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  • Laminarin Quantification in Microalgae with Enzymes from Marine Microbes.

Laminarin Quantification in Microalgae with Enzymes from Marine Microbes.

Bio-protocol (2018-04-20)
Stefan Becker, Jan-Hendrik Hehemann
ABSTRACT

The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via heterologous expression and purification. The assay can be used to monitor laminarin concentrations in environmental microalgae, which were concentrated from seawater by filtering, or in samples derived from algal lab cultures.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Imidazole, Molecular Biology, ≥99% (titration)
Sigma-Aldrich
4-Hydroxybenzhydrazide, ≥97%
Sigma-Aldrich
Agar, ash 2.0-4.5%
Sigma-Aldrich
DL-Dithiothreitol, ≥98% (HPLC), ≥99.0% (titration)
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Sodium citrate dihydrate, ≥99%, FG
Sigma-Aldrich
Sodium deoxycholate, ≥97% (titration)
Sigma-Aldrich
Ammonium sulfate, Molecular Biology, ≥99.0%
Sigma-Aldrich
Laminarin from Laminaria digitata, polysaccharide substrate for laminarinase
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Lysozyme from chicken egg white, powder or granules, ≥39,000 units/mg protein
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)