Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
J Biosci Bioeng. 2010 Feb;109(2):107-14. doi: 10.1016/j.jbiosc.2009.08.009. Epub 2009 Sep 18.
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.
巴西橡胶树是少数能产生用于许多重要应用的商业天然橡胶的高等植物之一。高等植物中高分子量橡胶分子的生物合成尚未阐明。在这里，使用来自巴西橡胶树的酶活性小分子橡胶粒子 (SRP) 进行体外橡胶生物合成。通过分子量分布 (MWD) 研究了体外橡胶合成的机制。在存在 Triton((R)) X-100 的情况下通过凝胶过滤和离心制备的高度纯化的 SRP 显示出低异戊二烯二磷酸 (IPP) 掺入，用于预存在橡胶的链延伸机制。首次分析了在没有添加任何引发剂的情况下在 SRP 中从预存在橡胶链延伸的体外橡胶的 MWD。当法呢基二磷酸作为烯丙基二磷酸引发剂存在时，获得了 70%掺入的添加 IPP（w/w）的橡胶转移酶活性。体外合成的橡胶在 GPC 分析中显示出典型的高分子量和低分子量部分的双峰 MWD，与体内橡胶相似，峰值约为 10^6 和 10^5 Da 或更低。高分子量和低分子量部分的分子量的反应时间独立性和依赖性分别表明，高分子量橡胶是通过预存在橡胶分子的链延伸合成的，而低分子量部分是由新合成的从添加的烯丙基底物延伸的橡胶分子合成的。
