JPS6236196A - Production of alanine - Google Patents

Abstract

PURPOSE:To form and to accumulate L-alanine in an aqueous medium efficiently, by collecting a D-alanine demanding variant by deactivating alanine racemase and treating the variant with lactic acid and an ammonia donor in an aqueous medium. CONSTITUTION:Lactic and an ammonia donor are treated with a bacterium having D-alanine demanding properties, lactic dehydrogenase activity and L- alanine dehydrogenase activity, capable of producing L-alanine from lactic acid and an ammonia donor, in an aqueous medium, to form and to accumulate L-alanine. A mold of Escherichia coli MK5013PICR301 having high L-alanine dehydrogenase (ADH) activity and high lactic dehydrogenase (LDH) activity obtained by taking out ADH gene from chromosome DNA of Bacillus sphaericus IFO3525 having active L-ADH and integrating the gene into a strain of variant Escherichia coli MK5013 having highly active LDH and deactivated alanine racemase is preferable as the bacterium.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing L-alanine.

L-7 ranin is one of the amino acids constituting living organisms, and is a substance in high demand in the pharmaceutical, food, and other fields.

[Conventional technology]

Several methods are known for producing alanine, including chemical synthesis method 1 fermentation method and enzymatic decarboxylation of as/ginate, but a cheaper and simpler method is desired.

[Problem that the invention seeks to solve]

The present inventor Hiromu previously prepared lactate dehydrogenase (hereinafter referred to as LJ) and L-alanine dehydrogenase (hereinafter referred to as LJ) in an aqueous medium containing lactic acid, an ammonia donor, and nicotinamide adenine nucleotide (hereinafter referred to as NAD). (hereinafter abbreviated as ADH)
Alternatively, he discovered that alanine could be efficiently produced and accumulated by reacting both enzymes with bacterial cells containing high titers or their crushed products, and filed a patent application (Japanese Patent Application No. 59-401).
41). However, when using bacterial cells, -11i fragments, or partially purified enzymes, D-alanine is produced as a by-product due to coexisting alanine racemase. Methods such as heat treatment and inhibitors have been proposed as methods to suppress this, but it is possible to completely suppress or eliminate alanine racemase and LDH.

Conditions that do not affect ADH activity sometimes give unsatisfactory results when scaled up, and there is a need to develop simpler and more reliable methods.

[Means for solving problems]

In view of the above-mentioned circumstances, the present inventors have conducted intensive studies on collecting mutant strains that are endowed with alanine auxotrophy, and have succeeded in collecting D-alanine auxotrophic mutant strains by deleting alanine racemase. We have established a simple and inexpensive method for producing L-alanine from lactic acid.

The present invention will be described in detail below.

As a result of various studies on methods of obtaining alanine from inexpensive raw materials using enzymes, the present inventors dehydrogenated lactic acid in an aqueous medium containing lactic acid, an ammonia donor, and nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD). enzyme (hereinafter abbreviated as LDH) and L-alanine dehydration enzyme (hereinafter A
Abbreviated as DH. ) was found to efficiently generate and accumulate alanine in the aqueous medium.

That is, both enzymes use NAD as a coenzyme, but as shown in the following formula (1), NAD becomes NADH from LDHK, and lactic acid is converted to pyruvate [Kf], and then converted to NADH by ADH in the presence of an ammonia donor, such as NH4. is N.A.D.
K is oxidized and at the same time pyruvate is converted to alanine.

Formula (1) Alanine can be produced from lactic acid more efficiently by using the system shown in Formula (1) that conjugates more NAD. K and NADH in this reaction need only be added at the start of the reaction, and the amount of NADH added may be small.

Ammonia or ammonium salts can be used as ammonia donors.

The LDH and ADH used in the present invention may be obtained from animals, plants, or microorganisms, but those from microorganisms are cheaper. LDH and A of microbial origin
When DH is used, alanine can be produced at low cost through the reaction of formula (1). However, since there is no microorganism that simultaneously produces LDH and ADH with high activity, microorganisms that produce each enzyme with high activity must be cultured individually.

As a result of further intensive studies regarding this point, we have decided to create a microorganism that has the ability to produce LDH, has DNA in which vector DNA and a gene encoding ADH are linked, and has the ability to produce ADH. Successful. In other words, Pachyrus sphaericus IFO3 has highly active ADH.
The ADH gene was extracted from the chromosomal DNA of 525 and inserted into the mutant strain E. coli MK5013, which has highly active LDH and lacks alanine racemase.
and E. coli MK5013 P with high LDH activity.
ICR301 bacteria was obtained.

Bacillus sphaericus IFO35, a genetic child 4 bacterium
For example, methods for extracting chromosomal DNA from 25 are described in J.B.
This can be carried out by conventional methods such as those described in Aet@ri/189 1065 (1965).

