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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   

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Applied and Environmental Microbiology
May 2017

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

  1. 2.0 ml reaction tubes (SARSTEDT, catalog number: 72.691 )
  2. Sterile toothpick
  3. Aluminium foil
  4. 5 ml HiTrap IMAC HP column (GE Healthcare, catalog number: 17-0920-05 )
  5. Polyethersulfone ultrafiltration membrane with 10-kDa cutoff value (Merck, catalog number: PBGC04310 )
  6. 5 ml HiTrap Desalting column (GE Healthcare, catalog number: 29-0486-84 )
  7. Whatman GF/F filters (GE Healthcare, catalog number: 1825-047 ), size is dependent on used filtration setup
  8. 50 ml centrifuge tubes (SARSTEDT, catalog number: 62.547.254 )
  9. Half-micro-cuvettes (BRAND, catalog number: 759015 )
  10. Petri dishes (SARSTEDT, catalog number: 82.1473 )
    Note: Alternatively a Pierce Protein Concentrator PES, 10 K MWCO, 5-20 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88527 ) (see Note 2).
  11. BL21 (DE3) competent E. coli (New England Biolabs, catalog number: C2527I )
  12. SOC-medium (is usually enclosed in BL21 (DE3) competent E. coli product)
  13. FaGH17A plasmid (Addgene, catalog number: 86462 ) (see Note 1)
  14. FbGH30 plasmid (Addgene, catalog number: 86463 ) (see Note 1)
  15. FbGH17A plasmid (Addgene, catalog number: 100911 ) (see Note 1)
  16. IPTG (Isopropyl β-D-thiogalactopyranoside from AppliChem, catalog number: A4773 )
  17. Lysozyme (from chicken egg white) (Sigma-Aldrich, catalog number: L6876 )
  18. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2 )
  19. Tryptone (Sigma-Aldrich, catalog number: 95039 )
  20. Yeast extract (Sigma-Aldrich, catalog number: 09182 )
  21. Agar (Sigma-Aldrich, catalog number: A7002 )
  22. Kanamycin sulfate (Sigma-Aldrich, catalog number: 60615 )
  23. Ammonium sulfate ((NH4)2SO4) (Sigma-Aldrich, catalog number: A4418 )
  24. Monopotassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 9791 )
  25. Disodium phosphate (Na2HPO4) (Sigma-Aldrich, catalog number: 71642 )
  26. Glucose (Sigma-Aldrich, catalog number: G8270 )
  27. Lactose (Sigma-Aldrich, catalog number: 17814 )
  28. Glycerol (Sigma-Aldrich, catalog number: G6279 )
  29. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: 208094 )
  30. Sucrose (Sigma-Aldrich, catalog number: 84100 )
  31. Tris(hydroxymethyl)aminomethane (Tris) (Sigma-Aldrich, catalog number: RDD008 )
  32. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  33. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  34. DNase I (from bovine pancreas) (Sigma-Aldrich, catalog number: 69182 )
  35. Nickel(II) sulfate hexahydrate (NiSO4·6H2O) (Sigma-Aldrich, catalog number: 227676 )
  36. Imidazole (Sigma-Aldrich, catalog number: I5513 )
  37. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  38. 3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: 69947 )
  39. Laminarin from Laminaria digitata (Sigma-Aldrich, catalog number: L9634 )
  40. Bovine serum albumin (BSA), lyophilized (Sigma-Aldrich, catalog number: A2153 )
  41. 4-Hydroxybenzhydrazide (Sigma-Aldrich, catalog number: H9882 )
  42. HCl (37%) (Sigma-Aldrich, catalog number: 320331 )
  43. Trisodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600 )
  44. Sodium hydroxide (NaOH) (VWR, catalog number: 28244.295 )
  45. Calcium chloride (CaCl2)
  46. LB-Kana-Agar-Plates (see Recipes)
  47. LB-Kana-Medium (see Recipes)
  48. 20x NPS stock (see Recipes)
  49. 50x 5052 stock (see Recipes)
  50. MgSO4 stock (see Recipes)
  51. ZY-Medium (see Recipes)
  52. ZYP-5052-Rich-Autoinduction-Medium (see Recipes)
  53. Sucrose stock (see Recipes)
  54. Deoxycholate stock (see Recipes)
  55. DNase I stock (see Recipes)
  56. NiSO4 stock (see Recipes)
  57. IMAC buffer A (see Recipes)
  58. IMAC buffer B (see Recipes)
  59. SEC buffer (see Recipes)
  60. SEC/DTT buffer (see Recipes)
  61. MOPS buffer (see Recipes)
  62. BSA stock solution (see Recipes)
  63. PAHBAH reagent A (see Recipes)
  64. PAHBAH reagent B (see Recipes)
  65. PAHBAH working reagent (see Recipes)

Equipment

  1. Bunsen burner or sterile hood
  2. 2 L Erlenmeyer flasks (or normal 2 L glass bottles)
  3. Rotating shaker for Erlenmeyer flasks (temperature control is recommended)
  4. Vortex mixer
  5. 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)
  6. Centrifuge for 15 and 50 ml tubes with cooling function, i.e., Centrifuge 5804 R (Eppendorf, model: 5804 R , catalog number: 5805000017)
  7. 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 ScientificTM, catalog number: 300-4100 )
  8. 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.
  9. Spectrometer, i.e., BioSpectrometer basic (Eppendorf, model: BioSpectrometer® basic , catalog number: 6135000009)
  10. Heated water bath, i.e., Thermolab (GFL, catalog number: 1070 )
  11. Two heat blocks, i.e., BioShake iQ (Analytik Jena, catalog number: 848-1808-0506 )
  12. Autoclave
    Note: Autoclave is strongly recommended but not essential, since one always uses the antibiotic kanamycin for the bacterial growth.
  13. 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 ScientificTM, catalog number: 88527 ) (see Note 2).

Procedure

  1. Transformation
    1. Transform each Addgene plasmid containing one enzyme into separate BL21 (DE3) competent E. coli according to the manufacturer’s instructions and by using LB-Kana-Agar-Plates (see Recipes). A common transformation protocol uses the following steps, which have to be carried out next to a Bunsen burner or in a sterile hood.
    2. Add 1 µl containing ca. 100 ng of plasmid DNA to the cell mixture and carefully invert the tube once. Do not vortex.
    3. Keep on ice for 5 min and do not mix.
    4. Heat shock at exactly 42 °C for exactly 10 sec and do not mix.
    5. Keep on ice for 5 min and do not mix.
    6. Add 500 µl SOC-medium, which is included in the E. coli BL21 (DE3) shipment.
    7. Incubate at 37 °C for 60 min and at 500 rpm.
    8. Spread 100 µl on an LB-Kana-Agar-Plate and incubate for at least 15 h at 37 °C.
    9. After picking a colony with a sterile toothpick and growing it for at least 8 h in a 3 ml LB-Kana-Medium (see Recipes) preculture at 37 °C and under constant stirring at 150 rpm, the cells can be stored at -80 °C after adding 25% (v/v) sterile glycerol (i.e., 200 µl of glycerol in 600 µl of preculture).

