JPWO2007105788A1 - Amino acid purification method - Google Patents

Abstract

Separating the amino acid from a culture solution containing a hydrophobic amino acid selected from the group consisting of alanine, leucine, valine, isoleucine, phenylalanine and proline or a neutral polar amino acid selected from the group consisting of serine, glycine, asparagine and glutamine, In the purification method, the culture solution containing the hydrophobic amino acid or neutral polar amino acid and the microbial cell is a column packed with a particle carrier having a particle diameter of 350 μm or more and capable of adsorbing the amino acid. By passing through the eluate after passing through the upper part, hydrophobic amino acids or neutral polar amino acids can be separated and purified.

Description

  The present invention relates to a method for purifying amino acids.

  As a method for separating and purifying hydrophobic amino acids or neutral polar amino acids produced by fermentation from the culture solution, microorganisms can be obtained by means such as centrifugation, condensation precipitation using a polymer coagulant, or ultrafiltration. A method is known in which a culture solution from which solid components such as microbial cells are removed is passed through an ion exchange resin so that the amino acid is adsorbed onto the resin and then the amino acid is eluted (Patent Document 1, etc.). Also, a method for purifying valine, characterized in that a culture solution containing valine is adjusted to around pH5.96 (the isoelectric point of valine) and fed to a strongly acidic cation exchange resin that has been previously converted to an ammonium salt form (Patent Document) 2) is also known. However, both methods require a pretreatment of removing microbial cells from the culture solution before bringing the ion exchange resin into contact with the culture solution. In addition, Patent Document 2 describes that cells may be removed as necessary, but an operation for accurately adjusting the pH of the culture solution, and when multiple types of cations are present as impurities in the culture solution Needs to be pretreated with a cation exchange resin in advance.

A culture solution containing microorganism cells is passed through a column filled with an ion exchange resin from above to adsorb amino acids to the resin, and then the cells attached to the resin by passing water from below the column. Is known, and the suspended cells are removed from above the column and amino acids are eluted (Patent Document 3). However, in this method, most of the cells contained in the culture solution together with the amino acids are converted into the resin. There is a problem that the purification efficiency of amino acids is poor due to adhesion.
Japanese Patent Publication No.39-5050 No. 6-17344 Japanese Patent Publication No. 4-53509

  An object of the present invention is to provide a method for easily and efficiently purifying a highly pure amino acid from a culture solution containing microbial cells.

The present invention relates to the following (1) to (3).
(1) Hydrophobic amino acid selected from the group consisting of alanine, leucine, valine, isoleucine, phenylalanine and proline, or the amino acid from a culture solution containing neutral polar amino acid selected from the group consisting of serine, glycine, asparagine and glutamine In the method for separating and purifying the above, the culture solution containing the hydrophobic amino acid or neutral polar amino acid and the microbial cell is filled with a particle carrier having a particle diameter of 350 μm or more and capable of adsorbing the amino acid. A method comprising separating and purifying a hydrophobic amino acid or a neutral polar amino acid by passing through the top of the column and then passing through the eluate.
(2) The method according to (1) above, wherein the particle carrier is a strongly acidic cation exchange resin.
(3) The method of (1) or (2) above, wherein the pH of the culture solution passing through the column is 1 to 4.

  According to the present invention, a highly pure amino acid can be purified easily and inexpensively.

  In the present invention, a hydrophobic amino acid selected from the group consisting of alanine, leucine, valine, isoleucine, phenylalanine and proline or a neutral polar amino acid selected from the group consisting of serine, glycine, asparagine and glutamine (hereinafter simply referred to as hydrophobic amino acid or As a culture solution containing a microbial cell of a microorganism and a neutral polar amino acid, a culture obtained by culturing a microorganism having the ability to produce the amino acid in a medium, and generating and accumulating the amino acid in the medium You can give liquid.

