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Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803
集胞藻PCC 6803中聚-β-羟基丁酸的定量测定

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Abstract

Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in (Damrow et al., 2016).

Keywords: Cyanobacteria(蓝藻), Synechocystis(集胞藻属), PHB(PHB), Nitrogen deprivation(氮缺乏)

Background

Non-diazotrophic cyanobacteria such as Synechocystis sp. PCC 6803 respond to the lack of combined nitrogen sources by bleaching, a process known as chlorosis (Allen and Smith, 1969). This acclimation response is characterized by four major structural and morphological changes: (i) a massive accumulation of electron-dense glycogen inclusions (approx. 40 nm in diameter) between the thylakoid layers accompanied by (ii) the degradation of the phycobilisome antenna complexes, (iii) the disassembling of the thylakoid membrane layers including a reduction by number and packing density, and (iv) the formation of distinct electron-transparent PHB granules (approx. 400-500 nm in diameter) (Damrow et al., 2016). The physiological function of cyanobacterial PHB metabolism, synthesized just in a few species, is quite opaque due to the absence of both catabolic enzymes and evident phenotype of PHB-deficient mutants (Beck et al., 2012; van der Woude et al., 2014; Damrow et al., 2016; Namakoshi et al., 2016).
   Facing the world’s trash and global warming crisis, the demands for durable, recyclable, biodegradable, and synthetic-alternative plastics such as PHB is enormous and focus attention to cyanobacterial producers (Asada et al., 1999; Ansari and Fatma, 2016). Various different techniques are published for the analysis of PHB molecules (for updated review see [Godbole, 2016]). We are presenting a combination of hydrolytic degradation of PHB to 3-hydroxybutyrate (3-HB) in alkaline regime, and a coupled colorimetric enzymatic assay. Here the coupling with a phenazine methosulphate-p-iodonitrotetrazolium violet (PMS-INT) system directs the enzymatic redox reaction of both NADH oxidation and 3-HB reduction by the 3-hydroxybutyrate dehydrogenase (HBDH) and thus precludes an interfering backward reaction. This rapid spectrophotometric quantification of PHB just needs very simple lab equipment, is not much time-consuming, and is yet both reliable and reproducible.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml centrifuge tubes (Eppendorf® Safe-Lock microcentrifuge tubes) (Eppendorf, catalog number: 0030120086 )
  3. 15 ml centrifuge tubes (TubeSpin® Bioreactor 15) (TPP Techno Plastic Products, catalog number: 87015 )
  4. 50 ml centrifuge tubes (TubeSpin® Bioreactor 50) (TPP Techno Plastic Products, catalog number: 87050 )
  5. Synechocystis sp. PCC 6803 cells (Pasteur culture collection of cyanobacteria) (Institut Pasteur, catalog number: PCC 6803 )
  6. Crushed ice
  7. Bi-distilled water
  8. 0.5 N sodium hydroxide (NaOH) (Carl Roth, catalog number: 9356.1 )
  9. 1 N hydrochloric acid (HCl-ROTIPURAN® 37%) (Carl Roth, catalog number: X942.1 )
  10. 50 mM Tris-hydrochlorid, pH 8.5 (PUFFERAN® ≥ 99%) (Carl Roth, catalog number: 9090.3 )
  11. 20 mM β-Nicotinamide adenine dinucleotide (NADH/H+), reduced disodium salt (Sigma-Aldrich, catalog number: N9785 )
    Note: This product has been discontinued.
  12. 1 mM β-Nicotinamide adenine dinucleotide (NAD+), reduced disodium salt (Sigma-Aldrich, catalog number: N0632 )
  13. 5 mM phenazine methosulfate (PMS) (Sigma-Aldrich, catalog number: P9625 )
  14. 5 mM p-Iodonitrotetrazolium violet (INT) (Sigma-Aldrich, catalog number: I8377 )
  15. 1 mM (R)-3-hydroxybutyric (R-3-HB) (Sigma-Aldrich, catalog number: 54920 ) with a molar mass of 104.10 g/mol
  16. 15 U/ml 3-hydroxybutyrate dehydrogenase (3-HBDH) grade II from Rhodobacter spheroides (Roche Diagnostics, catalog number: 10127833001 )
  17. BG11 stock solution ‘+N’; autoclaved (use 1:100) (see Recipes)
    1. Sodium nitrate (NaNO3 ≥ 99%, p.a., ACS, ISO) (Carl Roth, catalog number: A136.1 )
    2. Calcium chloride dihydrate (CaCl2·2H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 5239.1 )
    3. Citric acid (C6H8O7 ≥ 99.5%, p.a., ACS, anhydrous) (Carl Roth, catalog number: X863.1 )
    4. Magnesium sulfate heptahydrate (MgSO4·7H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: P027.1 )
    5. Ethylenediamine tetraacetic acid disodium salt dehydrate (EDTA ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 8043.2 )
  18. BG110 stock solution ‘-N’; autoclaved (use 1:100) (see Recipes)
    1. Calcium chloride (CaCl2·2H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 5239.1 )
    2. Citric acid (C6H8O7 ≥ 99.5%, p.a., ACS, anhydrous) (Carl Roth, catalog number: X863.1 )
    3. Magnesium sulfate (MgSO4·7H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: P027.1 )
    4. Ethylenediamine tetraacetic acid disodium salt dehydrate (EDTA ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 8043.2 )
  19. Trace Metal Mix for BG11; sterile filtrated (use 1:1,000) (see Recipes)
    1. Boric acid (H3BO3) (≥ 99.8%, p.a., ACS, ISO) (Carl Roth, catalog number: 6943.2 )
    2. Manganese(II) chloride tetrahydrate (MnCl2·4H2O ≥ 99%, p.a.) (Carl Roth, catalog number: T881.3 )
    3. Zinc sulfate heptahydrate (ZnSO4·7H2O ≥ 97%, extra pure) (Carl Roth, catalog number: 7316.1 )
    4. Sodium molybdate dihydrate (Na2MoO4·2H2O), ≥ 99.5% (Sigma-Aldrich, catalog number: M1651 )
    5. Cupric(II) sulfate pentahydrate (CuSO4·5H2O ≥ 99%, Ph.Eur., BP) (Carl Roth, catalog number: P025.1 )
    6. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Sigma-Aldrich, catalog number: 203106 )
  20. Extra solutions for BG11; sterile filtrated (use 1:1,000) (see Recipes)
    1. Ammonium iron(III) citrate (Carl Roth, catalog number: 9366.1 )
    2. Potassium phosphate dibasic (K2HPO4 ≥ 98%, Ph.Eur., BP) (Carl Roth, catalog number: T875.1 )
    3. Sodium carbonate carbonate monohydrate (Na2CO3·H2O ≥ 99.5%, p.a., ACS) (Carl Roth, catalog number: 2597.2 )
  21. Buffer solution for BG11; autoclaved (use 1:200) (see Recipes)
    1 M HEPES buffer (pH 8.0) (PUFFERAN® ≥ 99.5%) (Carl Roth, catalog number: 9105.4 )
  22. BG11 (‘+N’) medium (see Recipes)
  23. BG110 (‘-N’) medium (see Recipes)

