A pilot study was conducted into methods of synthesizing mimosine in a cell-free extract of Leucaena leucocephala leaflets. A set of procedures was developed that forms a basis for future studies. For most of the steps involved, existing procedures were adapted to the specific requirements of the experimental system.
The first step is the preparation of a cell-free Leucaena extract. Complexing of phenolic compounds with proteins after leaf maceration proved to be a major problem. Reducing agents and PVPP, both singly and in combination, were effective in minimizing this problem.
Passing the extract through a Sephadex G25 column proved to be a satisfactory way of removing mimosine, while retaining the proteins and large molecular weight compounds. This method also removed some pigments.
Two methods were used to attempt to provide the necessary precursors and energy sources for mimosine synthesis. One used the extract of another legume, phasey bean. The other relied on supplying necessary chemicals artificially.
An ion-exchange column was used to concentrate any mimosine present, and to remove anions and neutral compounds.
Thin-layer electrophoresis was found to be a very sensitive assay for mimosine and DHP, as little as 0.6 - 0.75 µg being detectable.
In none of the experiments was the cell-free synthesis of mimosine demonstrated. However a general methodology was developed. Associated problems were evaluated, and where possible, solutions proposed. Remaining problems are discussed, and recommendations for future study are given.