  2. Enzyme overexpression and purification
    1. Use 1 ml of the preculture from Step A9 to inoculate 1 L of ZYP-5052-Rich-Autoinduction-Medium (see Recipes) (Studier, 2005) in a 2 L Erlenmeyer flask to have enough headspace. Close the flask only with aluminum foil to allow gas exchange into the flask.
      Notes:
      1. Overexpression and purification are performed in almost the exact same way for all three enzymes (FbGH30, FaGH17A and FbGH17A). However, each purification has to be performed separately. It is not possible to transform all three enzymes into one expression system and purify all together. Nonetheless, after the overexpression, it is possible to purify all three enzymes within one working day.
      2. Overexpression can also be performed in IPTG-induced LB-Kana-Medium. The inoculated cultures need to grow for 8 h at 37 °C before 1 ml 1 M IPTG is added. After induction, the cultures need to be incubated for 8 h more at 16 °C. Subsequent harvesting and purification are performed as already described. This method is faster but yields lower protein amounts.
    2. Grow cultures for about 72 h at 20 °C [or room temperature (RT)] and with rotation at 150 rpm.
    3. Harvest cells by centrifugation at 4,500 x g for 25 min at 4 °C. Discard supernatant. The resulting pellet can be stored at -20 °C (for years) until further use.
    4. Resuspend the cell pellet in 15 ml sucrose stock solution (see Recipes).
    5. Add 5 mg of lysozyme and incubate for 10 min at RT under constant magnetic stirring at 500 rpm.
    6. Add 30 ml deoxycholate stock solution (see Recipes) and 0.2 ml MgSO4 stock solution (see Recipes).
    7. Add 1 ml DNase I stock solution (see Recipes) to liquefy the viscous solution.
    8. Centrifuge the lysate at 16,000 x g for 45 min at 4 °C. Transfer the supernatant containing the proteins of interest into a new tube.
    9. All steps during the following immobilized metal affinity chromatography (IMAC) are carried out with an FPLC at RT and a flow rate of 5 ml/min. Alternatively, the procedure can also be performed by using a peristaltic pump at the same or lower flow rates.
      Note: Steps B10-B14 can be performed manually using a peristaltic pump at 5 ml/min flow rate instead of an FPLC system. The column is then placed after the pump. The linear buffer gradient in Step B14 can be achieved similarly by using a stepwise increase of the imidazole concentration over the course of the total elution volume of 5 CV (~5 min). Starting with IMAC buffer A containing 50 mM imidazole, the buffer needs to be changed every 30 sec. At each step the imidazole concentrations increases by 50 mM (30 sec each): 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mM imidazole (the usual concentration for IMAC buffer A and B are 25 and 500 mM imidazole, respectively). To prevent air bubbles on the column, the flow needs to be paused every time the inlet is switching from one buffer to the other.
    10. Charge a 5 ml HiTrap IMAC HP column with 1 column volume (1 CV = 1 ml) of NiSO4 stock solution (see Recipes). Discard the flow that comes off the column.
    11. Equilibrate with 5 CVs of IMAC buffer A (see Recipes). Discard the flow that comes off the column.
    12. Inject the entire supernatant of the lysis onto the column. If the solution is too viscous, it needs to be diluted up to a volume of 50 ml using IMAC buffer A. Discard the flow that comes off the column.
    13. Wash the column with 15 CVs IMAC buffer A. Discard the flow that comes off the column.
    14. Elute the protein in a total number of 5 CVs of a continuous linear gradient from IMAC buffer A to IMAC buffer B (see Recipes). Collect the flow that comes off the column in thirty 1 ml fractions.
    15. For FbGH17A only: Add 50 µl of the reducing agent DTT (0.5 mg/ml) to each fraction immediately after the elution to prevent oxidation of the protein.
    16. Pool fraction 6 to 30 (if there is a UV detector, take only the samples corresponding to the major peak). Keep the protein on ice.
    17. The IMAC column has to be cleaned after each purification according to the manufacturer’s instruction.
    18. Assemble an ultrafiltration stirred cell together with a respective 10 kDa membrane. (alternatively, a centrifugation concentrator can be used, see Note 2).
    19. Concentrate the protein solution in the ultrafiltration chamber at 4 °C or on ice down to a volume of ≤ 1.5 ml.
    20. Centrifuge the protein solution for 20 min at 13,000 x g and 4 °C, to remove the precipitated, inactive protein.
    21. Transfer supernatant into a fresh tube and discard the pellet. Keep the protein on ice.
    22. The following use of a desalting column removes the imidazole. The procedure followed the manufacturer’s instructions and can be performed by using an FPLC system or a syringe at RT and a flow rate of 5 ml/min.
    23. For FbGH17A only: Use SEC/DTT buffer (see Recipes) instead of SEC buffer in Steps B24-26.
    24. Equilibrate a HiTrap desalting column with 5 CVs of SEC buffer. Discard the flow that comes off the column.
    25. Apply exactly 1.5 ml of the protein solution. Dilute the sample with SEC buffer if necessary. Do not exceed the sample volume of 1.5 ml. Discard the flow that comes off the column.
    26. Elute and collect the protein with 2 ml of SEC buffer and keep the protein on ice afterwards.
    27. The desalting column has to be cleaned after the purification according to the manufacturer’s instruction.
    28. Determine protein concentration by measuring the absorbance at 280 nm using a spectrometer and under consideration of the molecular weight and the extinction factor of each protein (FbGH30: MW = 54,700 Da, ε = 128,480 M-1 cm-1; FaGH17A: MW = 44,800 Da, ε = 87,905 M-1 cm-1; FbGH17A: MW = 46,600 Da, ε = 88,935 M-1 cm-1):

      where, c: protein concentration in mg/ml; A: absorbance at 280 nm; ε: extinction factor in M-1 cm-1; MW: molecular weight in Da; b: light path length in cm.
      Note: The success of overexpression (Steps B1-B3), lysis (Steps B4-B8), IMAC (Steps B9-B17), concentration (Steps B18-B21) and desalting (Steps B22-B27) can be verified by SDS-Polyacrylamide gel electrophoresis (He, 2011).
    29. Dilute the proteins in MOPS buffer (see Recipes) to a stock concentration of 10 µM. Keep protein on ice.
    30. For FbGH17A only: After dilution in MOPS buffer the enzyme must be used immediately, either for preparing aliquots (Step B31) or for hydrolysis reactions (Steps D1-D7).
    31. Prepare ready-to-use 50 µl aliquots and freeze them at -20 °C.
      Note: So far, there is no very long-term data on the shelf life of the enzymes at -20 °C, but they can be used for at least two years without any decrease in activity.

