Preparation and analysis of¹⁴C-lignin grass lignocellulose and DHP

Abstract

(¹⁴C-lignin) grass lignocellulose was prepared from wheat seedlings injected with (¹⁴C-uniform) phenylalanine. Seedlings were injected 2 wk after germination and grown for 3–4 wk in a diurnal light cycle before harvesting. The plant material was ground in liquid N₂, extracted with hot water, benzene-ethanol, and ethanol, and treated with protease. Treatment of the lignocellulose with acid, alkali, and cellulase solubilized¹⁴C, which was analyzed by HPLC and TLC. Reverse-phase HPLC demonstrated that¹⁴C-ferulic and coumaric acid were bound primarily to carbohydrate and lignin, respectively. Gel permeation chromatography by HPLC of¹⁴C solubilized by treatment with 1M NaOH confirmed that the majority of the¹⁴C was incorporated into high molecular weight material. No¹⁴C was detected in either hexoses or pentoses obtained from the lignocellulose and only a minor amount was present as¹⁴C-phenylalanine. These studies show that (¹⁴C-lignin) grass lignocelluloses must be carefully characterized before being used as defined substrates for biodegradation studies.

Coniferyl alcohol was synthesized by a route derived from those of Nakamura et al. (1974) and Nakamura and Higuchi (1976). DHP was then prepared by a modification of the method of Brunow and Wallin (1981) in which solutions of coniferyl alcohol and hydrogen peroxide were added alternately by a computer controlled HPLC system so that the coniferyl alcohol concentration was maintained below 1 mM throughout the synthesis. The DHP obtained was characterized by HPLC gel permeation chromatography and by NMR. The results of these analyses will be discussed.