JPH104967A - Dna coding maleic acid-isomerizing enzyme and its utilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new DNA coding a maleic acid-isomerizing enzyme which has a specific amino acid sequence and which may have the substitution, deletion or insertion of such an amino acid residue as substantially not damaging the activity for isomerizing the maleic acid to produce fumaric acid. SOLUTION: This new DNA codes a maleic acid-isomerizing enzyme which has an amino acid sequence of the formula and which may have the substitution, deletion or insertion of such an amino acid residue as substantially not damaging the activity for isomerizing the maleic acid to produce fumaric acid. The maleic acid isomerization enzyme is useful for efficiently isomerizing the maleic acid to produce the fumaric acid, etc. The new DNA coding the maleic acid-isomerizing enzyme is obtained by synthesizing a probe on the basis of the N-terminal sequence of the maleic acid- isomerizing enzyme produced by a microorganism belonging to the genus Bacillus circulans, subjecting the synthesizing probe to a Southern hybridization treatment with the fragment of the chromosome DNA of the microbial cell to form a λ-DNA library having the objective fragment, and subsequently subjecting the library to treatments such as a plaque hybridization treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA encoding maleate isomerase and its use, and more particularly to Bacillus circus.
ulans), which encodes an enzyme having an activity of isomerizing maleic acid to produce fumaric acid;
The present invention relates to a transformant carrying the DNA, a method for producing the enzyme using the transformant, and a method for producing fumaric acid. Fumaric acid, an isomer of maleic acid, is a useful organic acid that can be used as a raw material for various fine chemicals such as aspartic acid and malic acid.

[0002]

2. Description of the Related Art As a method for producing fumaric acid by isomerizing maleic acid, mainly a chemical method has been proposed (US Pat. Nos. 2,816,923 and 2,955).
No. 136, No. 2,332, 992), the conversion to fumaric acid is restricted by the reaction equilibrium, and this method is a high-temperature reaction, which causes the deterioration of maleic acid or fumaric acid, and produces by-products. There is a problem that the yield is reduced due to formation.

On the other hand, regarding the production of fumaric acid by an enzymatic reaction, maleic acid isomerase (male ci
It is known that s-trans-isomerase isomerizes maleic acid to fumaric acid. Examples of microorganisms that produce maleic acid isomerase include, for example, Pseudomonass sp.
p. ) [K. Otsuka, Agric. Bio
l. Chem. , Vol. 25, No. 9, p.
726 (1961)], Alcaligenes faecalis IB-
14 [Takamura, Agric. Bio
l. Chem. , Vol. 33, p. 718 (19
69)], Pseudomonas fluorescens (Pse
udomonas fluorescens) ATCC
23728 [Scher, J. et al. Biol. Che
m. , Vol. 244, p. 1878 (196
9)], the maleic acid isomerase produced by them has only been reported for a part of enzymatic properties, and the exact molecular weight, subunit structure, Details such as the amino acid sequence and the total DNA base sequence are not yet clear. For industrial use, it is necessary to obtain a transformant having a low activity and a high activity by using the conventional maleate isomerase as it is, and it has been desired to isolate a DNA encoding the enzyme.

[0004]

SUMMARY OF THE INVENTION If a maleate isomerase gene can be obtained and introduced into a microorganism by using an appropriate vector, a transformant capable of retaining multiple copies of the same gene in the cells can be obtained. In addition, it is possible to dramatically increase the catalytic ability of microorganisms as compared with conventional methods.

[0005] It is an object of the present invention to provide a DNA encoding a maleate isomerase derived from a microorganism, and a method for producing fumaric acid using the same.

[0006]

SUMMARY OF THE INVENTION The present inventors have proposed a method for preparing Bacillus circulans.
ns) As a result of intensive studies for the purpose of isolating a gene DNA fragment encoding maleic acid isomerase from microorganisms of the species, the gene DNA fragment was isolated and incorporated into a vector to form a recombinant vector. A microorganism is transformed with this recombinant vector to prepare a transformant having improved maleate isomerase activity, and this is used to efficiently produce fumaric acid or various derivatives derived from fumaric acid. Thus, the present invention was completed.

Thus, according to the present invention, (a) substitution or deletion of an amino acid residue having the amino acid sequence shown in SEQ ID NO: 1 which does not substantially impair the activity of isomerizing maleic acid to produce fumaric acid DNA encoding a maleate isomerase which may have an insertion, (b) a recombinant vector obtained by linking the DNA of the above (a) to a vector, and (c) a DNA obtained by introducing the DNA of the above (a). (D) culturing the transformant of (c) above in a medium, and collecting the maleate isomerase from the culture, thereby producing a maleate isomerase. Law, and (e)
Adding the crude transformant of maleic acid isomerase or the purified enzyme collected from the transformant of the above (c) or a culture of this transformant to an aqueous solution containing maleic acid;
A process for producing fumaric acid is provided, wherein maleic acid is isomerized to produce fumaric acid.

