海洋微生物酶法定量微藻中的昆布多糖

Stefan Becker; Jan-Hendrik  Hehemann

doi:10.21769/BioProtoc.2666

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Peer-reviewed

Laminarin Quantification in Microalgae with Enzymes from Marine Microbes

海洋微生物酶法定量微藻中的昆布多糖

SB Stefan Becker email

Jan-Hendrik Hehemann email

发布: 2018年04月20日第8卷第8期 DOI: 10.21769/BioProtoc.2666 浏览次数: 7421

评审: Anonymous reviewer(s)

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Cover of Applied and Environmental Microbiology, featuring study using the protocol.

May 2017

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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.

Keywords: Algae (藻类)

β-Glucan (β-葡聚糖)

Diatoms (硅藻)

Glycobiology (糖生物学)

Glycoside hydrolase (糖苷水解酶)

Laminarin (昆布多糖)

Chrysolaminarin (金藻昆布多糖)

Laminarinase (昆布多糖酶)

Marine organic matter (海洋有机物质)

Background

Marine polysaccharides play an important role in the marine carbon cycle and are a major part of the physiology of phytoplankton, but are severely understudied. For decades, the agro-food industry has been using ready-to-use kits based on enzymatic assays to analyse a wide range of different polysaccharides in their processes (Whitaker, 1974). These fast, robust and specific enzyme based methods assess polysaccharides originating from land-based plants, i.e., starch, as they are widely used in food, feed and other industrial applications (Brunt et al., 1998). However, similar assays for marine polysaccharides are still lacking. Inspired by the idea of using enzymes for polysaccharide quantification in algae, we developed an enzyme-based method to quantify the ecologically relevant beta-glucan laminarin, also known as chrysolaminarin, in diatoms and other microalgae.

The three glycoside hydrolases (GH) for this application are from Formosa spp. and they were characterized as follows: FbGH30 is an exo-acting β-1,6-glucanase of the GH30 family, specifically hydrolysing the β-1,6-linked glucose monomer branches attached to the laminarin backbone; and FaGH17A and FbGH17A are two endo-acting β-1,3-glucanases of the GH family 17, which acts specifically on the β-1,3-linked laminarin backbone (Becker et al., 2017)

This method enables the quantification of laminarin in crude substrate mixtures, without the need for purification of the laminarin. This enzymatic method is fast, does not require sophisticated instruments, the enzymes are stereospecific and they selectively cleave laminarin into glucose and oligosaccharides, which can be quantified with a common reducing sugar assay. The method can be easily applied in fieldwork. The assay itself comprises only the three steps of extraction, hydrolysis and the reducing sugar assay (Figure 1). It can be done within only a few hours. The limit of detection (LOD) of the assay is at 1.5 µg/ml. The three enzymes need to be produced only once and can be stored for years. After their production and purification, one has enough material to analyse thousands of samples. We decided to include the plasmid transformation and recombinant enzyme production part into the protocol, since we consider these steps feasible to be done by marine labs with less experience in biotechnology.

Figure 1. Schematic protocol after the production of the enzymes. A brief outline of the three main steps and their approximate duration.

