A purified preparation of the extracellular alginate lyase has been used to study kinetics and specificity towards purified, homopolymeric fragments of alginate. The enzyme preparation from Bacillus circulans 1351 degraded both block types, although with different efficiency, and thus appears to be nonspecific. Addition of calcium ions markedly enhanced the reaction rate for the polymannuronate block but had little or no effect on the reaction with polyguluronate. Michaelis-Menten kinetics are not obeyed in the absence of calcium ions and only for the polymannuronate in the presence of calciumThe study of progress curves in response to variation in substrate and enzyme concentrations strongly suggests that the abalone lyase is subject to a reversible product inhibition.

The action of alginate lyases may be easily followed in a UV-spectrophotometer, since each cut of the alginate chain will create an unsaturated unit at the non-reducing end with a strong absorbance at 230 nm. During prolonged incubation, this absorbance will approach an apparent endpoint level that reflects the initial substrate concentration. On this basis, a standardized assay has been developed. A combination of purified mannuronate lyase from Haliotis tuberculata and purified guluronate lyase from Klebsiella pneumoniae is applied to get quantitative concentration estimates that do not depend on alginate composition. The production of alginate in Azotobacter vinelandii is included as an example of application. Most important, by applying both enzymes alone and in combination, the block composition of the alginate may be estimated. Data for a series of widely different alginates have been compared with those obtained by NMR.

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Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.