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Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
乳杆菌属JCM 15040中具有异亮氨酸2-表异构酶活性的蛋白质的纯化

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Abstract

Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods for purification of this protein from L. otakiensis JCM 15040. Because no specific ligand that has high affinity for this enzyme has been identified, the purification was performed using ammonium sulfate fraction, four types of column chromatography and preparative Native-PAGE, not using an affinity column chromatography. We hope that the protocol will provide useful information for purification of an enzyme that cannot easily be purified using an affinity column chromatography.

Keywords: Lactic acid bacteria(乳酸菌), Protein purification(蛋白质纯化), Ion exchange chromatography(离子交换色谱法), Hydrophobic interaction chromatography(疏水相互作用色谱), Affinity chromatography(亲和层析)

Materials and Reagents

  1. 1.5 ml tube (ASONE Corporation, catalog number: 2-1998-02 )
  2. Dialysis membrane tube (molecular weight cutoff: 14,000) (EIDIA, catalog number: UC27-32-100 )
  3. Amicon Ultra 15 ml centrifugal filter 3 K device (Merck Millipore Corporation, catalog number: UFC500396 )
  4. Disposable homogenizer (1.5 ml scale) “Biomasher II” (NIPPI Corporation, catalog number: 320102 )
  5. Feeding tube (TERUMO CORPORATION, catalog number: SF-ET1725 )
  6. Syringe (10 ml scale) (TERUMO CORPORATION, catalog number: SS-10SZ )
  7. Plug silicon (ASONE Corporation, catalog number: 6-336-03 )
  8. Needle (TERUMO CORPORATION, catalog number: NN-2238R )
  9. Purification of the isoleucine 2-epimerase
    1. L. otakiensis JCM 15040 obtained from Japan Collection of Microorganisms (JCM)
    2. TOYOPEARL Phenyl-650M column
      Note: Pack 50 ml of TOYOPEARL Phenyl-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Bioscience LLC, catalog number: 14478 ).
    3. TOYOPEARL Butyl-650M column
      Note: Pack 50 ml of TOYOPEARL Butyl-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Bioscience LLC, catalog number: 07477 ).
    4. TOYOPEARL SuperQ-650M column
      Note: Pack 50 ml of TOYOPEARL SuperQ-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Corporation, catalog number: 17227 ).
    5. Acrylamide (Wako Pure Chemical Industries, Siyaku, catalog number: 016-00765 )
    6. N, N’-Methylenebisacrylamide (Nacalai Tesque, catalog number: 22402-02 )
    7. Ammonium persulfate (Wako Pure Chemical Industries, Siyaku, catalog number: 7727-54-0 )
    8. N, N, N’, N’-Tetramethylethylenediamine (Nacalai Tesque, catalog number: 33401-72 )
    9. Ammonium sulfate (Wako Pure Chemical Industries, Siyaku, catalog number: 019-03435 )
    10.  Sodium chloride (Wako Pure Chemical Industries, Siyaku, catalog number: 198-01675 )
    11. Lactobacilli MRS Broth (MRS medium powder) (BD, catalog number: 288130 ) (see Recipes)
    12. 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM dithiothreitol (see Recipes)
      Note: Unless otherwise indicated, this buffer is used as the standard buffer throughout the purification procedures.
      1. Sodium dihydrogenphosphate dihydrate (Wako Pure Chemical Industries,
        Siyaku, catalog number: 192-02815 )
      2. Disodium hydrogenphosphate 12-water (Wako Pure Chemical Industries,
        Siyaku, catalog number: 196-02835 )
      3. Ethylenediamine-N, N, N', N'-tetraacetic acid, disodium salt, dehydrate (EDTA)
        (Dojindo Molecular Technologies, catalog number: N001 )
      4. Dithiothreitol (Nacalai Tesque, catalog number: 14128-04 )
    13. Red Sepharose CL-4B column (Ohshima and Sakuraba, 1986) (see Recipes)
      Note: According to Recipes, prepare Red Sepharose CL-4B resin by attaching Reactive Red 120 (Sigma-Aldrich, catalog number: R0378-50G ) to Sepharose® CL-4B (Sigma-Aldrich, catalog number: CL4B200-100ML ). Pack 10 ml of Red Sepharose CL-4B resin in a chromatography column (diameter: 1.5 cm, length: 12 cm).
    14. 1.5 M Tris-HCl buffer (pH 8.8) (see Recipes)
      1. 2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base) (Wako Pure Chemical Industries, Siyaku, catalog number: 207-06275 )
      2. Hydrochloric acid (Nacalai Tesque, catalog number: 18402-45 )
    15. 0.5 M Tris-HCl buffer (pH 6.8) (see Recipes)
      1. 2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base)
      2. Hydrochloric acid
    16. Native-PAGE electrophoresis buffer (see Recipes)
      1. 2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base)
      2. Glycine (Wako Pure Chemical Industries, Siyaku, catalog number: 077-00735 )
    17. 2x Native-PAGE gel loading buffer (see Recipes)
      1. 2-Mercaptoethanol (Nacalai Tesque, catalog number: 21418-42 )
      2. Sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 193-00025 )
      3. Bromophenol blue (KANTO CHEMICAL, catalog number: 04319-30 )
  10. Isoleucine 2-epimerase activity assay
    1. Pyridoxal-5’-phosphate (Nacalai Tesque, catalog number: 29606-74 )
    2. L-isoleucine (PEPTIDE INSTITUTE, catalog number: 2712 )
    3. Flavin adenine dinucleotide disodium salt (Nacalai Tesque, catalog number: 16010-06 )
    4. 4-Aminoantipyrine (Nacalai Tesque, catalog number: 01907-52 )
    5. Phenol (Wako Pure Chemical Industries, Siyaku, catalog number: 168-12721 )
    6. D-Amino acid oxidase from porcine kidney (Sigma-Aldrich, catalog number: A5222-200UN )
    7. Peroxidase (TOYOBO BIO CHEMICAL DEPT, catalog number: PE0-301 )
    8. 0.5 M sodium phosphate buffer (pH 8.0) (see Recipes)
      1. Sodium dihydrogenphosphate dihydrate
      2. Disodium hydrogenphosphate 12-water