Hereinafter, the present invention will be described in detail. The "maleic acid isomerase" of the present invention
EC5. s-trans-isomerase;
“2.1.1)” means an enzyme that catalyzes a reaction for isomerizing maleic acid (cis type) to produce fumaric acid (trans type). In the present specification, a DNA encoding maleate isomerase may be referred to as a “maleate isomerase gene” for convenience.

The base sequence of the maleic acid isomerase gene of the present invention includes a base sequence capable of encoding the amino acid sequence represented by amino acid numbers 1 to 251 in the amino acid sequence of SEQ ID NO: 1. In addition, the DN of the present invention
A is not limited to this sequence, and is not limited to the amino acid sequence described above, as long as it does not substantially impair the activity of isomerizing maleic acid to generate fumaric acid, and is not limited to substitution, deletion, insertion or insertion of amino acid residues. DNAs encoding maleic acid isomerase having transposition, and Ds encoding maleic acid isomerase capable of hybridizing to these DNAs
All NAs are included in the gene DNA of the present invention.

[0010] More specifically, the base sequence of the maleate isomerase gene of the present invention includes the base sequence shown in SEQ ID NO: 1.

[0011] The maleate isomerase gene of the present invention comprises:
It was obtained for the first time by cloning from Bacillus circulans, but since its nucleotide sequence was determined by the present invention, it can be synthesized based on this sequence.

[0012] Bacillus circulans includes Bacillus circulans MI-113 strain.
This strain is a strain isolated by the present inventors as described in Example 1 below, and has been deposited at the Ministry of International Trade and Industry with the National Institute of Advanced Industrial Science and Technology under the accession number FERMP-15645. .

Hereinafter, an example of a method for obtaining a maleate isomerase gene from the microorganism, a method for producing the maleate isomerase which is the gene product, and a method for isomerizing maleic acid to produce fumaric acid will be described. I do.

[0014] The maleate isomerase gene is usually present on the chromosome of the above-mentioned source microorganism, and the chromosome-derived DN
It can be separated and obtained from the A library by the hybridization method described below.

(1) Purification of maleate isomerase, partial determination of its amino acid sequence and preparation of a DNA probe based thereon: Bacillus circulans MI-113
As a method for extracting and purifying maleate isomerase from the cells of the strain, any known method for purifying enzymes can be used.

For example, as a method for disrupting cells, ultrasonic disruption; mechanical disruption using a French press, a homogenizer, or the like; enzymatic disruption using lysozyme or the like can be used. The maleic acid isomerase can be obtained as a crude enzyme solution by separating a soluble fraction from the disrupted cells.

As a method for purifying the maleate isomerase from the obtained crude enzyme solution, generally, (a) separation by precipitation, for example, ammonium sulfate precipitation, (b) separation by chromatography, for example, ion exchange chromatography It can be carried out by a combination of (c) separation methods by electrophoresis, such as chromatography, affinity adsorption chromatography, gel filtration chromatography, etc., and one example is described in detail in Example 2.

The detection of the maleate isomerase fraction in the above-mentioned purification step is performed by the method of Otsuka et al. [Agric. Biol. Chem. , Vo
l. 25, p. 726 (1961)] in the presence of porcine heart-derived fumarase (manufactured by Boehringer Mannheim) by measuring the decrease in the maleic acid substrate by the maleate isomerase activity as a decrease in the absorbance at 240 nm.

The purified maleate isomerase was purified from Da
vis method [B. J. Davis, Ann. N.
Y. Acad. Sci. , Vol. 121,
p. 404 (1964)] or the Laemmli method [U. K. Laemmli, Nature, V
ol. 227, p. 680 (1970)], and subjected to coomassie blue dye solution [composition: 0.2% (w / v) Coomassie brilliant blue R250, 40% (v / v) methanol, 10%
% (V / v) acetic acid] or a silver staining kit (manufactured by Wako Pure Chemical Industries, Ltd.) to stain the enzyme protein.
The purification purity and the molecular weight of maleate isomerase can be known.

The partial determination of the amino acid sequence of the purified maleate isomerase is performed by separating the enzyme into its constituent subunits, followed by the amino acid sequence from the N-terminus of each subunit or an appropriate protease. The amino acid sequence from the N-terminus of the peptide fragment obtained by digesting each subunit with an amino acid sequencer by Edman degradation (Applied Biosystems, 473
(A type).

Next, a probe having a nucleotide sequence predicted from the obtained amino acid sequence is synthesized using a DNA synthesizer (Model 394, manufactured by Applied Biosystems).

An example of the partial amino acid sequence of the maleic acid isomerase obtained as described above is shown in SEQ ID NO: 2. Further, a probe DN deduced from the amino acid sequence
Examples of the sequence of A are shown in SEQ ID NOs: 3 and 4.

(2) Isolation of a chromosomal DNA fragment containing the maleate isomerase gene: Bacillus circulans
As a method for isolating the chromosomal DNA containing the maleate isomerase gene from the cells of the MI-113 strain, any known method for isolating the gene can be used. An example will be described below.

