JPH03112491A - Production of l-homophenylalanine - Google Patents

Abstract

PURPOSE:To produce L-homophenylalanine useful as a raw material for a precursor of ACE inhibitor, having high optical purity, efficiently and inexpensively by treating benzyllactic acid with a culture mixture or cells of bacterium having specific action. CONSTITUTION:Benzyllactic acid (substrate concentration: about 0.1-5%) is aerobically treated with a culture mixture of a bacterium such as Corynebacterium glutamicum ATCC 13032 belonging to at least one of Corynebacterium, Brevibacterium, Bacillus, Microbacterium, Citrobacter and Arthrobacter or, capable of converting benzyllactic acid into L- homophenylalanine, or cells or treated material thereof at preferably 25-35 deg.C at pH7.5-9.5 and L-homophenylalanine is isolated from the reaction solution and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing L-homophenylalanine. L-homophenylalanine is extremely useful as a raw material for a compound represented by the following formula (A), which is useful, for example, as a precursor of an ACE inhibitor.

<Conventional technology> Conventionally, as a method for producing L-homophenylalanine,
DL-homophenylalanine/optically active mandelic acid complex is optically resolved from optically active mandelic acid and DL-homophenylalanine, and L-homophenylalanine is collected from the precipitated L-homophenylalanine/D(-)-mandelic acid complex. N-acetyl-DL- using a method of
Asymmetric hydrolysis method of homophenylalanine (l1cm,
Fac, Set,, Kyssb* Uni,, S
er, Jin 13.89 <1981)) are known.

<Problem to be solved by the invention> However, the production of L-homophenylalanine using an enzyme has the disadvantage that the concentration of the reaction substrate is as low as 0.4 to 0.5%, and it is difficult to use an optical resolution agent. In the conventional separation method, the resolving agent is a natural product and is expensive, and in addition, L with high optical purity is used.
-The drawback is that homophenylalanine cannot be obtained in high yield.

An object of the present invention is to provide a method for efficiently and inexpensively producing L-homophenylalanine with high optical purity.

Means to solve problems〉 The above object is achieved by the present invention as described below.

That is, the present invention has the ability to convert benzyl lactic acid to L-homophenylalanine, and is capable of converting benzyl lactic acid into L-homophenylalanine.
Crobacterium genus or Arthrobacterium
(L-homophenylalanine is produced and accumulated by acting on benzyl lactic acid with a culture, bacterial cells, or a processed product thereof of at least one microorganism selected from the microorganisms belonging to the genus Artkrobacterl, and L-homophenylalanine is isolated from the reaction solution. This is a method for producing L-homophenylalanine, which is characterized by separate collection.

Hereinafter, the configuration of the present invention will be explained in detail.

The benzyl lactic acid used as a raw material in the present invention may be either a racemic form or a racemic form, which is usually an industrially advantageous racemic form.

In the present invention, Corynebacterium (C
ory++ebacterium), Brevibacterium [BrevibacteriumJ[, Bacillus genus, Microbacterium (Mic
robactcr1w+al or Arthrobacterium
(At least one type of microorganism selected from microorganisms belonging to the genus Artbrobactcrl is used.

Any such microorganism can be used.

For example, typical examples include Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 21651,
Corynebacterium acetoacidophilum ATC01
3870, Corynebacterium acetoacidophilum ATCC21270, Brevibacterium lactofermentum ATCC13869, Brevibacterium
flavum ATCC 14067, Brevibacterium flavum ATCC 13826, Arthrobacterium citreus ATCC 11624, Cyclobacterium ammoniaphyllum ATCC 15354, Bacillus subtilis ATCC 6051, and the like.

The above microorganisms can be cultured under normal conditions.

For culturing these microorganisms, a medium containing appropriate organic and inorganic carbon sources, nitrogen sources, vitamins, minerals, etc. that can be assimilated by these microorganisms is usually used. That is, as carbon sources for the nutrient medium, for example, sugars such as glucose, fructose, sucrose, and water-soluble starch, sugar alcohols such as glycerol, sorbitol, and mannitol, and organic acids such as fumaric acid and lactic acid can be used. . The amount of carbon source added is usually 0.
Examples of the nitrogen source, which is preferably about 1 to 20%, include ammonium salts of inorganic acids such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate, corn steep liquor, yeast extract,
Natural organic nitrogen sources such as casein hydrolyzate can be used.