Equipment

  1. Purification of the isoleucine 2-epimerase
    1. Incubator (TITEC CORPORATION, model: BR-43FM )
    2. Centrifuge (Hitachi Koki Co., model number: CR 21F and TOMY SEIKO CO., model number: MX-300 )
    3. Multi-Beads shocker (Yasui Kikai Corporation)
    4. Electrophoresis apparatus
    5. Column chromatography equipment (see Representative data)
      1. Magnetic stirrer (ASONE Corporation, model: HS-50E )
      2. Two-way stopcock (Bio-Rad Laboratories, catalog number: 7328102 )
      3. Three-way stopcock (TERUMO CORPORATION, catalog number: TS-TR1K )
      4. Chromatography column (diameter: 2.5 cm, length: 10 cm) (Bio-Rad Laboratories, catalog number: 7372512 )
      5. Chromatography column (diameter: 1.5 cm, length: 12 cm) (Bio-Rad Laboratories, catalog number: 7321010 )
      6. Fraction tube (15 ml scale) (ASONE Corporation, catalog number: 2-8007-02 )
  2. Isoleucine 2-epimerase activity assay
    1. Spectrophotometer (Shimadzu Scientific Instruments, model number: UVmini-1240 )
    2. Water bath (TOKYO RIKAKIKAI CO., model number: NTT-20S )
    3. Vortex (TAITEC CORPORATION, catalog number: 0061271-000 )

