JP3129180B2 - Method for producing D-histidine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing D-histidine using a microorganism.

[0002]

2. Description of the Related Art D-histidine is a compound useful as a raw material of pharmaceuticals such as antibiotics, a synthetic intermediate or an optical resolving agent. Conventionally, as a method for producing these D-histidines,
A physicochemical method such as a fractional crystallization method of a racemate, an optical resolution method by chromatography or an organic asymmetric synthesis method, or asymmetric hydrolysis of 5- (4-imidazolemethyl) -hydantoin using a microbial enzyme. Decomposition method (Ag
ric. Biol. Chem. , Vol. 51,715
-719 (1987)), DN-carbamoyl-α-
Method for hydrolyzing histidine (JP-A-4-81057)
9) Alternatively, a method of transferring an amino group to α-keto acid using a microbial enzyme (J. Biotechnol., Vo)
l. 8, 243-248 (1988)) and the like.

[0003]

The above-mentioned physicochemical methods are difficult to operate, and have disadvantages such as low product yield and low optical purity. (4
-Imidazolemethyl) -hydantoin and DN-carbamoyl-α-histidine are expensive and have the drawback that coenzyme regeneration is required. Therefore, development of a method for producing D-histidine that solves at least one of the above problems is desired.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found a microorganism having an ability to selectively degrade L-histidine in racemic histidine, and have completed the present invention. Was.

[0005] That is, the present invention provides a method wherein a culture of a microorganism having the ability to asymmetrically degrade L-histidine or a processed product thereof is allowed to act on racemic histidine, and the remaining D-histidine is then treated.
A method for producing D-histidine, comprising separating and collecting histidine.

The microorganism used in the present invention may be any microorganism that has the ability to asymmetrically degrade the L-type optically active substance in racemic histidine, ie, the ability to asymmetrically degrade L-histidine. Such microorganisms include, for example, Acinetobacter, Enterobacter, Klebsiella,
Microorganism belonging to the genus Corynebacterium, Pseudomonas, Serratia, Bacillus, Hafnia, Paracoccus, Flavobacterium, Brevibacterium, Proteus, Providencia, Morganella, Candida, Hansenula or Saccharomyces Is raised. Among these, microorganisms belonging to the genus Proteus or Providencia are preferred, and microorganisms belonging to the genus Proteus (eg, Proteus vulgaris) are particularly preferred. Specific examples of such microorganisms include Acinetobacter calcoacetics (Acinetoba).
cter calcoaceticus) IFO125
52, Enterobacter Aerogenes
Bacter aerogenes) IFO1201
0, Klebsiell
a pneumoniae) ATCC10031, I
FO3317, Corynebacterium alkanolitacum (Corynebacterium alkanol)
yticum) ATCC 21511, Pseudomonas
Aeruginosa (Pseudomonas aerug)
inosa) IAM1220, ATCC7700, IFO3918, Pseudomonas aureofacien (Pseudomonas aureofacien)
s) IAM1001, Pseudomonas obalis (Ps
eusomonas ovalis) IAM1177,
IAM1094, IAM1153, Pseudomonas sc
huylkillensis) IAM1126, Pseudomonas
gelidicola) OUT8116 (FERMP)
-14991), Pseudomonas Puchida (Pseu)
domonas putida) ATCC 33015,
Pseudomonas Fluorescence (Pseudomo)
nas fluorescens) IFO 3081, IAM 1219, Pseudomonas flagi (Pseud
omonasfragi) OUT8255, Serratia
Primurtica (Serratia plymutic)
a) IAM1255, Serratia marcessens (Se
rtia marcescens) ATCC147
64, ATCC 19180, ATCC 27117,
IAM1104, IAM12143, IAM12
359, IFO3735, IFO12648, Serratia liqienci
efaciens) ATCC 27592, Bacillus
Coagulans (Bacillus coagulan)
s) IFO 12714, Hafnia Albay (Hafn)
ia alvei) IFO3731, Paracoccus denit
rificans IFO12442, Flavobacterium sp.
p. ) FERM P-6901, Brevibacterium
Herbolum (Brevibacterium helv)
olum) IAM1637, Proteus vulgaris RIMD KS
(IAM12003), Proteus mirabilis (Pr
oteus mirabilis) IFO3849, Providencia alkaline faciliens (Provide)
ncia alcalifaiens) JCM167
3. Providencia Rittigeri (Providenc)
ia rettgeri) IFO13501, ATC
C21118, ATCC25932, ATCC99
19. Providencia Rusticiani (Provid
encia rusticianii) JCM395
3. Morganella Morgani
organii) IFO3848, Candida boidiniii IFO10
240, Hansenula polymorpha
apolymorpha IFO1024, Saccharomyces cerevisiae
revisiae) IFO2342, IFO234
5, the same IFO2114.