Equipment

  1. Wide-neck 500 ml Erlenmeyer flasks (Carl Roth, DURAN®, catalog number: C150.1 )
  2. Personal protective equipment (PPE)
    Note: These should be worn at all times when dealing with concentrated acids and alkali. See Note 3.
    1. Safety glasses (Sekuroka®-safety glasses EN166) (Carl Roth, catalog number: Y254.1 )
    2. Lab coat (Sekuroka®-lab coats) (Carl Roth, catalog number: T413.1 )
    3. Gloves (Rotiprotect®-nitrile evo) (Carl Roth, catalog number: CPX7.1 )
  3. Pipette set (Rainin Pipet-Lite LTS Starter Kit L-STARTXLS+) (Mettler-Toledo International, catalog number: 17014406 )
  4. Ice bucket
  5. Laminar flow bench (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II , catalog number: 51028225)
  6. Analytical balance (Mettler-Toledo International, model: XS105 )
  7. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM BiofugeTM Primo R , catalog number: 75005440)
  8. Light meter (LI-COR, model: LI-189 )
  9. Incubator (Infors, model: Multitron Pro , ‘Algae Special’)
  10. UV-Vis spectrophotometer (Analytic Jena, model: Specord® 50 PLUS )
  11. Bench pH/mV/°C meter (pH 1,000 L, pHenomenal®) (VWR, catalog number: 662-1422 )
  12. Thermomixer (Biometra, model: ThermoShaker TS1 , catalog number: 846-051-500)
  13. Vortex shaker (VWR, Peqlab, model: peqTWIST )
  14. Autoclave (Systec, model: Systec VX-150 )

Procedure

  1. Induction of PHB biosynthesis in Synechocystis sp. PCC 6803 upon nitrogen deprivation
    1. For nitrogen step down experiments as described in (Grundel et al., 2012; Damrow et al., 2016), an axenic pre-culture of Synechocystis sp. PCC 6803 (2 x 250 ml) grows in Erlenmeyer flasks to an OD750 of 0.5 at 28 °C under continuous illumination with white light of 80 µmol photons m-2 sec-1 in BG11 medium (see Recipes) containing 20 mM HEPES buffer (pH 8.0) and 17.6 mM sodium nitrate as the nitrogen source.
    2. For nitrogen deprivation, cells (500 ml) are pelleted by centrifugation at 4,000 x g for 10 min at room temperature and washed twice with 500 ml BG110 (see Recipes), which lacks sodium nitrate ‘-N’ (Stanier et al., 1971).
    3. The final cell suspension is split (2 x 250 ml) and both cultures ‘-N’ and ‘+N’ are supplemented with sodium acetate (final concentration 10 mM) as described by (Hein et al., 1998). Sodium nitrate is only added to the control ‘+N’ sample (final concentration 17.6 mM).
    4. The cultivation upon nitrogen deprivation are–likewise to the pre-culture cultivation–implemented in Erlenmeyer flasks at 28 °C under continuous shaking (80 rpm; Multitron Pro ‘Algae Special’) and continuous illumination using white light (80 µmol photons m-2 sec-1) for seven days.
  2. Quantification of dry weight
    1. 10 ml of each cell culture is pelleted by centrifugation (4,000 x g, 10 min), washed twice with 10 ml sterile water and finally concentrated up to 1 ml and transferred into a 1.5 ml reaction tube which has to be pre-weighted before.
    2. After another centrifugation step (10,000 x g, 3 min) the supernatant is entirely removed and the remaining cell pellet is dried completely at 80 °C in a thermomixer overnight (open cap).
    3. The dry weight of the cells (~5 mg) is determined by the weight of the ‘cell tubes’ subtracted from the dead weight of the tube (Table 1).

