JPH04271783A - New indole-3-pyruvic decarboxylase and its production - Google Patents

Abstract

PURPOSE:To provide a new indole-3-pyruvic decarboxylase and a method for producing the same. CONSTITUTION:It has been found that indoleacetic acid (IAA) is produced by Enterobacter cloacae through an indolepyruvic acid route which is one of biosynthetic routes for the IAA. Thereby, indole-3-pyruvic decarboxylase that is an entirely new enzyme catalyzing the second stage of the aforementioned route is isolated and a method for producing the enzyme is established.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is a typical plant hormone that plays the most important role in the biosynthetic pathway of indole acetic acid (IAA), which has various physiological activities such as promoting rooting and elongation of shoots and leaves. The present invention relates to a novel indole-3-pyruvate decarboxylase and a method for producing the same.

0002

[Prior art and problems to be solved by the invention] IAA
It is one of the representative plant hormones, and has been recognized to have effects such as promoting plant rooting, stem and leaf elongation, parthenocarpy, and inhibiting aging, and is a substance with important plant physiological meaning. on the other hand,
It has been revealed that IAA is produced not only by plants, but also by microorganisms and animals, and with regard to microorganisms in particular, various studies have been conducted on the meaning of the production of plant hormones by bacteria present in the leaves and rhizosphere of plants. It is progressing.

[0003] IAA is generally thought to be produced using the essential amino acid tryptophan as a precursor through three biosynthetic pathways as shown below. (Bio
l. Rev. , 48, 510-515 '73) 1) Tryptophan → indole acetamide → IAA 2) Tryptophan → tryptamine → indole acetaldehyde → IAA 3) Tryptophan → indole pyruvate → indole acetaldehyde → IAA In particular, the pathway 3) (indole pyruvate pathway) is thought to be the main biosynthetic pathway in plants, but because intermediates such as indolepyruvate and indoleacetaldehyde are unstable, the existence of the enzyme has not been confirmed, and it is believed that it is involved in plant growth. It has not been possible to find substances useful for agricultural production by controlling the biosynthetic pathway of IAA.

0004

[Means for solving the problem] The inventors isolated an enzyme that is involved in the indolepyruvate pathway and has the most important action in the biosynthetic pathway of IAA in plants, clarified its action, and further developed this enzyme. We conducted extensive research with the aim of using this enzyme as an industrially useful means by searching for the source of separation and establishing its production method. As a result, Enterobacter cloacae (this bacterium has been deposited as FERM-BP1529 at the National Institute of Microbiology, Agency of Industrial Science and Technology), which was isolated from the rhizosphere of a well-growing cucumber, was found to be one of the mechanisms of action for promoting plant growth. In addition to discovering that IAA is produced as a biosynthetic pathway, the conversion reaction of the precursor to IAA and the identification of intermediates revealed that the biosynthetic pathway is the indolepyruvate pathway, which is the same as the main biosynthetic pathway in plants. revealed. Furthermore, as a result of isolation of the IAA synthesis gene from Enterobacter cloacae (see Japanese Patent Application No. 2-45718), the gene encodes indole-3-pyruvate decarboxylase, which catalyzes the second step of the indolepyruvate pathway. I found out that This enzyme decarboxylates indole-3-pyruvate,
It generates indole acetaldehyde and CO2, and occupies the most important position in the IAA biosynthetic pathway. The inventors purified this enzyme and clarified its enzymatic chemical properties, established a new method for measuring enzyme activity, and established an enzyme reaction system with high industrial utility. Furthermore, the inventors conducted a wide search for the isolation source of this enzyme and found that a microorganism belonging to the genus Enterobacter produces IAA through the indolepyruvate pathway, thereby completing the present invention.

