Suspended chitin in test tubes was quantified by measuring its filling level as described previously (Jagmann et al., 2010). Samples for measuring chitin degradation products were centrifuged in 1.5-mL plastic MK-2206 research buy tubes at 16 100 g for 15 min at room temperature, and supernatants were stored at −20 °C until further analysis.
To determine chitin degradation products during incubation in cell-free supernatant of strain AH-1N, samples were centrifuged as described above. Supernatants were subsequently incubated at 100 °C for 5 min to inhibit chitinolytic enzymes. After a further centrifugation step, supernatants were transferred into new plastic tubes and stored at −20 °C until further analysis. Acetate, the monomer, dimer [N,N′-diacetylchitobiose
(Sigma)] and trimer [N,N′,N″-triacetylchitotriose (Sigma)] of GlcNAc were determined by ion-exclusion HPLC as described previously (Klebensberger et al., 2006). Ammonium Daporinad molecular weight was determined as described previously (Gesellschaft Deutscher Chemiker, 1996). Chitinolytic enzyme activities during growth of strains AH-1N and 4D9 with suspended or embedded chitin were determined indirectly with 4-methyl-umbelliferone (4-MU) derivatized substrates (Colussi et al., 2005). Assays were performed in 96-well black microtiter plates (Nunc) and contained 10 μL of the respective sample and 90 μL of McIlvaine buffer (pH 7). Cell-free culture supernatant was obtained by centrifugation at 16 100 g for 15 min. To measure chitinolytic enzyme activity in the biofilm fraction, single agarose beads were washed in 500 μL medium B and homogenized with a plastic pestle in 100 μL of the same IMP dehydrogenase medium. Assays were started by adding 25 μM
of either 4-MU-N′-acetyl-β-d-glucosaminide (4-MU-GlcNAc; Sigma) for measuring chitobiase activities or 4-MU-N′,N″-diacetyl-β-d-chitobioside [4-MU-(GlcNAc)2; Sigma] for measuring chitinase activities. Enzyme activities were determined at room temperature by measuring the fluorescence of released 4-MU at 465 nm after exciting at 340 nm in a microplate reader (Genios, Tecan) over a time period of 4 min. Activities were calculated using a 4-MU standard fluorescence curve in the range of 0–20 μM. Protein concentrations in culture supernatants were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). To confirm that A. hydrophila strain AH-1N and Flavobacterium sp. strain 4D9 employed different mechanisms of chitin degradation, both strains were incubated with suspended and embedded chitin, respectively, as the sole source of carbon, nitrogen, and energy. With suspended chitin, strain AH-1N grew concomitant with chitin degradation and reached numbers of 1.5 × 109 CFUs mL−1 within 120 h (Fig. 1). Cleavage of 4-MU-(GlcNAc)2 was detected in cell-free culture supernatants with a specific activity of 120 mU (mg protein)−1, indicating the presence of a released chitinase.