      Table 1. Determined dry weights


  3. Extraction and de-polymerization of PHB to (R)-3-hydroxybutyric acid (Figure 1) by alkaline hydrolysis.



    Figure 1. Hydrolytic degradation of poly-β-hydroxybutyrate (PHB) to (R)-3-hydroxybutyric acid (R-3-HB)

    1. 0.5 N NaOH (< 5 mg DW→300 µl 0.5 N NaOH; > 5 mg DW→400 µl 0.5 N NaOH) are added to the dried cell pellet from step 2 (‘+N’ and ‘-N’, separately) and incubated at 85 °C for 1 h by continuously shaking in a thermomixer and vigorously vortexing from time to time both to break the cells and to hydrolyze PHB into its monomer (R)-3-hydroxybutyrate (R-3-HB) as described by (Yu and Marchessault, 2000; Cui et al., 2006).
    2. After cooling on ice, the samples (‘+N’ and ‘-N’, separately) are neutralized by the addition of 1 N HCl in a ratio of four volumes 0.5 N NaOH (e.g., 300 µl) to one volume 1 N HCl (e.g., 100 µl). The samples (‘+N’ and ‘-N’, separately) are thoroughly mixed by vortexing.
    3. Afterwards the R-3-HB containing fraction (supernatant) of each sample (‘+N’ and ‘-N’, separately) is separated from the cell debris (pellet) by centrifugation (1 min, 4,500 x g, room temperature). The R-3-HB containing fraction (supernatant) is transferred to a new 1.5 ml reaction tube for each sample (‘+N’ and ‘-N’, separately).
    4. For further quantitative analysis, the R-3-HB containing samples (‘+N’ and ‘-N’, separately) are diluted in ratio of 1:10 dilution with bi-distilled water.
  4. Spectrophotometric quantification of R-3-HB monomers
    1. The R-3-HB content in each sample (‘+N’ and ‘-N’, separately) is finally spectrophotometrically quantified via an enzymatic assay. In a redox reaction, R-3-HB is oxidized to acetoacetate while NAD+ is reduced to NADH/H+ catalyzed in a 1:1 stoichiometry by 3-hydroxybutyrate dehydrogenase (HBDH) (Figure 2).


      Figure 2. Coupled enzymatic assay of R-3-HB oxidation by the 3-hydroxybutyrate dehydrogenase and simultaneous reoxidation of NADH/H+ to NAD+ by the PMS-INT system forming a stable color precipitate

    2. In order to prevent a backward reaction (reduction of acetoacetate to R-3-HB; oxidation of NADH/H+ to NAD+), the NADH/H+ synthesis is directly coupled to a phenazinemethosulphate-p-iodonitrotetrazolium violet (PMS-INT) colorimetric assay as described by (Lim and Buttery, 1977; Hinman and Blass, 1981). Here, the electron is transferred in a 1:1 stoichiometry from NADH/H+ via PMS to INT. The reduced form of INT is a stable formazan, which absorbs light at a wavelength of 500 nm (or 505 nm) (Lim and Buttery, 1977) (Figure 2).
    3. The level of R-3-HB is proportional both to the level of synthesized NADH/H+ and of formazan (Figure 2).
      100 µl of the diluted sample from step 3 or 100 µl 1 mM standard ((R)-3-hydroxybutyric) is added into a cuvette (1 ml assay volume) containing (triplicates for each sample):
      730 µl 50 mM Tris-HCl buffer, pH 8.5 (optimum pH for oxidation reaction)
      50 µl 1 mM NAD+
      10 µl 0.5 mM PMS
      100 µl 0.05 mM INT
    4. The reaction is monitored at 25 °C as the change (∆E = E2 - E1) in A500 (or 505 nm) on spectrophotometer before (E1) and ten min after (E2) initiation with 10 µl 15 U/ml HBDH according to the protocol as described in (Hinman and Blass, 1981) (Table 2). Tris-HCl buffer, pH 8.5 (50 mM) is used as a blank.

      Table 2. Absorption change at 500 nm (or 505 nm) for nitrogen-deprived samples


    5. The R-3-HB (and thus PHB) level is calculated via the calibration series of six different NADH/H+ concentrations ranging from 0, (0.1), 0.25, 0.5, 0.75, and 1.0 mM (triplicates for each concentration) in a PMS-INT formazan reaction. Tris-HCl buffer, pH 8.5 (50 mM) is used as a blank. Here, the ∆E (Table 3) is determined at 25 °C as the change in A500 (or 505 nm) on spectrophotometer before (E1) and three min after (E2) addition of 100 µl respective NADH/H+ standard solution to a cuvette containing (triplicates for each sample):
      730 µl 50 mM Tris-HCl buffer, pH 8.5
      60 µl bi-distilled water
      10 µl 0.5 mM PMS
      100 µl 0.05 mM INT

      Table 3. Absorption change at 500 nm (or 505 nm) for NADH calibration series

Data analysis

  1. NADH/H+ standard curve
    The calibration curve is plotted as a function of respective absorbance (∆E value) versus known concentration of NADH/H+ [mM]. The fitting curve and parameters are obtained by linear regression analysis. The linear regression coefficient should be in the range of 0.97 ≤ R2 ≤ 1 (Figure 3).


    Figure 3. NADH/H+ calibration curve

  2. R-3-HB standard control reaction
    Assuming a complete chemical conversion (1:1 stoichiometry), the respective absorbance of control reaction (0.1 mM R-3-HB) corresponds to 0.1 mM NADH/H+ (see NADH/H+ standard curve, Figure 3).
  3. PHB level quantification related to the dry weight
    The converted NADH/H+ and thus R-3-HB concentrations are calculated from the slope function of the linear regression analysis for each absorbance (∆E value). Including all dilution and concentration factors into the equations, the average R-3-HB amount of all three replicates is related to the determined dry weight (Table 4). Whereas in the control samples ‘+N’ no R-3-HB is detectable, the average R-3-HB (and thus PHB) content of the nitrogen-deprived cells of Synechocystis sp. PCC 6803 corresponds to 6-10% of the dry weight (Damrow et al., 2016).

