Determination of mannuronan C-5-epimerase activity in fermentation broth of Azotobacter vinelandii

Azotobacter vinelandii produces extracellular mannuronan C-5-epimerase activity. This activity appears during the biosynthesis of alginate and converts d-mannuronic acid residues into l-guluronic acid in the polymer chain. In order to study the production of mannuronan C-5-epimerase by fermentation of A. vinelandii, a rapid and reproducible assay for the analysis of epimerase activity in the fermentation broth is required. We have examined a method in which [ 3H]-alginate is used as a substrate and the tritium released into water is measured. Fermentation broths contain compounds and ions which may interfere with the analysis or affect the activity of the enzyme. The effects of potential interfering compounds have been characterized and the uncertainty of the analysis system has been determined. Furthermore, a standard method suitable for use directly on the fermentation broth has been developed. The standard deviation of this method within a single analysis is ± 2.5% whereas the standard deviation for samples measured several times in separate analyses on the average is 10%.