Procedure

  1. Purification of the isoleucine 2-epimerase
    1. Prepare 5 test tubes including 10 ml of MRS medium for preculture.
    2. Inoculate a part of a colony of L. otakiensis JCM 15040 into each MRS medium (10 ml) in the test tube. The preculture is performed statically at 30 °C for 36 h.
    3. The main culture is started by addition of the preculture media (10 ml x 5 tubes = 50 ml) into 10 L of MRS medium.
    4. L. otakiensis JCM 15040 is cultivated statically at 30 °C for 30 h in an incubator, after which cells are pelleted by centrifugation (8,000 x g for 30 min at 4 °C). The cell pellet (ca. 32.2 g, wet weight) is used as the starting material for purification of protein exhibiting isoleucine 2-epimerase activity. Unless otherwise indicated, all purification procedures are carried out at room temperature, and the enzyme solution is stored at 4 °C.
    5. To prepare a crude extract, the cells are washed twice with about four volumes (120 ml) of the standard buffer, and suspend in 120 ml of the same buffer. On the occasion of the washing, the cells are collected by centrifugation (8,000 x g for 30 min at 4 °C). Next, they are disrupted using a Multi-Beads Shocker (2,500 rpm for 60 sec at 2 °C, 5 times), and centrifuged (10,000 x g for 30 min at 4 °C). The resultant supernatant (100 ml) is used as the crude extract.
    6. The crude extract (100 ml) is mixed with two volumes of 3.6 M (NH4)2SO4 dissolved in the standard buffer. After incubation on a magnetic stirrer for 4 h at 4 °C, the mixture is centrifuged (10,000 x g for 30 min at 4 °C), and the supernatant (280 ml) is retrieved.
    7. The collected supernatant (280 ml) is applied to a TOYOPEARL Phenyl-650M column. This column chromatography separates proteins on the basis of hydrophobic interactions between the proteins and the resin. Before the supernatant is loaded, pre-equilibrate the column with 10 column volumes (500 ml) of 2.4 M (NH4)2SO4 dissolved in the standard buffer. After the loading, the column is washed once with three column volumes (150 ml) of the same buffer and proteins are eluted using a linear gradient of 2.4 to 0.4 M (NH4)2SO4 in the buffer [used buffer: 250 ml of the buffer with 2.4 M (NH4)2SO4 and 250 ml of the buffer with 0.4 M (NH4)2SO4]. The elution is performed at a flow rate of about 1 ml/min, and 50 fractions including about 10 ml of elute are collected.
    8. Assay the enzyme activity in each fraction (see section B “Isoleucine 2-epimerase activity assay” below), and choose five active fractions showing higher activity than other fractions. The five active factions (about 50 ml) are mixed, and then dialyzed against 100 volumes (5 L) of the standard buffer at 4 °C. After 4 h, the standard buffer (5 L) is changed and dialysis is continuously performed at 4 °C for 12 h.
    9. This first dialysate (60 ml) is mixed with two volumes of 3.6 M (NH4)2SO4 dissolved in the standard buffer, and the mixture is applied to a TOYOPEARL Butyl-650M column. This column chromatography separates proteins on the basis of hydrophobic interactions between the proteins and the resin. Before the supernatant is loaded, pre-equilibrate the column with 10 column volumes (500 ml) of 2.4 M (NH4)2SO4 dissolved in the standard buffer. After the loading, the column is washed once with three column volumes (150 ml) of the same buffer, and proteins are eluted using a linear gradient of 2.4 to 0.4 M (NH4)2SO4 in the buffer [used buffer: 250 ml of the buffer with 2.4 M (NH4)2SO4 and 250 ml of the buffer with 0.4 M (NH4)2SO4]. The elution is performed at a flow rate of about 1 ml/min, and 50 fractions including about 10 ml of elute are collected. The five active fractions are pooled and dialyzed as described above.
    10. This second dialysate (60 ml) is applied to a TOYOPEARL SuperQ-650M column. This column chromatography separates proteins on the basis of ionic interactions between the proteins and the resin. Before the supernatant is loaded, pre-equilibrate the column with 10 column volumes (500 ml) of the standard buffer. After the loading, the column is washed once with three column volumes (150 ml) of the buffer, and proteins are eluted using a linear gradient of 0 to 250 mM NaCl in the buffer (used buffer: 250 ml of the standard buffer and 250 ml of the buffer with 250 mM NaCl). The elution is performed at a flow rate of about 1 ml/min, and 50 fractions including about 10 ml of elute are collected. The five active fractions are pooled and dialyzed as described above.
    11. SDS-PAGE of this third dialysate shows that this dialysate includes not only isoleucine 2-epimerase, but also a putative NAD+-dependent alcohol dehydrogenase (data not shown). To remove this NAD+-dependent dehydrogenase, the third dialysate (50 ml) is applied to a Red Sepharose CL-4B column. Because the red dye immobilized in this column binds to a wide variety of NAD+- and NADP+-dependent enzymes but not isoleucine 2-epimerase, the NAD+-dependent enzyme and isoleucine 2-epimerase are separated by pooling the flow-through of this column chromatography. Before the supernatant is loaded, pre-equilibrate the column with 10 column volumes (100 ml) of the standard buffer. After the loading, the column is washed the column once with three column volumes (30 ml) of the standard buffer. During sample loading and column washing, the flow-through (about 80 ml) as the active fractions is pooled. The resultant enzyme solution is then concentrated to 300 μl using an Amicon Ultra centrifugal filter 3 K device, and then the concentrated enzyme solution is stored at 4 °C.
    12. Native-polyacrylamide gel electrophoresis (Native-PAGE) of the concentrated enzyme solution (300 μl) is performed on a polyacrylamide slab gel for further enzyme purification; 30 μl of the enzyme solution is loaded on each of 10 lanes. The electrophoresis is performed using the method of Laemmli (Laemmli, 1970) with some modifications; buffers without sodium dodecyl sulfate are used, and the protein sample is not heated during the pretreatment procedures. After electrophoresis using a constant current of 20 mA for 90 min, the gel is cut into 16 pieces using a cutter and a ruler. The gel pieces are individually crushed in 300 μl of the standard buffer using Biomasher II, and the resultant solutions are centrifuged (17,000 x g for 15 min at 4 °C) (Figure 2). The enzyme activity of the supernatants is assayed and the active enzyme solution is used as the final purified enzyme solution from L. otakiensis JCM 15040.

  2. Isoleucine 2-epimerase activity assay
    1. Prepare the first step reaction mixture in 1.5 ml tube. The composition of the first step reaction mixture is shown in the following table. Run this reaction for 1 h at 30 °C. The 1.5 ml tube containing the reaction mixture is incubated in a water bath.
      The first step reaction mixture (500 μl)
      0.5 M sodium phosphate buffer (pH 8.0)
      100 μl
      0.5 mM pyridoxal-5’-phosphate
      100 μl
      50 mM L-isoleucine
      100 μl
      Enzyme solution
      50 μl
      H2O
      150 μl
    2. Boil the first step reaction mixture in 1.5 ml tube for 10 min, and thus cool it to room temperature.
    3. Prepare the second step reaction mixture by adding reagents shown in the following table into the first step reaction mixture. Run this reaction for 15 min at 37 °C. The 1.5 ml tube containing the reaction mixture is incubated in a water bath.
      The second step reaction mixture (1,000 μl)
      The first step reaction mixture
      500 μl
      0.5 M sodium phosphate buffer (pH 8.0)
      100 μl
      0.4 mM fravin-adenine dinucleotide
      50 μl
      4 mM 4-aminoantipyrine
      50 μl
      40 mM phenol
      50 μl
      5 Unit/ml D-amino acid oxidase
      50 μl
      20 Unit/ml horseradish peroxidase
      50 μl
      H2O
      50 μl
    4. Apply the second step reaction mixture to a spectrophotometer, and thus measure absorbance at 500 nm. A measurement of the reaction mixture without the enzyme solution is used as a blank data.