  3. Sampling and extraction
    1. Environmental samples, i.e., seawater, or lab cultures of microalgae are filtered onto Whatman GF/F filters at 0.1-0.5 bar by using a standard membrane or peristaltic pump and a respective filtration setup. The required volume needs to be determined by experiments. In case of sea surface water, one needs at least 5 L, for highly concentrated lab cultures the demand is much smaller (~50 ml, depending on the cell numbers). The volume must be logged for each sample. The resulting filter can be stored at -20 °C until further use.
      Notes:
      1. Polycarbonate filters can be used as well. The diameter of the filters depends on the filtration setup.
      2. For concentrated lab cultures, centrifugation can be used instead of filtration as well. Diatom cultures need to be centrifuged at 4,500 x g for 20 min.
      3. The method can be applied to macroalgal samples as well (L. Scheschonk et al. in preparation).
    2. Place filter into a 15 or 50 ml centrifuge tube.
    3. Add 5-10 ml of MOPS buffer, just enough so that the entire filter is covered with liquid.
    4. Vortex every sample for 10 sec.
    5. Extract for 60 min at 60 °C in a heated water bath.
    6. Squeeze and take out the filter.
    7. Centrifuge for 15 min at 4,500 x g.
    8. Transfer the supernatant to a new tube. This extract can be stored for several weeks at -20 °C until further use.
      Note: There is no long-term data on the stability of laminarin samples.

  4. Hydrolysis
    1. Each sample is split up into six subsamples: three are hydrolyzed by the enzyme mixture and three are not hydrolyzed.
    2. Additionally, one has to prepare and treat (Steps C3 and C5-C7) samples for creating a calibration curve based on commercial laminarin from Laminarin digitata (Sigma-Aldrich). For microalgal samples, the following concentrations are suitable: 0, 7.8 µg/ml, 15.6 µg/ml, 31.3 µg/ml, 62.5 µg/ml, 125 µg/ml, 250 µg/ml, 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml (serial dilution in MOPS buffer).
    3. Thaw fresh aliquots of each enzyme stock solution and keep them on ice.
    4. Each hydrolyzation reaction mix consists of: 100 µl sample extract (or calibration standard), 1 µl FbGH30, 1 µl FaGH17A, 1 µl FbGH17A and 1 µl BSA stock solution (see Recipes) (see Note 3).
    5. Each non-hydrolyzation reaction mix consists of: 100 µl sample extract, 1 µl BSA stock solution and 3 µl of MOPS buffer (see Note 11).
    6. Mix all samples by shaking the reaction tubes.
    7. Incubate for 25 min at 37 °C in a heat block.
    8. Stop enzyme reaction by incubating the samples for 5 min at 99 °C in a second heat block. This extract can be stored for several weeks at -20 °C until further use.
      Note: There is no long-term data on the stability of laminarin samples.

  5. Reducing sugar assay
    1. Add 1 ml of PAHBAH working reagent (see Recipes) (Lever, 1972) to 0.1 ml of the sample.
    2. Heat each sample for exactly 5 min at 100 °C in a heat block.
      Note: The duration of boiling directly influences the absorbance and therefore the standard deviation of the measured triplicates. One should try to take the samples out of the heat block at the exact same speed that is used for putting them in. By doing this one can make sure that every sample is getting heated for the exact same duration.
    3. Determine absorbance at 410 nm using half-micro-cuvettes and a spectrometer.
    4. Use PAHBAH working reagent as blank and for the dilution of samples that are too concentrated.
      Note: The range of absorbance in which one can make reliable measurements depends on the spectrometer and must be checked in the manual in advance. The samples can be diluted in PAHBAH working reagent.

Data analysis

  1. Each sample is measured in six subsamples. The value of the non-hydrolyzed triplicates needs to be subtracted from the value of the hydrolyzed samples.
  2. The laminarin concentration in the respective sample can be determined by comparing this difference to the calibration curve.
  3. The concentration, which was measured in the extract, needs to be converted into the concentration of the original sample, by taking the exact filtration volume or the cell number into account.
  4. Based on the calibration below, the equation and the data for laminarin concentrations in samples of 20 L are as follows (Figure 2, Table 1): 



    Figure 2. Exemplary calibration

    Table 1. Exemplary data

Notes

  1. All three plasmid can be ordered by academics and non-profits via the Addgene plasmid repository (www.addgene.org). On the website, one can find protocols for the purification of the plasmid DNA if Addgene provides only bacterial stabs. The plasmids must get transformed into E. coli BL21 (DE3).
  2. For protein concentration, a centrifugation-based system can be used alternatively. These tubes are not reusable and the recovery rate is quite low. However, for a single usage, it is cheaper compared to the ultrafiltration system. The concentration has to be conducted according to the manufacturer’s instruction and at 4 °C. The volume of the protein solution needs to be decreased to a volume of ≤ 1.5 ml.
  3. It is possible and advisable to prepare master mixes containing all components that have to be added to each sample extract.

Recipes

Note: Milli-Q water was used to make up the following solutions unless otherwise indicated.