    Table 4. Determination of PHB content per dry weight

Notes

  1. All cell passages and media are implemented under sterile conditions using a laminar flow bench and an autoclave. The intensity of photosynthetically active radiation (PAR) is measured in µE m-2 sec-1 (μmol photons per m2 and sec) using a LI-189 light meter.
  2. The dried cell pellets are stored at 4 °C for at least three weeks.
  3. For safety reasons, adjust to protective measures and tight-closing tubes when handling acids and bases. For safety reasons, assure that the hot samples are cooled down before HCl addition. The supernatant containing R-3-HB monomers could be stored at -20 °C for at least three weeks.
  4. The PMS-INT reaction is light-sensitive. The samples are handled in darkness.

Recipes

Note: Unless otherwise indicated, bi-distilled water was used as a solvent in solutions.

  1. BG11 stock solution ‘+N’; autoclaved (use 1:100)
    17.65 M NaNO3
    0.18 M CaCl2·2H2O
    0.0031 M citric acid
    0.304 M MgSO4·7H2O
    0.0034 M EDTA
  2. BG11 stock solution ‘-N’; autoclaved (use 1:100)
    0.18 M CaCl2·2H2O
    0.0031 M citric acid
    0.304 M MgSO4·7H2O
    0.0034 M EDTA
  3. Trace Metal Mix for BG11; sterile filtrated (use 1:1,000)
    1.43 g/L H3BO3
    0.09 g/L MnCl2·4H2O
    0.11 g/L ZnSO4·7H2O
    0.195 g/L Na2MoO4·2H2O
    0.0395 g/L CuSO4·5H2O
    0.0247 g/L Co(NO3)2·6H2O
  4. Extra solutions for BG11; sterile filtrated (use 1:1,000)
    23 mM ferric ammonium citrate
    172 mM K2HPO4
    188 mM Na2CO3·H2O
  5. Buffer solution for BG11; autoclaved (use 1:200)
    1 M HEPES buffer (pH 8.0)
  6. BG11 (‘+N’)
    10 ml 100x BG11 (+N) stock solution
    1 ml Trace Metal Mix
    1 ml 23 mM ferric ammonium citrate
    1 ml 172 mM potassium phosphate dibasic
    1 ml 188 mM sodium carbonate
    5 ml 1 M HEPES buffer (pH 8.0)
    Add up to 1 L with deionized water
  7. BG110 (‘-N’)
    10 ml 100x BG11 (-N) stock solution diminished by sodium nitrate
    1 ml Trace Metal Mix
    1 ml 23 mM ferric ammonium citrate
    1 ml 172 mM potassium phosphate dibasic
    1 ml 188 mM sodium carbonate
    5 ml 1 M HEPES buffer (pH 8.0)
    Add up to 1 L with deionized water

Acknowledgments

We particularly acknowledge Wolfgang Lockau for his commitment and valuable discussions on this topic and for his sustained support of our research. We gratefully thank Gisa Baumert (Humboldt-Universität zu Berlin, Germany) for her excellent technical assistance. We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (to YZ) in the framework of the Collaborative Research Center 1078 on ‘Protonation Dynamics in Protein Function’ (SFB1078, project A4/Holger Dau).

References

  1. Allen, M. M. and Smith, A. J. (1969). Nitrogen chlorosis in blue-green algae. Arch Mikrobiol 69(2): 114-120.
  2. Ansari, S. and Fatma, T. (2016). Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PLoS One 11(6): e0158168.
  3. Asada, Y., Miyake, M., Miyake, J., Kurane, R. and Tokiwa, Y. (1999). Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling. Int J Biol Macromol 25(1-3): 37-42.
  4. Beck, C., Knoop, H., Axmann, I. M. and Steuer, R. (2012). The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms. BMC Genomics 13: 56.
  5. Cui, Y., Barford, J. P. and Renneberg, R. (2006). Determination of poly(3-hydroxybutyrate) using a combination of enzyme-based biosensor and alkaline hydrolysis. Anal Sci 22(10): 1323-1326.
  6. Damrow, R., Maldener, I. and Zilliges, Y. (2016). The multiple functions of common microbial carbon polymers, glycogen and PHB, during stress responses in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803. Front Microbiol 7: 966.
  7. Godbole, S. (2016). Methods for identification, quantification and characterization of polyhydroxyalkanoates. Int J Bioassays 5(04).
  8. Grundel, M., Scheunemann, R., Lockau, W. and Zilliges, Y. (2012). Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 158(Pt 12): 3032-3043.
  9. Hein, S., Tran, H. and Steinbuchel, A. (1998). Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria. Arch Microbiol 170(3): 162-170.
  10. Hinman, L. M. and Blass, J. P. (1981). An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates. J Biol Chem 256(13): 6583-6586.
  11. Lim, H. H. and Buttery, J. E. (1977). Determination of ethanol in serum by an enzymatic PMS-INT colorimetric method. Clin Chim Acta 75(1): 9-12.
  12. Namakoshi, K., Nakajima, T., Yoshikawa, K., Toya, Y. and Shimizu, H. (2016). Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803. J Biotechnol 239: 13-19.
  13. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2): 171-205.
  14. van der Woude, A. D., Angermayr, S. A., Puthan Veetil, V., Osnato, A. and Hellingwerf, K. J. (2014). Carbon sink removal: Increased photosynthetic production of lactic acid by Synechocystis sp. PCC6803 in a glycogen storage mutant. J Biotechnol 184: 100-102.
  15. Yu, G-e. and Marchessault, R. H. (2000). Characterization of low molecular weight poly(β-hydroxybutyrate)s from alkaline and acid hydrolysis. Polymer 41(3): 1087-1098.