Representative data


Figure 1. Image of the column chromatography equipment


Figure 2. Image of the purification procedure using Native-PAGE

Notes

Isoleucine 2-epimerase activity assay
For choosing active fraction, the enzyme activity is rapidly assayed by the following 2 step reactions (Figure 3). At first step, isoleucine 2-epimerase reaction with L-isoleucine as a substrate produces D-allo-isoleucine. At next step, D-amino acid oxidase-peroxidase coupling reaction is performed. In this coupling reaction, D-amino acid oxidase reaction produces H2O2 in oxidization of D-allo-isoleucine, and thus peroxidase reaction produces indophenol compound (λmax: 500 nm, ε: 6.39 mM-1.cm-1) from 4-aminoantipyrine and phenol using oxidation power of H2O2. The isoleucine 2-epimerase activity is indirectly assayed by increase in absorbance at 500 nm occurring from the indophenol compound production.


Figure 3. Principle of the assay method for isoleucine 2-epimerase activity

Recipes

  1. Purification of the isoleucine 2-epimerase
    1. MRS medium (1 L)
      Mix 55 g of MRS medium powder with 800 ml of dH2O
      Add dH2O to 1,000 ml
      Autoclave
      Stored at room temperature
    2. 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM dithiothreitol (1 L)
      Mix 3.90 g of NaH2PO4.2H2O and 8.95 g of Na2HPO4.12H2O with 800 ml of dH2O
      Add 2 ml of 0.5 M EDTA (pH 8.0)
      pH to 7.2 with HCl or NaOH (aq)
      Add dH2O to 1,000 ml
      Autoclave
      Add and mix 0.154 g of dithiothreitol
      Stored at 4 °C
    3. Red Sepharose CL-4B
      Add Reactive Red 120 (1 g in 100 ml of dH2O) and NaCl (22%, 20 ml) into slurry of Sepharose 4B (100 ml) previously washed with dH2O
      Tumble the mixture at 25 °C for 30 min slowly
      Add 2 g of Na2CO3 to the mixture
      Coupling is achieved by shaking gently for about 4 days at 25 °C
      Wash the slurry with 2 L of dH2O and 2 L of 1 M KCl
      Store the Red-Sepharose 4B at 4 °C
    4. 1.5 M Tris-HCl buffer (pH 8.8, 100 ml)
      Mix 18.2 g of Tris base with 80 ml of dH2O
      pH to 8.8 with HCl
      Add dH2O to 100 ml
      Autoclave
      Store at room temperature
    5. 0.5 M Tris-HCl buffer (pH 6.8, 100ml)
      Mix 6.1 g of Tris base with 80 ml of dH2O
      pH to 6.8 with HCl
      Add dH2O to 100 ml
      Autoclave
      Stored at room temperature
    6. Polyacrylamide slab gel for Native-PAGE (thickness, 1 mm; wide, 106 mm; high, 80 mm)
      Separate range (7.5% polyacrylamide gel, 6 ml)
      1.5 M Tris-HCl buffer (pH 8.8)
      1.5 ml
      29% (w/v) acrylamide + 1% (w/v) N, N’-methylene bis acrylamide solution
      1.5 ml
      10% (w/v) ammonium persulfate
      200 μl
      dH2O
      2.8 ml
      N, N, N’, N-Tetramethylethylenediamine
      3 μl

      Stacking range (4.5% polyacrylamide gel, 3 ml)

      0.5 M Tris-HCl buffer (pH 6.8)
      0.75 ml
      29% (w/v) Acrylamide + 1% (w/v) N’, N’-methylene bis acrylamide solution
      0.45 ml
      10% (w/v) Ammonium peroxodisulphate
      100 μl
      dH2O
      1.7 ml
      N, N, N’, N’-Tetramethylethylenediamine
      3 μl

    7. Native-PAGE electrophoresis buffer (1 L)
      Mix 3.03 g of Tris base, 14.4 g of glycine and 900 ml of dH2O
      Add dH2O to 1 L
      Autoclave
      Stored at room temperature
    8. 2x Native-PAGE gel loading buffer (10 ml)
      Mix 2.5 ml of 0.5 M Tris-HCl buffer (pH 6.8), 1 ml of 2-mercaptoethanol, 1 g of sucrose, 1 mg of bromophenol blue and 8 ml of dH2O
      Add dH2O to 10 ml
      Stored at −20 °C

  2. Isoleucine 2-epimerase activity assay
    1. 0.5 M sodium phosphate buffer (pH 8.0, 100 ml)
      Mix 1.06 g of NaH2PO4.2H2O and 15.5 g of Na2HPO4.12H2O with 80 ml of dH2O
      pH to 8.0 with HCl or NaOH (aq)
      Add dH2O to 100 ml
      Autoclave
      Stored at room temperature

Acknowledgments

In this method, we have modified Laemmli’s method for Native-PAGE. In addition, this work was supported by a grant for Promotion of Basic Research Activities for Innovate Bioscience from the Bio-oriented Technology Research Advancement Institution (BRAIN) and JSPS KAKENHI Grant Number 2402734.

References

  1. Ohshima, T. and Sakuraba, H. (1986). Purification and characterization of malate dehydrogenase from the phototrophic bacterium, Rhodopseudomonas capsulata. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 869(2): 171-177.
  2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.