The microorganism described above may be any microorganism as long as it has an ability to produce a hydrophobic amino acid or a neutral polar amino acid, preferably a prokaryote, more preferably a bacterium. As prokaryotes, Escherichia (Escherichia) genus Serratia (Serratia), Bacillus, Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus, the genus Brevibacterium (Microbacterium), Pseudomonas (Pseudomonas) genus, Agrobacterium (Agrobacterium) genus, belonging to the genus Alicyclobacillus (Alicyclobacillus), Anabaena (Anabena) genus Anashisutisu (Anacystis) genus Asuro Acetobacter (Arthrobacter) genus Azotobacter (Azotobacter) genus Kuromachiumu (Chromatium) genus Erwinia (Erwinia ) genus, Methylobacterium (Methylobacterium) genus, Phormidium (Phormidium) genus Rhodobacter (Rhodobacter) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Rodosupiriumu (Rhodospirillum) genus Scenedesmus (Scenedesmus) genus, stress Tomaisesu (Streptomyces) genus, Shinekokkasu (Synechoccus) genus Zymomonas (Zymomonas) microorganism belonging to the genus, such as, for example, Escherichia coli, Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus coagulans , Bacillus licheniformis , Bacillus pumilus , Brevibacterium ammoniagenes , Brevibacterium immariofilm ), Brevibacterium Sacca Lori tee cam (Brevibacterium saccharolyticum), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium Men Beam (Brevibacterium lactofermentum), Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium acetamide A Sid film (Corynebacterium acetoacidophilum), micro Corynebacterium ammoniagenes film (Microbacterium ammoniaphilum), Serratia Fikaria (Serratia ficaria), Serratia Fonchikora ( Serratia fonticola ), Serratia liquefaciens , Serratia marcescens , Pseudomonas aeruginosa , Pseudomonas putida, Pseudomonas putida , Agrobacterium radiobacter Umm Rizzo jeans (Agrobacter iumrhizogenes), Agrobacterium ruby (Agrobacterium rubi), Anabaena-Shirindorika (Anabaen a cylindrica ), Anabaena doliolum , Anabaena flos-aquae , Arthrobacter aurescens , Arthrobacter citreus , Arthrobacter citreus Miss ( Arthrobacter globformis ), Arthrobacter hydrocarboglutamicus , Arthrobacter mysorens , Arthrobacter nicotianae , Arthrobacter nicotianae , Arthrobacter paraffin , Arteubacter Robakuta proto folder Mie (Arthrobacter protophormiae), Arthrobacter Rose opal Rafi eggplant (Arthrobacter roseoparaffinus), Arthrobacter sulphureus (Arthrobacter sulfureus), Asurobakuta - urea tumefaciens (Arthrobacter ureafaciens), Kuromachiumu-Buderi (Chromatium buderi), Kuromachiumu-Tepidamu (Chromatium tepidum), Kuromachiumu-Binosamu (Chromatium vinosum), Kuromachiumu-Wamingi (Chromatium warmingii), Kuromachiumu-Furubiatatire (Chromatium fluviatile), Erwinia · Uredobara (Erwinia uredovora), Erwinia Karotobara (Erwinia carotovora), Erwinia Anas (Erwinia ananas), Erwinia Herikora (Erwinia herbicola), Erwinia Pankutata (Erwinia punctata), Erwinia terreus (Erwinia terreus), Mechirobakuteri um Rodeshianamu (Methylobacterium rhodesianum), Methylobacterium-exo torque Enns (Methylobacterium extorquens), Phormidium sp (Phormidium sp.) ATCC29409, Russia Dobakuta-Kapusuratasu (Rhodobacter capsulatus), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rod Pseudomonas Burasuchika (Rhodopseudomonas blastica), Rod Pseudomonas Marina (Rhodopseudomona smarina), Rod Pseudomonas pulse tris (Rhodopseudomonas palustris), Rodosupiriumu-Riburamu (Rhodospirillum rubrum ), Rodosupiriumu-Sarekishigensu (Rhodospirillum salexigens), Rodosupiriumu-Sarinaramu (Rhodospirillum salinarum), Streptomyces ambofaciens (Streptomyces ambofaciens), Streptomyces aureofaciens (Streptomyces aureofaciens), Streptomyces aureus (Streptomyces aureus ), Streptomyces fungicidicus ( Streptomyces fungicidicus ), Streptomyces grise Okuromogenasu (Streptomyces griseochromogenes), Streptomyces griseus (Streptomyces griseus), Streptomyces lividans (Streptomyces lividans), Streptomyces Oriboguriseusu (Streptomyces olivogriseus), Streptomyces Rameusu (Streptomyces rameus), Streptomyces Examples thereof include Streptomyces tanashiensis , Streptomyces vinaceus , and Zymomonas mobilis .

