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Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana
从小球藻Chlorella sorokiniana提取O-乙酰丝氨酸 (硫醇) 裂解酶 (OASTL) 并测定其活性   

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Abstract

O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between cysteine, the product of OASTL activity, and ninhydrin reagent, which forms a thiazolidine (Thz).

Keywords: Chlorella sorokiniana(Chlorella sorokiniana), Colorimetric assay(比色法), Cysteine(半胱氨酸), Microalgae(微藻类), Ninhydrin(茚三酮), O-acetylserine(thiol)lyase(O-乙酰(硫醇)裂解酶), Sulphur(硫)

Background

In archaea, bacteria, microalgae and plants, the synthesis of cysteine (Cys) represents a decisive stage of assimilatory sulphate reduction (Hell and Wirtz, 2008). Cys biosynthesis is the last step of sulphur assimilation and proceeds by two interconnected reactions catalysed by serine acetyltransferase (SAT, EC 2.3.1.30) and O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) (Salbitani et al., 2014; Carfagna et al., 2015).
  OASTLs catalyse the reaction between O-acetilserine (OAS) and sulphide to form Cys and acetate (Figure 1).


Figure 1. Schematic mechanism of cysteine biosynthesis catalyzed by O-acetylserine(thiol)lyase

   In vascular plants, OASTLs are localized in chloroplasts, mitochondria and the cytosol with different functions for Cys synthesis (Jost et al., 2000; Birke et al., 2013). In microalgae, OASTLs seem to be mainly localized essentially in the chloroplasts (Merchant et al., 2007; Bromke, 2013). However, in Chlorella sorokiniana two isoforms, chloroplastic and cytosolic OASTL, were found under S-deprivation conditions (Carfagna et al., 2011).

Many researchers have developed and modified protocols to determine OASTLs activity in plants and bacteria (Gaitonde, 1967; Burnell and Whatley, 1977; Lèon et al., 1987; Rolland et al., 1992). Here we describe a protocol for the determination of OASTL activity, optimized for the green microalga Chlorella sorokiniana 211-8K (Figure 2). This OASTL assay is a spectrophotometric analysis based on the colorimetric reaction of the formed L-cysteine with ninhydrin reagent to form a thiazolidine (Thz) (Prota and Posiglione, 1973).


Figure 2. Optical microscope image of Chlorella sorokiniana cells

Materials and Reagents

  1. Eppendorf tubes (1.5-2.0 ml)
  2. Cuvettes 1.5 ml (BRAND, catalog number: 759115 )
  3. CO2 tank
  4. Chlorella sorokininana Shihira & Krauss, strain 211/8K (CCAP, Cambridge University) (Figure 2)
  5. Liquid nitrogen
  6. Milli-Q water
  7. O-Acetyl-L-serine (OAS) (Sigma-Aldrich, catalog number: CDS020792 )
  8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  9. Sodium sulfide nonahydrate (Na2S·9H2O) (Sigma-Aldrich, catalog number: S2006 )
  10. Trichloracetic acid (TCA) (Sigma-Aldrich, catalog number: 91228 )
  11. Acetic acid (CH3COOH) (Avantor Performance Materials, J.T. Baker®, catalog number: 401424 )
  12. Ethanol (EtOH) (Avantor Performance Materials, J.T. Baker®, catalog number: 8007 )
  13. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  14. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  15. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 04248 )
    Note: This product has been discontinued.
  16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  17. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643 )
  18. Ethylenediaminetetraacetic acid ferric sodium salt (Fe-EDTA) (Sigma-Aldrich, catalog number: E6760 )
  19. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  20. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291 )
  21. Copper(II) sulfate (CuSO4) (Sigma-Aldrich, catalog number: 451657 )
  22. Ammonium molybdate tetrahydrate, (NH4)6Mo7O24·4H2O (Sigma-Aldrich, catalog number: M1019 )
  23. Manganese(II) chloride (MnCl2) (Sigma-Aldrich, catalog number: 13217 )
    Note: This product has been discontinued.
  24. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  25. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  26. Pyridoxal-phosphate (PLP) (Sigma-Aldrich, catalog number: P3657 )
  27. Ninhydrin (Sigma-Aldrich, catalog number: N4876 )
  28. Hydrochloric acid 37% (Avantor Performance Materials, J.T. Baker®, catalog number: 6012 )
  29. HEPES (Sigma-Aldrich, catalog number: H4034 )
  30. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
  31. Basal medium (see Recipes)
  32. Phosphate buffer (see Recipes)
  33. Extraction buffer (see Recipes)
  34. Ninhydrin solution (see Recipes)
  35. 1 M HEPES solution (see Recipes)

