JPH0347080A - Dna capable of coding tryptophanase - Google Patents

Abstract

PURPOSE:To efficiently produce tryptophanase by culturing an organic cell, etc., transformed with a recombinant plasmid containing a DNA sequence capable of coding the tryptophanase derived from a strain of the genus Proteus. CONSTITUTION:A DNA fragment mixture containing a gene capable of coding tryptophanase derived from a strain of the genus Proteus is linked with a vector DNA, such as plasmid vector DNA, to prepare a recombinant plasmid, which is subsequently introduced into an organic cell or microorganism to afford a transformant having a desired genetic character and a character of the vector DNA. The resultant transformant is then subjected to shaking culture with aeration, etc., in an LB culture medium, etc., to grow and accumulate the tryptophanase in the microbial cells. The recovered tryptophanase is subsequently purified by ultrasonic crushing, mechanical crushing using glass beads, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an expression vector in which a DNA sequence encoding tryptophanase derived from a strain of the genus Proteus is incorporated into an expression vector. The present invention relates to a recombinant plasmid, a transformant containing the &Il recombinant plasmid, and a method for producing tryptophanase using the transformant.

Tryptophanase is an enzyme that catalyzes the reaction of decomposing tryptophan into indole, pyruvate and ammonia, and the reaction formula is as follows.

Tryptophan + H! The above reaction is reversible, and tryptophan can be synthesized from indole, pyruvic acid and ammonia by using tryptophanase as a catalyst.

(Prior Art) Conventionally, tryptophanase has been synthesized from, for example, ProLeus rettogeri, Escherichia coli, K1
It is produced by culturing microorganisms capable of producing tryptophanase, such as strain 2, in the presence of tryptophan or its derivatives, and collecting tryptophanase from the culture. However, this method requires the addition of expensive substances such as tryptophan or its derivatives into the medium, and the yield of tryptophanase is not sufficient, so the development of an efficient method for producing tryptophanase is desired. was.

On the other hand, Proteus inconstance
It has been known that strains belonging to L. incons tans have the ability to produce L-tryptophan or its derivatives from an indole compound or its derivative, pyruvate ester, and ammonium ion (Japanese Patent Laid-Open No. 6
2-205791).

(Problems to be Solved by the Invention) An object of the present invention is to provide a new method for improving the productivity of tryptophanase without adding tryptophan or its derivatives to the culture medium using genetic engineering technology. It is in.

(Means for Solving the Problems) In order to solve the above problems, the present inventors have developed a DNA sequence encoding tryptophanase, a recombinant plasmid in which the DNA sequence is incorporated into an expression vector, and a recombinant plasmid containing the plasmid. The present inventors investigated a transformant and a method for producing tryptophanase using the transformant. First, the tryptophanase gene possessed by a tryptophanase-producing bacterium was cloned, and the DNA sequence of the gene was determined. Next, we obtained a recombinant plasmid in which the DNA sequence was incorporated into an expression vector, prepared a transformant containing the plasmid, studied a method for producing tryptophanase using the transformant, and completed the present invention. It's arrived.

Specifically, the present invention provides (1) a DNA sequence encoding tryptophanase derived from a strain of the genus Proteus.

(2) A recombinant plasmid containing the DNA sequence according to claim 1.

(3) A transformant transformed with the recombinant plasmid according to claim 2.

(4) Transforming biological cells or microorganisms with the recombinant plasmid incorporating the DNA sequence according to claim 1, culturing the obtained transformant, and collecting tryptophanase from the culture. A method for producing tryptophanase.

It consists of

The DNA sequence encoding tryptophanase derived from a strain of the genus Proteus of the present invention is shown in FIG.

Even if the DNA fragment encoding tryptophanase derived from a strain of the genus Proteus of the present invention has a part of the nucleotide sequence shown in Figure 1, the nucleotide sequence may be partially deleted, substituted, or inserted. Even if the addition of
As long as it encodes tryptophanase, it is within the scope of the present invention. Hereinafter, each step for carrying out the present invention will be explained in detail.

(a) DNA sequence encoding tryptophanase and preparation of $JI recombinant plasmid DNA encoding tryptophanase in the present invention
The Proteus strain from which the sequences were collected is Proteus inconstans (Proteus neon).
s tans ) is preferred, and the DNA sequence is preferably Proteus inconstans ).
stans) can be isolated from the chromosomal DNA. A suitable example is Proteus inconstans.
us 1nconstansc in nutrient medium, and this culture was cultured in 5. OOOrpm or more, preferably 8.00
0 to 10,000 rpm for 5 minutes or more, preferably 10
Centrifuge for ~15 minutes to obtain bacterial cells. From this bacterial cell, for example, Rodriguez R, L et al Rec.
ombinant DNA Techniques (＾
ddison-Wesley Pub! ishing
Company, 1983) or Koizumi
J, et al., Biotecb.

