Enzymatic Activity Assay for Invertase in Synechocystis Cells

Materials and Reagents

1. Pipette tips
   
2. 2 ml tubes (Cypress, China)
   
3. 10 ml conical tubes (Kangjian, China)
   
4. 1 ml syringe (Jianshi, China)
   
5. Syringe membrane filters, 0.22 μm (Jinteng, China)
   
6. Synechocystis sp. PCC 6803 (Tan et al., 2011)
   
7. Milli-Q water (Millipore, Germany)
   
8. Glass beads (Sigma-Aldrich, catalog number: G9018-250G )
   
9. Fructose standard (Sinopharm Chemical Reagent, catalog number: 63003034 )
   
10. Glucose standard (Sinopharm Chemical Reagent, catalog number: 10010518 )
    
11. Liquid nitrogen
    
12. 200 mM NaOH (prepared with Milli-Q water)
    
13. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
    
14. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sinopharm Chemical Reagent, catalog number: 10013018 )
    
15. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sinopharm Chemical Reagent, catalog number: 20011160 )
    
16. Critic acid monohydrate (C₆H₈O₇·H₂O) (Sinopharm Chemical Reagent, catalog number: 10007118 )
    
17. Ferric ammonium citrate ((NH₄)₃FeC₁₂H₁₀O₁₄/C₆H₈O₇·xFe₃·yNH₃) (Sinopharm Chemical Reagent, catalog number: 30011428 )
    
18. EDTA·2Na·2H₂O (Sinopharm Chemical Reagent, catalog number: 10009717 )
    
19. Sodium carbonate (Na₂CO₃) (Sinopharm Chemical Reagent, catalog number: 10019260 )
    
20. Boric acid (H₃BO₃) (Sinopharm Chemical Reagent, catalog number: 10004818 )
    
21. Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sinopharm Chemical Reagent, catalog number: 20026118 )
    
22. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sinopharm Chemical Reagent, catalog number: 10024018 )
    
23. Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sinopharm Chemical Reagent, catalog number: 10019818 )
    
24. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Sinopharm Chemical Reagent, catalog number: 10008218 )
    
25. Cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) (Sinopharm Chemical Reagent, catalog number: 10007216 )
    
26. Sodium nitrate (NaNO₃) (Sinopharm Chemical Reagent, catalog number: 10019918 )
    
27. Potassium dihydrogen phosphate (KH₂PO₄) (Sinopharm Chemical Reagent, catalog number: 10017618 )
    
28. Dipotassium hydrogen phosphate trihydrate (K₂HPO₄∙3H₂O) (Sinopharm Chemical Reagent, catalog number: 10017518 )
    
29. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
    
30. Isopropanol (Sinopharm Chemical Reagent, catalog number: 40064360 )
    
31. BG11 medium (see Recipes)
    
32. 100 mM potassium phosphate buffer (pH 7.0) (see Recipes)
    
33. 100 mM PMSF (see Recipes)
    

Equipment

1. 50 ml flasks
   
2. Pipettes (Eppendorf, Germany)
   
3. Shaker (Taicang Huamei, model: THZ-701B )
   
4. Water bath (Shanghai Yarong, model: B-260 )
   
5. Centrifuge (Beckman Coulter, model: Microfuge^® 22R )
   
6. -20 °C freezer (Haier, model: BCD-219D )
   
7. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
   
8. Thermal Cycler for PCR (Bio-Rad Laboratories, model: T-100 )
   
9. Ion chromatography (Thermo Fisher Scientific, Thermo Scientific^TM, model: Dionex^TM ICS-5000⁺ )
   
10. Dionex^TM CarboPac^TM PA10 analytical column (4 x 250 mm, Thermo Fisher Scientific, model: Dionex^TM CarboPac^TM PA10 )
    

Software

1. Chromeleon^TM software (Thermo Fisher Scientific, Thermo Scientific^TM, version 6.80; catalog number: CHROMELEON6)

Procedure

1. Cultivation of Synechocystis sp. PCC 6803 (Tan et al., 2011)
   Note: Monitor the growth of cyanobacterial cells by measuring the optical density (OD) at 730 nm with a spectrophotometer. Culture volume should be less than 30 ml in 50 ml flasks.
   1. Grow Synechocystis cells in 20 ml liquid BG-11 media (see Recipe 1) in 50 ml flasks with an initial OD₇₃₀ of 0.05.
      