(A) Preparation of chromosomal DNA library:
When extracting chromosomal DNA from the above strain, cells of the strain cultured in an appropriate medium can be used,
It is also possible to use a preserved sample obtained by freezing and preserving the cultured cells after collecting the cells.

The obtained chromosomal DNA is converted into an appropriate restriction enzyme,
For example, partial digestion is performed using Sau3AI, and a library of chromosomal DNA is prepared using a host-vector system such as Escherichia coli. Specific examples of usable vectors include lambda phage vectors such as λFIXII (manufactured by Toyobo Co., Ltd.), pUC118 (manufactured by Takara Shuzo), and pBR322.
(Manufactured by Takara Shuzo), Cosmid pWE-15 (Stratag
ene Co., Ltd.).

Various DNAs obtained by the above partial decomposition
Insertion of the fragment into the above vector, for example, into a phage vector λFIXII (manufactured by Toyobo Co., Ltd.) is performed by ligating a vector cleaved with an appropriate restriction enzyme, for example, Sau3AI, and a partially digested DNA fragment using T4 DNA ligase. It can be done by doing. Thus, chromosome DN
A library is obtained.

(B) Selection of a vector containing the maleate isomerase gene: chromosomal DNA prepared in the above (A)
To select a vector containing the maleate isomerase gene from the library, the chromosomal DNA library is used to select a host microorganism such as Escherichia coli (Es
In this case, clones carrying the maleate isomerase gene may be selected from the resulting transductants or transformants by appropriate means.

Specifically, the above phage vector was transformed into Escherichia coli (Escherichia coli).
i) For example, strain P2392 [Ausubel et a
l. , Nucleic Acids Res. , Vo
l. 7, p. 1513 (1979)]
This is overlaid on an agar medium to form plaque.

Next, the phage DNA in the plaque was transferred to a nitrocellulose membrane, the phage DNA was immobilized on the nitrocellulose membrane, and the probe prepared in the above item (1), for example, the nucleotide sequence shown in SEQ ID NOS: 3 and 4 was used. Hybridization using synthetic oligonucleotides having a molecular weight [Molecular Clonin
g, Cold Spring Harbor Lab
org. Press (1989)].

Thus, it is possible to detect and select a transductant containing a phage vector having a maleate isomerase gene derived from chromosomal DNA of Bacillus circulans.

Alternatively, when a chromosomal DNA library is prepared using the above plasmid vector, Escherichia coli (Escher
ichia coli) JM109 (manufactured by Takara Shuzo), and colony hybridization using the probe prepared in the above (1), for example, a synthetic oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 3 or 4 from the obtained transformant. Method [R. Bruce Wallac
e, et al. , Nucleic Acids
Res. , Vol. 9, p. 879 (198
1)] can be selected.

Further, phage DNA or plasmid DNA is extracted from the transductant or transformant selected as described above, and the inserted fragment is cut out of the vector with an appropriate restriction enzyme, for example, HindIII, to thereby obtain the present invention. DNA can be obtained.

The DNA fragments excised by the above operation are subjected to Southern hybridization using the probe prepared in the above (1), for example, the probe having the nucleotide sequence shown in SEQ ID NO: 3 or 4 [E. M. South
ern, J .; Mol. Biol. , Vol. 9
8, p. 503 (1975)]
It can be reconfirmed that the maleate isomerase gene is present in the inserted DNA fragment.

One of the thus obtained DNA fragments is the above-mentioned Bacillus circulans MI-113.
Examples include a DNA fragment of about 4 kb in size obtained by cutting the strain chromosomal DNA by complete digestion of the restriction enzyme PstI.

(3) Determination of base sequence: The entire base sequence of the 4 kb DNA fragment of PstI obtained in the above (2) is as follows:
For example, plasmid pUC118 vector (Takara Shuzo Co., Ltd.)
Dideoxynucleotide enzyme method using
eoxy chain termination method, S
angel et al. Proc. Natl.
Acad. Sci. USA, Vol. 74, p.
5463 (1977)].

The nucleotide sequence of the DNA fragment of about 4 kb determined in this manner confirmed the presence of an open reading frame, and confirmed that Bacillus circulans M
The maleic acid isomerase gene of the I-113 strain encodes a 251 amino acid sequence of amino acids 1 to 251 in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing below.
It was found to be composed of 3 base pairs. In addition, it was reconfirmed that the obtained nucleotide sequence (SEQ ID NO: 1) contained a sequence corresponding to the nucleotide sequence predicted from the amino acid sequence shown in SEQ ID NO: 2 determined in (1) above.

The maleic acid isomerase gene of the present invention containing the above base sequence is not limited to one isolated from natural chromosomal DNA of Bacillus circulans, but may also be a commonly used DNA synthesizer such as Applied Biosystems. Systems (Applied Biosystem)
s) It may be synthesized using a 394 DNA / RNA synthesizer manufactured by the company.

