Alginic acid

To ensure Smlt1473 was functional post-purification, the thiobarbituric acid (TBA) assay, with some adaptations, was used (46 (link)). Alginate lyases depolymerize alginate via a β-elimination mechanism, which results in the formation of a double bond, that is, an unsaturated product that ultimately reacts with thiobarbituric acid, leading to a pink chromogen with absorbance at 540 nm. Thus, absorbance at 540 nm correlates to alginate depolymerization (32 (link), 46 (link)).
Briefly, the assay used a 2 mg/mL alginate solution created from purified alginic acid (Sigma), 0.025 M periodic acid in 0.125 N sulfuric acid, 2% arsenite in 0.5 N HCl, and 0.45 µm filtered 0.71% 2-thiobarbituric acid in 0.7 mM NaOH. Due to light sensitivity, 0.71% TBA solution was stored in a dark container. Fifty microliters of 2 mg/mL alginate were added to a 1.5 mL microcentrifuge tube along with 40 μL of the enzyme, mixed, and left to incubate for 30 min at room temperature. After incubation, 15 μL of 0.025 M periodic acid was added and mixed, and the tubes were incubated for an additional 40 min at room temperature. To terminate the reaction, 30 μL of 2% sodium arsenite was added, mixed, and left to incubate for 2 min. For activity detection, 290 μL of 0.71% TBA reagent was added to each tube and placed in a heat block at 90°C for 10 min. Following incubation, tubes were centrifuged for 2 min, and then, 150 μL was added into a 96-well plate to measure absorbance at 540 nm.

P. aeruginosa isolates were streaked on LB agar from frozen stock and incubated at 37°C for 24 h. Contents of the agar plate were collected using 10 mL of 0.85% NaCl and vortexed thoroughly to ensure a homogenous solution. OD₆₀₀ was measured undiluted, 5-fold diluted, 10-fold diluted, and averaged for normalization purposes. Dilutions were used where undiluted sample OD₆₀₀ >0.8. To examine the degradation capability of Smlt1473 against alginate secreted by P. aeruginosa, the TBA assay described in Section 2.3 was used. Plate contents were collected in 3.3 mL of 0.85% NaCl (3× concentrated) so that less sample and more enzyme could be used while maintaining the same sample mass. Then, 16.7 μL of sample and 73.2 μL of buffer or treatment were used for final concentrations of 0.8 mg/mL WT Smlt1473, 0.65 mg/mL WT Smlt1473, 0.02 mg/mLWT Smlt1473, and 0.65 mg/mL Y222F. Since secreted fractions containing alginate from clinical isolates were used rather than purified alginic acid, we divided absorbance at 540 nm by OD₆₀₀ of the initial mucoid suspension to account for differences in bacterial growth and alginate production between the trials.
As a control, purified alginic acid (Sigma) was used in the TBA assay as well to prove Smlt1473 activity against alginate substrates (Fig. S1). In total, 73.2 μL of buffer or enzyme at various concentrations was added to 16.7 μL of 2 mg/mL alginate solution, corresponding to a final concentration of 370 μg/mL, which is in the range of alginate production for the mucoid P. aeruginosa isolates. The rest of the assay was conducted as described in Section 2.3.