The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

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Abstract
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 Mg2+ isopentenyl pyrophosphate, and an allylic pyrophosphate. The Km values for Mg2+, isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 .times. 10-4 molar, 8.3 .times. 10-5 molar, and 9.6 .times. 10-5 molar, respectively. The molecular characteristics of the rubber polymer synthesized from [14C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 .times. 106 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 .times. 106 compared to 1.49 .times. 106 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 .times. 105 after 3 hours of incubation with Mg2+ 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.