JPH02242690A - Production of l-alanine - Google Patents

Abstract

PURPOSE:To produce L-alanine in high yield without manifesting racemase activity by enzymatically reacting L-aspartic acid in the presence of a bacterium cell having enzymatic activity while maintaining pH 4.3-50 and 40-47 deg.C. CONSTITUTION:Pseudomonas dacunhae IAM1152 strain containing L-aspartic acid beta-decarboxylase is aerobically cultured in a medium containing a carbon source such as fumaric acid, a nitrogen source such as ammonium and an inorganic salt useful for ordinary microorganisms and the prepared cell is used for enzymatic reaction. Then an aqueous solution containing L-aspartic acid or a salt thereof is adjusted to proper pH in the presence of the bacterium cell and subjected to enzymatic reaction at pH 4.3-5.0 at 40-47 deg.C. Then separation and purification of formed alanine in the reaction solution can be carried out by well-known ion exchange resin treatment, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing alanine using an enzymatic method. According to the present invention, L-alanine can be efficiently produced with high yield.

As is well known, L-alanine is an important amino acid as a raw material for medicines, foods, or chemical industries, and its demand has been rapidly increasing in recent years.

(Prior art and problems) Pseudomonas dacu containing L-aspartate β-decarboxylase
The method of producing L-alanine by subjecting monoaspartic acid to an enzymatic reaction in the presence of microbial cells (nl+ae) or their crushed material has the problem that L-alanine is racemized by the alanine racemase activity present in the microbial cells. was.

In addition, in order to efficiently produce L-alanine, it is important to improve the reaction rate of β-decarboxylase reaction, but if the reaction temperature is raised to increase the reaction rate, It was also observed that the alanine racemase activity present was improved.

In order to solve this problem, a method has been proposed in which the alanine racemase activity is removed by pretreatment of the bacterial cells (Mochinan Sho 57-132882 Publication, Tokuma Sho 62-87088 Publication). is complicated, and simplification of the process as much as possible has a great effect on reducing manufacturing costs. Therefore, the present inventors developed a method for producing L-alanine at a high reaction rate without pretreating the bacterial cells. We have intensively investigated a method for efficiently producing L-alanine by suppressing racemase activity during the production reaction. As a result, the reaction at night l)H
The present invention has been completed by discovering that L-alanine can be produced in high yield without developing racemase activity by carrying out an enzymatic reaction in an acidic range while maintaining the reaction temperature at 40'C to 47C. reached.

(Structure and Effects of the Invention) The present invention involves reacting L-aspartic acid or a salt thereof in an aqueous solvent in the presence of microbial cells having L-aspartic acid β-decarboxylase activity or a crushed product thereof, When producing 14-alanine in the reaction solution, I) of reaction γα
The present invention provides a method for producing L-alanine, characterized in that H is maintained at 4.3 to 5.0 and the reaction temperature is maintained at 40 to 47°C.

(Detailed Description of the Invention) The microorganism used in the present invention is Pseudomonas dacnae, which contains L-aspartate β-decarboxylase.
Pseudoo+onas dacur+hae) I
AM 1152 is preferably used.

The above-mentioned microbial cells used in the present invention can be used as they are, or a crushed product obtained by crushing them by treatment such as ultrasonic crushing can also be used.

The Ig source used for preparing the above-mentioned microbial cells used in the method of the present invention is not particularly limited, and may be one used for general microorganisms.

1 of microbial cells containing L-aspartate β-decarboxylase! The carbon source of the medium used for I production is not particularly limited, and for example, fumaric acid, succinic acid, malic acid, L-aspartic acid, etc. can be used, and among them, fumaric acid is preferably used. As a nitrogen source for the culture medium, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used, and organic nutrient sources such as peptone, yeast extract, consteep liquor, casamino acids, etc. can also be used. I can do it. As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used.

Cultivation is performed under aerobic conditions such as aeration and shaking, and the culture temperature is 20°C to 40°C, preferably 28°C to 32°C.
I'll take 1 te. pH during cultivation is 5-10, preferably 7-10.
The pH is adjusted to around 8.0 by adding acid or alkali to adjust the pH during culturing. The concentration of carbon source in the medium at the start of culture is 0.05 to 10% by weight, and when fumaric acid is used as a specific example, the fumaric acid concentration is preferably 0.05% to 10% by weight. 1-5% by weight, more preferably 0.5-2% by weight
is suitable.

Culture intercostals for 10 hours to 4 days, with an optimal period of 1 to 3 days.

Collect bacterial cells from each culture obtained in this way,
It is washed with water or an appropriate buffer and used for the enzyme reaction of the present invention.