简介

蓝细菌合成各种化学上不同的高价值生物聚合物,如糖原(polyglucose),poly-β-β-羟基丁酸酯(PHB),蓝藻素(精氨酸和天冬氨酸的聚酰胺)和挥发物(多磷酸盐) 在超额条件下。 特别是在不平衡的C至N比下,糖原和一些蓝藻属中,PHB在一般氮应激反应进程中大量积累。 已经针对不同微生物来源的原位实验和/或从体外分析,建立了几种不同的技术。 在该协议中,描述了用于蓝藻(Stechocystis)的PHB定量的快速可靠的分光光度法。 如(Damrow等人,2016)中描述的氮缺乏的PCC 6803。
【背景】非重氮营养蓝细菌,例如集胞藻(Synechocystis) PCC 6803通过漂白来解决缺乏组合的氮源,这是一种被称为褪绿的过程(Allen和Smith,1969)。这种驯化反应的特征在于四个主要的结构和形态变化:(i)类囊体层之间的电子致密糖原夹杂物(直径约40nm)的大量积累伴随着(ii)藻糖酵母天线复合物的降解, (iii)类囊体膜层的拆卸,包括数量减少和包装密度,和(iv)形成不同的电子透明PHB颗粒(直径约400-500nm)(Damrow等人,2016)。由于不存在分解代谢酶和PHB缺陷型突变体的明显表型,所以在几种物种中合成的蓝细菌PHB代谢的生理功能是相当不透明的(Beck等人,2012; van ,2010; Damrow等人,2016; Namakoshi等人,2016)。
&NBSP;面对世界垃圾和全球变暖危机,对耐用,可回收,可生物降解和合成替代塑料(如PHB)的需求是巨大的,并将注意力集中在蓝藻生产商(Asada等人,1999年) ; Ansari和Fatma,2016)。公开了各种不同的技术来分析PHB分子(更新的评论参见[Godbole,2016])。我们在碱性条件下呈现了PHB与3-羟基丁酸酯(3-HB)的水解降解和偶联比色酶法的组合。在这里,与吩嗪甲基硫酸盐对碘硝基四唑紫(PMS-INT)系统的偶联引发了通过3-羟基丁酸脱氢酶(HBDH)的NADH氧化和3-HB还原的酶氧化还原反应,从而排除了干扰反向反应。 PHB的这种快速分光光度定量只需要非常简单的实验室设备,并不耗费时间,而且既可靠又可重复。

关键字:蓝藻, 集胞藻属, PHB, 氮缺乏

材料和试剂

  1. 移液器提示
  2. 1.5ml离心管(Eppendorf Safe-Lock微量离心管)(Eppendorf,目录号:0030120086)
  3. 15ml离心管(TubeSpin Bioreactor 15)(TPP Techno Plastic Products,目录号:87015)
  4. 50 ml离心管(TubeSpin ®/ > Bioreactor 50)(TPP Techno Plastic Products,目录号:87050)
  5. 集胞藻 sp。 PCC 6803细胞(巴斯德文化收集蓝细菌)(Institut Pasteur,目录号:PCC 6803)
  6. 碎冰
  7. 双蒸水
  8. 0.5N氢氧化钠(NaOH)(Carl Roth,目录号:9356.1)
  9. 1N盐酸(HCl-ROTIPURAN 37%)(Carl Roth,目录号:X942.1)
  10. 50mM Tris-盐酸盐,pH8.5(PUFFERAN≥99%)(Carl Roth,目录号:9090.3)
  11. 20mMβ-烟酰胺腺嘌呤二核苷酸(NADH / H + ),还原的二钠盐(Sigma-Aldrich,目录号:N9785)
    注意:本产品已停产。
  12. 1mMβ-烟酰胺腺嘌呤二核苷酸(NADH +),还原的二钠盐(Sigma-Aldrich,目录号:N0632)
  13. 5mM吩嗪甲硫酸盐(PMS)(Sigma-Aldrich,目录号:P9625)
  14. 5mM哌嗪 - 四氢呋喃(INT)(Sigma-Aldrich,目录号:I8377)
  15. 摩尔质量为104.10g / mol的1mM(R,R)-3-羟基丁酸(R-3-HB)(Sigma-Aldrich,目录号:54920)
  16. 15U / ml来自球状红细菌的3-羟基丁酸脱氢酶(3-HBDH)II级(Roche Diagnostics,目录号:10127833001)
  17. BG11储备液'+ N';高压灭菌(使用1:100)(见配方)
    1. 硝酸钠(NaNO 3 <99%,p.a.,ACS,ISO)(Carl Roth,目录号:A136.1)
    2. 氯化钙二水合物(CaCl 2·2H 2 O <99%,p.a.,ACS)(Carl Roth,目录号:5239.1)
    3. 柠檬酸(C 6 H 8 O 7≥99.5%,pa,ACS,无水)(Carl Roth,目录号:X863.1 )
    4. 硫酸镁七水合物(MgSO 4·7H 2 O≥99%,p.a.,ACS)(Carl Roth,目录号:P027.1)
    5. 乙二胺四乙酸二钠盐脱水(EDTA≥99%,p.a.,ACS)(Carl Roth,目录号:8043.2)
  18. BG11 <0>储备液'-N';高压灭菌(使用1:100)(见配方)
    1. 氯化钙(CaCl 2·2H 2 O <99%,p.a.,ACS)(Carl Roth,目录号:5239.1)
    2. 柠檬酸(C 6 H 8 O 7≥99.5%,pa,ACS,无水)(Carl Roth,目录号:X863.1 )
    3. 硫酸镁(MgSO 4·7H 2 O≥99%,p.a.,ACS)(Carl Roth,目录号:P027.1)
    4. 乙二胺四乙酸二钠盐脱水(EDTA≥99%,p.a.,ACS)(Carl Roth,目录号:8043.2)
  19. 用于BG11的痕量金属混合物无菌过滤(使用1:1,000)(见配方)
    1. 硼酸(H 3 3 BO 3)(≥99.8%,p.a.,ACS,ISO)(Carl Roth,目录号:6943.2)
    2. (II)氯化四水合物(MnCl 2·4H 2 O≥99%,p.a.)(Carl Roth,目录号:T881.3)
    3. 硫酸锌七水合物(ZnSO 4·7H 2 O≥97%,特别纯)(Carl Roth,目录号:7316.1)
    4. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O),≥99.5%(Sigma-Aldrich,目录号:M1651)
    5. 硫酸铜(II)五水合物(CuSO 4·5H 2 O≥99%,Ph.Eur。,BP)(Carl Roth,目录号:P025.1) br />
    6. 硝酸钴(II)六水合物(Co(NO 3 3)2·6H 2 O)(Sigma-Aldrich,目录号:203106) br />
  20. BG11额外解决方案;无菌过滤(使用1:1,000)(见配方)
    1. 柠檬酸铵(III)(Carl Roth,目录号:9366.1)
    2. 磷酸氢二钾(K 2 O 3 HPO 4%≥98%,Ph.Eur。,BP)(Carl Roth,目录号:T875.1)
    3. 碳酸钙一水合物(Na 2 CO 3·H 2 O≥99.5%,pa,ACS)(Carl Roth,目录号:2597.2 )
  21. BG11缓冲溶液;高压灭菌(使用1:200)(参见食谱)
    1 M HEPES缓冲液(pH 8.0)(PUFFERAN ≥99.5%)(Carl Roth,目录号:9105.4)
  22. BG11('+ N')介质(参见食谱)
  23. BG11 <0>('-N')介质(参见食谱)

