Preparation, Properties, and Enzymatic Degradation of Biochemically and Chemically C-Labelled Kraft Pulp, 1997 Biological Sciences Symposium Proceedings

A new in vitro test system suitable for testing ligninolytic enzymes on pulp was developed. The technique involves covalent coupling of P4 C-labelled synthetic lignin, so called DHP, or 14C-labelled coniferyl alcohol to kraft pulp or sulfite pulp using two methods: 1) Horseradish peroxidase and hydrogen peroxide at pH 6.5, and 2) potassium ferricyanide at pH 10. The technique is based on the simultaneous formation of phenoxy radicals from both lignin in unbleached pulp and the labelled DHP (or the labelled coniferyl alcohol). These radicals couple and form covalent bonds resulting in a i4C-labelled pulp. With potassium ferricyanide as oxidant at pH=10, the label penetrated deeper into the pulp fibre giving high specific activity and very little variation in measurements of released radioactivity after enzyme treatment. The results show that this chemical coupling technique is most useful for 14C-labelling of both kraft and sulfite pulps, although sulfite pulp gave a lower specific activity. Such labelled pulp was used in a small scale (30 mg pulp in 1.5 ml buffer) and tested for release of radioactivity during treatment with two diierent laccases in the presence of l-hydroxybenzotriazole. The labelled pulps could be bleached or extracted after enzyme treatment using standard procedures. Thus, the new i4C-pulp in vitro test system offers a great potential for rapid testing of a large number of ligninolytic enzymes, laccases as well as peroxidases, in a small scale.