  Preferred prokaryotes are more preferably bacteria belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Methylobacterium, Microbacterium, Pseudomonas or Streptomyces. May include bacteria belonging to the genus Escherichia, Corynebacterium, Methylobacterium or Microbacteria, such as Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Species belonging to the genus Methylobacterium, Microbacterium, Pseudomonas or Streptomyces, preferably the species belonging to the genus Escherichia, Corynebacterium, Methylobacterium or Microbacterium it can.

  More preferred bacteria include Escherichia coli, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium lactofermentum, Corynebacterium flavum, Corynebacterium efficasis, Methylobacterium rhodesianum, Micro Bacteria ammonia film, Bacillus subtilis, Bacillus megaterium, Serratia marcescens, Pseudomonas putida, Pseudomonas aeruginosa, Streptomyces sericolor and Streptomyces lividans, more preferably Escherichia coli, Coryne Examples include bacteria, glutamicum, methylobacterium rhodesianum, and microbacteria ammonia fill. .

The amino acid in the present invention may be any of L-form, DL-form, D-form and mixtures thereof.
As a medium for culturing the above-mentioned microorganism, any medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism, and can efficiently culture the microorganism. It may be used.
Any carbon source may be used as long as the microorganism can assimilate, glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol, Alcohols such as propanol can be used.

  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 6 hours to 14 days. The pH during the culture is preferably maintained at 4.0 to 10.0.

  The particle carrier used in the present invention is a microbial cell, preferably a prokaryotic cell, more preferably a bacterial cell, more preferably Escherichia or Corynebacterium, between the particles in a column packed with the particle carrier. Bacteria cells belonging to the genus Methylobacterium or Microbacteria, particularly preferably Escherichia coli, Corynebacterium glutamicum, Methylobacterium rhodesianum or microbacteria ammonia film Any particle carrier having a particle size capable of forming voids may be used, and the particle size may be non-uniform.

  Examples of the particle carrier include particle carriers having a particle diameter of 350 μm or more, preferably 400 μm or more, more preferably 420 μm or more, further preferably 500 μm or more, and most preferably 600 μm or more. Examples of the particle carrier having the above-described particle size include a particle carrier that can be obtained by screening a particle carrier having a non-uniform particle size through a sieve having an aperture of 0.35 mm, 0.40 mm, 0.42 mm, 0.50 mm, or 0.60 mm. be able to.

  In addition, since the microbial cells are more likely to pass between particles as the voids between particles are larger, the particle carrier used in the present invention has no particular upper limit on the particle size as long as the particle size is 350 μm or more, As a carrier that is easy to handle and has a high amino acid purification efficiency, the content of particles having a particle size of 2000 μm or more is 10% or less, preferably the content of particles having a particle size of 1500 μm or more is 10% or less, more preferably 1180 μm or more. Examples thereof include a carrier having a particle content of 10% or less.

  The particle carrier capable of adsorbing hydrophobic amino acids or neutral polar amino acids used in the present invention is obtained from a culture solution containing the amino acids and impurities such as by-product amino acids, sulfate ions, chloride ions and dyes. There is no particular limitation as long as it is a particle carrier that can selectively separate and purify hydrophobic amino acids or neutral polar amino acids based on the difference in adsorbability, and a strong acid cation exchange resin can be preferably mentioned.

  As the above-mentioned particle carrier, the Dowex series (HCR-S, HCR-W2, Marathon C, Monosphere 650C, MSC-1, Monosphere 88, 50W × manufactured by Dow Chemicals, which is a strongly acidic cation exchange resin, is used. 2, 50W × 4 and 50W × 8 etc.), Diaion SK series (SK1B, SK102, SK104, SK110, SK112 and SK116 etc.) and Diaion PK series (PK204, PK208 and PK212 etc.) and ROHM made by Mitsubishi Chemical A particle carrier obtained by adjusting the particle diameter from the Amberlite series (IR120B, IR122, IR124, XE-100, etc.) manufactured by And Haas can be mentioned.