Equipment

  1. Culture flask (WHEATON, catalog number: 356954 )
  2. Fluorescent lamps (Philips Lighting, model: TL-D 30W/55 )
  3. Bench centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: IEC CL30 )
  4. French pressure cell press (AMINCO RESOURCES, model: FA-078 )
  5. Superspeed centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall RC-5C Plus )
  6. Vortex mixer (Troemner, catalog number: 945302 )
  7. Eppendorf ThermoMixer® Comfort (Eppendorf, model: 5355 )
  8. Eppendorf MiniSpin® (Eppendorf, model: 5453000011 )
  9. Thermo bath (Labortechnik medingen, model: MWB 5 )
  10. Spectrophotometer (Cole-Parmer, JENWAY, model: 7315 )
  11. Optical microscope (Esselte, Leitz, model: Leitz Laborlux K )

Software

  1. SigmaPlot® 12 software

Procedure

  1. Algal culture condition
    Grow Chlorella sorokiniana culture in batch in basal medium (see Recipes) at 35 °C and under continuous illumination (fluorescent lamps, 250 μmol photons m-2 sec-1). Bubble the culture with air containing 5% CO2. Under these conditions, the algal growth rate constant (µ) is 3 d-1.

  2. Preparation of microalgae extracts
    1. Harvest 200 ml of algal culture by centrifugation (4,500 x g for 10 min). Collect the cells during the exponential growth phase (culture OD550 between 0.5 and 1.0).
    2. Discard the supernatant and re-suspend the pellet in 5 ml of cold (4 °C) extraction buffer (see Recipes).
    3. Lyse the cells by passing twice through a French pressure cell (1,100 psi).
      Note: Other methods and procedures can be used to lyse microalgae; among the most common methods, there are the use of magnetic stirrer, microwave radiation, ultrasonication and enzyme treatment (Dvoretskyet al., 2016; Farooq et al., 2016; Huang et al., 2016). The breaking of the cells can be observed with an optical microscope.
    4. Centrifuge cell homogenate at 11,000 x g for 15 min at 4 °C. Use the resulting supernatant as crude extract (CE) and assay it for enzyme activity.
    5. Keep the crude extract on ice or freeze the sample in liquid nitrogen and store at -80 °C for future assays.
      Note: Store the samples at -80 °C for a maximum period of six months. The frozen sample can be thawed at room temperature.

  3. OASTL assay
    1. Add the following solutions as shown in Table 1 to Eppendorf tubes (1.5-2.0 ml)

      Table 1. OASTL protocol assay

      Notes:
      1.  For the preparation of the reaction mix, it is important to respect the order of the solutions as shown in Table 1.
      2. The volume of Milli-Q water in the sample depends on the amount of CE used, considering a final volume of the reaction mix of 100 µl.
      3.  OAS (O-acetylserine) needs to be prepared fresh before use. DTT and Na2S can be prepared, aliquoted and stored at -20 °C for six months.

    2. After preparation of the reaction mix, vortex the samples and incubate the tubes in a thermomixer at 50 °C for 5 min.
    3. Stop the reaction by adding 50 µl of 20% (v/v) trichloracetic acid (TCA). Spin at 13,000 x g and transfer the supernatants to clean tubes.
      Note: TCA can be stored at 4 °C for six months.
    4. Add to the tubes 100 µl of glacial acetic acid and 200 µl of ninhydrin solution (see Recipes).
    5. Incubate the samples at 100 °C for 5 min and then cool them in a Thermo bath at 10 °C for 5 min.
      Note: As an alternative to Thermo bath, the samples can be incubated in a thermomixer at 99 °C.
    6. Add 550 µl of ethanol, vortex and read the absorbance spectrophotometrically at 560 nm.

