dest.) to remove NaCl. Afterwards 20 μl of purified lipase LipA from

P. aeruginosa PABST7.1/pUCPL6A (72 ng/ml A. dest.) was added and incubated at 30°C for 30 min. Non-bound lipase was removed by two washing steps with 100 μl A. dest. each. Bounded lipase was detected via activity measurement in the microtiter plate using pNPP as substrate. The cleavage of the substrate was monitored at 405 nm in a microtiter plate reader. All experiments were performed in duplicates and repeated three times. Heat treatment of lipase The stabilization of lipases through the EPZ004777 nmr interaction with alginate was investigated by heat treatment of purified lipase in presence and absence of polysaccharides. One volume purified lipase LipA from P. aeruginosa PABST7.1/pUCPL6A (36 μg/ml A. dest.) was mixed with one CRT0066101 cost volume purified polysaccharides (2 mg/ml in 100 mM Tris–HCl buffer, pH 7.5), which were previously heated (15 min, 90°C) and afterwards cooled on ice to room temperature. After pre-incubation for 30 min at room temperature the samples were incubated for 0–60 min at 70°C to determine lipase inactivation kinetics. Moreover, the samples were incubated for 20 min at different temperatures (37°C; 50°C; 60°C; 70°C; 80°C; 90°C) to determine T50. T50 represents the temperature at see more which incubation for 20 minutes reduces the enzymes activity by half. Every

10 min the residual lipase activities were detected after cooling on ice, using pNPP as substrate. All experiments were performed in duplicates and repeated three times. Degradation of lipase by proteases The protection of lipase from proteolytic degradation through the interaction with alginate was studied by using purified elastase LasB from Amylase P. aeruginosa (EMD4Biosciences, San Diego, USA). Briefly, 0.5 ml purified lipase LipA from P. aeruginosa PABST7.1/pUCPL6A (36 μg/ml A. dest.) was mixed with 0.5 ml purified polysaccharides (2 mg/ml

in 100 mM Tris–HCl buffer, pH 7.5) previously heated for 15 min and 90°C and afterwards cooled on ice to room temperature. After pre-incubation for 30 min at room temperature, 20 μl purified elastase (0.1 mg/ml with 25 U/ml in A. dest.) were added. After 24 h incubation at 37°C the residual lipase activity was detected, using pNPP as a substrate. All experiments were performed in duplicates and repeated three times. Modeling of lipase-alginate interaction The protein structure was based on the crystal structure of the lipase protein resulting from the X-ray diffraction analysis of a lipase protein [37]. The crystal structure was obtained from the RCSB protein data bank [72]. The hydrogens of the amino acids were adjusted according to the pKs values of the amino acids at a pH value of 7.0. Therefore, the resulting net charge of the protein was in accordance to an aqueous solution of pH = 7.0. Inter- and intramolecular interactions were calculated by a molecular mechanics force field approach.