Chromosomal DNA can be obtained by the method described in Bioeng, 27, 721-728, 1985.

This chromosomal DNA is cleaved with a restriction enzyme such as EcoRI (manufactured by Toyobo) at 30°C or higher, preferably 37°C, for 30 minutes or more, preferably 1 to 2 hours, to obtain chromosomal DNA fragments of various sizes. Obtain a mixture of pieces. From the DN fragment mixture obtained in this way, IINA fragments with low molecular weights (for example, less than IKbρ) are removed by, for example, sig sugar density gradient centrifugation, and then subjected to fragmentation using ethanol precipitation, etc.
A mixture of DNA fragments containing the gene encoding tributophanases is obtained by condensation using an i-reduction method.

The recombinant plasmid of the present invention is a Proteus plasmid.
s) DN encoding tryptophanase derived from a strain of the genus
The A sequence is integrated into vector DNA.

The vector DNA used in the present invention is one that allows tryptophan to be expressed in host biological cells or microorganisms without requiring the addition of tryptophan or its derivatives. For example, plasmid vector D
NA, bacteriophage vector DNA, etc., and a preferred example is plasmid vector pBR32.
2, ρtlc19 (Toyobo 51), bacteriophage vector λZAP, E gate BL3 (Strage
(manufactured by me), etc.

In the present invention, a DNA fragment mixture containing a gene encoding tryptophanase is ligated to the vector DNA described above. For ligation, use E. coli DNAU gauze, T4DN, etc.
A ligase (manufactured by Toyobo), preferably T4 DNA ligase, at 4 to 17°C, preferably 4 to 16°C.
Recombinant I
Obtain INA. Using this M-transformation [lNA, host microorganisms such as [! , colt-12, E, coli J
M109 strain, E. coli IIBIOI strain, E. coli
li C600 strain, 14. E. coli This transformation and transduction is described, for example, in Maniatis T, et al.

Molecular Cloning (Cold Sp
This method can be carried out by the method described in J.D. Ring 11 Arbor, 1982).

Then, a strain capable of producing tryptophanase is screened from the transformant or transductant by an enzyme immunoassay or the like, and a recombinant DNA containing a gene encoding tryptophanase is obtained. Furthermore, the DNA fragment containing the gene encoding tryptophanase in the recombinant DNA is fragmented into small fragments according to a conventional method and ligated with the vector DNA to obtain a recombinant plasmid having a DNA sequence encoding tryptophanase. I can do it.

The nucleotide sequence of the D) JA fragment inserted into the G-replacement plasmid was determined, and the D) JA fragment encoding tryptophanase was determined.
Obtain NA fragment.

(b) Preparation of transformants D encoding tryptophanase obtained in (a)
By introducing the Mi recombinant plasmid into which the NA sequence has been incorporated into a vector into biological cells or microorganisms, a transformant having both the desired genetic traits and the traits of the vector DNA can be obtained. Host microorganisms include E. coli K1
2 strains, E. coli JM109 strain, E. coli H
8101 strain, E. coli C600 strain, E. coli
Examples include the Dll-1 strain. Suitable examples obtained in this way include, for example, E. coli IIBIO
I (pTE1920).

(C) Production of tryptophanase To culture the transformant obtained in (b) above, a medium suitable for the production of tryptophanase and suitable for the growth of the host microorganism is used. LB medium (tryptone, yeast extract, salt), BPB medium (Dirco; polypeptone, yeast extract, potassium phosphate), etc., which are used as a growth medium for Escherichia, may be used as the basic medium. In addition, other carbon sources, nitrogen sources, and nutrients such as amino acids and vitamins may be added as necessary. The culture method is usually shaking culture or aerated agitation culture. The culture temperature is in the range of 20 to 45°C, preferably around 37°C, and the initial culture pH is pH 6.0 to 7.5.
, preferably around pH 6.2 to 6.8.

It can be carried out under conditions other than these if the strain used grows.

The culture period is 10 to 40 hours, and tryptophanase grows and accumulates within the bacterial cells.