Data analysis

  1. To evaluate the range of reliable activity, it is necessary to make a calibration curve for L-cysteine using known concentrations of the amino acid (0.1-3.0 mM).
    Note: We estimated valid an absorbance range at 560 nm between 0.5-1.5.
  2. Enzymatic units were calculated using the following formula:
    Enzymatic units (U) = A560V2V0/εt1V1Ve
    where, A560 is the absorbance at 560 nm; V2 is the final volume (ml) including ethanol; V0 is the volume of the reaction mix incubated at 50 °C; ε is the OAS molar extinction coefficient (mM-1 cm-1); t1 is the time (min) of incubation at 100 °C; V1 is the volume of the reaction mix incubated at 50 °C included TCA; Ve is the crude extract volume used for the assay (ml).
    Note: We estimated ε = 6.4 mM-1 cm-1.
  3. OASTL activity was expressed in units that correspond to the formation of 1 µmol of cysteine min-1. The OASTL activity in each sample should be correlated with the soluble protein content (mg ml-1 extract) that was determined by the Bio-Rad Protein Assay based on the Bradford method (Bradford, 1976), using bovine serum albumin as the standard. The enzymatic unit correlated with the protein content is U mg-1 protein.
    Note: The use of other methods for protein determination is possible.
  4. Data of the mean ± SE of 3-6 independent experiments should be presented (Figure 3).
  5. Experimental data analyses and graphs could be carried out using SigmaPlot® 12 software (Carfagna et al., 2016).
    Note: The use of other software to analyze data is possible.


    Figure 3. OASTL activity in Chlorella sorokiniana. Effect of 24 h sulphur-starvation on enzymatic activity (Salbitani et al., 2014).

Recipes

  1. Basal medium
    13 mM KH2PO4
    4.3 mM K2HPO4
    0.35 mM NaCl
    1.2 mM MgSO4
    0.35 mM Fe-EDTA
    0.18 mM CaCl2
    5 mM KNO3
    Oligoelements: 0.31 mM CuSO4, 0.12 mM (NH4)6Mo7O24, 14 mM MnCl2, 0.76 mM ZnSO4, 46 mM H3BO3
    Adjust the pH to 6.5 and sterilize by autoclaving. The medium is stored at 2-8 °C, in the dark
  2. Phosphate buffer, pH 7.5
    To prepare 50 mM phosphate buffer pH 7.5, mix 0.94 ml of 1 M KH2PO4 with 4.06 ml of 1 M K2HPO4 and add distilled water up to a final volume of 100 ml
    Note: Store the buffer at 2-8 °C for three months. The pH should be measured prior to use and adjusted, if necessary.
  3. Extraction buffer
    50 mM phosphate buffer pH 7.5
    10 μM PLP (pyridoxal-phosphate)
    1 mM DTT (dithiothreitol)
    Note: Prepare fresh before use.
  4. Ninhydrin solution
    To prepare the ninhydrin solution, dissolve 0.12 g of ninhydrin in 3 ml of glacial acetic acid and 2 ml of hydrochloric acid 37%
    Note: The ninhydrin solution can be stored in the dark at room temperature for five days.
  5. 1 M HEPES solution
    Dissolve 11.9 g of HEPES in Milli-Q water up to a final volume of 50 ml
    Adjust the pH to 7.20 with KOH
    Note: Store the buffer at 2-8 °C for three months. The pH should be measured prior to use and adjusted, if necessary.

Acknowledgments

We thank the anonymous reviewers for helpful comments. This study was financed by Regione Campania (PON-Smart Generation and LR 5/2002).

