Washed Rubber Particles Research Articles

Study on in vitro NR biosynthesis by rapid quantitative determination of substrate depletion

A convenient and nonradioactive method for quantifying in vitro NR biosynthesis is presented that is based upon the quantitation of substrate depletion by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). NR oligomers could be in vitro biosynthesized with the enzyme source from Hevea brasiliensis (Hevea) or Taraxacum kok-saghyz (TKS) by exogenous monomers (IPP) and initiators (FPP). The IPP incorporation rate and FPP consumption rate were 62.24% and 51.14% respectively when the washed rubber particles (WRP) of Hevea was the enzyme source. The IPP incorporation rate and the FPP consumption rate were 74.49% and 95.90% respectively when the sediment bottom fraction (BF) of Hevea was the enzyme source. The in vitro NR biosynthesis can be divided into two stages:(1) the initiation reaction of FPP, which occurs more in BF, and (2) the growth reaction of IPP, which occurs more in WRP. In addition, the IPP incorporation and FPP consumption rates were 59.39% and 34.15% respectively when the BF of TKS was selected as an enzyme source.

Euphorbia characias latex: Micromorphology of rubber particles and rubber transferase activity

We have recently characterized a natural rubber in the latex of Euphorbia characias. Following that study, we here investigated the rubber particles and rubber transferase in that Mediterranean shrub. Rubber particles, observed by scanning electron microscopy, are spherical in shape with diameter ranging from 0.02 to 1.2 μm. Washed rubber particles exhibit rubber transferase activity with a rate of radiolabeled [14C]IPP incorporation of 4.5 pmol min−1mg−1.Denaturing electrophoresis profile of washed rubber particles reveals a single protein band of 37 kDa that is recognized in western blot analysis by antibodies raised against the synthetic peptide whose sequence, DVVIRTSGETRLSNF, is included in one of the five regions conserved among cis-prenyl chain elongation enzymes.The cDNA nucleotide sequence of E. characias rubber transferase (GenBank JX564541) and the deduced amino acid sequence appear to be highly homologous to the sequence of several plant cis-prenyltransferases.

UNRAVELING THE MYSTERY OF NATURAL RUBBER BIOSYNTHESIS. PART II: COMPOSITION AND GROWTH OF IN VITRO NATURAL RUBBER USING HIGH-RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY

ABSTRACT The superior properties of natural rubber (cis-1,4-polyisoprene [NR]) are a function of its structure and composition, properties that still remain a mystery and that are irreplaceable by any synthetic rubber. NR from guayule (Parthenium argentatum) has been gaining special interest for its hypoallergenic properties while maintaining superior mechanical properties that are commonly associated with the Brazilian rubber tree (Hevea brasiliensis), the most common source of NR. Techniques exist to isolate washed rubber particles (WRPs) that contain enzymatically active rubber transferase, to study NR biosynthesis, and previous work on the in vitro NR growth in Hevea has demonstrated the presence of around 50 wt% of a low molecular weight ([MW], Mn <10 000 g/mol) fraction. Structural and compositional analyses of this low MW fraction in Hevea are challenging due to the high protein content. We discuss the analysis and composition of guayule latex and WRPs using high-resolution Size Exclusion Chromatography. We also discuss the composition of the soluble fraction of inactive guayule latex using matrix-assisted laser desorption ionization/time of flight mass spectrometry.