The ion form of the strongly acidic cation exchange resin can be appropriately selected according to the amino acid to be separated and purified.
As a method for adjusting the particle diameter of the particle carrier, the particle carrier is sieved with a sieve having an opening of 0.35 mm, preferably 0.40 mm, more preferably 0.42 mm, more preferably 0.50 mm, and most preferably 0.60 mm. For example, a method of collecting a particle carrier that does not pass through a sieve mesh can be used. In the above particle carrier, when a commercially available product is a carrier composed of particles having a particle size of 350 μm or more, such as Marathon C, it can be used in the present invention without adjusting the particle size.

The strongly acidic cation exchange resin may be a gel type or a porous type, and the degree of crosslinking of the resin is not particularly limited, but the degree of crosslinking is preferably 4 to 16%, more preferably 6 to 10%.
The concentration of the hydrophobic amino acid or neutral polar amino acid in the culture solution passing through the column packed with the particle carrier is not particularly limited as long as the amino acid is dissolved. When crystals of hydrophobic amino acids or neutral polar amino acids are precipitated in the culture medium after completion of the culture, the amino acid crystals are dissolved by adding water, warming or adding an acid, or The separated culture solution can be passed through.

  The pH of the culture solution passing through the column is not limited as long as the particle carrier used in the method of the present invention can adsorb hydrophobic amino acids or neutral polar amino acids, but the range is preferably 1 to 1. 4, more preferably 1 to 3.5. If necessary, the pH of the culture solution is adjusted to an inorganic or organic acid such as hydrochloric acid, sulfuric acid, acetic acid or malic acid, an alkaline solution such as sodium hydroxide, urea or carbonic acid. It can adjust within the said range using calcium, ammonia, etc.

The column used in the present invention may be any column that is usually used for purification of chemical substances.
The amount of the particle carrier used in the present invention can be appropriately set according to the type of the hydrophobic amino acid or neutral polar amino acid to be purified, the pH of the culture medium to be passed, for example, the amino acid concentration in the culture liquid Can be increased by 1 to 2 times the amount of the culture solution.

In the method of the present invention, a culture solution containing hydrophobic amino acids or neutral polar amino acids and microbial cells has a particle diameter of 350 μm or more and has the ability to adsorb hydrophobic amino acids or neutral polar amino acids. It passes through the upper part of the column packed with the particle carrier, that is, the so-called upper layer of the column bed.
The tower speed is preferably 0.3 to 10 m / h, more preferably 0.5 to 7 m / h.

After passing the culture solution, the culture solution remaining in the column can be pushed out and washed by passing water or the like from the upper part or the lower part of the column as necessary.
The eluate is preferably passed continuously from the top of the column, and the amino acids can be separated and purified by eluting hydrophobic amino acids or neutral polar amino acids.
The eluate used in the present invention is not particularly limited in type and concentration as long as it is a solution capable of eluting hydrophobic amino acids or neutral polar amino acids bound to the particle carrier. May be 0.2 to 6 mol / L, more preferably 0.5 to 3 mol / L of an aqueous ammonia solution, an aqueous sodium hydroxide solution or the like.

The column speed of the eluate is preferably 0.3 to 10 m / h, more preferably 0.5 to 7 m / h.
The hydrophobic amino acid or neutral polar amino acid separated and purified as described above can be further purified by means such as decolorization, concentration and crystallization.
The particle carrier in the column after eluting the hydrophobic amino acid or neutral polar amino acid can be regenerated by passing an appropriate solvent such as water from the top of the column and pushing out the eluate inside the column. When a strongly acidic cation exchange resin is used as a particle carrier, it is repeatedly used in the method of the present invention by extruding the eluate inside the column by passing water through the column without performing a special resin regeneration operation. be able to.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to an Example.