References

  1. Birke, H., Heeg, C., Wirtz, M. and Hell, R. (2013). Successful fertilization requires the presence of at least one major O-acetylserine(thiol)lyase for cysteine synthesis in pollen of Arabidopsis. Plant Physiol 163(2): 959-972.
  2. Bradford, M. A. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72: 248-254.
  3. Bromke, M. A. (2013). Amino acid biosynthesis pathways in diatoms. Metabolites 3(2): 294-311.
  4. Burnell, J. N. and Whatley, F. R. (1977). Sulphur metabolism in Paracoccus denitrificans. purification, properties and regulation of serine transacetylase, O-acetylserine sulphydrylase and β-cystathionase. Biochim Biophys Acta 481(1): 246-265.
  5. Carfagna, S., Bottone, C., Cataletto, P. R., Petriccione, M., Pinto, G., Salbitani, G., Vona, V., Pollio, A. and Ciniglia, C. (2016). Impact of sulfur starvation in autotrophic and heterotrophic cultures of the extremophilic microalga Galdieria phlegrea (Cyanidiophyceae). Plant Cell Physiol 57(9): 1890-1898
  6. Carfagna, S., Salbitani, G., Bottone, C., De Marco, A. and Vona, V. (2015). Cross-effects of nitrogen and sulphur starvation in Chlorella sorokiniana 2112/8K. Nat Resour 6: 221-229.
  7. Carfagna, S., Salbitani, G., Vona, V. and Esposito, S. (2011). Changes in cysteine and O-acetyl-L-serine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status. J Plant Physiol 168(18): 2188-2195.
  8. Dvoretsky, D., Dvoretsky, S., Temnov, M., Akulinin, E. and Peshkova, E. (2016). Enhanced lipid extraction from microalgae Chlorella vulgaris biomass: experiments, modelling, optimization. Chem Eng Trans 49: 175-180.
  9. Farooq, W., Mishra, S. K., Moon, M., Suh, W. I., Shrivastav, A., Kumar, K., Kwon, J. H., Park, M. S. and Mu, Y. (2016). Energy efficient process for microalgae cell disruption for oil recovery using triiodide resin. Algal Res 13: 102-108.
  10. Gaitonde, M. K. (1967). A spectrometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 104: 627–633.
  11. Hell. R. and Wirtz, M. (2008). Metabolism of cysteine in plants and phototrophic bacteria. In: Hell, R., Dahl, C. and Leustek, T. (Eds.). Sulfur metabolism in phototrophic organisms. Springer pp: 61-94.
  12. Huang, Y., Qin, S., Zhang, D., Li, L. and Mu, Y. (2016). Evaluation of cell disruption of Chlorella vulgaris by pressure-assisted ozonation and ultrasonication. Energies 13: 173-184.
  13. Jost, R., Berkowitz, O., Wirtz, M., Hopkins, L., Hawkesford, M. J. and Hell, R. (2000). Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana. Gene 253(2): 237-247.
  14. Lèon, J., Romero, L. C., Galvan, F. and Vega, J. M. (1987). Purification and physicochemical characterization of O-acetyl-l-serine sulfhydrylase from Chlamydomonas reinhardtii. Plant Sci 53(2): 93-99.
  15. Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Marechal-Drouard, L., Marshall, W. F., Qu, L. H., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Cardol, P., Cerutti, H., Chanfreau, G., Chen, C. L., Cognat, V., Croft, M. T., Dent, R., Dutcher, S., Fernandez, E., Fukuzawa, H., Gonzalez-Ballester, D., Gonzalez-Halphen, D., Hallmann, A., Hanikenne, M., Hippler, M., Inwood, W., Jabbari, K., Kalanon, M., Kuras, R., Lefebvre, P. A., Lemaire, S. D., Lobanov, A. V., Lohr, M., Manuell, A., Meier, I., Mets, L., Mittag, M., Mittelmeier, T., Moroney, J. V., Moseley, J., Napoli, C., Nedelcu, A. M., Niyogi, K., Novoselov, S. V., Paulsen, I. T., Pazour, G., Purton, S., Ral, J. P., Riano-Pachon, D. M., Riekhof, W., Rymarquis, L., Schroda, M., Stern, D., Umen, J., Willows, R., Wilson, N., Zimmer, S. L., Allmer, J., Balk, J., Bisova, K., Chen, C. J., Elias, M., Gendler, K., Hauser, C., Lamb, M. R., Ledford, H., Long, J. C., Minagawa, J., Page, M. D., Pan, J., Pootakham, W., Roje, S., Rose, A., Stahlberg, E., Terauchi, A. M., Yang, P., Ball, S., Bowler, C., Dieckmann, C. L., Gladyshev, V. N., Green, P., Jorgensen, R., Mayfield, S., Mueller-Roeber, B., Rajamani, S., Sayre, R. T., Brokstein, P., Dubchak, I., Goodstein, D., Hornick, L., Huang, Y. W., Jhaveri, J., Luo, Y., Martinez, D., Ngau, W. C., Otillar, B., Poliakov, A., Porter, A., Szajkowski, L., Werner, G., Zhou, K., Grigoriev, I. V., Rokhsar, D. S. and Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318(5848): 245-250.
  16. Prota, G. and Ponstglione, E. (1973). On the reaction of ninhydrin with cysteine and its analogues: A revision. Tetrahedron 29(24): 4271-4274.
  17. Rolland, N., Droux, M. and Douce, R. (1992). Subcellular distribution of O-acetylserine(thiol)lyase in cauliflower (Brassica oleracea L.) inflorescence. Plant Physiol 98(3): 927-935.
  18. Salbitani, G., Wirtz, M., Hell, R. and Carfagna, S. (2014). Affinity purification of O-acetylserine(thiol)lyase from Chlorella sorokiniana by recombinant proteins from Arabidopsis thaliana. Metabolites 4(3): 629-639.