设备

  1. 宽颈500ml锥形瓶(Carl Roth,DURAN ,目录号:C150.1)
  2. 个人防护装备(PPE)
    注意:处理浓缩酸和碱时,应始终佩戴这些。请参阅注3。
    1. 安全眼镜(Sekuroka ®安全眼镜EN166)(Carl Roth,目录号:Y254.1)
    2. 实验室外套(Sekuroka ® -lab大衣)(Carl Roth,目录号:T413.1)
    3. 手套(Rotiprotect ® -nitrile evo)(Carl Roth,目录号:CPX7.1)
  3. 移液器套件(Rainin Pipet-Lite LTS入门套件L-STARTXLS +)(Mettler-Toledo International,目录号:17014406)
  4. 冰桶
  5. 层流流量台(Thermo Fisher Scientific,Thermo Scientific TM,型号:MSC-Advantage TM II类,目录号:51028225)
  6. 分析天平(Mettler-Toledo International,型号:XS105)
  7. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Biofuge TM Primo R,目录号:75005440)
  8. 光度计(LI-COR,型号:LI-189)
  9. 孵化器(Infors,型号:Multitron Pro,'Algae Special')
  10. UV-Vis分光光度计(分析耶拿,型号:Specord ® 50 PLUS)
  11. 台式pH / mV /℃表(pH 1,000L,pHenomenal )(VWR,目录号:662-1422)
  12. Thermomixer(Biometra,型号:ThermoShaker TS1,目录号:846-051-500)
  13. 涡旋振动筛(VWR,Peqlab,型号:peqTWIST)
  14. 高压灭菌器(Systec,型号:Systec VX-150)

程序

  1. 诱导集落藻的PHB生物合成PCC 6803脱氮
    1. 对于如(Grundel等人,2012; Damrow等人,2016)中所述的氮降压实验,将集胞藻的无菌预培养物 sp。 PCC 6803(2×250ml)在锥形烧瓶中在28℃下连续照射80微摩尔光子的光 秒,在OD 750在含有20mM HEPES缓冲液(pH8.0)和17.6mM硝酸钠作为氮源的BG11培养基(参见食谱)中 -1 。
    2. 对于氮缺乏,通过在室温下以4,000xg离心10分钟使细胞(500ml)沉淀,并用500ml BG11.0(参见食谱)洗涤两次,其中缺乏硝酸钠“-N”(Stanier等人,1971)。
    3. 将最终的细胞悬浮液分开(2×250ml),并且如(Hein等人)所述,用乙酸钠(终浓度为10mM)补充两种培养物的“N”和“+ N” ,1998)。硝酸钠仅加入对照'+ N'样品(终浓度17.6mM)。
    4. 在氮缺乏条件下的培养同样适用于在锥形培养瓶中,在28℃,连续振荡(80rpm; Multitron Pro'Algae Special')下进行的培养前培养,并使用白光(80μmol光子m -2 sec -1 )七天。
  2. 干重定量
    1. 通过离心(4,000 xg,10分钟)将10ml细胞培养物沉淀,用10ml无菌水洗涤两次,最后浓缩至1ml,并转移到1.5ml反应管中,该反应管必须预先加权。
    2. 在另一个离心步骤(10,000×g,3分钟)后,将上清液完全除去,剩余的细胞沉淀物在80℃下在温热混合器中完全干燥过夜(开盖)。
    3. 细胞的干重(〜5mg)由管子的体重减去的“细胞管”的重量确定(表1)。