(4) Preparation of a recombinant vector having a maleate isomerase gene and a transformant carrying this recombinant vector:

The maleic acid isomerase gene of the present invention or a DNA fragment containing the same is ligated to an appropriate vector to prepare a recombinant vector, and this recombinant vector is used to transform an appropriate host microorganism. Thus, it can be expressed. When preparing a recombinant vector, usually, the maleic acid isomerase gene together with a promoter suitable for the host microorganism, such that the 5 'end of the coding region of the gene is ligated downstream of the promoter, the vector Insert Alternatively, a maleate isomerase gene may be inserted into an expression vector containing a promoter.

In addition to the expression method using such an expression vector, a homologous recombination technique for directly introducing a DNA fragment containing a maleate isomerase gene linked to a promoter into the chromosome of a host microorganism [A. A. Verte
s, et al. , Biosci. Biotec
hnol. Biochem. , Vol. 57,
p. 2036 (1993), etc.] or a technique of introducing using a transposon, insertion sequence, or the like [A. A.
Vertes et al. , Molecular
Microbiol. , Vol. 11, p. 73
9 (1994), etc.].

The expression vector is not particularly limited as long as it can be replicated and propagated in a host microorganism, and any of a plasmid vector and a phage vector can be used. Specific plasmid vectors include pBR322, pUC18, pHSG298, pU
C118, pSTV28, pTWV228, pHY30
0PLK (the above plasmid vectors can be purchased, for example, from Takara Shuzo Co., Ltd.), pKK223-3, pPL-
Lambda Inducible Expressi
on Vector (these can be purchased from, for example, Pharmacia), pCRY31, pCRY3KE, pC
RY3KX (US Pat. No. 5,185,262), or derivatives thereof, and the like.

As a phage vector, (λF
ixII vector (available from Stratagene) and the like. As the promoter for expressing the maleic acid isomerase gene of the present invention, a promoter possessed by a host microorganism can be generally used, but the promoter is not limited thereto. Any promoter may be used as long as it is a nucleotide sequence derived from a prokaryote. Specifically, the promoter of the lactose operon,
Tryptophan operon promoter, phage lambda derived PL promoter, tryptophan lactose hybrid (tac) promoter [H. A. Bose et
al. , Proc. Natl. Acad. S
ci. U. S. A. , Vol. 80, p. 2
1 (1983)]. When a microorganism capable of functioning as a host derived from a bacterium belonging to the genus Bacillus is used as a host, a promoter specific to the obtained maleate isomerase gene may be used. Furthermore, when the base sequence of the promoter is known, a commonly used DNA synthesizer such as Applied Biosystems 3
It may be synthesized using a 94 DNA / RNA synthesizer.

Among these promoters, an inducible promoter can be used for the purpose of improving expression efficiency. For example, in the case of the lactose operon promoter, lactose or isopropyl-
Gene expression can be induced by adding β-D-thiogalactoside (IPTG).

The host into which the maleate isomerase gene is introduced is not particularly limited, but Escherichia bacteria, Bacillus (Ba)
bacillus, Serratia
Genus bacteria, Pseudomonas genus bacteria, Corynebacterium (Corynebacter)
ium) bacteria, Breviba
cterium), Rhodococcus (Rhodoc)
occus), Lactobacillus (Lactobaba)
bacteria, Streptomyces (Stre)
ptomyces bacterium, Thermu
s) Bacteria, Streptococcus
ccus) bacteria and the like can be suitably used.

Specifically, Escherichia coli (Esh
erichia coli), Bacillus subtilis (B)
A. subtilis), Bacillus brevis, Bacillus subtilis
Stearothermophilus (Bacillus stea)
rothermophilus), Seratia marcescens, Pseudomonas pu
tida), Pseudomonas aeruginosa (Pseu)
domonas aeruginosa), Corynebacterium glutamicum (Corynebacteri)
um glutamicum), Brevibacterium
Flavum (Brevacterium flavu)
m), Brevibacterium lactofermentum (B
revacterium lactofermen
tun), Rhodococcus erythropolis (Rhodo)
coccus erythropolis, Thermus thermophilus
ilus), Streptococcus lactis (Str)
eptococcus lactis, Lactobacillus casei
i), Streptomyces lividans (Strept
omyces lividans) can be used.

As a method for introducing a gene into the host microorganism, a competent cell method [Journal of Mol] is used.
ecological Biology, Vol. 53, p.
159 (1970)], a pulse wave energization method [J. In
dust. Microbiol. , Vol. 5,
p. 159 (1990)] and a transduction method using phage [E. Ohtsubo, G
enetics, Vol. 64, p. 189 (1
970)] and the joint transfer method [J. G. FIG. C. Otto
w, Ann. Rev .. Microbiol. ,
Vol. 29, p. 80 (1975)], cell fusion method [M. H. Gabor, J.M. cterio
l. , Vol. 137, p. 1346 (197
9)] can be used.

(5) Production of maleic acid isomerase using the transformant of the present invention, and isomerization reaction of maleic acid to fumaric acid using maleic acid isomerase: (4)
The maleic acid isomerized by adding a partially purified fraction of the maleic acid isomerase or a purified enzyme obtained from the transformant obtained as described above or a culture of this transformant to an aqueous solution containing maleic acid. To produce fumaric acid. Here, the culture refers to the cells of the transformant or / and the medium after culturing (the culture supernatant when a liquid medium is used for culturing).