简介

O-乙酰丝氨酸(硫醇)裂解酶(OASTL)是催化无机硫化物与O-乙酰丝氨酸反应形成含S氨基酸L-半胱氨酸的酶。 在这里我们描述一个改进的方案来评估这种酶从微藻小球藻的活性。 它是基于半胱氨酸(OASTL活性的产物)和形成噻唑烷(Thz)的茚三酮试剂之间的反应的比色测定。
【背景】在古代,细菌,微藻和植物中,半胱氨酸(Cys)的合成代表了同化硫酸盐还原的决定性阶段(Hell和Wirtz,2008)。 Cys生物合成是硫同化的最后一步,由丝氨酸乙酰转移酶(SAT,EC 2.3.1.30)和O-乙酰丝氨酸(硫醇)裂解酶(OASTL,EC 4.2.99.8)催化的两个相互连接的反应进行(Salbitani等,2014 ; Carfagna等,2015)。
OASTLs催化O-乙酰丝氨酸(OAS)和硫化物之间的反应形成Cys和乙酸酯(图1)。
在血管植物中,OASTLs位于叶绿体,线粒体和胞质溶胶中,具有不同的Cys合成功能(Jost等,2000; Birke等,2013)。在微藻中,OASTLs主要在叶绿体中主要定位(Merchant et al。,2007; Bromke,2013)。然而,在小球藻Sorokiniana中,发现两种同种型,氯代和胞质OASTL,在S剥夺条件下(Carfagna等,2011)。
许多研究人员已经制定和修改了确定植物和细菌中OASTLs活性的方案(Gaitonde,1967; Burnell和Whatley,1977;Lèon等,1987; Rolland等,1992)。在这里,我们描述了用于测定OASTL活性的方案,针对绿色微藻(Chlorella sorokiniana)211-8K进行了优化(图2)。该OASTL测定是基于形成的L-半胱氨酸与茚三酮试剂的比色反应以形成噻唑烷(Thz)(Prota和Posiglione,1973)的分光光度分析。

关键字Chlorella sorokiniana, 比色法, 半胱氨酸, 微藻类, 茚三酮, O-乙酰(硫醇)裂解酶, 硫

材料和试剂

  1. Eppendorf管(1.5-2.0毫升)
  2. 比色皿1.5毫升(BRAND,目录号:759115)
  3. CO 2 tank
  4. 小球藻sorokininana Shihira&克劳斯,应变211/8K(剑桥大学CCAP)(图2)
  5. 液氮
  6. Milli-Q水
  7. O-乙酰丝氨酸(OAS)(Sigma-Aldrich,目录号:CDS020792)
  8. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  9. 硫化钠九水合物(Na 2 S·9H 2 O)(Sigma-Aldrich,目录号:S2006)
  10. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:91228)
  11. 乙酸(CH 3 COOH)(Avantor Performance Materials,J.T.Baker ,目录号:401424)
  12. 乙醇(EtOH)(Avantor Performance Materials,J.T.Baker ,目录号:8007)
  13. Bio-Rad蛋白测定染料浓缩物(Bio-Rad Laboratories,目录号:5000006)
  14. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  15. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:04248)
    注意:本产品已停产。
  16. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  17. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M2643)
  18. 乙二胺四乙酸铁盐钠盐(Fe-EDTA)(Sigma-Aldrich,目录号:E6760)
  19. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  20. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291)
  21. 硫酸铜(II)(CuSO 4)(Sigma-Aldrich,目录号:451657)
  22. 钼酸铵四水合物,(NH 4)6族Mo 7 O 24·4H 2/sub > O(Sigma-Aldrich,目录号:M1019)
  23. 氯化锰(II)(MnCl 2)(Sigma-Aldrich,目录号:13217)
    注意:本产品已停产。
  24. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z0251)
  25. 硼酸(H 3 O 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  26. 吡哆醛 - 磷酸(PLP)(Sigma-Aldrich,目录号:P3657)
  27. 茚三酮(Sigma-Aldrich,目录号:N4876)
  28. 盐酸37%(Avantor Performance Materials,J.T.Baker ®,目录号:6012)
  29. HEPES(Sigma-Aldrich,目录号:H4034)
  30. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)
  31. 基础培养基(见食谱)
  32. 磷酸盐缓冲液(见配方)
  33. 提取缓冲液(见配方)
  34. 茚三酮溶液(参见食谱)
  35. 1 M HEPES溶液(参见食谱)