[0005]

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to the following Examples. [Example 1] [1] Enterobacter cloacae (FERM-BP1529, deposited with the Institute of Microbiology, Agency of Industrial Science and Technology)
Culture method for producing indole-3-pyruvate decarboxylase from
l, 0.5%, 0.1mM thiamine pyrophosphate (TPP
), aerobically cultured at 30°C for 24 hours using a liquid medium containing 0.2M phosphate buffer (pH 6.5),
Bacteria were collected by centrifugation at 00 rpm for 40 minutes. PMT solution (50mM
Phosphate buffer, 5mM MgCl2, 1mM TPP, p
After adding 200 ml of H6.5) and suspending,
Ultrasonic disruption for 10 minutes was performed 10 times. This solution was centrifuged at 4°C and 18,000 rpm for 40 minutes to remove the precipitate to obtain a crude enzyme extract.

[2] Purification of indole-3-pyruvate decarboxylase from Enterobacter cloacae (1) Method for measuring indole-3-pyruvate decarboxylase activity Enzyme solution 0.1ml, 0.1M phosphate buffer (pH 6.5
)0.1ml, 50mM MgCl2 0.1ml, 1m
Mix 0.1 ml of MTPP, 0.1 ml of 10 mM indole-3-pyruvic acid, and 0.5 ml of water, and
After reacting at ℃ for 30 minutes, 4 ml of 0.1N HCl was added to inactivate the enzyme. Thereafter, indole-3-acetaldehyde produced in the reaction solution was quantified by high performance liquid chromatography. The amount of enzyme that produces 1 μmol of indole-3-acetaldehyde in 1 minute was added to the reaction solution.
It was taken as a unit. (2) Purification of indole-3-pyruvate decarboxylase 800 ml of PMT solution was added to the crude enzyme solution obtained in [1], and after mixing, 400 g of ammonium sulfate was added and the resulting precipitate was removed. 200 g of ammonium sulfate was added to the supernatant, and the resulting precipitate was collected by centrifugation. The precipitate is 100ml of PMT.
After dissolving in a solution, desalting was performed by dialysis. Then P.M.
This crude enzyme solution was charged into a column filled with 300 ml of MONO-Q anion exchange resin (manufactured by Pharmacia) equilibrated with T solution, and the initial reaction was PMT solution and finally 0.
．． A concentration gradient was created to obtain a PMT solution containing 5M NaCl for elution. The obtained active fraction was concentrated to 100 ml by ultrafiltration (manufactured by Asahi Kasei Corporation, using Mini Module NM-3). Phenyl-Superos was prepared by equilibrating this concentrate with a PMT solution containing 1M ammonium sulfate.
Charge the e-column (manufactured by Pharmacia), and the first shot is 1
Elution was performed by creating a concentration gradient using a PMT solution containing ammonium sulfate (M) so that a final PMT solution was obtained. The active fraction obtained from this was concentrated to 20 ml by ultrafiltration method, and the PM
DEAE-TOYOPEARL65 equilibrated with T solution
Charge the 0S column (manufactured by Tosoh Corporation), starting with PMT solution and finally PMT containing 0.5M NaCl.
A concentration gradient was created to create a solution and elution was performed. The active fraction thus obtained was concentrated by ultrafiltration to obtain purified indole-3-pyruvate decarboxylase. This enzyme was determined to have a molecular weight of 60,000 by SDS electrophoresis.
It was revealed that this is a single enzyme.

[3] Properties of indole-3-pyruvate decarboxylase derived from Enterobacter cloacae [
The physicochemical properties of the purified indole-3-pyruvate decarboxylase obtained in step 2] will be described. The enzymatic activity measurement method for indole-3-pyruvate decarboxylase was carried out according to the method described in [2] (1). (1) Action: Acts on indole-3-pyruvic acid, decarboxylates the carboxylic acid moiety, and produces indole-3-acetaldehyde and carbon dioxide. (2) Substrate specificity: acts well on indole-3-pyruvate and slightly acts on pyruvic acid. However, it has little effect on oxalic acid, oxaloacetic acid, acetoacetic acid, tryptophan, and indole-lactic acid. (3) Optimum pH: The optimum pH is around pH 6.5, and there is almost no effect on the alkaline side. (4) pH stability: It is quite stable around pH 6.5, but unstable on the alkaline side. (5) Heat resistance: 30 in the range of 10 to 50°C at pH 6.5
It is hardly inactivated under heating conditions for 1 minute. (6) Effects of metal ions and coenzymes: TPP and M
When g ions are added, the reaction is activated. (7) km value 10 for indole-3-pyruvic acid
The km value for indole-3-pyruvate is approximately 0.16 mM when operated in mM phosphate buffer at pH 6.5 and 25°C. (8) Effect of inhibitor: Activity is inhibited by α-ketoglutaric acid. (9) Molecular weight: 60,000 (by SDS electrophoresis)