Materials and Reagents

2.0 ml reaction tubes (SARSTEDT, catalog number: 72.691 )
Sterile toothpick
Aluminium foil
5 ml HiTrap IMAC HP column (GE Healthcare, catalog number: 17-0920-05 )
Polyethersulfone ultrafiltration membrane with 10-kDa cutoff value (Merck, catalog number: PBGC04310 )
5 ml HiTrap Desalting column (GE Healthcare, catalog number: 29-0486-84 )
Whatman GF/F filters (GE Healthcare, catalog number: 1825-047 ), size is dependent on used filtration setup
50 ml centrifuge tubes (SARSTEDT, catalog number: 62.547.254 )
Half-micro-cuvettes (BRAND, catalog number: 759015 )
Petri dishes (SARSTEDT, catalog number: 82.1473 )
Note: Alternatively a Pierce Protein Concentrator PES, 10 K MWCO, 5-20 ml (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 88527 ) (see Note 2).
BL21 (DE3) competent E. coli (New England Biolabs, catalog number: C2527I )
SOC-medium (is usually enclosed in BL21 (DE3) competent E. coli product)
FaGH17A plasmid (Addgene, catalog number: 86462 ) (see Note 1)
FbGH30 plasmid (Addgene, catalog number: 86463 ) (see Note 1)
FbGH17A plasmid (Addgene, catalog number: 100911 ) (see Note 1)
IPTG (Isopropyl β-D-thiogalactopyranoside from AppliChem, catalog number: A4773 )
Lysozyme (from chicken egg white) (Sigma-Aldrich, catalog number: L6876 )
Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2 )
Tryptone (Sigma-Aldrich, catalog number: 95039 )
Yeast extract (Sigma-Aldrich, catalog number: 09182 )
Agar (Sigma-Aldrich, catalog number: A7002 )
Kanamycin sulfate (Sigma-Aldrich, catalog number: 60615 )
Ammonium sulfate ((NH₄)₂SO₄) (Sigma-Aldrich, catalog number: A4418 )
Monopotassium phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: 9791 )
Disodium phosphate (Na₂HPO₄) (Sigma-Aldrich, catalog number: 71642 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Lactose (Sigma-Aldrich, catalog number: 17814 )
Glycerol (Sigma-Aldrich, catalog number: G6279 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: 208094 )
Sucrose (Sigma-Aldrich, catalog number: 84100 )
Tris(hydroxymethyl)aminomethane (Tris) (Sigma-Aldrich, catalog number: RDD008 )
Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
Triton X-100 (Sigma-Aldrich, catalog number: X100 )
DNase I (from bovine pancreas) (Sigma-Aldrich, catalog number: 69182 )
Nickel(II) sulfate hexahydrate (NiSO₄·6H₂O) (Sigma-Aldrich, catalog number: 227676 )
Imidazole (Sigma-Aldrich, catalog number: I5513 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: 69947 )
Laminarin from Laminaria digitata (Sigma-Aldrich, catalog number: L9634 )
Bovine serum albumin (BSA), lyophilized (Sigma-Aldrich, catalog number: A2153 )
4-Hydroxybenzhydrazide (Sigma-Aldrich, catalog number: H9882 )
HCl (37%) (Sigma-Aldrich, catalog number: 320331 )
Trisodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600 )
Sodium hydroxide (NaOH) (VWR, catalog number: 28244.295 )
Calcium chloride (CaCl₂)
LB-Kana-Agar-Plates (see Recipes)
LB-Kana-Medium (see Recipes)
20x NPS stock (see Recipes)
50x 5052 stock (see Recipes)
MgSO₄ stock (see Recipes)
ZY-Medium (see Recipes)
ZYP-5052-Rich-Autoinduction-Medium (see Recipes)
Sucrose stock (see Recipes)
Deoxycholate stock (see Recipes)
DNase I stock (see Recipes)
NiSO₄ stock (see Recipes)
IMAC buffer A (see Recipes)
IMAC buffer B (see Recipes)
SEC buffer (see Recipes)
SEC/DTT buffer (see Recipes)
MOPS buffer (see Recipes)
BSA stock solution (see Recipes)
PAHBAH reagent A (see Recipes)
PAHBAH reagent B (see Recipes)
PAHBAH working reagent (see Recipes)

Equipment

Bunsen burner or sterile hood
2 L Erlenmeyer flasks (or normal 2 L glass bottles)
Rotating shaker for Erlenmeyer flasks (temperature control is recommended)
Vortex mixer
Microcentrifuge for 1.5 or 2.0 ml reaction tubes with cooling function, i.e., Centrifuge 5418 R (Eppendorf, model: 5418 R , catalog number: 5401000013)
Centrifuge for 15 and 50 ml tubes with cooling function, i.e., Centrifuge 5804 R (Eppendorf, model: 5804 R , catalog number: 5805000017)
Standard membrane or peristaltic pump and respective filtration setup, i.e., ME 1 (Vacuubrand, model: ME 1 ) and Nalgene filter holder receiver (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 300-4100 )
Standard fast protein liquid chromatography system (FPLC), i.e., ÄKTA start (GE Healthcare, model: ÄKTA start, catalog number: 29-0220-94 )
Note: Alternatively a peristaltic pump with a flow rate of ≤ 5 ml/min.
Spectrometer, i.e., BioSpectrometer basic (Eppendorf, model: BioSpectrometer^® basic , catalog number: 6135000009)
Heated water bath, i.e., Thermolab (GFL, catalog number: 1070 )
Two heat blocks, i.e., BioShake iQ (Analytik Jena, catalog number: 848-1808-0506 )
Autoclave
Note: Autoclave is strongly recommended but not essential, since one always uses the antibiotic kanamycin for the bacterial growth.
Ultrafiltration stirred cell, i.e., Amicon (Merck, catalog number: UFSC05001 )
Note: Alternatively a Pierce Protein Concentrator PES, 10 K MWCO, 5-20 ml (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 88527 ) (see Note 2).

Procedure

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Becker, S. and Hehemann, J. (2018). Laminarin Quantification in Microalgae with Enzymes from Marine Microbes. Bio-protocol 8(8): e2666. DOI: 10.21769/BioProtoc.2666.