The present enzyme may be purified by any commonly used purification method. For example, for the extraction method, any method such as ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant, etc. may be used. Furthermore, the extract can be prepared using known salting-out methods such as ammonium sulfate or sulfuric acid, metal aggregation methods such as magnesium chloride or calcium chloride, aggregation methods such as protamine or polyethyleneimine, and DE8 (diethylaminoethyl) Sepharose (Pharmacia). It can be purified by ion exchange chromatography using CM (carboxymethyl), gel filtration, etc.

(Effect of the invention) When a transformant having a recombinant plasmid containing the DNA sequence encoding tryptophanase of the present invention is cultured, tryptophanase can be efficiently produced without adding tryptophan to the medium. Become.

(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.

Proteus inconstance
onstans) strain IP012931 in a nutrient medium (polypeptone 1%, yeast x-jr 0.59A, salt 0.
5%) 1ooIIJffi and 1 at 37℃.
A culture solution was obtained by culturing with shaking for 0 hours.

This culture solution was centrifuged at I2.00 rpm for 1 u to obtain wet bacterial cells. From the bacterial cells, Rodriguez R,
I, et al.

Recombinant DNA Technique
s (Addison-Wesley Publishing
Chromosomal DNA was prepared by the method described in J. G. Company, 1983).

The chromosomal DNA was treated with the restriction enzyme EcoRI (manufactured by Toyobo) 2
5 units were cut at 37°C for 11 hours. 5-2 after cutting
0% sucrose density gradient centrifugation was performed to prepare DNA fragments of 2 to 20 Kbp fractions.

Bacterial Phage Hector λ-ZAP (EcoRT
1 μg (cut, Stratagene) and 0.5 μg of the chromosomal DNA cut with EcoRJ obtained in (1) above.
μg was reacted with 1 unit of T, I) NAIJ gauze (manufactured by Toyobo) at 16°C for 112 hours to ligate the DNA.

The ligated DNA was prepared using the λ DNA in vitro packaging kit Gigapack Gold (Stratege
Pancakes were prepared using a pancake (manufactured by NE). The recombinant phage obtained by packaging was Maniatis T
, et al Molecular Cloning (C
E. coli XLI-Blue strain (S L
(manufactured by Ratagene) was transfected. Use (INA
Approximately 2 x 1O6 phage plaques were obtained per ug.

Search for recombinant phage having a DNA sequence encoding tryptophanase was performed by enzyme immunoassay. The enzyme immunotest was performed using the Gene Expression kit (manufactured by Boehringer Mannheim). This enzyme immunotest showed a detection limit of about 10 pg of tryptophanase, and 1 to 3 positive colors were obtained per 1000 phage plaques.

S tra from 3 positive plaques in enzyme immunoassay
A recombinant plasmid was cut out according to the instructions in the λZAP usage manual manufactured by Tagene. When the three types of recombinant plasmids obtained were cut with EcoRI, a DNA fragment of 8.3 KbP was found in common among the three types of recombinant plasmids.

This 8.3 Kbp DNA fragment was characterized using various restriction enzymes (Figure 3).

From this 8.3Kbp EcoRI fragment approximately 47/g to old n
dl [[ (manufactured by Toyobo) to obtain an EcoRI-Hind■ fragment of about 4 Kbp. The resulting DNA fragments were subjected to agarose electrophoresis using a DNA purification kit, GENE.
Extraction and purification from agarose gel were performed using CLEANTM (manufactured by BIo 101).

Approximately 1 ug of the approximately 4 Kbp EcoRI-11ind m DNA fragment was cut into a plasmid vector BIuescriptKS
(Gate-13) (Manufactured by StrataBene) Approx. 0.
1 unit of T4 ligase at 16°C.
React for 2 hours to ligate the DNA and create a recombinant plasmid p.
Obtained TE201.

Kil to further miniaturize the obtained DNA fragments.

Using Deletion Kit for 5equence (manufactured by Takara Shuzo), the inserted DNA fragment was delayed by about 1.7 bp from the EcoRI cleavage site and about 0.7 Kbp from the Hind III site according to the manual, so that the inserted DNA fragment was about 1.7 bp from the Hind III site. A recombinant plasmid pTE202 with a size of 6 Kbp was obtained.

Next, plasmid pTE202 was digested with pnl and 5acT to cut out a desired DNA fragment using its multi-cloning site.

The obtained approximately 1.6 Kbp pnl-3acl fragment
μg was mixed with about 0.5 μg of expression vector pUc19 which had been cut and circularized with Kpnl-5acl, and reacted with 1 unit of T4 ligase for 16 hours (12 hours) to ligate the DNA to obtain recombinant plasmid pTE1920.

