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

Abstract

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 °C; Km was 0.12 ± 0.01 mg/ml, and half-inactivation time was 30 min at 42 °C. Recombinant ALFA4 was active predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 °C, Km was 3.01 ± 0.05 mg/ml. It was stable up to 30 °C; half-inactivation time was 1h 40 min at 37 °C. ¹H 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 µg/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.

Keywords: alginate lyase; anticancer activity; enzyme specificity; oligosaccharide; polysaccharide.

Figure 1

Optimum pH of ALFA3 when acting on different substrates. M – sodium mannuronate, MG – mannuronate-enriched sodium alginate, G – guluronate-enriched sodium alginate. Standard deviations are given.

Figure 2

Kinetic analysis of digestion of different substrates by ALFA3 (A) and final products of digestion of different substrates by ALFA4 (B). M – sodium polymannuronate, MG – mannuronate-enriched sodium alginate, G – guluronate-enriched sodium alginate. Time of reaction is given in h. Each lane was loaded with 60 µg of sample.

Figure 3

Products of action ALFA3 on mannuronate-enriched substrate precipitated by ethanol in ratios from 8.6 to 45.0% (low-molecular fraction). Numbers correspond to ethanol percentage in precipitation mixtures. Precipitation was run during 18 h at 4 °C.

Figure 4

¹H NMR analysis of products after action of ALFA3 on guluronate-enriched substrate. (A) mannuronate-enriched sodium alginate, (B) total products after action of ALFA3, (C) high-molecular weight fractions after action of ALFA3, and (D) low-molecular weight fractions after action of ALFA3.

Figure 5

¹H NMR analysis of action products of ALFA3 (A) and ALFA4 (B) on mannuronate-enriched substrate, as well as products of their joint action (C). M—sodium polymannuronate, MG—mannuronate-enriched sodium alginate, G—guluronate-enriched sodium alginate.

Figure 6

The effect of alginates and products of their digestion on colony formation of human cancer cells. All concentrations are 200 µg/mL.