Functional identification of alginate lyase from the brown alga Saccharina japonica

Abstract

Despite the progress in massive gene analysis of brown algal species, no alginate-degrading enzyme from brown alga has been identified, impeding the understanding of alginate metabolism in brown alga. In the current study, we identified and characterized alginate lyase from Saccharina japonica using a protein-based approach. First, cDNA library was prepared from the S. japonica sporophyte. Expression screening was then performed; the encoding gene was identified and cloned; and the recombinant enzyme was purified and characterized. Alginate lyase production in algal tissues was evaluated by western blotting. The identified alginate lyase, SjAly (359 amino acids, with a predicted N-terminal secretion signal of 27 residues), is encoded by an open reading frame comprising seven exons. Recombinant SjAly exhibited endolytic alginate lyase activity, specifically toward stretches of consecutive β-D-mannuronic acid units. The optimum temperature, pH, and NaCl concentration were 30 °C, pH 8.0, and 100 mM, respectively. SjAly exhibited pronounced activity below 20 °C, the S. japonica growth temperature. SjAly was highly expressed in the blade but not the stipe and rhizoid. The data indicate that S. japonica possesses at least one active alginate lyase. This is the first report of a functional alginate lyase from brown alga, the major natural alginate producer.

Figure 1

Sequence information of SjAly, the candidate protein for an alginate degradation enzyme of Saccharina japonica. (a) Comparison of amino acid sequences of SjAly and Ectocarpus siliculosus homologs CBJ32331, CBJ32332, CBJ32749, CBN79456, CBN76926, and CBN76927. The numbers correspond to GenBank accession numbers. Residues identical to SjAly are boxed. (b) Predicted genomic structure of SjAly gene. The exons and introns are shown by red boxes and blue lines, respectively.

Figure 2

Alginate degradation by purified rSjAly. (a) Schematic drawing of rSjAly. (b) SDS-PAGE of purified rSjAly. Left lane, marker protein; right lane, purified rSjAly. Full-length gel is presented here. (c) TLC analysis of rSjAly reaction degradation products. ALG, alginate; PEC, pectin; GAL, polygalacturonic acid; XG, xanthan gum; HEP, heparin; HA, hyaluronic acid; CSA, chondroitin sulfate A; CSB, chondroitin sulfate B; CSC, chondroitin sulfate C. ΔMM and ΔMMM are unsaturated tri- and tetramannuronic acids, respectively, prepared by digestion of polyM with HULK alginate lyase. Enzyme reaction was conducted in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.05 mg/mL rSjAly, and 0.2% (w/v) each polysaccharide, at 20 °C for 24 h. (d) Evaluation of the alginate lyase activity of rSjAly. The relative viscosity (open circles) and absorbance at 548 nm after 2-thiobarbituric acid (TBA) reaction (closed circles) were determined in a mixture of 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.05 mg/mL rSjAly, and 1% (w/v) sodium alginate, at 20 °C at the indicated time points. (e) Substrate preference of rSjAly. Enzyme reaction was conducted in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.02 mg/mL rSjAly, and 0.2% (w/v) each substrate, at 20 °C for 10 min. The relative activity of 100% was equivalent to 9.1 U/mg. An inset shows TLC analysis of rSjAly degradation products. Enzyme reaction was conducted in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.02 mg/mL rSjAly, and 0.2% (w/v) each substrate, at 20 °C for 24 h. (f) TLC analysis of fractionated polyM products of degradation by rSjAly. Before, the sample before column chromatography. A, B, C, and D, degradation products eluted at 50 mM, 250 mM, 350 mM, and 450 mM NH₄Cl (Supplementary Fig. S6), respectively. Each determined mass by ESI-MS (Supplementary Fig. S7) and predicted possible structure are shown below the lane of each sample. ΔM, ΔMM, and ΔMMM are mean unsaturated di-, tri-, and tetramannuronic acids, respectively.

Figure 3

Biochemical characterization of the alginate lyase activity of rSjAly. (a) The effect of temperature on rSjAly activity. Enzyme reaction was conducted in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.02 mg/mL rSjAly, and 0.2% (w/v) polyM, at the indicated temperature for 10 min. The relative activity of 100% was equivalent to 13.8 U/mg. (b) The effect of temperature on rSjAly stability. A mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, and 0.1 mg/mL rSjAly was incubated at the indicated temperature for 30 min and placed on ice for 10 min. Then, enzyme activity was assayed in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.1 M NaCl, 0.02 mg/mL rSjAly, and 0.2% (w/v) polyM, at 20 °C for 10 min. The relative activity of 100% was equivalent to 8.9 U/mg. The dotted line indicates the temperature threshold for 50% loss of activity. (c) The effect of pH on rSjAly activity. Enzyme reaction was conducted in a mixture containing 0.1 M NaCl, 0.02 mg/mL rSjAly, 0.2% (w/v) polyM, 10 mM sodium acetate (pH 4.2–5.6, open circles), 10 mM sodium phosphate (pH 6.0–8.2, closed circles), 10 mM imidazole-HCl (pH 6.4–8.5, open squares), and 10 mM glycine-NaOH (pH 9.2–9.8, closed squares), at 20 °C for 10 min. The relative activity of 100% was equivalent to 9.1 U/mg. (d) The effect of NaCl or rSjAly activity. Enzyme reaction was conducted in a mixture containing 10 mM sodium phosphate (pH 8.0), 0.02 mg/mL rSjAly, 0.2% (w/v) polyM, and 0.01–1.0 M NaCl, at 20 °C for 10 min. The relative activity of 100% was equivalent to 9.4 U/mg. (e) The effect of various compounds on rSjAly activity. To investigate the effect of monovalent cations, a mixture of 10 mM imidazole-HCl (pH 8.0), 0.1 mg/mL rSjAly, and 100 mM each monovalent cation was incubated on ice for 1 h. The enzyme reaction was then assayed in a mixture containing 10 mM imidazole-HCl (pH 8.0), 0.02 mg/mL rSjAly, 0.2% (w/v) polyM, and 100 mM each monovalent cation, at 20 °C for 10 min. For other compounds, the incubation and assay conditions were the same except that 100 mM NaCl and the indicated concentration of each compound were used. The relative activity of 100% was equivalent to 9.5 U/mg.

Figure 4

Western blot analysis of SjAly from Saccharina japonica. Each sample was applied to one gel in order of symmetry. After electrophoresis, the gel was cut in the middle, one stained with Coomassie Brilliant Blue and the other transferred to a nitrocellulose filter for western blotting. The primary antibody was diluted 1,000 times with 1 × TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) and used, and the secondary antibody was similarly diluted 10,000 times and used. Protein bands were visualized using an ECL prime western blotting detection reagent (GE Healthcare Life Sciences, Pittsburgh, PA) and chemi-luminescence imager (EZ-capture MG, ATTO, Tokyo, Japan) in an auto exposure mode. An overexposed image is represented in Supplementary Fig. S8. (a) SDS-polyacrylamide gel stained using Coomassie Brilliant Blue. (b) Western blot analysis using anti-SjAly antibodies. rSjAly, purified recombinant SjAly; Marker, protein ladder; Blade, protein extract from the blade; Stipe, protein extract from the stipe; Rhizoid, protein extract from the rhizoid. Full-length gel and blot are presented here.