and poinsettia as a model system, a negative correlation was observed between the expression level of a plant defense-related gene and the pathogen's titer. The findings provide potential explanations to PoiBI's distribution patterns in the plant and highlight the importance of studying phytoplasma-plant interactions in regard to the pathogen's population dynamics in other pathosystems.

De Novo Biosynthesis of the Anticancer Compound Euphol in Saccharomyces cerevisiae.

Euphol is a euphane-type tetracyclic triterpene which is primarily found in the Euphorbia genus. Euphol has been renowned because of its great potential as a promising anticancer drug. Surprisingly, despite its diverse antitumor effects, the respective gene for euphol biosynthesis had not been identified until this study. In our experiments with Euphorbia tirucalli, euphol was detected predominantly in latex, the element that is often used for cancer treatments in Brazil. Two latex-specifically expressed oxidosqualene cyclases (OSCs) from E. tirucalli, designated as EtOSC5 and EtOSC6, were functionally characterized by expression in a lanosterol synthase knockout yeast strain GIL77. EtOSC5 produces euphol and its 20S-isomer tirucallol as two of the major products, while EtOSC6 produces taraxasterol and ß-amyrin as the major products. These four compounds were also detected as the major triterpenes in the E. tirucalli latex, suggesting that EtOSC5 and EtOSC6 are the primary catalysts for the formation of E. tirucalli latex triterpene alcohols. Based on a model structure of EtOSC5 followed with site-mutagenesis experiments, the mechanism for the EtOSC5 activity was proposed. By applying state-of-the-art engineering techniques, the expression of EtOSC5 together with three other known precursor genes were chromosomally integrated into Saccharomyces cerevisiae. The resulting engineered yeast strain YS5E-1 produced 1.84 ± 0.17 mg/L of euphol in shake flasks.

Characterization of a Sesquiterpene Synthase Catalyzing Formation of Cedrol and Two Diastereoisomers of Tricho-Acorenol from Euphorbia fischeriana.

A sesquiterpene synthase gene was identified from the transcriptome of Euphorbia fischeriana Steud, and the function of its product EfTPS12 was characterized by in vitro biochemical experiments and synthetic biology approaches. EfTPS12 catalyzed conversion of farnesyl diphosphate into three products, including cedrol (1) and eupho-acorenols A (2) and B (3) (two diastereoisomers of tricho-acorenol), thereby being named EfCAS herein. The structures of 2 and 3 were determined by spectroscopic methods and comparison of experimental and calculated electronic circular dichroism spectra. EfCAS is the first example of a plant-derived sesquiterpene synthase that is capable of synthesizing acorane-type alcohols. This study also documents that synthetic biology approaches enable large-scale preparation of volatile terpenes and thereby substantially facilitate characterization of corresponding terpene synthases and elucidation of the structures of their products.

Structure of a nucleotide pyrophosphatase/phosphodiesterase (NPP) from Euphorbia characias latex characterized by small-angle X-ray scattering: clues for the general organization of plant NPPs.

Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrub Euphorbia characias (ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.

Roles of a protease from Euphorbia resinifera latex in human anticoagulant and antithrombotic activities.

Thromboembolism is a major cause of morbidity and mortality worldwide. Most therapeutic drugs for treating thrombosis can cause hemorrhage and have short half-lives within human blood circulation resulting in a need to discover and develop novel anticoagulants/antithrombotics. EuRP-61 has been isolated from a plant latex (Euphorbia resinifera) and characterized as a serine protease. In this study, EuRP-61 was able to hydrolyze all chains of human fibrin clots. The enzyme may have long term stability in blood circulation as its fibrinogenolytic activity was not affected by human blood circulating inhibitors such as α2-macroglobulin and antithrombin III. The enzyme may affect the extrinsic, intrinsic or common pathways of the human blood coagulation cascade as evidenced by its prolonged of both prothrombin (PT) and activated partial thromboplastin (APTT) time. Moreover, the enzyme inhibited platelet aggregation via the ADP-receptor pathway. EuRP-61 was not toxic to human red blood cells in the 4 common blood groups (A, B, O and AB) (all Rh+) or human peripheral blood mononuclear cells (hPBMCs). The enzyme may protect human peripheral blood cells from aggregation without destroying them. This study provides evidence that EuRP-61 may have potential as an agent for the treatment of thrombosis.

