Two New Alginate Lyases of PL7 and PL6 Families from Polysaccharide-Degrading Bacterium Formosa algae KMM 3553T: Structure, Properties, and Products Analysis

A bifunctional alginate lyase (ALFA3) and mannuronate-specific alginate lyase (ALFA4) genes were found in the genome of polysaccharide-degrading marine bacterium Formosa algae KMM 3553(T). They were classified to PL7 and PL6 polysaccharide lyases families and expressed in E. coli. The recombinant ALFA3 appeared to be active both on mannuronate- and guluronate-enriched alginates, as well as pure sodium mannuronate. For all substrates, optimum conditions were pH 6.0 and 35 degrees C; Km was 0.12 +/- 0.01 mg/mL, and half-inactivation time was 30 min at 42 degrees C. Recombinant ALFA4 was active predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 degrees C, Km was 3.01 +/- 0.05 mg/mL. It was stable up to 30 degrees C; half-inactivation time was 1 h 40 min at 37 degrees C. H-1 NMR analysis showed that ALFA3 degraded mannuronate and mannuronate-guluronate blocks, while ALFA4 degraded only mannuronate blocks, producing mainly disaccharides. Products of digestion of pure sodium mannuronate by ALFA3 at 200 mu g/mL inhibited anchorage-independent colony formation of human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 up to 17% stronger compared to native polymannuronate. This fact supports previous data and suggests that mannuronate oligosaccharides may be useful for synergic tumor therapy.