The enzymatic incorporation of isopentenyl pyrophosphate into polyisoprene in rubber particles from Parthenium argentatum Gray

Studies have indicated the presence of both a de novo biosynthesis of new polyisoprene chains and the elongation of pre-existing rubber-PP chains in rubber particles from Ficus elastica Roxb. and small rubber particles from Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. In this paper, we have isolated rubber particles from the cortical parenchyma of Parthenium argentatum Gray with an adsorbed isopentenyl pyrophosphate isomerase that was competent to form polyisoprene to determine the presence of de novo biosynthesis of polyisoprene and the elongation of pre-existing rubber-PP. The gel filtrated rubber particles were supplied saturating concentrations of [ 14C]-isopentenyl pyrophosphate for 1 h and the resulting concentrations of [ 14C]-polyisoprene were isolated and analyzed with a Waters 150C Gel Permeation Chromatograph. The mol wt distribution of the [ 14C]-polyisoprene ranged from 10 3 to 10 6 Da with no formation of high mol wt chains equal to the polyisoprene in the mol wt distribution of natural rubber from P. argentatum. The mol wt distribution of [ 14C]-polyisoprene following 1 h of incubation of the reaction mixture was similar to the mol wt distribution formed by the bound rubber transferase in washed rubber particles of H. brasiliensis and the time-dependent formation of polyisoprene by washed rubber particles from P. argentatum. The data support the conclusion that the washed rubber particles from P. argentatum contain a bound rubber transferase that initiates the formation of new chains of polyisoprene from the dimethylallyl pyrophosphate initiator generated from the added isopentenyl pyrophosphate isomerase followed by the extensive polymerization of isopentenyl pyrophosphate to form a monomodal rubber polymer. There was no evidence in these washed rubber particles of a chain elongation of pre-existing rubber-PP.

UNRAVELING THE MYSTERY OF NATURAL RUBBER BIOSYNTHESIS. PART I: INVESTIGATION OF THE COMPOSITION AND GROWTH OF IN VITRO NATURAL RUBBER USING HIGH RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY

Abstract Monitoring the growth of in vitro natural rubber was accomplished by high resolution size exclusion chromatography, SEC. Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphate monomer in the presence of farnesyl pyrophosphate initiator. The high-resolution SEC was able to detect the formation of new rubber. Changes in the low molecular weight fraction were also detected. Gravimetric analysis revealed ∼30% mass gain after the in vitro synthesis. The overall gel content was found to be reduced, which further supported the formation of new rubber. This is the first report that utilizes high-resolution SEC to monitor the in vitro NR growth without the use of radiolabeling.

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Hevea brasiliensis and Parthenium argentatum. Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz–DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz–DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.

Structural characterization of natural rubber based on recent evidence from selective enzymatic treatments

A structural characterization of natural rubber (NR) was carried out by a combination of selective enzymatic treatment and high-resolution characterization techniques. The nitrogenous groups in NR from Hevea brasiliensis have been regarded as an important factor governing cured rubber properties, although there is no structural evidence to prove that relationship. The nitrogenous compounds in highly purified NR were characterized to determine the origin of the nitrogenous groups in NR. The rubber particles of fresh latex were purified by washing, i.e., a successive dilution of the cream fraction and concentration by centrifugation in the presence of a surfactant. The nitrogen content of the NR formed from the washed rubber particles decreased from 0.281% to 0.015% after washing 3-5 times; this content was similar to that of NR deproteinized by a proteolytic enzyme. The presence of 1-2 nitrogen atoms per rubber chain was estimated on the basis of nitrogen content and number-average molecular weight (Mn). The nitrogen content of fractionated rubber fractions from rubber particles washed for five times increased with increasing Mn. The nitrogenous groups in rubber chains are postulated to mostly originate from phospholipids associating with branch points and functional groups formed by oxidation and successive reactions in NR latex. An analysis of the structure of the chain end group (alpha-terminal) was carried out by treating the deproteinized NR (DPNR) latex with lipase, phosphatase, and phospholipases A(2), B, C and D. The enzymatic treatment of DPNR latex significantly decreased the long-chain fatty acid ester content in DPNR, and also the Mn and Higgins'k' constant, except for phospholipase D treatment. This indicates the presence of phospholipid molecules in NR, which connect rubber molecules together. (1)H-NMR results of rubber obtained from DPNR latex showed weak signals due to monophosphate, diphosphate and phospholipids at the alpha-terminal. Treating DPNR latex with lipase and phosphatase decreased the relative intensity of the (1)H-NMR signals corresponding to phospholipids; however, with the same treatment, no change in the signals due to mono- and diphosphates was observed. The presence of mono- and diphosphates as well as some phospholipid signals after lipase and phosphatase treatment indicates that mono- and diphosphate groups are directly linked to the modified structure at their alpha-terminals; such connections are considered to be due to the association or linkage of such groups with phospholipid.