Identification and characterization of a protease (EuRP-61) from Euphorbia resinifera latex.

A serine protease designated as EuRP-61 was purified from Euphorbia resinifera latex. The N-terminal sequence of 15 amino acids of EuRP-61 supported the conclusion that the enzyme was a serine protease because its amino acid sequence had homology (between 50 and 70% identities) with the subtilisin-like proteases of other plants. EuRP-61 had a molecular weight estimated at 61 kDa analyzed by MALDI-TOF MS. The enzyme could cleave human fibrinogen with optimal conditions at pH 5.0 and 45 °C. The enzyme had a broad range of pH stability from 1 to 14 and tolerance to denaturation up to a temperature of approximately 65-66 °C. EuRP-61 hydrolyzed fibrinogen with a Michaelis constant (Km) of 4.95 ±â€¯0.1 µM; a maximal velocity (Vmax) of 578.1 ±â€¯11.81 ng min-1; and a catalytic efficiency (Vmax/Km) of 116.8 ±â€¯1 ng µM-1 min-1. EuRP-61was crystallized under the condition of sodium iodide (0.2 M), Bis-Tris propane (0.1 M, pH 8.5) and PEG3350 (20%) by the sitting-drop method. The crystal belonged to space group P212121, with unit cell dimension a = 109.91, b = 67.38 and c = 199.45 Šand diffracted X-ray to 2.53 Šresolution. The crystal structure of EuRP-61 will be explored further by special phase solving techniques.

Target site as the main mechanism of resistance to imazamox in a Euphorbia heterophylla biotype.

Euphorbia heterophylla is a weed species that invades extensive crop areas in subtropical regions of Brazil. This species was previously controlled by imazamox, but the continuous use of this herbicide has selected for resistant biotypes. Two biotypes of E. heterophylla from southern Brazil, one resistant (R) and one susceptible (S) to imazamox, were compared. The resistance of the R biotype was confirmed by dose-response assays since it required 1250.2 g ai ha-1 to reduce the fresh weight by 50% versus 7.4 g ai ha-1 for the S biotype. The acetolactate synthase (ALS) enzyme activity was studied using ALS-inhibiting herbicides from five different chemical families. The R biotype required the highest concentrations to reduce this enzyme activity by 50%. A Ser653Asn mutation was found in the ALS gene of the R biotype. The experiments carried out showed that imazamox absorption and metabolism were not involved in resistance. However, greater 14C-imazamox root exudation was found in the R biotype (~70% of the total absorbed imazamox). Target site mutation in the ALS gene is the principal mechanism that explains the imazamox resistance of the R biotype, but root exudation seems to also contribute to the resistance of this biotype.

Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

Serine Protease Mauritanicain from Euphorbia mauritanica and Phorbol-12-myristate-13-acetate Modulate the IL-8 Release in Fibroblasts and HaCaT Keratinocytes.

In recent years, skin reactions such as phytophotodermatitis, contact dermatitis, and other inflammatory responses after contact with chemicals from various plants, e.g., Heracleum mantegazzianum or Hippomane mancinella, are one of the hot topics in phytobiology. Occupational skin inflammation after contact with latices of plants from Euphorbiaceae are common among people who work with plants of this family. Activation of protein kinase C by G protein-coupled receptors such as protease-activated receptors is associated with skin inflammation. In this study, we focused on the inflammatory modulation potential of proteases combined with diterpenes on human skin. Because of its role as a proinflammatory cytokine, we concentrated on the release of IL-8 by fibroblasts and keratinocytes. Therefore, primary human dermal fibroblasts and the HaCaT keratinocytes cell line were used as a model. The results indicated that the combination of the protease mauritanicain from Euphorbia mauritanica and phorbol-12-myristate-13-acetate induced a significantly increased IL-8 release in HaCaT keratinocytes compared to single treatments. The obtained results also suggest that mauritanicain has an anti-inflammatory effect on primary human dermal fibroblasts.