The above transformant can be cultured in a conventional nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, various vitamins, etc. Examples of the carbon source include glucose, sucrose, fructose, maltose and the like. Sugars, alcohols such as ethanol and methanol, organic acids such as citric acid, malic acid, succinic acid, maleic acid and fumaric acid, and molasses. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like are used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, various vitamins such as casamino acid and biotin can be added to the medium.

The cultivation is usually carried out under aerobic conditions such as aeration and stirring and shaking. The culture temperature is not particularly limited as long as the host microorganism can grow, and the pH during the culture is not particularly limited as long as the host microorganism can grow. The pH adjustment during the culture can be performed by adding an acid or an alkali.

By collecting the cells from the culture thus obtained by centrifugation or the like, the expression product of the maleate isomerase gene, that is, the cells containing the maleate isomerase can be obtained.

The transformants used in the method for producing fumaric acid of the present invention include, but are not limited to, cells isolated from a culture solution in which the transformants have been cultured, as well as culture solutions, crushed cells, and extracts. Alternatively, a crude enzyme preparation obtained from a transformant or a purified enzyme preparation may be used. Furthermore, those obtained by immobilizing the above transformant, its crushed product, extract or purified enzyme on a carrier can also be used.

When the transformant is immobilized on a carrier,
As collected from the culture, or a suitable buffer, for example, a phosphate buffer of about 0.02 to 0.2 M (pH 6 to
The cells washed in 10) and the like can be used. Further, the cells recovered from the culture, ultrasonic, crushed product obtained by crushing by means such as squeezing, an extract containing maleate isomerase obtained by extracting the crushed product with water or the like An extract obtained by further performing a treatment such as ammonium sulfate salting-out or column chromatography on the extract and immobilizing a partially purified component of maleate isomerase on a carrier is also used for the production of fumaric acid of the present invention. be able to.

The immobilization of the cells, the disrupted cells, the extract, or the purified enzyme is carried out according to a commonly used method known per se in a suitable carrier such as acrylamide monomer, alginic acid or carrageenan. Can be performed by a method of immobilizing

The aqueous solution used for the reaction may be an aqueous solution containing maleic acid or a suitable buffer such as 0.02
A phosphate buffer of about 0.2 M (pH 6 to 10) can be used. When it is necessary to further increase the material permeability of the cell membrane of the bacterial cells, the aqueous solution contains 0.05 to 2% (w / w) of toluene, xylene, a nonionic surfactant and the like.
v) It can also be added.

The concentration of maleic acid as a reaction raw material in the aqueous solution is suitably about 0.1 to 2M. As the form of maleic acid, not only the form of acid or salt but also its anhydride can be used. Maleic anhydride is easily converted to maleic acid by converting it into an aqueous solution. Examples of the maleic acid salt include a sodium salt, a potassium salt and the like.

The temperature and pH of the enzyme reaction in the above aqueous solution are not particularly limited, but are usually from 10 to 60 ° C., preferably from 15 to 50 ° C., and the pH in the reaction solution is 5 to 50 ° C.
-10, preferably around 6-9. Further, the pH can be adjusted by adding an acid or an alkali. The aqueous solution containing fumaric acid thus obtained can be directly used as a raw material for producing malic acid, aspartic acid and the like. The present invention has been described above, and will be described more specifically with reference to the following examples. However, the examples are to be regarded as helping to obtain a specific recognition of the present invention, and do not limit the scope of the present invention.

[0057]

【Example】

[Example 1] Isolation of microorganisms having maleate isomerase activity (1) Isolation of maleate-assimilating microorganisms A soil sample collected from nature was used as a medium A [composition: disodium maleate 5 g, KH ₂ PO ₄ 0.7 g, K _{2
HPO 4 1.4g, NH 4 NO 3} 1g, MgSO 4 · 7
H ₂ O 0.2 g, biotin 0.2 mg, thiamine
Hydrochloride _{0.2mg, FeSO 4 · 7H 2 O} 20mg,
MnSO ₄ 20 mg, distilled water 1 L (pH 7.2)]
The mixture is poured into 10 ml, and cultured with shaking aerobically at 40 ° C. for 2 days. 1 ml of this culture was subcultured into 10 ml of the above-mentioned medium A, and further cultured for 2 days. After performing the operation of this enrichment culture twice, it was spread on a plate medium having the composition of the above-mentioned medium A containing 2% of agar, and cultured at 40 ° C for 2 to 4 days. Colonies that grew on this plate medium were isolated.

(2) Selection of microorganism having maleate isomerase activity The microorganism isolated in (1) above was shake-cultured at 40 ° C. for 1 day in 5 ml of the above-mentioned medium A, and centrifuged (3, 3). 00
(0 × g, 4 ° C., 20 minutes). Next, the obtained cells were suspended in 1 ml of a 0.1 M phosphate buffer (pH 7) containing dithiothreitol (1 mM), and the cells were sonicated (Cosmo Bioruptor).
UCD-200T) to disrupt the cells (4 ° C, 15 ° C).
), And the supernatant obtained by centrifuging the crushed product was collected and used as an enzyme solution.