Investigation of the polycarbonate/crushed‐rubber‐particle interphase by nanoindentation

AbstractNanoindentation tests were carried out to determine the nanomechanical characteristics of recycled polycarbonate (PC)/crushed‐rubber blends. Both the modulus and hardness of the matrix, particles, and PC/rubber interphase were obtained. Different blends with untreated, flamed, and washed rubber particles were characterized. The results proved the good recyclability of PC. Indentations performed in the PC matrix showed that the rubber acted as a plasticizer for the PC matrix, probably because of a diffusion of free rubber chains. This plasticizing effect was accentuated by flamed rubber particles in the blends. The results showed that flame and methanol treatments modified the morphology of the rubber particles. These treatments induced a significant increase in the nanomechanical properties of the rubber particles. In the interface region between the PC matrix and rubber particles, a gradual change in the mechanical properties was confirmed. Profiles of the interface modulus and hardness helped to determine the interface width according to the particle treatment. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2687–2694, 2007

Structural Characterization of Terminal Groups in Natural Rubber: Origin of Nitrogenous Groups

Abstract The nitrogenous groups in natural rubber (NR) from Hevea brasiliensis have been regarded as an important factor governing cured rubber properties, although there is no structural evidence. The nitrogenous compounds in highly purified NR were characterized to disclose the origin of nitrogenous groups. The rubber particles in fresh latex were purified by washing, i.e., successive dilution of the cream fraction and concentration by centrifugation in the presence of surfactant. The nitrogen content of natural rubber from the washed rubber particles decreased from 0.281% to 0.015% level after washing 3–5 times, the level of which was similar to that of deproteinized natural rubber with a proteolytic enzyme. The presence of 1–2 nitrogen atoms per rubber chain was presumed based on the nitrogen content and Mn value. The nitrogen content of fractionated rubber fractions from rubber particles washed 5 times increased with increasing molecular weight. The nitrogenous group in the rubber chain is postulated to originate from mostly phospholipids associating with branch-points and from functional groups formed by oxidation and successive reactions in latex.

Significant Role of Bacterial Undecaprenyl Diphosphate (C55‐UPP) for Rubber Synthesis by Hevea Latex Enzymes

Washed bottom fraction (BF) membrane-bound particles of centrifuged fresh Hevea latex were found to be very active in rubber biosynthesis (RB). The washed BF membrane (WBM) showed higher RB activity and is strongly stimulated by anionic surfactants--more by DOC than SDS. WBM enzymes system can synthesize rubber either with allylic isoprenes (higher RB) or without (lower RB). Washed rubber particles (WRP), used generally in RB assays, had very low RB activity compared to the much higher activity observed for WBM. Bacterial undecaprenyl diphoshate (C(55)-UPP) was very active allylic initiator for rubber synthesis by WBM. Comparisons of allylic UPP with the shorter ones (C(15)-FPP, C(20)-GGPP) showed that UPP was the most effective. WBM activity orders were UPP >> GGPP > FPP. The DOC activated WBM synthesized less polyprenyl intermediates (butanol extractable) but more final rubber product (toluene/hexane extract), different than FPP and GGPP. WBM enzymes were highly versatile in using diverse different allylics, but UPP was most preferable. WRP was found a little active for UPP with DOC, but still much lower than WBM. Rubber product analysis by RP-TLC with acetone/hexane solvent system showed that WBM was mostly rubber, but WRP was mainly the intermediates. Quantitative analysis showed that WBM labeled rubber was confined to the origin spot, different than WRP as mainly labeled intermediates. It was thus confirmed that the WBM plays the key role in RB functions, and not WRP as mostly reported. WBM served as the actual rubber synthesis site, and bacterial UPP was very good RB initiator.

Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis

Summary Plants produce many isoprenoid compounds of biological and/or economic importance. The first step in isoprenoid biosynthesis is the isomerization of isopentenyl diphosphate (IPP) to its electrophilic isomer, dimethylallyl diphosphate (DMAPP), by the enzyme IPP isomerase (EC 5.3.3.2). The following successive head-to-tail condensation reactions of the five-carbon intermediates lead to the synthesis of a variety of isoprenoids. Among these polyisoprenes, natural rubber is an important raw material with many industrial uses. In order to build our knowledge base of rubber biosynthesis, we cloned and characterized two cDNA clones encoding the IPP isomerase from the latex of Hevea brasiliensis , where a large quantity of natural rubber ( cis -1,4-polyisoprene) is produced. The two clones differ only in the untranslated regions, and have a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. The deduced protein is acidic, with an isoelectric point of 4. 7, and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particles deprived of initiating allylic diphosphates, revealed that the recombinant IPP isomerase is functional in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in isoprenoid biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree. Analyses of RNA extracted from extruded latex of the trees, wounded with nails, showed that wounding did not change the transcript level of IPP isomerase.

Multiwell filtration system results in rapid, high-throughput rubber transferase microassay

The amount of rubber synthesised by washed rubber particles (WRP) has been measured by the incorporation of radiolabelled isopentenyl diphosphate in enzymatic reactions assembled in a MultiScreen® 96-well plate with the wells sealed underneath with a membrane which prevented leakage but allowed rapid harvesting. After stopping the reactions, the WRP were trapped on the underlying membrane by applying vacuum and, after appropriate processing, the radioactive rubber on the filters was determined by liquid scintillation counting. The processing time for 96 samples was less than 30 min (a 20-fold reduction compared with our previous published protocol) and, compared with another multiwell rubber transferase assay, the present method yielded a 4-fold saving in time and eliminated the requirement for carrier rubber. The described assay can measure accurately the activity present in 0.07 mg of WRP and can be performed in a volume of 5 µL, which is smaller than any other assay reported. This assay can also be used to measure rubber transferase activity in divergent species, such as Hevea brasiliensis, Parthenium argentatum and Ficus elastica.

The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg(2+) isopentenyl pyrophosphate, and an allylic pyrophosphate. The K(m) values for Mg(2+), isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 x 10(-4) molar, 8.3 x 10(-5) molar, and 9.6 x 10(-5) molar, respectively. The molecular characteristics of the rubber polymer synthesized from [(14)C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 x 10(6) to 500 Angstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 x 10(6) compared to 1.49 x 10(6) for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 x 10(5) after 3 hours of incubation with Mg(2+) and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer.

Rubber transferase activity in rubber particles of guayule

Rubber transferase (RuT) activity, measured as incorporation of radiolabelled isoprene from [14C]isopentenyl pyrophosphate (IPP) into rubber, was assayed in suspensions of washed rubber particles (WRPs) purified from stembark tissue of Parthenium argentatum. Isolated WRPs had high RuT activity which was not diminished even after repeated washing, demonstrating the firm association of the enzyme with the particles. The activity of RuT was characterized with respect to substrate and WRP concentration. The rate of IPP-incorporation was dependent upon the concentration of two substrates, IPP and the allylic pyrophosphate starter molecule E,E-farnesyl pyrophosphate (FPP). The enzyme present in 6 × 1010 WRPs per ml was saturated by 1 mM IPP and 20 μM FPP. Under saturating cosubstrate concentrations the apparent Km of RuT was ca 300 μM IPP and 3 μM FPP. Analysis of WRPs by SDS-PAGE revealed a simple protein profile characteristic of guayule rubber particles. A successful and facile assay for IPP-polymerization by isolated rubber particles is described.