The amount of nitrogen source added is usually preferably about 0.1 to 10% based on the medium. Examples of inorganic salts include alkali metal phosphates such as potassium phosphate and sodium phosphate, potassium chloride, and sodium chloride. It is preferable to use metal sulfates such as alkali metal chloride, magnesium sulfate, ferrous sulfate, etc., and the amount of inorganic salts added is usually about 0.001 to 1% based on the medium. The culture conditions for microorganisms depend on the type of bacteria, but the pH is usually 5.0.
-9.0°C, preferably 7.0-8°C, and the temperature is usually 20-40°C, preferably 30-37°C, and the culture is usually carried out aerobically for 20-72 hours.

In the reaction of the present invention, cultures, cells, or processed products of these microorganisms are used. Preferably, a bacterial cell suspension or a bacterial cell treatment product is used.

The bacterial cell suspension referred to here refers to the bacterial cells obtained by culturing them by centrifugation or filtration, and the treated bacterial cell material refers to the bacterial cells obtained by culturing them by ultrasonication. Examples include those immobilized on an acrylamide gel carrier by a known method, washed bacterial cells, freeze-dried bacterial cells, and bacterial cell extracts.

The concentration of benzyl lactic acid, which is a reaction substrate, in the reaction solution can generally be about 0.1 to 5%. Regarding the addition method, it may be added all at once or in parts.

The reaction temperature is usually 20-40°C, preferably 25-35°C.
The pH of the reaction solution is usually maintained at 5 to 11.0, preferably 7.5 to 9.0.

The reaction time varies depending on the reaction temperature, but is usually 30℃ at 30℃.
0 to 90 hours.

There are two reaction methods: one is to add a substrate to the cultured solution and shake aerobically, and the other is to add a substrate to a bacterial cell suspension or treated bacterial cell material and shake aerobically. , both can be adopted, but the latter gives better results.

In addition, in order to shorten the reaction time or increase the amount of L-homophenylalanine accumulated, it is preferable to carry out the reaction in the presence of a surfactant and a coenzyme. Examples of the surfactant include polyethylene glycol, p
-Isooctyl phenyl ether, cetyltrimethylammonium bromide, etc. can be used, and the amount used is preferably about 0.0001 to 1% based on the reaction solution.As the coenzyme, for example, Pyridoxal phosphoric acid can be mentioned, and it is preferable to use the amount thereof in a concentration of about 1 to 1,000 μM based on the reaction solution.

The reaction of the present invention proceeds more efficiently when carried out in the presence of L-glutamic acid as an amino group donor. When L-glutamic acid is added, the amount added is preferably 0.1 to 20% based on the medium.

Thus, by the reaction of the present invention, L-
Homophenylalanine is produced.

To isolate the L-homophenylalanine thus produced from the reaction solution, a general separation and purification method may be used. For example, since L-homophenylalanine itself is almost insoluble in water, acid or alkali is added to dissolve L-homophenylalanine, impurities are removed from the reaction solution by a conventional method such as filtration or centrifugation, and then the L-homophenylalanine can be isolated and collected by neutralizing the solution.

In addition, when (R8)-benzyl lactic acid is used as a raw material in the present invention, only 8 forms of benzyl lactic acid as a substrate are converted to L-homophenylalanine, so if L-homophenylalanine accumulates in the reaction solution, at the same time(
R)-benzyl lactic acid can also be obtained.

<Examples> Hereinafter, the present invention will be explained in more detail with reference to Examples.

In the Examples, the production amount and optical purity of L-homophenylalanine were determined by high performance liquid chromatography (HPL).
C) under the following conditions (optical purity analysis was carried out by Jo
urnal of chromatography,
202 (1980) 375-379)).

L-homophenylalamine production amount column 2 Capsule pack Cps (Shiseido) liquid extract
: 0.01% H3PO4/acetonitrile = 83/17 Flow rate: Detection: 1.5 ml/llin UV214nm L-homophenylalamine optical purity column: A-3020DS (MMC) eluate
:0.05%H3PO4/acetonitrile=65/35 Flow rate: 1. Oml/l1lin derivatization reagent: 2.3.4.6-tetra-0-a
cct71-β-gl+cop7ranos711so
thioc7a++ate (GITC') Detection: UV254nn+ In the examples, the yield is expressed as mol% of L-homophenylalanine produced relative to the reaction substrate.