Fabrication of silver chloride nanoparticles using a plant serine protease in combination with photoactivation and investigation of their biological activities.

Recently, the development of "green" methods for fabrication of silver nanoparticles (Ag-NPs) has been emphasized, in view of their environmental safety, feasibility, and low cost. In this study, a serine protease, EuP-82 from Euphorbia cf. lactea latex, was used to fabricate silver chloride nanoparticles (AgCl-NPs) in phosphate-buffered saline (pH 7.2), under the influence of visible light. The fabricated nanoparticles had a maximal surface plasmon resonance absorption peak at 435 nm. The size of the AgCl-NPs, estimated by scanning electron microscopy, was 57 ± 14.7 nm. Energy dispersive X-ray spectroscopy, X-ray absorption spectroscopy, and X-ray diffraction analysis confirmed that the fabricated Ag-NPs were of the AgCl type. The fabricated nanoparticles had antioxidant activity, scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals with IC50 of 204 ± 1.8 µg/mL. The fabricated AgCl-NPs had broad-spectrum in vitro antimicrobial activities, acting against the Gram-positive bacteria Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Bacillus cereus, and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. AgCl-NPs also showed antifungal activity against Candida albicans and C. tropicalis. In addition, AgCl-NPs showed antiprotozoal activity against Giardia lamblia, with IC50 202 ± 2.1 µg/mL. Based on the biological activities of the fabricated AgCl-NPs, they have the potential for widespread application in medicine and industry.

Amine oxidase from Euphorbia characias: Kinetic and structural characterization.

This minireview focuses on a plant copper/2,4,5-trihydroxyphenyl alanine quinone amine oxidase isolated from the latex of the shrub Euphorbia characias (ELAO). This enzyme has been investigated in terms of both molecular structure and kinetic mechanism. The characterization of this enzyme allowed us to identify specific amino acids and domains that play a key role in modulating substrate access into the active site not only for ELAO but also for other plant and mammalian amine oxidases. As mammalian amine oxidases are implicated in several physiological and pathological conditions, the deep structural characterization of their active site accession mechanisms could be the starting point for the development of enzyme modulators with high therapeutic potential. Thus, this paper gives structural/functional insights that open new perspectives in the research about the whole amine oxidase family.

Strictly Conserved Residues in Euphorbia tirucalli ß-Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation-π Interaction and CH-π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture.

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli ß-amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild-type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π-electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation-π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E-ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56-78 % of wild-type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH-π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation-π interaction.

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae.

Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery.

Euphorbia tirucalli ß-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH-π Interaction between Val483 and Trp534 for Catalytic Action.

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli ß-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, afforded ß-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation-π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH-π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.

Immobilization of Euphorbia tirucalli peroxidase onto chitosan-cobalt oxide magnetic nanoparticles and optimization using response surface methodology.

Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50°C and 55°C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2mg/286mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability.

ß-Amyrin biosynthesis: catalytic mechanism and substrate recognition.

The enzymatic polycyclization reactions catalyzed by oxidosqualene (OXSQ) cyclases (OSCs) proceed with complete regio- and stereospecificity, leading to the formation of new C-C bonds and chiral centers and to the generation of diverse polycyclic sterols and triterpenoids. The diverse structural array is remarkable, and approximately 150 different carbon frameworks have been found. Detailed investigations on squalene-hopene cyclase (SHC) and lanosterol synthase (LaS) have been reported, but progress in the study of ß-amyrin synthase, which is ubiquitously found in plants, has lagged in comparison. In the past several years, remarkable advances in ß-amyrin biosynthetic studies have been made. In this review, the catalytic mechanism and substrate recognition of ß-amyrin synthase, as revealed by site-directed mutagenesis and substrate analog experiments, are outlined and compared with those of LaS and SHC to highlight the features of ß-amyrin synthase.

