Micro-analytical investigations on lignin in enzyme-digested tobacco lamina and midrib using pyrolysis-mass spectrometry and Curie-point pyrolysis-gas chromatography / mass spectrometry

An isolation procedure is presented for enrichment of lignin in lamina and midrib of tobacco by digestion with the enzymes pronase (protease) and cellulase (polysaccharidase). Digestion by cellulase was successful only after mechanical fracturing of the wall matrix, extensive removal of cytosolic and extraneous wall components, and after the chemical disclosure of the wall by pronase digestion. The purity of the enzyme digested residues and controls was checked by pyrolysis—mass spectrometry (Py-MS) and Curie-point pyrolysis—gas chromatography / mass spectrometry (CuPy-GC/MS). CuPy-GC/MS showed exclusively markers for lignin and carbohydrates in the pyrolysates of the enzyme digested tobacco samples. Lignin was clearly enriched in the enzyme digested residues. The relative distribution of monomeric and dimeric lignin pyrolysis products in the pyrolysis-mass spectra was comparable in lamina and midrib material. The lignin in both tobacco lamina and midrib material can be classified as a GS lignin with low amounts of coumaryl and syringyl units. Substantial amounts of carbohydrates were shown to be remaining in the residues. Cellulase digestion resulted in a reduction of 60% of the hexose markers in the pyrolysate of the lamina material and of 40% in the midrib material.

The MS data on the lignin in enzyme digested cell walls of tobacco lamina material were compared with those from the Björkman lignin of the same tobacco sample. The Björkman tobacco lignin was a highly purified soluble lignin isolate contaminated with minor amounts of carbohydrates. CuPy-GC/MS indicated a higher abundance of syringyl units in the Björkman lignin compared to enzyme digested residues of the lamina material. This difference is presumably caused by a selective (Björkman) extraction of lignin from the secondary cell wall. The enzyme prepared lignin not only uses a milder preparation method but also gives a more complete view into the cell wall lignin of tobacco.