Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria,
e.g.,, the marine Bacteroidetes
Zobellia galactanivorans (
Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of
Zobellia galactanivorans. The
ZgFK enzyme catalyzes the conversion of fructose to fructose-6-phosphate using ATP as a cofactor.
[Principle] Fructokinase (FK) activity was determined by an enzyme-coupled assay (Figure 1). ADP formed through the FK reaction,
i.e., phosphorylation of fructose to fructose-6-phosphate (F6P), is used by the pyruvate kinase (PK) which transforms phosphoenolpyruvate (PEP) to pyruvate. Then, lactate dehydrogenase (LDH) converts pyruvate to lactate using NADH as a cofactor. FK activity is measured by following the decrease in absorbance at 340 nm which corresponds to the transformation of NADH to NAD
+.
Figure 1. Enzyme-coupled reaction used for determination of fructokinase (FK) activity