Preparation and analysis of14C-lignin grass lignocellulose and DHP
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(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.