设备

  1. 培养瓶(WHEATON,目录号:356954)
  2. 荧光灯(飞利浦照明,型号:TL-D 30W/55)
  3. 台式离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:IEC CL30)
  4. 法国压力机(AMINCO RESOURCES,型号:FA-078)
  5. 超速离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall RC-5C Plus)
  6. 涡街搅拌机(Troemner,目录号:945302)
  7. Eppendorf ThermoMixer ® Comfort(Eppendorf,型号:5355)
  8. Eppendorf MiniSpin ®(Eppendorf,型号:5453000011)
  9. 热浴(Labortechnik medingen,型号:MWB 5)
  10. 分光光度计(Cole-Parmer,JENWAY,型号:7315)
  11. 光学显微镜(Esselte,Leitz,型号:Leitz Laborlux K)

软件

  1. SigmaPlot ® 12软件

程序

  1. 藻类培养条件
    在基础培养基(见食谱)中,在35℃和连续照射下(荧光灯,250μmol光子)在 秒下生长小球藻sorokiniana 1 )。用含有5%CO 2的空气鼓泡培养物。在这些条件下,藻类生长速率常数(μ)为3 d -1
  2. 微藻提取物的制备
    1. 通过离心(4,500×g×10分钟)收获200ml藻类培养物。在指数生长阶段(培养物OD 550在0.5和1.0之间)收集细胞。
    2. 丢弃上清液,并将沉淀重新悬浮在5ml冷(4℃)提取缓冲液中(见食谱)。
    3. 通过两次通过法国压力池(1,100psi)使细胞裂解。
      注意:其他方法和程序可用于裂解微藻;在最常见的方法中,使用磁力搅拌器,微波辐射,超声波和酶处理(Dvoretskyet al。,2016; Farooq等,2016; Huang等,2016)。可以用光学显微镜观察细胞的破裂。
    4. 离心细胞匀浆在11,000xg下于4℃15分钟。使用所得上清液作为粗提物(CE),并测定其酶活性
    5. 将粗提物保存在冰上,或将样品冷冻在液氮中,储存于-80°C,以备将来测定 注意:将样品储存在-80°C最多6个月。冻结的样品可在室温下解冻。

  3. OASTL分析
    1. 将如表1所示的以下解决方案添加到Eppendorf管(1.5-2.0 ml)

      表1. OASTL协议测定

      注意:
      1. 对于制备反应混合物,重要的是遵守表1所示的解决方案顺序。
      2. 考虑到100μl的反应混合物的最终体积,样品中的Milli-Q水的体积取决于所用的CE的量。
      3. 在使用前,需要准备新鲜的OAS(O-乙酰丝氨酸)。 DTT和Na 2 S可以制备,等分并在-20℃下储存六个月。

    2. 在制备反应混合物后,旋转样品并在50℃的温热混合器中孵育管5分钟。
    3. 加入50μl20%(v/v)三氯乙酸(TCA)停止反应。旋转13,000 x g,并将上清液转移至干净的管中 注意:TCA可以在4°C储存六个月。
    4. 加入管100μl冰醋酸和200μl茚三酮溶液(参见食谱)。
    5. 将样品在100℃孵育5分钟,然后在10℃的Thermo浴中冷却5分钟。
      注意:作为Thermo bath的替代品,样品可以在99℃的温热混合器中孵育。
    6. 加入550μl乙醇,旋转,并在560 nm下用光度法测定吸光度。