简介

在异质发酵细菌物种,例如otakiensis的乳酸杆菌(Lactobacillus otakiensis)的培养基中观察到D-亮氨酸,D-异亮氨酸 - 异亮氨酸和D-缬氨酸的显着积累。 otakiensis)JCM 15040.从细菌细胞中纯化导致这种积累的消旋酶,异亮氨酸2-差向异构酶。这是首次报道的在乳酸菌中这种D-支链氨基酸的产生的观察结果,以及在任何生物体中具有异亮氨酸2-差向异构酶活性的消旋酶的第一个实例。在所述的方案中,我们介绍从L中纯化该蛋白质的方法。因为没有鉴定到对该酶具有高亲和力的特异性配体,所以使用硫酸铵级分,四种类型的柱层析和制备型Native-PAGE,不使用亲和柱层析进行纯化。我们希望协议将提供有用的信息用于纯化不能容易地使用亲和柱层析纯化的酶。

关键字:乳酸菌, 蛋白质纯化, 离子交换色谱法, 疏水相互作用色谱, 亲和层析

材料和试剂

  1. 1.5ml管(ASONE Corporation,目录号:2-1998-02)
  2. 透析膜管(截留分子量:14,000)(EIDIA,目录号:UC27-32-100)
  3. Amicon Ultra 15ml离心过滤器3K装置(Merck Millipore Corporation,目录号:UFC500396)
  4. 一次性均化器(1.5ml规模)"Biomasher II"(NIPPI Corporation,目录号:320102)
  5. 供料管(TERUMO CORPORATION,目录号:SF-ET1725)
  6. 注射器(10ml规模)(TERUMO CORPORATION,目录号:SS-10SZ)
  7. 插塞硅(ASONE公司,目录号:6-336-03)
  8. 针(TERUMO CORPORATION,目录号:NN-2238R)
  9. 异亮氨酸2-差向异构酶的纯化
    1. L。 otakiensis JCM 15040获自日本微生物保藏中心(JCM)
    2. TOYOPEARL Phenyl-650M柱
      注意:在色谱中包装50ml TOYOPEARL Phenyl-650M树脂 柱(直径:2.5cm,长度:10cm)(Tosoh Bioscience LLC, 号码:14478)。
    3. TOYOPEARL丁基-650M柱
      注意: 在色谱柱中装入50ml TOYOPEARL Butyl-650M树脂 (直径:2.5cm,长度:10cm)(Tosoh Bioscience LLC,目录号:07477)。
    4. TOYOPEARL SuperQ-650M列
      注意:Pack 50 ml TOYOPEARL SuperQ-650M树脂在色谱柱(直径: ?2.5cm,长度:10cm)(Tosoh Corporation,目录号:17227)。
    5. 丙烯酰胺(Wako Pure Chemical Industries,Siyaku,目录号:016-00765)
    6. N,N' - 亚甲基双丙烯酰胺(Nacalai Tesque,目录号:22402-02)
    7. 过硫酸铵(Wako Pure Chemical Industries,Siyaku,目录号:7727-54-0)
    8. N,N,N',N' - 四甲基乙二胺(Nacalai Tesque,目录号:33401-72)
    9. 硫酸铵(Wako Pure Chemical Industries,Siyaku,目录号:019-03435)
    10. 氯化钠(Wako Pure Chemical Industries,Siyaku,目录号:198-01675)
    11. 乳杆菌MRS肉汤(MRS培养基粉末)(BD,目录号:288130)(参见配方)
    12. 含有1mM EDTA和1mM二硫苏糖醇的50mM磷酸钠缓冲液(pH7.2)(见配方)
      注意:除非另有说明,该缓冲液在整个纯化程序中用作标准缓冲液。
      1. 磷酸二氢钠二水合物(Wako Pure Chemical Industries,
      2. 磷酸氢二钠12-水(Wako Pure Chemical Industries,
        ) Siyaku,目录号:196-02835)
      3. 乙二胺-N,N,N',N'' - 四乙酸二钠盐,脱水物(EDTA)
        (Dojindo Molecular Technologies,目录号:N001)
      4. 二硫苏糖醇(Nacalai Tesque,目录号:14128-04)
    13. 红色Sepharose CL-4B柱(Ohshima和Sakuraba,1986)(参见Recipes)
      注意:根据食谱,准备红色Sepharose CL-4B树脂 附着活性红120(Sigma-Aldrich,目录号:R0378-50G) ?Sepharose CL-4B(Sigma-Aldrich,目录号:CL4B200-100ML)。包 将10ml红色琼脂糖CL-4B树脂在色谱柱(直径: ?1.5厘米,长度:12厘米)。
    14. 1.5 M Tris-HCl缓冲液(pH 8.8)(见配方)
      1. 2-氨基-2-羟甲基-1,3-丙二醇(Tris碱)(Wako Pure Chemical Industries,Siyaku,目录号:207-06275)
      2. 盐酸(Nacalai Tesque,目录号:18402-45)
    15. 0.5 M Tris-HCl缓冲液(pH 6.8)(见配方)
      1. 2-氨基-2-羟甲基-1,3-丙二醇(Tris碱)
      2. 盐酸
    16. Native-PAGE电泳缓冲液(参见配方)
      1. 2-氨基-2-羟甲基-1,3-丙二醇(Tris碱)
      2. 甘氨酸(Wako Pure Chemical Industries,Siyaku,目录号:077-00735)
    17. 2x Native-PAGE凝胶上样缓冲液(参见Recipes)
      1. 2-巯基乙醇(Nacalai Tesque,目录号:21418-42)
      2. 蔗糖(Wako Pure Chemical Industries,Siyaku,目录号:193-00025)
      3. 溴酚蓝(KANTO CHEMICAL,目录号:04319-30)
  10. 异亮氨酸2-差向异构酶活性测定
    1. 5'-磷酸吡哆醛(Nacalai Tesque,目录号:29606-74)
    2. L-异亮氨酸(PEPTIDE INSTITUTE,目录号:2712)
    3. 黄素腺嘌呤二核苷酸二钠盐(Nacalai Tesque,目录号:16010-06)
    4. 4-氨基安替比林(Nacalai Tesque,目录号:01907-52)
    5. 苯酚(Wako Pure Chemical Industries,Siyaku,目录号:168-12721)
    6. 来自猪肾的D-氨基酸氧化酶(Sigma-Aldrich,目录号:A5222-200UN)
    7. 过氧化物酶(TOYOBO BIO CHEMICAL DEPT,目录号:PE0-301)
    8. 0.5 M磷酸钠缓冲液(pH 8.0)(参见配方)
      1. 磷酸二氢钠二水合物
      2. 磷酸氢二钠12-水