[0007] These microorganisms may be any strains as long as they have the necessary ability for the present invention.
It may be a mutant obtained by artificial treatment such as ultraviolet irradiation or treatment with a mutagen. Further, those derived from these microorganisms by genetic engineering or biotechnological techniques such as a recombinant DNA method and a cell fusion method may be used. For example, according to a genetic engineering technique, a gene of the target enzyme is isolated from a chromosome fragment of a microorganism producing an asymmetric degrading enzyme, and a recombinant plasmid is prepared by inserting the gene into an appropriate plasmid vector. By transforming an appropriate host microorganism with the above, a microorganism having an enzyme-producing ability or a microorganism having further improved enzyme-producing ability can be obtained. Furthermore, the recombinant plasmid may be used to transform a host microorganism having other excellent properties (for example, easy cultivation), if necessary.

[0008] As the culture in the present invention, for example,
Examples include a culture solution or viable cells of the microorganism,
Examples of the processed product of the culture include a processed product of a culture solution (such as a culture supernatant) and a processed product of cells.

The above-mentioned culture (culture solution, cells, etc.) can be prepared, for example, by subjecting the microorganism to a medium usually used in this field (a conventional medium containing a carbon source, a nitrogen source and inorganic salts) at normal temperature or under heating ( (Preferably about 20 ° C. to about 40 ° C.) and cultured under aerobic conditions at a pH of about 5 to about 8, and if necessary, the cells are separated and collected from the thus obtained culture by a conventional method. Can be obtained. In culturing, histidine can be added to the medium in an amount of about 0.001 to about 10%, particularly about 0.1 to about 1% to increase the enzyme activity.

[0010] As the treated cells of microorganisms, the above culture is subjected to various physicochemical methods, for example, ultrasonic wave, French press, osmotic pressure, freeze-thaw, freeze-drying, alumina destruction, lytic enzyme, surfactant or In addition to the cells treated with an organic solvent or the like, a partial purification prepared by a known method from the culture (culture solution, cells, etc.) or its treated product (culture supernatant, treated cells, etc.) An enzyme or a purified enzyme having an ability to asymmetrically degrade L-histidine in racemic histidine can be mentioned.

Further, the microbial cells, treated cells or enzymes of the present invention may be immobilized by, for example, a polyacrylamide method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginic acid gel method, or an agar gel method. Can also be used.

Further, a microorganism having the ability to asymmetrically degrade L-histidine can be cultured in a medium containing racemic histidine, and D-histidine remaining in the medium can be separated and collected.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The asymmetric decomposition reaction according to the present invention comprises:
It can be carried out by bringing a culture of a microorganism having the ability to asymmetrically degrade L-histidine or a processed product thereof into a solution and incubating the racemic histidine as a raw material compound with a solution. In addition, the reaction may be performed in parallel with the culture of the microorganism.If the reaction is performed in parallel with the culture of the microorganism, the reaction is performed under the same conditions as the culture using a medium to which racemic histidine is added in advance. Just do it.

The reaction of the present invention can be suitably carried out in an aqueous solution, and the reaction is carried out at room temperature to under heating, preferably about 10 to 10 hours.
The process preferably proceeds at about 50 ° C, particularly preferably at about 25 to about 40 ° C. The reaction solution has a pH of about 5 to about 11, especially pH.
Preferably, it is adjusted to be about 6 to about 9.

The feed concentration of the racemic histidine, which is a starting compound serving as a reaction substrate, is usually about 0.05 to about 30.
%, Especially about 1 to about 20%. At that time, the reaction substrate may be added all at once, or may be added in several portions during the reaction.

As the racemic histidine as a raw material compound, not only those containing D-histidine and L-histidine in equal amounts but also those containing both optically active substances can be used.

When a viable cell is used in the present invention, the reaction time can be reduced by adding a surfactant to the reaction solution. Examples of the surfactant used for this purpose include cetylpyridinium bromide, cetyltrimethylammonium bromide and p-isooctylphenyl ether (trade name: Triton X-100, manufactured by Rohm and Haas Company, USA). About 0.
It is preferable to use about 0001 to about 0.1%.

After completion of the reaction, D-histidine can be easily collected and isolated from the reaction solution according to a conventional method. For example, after removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, the solution obtained by adsorbing and removing dyes and the like with activated carbon is concentrated under reduced pressure, and cooled and crystallized to obtain D-.
Histidine crystals can be obtained.

Further, the culture of the microorganism or the processed product thereof may be:
Assaying whether or not it has the ability to asymmetrically degrade L-histidine in racemic histidine can be easily carried out according to the above reaction method, for example, as follows. That is, a culture or a processed product of a microorganism to be assayed is added to a medium or an aqueous solution containing racemic histidine,
React at 120 ° C for 120 hours. The reaction-terminated liquid is applied to an optically active column (for example, SUMICHI, manufactured by Sumika Chemical Analysis Service, Japan)
(RAL OA-5000), and can be analyzed and quantified by high performance liquid chromatography to measure the content of each of D-histidine and L-histidine. By measurement, for example, when the L-type optically active substance decreases and D-histidine remains, it is determined that the substance has the ability to asymmetrically degrade L-histidine.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

In this specification, "%" means "weight / volume (g / dl)". In this example, the quantification of the optically active form of histidine was determined by S
This was performed by high performance liquid chromatography using UMICHIRAL OA-5000 (manufactured by Sumika Chemical Analysis Service).