  1. LB-Kana-Agar-Plates
    1. Dissolve:
      10 g NaCl
      10 g tryptone
      5 g yeast extract
      12.5 g agar
      Complete the volume to 1 L with non-sterile water
    2. Autoclave or boil the solution
    3. After letting the solution cool down to a temperature below 50 °C, add 1 ml of prepared kanamycin sulfate (50 mg/ml)
    4. After stirring the solution, the plates need to be poured next to a Bunsen burner or in a sterile hood
    5. When the plates are solid, they can be stored at 4 °C for several weeks
  2. LB-Kana-Medium
    1. Dissolve:
      10 g NaCl
      10 g tryptone
      5 g yeast extract
      Complete the volume to 1 L with non-sterile water in a 2 L Erlenmeyer flask or bottle
    2. Close the flask with aluminum foil but keep it air-penetrable
    3. It is recommended to autoclave the solution
    4. After letting the solution cool down to a temperature below 50 °C, add 1 ml of prepared kanamycin sulfate (50 mg/ml)
  3. 20x NPS stock
    Dissolve:
    66 g (NH4)2SO4
    136 g KH2PO4
    142 g Na2HPO4
    Complete the volume to 1 L with non-sterile water
    It is recommended to autoclave the solution before use
  4. 50x 5052 stock
    Dissolve:
    25 g glucose
    100 g lactose
    250 g glycerol (weigh in a beaker) and complete the volume to 1 L with non-sterile water
    It is recommended to autoclave the solution before use
  5. MgSO4 stock
    Dissolve 12 g MgSO4 and complete the volume to 100 ml with non-sterile water
    It is recommended to autoclave the solution before use
  6. ZY-Medium
    Dissolve 10 g tryptone and 5 g yeast extract in 928 ml H2O
    It is recommended to autoclave the solution before use
  7. ZYP-5052-Rich-Autoinduction-Medium
    Mix 928 ml ZY-Medium
    1 ml 1 M MgSO4
    20 ml 50x 5052
    50 ml 20x NPS and 2 ml kanamycin sulfate (50 mg/ml) next to a Bunsen burner
  8. Sucrose stock
    1. Dissolve 250 g sucrose and 6 g Tris
    2. Adjust to pH 7.5 and complete the volume to 1 L with non-sterile water
    3. It is recommended to autoclave the solution before use
  9. Deoxycholate stock
    1. Dissolve:
      10 g sodium deoxycholate
      10 ml Triton X-100
      5.8 g NaCl
      2.4 g Tris
    2. Adjust to pH 7.5 and complete the volume to 1 L with non-sterile water
    3. It is recommended to autoclave the solution before use
  10. DNase I stock
    Dissolve 50 mg DNase I in 35 ml IMAC buffer A and 15 ml glycerol
    The solution can be stored at -20 °C
  11. NiSO4 stock
    Dissolve 3.9 g NiSO4 and complete the volume to 50 ml
    Note: NiSO4 is harmful. Protect yourself by using gloves, eye and mouth protection. Discard contaminated solutions in an appropriate heavy metal waste.
  12. IMAC buffer A
    1. Dissolve 29.2 g NaCl, 2.4 g Tris and 1.7 g imidazole
    2. Adjust to pH 7.5 and complete the volume to 1 L with non-sterile water
    3. It is recommended to sterile filter the solution before use
  13. IMAC buffer B
    1. Dissolve 29.2 g NaCl, 2.4 g Tris and 34 g imidazole
    2. Adjust to pH 7.5 and complete the volume to 1 L with non-sterile water
    3. It is recommended to sterile filter the solution before use
  14. SEC buffer
    1. Dissolve 29.2 g NaCl and 2.4 g Tris
    2. Adjust to pH 7.5 and complete the volume to 1 L with non-sterile water
    3. It is recommended to sterile filter the solution before use
  15. SEC/DTT buffer
    Dissolve 23 mg dithiothreitol in 50 ml SEC buffer
  16. MOPS buffer
    1. Dissolve 5.2 g 3-(N-Morpholino)propanesulfonic acid
    2. Adjust to pH 7.0 and complete the volume to 500 ml
    3. It is recommended to sterile filter the solution before use
    4. Use a brown glass bottle and protect from light
  17. BSA stock solution
    Dissolve 100 mg BSA in 1 ml of H2O
    It is recommended to sterile filter the solution before use
  18. PAHBAH reagent A
    1. Dissolve 10 g 4-hydroxybenzhydrazide in 60 ml H2O
    2. Add 10 ml concentrated HCl (37%) and complete the volume to 200 ml by adding more H2O
    Note: The solution can be stored at RT. Do not use if precipitate is present in the solution.
  19. PAHBAH reagent B
    1. Dissolve 24.9 g trisodium citrate dehydrate in 500 ml H2O
    2. Add 2.2 g CaCl2 and mix
    3. Dissolve 40 g NaOH in a separate 1 L bottle
    4. Mix both solutions slowly and under constant stirring and complete the volume to 2 L with non-sterile water
    5. The solution must become clear within several minutes
    Note: The solution can be stored at RT. Do not use if precipitate is present in the solution.
  20. PAHBAH working reagent
    Prepare a fresh 9:1 mixture of PAHBAH reagent B and PAHBAH reagent A
    Can be used for several hours, but needs to be kept on ice

Acknowledgments

This protocol was adapted and modified from Becker et al., 2017. The research was supported by the Deutsche Forschungsgemeinschaft (grant HE 7217/1-1 to Jan-Hendrik Hehemann) and by the Max Planck Society.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Becker, S., Scheffel, A., Polz, M. F. and Hehemann, J. H. (2017). Accurate quantification of laminarin in marine organic matter with enzymes from marine microbes. Appl Environ Microbiol 83(9).
  2. Brunt, K., Sanders, P. and Rozema, T. (1998). The enzymatic determination of starch in food, feed and raw materials of the starch industry. Starch - Stärke 50(10): 413-419.
  3. He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80.
  4. Lever, M. (1972). A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47(1): 273-279.
  5. Myklestad, S. (1974). Production of carbohydrates by marine planktonic diatoms. I. Comparison of nine different species in culture. J Exp Mar Biol Ecol 15(3): 261-74.
  6. Painter, T. J. (1983). Algal polysaccharides. In: The polysaccharides. Elsevier 195-285.
  7. Percival, E. and Ross, A. (1951). 156. The constitution of laminarin. Part II. The soluble laminarin of laminaria digitata. J Chem Soc (Resumed) 0: 720-6.
  8. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234.
  9. Whitaker, J. R. (1974). Analytical applications of enzymes. In: Whitaker, J. R. (Ed.). Adv Chem Ser 136: 31-78.

简介

海洋β-葡聚糖昆布多糖是微藻中丰富的储存多糖。高生产率和异养细菌的快速消化将昆布多糖转化为理想的碳源和能源,因此它是海洋碳循环的关键参与者。作为主要的储存葡聚糖昆布多糖也在微藻的能量代谢中发挥核心作用(Percival and Ross,1951; Myklestad,1974; Painter,1983)。我们利用可以选择性消化昆布多糖的酶,从而可以对环境样品中的这种多糖进行定量。这些酶将昆布多糖水解成葡萄糖和寡糖,用标准的还原糖测定法测定得到昆布多糖浓度。在此测定之前,需要通过异源表达和纯化产生三种酶。该测定可用于监测环境微藻中的昆布多糖浓度,其通过过滤从海水中浓缩,或用来自藻类实验室培养物的样品中浓缩。

【背景】海洋多糖在海洋碳循环中起着重要作用,是浮游植物生理学的重要组成部分,但受到严重影响。几十年来,农业食品工业一直使用基于酶分析的即用试剂盒来分析各种不同的多糖(Whitaker,1974)。这些快速,稳健和特异性的基于酶的方法评估源自陆地植物即淀粉的多糖,因为它们广泛用于食品,饲料和其他工业应用中(Brunt等人, ,1998)。然而,海洋多糖的类似测定仍然缺乏。受到使用酶在藻类中进行多糖定量的想法的启发,我们开发了一种基于酶的方法来量化在硅藻和其他微藻中生态相关的β-葡聚糖昆布氨酸,也称为菊科金刚烷。

