Dataset: Polysaccharide Hydrolase activities in Danish coastal seawater and sediments under varying hydrostatic pressures on samples collected in September 2023

Sample water for incubation and filtration was collected via Niskin bottles mounted on a rosette, equipped with a CTD, as part of a routine collection at a coastal station off the coast of Helsingor, Denmark in September, 2023, aboard the R/V Ophelia.

 

For each substrate, three 50 mL falcon tubes were filled with seawater and one 50 mL falcon tube was filled with autoclaved seawater to serve as a killed control. Substrate was added at 3.5 μM monomer-equivalent concentrations, except for fucoidan, which was added at 5 μM concentrations (a higher concentration was necessary for sufficient fluorescence signal). Two 50 mL falcon tubes – one with seawater and one with autoclaved seawater – with no added substrate served as blank controls. Incubations were stored in the dark at as close to in situ temperature as possible.

 

For some of the samples (i.e., amended samples), they were amended with high-molecular-weight organic matter extracted from freeze-dried Thalassiosira weisflogii (following Balmonte et al., 2019) to enhance enzymatic activity. Some of these samples were either immediately pressurized after polysaccharide addition, or incubated for 7 days prior to adding polysaccharide and pressurizing. A subset of this 7-day amended seawater was filtered (0.2 µm) to obtain the dissolved enzyme fraction, and incubated with polysaccharides and pressurized to different pressures. 

 

Incubations were sub-sampled at different timepoints.  

 

The hydrolysis of high molecular weight substrate to lower molecular weight hydrolysis products was measured using gel permeation chromatography with fluorescence detection, after the method of Arnosti [1996, 2003]. In short, the subsample was injected onto a series of columns consisting of a 21 cm column of G50 and a 19 cm column of G75 Sephadex gel. The fluorescence of the column effluent was measured at excitation and emission wavelengths of 490 and 530 nm, respectively.

 

Subsamples were run on a gel permeation chromatography instrument to separate out size classes of fluorescently-labeled polysaccharides. Hydrolysis is calculated as a change in the size distribution of polysaccharide with time.

 

Polysaccharide used for incubation:

• ara = arabinogalactan
• chn = chondroitin sulfate
• fuc = fucoidan
• lam = laminarin
• man = mannan
• pul = pullulan
• xyl = xylan