   2. Cultivate the Synechocystis cultures at 30 °C with shaking (150 rpm) and constant white light illumination (50 μE/m²/sec) for ~4 days to reach the exponential growth phase.
      
2. Preparation of cell crude extracts from Synechocystis cells
   1. Collect 8 ml of the exponential phase culture (OD₇₃₀ ≈ 2) of Synechocystis by centrifugation at 8,000 x g for 10 min at room temperature.
      
   2. Resuspend Synechocystis cells in 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) in a 2 ml tube.
      
   3. Add 1 g of glass beads (150-212 μm; Sigma-Aldrich) into the 2 ml tube containing cell suspensions and 100 mM Phenylmethanesulfonyl fluoride (PMSF) to reach a final concentration of 1 mM. Then, vortex the tube at the highest speed for 1 min. For each 10 sec vortexing, cool the tube on ice for 5 sec.
      
   4. After centrifugation at 12,000 x g, 4 °C for 30 min, transfer the supernatant into a new tube and store on ice before use.
      Note: The cell crude extracts should be used immediately.
      
3. Detection of the in vitro invertase activity
   1. For 50 µl in vitro invertase reactions, mix potassium phosphate buffer (pH 7.0), sucrose and crude extracts together (The final concentrations of potassium phosphate and sucrose are 100 mM and 2 g/L respectively. Whereas, the protein concentrations of crude extracts are 2.5~4.0 g/L.) Use the reaction mixture without the cell crude extract as a negative control.
      
   2. Incubate the mixtures at 30 °C in a thermal cycler (T100, Bio-Rad, USA) for 1.5 h. Then, stop the reaction by three freeze-thaw cycles. In each freeze-thaw cycle, freeze the reaction mixtures in liquid nitrogen and next thaw by incubation in a 65 °C water bath. Measure the amount of the resulting glucose and fructose as well as the rest of the sucrose substrate using ion chromatography (Figure 1).
      
   3. Spin down the reaction mixtures at 12,000 x g for 10 sec. Then, subject 25 μl of the diluted mixtures to ICS-5000⁺ ion-exchange chromatography system equipped with an electrochemical detector and a Dionex^TM CarboPac^TM PA10 analytical column (4 x 250 mm, ThermoFisher, Waltham, MA, USA). Elute the column with 200 mM NaOH at a flow rate of 1.0 ml/min.
      Note: The reaction mixtures should be diluted to at least 50 folds before analyzed by ion chromatography.
      
   4. Prepare a series of glucose standard solutions (0.01, 0.05, 0.1, 0.5, 1, 2 mg/L), establish the standard curve by using ion chromatography (see below).
      
4. Quantification of glucose resulted from the sucrose degradation catalyzed by invertase
   1. Retention times of glucose and fructose using the method described above are 3.5 and 3.8 min respectively (Figure 1).
      
   2. Establish the glucose standard curve by using the peak area calculated by the Chromeleon software (Figure 2).
      
      
      Figure 1. Ion chromatography profiles of standards and the in vitro invertase reaction mixtures. A. Chromatogram of the glucose-fructose standard mixture. 100 mg/L of the glucose and fructose standards were mixed together and analyzed by ion chromatography. B. Chromatogram of the in vitro invertase reaction mixture without the cell crude extract (negative control). C. Chromatogram of the in vitro invertase reaction mixture. Glu, Glucose; Fru, Fructose; Suc, Sucrose.
      
      
   3. For quantification of glucose in reaction mixtures, calculate the areas of the target peak obtained by ion chromatography by the Chromeleon software, and then determine the glucose concentration using the standard curve of the glucose standards (Figure 2).
      
      
      Figure 2. The glucose standard curve
      

Data analysis

Calculate the in vitro invertase enzymatic activities according to equations:

Invertase activities [nmol glucose/min/mg protein] = C_glucose x D/MW_glucose/RT/C_protein

where, C_glucose, the glucose resulted from the invertase reactions, unit: nmol/L;
D, dilution ratio for the cell crude extract, namely 50 in this protocol;
MW_gluose, molecular weight of glucose, namely 180;
RT, reaction time, namely 90 min in this protocol;
C_protein, the protein concentration of the cell crude extract, unit: mg/L.