[Example 2] Enterobacter cloacae (type stra) other than FERM-BP1529
in IAM12349 and IAM1615, IA
M1624) and Enterobacter amnigena (E
nterobacter amnigena: typ.
Regarding e strain JCM1237),
Indole-3-pyruvate decarboxylase activity was observed. Therefore, it was possible to produce and purify indole-3-pyruvate decarboxylase using the same method as in Example 1 for the above bacterial strain. The physicochemical properties of these enzymes also differ from indole-3 possessed by Enterobacter cloacae (FERM-BP1529).
-The physicochemical properties were the same as those of pyruvate decarboxylase.

[Example 3] [1] Isolation of nuclear DNA from Enterobacter cloacae Enterobacter cloacae was cultured with liquid shaking in 100 ml of LB medium at 37°C for 1 day, and the culture solution was
The cells were centrifuged at 0 rpm for 20 minutes and the bacterial cells were collected. 16ml of bacterial cells
TESH solution (0.2M Tris-HCl, 0.
02M EDTA, 0.05M NaCl pH
8.0) and 4 ml of 0.5 M EDTA, 0
．． 2 ml of 0.5%, RNase A (manufactured by Sigma) 0.
2 ml of egg white lysozyme (manufactured by Sigma) was added, mixed, and reacted at 37° C. for 2 hours. Next, 1 ml of 5% SDS
The solution was added and reacted overnight at 37° C. with slow stirring. The same amount of phenol-saturated TESH solution was added to this solution, shaken at room temperature for 10 minutes, and then rotated at 3500 rpm for 10 minutes at room temperature. The aqueous layer was transferred so as not to remove the intermediate layer, and the same phenol treatment as above was repeated three times. Add twice the amount of ethanol to the resulting aqueous layer, roll up the nuclear DNA while stirring with a glass rod, and add TE solution (0.01M
Tris-HCl, 0.001M EDTA
(pH 8.0) to prepare a nuclear DNA solution. [2] Preparation of genomic library from Enterobacter cloacae (1) 100 μl (450 μg) of nuclear DNA solution prepared from Enterobacter cloacae obtained in [1] above
/ml) and Sau3AI (manufactured by Toyobo Co., Ltd.)
0.25, 0.5, 1, 2, 4, 8, 16 units of were added and reacted at 37°C for 30 minutes. Various reaction solutions were run on agarose gel electrophoresis, and DNA with a length of 1 to 20 kbp was detected.
Only the reaction solution containing A was pooled and subjected to phenol extraction (phenol: chloroform isoamyl alcohol = 50:
49:1 Vol ratio) three times, then 1/1 of this solution
Add 0 amount of 3NCH3 COONa and 2 times the amount of ethanol, mix, cool at -20°C for 20 minutes, and centrifuge.
DNA was collected. The DNA was washed with 90% cold ethanol, dried under reduced pressure, and dissolved in 100 μl of TE solution.

