Purification and Characterization of a Novel Alginate Lyase from the Marine Bacterium Bacillus sp. Alg07

Abstract

Alginate oligosaccharides with different bioactivities can be prepared through the specific degradation of alginate by alginate lyases. Therefore, alginate lyases that can be used to degrade alginate under mild conditions have recently attracted public attention. Although various types of alginate lyases have been discovered and characterized, few can be used in industrial production. In this study, AlgA, a novel alginate lyase with high specific activity, was purified from the marine bacterium Bacillus sp. Alg07. AlgA had a molecular weight of approximately 60 kDa, an optimal temperature of 40 °C, and an optimal pH of 7.5. The activity of AlgA was dependent on sodium chloride and could be considerably enhanced by Mg²⁺ or Ca²⁺. Under optimal conditions, the activity of AlgA reached up to 8306.7 U/mg, which is the highest activity recorded for alginate lyases. Moreover, the enzyme was stable over a broad pH range (5.0-10.0), and its activity negligibly changed after 24 h of incubation at 40 °C. AlgA exhibited high activity and affinity toward poly-β-d-mannuronate (polyM). These characteristics suggested that AlgA is an endolytic polyM-specific alginate lyase (EC 4.2.2.3). The products of alginate and polyM degradation by AlgA were purified and identified through fast protein liquid chromatography and electrospray ionization mass spectrometry, which revealed that AlgA mainly produced disaccharides, trisaccharides, and tetrasaccharide from alginate and disaccharides and trisaccharides from polyM. Therefore, the novel lysate AlgA has potential applications in the production of mannuronic oligosaccharides and poly-α-l-guluronate blocks from alginate.

Keywords: Bacillus sp. Alg07; alginate lyase; alginate oligosaccharides; marine bacterium; purification.

Figure 1

Neighbor-joining phylogenetic tree generated on the basis of the 16S rRNA gene sequences of strain Alg07 and other known Bacillus species.

Figure 2

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result for AlgA. Lane M, protein ladder; Lane 1, purified AlgA.

Figure 3

Effects of temperature and pH on the relative activity of AlgA. (A) Optimal temperature of AlgA. (B) Thermostability of AlgA at 40 °C (filled square), 45 °C (filled circle), and 50 °C (filled triangle). (C) Optimal pH for the relative activity of AlgA was determined in 20 mM CH₃COOH-CH₃COONa buffer (filled square), 20 mM Tris-HCl buffer (filled circle), or 20 mM Glycine-NaOH buffer (filled triangle). (D) pH stability of AlgA in 20 mM CH₃COOH-CH₃COONa buffer (filled square), 20 mM Tris-HCl (filled circle), and 20 mM Glycine-NaOH (filled triangle).

Figure 4

Effect of NaCl (A) and metal ions (B) on the activity of AlgA.

Figure 5

Relative activities of AlgA toward alginate, polyM, and polyG.

Figure 6

Patterns of the polysaccharide degradation products of AlgA. Enzymatic degradation products collected at 0.5, 2, 4, 6, and 10 h were subjected to gel filtration with a Superdex peptide 10/300 GL column. The absorbances of the products were monitored at 235 nm.

Figure 7

Final products of alginate, polyM, and polyG after degradation by AlgA. Oligosaccharide products were gel-filtered through a Superdex peptide 10/300 GL column and monitored at a wavelength of 235 nm.

Figure 8

Electrospray ionization mass spectrometry (ESI-MS) analysis of the final oligosaccharide products. (A) Fraction peak 1 separated through fast protein liquid chromatography (FPLC), (B) fraction peak 2 separated through FPLC, and (C) fraction peak 3 separated through FPLC.