这种应用的三种糖苷水解酶(GH)来自福尔摩沙(Formosa)。并且它们的特征如下:FbGH30是GH30家族的外切型β-1,6-葡聚糖酶,特别是水解与昆布多糖骨架连接的β-1,6-连接的葡萄糖单体分支;并且FaGH17A和FbGH17A是GH家族17的两种内作用β-1,3-葡聚糖酶,其特异性地作用于β-1,3-连接的昆布多糖主链上(Becker等人,2017年, )

该方法能够在粗制底物混合物中定量海带多糖,而不需要纯化海带多糖。这种酶法快速,不需要复杂的仪器,酶是立体定向的,它们选择性地将昆布多糖切成葡萄糖和寡糖,可以用普通的还原糖测定法定量。该方法可以在现场工作中轻松应用。该测定本身仅包含提取,水解和还原糖测定的三个步骤(图1)。它可以在几个小时内完成。检测限(LOD)为1.5μg/ ml。这三种酶只需要生产一次,可以储存多年。在生产和纯化后,人们有足够的材料来分析数千个样品。我们决定将质粒转化和重组酶生产部分纳入协议中,因为我们认为这些步骤可行,而生物技术经验较少的海洋实验室可以完成这些步骤。


图1.生产酶之后的示意图。简要概述三个主要步骤及其大致的持续时间。

关键字:藻类, β-葡聚糖, 硅藻, 糖生物学, 糖苷水解酶, 昆布多糖, 金藻昆布多糖, 昆布多糖酶, 海洋有机物质

材料和试剂

  1. 2.0 ml反应管(SARSTEDT,目录号:72.691)
  2. 无菌牙签
  3. 铝箔
  4. 5 ml HiTrap IMAC HP色谱柱(GE Healthcare,目录号:17-0920-05)
  5. 具有10-kDa截止值的聚醚砜超滤膜(Merck,目录号:PBGC04310)
  6. 5 ml HiTrap脱盐柱(GE Healthcare,目录号:29-0486-84)
  7. Whatman GF / F过滤器(GE Healthcare,目录号:1825-047),尺寸取决于使用的过滤设置

  8. 50 ml离心管(SARSTEDT,目录号:62.547.254)
  9. 半微量比色杯(品牌,目录号:759015)
  10. 培养皿(SARSTEDT,目录号:82.1473)
    备注:另一种Pierce蛋白浓缩剂PES,10K MWCO,5-20ml(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88527)(见注2)。
  11. BL21(DE3)胜任 E。 (新英格兰生物实验室,目录号:C2527I)
  12. SOC-培养基(通常封装在BL21(DE3)主管大肠杆菌产品中)
  13. FaGH17A质粒(Addgene,目录号:86462)(参见注释1)
  14. FbGH30质粒(Addgene,目录号:86463)(参见注释1)
  15. FbGH17A质粒(Addgene,目录号:100911)(见注1)
  16. IPTG(来自AppliChem的异丙基β-D-硫代吡喃半乳糖苷,目录号:A4773)
  17. 溶菌酶(来自鸡蛋清)(Sigma-Aldrich,目录号:L6876)
  18. 氯化钠(NaCl)(Carl Roth,目录号:3957.2)
  19. 胰蛋白胨(Sigma-Aldrich,目录号:95039)
  20. 酵母提取物(Sigma-Aldrich,目录号:09182)
  21. 琼脂(Sigma-Aldrich,目录号:A7002)
  22. 硫酸卡那霉素(Sigma-Aldrich,目录号:60615)
  23. 硫酸铵((NH 4)2 SO 4)(Sigma-Aldrich,目录号:A4418)
  24. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:9791)
  25. 磷酸二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:71642)
  26. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  27. 乳糖(Sigma-Aldrich,目录号:17814)
  28. 甘油(Sigma-Aldrich,目录号:G6279)
  29. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:208094)
  30. 蔗糖(Sigma-Aldrich,目录号:84100)
  31. 三(羟甲基)氨基甲烷(Tris)(Sigma-Aldrich,目录号:RDD008)
  32. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  33. Triton X-100(Sigma-Aldrich,目录号:X100)
  34. DNA酶I(来自牛胰腺)(Sigma-Aldrich,目录号:69182)
  35. 硫酸镍(II)六水合物(NiSO 4·6H 2 O)(Sigma-Aldrich,目录号:227676)
  36. 咪唑(Sigma-Aldrich,目录号:I5513)
  37. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  38. 3-(N-吗啉代)丙磺酸(MOPS)(Sigma-Aldrich,目录号:69947)
  39. 来自海带的海带酸(Sigma-Aldrich,目录号:L9634)
  40. 牛血清白蛋白(BSA)冻干(Sigma-Aldrich,目录号:A2153)
  41. 4-羟基苯甲酰肼(Sigma-Aldrich,目录号:H9882)
  42. HCl(37%)(Sigma-Aldrich,目录号:320331)
  43. 柠檬酸三钠二水合物(Sigma-Aldrich,目录号:W302600)
  44. 氢氧化钠(NaOH)(VWR,目录号:28244.295)
  45. 氯化钙(CaCl 2)
  46. LB-Kana-Agar-Plates(见食谱)
  47. LB-Kana-Medium(见食谱)
  48. 20倍NPS股票(见食谱)
  49. 50x 5052股票(见食谱)
  50. MgSO 4储备(见食谱)
  51. ZY-Medium(见食谱)
  52. ZYP-5052-Rich-Autoinduction-Medium(见食谱)
  53. 蔗糖库存(见食谱)
  54. 脱氧胆酸股票(见食谱)
  55. DNase I库存(见食谱)
  56. NiSO <4>股票(见食谱)
  57. IMAC缓冲区A(请参阅食谱)
  58. IMAC缓冲区B(请参阅食谱)
  59. SEC缓冲液(见食谱)
  60. SEC / DTT缓冲区(请参阅食谱)
  61. MOPS缓冲区(请参阅食谱)
  62. BSA储备溶液(见食谱)
  63. PAHBAH试剂A(见食谱)
  64. PAHBAH试剂B(见食谱)
  65. PAHBAH工作试剂(见食谱)

设备

  1. 本生燃烧器或无菌罩
  2. 2升锥形瓶(或2L普通玻璃瓶)

  3. 旋转摇瓶用于锥形瓶(推荐使用温度控制)
  4. 涡旋混合器
  5. 微量离心用于1.5或2.0ml具有冷却功能的反应管,即离心机5418R(Eppendorf,型号:5418R,目录号:5401000013)
  6. 离心机用于冷却功能为15和50毫升的离心管,即离心机5804 R(Eppendorf,型号:5804 R,目录号:5805000017)
  7. 标准膜或蠕动泵和相应的过滤装置ME1(Vacuubrand,型号:ME1)和Nalgene过滤器固定器接收器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:300-4100)
  8. 标准快速蛋白液相色谱系统(FPLC),即,ÄKTAstart(GE Healthcare,型号:ÄKTAstart,目录号:29-0220-94)
    注意:另一种蠕动泵的流速≤5 ml / min。

