Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040

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