This recombinant plasmid pTE1920 is an expression vector p
The rank promoter of Uc19 (Lac Pro
It encodes tryptophanase, which has biological activity under the control of moter).

On the other hand, single-strand D
Prepare NA and use 7-De for the single-stranded DNA obtained
aza-3equencing Kit (manufactured by Toyobo)
The base sequence was determined using Next, the base sequence of the DNA fragment inserted into the recombinant plasmid pTE1920 was determined. The nucleotide sequence encoding tryptophanase is shown in FIG.

Mania using the recombinant plasmid pTE1920 having the DNA sequence encoding the above tryptophanase.
tis T, et al Molecular Clo
ning (Cold Spring flabor,
E. coli IIBIO by the method described in 19B2)
Transform E. coli) IBIOI (pTE
1920) was adjusted.

This transformant contains approximately 6 soluble proteins without an inducer.
The target tryptophanase was produced down to 0%.

The tryptophanase activity measurement method is to react tryptophan in a solution containing 0.2x indole, 1.1x sodium pyruvate, 1,1x ammonium chloride, and 0.1 mM pyridoxal phosphate at 20°C for 30 minutes. The amount produced was measured by HPLC (Hitachi Manufacturing Co., Ltd.). Enzyme activity was defined as 1 unit of enzyme activity that produced 1 micromole of tryptophan per minute at 20°C.

■ Donor strain Proteus inconstance (Proteu
s 1n constants) and E. coli HBI
The enzyme productivity of the OI (pTE1920) strain was compared in a medium (2% polypeptone, 2% yeast extract, 0.3% lactose, 0.5% sodium chloride, 50 μg/ml ambicicillin).

The culture activity of each strain is shown in Table 1 below.
It can be seen that tryptophanase is highly produced in the li HBLOI (pTE1920) strain without induction of tryptophan or the like.

Table 1 Medium 6 similar to the medium used for the comparison of enzyme productivity above!
Dispense into a 102-jar fermenter and heat at 121°C.
After sterilizing in an autoclave for 15 minutes and cooling, 50 mg
6d/d ampicillin was added. E and c were cultured in this medium with shaking at 37°C for 8 hours in the same medium as above.
oli IIBIOI (pTE1920)'? t
60 tnl of a. Tryptophanase activity at the end of culture was 1.050/d-Broth.

Culture solution 6I! Collect bacterial cells by centrifugation from
The cells were suspended in MK-Lin #t1M-gai'e pH 7,5,500d, and the cells were crushed by a conventional mechanical crushing method using glass beads. A crude enzyme solution of tryptophanase was obtained from this cell suspension by centrifugation.

The thus obtained crude enzyme solution was subjected to nucleic acid removal treatment with polyethyleneimine, and then subjected to salting out fractionation using ammonium sulfate. 50 d of the salt-folded precipitate obtained here.
5011IM was redissolved in monophosphate buffer, pH 7.5, and subjected to DEAE-Sepharose (manufactured by Pharmacia) column chromatography equilibrated with the same buffer. Approximately 0.1~ by needle salt concentration gradient elution method
Tryptophanase active fractions were obtained in a salt concentration range of 0.3M. After concentrating this tryptophanase active fraction using an ultrafilter, 50s+M K-phosphate buffer, pl+
Gel filtration was performed using Sephadex G-25 (Pharmacia!!i) column chromatography equilibrated with 7.5. In this way, 365 units of purified tryptophanase were obtained. The yield was 58 %Met.

[Brief explanation of drawings]

Figure 1 Huff “Lotius inconstans” (Prot.
Fig. 2 shows the corresponding amino acid sequence. FIG. 3 shows the construction progress of recombinant plasmid pTE1920. Figure 1 (a) ATCGCT AAA AGA ATT GT
A GAA CCA TTCCGC0 ATT AAA ATG GTA GAA
AAT ATCCGT ATT CCA0

Claims (4)

[Claims] (1) A DNA sequence encoding tryptophanase derived from a strain of the genus Proteus. (2) A recombinant plasmid containing the DNA sequence according to claim 1. (3) A transformant transformed with the recombinant plasmid according to claim 2. (4) Transforming biological cells or microorganisms with the recombinant plasmid incorporating the DNA sequence according to claim 1, culturing the obtained transformant, and collecting tryptophanase from the culture. A method for producing tryptophanase.