设备

  1. 异亮氨酸2-差向异构酶的纯化
    1. 孵育器(TITEC CORPORATION,型号:BR-43FM)
    2. 离心机(Hitachi Koki Co.,型号:CR 21F和TOMY SEIKO CO。,型号:MX-300)
    3. 多珠冲击器(Yasui Kikai Corporation)
    4. 电泳仪
    5. 柱色谱设备(见代表数据)
      1. 磁力搅拌器(ASONE Corporation,型号:HS-50E)
      2. 双向旋塞(Bio-Rad Laboratories,目录号:7328102)
      3. 三通旋塞(TERUMO CORPORATION,目录号:TS-TR1K)
      4. 色谱柱(直径:2.5cm,长度:10cm)(Bio-Rad Laboratories,目录号:7372512)
      5. 色谱柱(直径:1.5cm,长度:12cm)(Bio-Rad Laboratories,目录号:7321010)
      6. 分馏管(15ml规模)(ASONE Corporation,目录号:2-8007-02)
  2. 异亮氨酸2-差向异构酶活性测定
    1. 分光光度计(Shimadzu Scientific Instruments,型号:UVmini-1240)
    2. 水浴(TOKYO RIKAKIKAI CO。,型号:NTT-20S)
    3. Vortex(TAITEC CORPORATION,目录号:0061271-000)