  9. 光谱仪,也就是说,BioSpectrometer basic(Eppendorf,型号:BioSpectrometer Basic,目录号:6135000009)。
  10. 加热水浴,即,Thermolab(GFL,目录号:1070)
  11. 两个加热块,即BioShake iQ(Analytik Jena,目录号:848-1808-0506)
  12. 高压灭菌器
    注意:强烈推荐高压灭菌器,但不是必需的,因为人们总是使用抗生素卡那霉素来促进细菌生长。
  13. 超滤搅拌室,即Amicon(Merck,目录号:UFSC05001)
    备注:另一种Pierce蛋白浓缩剂PES,10K MWCO,5-20ml(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88527)(见注2)。

程序

  1. 转型
    1. 将每个含有一种酶的Addgene质粒转化到单独的BL21(DE3)感受态E中。根据制造商的说明并使用LB-假名琼脂平板(参见食谱)来制备大肠杆菌。常见的转化方案采用以下步骤,必须在本生灯旁边或无菌罩内进行。
    2. 添加1微升含有 ca 。将100ng质粒DNA加入到细胞混合物中并小心倒转一次。不要漩涡。
    3. 保持冰上5分钟,不要混合。
    4. 正好在42°C的热冲击正好10秒,不混合。
    5. 保持冰上5分钟,不要混合。
    6. 添加500μlSOC-培养基,包含在 E中。大肠杆菌BL21(DE3)出货。

    7. 在37°C孵育60分钟和500转。
    8. 在LB-Kana琼脂平板上扩散100μl,37°C孵育至少15 h。
    9. 在用无菌牙签挑取菌落并在37℃下在3ml LB-Kana-Medium(参见食谱)预培养中生长至少8小时并在150rpm下持续搅拌的情况下,细胞可以储存在-80在添加25%(v / v)无菌甘油(em-ie,200μl甘油在600μl预培养物中)之后加入5%

  2. 酶的过表达和纯化
    1. 使用1ml来自步骤A9的预培养物在2L锥形瓶中接种1L ZYP-5052-Rich-Autoinduction-Medium(见配方)(Studier,2005)以具有足够的顶部空间。
      只需用铝箔封闭烧瓶,使气体能够进入烧瓶。
      注意:
      1. 对于所有三种酶(FbGH30,FaGH17A和FbGH17A),几乎以完全相同的方式进行过表达和纯化。但是,每种纯化都必须分开进行。将所有三种酶转化为一种表达系统并一起纯化是不可能的。尽管如此,在过表达之后,可能在一个工作日内纯化所有三种酶。
      2. 过表达也可以在IPTG诱导的LB-Kana培养基中进行。接种的培养物需要在37℃下生长8小时,然后加入1ml 1M IPTG。诱导后,培养物需要在16℃下再培养8小时。如已经描述的那样进行后续的收获和纯化。这种方法速度更快,但蛋白质含量更低。
    2. 在20°C [或室温(RT)]下培养培养物约72小时,并以150 rpm的转速培养。
    3. 通过在4,500℃下4,500g离心25分钟来收获细胞。弃上清。产生的颗粒可以在-20°C(多年)储存直至进一步使用。

    4. 在15毫升蔗糖原液中重悬细胞沉淀(请参阅食谱)。
    5. 加入5毫克的溶菌酶,并在室温下恒温磁力搅拌下以500转/分孵育10分钟。
    6. 加入30ml脱氧胆酸盐原液(参见食谱)和0.2ml MgSO 4储备溶液(参见食谱)。
    7. 加入1 ml DNase I储备溶液(参见食谱)以液化粘性溶液。
    8. 在4℃下将溶解产物在16,000×gg下离心45分钟。将含有感兴趣蛋白质的上清转移到新管中。
    9. 以下固定化金属亲和色谱(IMAC)期间的所有步骤在室温下以FPLC进行,流速为5ml /分钟。另外,该程序也可以通过蠕动泵以相同或更低的流量进行。
      注意:步骤B10-B14可以使用蠕动泵以5毫升/分钟的流速手动执行,而不是FPLC系统。然后将该柱放置在泵之后。步骤B14中的线性缓冲剂梯度可以类似地通过使用在5CV(〜5分钟)的总洗脱体积的过程中逐步增加咪唑浓度来实现。从含有50mM咪唑的IMAC缓冲液A开始,缓冲液需要每30秒更换一次。在每个步骤中,咪唑浓度增加50mM(各30秒):50,100,150,200,250,300,350,400,450,500mM咪唑(IMAC缓冲液A和B的通常浓度为25和500mM咪唑)。为防止色谱柱上出现气泡,每当进样口从一个缓冲液切换到另一个缓冲液时,需要暂停流量。
    10. 用1个柱体积(1 CV = 1 ml)的NiSO 4原液(见配方)加入5 ml HiTrap IMAC HP柱。放弃离开柱子的流量。
    11. 用5 CV的IMAC缓冲液A平衡(见食谱)。放弃离开柱子的流量。
    12. 将整个溶胞上清液注入柱子。如果溶液过于粘稠,则需要使用IMAC缓冲液A稀释至50 ml的体积。丢弃离开柱子的流量。
    13. 用15个CV IMAC缓冲液A清洗色谱柱。丢弃离开色谱柱的流量。
    14. 从IMAC缓冲液A到IMAC缓冲液B(参见食谱)以总数为5个CV的连续线性梯度洗脱蛋白质。
      收集三十毫升馏分中流出的色谱柱。
    15. 仅限FbGH17A:在洗脱后立即向每个级分添加50μl还原剂DTT(0.5 mg / ml)以防止蛋白质氧化。
    16. 泳池分数为6到30(如果有紫外检测器,只取对应主峰的样品)。保持蛋白质在冰上。
    17. 根据制造商的说明,每次净化后都必须清洁IMAC色谱柱。
    18. 与各自的10kDa膜一起组装超滤搅拌的细胞。 (或者可以使用离心浓缩器,见注2)。

    19. 在4°C或冰上浓缩超滤室中的蛋白质溶液至体积≤1.5 ml。
    20. 在13,000×gg和4℃下离心蛋白质溶液20分钟以除去沉淀的无活性蛋白质。
    21. 将上清转移到新鲜试管中并丢弃沉淀。保持蛋白质在冰上。
    22. 以下使用脱盐塔除去咪唑。该程序遵循制造商的说明并且可以通过使用FPLC系统或注射器在RT和5ml / min的流速下进行。
    23. 仅适用于FbGH17A:在步骤B24-26中使用SEC / DTT缓冲液(请参阅配方)而不是SEC缓冲液。
    24. 用5个CV缓冲液平衡HiTrap脱盐柱。放弃离开柱子的流量。
    25. 应用恰好1.5毫升的蛋白质溶液。必要时用SEC缓冲液稀释样品。不要超过1.5毫升的样本量。放弃离开柱子的流量。