Vector DNA may be any DNA as long as it can self-replicate within the body of a microorganism. One example is P.
There is Bl 322. Chromosomal DNA and vector DNA
are cut using restriction endonuclease and clease, respectively.
For chromosomal DNA K, many types of restriction endonucleases can be used by adjusting the reaction conditions so that partial cleavage occurs with restriction endonucleases. A conventional method using ribose can be used to link the friend chromosomal DNA fragment thus obtained and the cut vector DNA. The chromosomal DNA fragment thus obtained and the vector DNA are ligated, and the recipient strain of the DNA is LDf (any bacterial strain with high activity may be used).

The introduction into recipient bacteria is carried out according to, for example, J, Mo1. Blol 5
3159 (1970) K can be used. Among the transformed strains, those containing the ADH gene may be selected using the marker properties of the vector DNA and ADH activity as indicators. Through such operations, a strain having high LDH and ADH activities can be created. A method for constructing a strain having high ADH activity will be specifically described below.

1) Preparation of chromosomal DNA Bacillus sphaericus IFO3525 was isolated from 11 “B8
ct-penassay Broth J (Product name D
lfeo1JA) for approximately 2 hours at 45°C with shaking to collect cells in the logarithmic growth phase, followed by the usual DNA extraction method (
Chromosomal DNA was extracted and purified from the button (such as L Bacterio189.1065 (1965)) and 2.6 da was collected.

2) Insertion of chromosomal DNA fragment into vector DNA In order to clone the genetic region containing at least the ADH gene, ampicillin resistance is required as the self-replicating DNA that serves as the vector! Lasmid PBR322 was used.

1) 10μy of nine DNA and 5μI of vector DNA
The DNA strands were cleaved by using the restriction enzyme Sat-1, a type of crease, at 37°C for 1 hour. After heat treatment at 65°C for 10 minutes, both reaction solutions were mixed and A
In the presence of TP and dithiothreitol.

10°C using DNA ligase derived from T4 phage.

Do not perform the DNA strand ligation reaction for 24 hours.

3) Estherichia coli MK5013 for transformation with f2 zumid carrying the ADH gene: If
Competent state (state with the ability to take in DNA)
K is J, Mo1. Blot 53 159 (
Cells were prepared as described in (1970).

Vector D obtained in 2] was added to this competent cell suspension.
Add a solution of DNA containing DNA in which genes encoding NA and ADH are linked, keep it cooled in water for 30 minutes, and immediately add
Heat treatment was applied at 2°C for 2 minutes.

Next, take a certain amount of this cell suspension and add a new medium (14 trigton, 0.5 yeast extract, 0.5 salt.

The cells were inoculated with 0.05% magnesium sulfate heptahydrate, pH 7, 2)K, and cultured with shaking at 37°C for 1 hour to complete the transformation.

Transfer the culture solution to agar containing Ambishi IJ 750μ9/Go.
51 was added to the above medium and kept warm at 37°C.

After 1 to 2 days, 210 colonies that appeared on the medium were picked, each clone was purified, and strain MK5013-PICR-3 expressing ADH activity was selected. Present in this strain! Lasmid PICR-3 is IIK Pace (
It was ah.

Next, we attempted to downsize the recombinant plasmid using the MK5013-PICR-3 strain. i.e. MK5013-PI
After completely decomposing the plasmid PICR-3 present in CR-3 strain with restriction enzyme H1nd III, PBR322 was added again.
Recombinant derasmid PICR- connected to plasmid
I got 301. The present grasmid PICR-301 was paced at 8. The glasmid was introduced into Esserichia coli MK-5013 by the same treatment as in 3), resulting in an E. coli MK5013>PICR-301eAJ.

1'221<k, rE12z-PQ+Wawa Now, the nine bacterial strains obtained in this way and their siblings were cultured using the usual bacterial culture method.
In other words, as a medium, it is a carbon source.

The nitrogen source may contain inorganic ions and, depending on the heart wall, organic trace metals such as amino acids and vitamins. As the carbon source, glucose, fructose, dextrin, etc., organic acids such as lactic acid, etc. can be used, and as the nitrogen source, begtone, yeast extract, ammonia and its salts, etc. can be used.

Culture was carried out under aerobic conditions, with appropriate humidity and temperature adjustment.
Continue until ADH activity reaches its maximum.

To use for enzymatic reactions, cultured bacteria can be used as is, or can be collected and washed. - Cell extracts disrupted using a Lumil, French press, or ultrasonic disruption device may also be used. Further, specimens prepared by ammonium sulfate fractionation, ultrafiltration, reflux filtration, ion exchange chromatography, etc. can also be used. Furthermore, L
D) Immobilized ADHIL1+i enzymes, bacterial cells containing them, or crushed products can also be used.