For the detection of maleate isomerase activity, the enzyme solution obtained above was mixed with 0.5 ml of reaction solution A [composition: sodium maleate 50 mM, dithiothreitol 1 mM, Tris-HCl buffer 100 mM (pH 8)]. And reacted at 40 ° C. for 24 hours. The obtained reaction solution was analyzed using an organic acid analysis column [SequeTag ™
(0.5x25cm MilliGen / Biosea
rch, U.S. S. A. ], UV detector (240 nm)
High performance liquid chromatography analysis using
LC-5A) (mobile phase: 35% methanol solution containing 5 mM KH ₂ PO ₄ and 5 mM H ₃ PO ₄ ). Among the microorganisms isolated in the above (1), those having maleic acid isomerase activity were selected as microorganisms having a fumaric acid peak in addition to the raw material maleic acid peak. One strain of a microorganism having maleic acid isomerase activity, MI-113, obtained by the above separation operation was obtained. As a result of further examination of mycological properties of the isolated strain as described above, it was found that the strain was a novel strain, and Bacillus circulans (Bacillus circulans) was identified.
s) It was named MI-113 strain. This bacterium has been deposited with the Biotechnology Research Institute under the accession number: FERM P-15645.

1. Morphological properties Morphology: Bacillus spores: not formed (atypical) Gram stain: positive Motility: none

[0061] 2. Physiological properties (+: positive,-: negative) Attitude to oxygen: facultative anaerobic Growth under anaerobic: + catalase: + VP reaction:-pH in VP broth: 5.7 Acid production :-Glucose: +-Arabinose: +-Xylose: +-Mannitol: + Production of gas from glucose:-Liquefaction of gelatin: + Decomposition of starch: + Use of citrate: + Use of propionate:-Egg yolk Reaction:-Reduction of nitrate: + Growth at pH 6.8 (Nutrient broth): + Growth at pH 5.7:-Growth in the presence of 5% NaCl:-Growth in the presence of 7% NaCl: -10 C. Growth at -30.degree. C .: + Grow at 40.degree. C .: + Grow at 50.degree. C .:-Maleate isomerase activity: +3. Characteristics of bacterial cell components GC content (mol%) of intracellular DNA: 39% Cell wall diamino acid: meso-diaminopimelic acid quinone system: MK-7

Example 2 Bacillus circulans
Cloning of DNA Fragment Containing Maleate Isomerase Gene Derived from MI-113 Strain (1) Purification of Maleate Isomerase, Partial Determination of Amino Acid Sequence and Preparation of DNA Probe Based thereon: Medium A [Composition: Maleic Disodium acid 5g, KH ₂
PO ₄ 0.7 g, K ₂ HPO ₄ 1.4 g, NH ₄ NO ₃
1 g, MgSO ₄ .7H ₂ O 0.2 g, biotin
0.2 mg, thiamine hydrochloride 0.2 mg, FeSO ₄
・ 7H ₂ O 20mg, MnSO ₄ 20mg, distilled water
1L (adjusted to pH 7.2)] 100 ml into a 500 ml Erlenmeyer flask, sterilized at 120 ° C for 20 minutes, and mixed with Bacillus circulans MI-113.
The strain was inoculated and cultured with shaking at 40 ° C. for 24 hours.

Next, 1000 ml of the same medium as above was placed in a 3 L jar fermenter,
After 30 minutes of sterilization, 30 ml of the above culture was inoculated, and cultured under aeration and stirring at 40 ° C. for 24 hours. The culture solution after the culture was centrifuged (3,500 × g, 4 ° C., 20 minutes) to collect the cells.

The maleic acid isomerase was extracted from the obtained cells by suspending the cells in a phosphate buffer solution (20 mM, pH 7.2) containing 0.5 mM dithiothreitol, and then using an ultrasonic crusher ( (Branson) to disrupt the cells. Centrifuged cells (10,000 ×
g, 4 ° C., 30 minutes) to obtain a soluble fraction of the supernatant as a crude enzyme solution.

Next, this crude enzyme solution was subjected to ammonium sulfate fractionation (35 to 7).
0%) and dialyzed against the above phosphate buffer at 4 ° C. overnight. Then, the dialysate was subjected to DEAE Sepharose (Pharmacia) column chromatography, gel filtration (Sephacryl S-200, Pharmacia) column chromatography, and Mono Q (Pharmacia) column chromatography to obtain maleic acid isomerase. An active fraction was obtained.

The activity of maleate isomerase was detected by the method of Otsuka et al. [Agric. Biol. Chem. , V
ol. 25, p. 726 (1961)] and porcine heart-derived fumarase (Boehringer Mannheim)
In the presence, the decrease in maleic acid was measured as the change in absorbance at 240 nm.