    26. 用2毫升SEC缓冲液洗脱并收集蛋白质,然后将蛋白质保存在冰上。
    27. 根据制造商的说明,净化后的清洁柱必须经过净化。
    28. 通过使用分光计测量280nm处的吸光度并且考虑每种蛋白质的分子量和消光因子(FbGH30:MW = 54,700Da,ε= 128,480M -1 cm -1)来测定蛋白质浓度, -1; FaGH17A:MW = 44,800Da,ε= 87,905M -1 -1 cm -1; FbGH17A:MW = 46,600Da,ε= 88,935 M <-1> cm -1 ):

      其中,c:以mg / ml为单位的蛋白质浓度; A:在280nm处的吸光度; ε:在M -1 -1 cm -1 -1范围内的消光系数; MW:Da中的分子量; b:以cm为单位的光路长度。
      注意:可以验证过表达(步骤B1-B3),裂解(步骤B4-B8),IMAC(步骤B9-B17),浓度(步骤B18-B21)和脱盐(步骤B22-B27)的成功通过SDS-聚丙烯酰胺凝胶电泳(He,2011)。
    29. 将MOPS缓冲液中的蛋白质稀释(见配方)至10μM的储存浓度。保持蛋白质在冰上。
    30. 仅适用于FbGH17A:在MOPS缓冲液中稀释后,必须立即使用该酶来制备等分试样(步骤B31)或进行水解反应(步骤D1-D7)。
    31. 准备好即用的50μl等分试样,并在-20°C冷冻。
      注意:迄今为止,在-20°C没有关于酶的保存期限的非常长期的数据,但它们可以使用至少两年而没有任何活性降低。

  3. 采样和提取
    1. 通过使用标准膜或蠕动泵以及相应的过滤设置,将环境样品,即,海水或微藻的实验室培养物过滤到0.1-0.5巴的Whatman GF / F过滤器上。所需的体积需要通过实验确定。对于海水表面,至少需要5升,对于高浓度的实验室培养,需求量要小得多(根据细胞数量,约50毫升)。必须记录每个样品的体积。生成的过滤器可以保存在-20°C直到进一步使用。
      备注:
      1. 聚碳酸酯过滤器也可以使用。过滤器的直径取决于过滤设置。
      2. 对于浓缩的实验室培养物,离心也可以用来代替过滤。硅藻培养需要在4,500 x g下离心20分钟。
      3. 该方法也可应用于大型海藻样品(L.Scheschonk等准备中)。
    2. 将过滤器放入15或50毫升的离心管中。

    3. 添加5-10毫升MOPS缓冲液,足以让整个过滤器都被液体覆盖。
    4. 漩涡每个样本10秒。

    5. 在60°C的热水浴中提取60分钟
    6. 挤压并取出过滤器。

    7. 在4,500 em x em下离心15分钟
    8. 将上清转移到新管中。这种提取物可以在-20°C下储存数周,直至进一步使用。
      注:没有关于昆布多糖样品稳定性的长期数据。

  4. 水解
    1. 每个样品分成六个子样品:三个被酶混合物水解,三个不被水解。
    2. 此外,必须准备和处理(步骤C3和C5-C7)样品,以根据来自 Laminarin digitata (Sigma-Aldrich)的商业昆布多糖创建校准曲线。对于微藻样品,以下浓度是合适的:0,7.8μg/ ml,15.6μg/ ml,31.3μg/ ml,62.5μg/ ml,125μg/ ml,250μg/ ml,0.5mg / ml,1.0mg / ml和2.0mg / ml(在MOPS缓冲液中连续稀释)。

    3. 解冻每一种酶原液的新鲜等分试样并保存在冰上。
    4. 每种水解反应混合物包括:100μl样品提取物(或校准标准品),1μlFbGH30,1μlFaGH17A,1μlFbGH17A和1μlBSA储备溶液(见配方)(见注3)。
    5. 每种非水解反应混合物包括:100μl样品提取物,1μlBSA储备溶液和3μlMOPS缓冲液(参见注释11)。
    6. 通过摇动反应管混合所有样品。

    7. 在37°C孵育25分钟
    8. 通过在99℃在第二个加热块中温育样品5分钟停止酶反应。这种提取物可以在-20°C下储存数周,直至进一步使用。
      注:没有关于昆布多糖样品稳定性的长期数据。

  5. 还原糖测定

    1. 添加1 ml PAHBAH工作试剂(见配方)(Lever,1972)至0.1 ml样品。

    2. 每个样品在100°C加热5分钟 注意:沸腾的持续时间直接影响吸光度,因此会影响测量的一式三份的标准偏差。人们应该尝试以与用于加热的速度完全相同的速度将样品从加热块中取出。通过这样做,可以确保每个样品在相同的持续时间内被加热。 />
    3. 使用半微量比色杯和分光计测定410 nm处的吸光度。

    4. 使用PAHBAH工作试剂作为空白,并稀释太浓的样品。
      注意:可以进行可靠测量的吸光度范围取决于光谱仪,必须事先在手册中进行检查。样品可以用PAHBAH工作试剂稀释。

数据分析

  1. 每个样本在六个子样本中测量。未水解的一式三份的值需要从水解样品的值中减去。
  2. 通过将该差异与校准曲线进行比较可以确定各个样品中的昆布多糖浓度。
  3. 通过精确过滤体积或细胞数量,需要将提取物中测得的浓度转换为原始样品的浓度。
  4. 根据以下校准,20 L样品中昆布多糖浓度的方程和数据如下(图2,表1):&nbsp;



    图2.示例性校准

    表1.示例性数据

笔记

  1. 所有这三种质粒都可以通过Addgene质粒库( www.addgene.org )由学者和非营利组织订购。 。在网站上,如果Addgene只提供细菌刺,可以找到纯化质粒DNA的方案。质粒必须转化为E.E。大肠杆菌BL21(DE3)。
  2. 对于蛋白质浓度,可以选择使用基于离心的系统。这些管不可重复使用,回收率很低。但是,单次使用比超滤系统便宜。浓度必须根据制造商的说明在4°C下进行。
    蛋白质溶液的体积需要减少到≤1.5毫升。
  3. 准备包含必须添加到每个样品提取物中的所有组分的主混合物是可能的和可取的。

