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Merck

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.

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Sigma-Aldrich
Acido cloridrico, ACS reagent, 37%
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D-(+)-Glucosio, ≥99.5% (GC)
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Sieroalbumina, lyophilized powder, ≥96% (agarose gel electrophoresis)
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Triton X-100, laboratory grade
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Lisozima, powder or granules, ≥39,000 units/mg protein
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DL-Ditiotreitolo, ≥98% (HPLC), ≥99.0% (titration)
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Solfato d′ammonio, Molecular Biology, ≥99.0%
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Desossicolato di sodio, ≥97% (titration)
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Sodio citrato tribasico, ≥99%, FG
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Imidazolo, Molecular Biology, ≥99% (titration)
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Kanamycin sulfate, mixture of Kanamycin A (main component) and Kanamycin B and C
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Glicerolo, BioXtra, ≥99% (GC)
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4-Hydroxybenzhydrazide, ≥97%
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Laminarin from Laminaria digitata, polysaccharide substrate for laminarinase
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Agar, ash 2.0-4.5%