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Extraction and Purification of Laccases from Rice Stems

水稻茎秆酶的提取与纯化

CS Chenna Swetha
PS P. V. Shivaprasad

发布: 2019年04月05日第9卷第7期 DOI: 10.21769/BioProtoc.3208 浏览次数: 6758

评审: eman ahmed ibrahimkaushik ghoseAnonymous reviewer(s)

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Abstract

Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.

Keywords: Laccase (漆酶) search Lignin (木质素) search Cell wall (细胞壁) search Phenylpropanoid pathway (苯丙烷途径) search Rice (水稻) search Plants (植物) search

Background

Laccases are oxidases ubiquitously present in bacteria, fungi, animals, and plants. They are some of the oldest enzymes identified. Laccase is involved in diverse functions such as pigmentation of fungal spores, regeneration of plants, as fungal virulence factors, and in lignification of cell walls and delignification during wood rotting. They oxidize the biosynthesis of secondary metabolite called lignin in vascular tissues of plants. Purification of laccases is very challenging as laccases are expressed in very low amounts. Existing protocols to purify laccases are from either microorganism or softer tissues of plants such as leaves. This protocol provides an efficient approach to extract and purify laccases from harder tissues of plants such as rice stems.

Materials and Reagents

  1. Pipette tips (Tarsons)
  2. Spatula (Fisher Scientific, catalog number: 11533462)
  3. Filter paper (Whatman filters, No. 1)
  4. 15 ml and 50 ml centrifuge tubes (Tarsons)
  5. Dialysis membrane (Thermo Fisher Scientific, 10 kDa molecular weight cut-off (MWCO); catalog number: 68100)
  6. Econo-column glass chromatography columns (Bio-Rad, catalog number: 7371512)
  7. O. sativa var. Pusa Basmati-1 (PB-1)
    Note: PB-1 is an aromatic variety of domesticated rice.
  8. Liquid nitrogen
  9. Concanavalin-A beads (Sigma-Aldrich, catalog number: C7555-5ML)
  10. Sodium chloride (NaCl) (HiMedia, catalog number: MB023-1Kg)
  11. Magnesium chloride (MgCl2) (Fischer Scientific, catalog number: 15535-500G)
  12. Manganese (II) chloride (MnCl2) (Sigma, catalog number: M8054)
  13. Calcium chloride (CaCl2) (Sigma, catalog number: C3306-250G)
  14. Tris-base (Fisher Scientific, catalog number: 15965-500G)
  15. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 29505)
  16. Tris-HCl (pH 6.8 and 8.8) (Bio-Rad)
  17. Sodium acetate (CH3COONa) (HiMedia, catalog number: MB048-500G)
  18. Polyvinylpyrrolidone (PVP) (Sigma, catalog number: P5288-100G)
  19. Phenylmethylsulfonyl fluoride (PMSF) (Sigma, catalog number: P7626-5G)
  20. Dithiothreitol (DTT) (Roche, catalog number: 11096176001)
  21. Glycine (Sigma, catalog number: G7126-100G)
  22. Protein cocktail inhibitor (Sigma-Aldrich, catalog number: 5056489001)
  23. 4-hydroxyindole (Sigma-Aldrich, catalog number: 219878)
  24. Mannose (Sigma-Aldrich, catalog number: 112585)
  25. Protein concentrator (Sartorius, Vivaspin, 30,000 d MWCO, catalog number: VS15T21)
  26. 30% acrylamide (Bio-Rad, catalog number: 161-0156)
  27. 10% sodium dodecyl sulfate (SDS) (Invitrogen, catalog number: 15525-017)
  28. 10% ammonium persulfate (Sigma, catalog number: 161-0700)
  29. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma, catalog number: T7024-25ml)
  30. Coomassie brilliant blue (CBB-R-250) (Sigma, catalog number: B-7920-10G)
  31. Bromophenol blue (Sigma, catalog number: B5525-5G)
  32. Glycerol (Millipore, catalog number: DC4P640148)
  33. Ethanol (Emsure, catalog number: 1.00983.0511)
  34. Acetic acid (Fisher Scientific, catalog number: 11005)
  35. Methanol (Merck, catalog number: 60600905001730)
  36. Isopropanol (Fisher Scientific, catalog number: 13825)
  37. Dialysis buffer (see Recipes)
  38. Extraction buffer (see Recipes)
  39. Equilibration buffer (see Recipes)
  40. Wash buffer (see Recipes)
  41. Regeneration buffer (see Recipes)
  42. Storage buffer (see Recipes)
  43. 50 mM Mannose (see Recipes)
  44. 12% SDS-PAGE gel (see Recipes)
  45. TGS buffer (see Recipes)
  46. Native-PAGE gel (see Recipes)
  47. Native gel sample loading dye (6x) (see Recipes)
  48. TG buffer (see Recipes)
  49. CBB-R-250 staining solution (see Recipes)
  50. Destaining solution (see Recipes)
  51. 5 mM 4-hydroxyindole (see Recipes)

Equipment

  1. Scissors
  2. Spatula
  3. Conical flask
  4. Mortar and pestle (Sigma, catalog numbers: Z247472 and Z247510)
  5. Pipettes (Gilson, models: P1000, P200, P100, P20 and P10)
  6. Cold centrifuge (Thermo Scientific, Heraeus Megafuge 16R, 75003694)
  7. -20 °C refrigerator (Panasonic)
  8. Thermomixer (Eppendorf, Thermomixer comfort)
  9. Chemical fume hood
  10. Rocker 
  11. Magnetic stirrer
  12. Magnetic beads
  13. Peristaltic pump (Cole Parmer 7520-67 Masterflex Console Drive, model: 77200-52)

Note: The materials, reagents and equipment not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

Procedure

English
中文翻译

文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Swetha, C. and Shivaprasad, P. V. (2019). Extraction and Purification of Laccases from Rice Stems. Bio-protocol 9(7): e3208. DOI: 10.21769/BioProtoc.3208.

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分类

植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化

植物科学 > 植物分子生物学 > 蛋白质

生物化学 > 蛋白质 > 分离和纯化

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