食谱

注意:除非另有说明,Milli-Q水用于组成以下溶液。

  1. LB-假名琼脂平板
    1. 解散:
      10克NaCl
      10克胰蛋白胨
      5克酵母提取物
      12.5克琼脂

      使用非无菌水完成体积至1L
    2. 高压灭菌器或煮解决方案
    3. 让溶液冷却至50°C以下后,加入1 ml制备好的硫酸卡那霉素(50 mg / ml)。
    4. 在搅拌溶液之后,需要将平板倒在本生灯旁边或无菌通风橱里
    5. 当盘子是坚实的,他们可以被存放在4°C几个星期
  2. LB-假名中等
    1. 解散:
      10克NaCl
      10克胰蛋白胨
      5克酵母提取物

      用2升锥形瓶或瓶子中的非无菌水将体积加至1L
    2. 用铝箔封闭烧瓶,但保持空气透过
    3. 建议高压灭菌解决方案
    4. 让溶液冷却至50°C以下后,加入1 ml制备好的硫酸卡那霉素(50 mg / ml)。

  3. 20x NPS股票 解散:
    66克(NH 4)2 SO 4←>
    136克KH 2 PO 4 4 142克Na 2 HPO 4 4/2
    使用非无菌水完成体积至1L 建议在使用前高压灭菌溶液
  4. 50x 5052股票
    解散:
    25克葡萄糖
    100克乳糖
    250克甘油(在烧杯中称量)并用非无菌水将体积加至1升
    建议在使用前高压灭菌溶液
  5. 硫酸镁4股票
    将12克MgSO 4溶解并用非无菌水将体积加至100毫升。
    建议在使用前高压灭菌溶液
  6. ZY-中等
    将10g胰蛋白胨和5g酵母提取物溶解在928ml H 2 O中 建议在使用前高压灭菌溶液
  7. ZYP-5052-Rich-Autoinduction-Medium

    混合928毫升ZY-中等 1毫升1M MgSO 4
    20毫升50x 5052
    50毫升20x NPS和2毫升硫酸卡那霉素(50毫克/毫升)旁边的本生燃烧器
  8. 蔗糖库存
    1. 溶解250克蔗糖和6克Tris
    2. 调整至pH 7.5,用未消毒的水将体积调整至1 L
    3. 建议在使用前高压灭菌溶液
  9. 脱氧胆酸股票
    1. 解散:
      10克脱氧胆酸钠
      10毫升Triton X-100
      5.8克NaCl
      2.4克Tris
    2. 调整至pH 7.5,用未消毒的水将体积调整至1 L
    3. 建议在使用前高压灭菌溶液
  10. DNase I股票
    将50 mg DNA酶I溶解在35 ml IMAC缓冲液A和15 ml甘油中
    该解决方案可以存储在-20°C
  11. NiSO <4>股票
    溶解3.9 g的NiSO 4并将体积完全溶解至50 ml
    注意:NiSO 4 是有害的。通过使用手套,眼睛和嘴巴保护自己。在适当的重金属废物中丢弃受污染的溶液。
  12. IMAC缓冲区A

    1. 溶解29.2克氯化钠,2.4克Tris和1.7克咪唑
    2. 调整至pH 7.5,用未消毒的水将体积调整至1 L
    3. 建议在使用前无菌过滤溶液
  13. IMAC缓冲区B

    1. 溶解29.2克氯化钠,2.4克Tris和34克咪唑
    2. 调整至pH 7.5,用未消毒的水将体积调整至1 L
    3. 建议在使用前无菌过滤溶液
  14. SEC缓冲液
    1. 溶解29.2克NaCl和2.4克Tris
    2. 调整至pH 7.5,用未消毒的水将体积调整至1 L
    3. 建议在使用前无菌过滤溶液
  15. SEC / DTT缓冲区

    溶解23毫克二硫苏糖醇在50毫升SEC缓冲液中
  16. MOPS缓冲液
    1. 溶解5.2克3-(N-吗啉代)丙磺酸
    2. 调整到pH值7.0,并完成体积到500毫升
    3. 建议在使用前无菌过滤溶液
    4. 使用棕色玻璃瓶并避光。
  17. BSA库存解决方案
    将100mg BSA溶解在1ml H 2 O中 建议在使用前无菌过滤溶液
  18. PAHBAH试剂A
    1. 将10g 4-羟基苯甲酰肼溶于60ml H 2 O中
    2. 加入10ml浓盐酸(37%),并通过加入更多的H 2 O 2完成体积至200ml。
    注意:该解决方案可以存储在室温下。如果溶液中含有沉淀物,请勿使用。
  19. PAHBAH试剂B
    1. 将24.9克柠檬酸三钠脱水物溶于500毫升H 2 O中
    2. 加入2.2克CaCl 2 2并混合
    3. 将40克NaOH溶解在单独的1升瓶中
    4. 缓慢搅拌两种溶液并不断搅拌,用无菌水将体积加至2L。
    5. 解决方案必须在几分钟内变清楚
    注意:解决方案可以存储在RT处。如果溶液中含有沉淀物,请勿使用。
  20. PAHBAH工作试剂
    准备PAHBAH试剂B和PAHBAH试剂A
    新鲜的9:1混合物 可以使用几个小时,但需要保存在冰上

致谢

该协议是在2017年由Becker等人修改和修改的。该研究得到了德意志联邦理工学院(授予HE 7217 / 1-1给Jan-Hendrik Hehemann)和马克斯普朗克协会的支持。
作者声明,该研究是在没有任何商业或财务关系的情况下进行的,可能会被视为潜在的利益冲突。

参考

  1. Becker,S.,Scheffel,A.,Polz,M.F。和Hehemann,J.H。(2017)。 利用海洋微生物酶精确定量海洋有机物中的昆布多糖 Appl Environ Microbiol 83(9)。
  2. Brunt,K.,Sanders,P.和Rozema,T。(1998)。 淀粉工业中食物,饲料和原料中淀粉的酶法测定。淀粉 - Stärke 50(10):413-419。 >
  3. 他,楼(2011年)。 Laemmli-SDS-PAGE 。 Bio-protocol Bio101:e80。 br />
  4. Lever,M.(1972)。 比色法测定碳水化合物的新反应。
  5. Myklestad,S.(1974)。 海洋浮游硅藻生产碳水化合物。 I.文化中九种不同物种的比较。 J Exp Mar Biol Ecol 15(3):261-74。
  6. Painter,T.J。(1983)。 海藻多糖中:多糖。 Elsevier 195-285。
  7. Percival,E.和Ross,A.(1951)。 156。海带多糖的构成。第二部分。海带昆布多糖的可溶性昆布多糖。 J Chem Soc(Resumed) 0:720-6。
  8. Studier,F. W.(2005)。 在高密度振荡培养中通过自动诱导产生蛋白质 Protein Expr Purif 41(1):207-234。
  9. Whitaker,J.R。(1974)。 酶的分析应用在:Whitaker,JR(版)。 Adv Chem Ser 136:31-78。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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