In the method of the present invention, an enzymatic reaction is carried out in an aqueous solution containing at least L-aspartic acid or a salt thereof in the presence of the microbial cells prepared above or a crushed product thereof. Here, the concentration of L-aspartic acid or its salt added to the aqueous solution is 0.5 to 50 M11%, preferably 3.
~30% by weight. In addition, [,-aspartic acid is
Due to the solubility during the reaction, it may exist in a dissolved state or as a powder (insoluble state B). To adjust the pH of the reaction solution, an alkaline solution such as aqueous ammonia, sodium hydroxide, potassium hydroxide, etc. is preferably used.

The aqueous solution was further added with 0.0% pyridoxal 5' phosphoric acid.
0005-0.05% by weight, preferably 0.001-0
．． It can be used by adding 0.01% by weight. Furthermore, if necessary, a nonionic surfactant such as polyoxyethylene sorbitan monooleate i. 5
It can be used in an amount of 0.03 to 0.2% by weight, preferably 0.03 to 0.2% by weight. Also, if necessary, pyruvate,
α-keto acids such as α-ketobutyric acid from 0.0001 to 0°5
! Ii%, good mat,,<ha0. 002~0. 2mj
It can be used by adding 1%. In the present invention,
pH during enzyme reaction is 4.3 to 5.0, preferably 4.5
-4.8, the reaction temperature is 40-47°C, preferably 42-45°C, and the reaction is usually carried out for about 3 to about 48 hours.

Separation and purification of L-alanine produced in the reaction solution obtained by the above reaction method can be carried out by a known ion exchange resin treatment or the like.

Experimental Examples In the following experimental examples, the quality of L-alanine was confirmed by the Rf value on a paper chromatograph, the retention time on a high performance liquid chromatograph, and the specific rotation of the purified product. Quantification is
It was performed using high performance liquid chromatography (Shimadzu LC-5A).

The amount of D-alanine produced was determined by measuring pyruvic acid produced from D-alanine as hydrazone using D-amino acid oxidase derived from pig kidney (manufactured by Boehringer Mannheim Yamanouchi Pharmaceutical Co., Ltd.).
ds in Enzymology, vol, XV
U, Part A, Edited by Her
bert Tabor and Ce1a White
Tabor, Academic Press, N.
ew Vork, 1970P, 17! -170
).

In addition, in the following experimental examples, % means weight %.

The present invention will be explained in more detail with reference to Examples below.

Example (1) Preparation medium for microorganisms (sodium fumarate 0.5%, ammonium fumarate 1.0%, yeast extract 0.5%, potassium phosphate 0.05%, Mg SO4.7 H200, 05
% content, pH 7, (1) rooms! After dispensing into a 500 mg Erlenmeyer flask and sterilizing it, Pseudomonas dacunbae J
AM 1152 was inoculated and cultured with shaking at 30°C for 1 day (preculture).Next, a medium 12 similar to the above medium was charged into a 22 volume aerated stirring tank and sterilized (120°C, 20 minutes). After that, add 20 m 12 of preculture and rotate at 1 rpm.
Culture was carried out for 1 day at 00 Or p m, aeration ffi 1 v v m, temperature 30° C., and R) 17.3.

After culturing, the culture was centrifuged from 100 mR to collect bacteria, and the facepiece was washed with 0.9% NaC. After washing once with a solution, the washed bacterial cells were used for an enzyme reaction.

(2) Experimental method The bacterial cells obtained above were treated with an aqueous reaction solution [30% of aspartic acid, 0.05% of polyoxyethylene sorbitan monooleate, 5% of pyridoxal] in the experimental areas shown in Table 1. '-Phosphoric acid 0.05%, sodium pyruvate 0.0
Contains 2%, p I (adjusted with ammonia water (25% NHJ)
The total amount of alanine produced and the amount of alanine produced after suspension in 200 m 11 of water and shaking at each temperature for 5 hours were measured.

As a control, the total amount of alanine produced and the amount of D-alanine produced when the reaction was carried out at a temperature of 37°C11)H5.5 were set as 100.

(3) Results The results are shown in the following table, and by the method of the present invention,
It became possible to react monoaspartate β-decarboxylase at a high reaction rate without expressing alanine racemase activity.

Claims (1)

[Claims] (1) In the presence of microbial cells containing L-aspartate β-decarboxylase or a crushed product thereof, L-aspartic acid or its salt is reacted in an aqueous solvent, and L-aspartic acid or its salt is reacted in the reaction solution.
- When producing alanine, adjust the pH of the reaction solution to 4.3~
5.0 and maintaining the reaction temperature at 40 to 47°C.