Screening Polysaccharide-Degrading Enzymes

1% agar (catalog number A1296, Sigma-Aldrich Corp., St. Louis, MO, USA) containing 0.2% carboxymethylcellulose (catalog number C4888, Sigma-Aldrich Corp., St. Louis, MO, USA) was poured into petri dishes or into the wells of a 96-well plate, respectively. Stock solutions of polymer-degrading enzymes were dissolved in 1× Tris-buffered saline (TBS) buffer or deionized water, following the suppliers’ instructions for reconstitution of the enzyme. Spot plating was performed with 5 μL of commercial cellulase from Aspergillus niger (catalog number C1184, Sigma-Aldrich Corp., St. Louis, MO, USA) at different concentrations (0.1 and 5 μg/μL). Agarase (1 μg/μL) (catalog number EO0461, Fermentas, Burlington, ON, Canada), amylase (1 μg/μL) (catalog number 10065, Sigma-Aldrich Corp., St. Louis, MO, USA) and proteinase K (1 μg/μL) (catalog number EO0491, Fermentas, Burlington, ON, Canada) were used as control enzymes. In some cases, agar was replaced by 1% agarose or 0.75% Gelrite with or without 0.2% CMC, respectively. All plates were incubated at 27 °C for 12–16 h after which hydrolysis zones were visualized by flooding of the plates/wells with Gram’s iodine (2 g potassium iodide and 1 g iodine in 300 mL water) for 5 min followed by a rinses with deionized water. For detection with Congo Red, the plates were flooded with 0.1% Congo Red for 15–20 min and then rinsed with 1 M NaCl. Plates where CMC was omitted were used as non-substrate controls in all experiments.