Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

Abstract

Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ϵ-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 °C and around 6, respectively. A high concentration of NaCl (0.8-1.4 m) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 °C for 30 min. Heat stability was decreased in the presence of 5 mm DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.

Keywords: algae; alginate; alginate lyase; bacteria; brown algae; carbohydrate; carbohydrate processing; extremophile; heat stability.

FIGURE 1.

Comparison among the amino acid sequences of NitAly, its homologous proteins, and several characterized family PL-7 alginate lyases. NitAly, Nitratiruptor sp. SB155-2 hypothetical protein NIS_0185 (GenBank^TM accession no. WP_012081562); Caminibacter, C. mediatlanticus hypothetical protein (GenBank^TM accession no. WP_007473671); Hippea, H. jasoniae hypothetical protein (GenBank^TM accession no. WP_051904545); Nautilia, N. profundicola predicted alginate lyase (GenBank^TM accession no. WP_015901805); PyAly, P. yezoensis alginate lyase PyAly (GenBank^TM accession no. BAI66416) (39); PA1167, P. aeruginosa PAO1 alginate lyase PA1167 (GenBank^TM accession no. AAG04556) (15); A1m, Agarivorans sp. JAM-A1m alginate lyase A1m (GenBank^TM accession no. AB426616) (31); A9mT, Vibrio sp. alginate lyase A9mT (GenBank^TM accession no. AB473598) (25); FlAlyA, Flavobacterium sp. UMI-01 alginate lyase FlAlyA (18). Residues invariant among all listed proteins are indicated with blue boxes. Residues identical with those of NitAly are indicated with yellow boxes. Cys-80 and Cys-232 in NitAly and their corresponding conserved Cys residues in other proteins are highlighted with red. An arrowhead shows the cleavage site of the signal peptide in NitAly predicted by the SignalP 3.0 software program. Catalytic residues proposed in PL-7 alginate lyases (15, 21, 65) are shown by solid circles. The level of identity of each other protein with NitAly is shown at the end of each sequence, respectively.

FIGURE 2.

Bacterial expression and purification of rNitAly. A, schematic drawing of rNitAly. B, SDS-PAGE of purified rNitAly. Reduced and non-reduced indicate that the samples were prepared in the presence and absence of 20 mm DTT, respectively. C, SDS-PAGE of PEG-Mal-labeled rNitAly pretreated in the presence and absence of 20 mm DTT.

FIGURE 3.

Evaluation of an alginate lyase activity of rNitAly. Relative viscosity (closed circles) and absorbance at 235 nm (open circles) were measured in a mixture of 10 mm sodium phosphate (pH 6.0), 1 m NaCl, 0.1 mg/ml BSA, 1% (w/v) sodium alginate, and 0.005 mg/ml rNitAly at 30 °C at the indicated time points.

FIGURE 4.

Substrate preferences of rNitAly and degradation products. A, substrate preferences of rNitAly. Enzyme reactions were conducted in a solution containing 10 mm sodium phosphate buffer (pH 6.0), 1 m NaCl, 0.1 mg/ml BSA, 0.005 mg/ml rNitAly, and 0.5% (w/v) substrate (polyM, polyG, polyMG, or sodium alginate) at 50 °C for 15 min. A relative activity of 100% was equivalent to 1,290 units/mg. Assays were done three times, and the data were shown as mean ± S.D. B, thin layer chromatography analysis of the degradation products of polyM following incubation with rNitAly. The enzyme reaction was conducted using polyM as the substrate under the same conditions described for A, and was stopped by the addition of 4 volumes of ice-cold ethanol at the indicated time point.

FIGURE 5.

Effects of temperature, pH, NaCl, and divalent metals on rNitAly activity. A, temperature dependence of alginate lyase activity of rNitAly. Enzyme reactions were conducted in a solution containing 10 mm sodium phosphate (pH 6.0), 1 m NaCl, 0.1 mg/ml BSA, 0.005 mg/ml rNitAly, and 0.5% (w/v) polyM for 15 min at the indicated temperatures. Relative activity of 100% was equivalent to 1,620 units/mg. B, pH dependence of the alginate lyase activity of rNitAly. Assays were performed as described for A, except that reaction mixtures were incubated at 50 °C for 15 min in 10 mm sodium acetate for pH 4.0–5.1 (open circles), 10 mm sodium phosphate for pH 5.7–7.4 (closed circles), 10 mm Tris-HCl for pH 8.1–8.5 (closed triangles), and 10 mm glycine-NaOH for pH 9.2–10.4 (open triangles). Relative activity at 100% was equivalent to 1,230 units/mg. C, NaCl dependence of the alginate lyase activity of rNitAly. Assays were performed as described for A at 50 °C, except that different concentrations of NaCl were used as indicated. Relative activity at 100% was equivalent to 1,430 units/mg.

FIGURE 6.