程序

  1. 异亮氨酸2-差向异构酶的纯化
    1. 准备5个试管,包括10ml MRS培养基用于预培养
    2. 接种 L的菌落的一部分。 otakiensis JCM 15040到每个MRS 培养基(10ml)。预培养是静态进行的 在30℃下保持36小时
    3. 通过向10L MRS培养基中加入预培养培养基(10ml×5管= 50ml)开始主培养。
    4. L。 otakiensis JCM 15040在30℃下静置30小时 在培养箱中,然后通过离心(8,000xg,在4℃下30分钟)沉淀细胞。细胞沉淀( c a。 32.2g,湿重) 用作纯化蛋白质的起始原料 异亮氨酸2-差向异构酶活性。除非另有说明,所有 纯化程序在室温下进行, 酶溶液贮存在4℃
    5. 为了制备粗提取物, 用约四体积(120ml)洗涤细胞两次 标准缓冲液,并悬浮在120ml相同的缓冲液中。上的 洗涤时,通过离心收集细胞 (在4℃下8,000×g/30分钟)。接下来,使用a 多珠冲击器(2,500rpm,60℃,2℃,5次),和 离心(10,000×g在4℃下30分钟)。得到的上清液 (100ml)用作粗提取物
    6. 将粗提取物(100ml) ?与溶解在标准溶液中的两倍体积的3.6M(NH 4)2 SO 4 SO 4混合, 缓冲。在磁力搅拌器上在4℃温育4小时后, 混合物离心(10,000×g,在4℃下30分钟),并且 回收上清液(280ml)
    7. 收集上清液(280 ?ml)施加到TOYOPEARL Phenyl-650M柱上。此列 色谱法基于疏水性分离蛋白质 蛋白质和树脂之间的相互作用。之前的上清液 ,用10个柱体积(500ml)的柱预平衡柱 ?溶解在标准缓冲液中的2.4M(NH 4)2 SO 4 SO 4。装载后, 将柱用三个柱体积(150ml)洗涤一次 ?缓冲液和蛋白质使用2.4至0.4M的线性梯度洗脱 (NH 4)2 SO 4 SO 4在缓冲液中[使用的缓冲液:250ml具有2.4M的缓冲液 (NH 4)2 SO 4和250ml具有0.4M(NH 4)亚基的缓冲液。 > 2 SO 4]。洗脱是 ?以约1ml/min的流速进行,50个级分包括 收集约10ml洗脱液。
    8. 测定酶活性 ?每个级分(参见下面的B部分"异亮氨酸2-差向异构酶活性测定"), 并选择五个活性级分显示比其他活性更高 分数。将五个活性级分(约50ml)混合,然后 在4℃下相对于100体积(5L)的标准缓冲液透析。后 ?4 h,更换标准缓冲液(5 L),连续进行透析 在4℃下进行12小时
    9. 将该第一透析液(60ml)混合 ?使用溶解在标准缓冲液中的两倍体积的3.6M(NH 4)2 SO 4 SO 4, 并将混合物施加到TOYOPEARL Butyl-650M柱上。此列 ?色谱法基于疏水性分离蛋白质 蛋白质和树脂之间的相互作用。之前的上清液 ?,用10个柱体积(500ml)预平衡柱, 的溶于标准缓冲液中的2.4M(NH 4)2 SO 4 SO 4。装载后, 将柱用三个柱体积(150ml)洗涤一次 ?缓冲液,使用2.4?0.4M的线性梯度洗脱蛋白质 ?(NH 4)2 SO 4 SO 4在缓冲液中[使用的缓冲液:250ml具有2.4M的缓冲液 (NH 4)2 SO 4和250ml具有0.4M(NH 4)亚基的缓冲液。 > 2 SO 4]。洗脱是 ?以约1ml/min的流速进行,50个级分包括 收集约10ml洗脱液。合并五种活性级分 ?并如上所述进行透析
    10. 将该第二透析液(60ml) 应用于TOYOPEARL SuperQ-650M柱。此柱色谱 ?分离蛋白质之间的离子相互作用 蛋白质和树脂。在装载上清液之前, 用10个柱体积(500ml)的柱预平衡柱 标准缓冲液。装载后,用三次洗涤柱一次 ?柱体积(150ml)的缓冲液,并使用a。洗脱蛋白质 线性梯度的0至250mM NaCl的缓冲液(使用的缓冲液:250ml 的标准缓冲液和250ml含有250mM NaCl的缓冲液)。的 以约1ml/min的流速和50个级分进行洗脱 包括约10ml的洗脱物。五个活性级分 合并并如上所述进行透析
    11. SDS-PAGE 第三透析液显示该透析液不仅包括异亮氨酸 2-差向异构酶,还包括推定的NAD +依赖性醇脱氢酶 (数据未示出)。为了去除这种NAD + - 依赖性脱氢酶,第三 ?透析液(50ml)加到Red Sepharose CL-4B柱上。因为 固定在该柱中的红色染料结合各种各样的NAD + 和NADP +依赖性酶,但不是异亮氨酸2-差向异构酶 NAD +依赖性酶和异亮氨酸2-差向异构酶被分开 合并该柱色谱的流通液。之前 装载上清液,用10个柱体积预平衡柱 ?(100ml)的标准缓冲液。装载后,柱子是 用三个柱体积(30ml)的标准物洗涤柱一次 ?缓冲。在样品加载和柱洗涤期间,流过 (约80ml),作为活性级分。所得酶 然后使用Amicon Ultra将溶液浓缩至300μl 离心过滤器3 K装置,然后浓缩酶溶液 ?储存在4℃
    12. 聚丙烯酰胺凝胶电泳 (Native-PAGE)浓缩酶溶液(300μl) 在聚丙烯酰胺板凝胶上进一步酶纯化; 30μl 将酶溶液装载在10个泳道中的每一个上。电泳 使用Laemmli(Laemmli,1970)的方法进行 修改;使用不含十二烷基硫酸钠的缓冲液, 蛋白质样品在预处理程序期间不被加热。后 电泳使用20mA的恒定电流90分钟,凝胶为 ?用切刀和尺子切成16块。凝胶块 使用Biomasher在300μl标准缓冲液中单独破碎 II,并将所得溶液离心(17,000×g)15分钟 ?4℃)(图2)。测定上清液的酶活性 并将活性酶溶液用作最终的纯化酶 来自 L的解。 otakiensis JCM 15040。

  2. 异亮氨酸2-差向异构酶活性测定
    1. 在1.5ml管中制备第一步反应混合物。组成 的第一步反应混合物示于下表中。跑 该反应在30℃下进行1小时。含有1.5ml管的反应管 混合物在水浴中孵育
      第一步反应混合物(500μl)
      0.5M磷酸钠缓冲液(pH8.0) 100微升
      0.5mM吡哆醛-5'-磷酸 100微升
      50mM L-异亮氨酸 100微升
      酶溶液
      50微升
      H sub 2 O
      150μl
    2. 将第一步反应混合物在1.5ml管中煮沸10分钟,然后冷却至室温
    3. 通过加入如下所示的试剂制备第二步反应混合物: ?下表进入第一步反应混合物。运行此 在37℃反应15分钟。含有1.5ml管的反应管 混合物在水浴中孵育
      第二步反应混合物(1000μl)
      第一步反应混合物
      500微升
      0.5M磷酸钠缓冲液(pH8.0) 100微升
      0.4mM酪蛋白 - 腺嘌呤二核苷酸 50微升
      4mM 4-氨基安替比林 50微升
      40mM苯酚
      50微升
      5单位/ml D-氨基酸氧化酶
      50微升
      20单位/ml辣根过氧化物酶
      50微升
      H sub 2 O
      50微升
    4. 将第二步反应混合物施加到分光光度计,和 从而测量500nm处的吸光度。反应混合物的测量 ?没有使用酶溶液作为空白数据。

代表数据


图1.柱色谱设备的图像


图2.使用Native-PAGE进行纯化的图像

笔记

异亮氨酸2-差向异构酶活性测定
为了选择活性级分,通过以下两步反应快速测定酶活性(图3)。在第一步,异亮氨酸2-差向异构酶与L-异亮氨酸作为底物反应产生D-异亮氨酸 - 异亮氨酸。在下一步,进行D-氨基酸氧化酶 - 过氧化物酶偶联反应。在该偶联反应中,D-氨基酸氧化酶反应在D-异亮氨酸 - 异亮氨酸的氧化中产生H 2 O 2 - 2,因此过氧化物酶反应使用H 2 O 2的氧化能力从4-氨基安替比林和苯酚制备靛酚化合物(λmax:500nm,ε:6.39mM s -1 -1 cm -1 -1)/sub> O 。异亮氨酸2-差向异构酶活性通过由靛酚化合物生产产生的500nm处吸光度的增加来间接测定。