(2) Plasmid vector PUC119
Add 160 units of BamHI (manufactured by Toyobo Co., Ltd.) to 500 μl (40 μg/ml) and incubate at 37°C/4 hr.
After reaction and two phenol extractions, ethanol precipitation was performed and the mixture was dried under reduced pressure. Add 40% sterile water to the recovered DNA.
0μl, pH 8.0 0.5M Tris-HC
Add 50 μl of alkaline phosphatase (manufactured by Boehringer) (1 unit/μl),
After mixing, the mixture was reacted at 37°C for 3 hours. After that, phenol extraction and ethanol precipitation were performed three times to recover the DNA.
Dissolved in 0 μl of TE solution. (3) 10 μl of a solution where genomic DNA was partially digested with Sau3AI, 10 μl of a solution where PUC119 was digested with BamHI, T4 ligase (manufactured by Toyobo Co., Ltd.) 7
μl (5 units/μl), 3 μl buffer (66
0mM Tris-HCl, pH 7.6, 66mM
gCl2, 100mM dithiothreitol, 660μ
After mixing, ligase reaction was performed at 16° C. for 16 hours.

[3] Identification of a clone encoding the IAA synthetic gene (1) The solution obtained by the above ligase reaction was subjected to the Hanahan method (J. Mol. Biol., 166, 557, '
In addition to the competent cells DH5α prepared in
The cells were plated on L agar medium containing mXgal and cultured at 37°C for 24 hours. The obtained white colonies were picked and 100p
When cultured in a liquid medium containing pm ampimiline,
A strain producing IAA was selected as a clone carrying the IAA synthesis gene. IAA production ability was determined by culturing the transformant in a conventional manner and determining whether IAA was produced in the culture solution using high performance liquid chromatography. (2) The above IAA-producing strain was cultured in liquid medium in L medium, and the resulting bacterial cells were subjected to alkaline minipreparation method (Nucleic Acids Res).
．． , 7, 6, 1513-1523 '79). When this DNA was cut with BamHI and subjected to 0.8% agarose gel electrophoresis, it was confirmed that about 4 kbp of foreign DNA had been inserted. PU with this approximately 4kbp insert
C119 was changed to PIA119 (Patent application Hei 2-45718
reference).

[4] Purification and properties of indole-3-pyruvate decarboxylase from Escherichia coli harboring the IAA synthesis gene. As a result of producing and purifying acid decarboxylase, its physicochemical properties were determined to be similar to that of Endobacter cloacae (FERM).
BP-1529).

[0013]

[Effects of the Invention] By using this enzyme, it becomes easy to search for substances that control the IAA biosynthetic pathway, and by using this enzyme in a bioreactor, a new method for producing IAA becomes possible.

Claims (3)

[Claims] Claim 1: A novel indole-3-pyruvate decarboxylase having the following physicochemical properties. (a) Action: The carboxylic acid part of indole-3-pyruvic acid is decarboxylated to produce indole-3-acetaldehyde and carbon dioxide. (b) Substrate specificity: acts well on indole-3-pyruvic acid, and acts slightly on pyruvic acid. However, it has little effect on oxalic acid, oxaloacetic acid, acetoacetic acid, tryptophan, and indole-lactic acid. (c) Optimal pH and stable pH range: Optimal pH is 6.4
~6.8, and when reacted at 37°C for 24 hours, the pH
It is stable in the range of 6.0 to 7.5. (d) Range of suitable temperature for action: There is an optimum temperature for action in the range of 40°C to 50°C. (e) Heat resistance: 30 in the range of 10 to 50°C at pH 6.5
It is hardly inactivated under heating conditions for 1 minute. (f) Effects of metal ions and coenzymes: When Mg ions and thiamine pyrophosphate are added, the reaction is activated. (g) Effect of inhibitors: Activity is inhibited by α-ketoglutaric acid. (h) Molecular weight: 60,000 (according to SDS electrophoresis method) [Claim 2] A microorganism belonging to the genus Enterobacter is cultivated, and the indole-3 according to claim 1 is obtained from the culture solution.
- The method for producing indole-3-pyruvate decarboxylase according to claim 1, characterized in that the pyruvate decarboxylase is purified and isolated. 3. Cultivating a microorganism obtained by introducing a gene encoding the enzyme according to claim 1, and purifying and isolating the indole-3-pyruvate decarboxylase according to claim 1 from the culture solution. 2. The method for producing indole-3-pyruvate decarboxylase according to claim 1.