      表1.确定的干重


  3. 通过碱性水解将PHB萃取和去聚合((R))-3-羟基丁酸(图1)。



    图1.聚 - β-羟基丁酸酯(PHB)的水解降解( R-3-HB)
    1. 将0.5N NaOH(<5mg DW→300μl0.5N NaOH;> 5mg DW→400μl0.5N NaOH)加入到步骤2('+ N'和'-N'的干燥的细胞沉淀中,分别),并在85℃下在恒温混合器中连续摇动孵育1小时,并随时剧烈涡旋以破坏细胞并将PHB水解成其单体(R)-3-羟基丁酸酯( (Yu和Marchessault,2000; Cui等人,2006)所述的R-3-HB)。
    2. 在冰上冷却后,样品('+ N'和'-N'分别)通过加入1N HCl以四体积0.5N NaOH(例如,300μl )至一体积1N HCl(例如,100μl)。样品('+ N'和'-N'分别)通过涡旋充分混合。
    3. 然后通过离心(1分钟,4500分钟)从每个样品('+ N'和'-N'分开地)含有R-3-HB的级分(上清液)分离出细胞碎片,室温)。将含有R-3-HB的级分(上清液)转移到每个样品的新的1.5ml反应管(分别为'+ N'和'-N')。
    4. 对于进一步的定量分析,将含有R-3-HB的样品('+ N'和'-N'分别)以1:10稀释的比例用双蒸水稀释。
  4. R-3-HB单体的分光光度定量
    1. 每个样品中的R-3-HB含量('+ N'和'-N'分别)最终通过酶测定法进行分光光度法定量。在氧化还原反应中,R 3-HB被氧化成乙酰乙酸酯,而NAD + 被还原成NADH / H +催化的3-羟基丁酸脱氢酶(HBDH)(图2)

      图2.通过3-羟基丁酸脱氢酶的R-3-HB氧化的偶联酶测定和NADH / H的同时再氧化 >到达 由PMS-INT系统形成稳定的颜色沉淀 >
    2. 为了防止反应(乙酰乙酸酯还原为R-3-HB; NADH / H + 氧化成NAD ),NADH / H 如(Lim and Buttery,1977; Hinman and Blass,1981)所述,合成直接与吩嗪亚硫酸氢盐 - 对碘硝基四唑紫(PMS-INT)比色测定法结合。这里,电子以1:1的化学计量从NADH / H + 通过PMS转移到INT。 INT的还原形式是稳定的甲an,其吸收波长为500nm(或505nm)的光(Lim和Buttery,1977)(图2)。
    3. R-3-HB的水平与合成的NADH / H + 和甲the的水平成正比(图2)。
      将100μl来自步骤3的稀释样品或100μl1mM标准((R)-3-羟基丁酸)加入到含有(每个样品一式三份)的比色皿(1ml测定体积)中:
      730μl50 mM Tris-HCl缓冲液,pH 8.5(氧化反应的最适pH)
      50μl1mM NAD +
      10μl0.5 mM PMS
      100μl0.05 mM INT
    4. 在分光光度计之前(E1 )和10分钟后,在25℃下将反应监测为A500(或505nm)中的变化(ΔE= E2-E1 ) (Hinman和Blass,1981)中描述的方案(表2),用10μl15U / ml HBDH引发(E2)启动。使用Tris-HCl缓冲液,pH8.5(50mM)作为空白
      表2.缺氮样品的500nm(或505nm)吸收变化


    5. 通过六个不同的NADH / H + 浓度范围为0,(0.1),0.25,0.5,0.75和1.0mM的校正系列计算R-3-HB(和因此PHB)水平(每种浓度一式三份)在PMS-INT形式反应中。使用Tris-HCl缓冲液,pH8.5(50mM)作为空白。这里,ΔE(表3)在25℃下作为在分光光度计之前(E1 )和3分钟后((E1))的A500(或505nm) E2)将100μl各自的NADH / H +标准溶液加入到含有(每个样品一式三份)的比色皿中:
      730μl50mM Tris-HCl缓冲液,pH8.5
      60μl双蒸水
      10μl0.5 mM PMS
      100μl0.05 mM INT

      表3. NADH校准系列的500nm(或505nm)吸收变化

数据分析

  1. NADH / H + 标准曲线
    将校准曲线作为各自吸光度(ΔE值)与已知浓度的NADH / H + [mM]的函数作图。拟合曲线和参数通过线性回归分析得到。线性回归系数应在0.97≤R 2 <1> <1>的范围内(图3)。 br />

    图3. NADH / H + 校准曲线

  2. R-3-HB标准对照反应
    假设完全化学转化(1:1化学计量),对照反应(0.1mM R-3-HB)的相应吸光度对应于0.1mM NADH / H +(参见NADH / H + 标准曲线,图3)。
  3. PHB水平量化与干重相关
    从每个吸光度的线性回归分析的斜率函数(ΔE值)计算转化的NADH / H + ,从而计算R-3-HB浓度。将所有稀释度和浓度因子包括在方程式中,所有三个重复的平均R-3-HB量与确定的干重相关(表4)。而在对照样品中,+ N'不能检测到R-3-HB,所以集胞藻的缺氮细胞的平均R-3-HB(因此PHB)含量。 PCC 6803对应于干重的6-10%(Damrow等人,2016)。

    表4.每干重PHB含量的测定

笔记

  1. 所有细胞通道和培养基在无菌条件下使用层流台和高压釜进行。光合有效辐射(PAR)的强度以μEm -2 sec -1 (μmol光子/ m 2 /秒和秒)测量,使用一个LI-189光度计。
  2. 干燥的细胞沉淀物在4℃下储存至少三周。
  3. 出于安全考虑,在处理酸碱时应调整保护措施和紧闭管。为安全起见,请确保在添加HCl之前将热样冷却下来。含有R-3-HB单体的上清液可以在-20°C储存至少三周
  4. PMS-INT反应是光敏感的。样品在黑暗中处理。