程序

  1. 藻类培养条件
    在基础培养基(见食谱)中,在35℃和连续照射下(荧光灯,250μmol光子)在秒下生长小球藻sorokiniana 1 )。用于含有5%CO 2的空气鼓泡培养物。在这些条件下,藻类生长速率常数(μ)为3 d -1
  2. 微藻提取物的制备
    1. 通过离心(4,500×g×10分钟)收获200ml藻类培养物。在指数生长阶段(培养物OD 550在0.5和1.0之间)收集细胞。
    2. 丢弃上清液,并将沉淀重新悬浮在5ml冷(4℃)提取缓冲液中(见食谱)。
    3. 通过两次通过法国压力池(1,100psi)使细胞裂解。
      注意:其他方法和程序可用于裂解微藻;在最常见的方法中,使用磁力搅拌器,微波辐射,超声波和酶处理(Dvoretskyet al。,2016;法鲁克等2016;黄等,2016)。可以用光学显微镜观察细胞的破裂。
    4. 离心细胞匀浆在11,000xg下于4℃15分钟。使用所得上清液作为粗提物(CE),并测定其酶活性
    5. 将粗提物保存在冰上,或将样品冷冻在液氮中,储存于-80℃,以备将来测定 注意:将样品储存在-80°C最多6个月。冻结的样品可在室温下解冻。

  3. OASTL分析
    1. 将如表1所示的以下解决方案添加到Eppendorf管(1.5-2.0 ml)

      表1。 OASTL协议测定

      注意:
      1. 对于制备反应混合物,重要的是遵守表1所示的解决方案顺序。
      2. 考虑到100μl的反应混合物的最终体积,样品中的Milli-Q水的体积取决于所用的CE的量。
      3. 在使用前,需要准备新鲜的OAS(O-乙酰丝氨酸)。DTT和Na 2 S可以制备,等分并在-20℃下储存六个月。

    2. 在制备反应混合物后,旋转样品并在50℃的温热混合器中孵育管5分钟。
    3. 加入50μl20%(v/v)三氯乙酸(TCA)停止反应旋转13,000 x g,并将上清液转移至干净的管中 注意:TCA可以在4°C储存六个月。
    4. 加入管100μl冰醋酸和200μl茚三酮溶液(参见食谱)
    5. 将样品在100℃孵育5分钟,然后在10℃的热浴中冷却5分钟。
      注意:作为Thermo bath的替代品,样品可以在99℃的温热混合器中孵育。
    6. 加入550μl乙醇,旋转,并在560 nm下用光度法测定吸光度

食谱

  1. 基础培养基
    13mM KH PO 4
    4.3mM K 2 HPO 4
    0.35 mM NaCl
    1.2mM MgSO 4
    0.35 mM Fe-EDTA
    0.18mM CaCl 2
    5mM KNO 3
    Oligoelements:0.31mM CuSO 4,0.12mM(NH 4)6 6 Mo 7 O 24,/sub> 14mM MnCl 2,0.76mM ZnSO 4,46mM H 3 BO 3/Br /> 调节pH至6.5并通过高压灭菌消毒。介质储存在2-8°C,黑暗中
  2. 磷酸盐缓冲液,pH 7.5 为了制备pH 7.5的50mM磷酸盐缓冲液,将0.94ml 1M KH 2 PO 4与4.06ml 1KH 2 HPO > 4 ,加入蒸馏水至最终体积为100 ml 注意:将缓冲液存放在2-8°C三个月。使用前应测量pH值,必要时进行调整。
  3. 提取缓冲区
    50mM磷酸盐缓冲液pH7.5
    10μMPLP(吡哆醛 - 磷酸盐)
    1mM DTT(二硫苏糖醇)
    注意:使用前请准备好。
  4. 茚三酮溶液
    为了制备茚三酮溶液,将0.12g茚三酮溶于3ml冰醋酸和2ml盐酸中37%
    注意:茚三酮溶液可以在室温黑暗中储存五天。
  5. 1 M HEPES解决方案
    将11.9g HEPES溶于Milli-Q水中,最终体积为50ml ml 用KOH调节pH至7.20 注意:将缓冲液存放在2-8°C三个月。使用前应测量pH值,必要时进行调整。

致谢

我们感谢匿名评论者有用的评论。这项研究由Regione Campania(PON-Smart Generation和LR 5/2002)资助。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Salbitani, G. and Carfagna, S. (2017). Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana. Bio-protocol 7(12): e2342. DOI: 10.21769/BioProtoc.2342.
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