In vitro synthesis of high molecular weight rubber by Hevea small rubber particles.

Hevea brasiliensis is one of few higher plants producing the commercial natural rubber used in many significant applications. The biosynthesis of high molecular weight rubber molecules by the higher plants has not been clarified yet. Here, the in vitro rubber biosynthesis was performed by using enzymatically active small rubber particles (SRP) from Hevea. The mechanism of the in vitro rubber synthesis was investigated by the molecular weight distribution (MWD). The highly purified SRP prepared by gel filtration and centrifugation in the presence of Triton((R)) X-100 showed the low isopentenyl diphosphate (IPP) incorporation for the chain extension mechanism of pre-existing rubber. The MWD of in vitro rubber elongated from the pre-existing rubber chains in SRP was analyzed for the first time in the case of H. brasiliensis by incubating without the addition of any initiator. The rubber transferase activity of 70% incorporation of the added IPP (w/w) was obtained when farnesyl diphosphate was present as the allylic diphosphate initiator. The in vitro synthesized rubber showed a typical bimodal MWD of high and low molecular weight fractions in GPC analysis, which was similar to that of the in vivo rubber with peaks at around 10(6) and 10(5) Da or lower. The reaction time independence and dependence of molecular weight of high and low molecular weight fractions, respectively, indicated that the high molecular weight rubber was synthesized from the chain extension of pre-existing rubber molecules whereas the lower one was from the chain elongation of rubber molecules newly synthesized from the added allylic substrates.

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.