Effects of temperature and pH on rNitAly stability. A, heat stability of rNitAly. For this assay, 0.2 mg/ml rNitAly in 10 mm sodium phosphate (pH 6.0) and 1 m NaCl were incubated for 30 min at the indicated temperatures and placed on ice. Enzyme activity was then assayed as described in Fig. 5A at 50 °C. Relative activity at 100% was equivalent to 1,230 units/mg. B, effects of time extension of heat treatment on the heat stability of rNitAly. For this assay, 0.2 mg/ml rNitAly in 10 mm sodium phosphate (pH 6.0) and 1 m NaCl were incubated for the indicated time at 20 °C (open circles), 30 °C (closed circles), 40 °C (open triangles), and 50 °C (closed triangles) and placed on ice. Assays were performed as described for A. Relative activity at 100% was equivalent to 1,210 units/mg. C, pH stability of rNitAly. For this assay, 0.2 mg/ml rNitAly in 10 mm sodium phosphate (pH 6.0) and 1 m NaCl was dialyzed against 1,000 volumes of 1 m NaCl and 10 mm sodium acetate (pH 4.0 or 5.0) (open circles), 10 mm sodium phosphate (pH 6.0 or 7.0) (closed circles), 10 mm Tris-HCl (pH 8.0) (open triangle), and 10 mm glycine-KOH (pH 9.0) (closed triangle) at 4 °C for 8 h. Assays were performed as described for A. Relative activity at 100% was equivalent to 1,410 units/mg. All assays were repeated three times, and the data are shown as mean ± S.D.

FIGURE 7.

Effects of DTT on the temperature dependence and heat stability of NitAly. A, temperature dependence of alginate lyase activity of rNitAly in the presence of DTT. Assays were performed as described for Fig. 5A, except that DTT was added to the reaction mixture at a final concentration of 5 mm. Relative activity at 100% was equivalent to 1,590 units/mg. B, heat stability of rNitAly in the presence of DTT. Samples were preincubated as described for Fig. 6A, except that DTT was added at a final concentration of 5 mm. Assays were performed as described in A at 50 °C. Relative activity at 100% was equivalent to 1,080 units/mg. All assays were repeated three times, and the data are shown as mean ± S.D.

FIGURE 8.

Homology modeling of NitAly and PyAly. The structures of NitAly (A, residues 34–242) and PyAly (B, residues 13–216) were predicted using the PHYRE2 program (63) and P. aeruginosa alginate lyase PA1167 (Protein Data Bank code 1vav) as the template (15) with 100% confidence, respectively. A, Cys-80 and Cys-232 are shown by red globules. The residues Arg-85, Gln-123, His-125, and Tyr-226, which correspond to the suggested catalytic residues in PA1167 (15, 21), are shown by pink sticks. B, Gly-79 and Asp-230 are shown by red globules.

FIGURE 9.

Effects of cysteine residue mutations in rNitAly on temperature dependence and heat stability. A, C, and E, temperature dependence of alginate lyase activity of rNitAlyC80A (A), rNitAlyC232A (C), and rNitAlyC80A/C232A (E). Assays were performed as described for Fig. 5A. Relative activities at 100% were equivalent to 1,520 units/mg (A), 1,630 units/mg (C), and 1,480 units/mg (E). B, D, and F, heat stability of rNitAlyC80A (B), rNitAlyC232A (D), and rNitAlyC80A/C232A (F). Assays were performed as described for Fig. 6A. Relative activity at 100% was equivalent to 970 units/mg (B), 1,060 units/mg (D), and 960 units/mg (F). All assays were repeated three times, and the data are shown as mean ± S.D.

FIGURE 10.

Effects of replacing the residues of rPyAly with cysteine on its temperature dependence. Enzyme reactions were conducted in a solution containing 10 mm sodium phosphate (pH 8.0), 0.1 m NaCl, 0.1 mg/ml BSA, 0.25% (w/v) polyM, and 0.005 mg/ml rPyAly (A), rPyAlyG79C (B), rPyAlyD230C (C), or rPyAlyG79C/D230C (D) for 15 min at the indicated temperatures. Relative activity at 100% was equivalent to 1,820 units/mg (A), 1,740 units/mg (B), 1,710 units/mg (C), and 1,780 units/mg (D). All assays were repeated three times, and the data are shown as mean ± S.D.

FIGURE 11.

Effects of replacing residue(s) of rPyAly with cysteine on its heat stability. Enzyme solution containing 10 mm sodium phosphate (pH 8.0), 0.1 m NaCl, and 0.15 mg/ml rPyAly (A), rPyAlyG79C (B), rPyAlyD230C (C), or rPyAlyG79C/D230C (D, −DTT) was incubated for 30 min at the indicated temperatures and placed on ice. For rPyAlyG79C/D230C, 5 mm DTT was also added during incubation (D, +DTT). Enzyme activity was then assayed as described for Fig. 10 at 30 °C. Relative activity at 100% was equivalent to 1,730 units/mg (A), 1,620 units/mg (B), 1,540 units/mg (C), 1,600 units/mg (D, −DTT), and 1,420 units/mg (D, +DTT). All assays were repeated three times, and the data are shown as mean ± S.D.

FIGURE 12.

Assignment of genes in the alginate biosynthesis operon of P. aeruginosa and their homologs in Nitratiruptor sp. SB155-2. Genes encoding proteins with homology between P. aeruginosa and Nitratiruptor sp. SB155-2 are connected by dotted lines. Each pentagon is classified by a different color based on known function in P. aeruginosa as follows: blue, precursor biosynthesis; green, alginate polymerization; brown, alginate export; violet, epimerization of mannuronic acid to guluronic acid; red, alginate degradation; gray, O-acetylation. Yellow pentagons show genes with unidentified functions in Nitratiruptor sp. SB155-2.