[0022]

EXAMPLES Example 1 DL-histidine 2%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.0
3 ml of medium consisting of 5% and 0.02% yeast extract (pH
7.0) was placed in a test tube, and sterilized at 120 ° C. for 10 minutes. Microorganisms shown in Tables 1 and 2 below were inoculated into this medium, and after shaking culture at 30 ° C. for 168 hours, D-histidine remaining in the culture solution was quantified. The contents of D-histidine are shown in Tables 1 and 2 below, and L-histidine, which is an enantiomer thereof, was hardly detected in the culture solution.

[0023]

[Table 1]

[0024]

[Table 2]

Example 2 100 ml of a medium (pH 7.0) consisting of 0.5% of DL-histidine, 1.0% of polypeptone, 1.0% of yeast extract and 0.5% of sodium chloride were placed in a 500 ml shake flask. Sterilized at 120 ° C. for 10 minutes. Proteus vulgaris (Proteus vulgar) is added to this medium.
is) One loopful of RIMD KS (IAM12003) was inoculated and cultured at 30 ° C for 20 hours. The above culture solution 200
The cells collected from 0 ml by centrifugation were suspended in physiological saline, and then collected by centrifugation. DL
-800 mM 50 mM phosphate buffer containing 80 g histidine
Then, L-histidine was completely decomposed by adding an asymmetric decomposition reaction at 30 ° C. for 48 hours. After the reaction, the bacteria were removed by centrifugation to obtain a supernatant. The supernatant was subjected to ultrafiltration to remove proteins and the like to obtain a filtrate. The filtrate was decolorized with activated carbon to obtain a decolorized liquid. The decolorizing solution is an ion exchange resin (manufactured by Mitsubishi Kasei, Diaion SK11).
6) and adsorbed the product. The adsorbate was eluted with aqueous ammonia, and the eluate was concentrated under reduced pressure and crystallized by cooling to obtain 13.6 g of D-histidine crystals.

Optical rotation: [α] _D ²⁰ = 11.9 ° (C
= 11.6N HCl) Optical purity: 100% Example 3 3 ml of a medium consisting of 1.0% DL-histidine, 1.0% polypeptone, 1.0% yeast extract and 0.5% sodium chloride (pH 7.0) Were inoculated with the bacteria shown in Table 3 below, and cultured at 30 ° C. for 20 hours. The cells collected by centrifugation from 3 ml of the above culture solution were suspended in physiological saline, and then collected by centrifugation. 2 ml of a 50 mM phosphate buffer containing 10% DL-histidine (pH 7.
0) was added, and an asymmetric decomposition reaction was performed at 30 ° C. for 168 hours.
The D-histidine content of this reaction solution is as shown in Table 3 below, and its enantiomer, L-histidine, was hardly detected in the reaction solution.

[0027]

[Table 3]

[0028]

The method of the present invention is an industrially advantageous production method because D-histidine can be efficiently produced from industrially inexpensive racemic histidine with high optical purity.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI (C12P 41/00 C12R 1: 385) (C12P 41/00 C12R 1: 385) (C12P 41/00 C12R 1:38) (C12P 41/00 C12R 1:40) (C12P 41/00 C12R 1:39) (C12P 41/00 C12R 1:43) (C12P 41/00 C12R 1: 425) (C12P 41/00 C12R 1:07) (C12P 41/00 C12R 1:13) (C12P 41/00 C12R 1:37) (C12P 41/00 C12R 1:72) (C12P 41/00 C12R 1:78) (C12P 41/00 C12R 1: 865) (56 References JP-A-49-61390 (JP, A) JP-A-48-10290 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 41/00 BIOSIS (DIALOG) CA (STN)

Claims (4)

(57) [Claims] To 1. A racemic histidine, L- histidine a microorganism having ability of asymmetrically degrading Ashinetoba
Genus, Enterobacter, Klebsiella, coli
Nebacterium, Pseudomonas, Serratia, Ba
Shirasu, Hafnia, Brevibacterium, Prote
Usus, Providencia, Morganella, Candida
Genus, a culture of a microorganism belonging to the genus Hansenula or the genus Saccharomyces or a treated product thereof, and the remaining D-
A method for producing D-histidine, comprising separating and collecting histidine. 2. The method according to claim 1, wherein the microorganism capable of asymmetrically degrading L-histidine is a microorganism belonging to the genus Proteus or Providencia. 3. L- histidine a microorganism having ability of asymmetrically degrading the claim 2 is a Proteus vulgaris
The manufacturing method described. 4. In a medium containing racemic histidine, L
The method according to any one of claims 1 to 3, wherein a microorganism having an ability to asymmetrically degrade histidine is cultured, and D-histidine remaining in the medium is separated and collected.