Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae

Abbreviations: CFE, cell free extract; ESI-MS, electrospray ionization mass spectrometry; HPLC, high-performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; polyG, polyguluronic acid-rich region; polyM, polymannuronic acid-rich region; PL, polysaccharide lyase; polyMG, random region; SEC, size exclusion chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Figures & data

Fig. 1. Results of SDS-PAGE and the AlgC-PL7 amino acid sequence.

Notes: (A) Analysis of AlgC-PL7 by SDS-PAGE. Lane M, molecular weight markers; Lane 1, purified AlgC-PL7 (35 kDa). (B) Amino acid sequence of AlgC-PL7. The AlgC-PL7 signal sequence was analyzed by Signal IP, which suggested that AlgC-PL7 was located in the periplasm (dashed line). The N-terminal sequence of purified AlgC-PL7 was determined by N-terminal amino acid analysis (line). AlgC-PL7 has three conserved amino acid sequences (RTEL, QIH, and YFKAGSYNQ), indicating it should be classified in the PL-7 family.

Fig. 2. Effects of temperature, pH, and salt on AlgC-PL7 activity.

Notes: (A) Optimal temperature for AlgC-PL7. (B) Optimal pH for AlgC-PL7. Lyase activity was measured in the presence of sodium citrate buffer (pH 4–5), potassium phosphate buffer (pH 6–8), and Tris buffer (pH 7.5–9.5). (C) Salt-dependent lyase activity of AlgC-PL7. Relative activities in the absence of salt are presented. Lyase activities were measured at 0.1–2.0 M NaCl (black) and KCl (white). Error bars represent standard errors of at least three independent measurements.

Fig. 3. Thermostability of Cobetia sp. NAP1-produced AlgC-PL7 and recombinant AlgC-PL7.

Notes: (A) Thermostability of AlgC-PL7. Samples were incubated at each temperature for 1 h and then cooled on ice. (B) Time-lapse measurements (every 15 min) of AlgC-PL7. (C) Analysis of heat-treated cell-free extract containing recombinant AlgC-PL7 by SDS-PAGE. Lane M, molecular weight markers; Lanes 1 and 2, before (Lane 1) and after (Lane 2) the supernatant fraction was heated; Lanes 3 and 4, before (Lane 3) and after (Lane 4) the precipitated fraction was heated. (D) Lyase activity (by kinetic measurement) of the cell-free extract precipitated fraction before (dashed line) and after (line) the heat treatment. Error bars represent standard errors of at least three independent measurements.

Fig. 4. Substrate specificity of Cobetia sp. NAP1-produced AlgC-PL7 (A) and recombinant AlgC-PL7 (B).

Notes: PolyMG, polyM, and polyG were prepared using a published method.²⁴⁾ Error bars represent standard errors of at least three independent measurements.

Fig. 5. ESI-MS analysis of the degradation products resulting from AlgC-PL7 activity.

Notes: (A) 15 min reaction, (B) 110 min reaction, and (C) 48 h reaction. Molecular peaks of (m/z) 175 and 351 were detected, which are consistent with the presence of alginate mono- and disaccharides.²⁷⁾ The ESI-MS analysis was conducted in the negative mode.

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