Recipes

1. BG11 medium (Rippka et al., 1979)
   1. 8 kinds of 10x stocks were prepared as follows:
      Stock 1 (40 g/L K₂HPO₄·3H₂O)
      Stock 2 (75 g/L MgSO₄·7H₂O)
      Stock 3 (36 g/L CaCl₂·2H₂O)
      Stock 4 (6 g/L Critic acid)
      Stock 5 (6 g/L Ferric ammonium citrate)
      Stock 6 (1 g/L EDTA·2Na·2H₂O)
      Stock 7 (20 g/L Na₂CO₃)
      Stock A5 (2.86 g/L H₃BO₃, 1.81 g/L MnCl₂·4H₂O, 0.22 g/L ZnSO₄·7H₂O, 0.39 g/L NaMoO₄·2H₂O, 0.08 mg/L CuSO₄·5H₂O, 0.01 g/L CoCl₂·6H₂O)
      Autoclave Stocks 1 and 5 at 121 °C for 20 min. Store the autoclaved stocks and other stocks at 4 °C before use
      
   2. Dissovle 1.5 g NaNO₃ in 992 ml of ddH₂O, and add 1 ml of Stock 2, 3, 4, 6, 7 and A5 into the medium. Autoclave the medium at 121 °C for 20 min, and supplement with 1 ml of Stocks 1 and 5
      
2. 100 mM potassium phosphate buffer (pH 7.0)
   1. Prepare 0.2 M K₂HPO₄ solution by dissolving 45.6 g K₂HPO₄∙3H₂O in ddH₂O and adjusting the volume to 1 L
      
   2. Prepare 0.2 M KH₂PO₄ solution by dissolving 27.2 g KH₂PO₄ in ddH₂O and adjusting the volume to 1 L
      
   3. Prepare 100 mM potassium phosphate buffer (pH 7.0) by mixing 38 ml of 0.2 M KH₂PO₄ solution, 62 ml of 0.2 M KH₂PO₄ solution and 100 ml of ddH₂O
      
3. 100 mM PMSF
   Dissolve 0.174 g PMSF into 10 ml of isopropanol, and store at -20 °C before use
   

Acknowledgments

This work was supported by the National Science Fund for Distinguished Young Scholars of China (31525002 to X. Lu), Shandong Key basic Research project (ZR2017ZB0211), the Joint Sino-German Research Project (grant GZ 984 to X. Lu), the Shandong Taishan Scholarship (X. Lu), the National Science Foundation of China (31301018 to X. Tan), the Key Research Program of the Chinese Academy of Sciences (ZDRW-ZS-2016-3 to X. Tan) and Qingdao Innovative Leading Talent (15-10-3-15-(31)-zch).

References

1. Curatti, L., Giarrocco, L. E., Cumino, A. C. and Salerno, G. L. (2008). Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria. Planta 228(4): 617-625.
   
2. Du, W., Liang, F., Duan, Y., Tan, X. and Lu, X. (2013). Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metab Eng 19: 17-25.
   
3. Kolman, M. A., Nishi, C. N., Perez-Cenci, M. and Salerno, G. L. (2015). Sucrose in cyanobacteria: from a salt-response molecule to play a key role in nitrogen fixation. Life (Basel) 5(1): 102-126.
   
4. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1):1-61.
   
5. Tan, X., Yao, L., Gao, Q., Wang, W., Qi, F. and Lu, X. (2011). Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab Eng 13(2): 169-176.
   
6. Tan, X., Du, W. and Lu, X. (2015). Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria. Appl Microbiol Biotechnol 99(5): 2147-2154.
   
7. Vargas, W., Cumino, A. and Salerno, G. L. (2003). Cyanobacterial alkaline/neutral invertases. Origin of sucrose hydrolysis in the plant cytosol? Planta 216(6): 951-960.
   
8. Vargas, W. A. and Salerno, G. L. (2010). The Cinderella story of sucrose hydrolysis: Alkaline/neutral invertases, from cyanobacteria to unforeseen roles in plant cytosol and organelles. Plant Sci 178(1):1-8.