The DEH solution was prepared and quantitated as described previously13 (link), but without endo-type alginate lyase A1-I, except when stated. Briefly, an alginate solution [1% (w/v)] was prepared by autoclaving 1.0 g of sodium alginate (Nacalai Tesque) in 100 mL of pure water (Elix, Millipore). To this alginate solution, 1.0 mL of the purified exo-type alginate lyase Atu3025 (2.15 mg/mL, 8.57 U/mg), prepared as described previously15 (link), was added, and the reaction mixture was incubated at 30 °C and 100 spm for 18 h. The mixture was filtered through a Centriprep-10K centrifugal filter at 4 °C and 1,600 × g for 30 min, and then the filtrate was freeze-dried, adjusted to a concentration of 2.5% (w/v) with pure water, and sterilized using a 0.22-μm filter.
When DEH reactivity was examined, the DEH solution with other compounds to be tested was incubated at 30 °C and the reaction stopped by immersion in ice water.
When DEH reactivity was examined, the DEH solution with other compounds to be tested was incubated at 30 °C and the reaction stopped by immersion in ice water.
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