Example 1 Glucose 2%, Polypeptone 1%, Polypeptone 81
．． 5% yeast extract, 0.15% yeast extract, and 0.4% potassium dihydrogen phosphate (pH 7.5).
The mixture was placed in a 0ml shaking flask and sterilized at 120°C for 10 minutes. Corynebacterium glutamicum A was added to this medium.
TCC13032, ATCC21651, Corynebacterium acetoacidophilum ATCC13870, A
TCC21270, Brevibacterium lactofermentum ATCC13869, Brevibacterium divalicatum ATCC14020, Arthrobacterium
・Citreum ATCC11624, Cyclobacterium ammoniafilmi ATCC15354, Bacillus・
Satiris ATCC 6051, 1 platinum postspecies, 3 each
After culturing with shaking at 0°C for 72 hours, the bacterial cells collected from the culture solution by centrifugation were washed once with ion-exchanged water.
Do not store frozen.

(R3)-benzyl lactic acid 1.5%, sodium L-glutamate 3%, pyridoxal phosphate 50 μM, polyethylene glycol p-yne octylphenyl ether 1 v/v% in 0.1 M phosphate buffer y-(pH
7,5) The obtained cryopreserved bacterial cells were sufficiently suspended in 50 ml of solution. Table 1 shows the amount of homophenylalanine produced after reacting this suspension at 30° C. for 100 hours.

Note that the optical purity of the produced L-homophenylalanine was approximately 100% in all cases.

Surface practical example 2 Glucose 40t, D L-lactic acid 20. G,! 20g of ammonium acid, 2g of potassium dihydrogen phosphate, Ig of sodium chloride, 5g of urea, 20g of ferrous sulfate heptahydrate.
■, 2I containing manganese sulfate 20■, biotin 50a
Pour 1 liter of liquid culture medium into a 51 jar fermenter,
Sterilization was performed at 5°C for 20 minutes. This 5J! In advance, add 100 ml of the same medium to a jar fermenter at 30℃ for 7 days.
Corynebacterium glutamicum ATCC 13032, which had been cultured with shaking for 2 hours, was inoculated and cultured with aeration at 30° C. for 48 hours. After culturing, the bacterial cells collected from the culture solution by centrifugation were washed once with ion-exchanged water, divided into two equal parts, and stored frozen.

(R8)-Benzyl lactic acid 20K was dissolved in 50 μM phosphate buffer (pH 9,0) 141, and Flfid to pH 9,0 with an aqueous sodium hydroxide solution.

After fully suspending half of the cryopreserved cells obtained in this substrate solution, the mixture was stirred at 30°C for 24 hours in a 51 jar fermenter. Acid 50μM, polyethylene glycol p-isooctylphenyl ether 1
V/■% aqueous solution 11 was added. After adjusting the pH to 7°5 with hydrochloric acid and thoroughly suspending the remaining half of the bacterial cells obtained in Example 2, the mixture was stirred at 30°C for 72 hours. After a zero reaction, solid matter was separated by centrifugation. After that, the obtained solid material was reduced to 0. I
It was thoroughly washed with 21 liters of N-hydrochloric acid. Furthermore, after separating the bacterial cells by centrifugation, the solution side was completely concentrated and excess hydrochloric acid was distilled off.

Dissolve the residue in 1j of water and dilute with aqueous sodium hydroxide solution.
Adjusted to H5-6. After filtering the precipitate, it was washed with water and
By drying under reduced pressure at °C, 7.8 g of L-homophenylalanine was obtained (isolated yield 78%, optical purity 99%).

<Effects of the Invention> According to the present invention, L-homophenylalanine can be efficiently obtained in high yield and purity from benzyl lactic acid. Furthermore, since the concentration of the reaction substrate can be increased, the reaction time can be shortened and the yield can also be improved. Furthermore, there is no need for an expensive optical resolution agent, and it can be manufactured at low cost.

Patent application Daitoshi Co., Ltd.

Claims (1)

[Claims] Corynebacterium has the ability to convert benzyl lactic acid to L-homophenylalanine and
terium), Brevibacterium (Brevib)
genus acterium, Bacillus
Genus, Microbacterium
m) Genus or Arthrobacter
er) A culture, bacterial cells, or a processed product thereof of at least one type of microorganism selected from the microorganisms belonging to the genus genus is allowed to act on benzyl lactic acid to produce and accumulate L-homophenylalanine, and L-homophenylalanine is isolated from the reaction solution. 1. A method for producing L-homophenylalanine, which is characterized by separate collection.