The purified fraction of the maleate isomerase was subjected to polyacrylamide gel (4 to 20% gradient gel).
Separation by electrophoresis [running in a buffer solution (25 mM Tris-192 mM glycine, pH 8.4) at a constant current of 40 mA for 1 hour], Coomassie blue dye [composition:
0.2% (w / v) Coomassie Brilliant Blue R25
0, 40% (v / v) methanol, 10% (v / v) acetic acid], a single band having a molecular weight of about 60 kDa was detected. Further, the purified fraction was treated with sodium dodecyl sulfate (SDS) -treated solution [composition: 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol] Blue (BPB)] and heat denaturation at 95 ° C. for 3 minutes, followed by polyacrylamide gel (10
-20% gradient gel) electrophoresis [electrophoresis in a buffer (25 mM Tris-192 mM glycine-0.1% SDS, pH 8.4) at 40 mA constant current for 1 hour]
As a result, only a band having a molecular weight of about 30 kDa was detected. From these results, the structure of the maleate isomerase was found to have a molecular weight of about 30 k.
It was estimated to be a dimer composed of a Da subunit and having a molecular weight of about 60 kDa.

The amino acid sequence from the N-terminus of this purified isomerase was determined using an amino acid sequencer (manufactured by Applied Biosystems, Model 473A), and the result is shown in SEQ ID NO: 2. Next, a probe DNA having the nucleotide sequences shown in SEQ ID NOS: 3 and 4 predicted from the obtained amino acid sequence was synthesized using a DNA synthesizer (Applied Biosystems, Inc., Model 394).

(2) Preparation of chromosomal DNA fragment containing maleate isomerase gene: Bacillus circulans
The MI-113 strain was cultured by shaking the medium A at 40 ° C. until the late logarithmic growth phase, and the cells were collected.

The obtained bacterial cells were adjusted to a concentration of 10 mg / ml with 10 mM NaCl-20 mM containing lysozyme.
Tris buffer (pH 8.0)-1 mM EDTA · 2N
a. Next, proteinase K (manufactured by Takara Shuzo Co., Ltd.) was added to a final concentration of 100 μg / ml, and the mixture was kept at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate (SDS) was added to a final concentration of 0.5%, and the cells were lysed at 50 ° C. for 6 hours.
To this lysate, an equal amount of a phenol / chloroform solution was added, and the mixture was gently shaken at room temperature for 10 minutes, followed by centrifugation (5,000 × g, 20 minutes, 10 to 12 ° C.).
Then, the supernatant fraction was collected. After sodium acetate was added to the supernatant to 0.3 M, a 2-fold amount of ethanol was slowly added. DNA existing between the aqueous layer and the ethanol layer was taken out with a glass rod, washed with 70% ethanol, and air-dried. A 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution 5 was added to the obtained DNA.
Then, the mixture was allowed to stand at 4 ° C. overnight to obtain a chromosomal DNA solution.

Next, 50 U (units) of the restriction enzyme PstI was added to 90 μl of the chromosomal DNA solution, reacted at 37 ° C. for 1 hour to completely degrade, and subjected to agarose electrophoresis. The DNA was transferred from the agarose gel onto a nylon membrane, and the 5 'terminal phosphate group was radioisotope labeled with T4 polynucleotide kinase (manufactured by Takara Shuzo) and [γ- ³² P] ATP [Anal. Biochem. ,
158, 307-315 (1986)]
Using the synthetic probe shown in FIGS.
ullar Cloning, Cold Spring
Harbor Laboratory Press (19
89)] and Southern hybridization was performed.

As a result, D at which the synthetic probe strongly hybridizes at a position of about 4 kb on the nylon membrane.
The presence of the NA fragment was confirmed.

(3) Insertion of chromosomal DNA into vector:
Bacillus circulans MI-1 obtained in the above item (2)
Restriction enzyme Sau was added to 90 μl of the chromosomal DNA solution of 13 strains.
5 Units of 3AI were added, and reacted at 37 ° C. for 10 minutes to partially decompose. Partially digested DNA of various lengths
And Klenow fragment (Klenow fragment) after digestion with restriction enzyme XhoI.
Using t), T (thymidine), C (deoxycytosine)
Vector λFIXII (λFIXII)
/ XhoI-partial fill-in tr
atated DNA: manufactured by Toyobo Co., Ltd.)
50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂
And 1 unit of T4 DNA ligase 1 unit (the concentration of each component is the final concentration), reacted at 16 ° C. for 10 hours to ligate the partially degraded DNA and the vector,
A λDNA library of chromosomal DNA was obtained.

(4) Preparation of Transformant and Selection of Target Recombinant DNA: The λ DNA library phage solution (2 to 5 × 10 ⁴ pfu; diluted with SM buffer) prepared in the above (3) and Escherichia An equivalent amount of the culture solution of E. coli P2392 was mixed and kept at 37 ° C for 15 minutes. 3 to 4 ml of λ medium (1% Try
pton, 0.5% Yeast extract, 0.
_{5% NaCl, 0.2% MgSO 4} · 7H 2 O, 0.5
% Agar) and add λ plate (1% Trypto)
n, 0.5% NaCl, 0.2% MgSO 4 · 7H 2
O, 1% Agar) at 37 ° C for 12-1.
The cells were cultured for 6 hours. A nitrocellulose filter was placed on this medium, the plaque formed on the medium was adsorbed to the filter, and the filter was placed on a filter paper immersed in the reagents of (a) to (c) below for 5 minutes each, followed by treatment.