图3.异亮氨酸2-差向异构酶活性的测定方法的原理

食谱

  1. 异亮氨酸2-差向异构酶的纯化
    1. MRS介质(1 L)
      将55g的MRS介质粉末与800ml的dH 2 O混合 将dH <2> O添加至1,000 ml
      高压灭菌器
      在室温下贮存
    2. 含有1mM EDTA和1mM二硫苏糖醇(1L)的50mM磷酸钠缓冲液(pH7.2) 将3.90g的NaH 2 PO 4·4·2H 2·2H 2 O和8.95g的Na 2 SO 4·H 2 O混合, /sub>HPO412H<2O与800ml dH 2 O
      加入2ml 0.5M EDTA(pH 8.0)
      用HCl或NaOH(水溶液)将pH调节至7.2 将dH <2>
      O添加至1,000 ml
      高压灭菌器
      加入并混合0.154g二硫苏糖醇 储存在4°C
    3. 红色琼脂糖CL-4B
      将活性红120(1g,在100ml dH 2 O中)和NaCl(22%,20ml)加入到 ?预先用dH 2 O洗涤的Sepharose 4B(100ml)的淤浆 将混合物在25℃下缓慢搅拌30分钟
      向混合物中加入2g Na 2 CO 3 sub混合物
      通过在25℃下轻轻摇动约4天来实现偶联 用2L的dH 2 O和2L的1M KCl洗涤浆料 将红 - 琼脂糖4B储存在4℃下
    4. 1.5M Tris-HCl缓冲液(pH 8.8,100ml) 将18.2g Tris碱与80ml dH 2 O混合 用盐酸将pH调节至8.8 将dH <2> O添加到100 ml
      高压灭菌器
      在室温下贮存
    5. 0.5M Tris-HCl缓冲液(pH 6.8,100ml) 将6.1g Tris碱与80ml dH 2 O混合 用盐酸将pH调节至6.8 将dH <2> O添加到100 ml
      高压灭菌器
      在室温下贮存
    6. 用于Native-PAGE(厚度,1mm;宽,106mm;高,80mm)的聚丙烯酰胺板状凝胶
      分离范围(7.5%聚丙烯酰胺凝胶,6ml)
      1.5M Tris-HCl缓冲液(pH 8.8)
      1.5 ml
      29%(w/v)丙烯酰胺+ 1%(w/v)N,N' - 甲基双丙烯酰胺溶液
      1.5 ml
      10%(w/v)过硫酸铵
      200μl
      dH 2 2 O 2.8 ml
      N,N,N',N''''''' - 四甲基乙二胺
      3微升

      堆叠范围(4.5%聚丙烯酰胺凝胶,3ml)
      0.5M Tris-HCl缓冲液(pH 6.8)
      0.75 ml
      29%(w/v)丙烯酰胺+ 1%(w/v)N',N' - 甲基双丙烯酰胺溶液
      0.45 ml
      10%(w/v)过氧二硫酸铵
      100微升
      dH 2 2 O 1.7 ml
      N,N,N',N' - 四甲基乙二胺
      3微升

    7. Native-PAGE电泳缓冲液(1L)
      将3.03g Tris碱,14.4g甘氨酸和900ml dH 2 O混合。 将dH <2> O添加到1 L
      高压灭菌器
      在室温下贮存
    8. 2x Native-PAGE凝胶上样缓冲液(10ml) 混合2.5ml 0.5M Tris-HCl缓冲液(pH6.8),1ml 2-巯基乙醇,1g蔗糖,1mg溴酚蓝和8ml dH 2 2 O 将dH 2加到10ml ml/h 储存于-20°C

  2. 异亮氨酸2-差向异构酶活性测定
    1. 0.5M磷酸钠缓冲液(pH8.0,100ml) 将1.06g的NaH 2 PO 4·4·2H 2·2H 2 O和15.5g的Na 2 PO 4混合, /sub>HPO<4.12H<2O与80ml dH 2 O
      用HCl或NaOH(水溶液)将pH调节至8.0 将dH <2> O添加到100 ml
      高压灭菌器
      在室温下储存

致谢

在这个方法中,我们修改了Native-PAGE的Laemmli的方法。此外,这项工作得到了来自生物技术研究推进机构(BRAIN)和JSPS KAKENHI Grant号2402734的用于促进创新生物科学的基础研究活动的赠款的支持。

参考文献

  1. Ohshima,T。和Sakuraba,H。(1986)。 来自光养细菌,红假单胞菌(Rhodopseudomonas capsulata)的苹果酸脱氢酶的纯化和表征。 Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology 869(2):171-177。
  2. Laemmli,U.K。(1970)。 在噬菌体T4头部装配过程中切割结构蛋白。 自然 227(5259):680-685。
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引用:Mutaguchi, Y. and Ohshima, T. (2015). Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040. Bio-protocol 5(20): e1632. DOI: 10.21769/BioProtoc.1632.
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