Purification of L-alanine
After adjusting the pH of the culture solution 24L containing 25g / l of L-alanine and 720g of microorganisms belonging to the genus Microbacteria by wet cell weight to pH 2.0 with sulfuric acid, particles with a particle size of less than 420μm were removed by sieving Furthermore, L-alanine was adsorbed by passing through a column (packing height 6 m) packed with 12 L of SK1B (Mitsubishi Chemical Co., Ltd. H type) at 30 ° C. and a linear velocity of 6 m / h. Next, 10 L of water was passed from above to extrude the culture solution remaining in the column. At this time, the cell removal rate was 90%. The column was washed by passing water from the bottom of the column and then eluted with a 1 mol / L aqueous ammonia solution to obtain 15 L of an eluted fraction.

The obtained elution fraction was concentrated to 6 L to remove ammonia. After adding 60 g of activated carbon to the concentrate and stirring for 30 minutes, the mixture was decolored and filtered, and then concentrated to 1 L to obtain an L-alanine crystal slurry.
After separating the crystals from the crystal slurry using a basket type separator, the crystals were washed with water and dried. As a result, 300 g of L-alanine crystals (yield 50%, purity 99%, no bacterial contamination) were obtained.

Purification of L-valine
SK1B in which 25L of a culture solution containing L-valine 40g / l and microbial cells belonging to the genus Escherichia in a wet cell weight of 2500g was adjusted to pH 2.0 with sulfuric acid, and particles with a particle diameter of less than 420μm were removed by sieving (H type) L-valine was adsorbed by passing through a column packed with 12 L (packing height 6 m) at 30 ° C. and a linear velocity of 6 m / h. Next, 10 L of water was passed from above to extrude the culture solution remaining in the column. At this time, the cell removal rate was 90%. The column was washed by passing water from the bottom of the column and then eluted with a 0.5 mol / L aqueous ammonia solution to obtain an elution fraction of 40 L.

The obtained elution fraction was concentrated to 25 L to remove ammonia. 100 g of activated carbon was added to the concentrated liquid, and after decoloring and filtering by stirring for 30 minutes, L-valine crystal slurry was obtained by concentrating to 3 L.
After separating the crystals from the crystal slurry using a basket type separator, the crystals were washed with water and dried. As a result, 700 g of L-valine crystals (yield 70%, purity 98%, no contamination with bacterial cells) were obtained.

Purification of L-serine
After adjusting the pH of the culture solution 12L containing 50g / l of L-serine and 1560g of microbial cells belonging to the genus Methylobacterium to pH 3.0 with sulfuric acid, particles with a particle size of less than 420μm were screened. L-serine was adsorbed by passing through a column (packing height: 6 m) packed with SK1B (H type) 12 L removed at 30 ° C. and a linear velocity of 6 m / h. Next, 10 L of water was passed from above to extrude the culture solution remaining in the column. At this time, the cell removal rate was 90%. The column was washed by passing water from below the column, and then eluted with a 2 mol / L aqueous ammonia solution to obtain 15 L of an elution fraction.

The obtained elution fraction was concentrated to 6 L to remove ammonia. After adding 60 g of activated carbon to the concentrate and stirring for 30 minutes, the mixture was decolored and filtered, and 1 L of methanol was added to obtain an L-serine crystal slurry.
After separating the crystals from the crystal slurry using a basket type separator, the crystals were washed with water and dried. As a result, 300 g of L-serine crystals (yield 50%, purity 98%, no contamination with bacterial cells) were obtained.

Purification of L-glutamine (1)
After adjusting the pH to 3.5 with sulfuric acid, 12L of the culture solution containing 50g / l of L-glutamine and 480g of microbial cells belonging to the genus Corynebacterium was removed to particles of less than 420μm by sieving. L-glutamine was adsorbed by passing through a column (packing height 6 m) packed with SK1B (H type) 12 L at 30 ° C. and a linear velocity of 6 m / h. Next, 10 L of water was passed from above to extrude the culture solution remaining in the column. At this time, the cell removal rate was 90%. The column was washed by passing water from below the column, and then eluted with a 0.5 mol / L aqueous ammonia solution to obtain 25 L of an elution fraction.

