Laminarin

The interaction between NaD1 and polysaccharides was characterized by NMR using ¹⁵N labelled NaD1 with laminarin (Sigma) as a source of β-glucan or chitohexaose (Megazyme) as a source of oligosaccharide from chitin. Spectra recorded at 600 MHz on a Bruker Avance I spectrometer included 2D ¹⁵N-Heteronuclear Single Quantum Coherence (HSQC), 3D ¹⁵N-HSQC-Nuclear Overhauser Effect spectroscopy (NOESY) and 3D ¹⁵N-HSQC-Total Correlation Spectroscopy (TOCSY). Since chemical shifts are indicative of local environment, titration experiments were conducted to determine changes that occurred upon the interaction of ¹⁵N-labelled NaD1 with laminarin or chitohexaose. Aliquots of a stock solution of laminarin (146 mg/mL H₂O, 110 μL Na₃PO₄, 110 μL D₂O) were sequentially titrated into an NMR tube containing the ¹⁵N-labelled NaD1 (400 μL ¹⁵N-NaD1 4 mg/mL 90% H₂O/10% D₂O v/v, 50 μL 0.5 M Na₃PO₄, pH 6.3). For the chitohexaose experiments, a stock solution of hexaacetyl chitohexaose (3.3 mg/mL H₂O) was added similarly to a sample of ¹⁵N-labelled NaD1 (450 μL ¹⁵N-NaD1 1.1 mg/mL). Chemical shift differences of greater than 0.1 ppm (for ¹⁵N shifts) and 0.01 ppm (for proton shifts) were considered significant.

The triple helical conformation of polysaccharide was investigated using the azo dye Congo red as described [20 (link)]. Briefly, 2 mL of 1 mg/mL nostoglycan solution was mixed with 2 mL of 100 μM Congo red solution, and then 1 mL of NaOH solution with different concentrations was added to achieve a final NaOH concentration of 0–0.5 M. Laminarin and dextran (Sigma, St. Louis, MO, USA) were used as controls for triple helical and random coil conformations, respectively. The λ_max of the mixture was recorded by wavelength scanning from 400 to 600 nm using a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

The E. coli strain BL21 (DE3) pLyS, containing pPinsEXO1 or pRSET-C empty plasmid, were grown in LB broth supplemented with ampicillin (50 μg/ml). IPTG (final concentration, 1 mM) was added to the culture to induce protein expression. After incubation at 37°C for 4 hr, several cell suspensions (bacteria with pPinsEXO1) were prepared, ranging from 1 x 10² to 1 x 10⁹ cells/ml (1 OD₆₀₀ = 10⁹ cells/ml), and 100 μl of each cell suspension were spotted onto a 6-mm-diameter antibiotic assay disc (Whatman) placed on a LB agar mixed with 1.5% (w/v) of Avicel PH-101 (Fluka-Sigma), or a LB agar overlaid with 100 μl of 0.5% (w/v) Laminarin (Sigma). Positive controls included either 100 μl (concentration: 1 x 10²–1 x 10⁻⁹ mg/ml) of Trichoderma reesei’s cellulase (Sigma) or Trichoderma harzianum’s lysing enzyme (containing β-glucanase, cellulase, protease, and chitinase activities; Sigma). Negative controls included 100 μl of plain LB broth, or bacteria with pRSET-C (1 x 10⁹ cells/ml). After incubation at 37°C overnight, LB agar was stained with iodine-staining solution [2 g potassium iodide (Carlo Erba) and 1 g iodine (BDH Laboratory) in 300 ml of distilled water] [36 (link)]. Diameter of the hydrolytic (clear) zone generated by each sample was measured. All samples were tested in triplicate.