To convert alanine from lactic acid, use the created bacterial strain and culture it in a medium containing lactic acid for 24 to 120 hours, or add the bacterial cells or enzyme preparation to an aqueous medium containing NAD, lactic acid, and an ammonia donor at pH 4.0-10. 0 preferably 7.0 to 8.0
20°C to 80°C, preferably 30°C to 50°C
By reacting with K, more alanine can be produced.

4) Induction of D-alanine auxotrophic strain Esthelichia coli C-600 strain is inoculated onto a bouillon agar medium and cultured overnight at 30°C.
Suspend in 1M phosphate buffer at 30°C.

Treat for 30 minutes, remove nitrosoguanidine by centrifugal washing (10,000 rpm), dilute appropriately, and plate on a bouillon agar plate at approximately 100 colonies/plate.
Grow the colonies at 30°C overnight.

On the other hand, glucose 0.5%, ammonium sulfate 0.54, phosphoric acid 1
Potassium 0.14, dipotassium phosphate magnesium monate 0.0
5%, salt 0.1% t FeSO4 and VLn, S
Agar plates were prepared by dividing a medium containing 1.5 parts of 04 and 2 p'pm of e-agar into two, adding 0.11 parts of L-alanine to one side and 0.11 parts of D-alanine to the other, and growing them on bouillon agar plates. Replica the nitronganinon-treated bacteria. Heat the replica grate to 30℃.

After culturing overnight, colonies that did not grow on the L-alanine-containing medium but grew on the D-alanine-containing medium were isolated.

Through the above operations, four mutant strains shown in Table 1 were obtained.

Etherichia coli C-600+ +
+MK5013 − + −50
14-+ - 5041-+ - 5043-+ - 5061-+ - Alanine racemase activity is 0.1 M D or L-alanine, 0.1 M) Six bacteria were suspended in lithium-hydrochloric acid (pH 8.5) and incubated at 37°C. After reacting for 60 minutes, the presence or absence of racemase activity was determined using a commercially available Chiral Pack.

Of these four strains, MK5013 was used as a representative strain.

This operation for imparting D-A, ranin auxotrophy is carried out on the DNA in which the vector and the gene encoding ADH are linked.
This may be done before or after incorporating.

Quantification of LDH, ADH activity, and alanine was performed as follows.

LDH activity was determined by Tolzenbach, F (196
6) M@thod in Enzymology 92
0 ADf according to the method described in 78 (activity was determined according to the method described in Kenji Soda and Toshihisa Oshima (1976) Biochemistry Experimental Course II, Volume 193).

For the quantitative determination of alanine, DL-alanine was determined using an ordinary amino acid analyzer, and the separate quantitative determination of D and L was performed by P, Ee Hare et al., Scl@nee 204
1226 (1979) using high performance liquid chromatography.

I can do it.

A more detailed explanation will be given below with reference to Examples.

Example-1 Eserichia coli AJ l'Z-'Zll? Gla stock
Coss 196 ep@pton 0.14 m It is inoculated into a liquid medium containing 0.5% yeast extract, DL-alanine o, ooss, and 0.54 ml of sodium chloride, and pre-cultured at 37°C overnight.

On the other hand, DL-ammonium lactate 10%, phosphoric acid-potassium 0
．． 1%, dipotassium phosphate 0.2%, magnesium sulfate heptahydrate O, OS*, salt 0.1, yeast extract 0.054. Dispense 5Qrtrl of a liquid medium containing 0.005% of L-threonine, 0.0025% of L-leucine, 0.0025% of DL-alanine, 0.0001% of thiamine, and pi-17,2 into a 500D shoulder flask. 54 cultures were inoculated with autoclave sterilization (120°C, 10 minutes) and precultured, and cultured with shaking at 37°C for 48 hours. As a result, 2.8 I/lit of L-alanine was accumulated.

Example 2 A culture solution cultured overnight at 37°C in the preculture medium of Example 1 was centrifuged at 10,000 rpm for 10 minutes to collect bacterial cells, washed twice with 0.1M phosphate buffer, and diluted with the same buffer. Suspend in

On the other hand, 10 DL-lactic acid, 5 NH4CL, 0.
10 to the reaction solution containing 15 NAD! Bacterial cells were added so that n9/rR1 (manga) was obtained, the - was adjusted to 8.0 with ammonia, and the reaction was performed while stirring slowly at 37°C for 24 hours.
L-alanine of 21/dt was produced.

Claims (1)

[Claims] 1) D-alanine requirement, lactate dehydrogenase activity and L-
A microorganism having alanine dehydrogenase activity and the ability to produce L-alanine from lactic acid and ammonia donors is allowed to act on lactic acid and ammonia donors in an aqueous medium to produce L-alanine.
- A method for producing L-alanine, which comprises producing and accumulating alanine and collecting it. 2) Claim 1, wherein the microorganism is a microorganism that has D-alanine auxotrophy and lactate dehydrogenase activity, and has DNA in which vector DNA and a gene encoding L-alanine dehydrogenase are linked. The method for producing alanine described in Section 1.