The obtained elution fraction was concentrated to 15 L to remove ammonia. After adding 60 g of activated carbon to the concentrate and stirring for 30 minutes, the mixture was decolored and filtered, and then concentrated to 1 L to obtain an L-glutamine crystal slurry.
After separating the crystals from the crystal slurry using a basket type separator, the crystals were washed with water and dried. As a result, 400 g of L-glutamine crystals (yield 67%, purity 99%, no contamination with bacterial cells) were obtained.

Purification of L-glutamine (2)
After adjusting 12L of a culture solution containing L-glutamine 50g / l and microorganisms belonging to the genus Corynebacterium in a wet cell weight of 480g to pH 3.5 with sulfuric acid, Marathon C (Dow) -Chemicals Co., Ltd. Particle size 535-635μm, Uniformity coefficient 1.1 or less, H-type) Adsorbed L-glutamine by passing through a column (packing height 6m) at 30 ° C and linear speed 6m / h. I let you. Next, 10 L of water was passed from above to extrude the culture solution remaining in the column. At this time, the cell removal rate was 90%. The column was washed by passing water from below the column, and then eluted with a 0.5 mol / L aqueous ammonia solution to obtain 25 L of an elution fraction.

The obtained elution fraction was concentrated to 15 L to remove ammonia. After adding 60 g of activated carbon to the concentrate and stirring for 30 minutes, the mixture was decolored and filtered, and then concentrated to 1 L to obtain an L-glutamine crystal slurry.
After separating the crystals from the crystal slurry using a basket type separator, the crystals were washed with water and dried. As a result, 400 g of L-glutamine crystals (yield 67%, purity 99%, no contamination with bacterial cells) were obtained.
Comparative Example 1
Purification of L-alanine A column (packing height) packed with 12 L of SK1B (particle size 297 to 1190 μm, type H) that had not been adjusted for particles by sieving 24 L of the culture solution adjusted to pH by the same method as in Example 1. 6 m) was passed under the same conditions as in Example 1, but when 5 L was passed, the column was blocked.
Comparative Example 2
Purification of L-valine A column (packing height) filled with 12 L of SK1B (particle size 297 to 1190 μm, H type) without adjusting the particle size by sieving 25 L of the culture solution adjusted to pH by the same method as in Example 2. 6 m) was passed in the same manner as in Example 2, but when 3 L was passed, the column was blocked.
Comparative Example 3
Purification of L-Serine A column (packing height) packed with 12 L of SK1B (particle size 297 to 1190 μm, type H) without adjusting the particle size by sieving 12 L of the culture solution adjusted to pH by the same method as in Example 3. 6m) was passed in the same manner as in Example 3, but when 6L was passed, the column was blocked.
Comparative Example 4
Purification of L-glutamine A column packed with 12 L of SK1B (particle size of 297 to 1190 μm, type H) that is not adjusted for particles by sieving 12 L of L-glutamine culture solution adjusted to pH in the same manner as in Example 4. The column was passed through (packing height 6 m) in the same manner as in Example 4, but when 6 L was passed, the column was blocked.

  According to the present invention, amino acids can be easily purified from a culture solution containing hydrophobic amino acids or neutral polar amino acids and microorganism cells.

Claims (3)

  Separating the amino acid from a culture solution containing a hydrophobic amino acid selected from the group consisting of alanine, leucine, valine, isoleucine, phenylalanine and proline or a neutral polar amino acid selected from the group consisting of serine, glycine, asparagine and glutamine, In the purification method, the culture solution containing the hydrophobic amino acid or neutral polar amino acid and the microbial cell is a column packed with a particle carrier having a particle diameter of 350 μm or more and capable of adsorbing the amino acid. A method comprising separating and purifying a hydrophobic amino acid or a neutral polar amino acid by passing through an eluate after passing through an upper part.   The method of claim 1, wherein the particle carrier is a strongly acidic cation exchange resin.   The method according to claim 1 or 2, wherein the pH of the culture solution passing through the column is 1 to 4.