食谱

注意:除非另有说明,否则使用双蒸水作为溶液中的溶剂

  1. BG11储备液'+ N';高压灭菌(使用1:100)
    17.65 M NaNO 3
    0.18M CaCl 2·2H 2 O
    0.0031 M柠檬酸
    0.304M MgSO 4·7H 2 O
    0.0034 M EDTA
  2. BG11储备液'-N';高压灭菌(使用1:100)
    0.18M CaCl 2·2H 2 O
    0.0031 M柠檬酸
    0.304M MgSO 4·7H 2 O
    0.0034 M EDTA
  3. 用于BG11的痕量金属混合物无菌过滤(使用1:1,000)
    3.43g / L H 3/3 3/3
    0.09g / L MnCl 2·4H 2 O
    0.11g / L ZnSO 4·7H 2 O
    0.195g / L Na 2 MoO 4·2H 2 O
    0.0395g / L CuSO 4·5H 2 O
    0.0247g / L Co(NO 3 3)2·6H 2 O
  4. BG11额外解决方案;无菌过滤(使用1:1,000)
    23 mM柠檬酸铁铵
    172mM K 2 HPO 4
    188mM Na 2 CO 3 / H 2 O 2 / /
  5. BG11缓冲溶液;高压灭菌(使用1:200)
    1 M HEPES缓冲液(pH 8.0)
  6. BG11('+ N')
    10 ml 100x BG11(+ N)储备溶液
    1毫升痕量金属混合物
    1毫升23毫克柠檬酸铵柠檬酸铵 1毫升172毫克磷酸氢钾二价 1ml 188mM碳酸钠
    5 ml 1 M HEPES缓冲液(pH 8.0)
    用去离子水加至1升
  7. BG11 0 ('-N')
    10 ml 100x BG11(-N)储备溶液减少了硝酸钠 1毫升痕量金属混合物
    1毫升23毫克柠檬酸铵柠檬酸铵 1毫升172毫克磷酸氢钾二价 1ml 188mM碳酸钠
    5 ml 1 M HEPES缓冲液(pH 8.0)
    用去离子水加至1升

致谢

我们特别感谢沃尔夫冈·洛克对他的承诺和宝贵的讨论,并持续支持我们的研究。我们非常感谢Gisa Baumert(德国柏林洪堡大学)的出色技术援助。我们感谢Deutsche Forschungsgemeinschaft(DFG)在“关于蛋白质功能的质子化动力学”(SFB1078,A4 / Holger Dau)的协作研究中心1078框架下的财政支持(YZ)。

参考

  1. Allen,MM和Smith,AJ(1969)。&nbsp; 氮蓝绿色藻类中的绿脓病。 Arch Mikrobiol 69(2):114-120。
  2. Ansari,S.和Fatma,T.(2016)。蓝藻多羟基丁酸酯(PHB):筛选,优化和表征。 PLoS One 11(6):e0158168。
  3. Asada,Y.,Miyake,M.,Miyake,J.,Kurane,R。和Tokiwa,Y。(1999)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/10416648“target =”_ blank“>通过蓝细菌的聚(羟基丁酸酯)的光合积累 - CO 2回收的代谢和潜力 Int J Biol Macromol 25(1-3):37-42。
  4. Beck,C.,Knoop,H.,Axmann,IM and Steuer,R。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 22300633“target =”_ blank“>蓝藻代谢的多样性:多种光营养微生物的基因组分析。 BMC Genomics 13:56.
  5. 崔,Y.,Barford,JP和Renneberg,R。(2006)。使用基于酶的生物传感器和碱性水解的组合测定聚(3-羟基丁酸酯)。 22(10):1323-1326。 >
  6. Damrow,R.,Maldener,I。和Zilliges,Y。(2016)。&nbsp; 常用微生物碳聚合物,糖原和PHB的多重功能在非重氮营养蓝藻中的应答反应中。 PCC 6803。前微生物 7:966.
  7. Godbole,S.(2016)。&nbsp; 多羟基链烷酸酯的鉴定,定量和表征方法。 Int J Bioassays&nbsp; 5(04)。
  8. Grundel,M.,Scheunemann,R.,Lockau,W.和Zilliges,Y。(2012)。受损的糖原合成引起代谢溢出反应并影响蓝藻中的应激反应。 PCC 6803。微生物学158(Pt 12):3032-3043。
  9. Hein,S.,Tran,H。和Steinbuchel,A.(1998)。 Synechocystis sp。 PCC6803具有与无氧紫色硫细菌类似的双组分聚羟基链烷酸合成酶。 180微量元素170(3):162-170。
  10. Hinman,LM和Blass,JP(1981)。&nbsp; An NADH连接的分光光度法测定粗制组织匀浆中的丙酮酸脱氢酶复合物。生物化学256(13):6583-6586。
  11. Lim,HH和Buttery,JE(1977)。&nbsp; 确定通过酶促PMS-INT比色法测定血清中的乙醇。 75(1):9-12。
  12. Namakoshi,K.,Nakajima,T.,Yoshikawa,K.,Toya,Y.和Shimizu,H。(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/27693092“target =”_ blank“> glgC和phaCE的组合缺失增强了集胞藻中的乙醇生产 PCC 6803。 J Biotechnol 239:13-19。
  13. Stanier,RY,Kunisawa,R.,Mandel,M.和Cohen-Bazire,G.(1971)。单细胞蓝绿色藻类的纯化和性质(顺序为Chroococcales )。细菌感染 v 35(2 ):171-205。
  14. van der Woude,AD,Angermayr,SA,Puthan Veetil,V.,Osnato,A.and Hellingwerf,KJ(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/24858679“target =”_ blank“>碳水槽去除:通过集胞藻增加乳酸的光合产生。 PCC6803在糖原储存突变体中。生物技术184:100-102。
  15. 俞,G-e。和Marchessault,R.H。(2000)。 从碱性表征低分子量聚(β-羟基丁酸酯)和酸水解。聚合物 41(3):1087-1098。
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引用:Zilliges, Y. and Damrow, R. (2017). Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803. Bio-protocol 7(14): e2402. DOI: 10.21769/BioProtoc.2402.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。