Characterization of the binding of a glycosylated serine protease from Euphorbia cf. lactea latex to human fibrinogen.

In this study, the binding of a glycosylated serine protease (EuP-82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP-82 to fibrinogen in the conditions with or without the activator (Ca2+ ) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen-EuP-82 in the condition with the inhibitor (Zn2+ ) was an unfavorable endothermic reaction. EuP-82 could not inhibit the platelet activity in citrated whole blood via the ADP-receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP-82 provided multiple fibrinogen-binding sites that were not related to the arginine-glycine-aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP-82 had neither thrombin-like activity nor anticoagulant activity. The SR-FTIR spectra revealed that EuP-82 was a glycoprotein. Deglycosylation of EuP-82 did not affect its proteolytic activity. Moreover, EuP-82 did not exhibit any toxicity to the living cells (NIH-3T3). This study supports that EuP-82 may be useful for wound-healing material through stabilizing the clot via the platelet induction for the first process.

ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

Many of the functions of the active site residues in ß-amyrin synthase and its catalytic mechanism remain unclear. Herein, we examined the functions of the highly conserved Phe413, Tyr259, and Trp257 residues in the ß-amyrin synthase of Euphorbia tirucalli. The site-specific mutants F413V and F413M [corrected] showed nearly the same enzymatic activities as the wild type, indicating that π-electrons are not needed for the catalytic reaction. However, the F413A [corrected] mutant yielded a large amount of the tetracyclic dammarane skeleton, with decreased production of ß-amyrin. This indicates that the Phe413 [corrected] residue is located near the D-ring formation site and works to position the oxidosqualene substrate correctly within the reaction cavity. On the other hand, the major catalysis-related function of the Tyr259 and Trp257 residues is to yield their π-electrons to the cationic intermediates. The Y259F variant showed nearly equivalent activity to that of the wild type, but aliphatic mutants such as the Ala, Val, and Leu variants showed significantly decreased the activity and yielded the tetracyclic dammarane scaffold, strongly demonstrating that the Tyr259 residue stabilizes the baccharenyl secondary cation via cation-π interaction. The aliphatic variants of Trp257 exhibited remarkably decreased enzymatic activity, and lupeol was produced in a high production ratio, indicating that Trp257 stabilizes the oleanyl cation via cation-π interaction. The aromatic Phe and Tyr mutants exhibited high activities owing to their more increased π-electron density relative to that of the aliphatic mutants, but lupeol was produced in a significantly high yield besides ß-amyrin. The Trp residue is likely to be responsible for the robust binding of Me-30 through CH-π interaction. The decreased π-electron density of the Phe and Tyr mutants compared to that of Trp would have resulted in the high production of lupeol.

Chitinase III in Euphorbia characias latex: Purification and characterization.

This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Purification and Characterization of a Thermostable Caseinolytic Serine Protease from the Latex of Euphorbia heterophylla L.

A new thermostable caseinolytic serine protease was purified from the latex of Euphorbia heterophylla L. to electrophoretic homogeneity by a procedure involving successive steps of pretreatment of the latex, PEG fractionation, CM-cellulose chromatography and DEAE-cellulose chromatography. The purified protease was found to be a monomeric protein of molecular weight 77.2 kDa. It exhibited caseinolytic activity with hyperbolic azocasein saturation with Vmax and Km values of 0.11 units.mL(-1) and 0.55 mg.mL(-1) respectively. Specific inhibitory studies revealed the enzyme to be a serine protease. The protease was characterized by pH optimum of 8.0 and high thermostability with T1/2 of 75°C. Based on the results of peptide mass fingerprinting analysis, the protease was shown to be a new protein not characterized earlier.