A) 0.5M NaOH, 1.5M Na
Cl b) 0.5 M Tris-HCl (pH 7.5),
5 M NaCl, 0.001 M EDTA c) 2 x SSC (20 x SSC; NaCl 175.3)
g, 88.2 g of trisodium citrate dihydrate in 1 L of distilled water)

After the filter was air-dried, the DNA was fixed by performing a dry heat treatment at 80 ° C. for 2 hours. Using the probes shown in SEQ ID NOs: 3 and 4 prepared in the above (1), plaque hybridization was carried out on the filter prepared above in a conventional manner [Molecular Cloning,
Cold Spring Harbor Labora
tory Press (1989)].
As a result, hybridization-positive plaques were selected for both probes, and phage DNA was selected therefrom.
And the length cut out by the restriction enzyme PstI is about 4
The kb insert was confirmed by agarose gel electrophoresis.

(5) Preparation of Restriction Enzyme Map of Recombinant DNA: About 4 kb long DNA (P
In order to further identify the position of the maleate isomerase gene present on the (stI-PstI) fragment, the DNA fragment was subcloned into plasmid pUC118 (Takara Shuzo) as described below.

The DNA fragment cut out with PstI and the cloning vector pUC118 (Takara Shuzo Co., Ltd.)
Was cleaved with the restriction enzyme PstI and then dephosphorylated, mixed, and mixed with 50 mM Tris buffer (pH
7.6) 10 mM dithiothreitol, 1 mM AT
P, 10 mM MgCl ₂ and T4 DNA ligase 1
Each component of the unit was added (the concentration of each component was the final concentration), and reacted at 16 ° C. for 10 hours.
The stI fragment and the vector were ligated.

Using the ligation reaction solution obtained, a calcium chloride method [Journal of Molecular B]
ology, Vol. 53, p. 159 (197
0)] to transform Escherichia coli JM109 (Takara Shuzo Co., Ltd.) into 50 μg / ampicillin /
ml of medium [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with the restriction enzyme PstI, and the inserted fragment was examined by the hybridization method. In addition to the 3.4 kb DNA fragment of pUC118, a DNA fragment of about 4 kb length was confirmed. The plasmid obtained above was named pMI113, and the Escherichia coli JM109 strain (Takara Shuzo) transformed with the plasmid was named ECMI113.

Example 3 Determination of Nucleotide Sequence: The DNA (PstI-PstI) fragment of about 4 kb in length containing the maleate isomerase gene obtained in item (5) of Example 2 was analyzed for its nucleotide sequence. The sequence was determined by the dideoxynucleotide enzymatic method (dide
oxychain termination method) [Sa
nger, F .; et al. Proc. Natl.
Acad. Sci. U. S. A. , Vol. 74, p.
5463, (1977)]. Due to the presence of the open reading frame in the nucleotide sequence, the maleic acid isomerase gene has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing below, and is composed of 753 base pairs encoding 251 amino acids. There was found. In addition, it was confirmed that this nucleotide sequence contained a sequence corresponding to all the nucleotide sequences predicted from the amino acid sequence (SEQ ID NO: 2) determined in item (1) of Example 2. .

Example 4 Production of Maleic Acid Isomerase and Confirmation of Activity The ECMI113 strain prepared in Example 2 was transformed into an LB medium containing 50 μg / ml of ampicillin [10 g of tryptone,
(5 g of yeast extract, 5 g of NaCl) and aerobically shake-cultured at 30 ° C. for 15 hours. The obtained culture was centrifuged (3,000 × g, 4 ° C., 20 minutes) to collect the cells, and then a phosphate buffer [composition: 20 mM
Potassium phosphate buffer (pH 7.2), 0.5 mM dithiothreitol]. Then, the washed cells were added.
5 g (wet weight) was suspended in 2 ml of the above-mentioned phosphate buffer solution and subjected to an ultrasonic crusher (manufactured by Branson) under ice cooling to obtain a crushed bacterial cell. The crushed material is centrifuged (10,000 × g, 4
C. for 30 minutes) to obtain a supernatant as a crude enzyme solution.

As a control, a plasmid pUC118 containing no foreign gene was transformed into Escherichia coli JM109 strain in the same manner as in Example 2 (5), and a crude enzyme solution was prepared from the transformant in the same manner as described above. And the following activity measurement. Confirmation of maleate isomerase activity
For these crude enzyme solutions, the decrease in maleic acid was measured as a change in absorbance at 240 nm in the presence of porcine heart-derived fumarase (Boehringer Mannheim) in the same manner as in item (1) of Example 2 [K. Otsuka, Agri
c. Biol. Chem. , 25, 726 (196
1)]. As a result, the absorbance at 240 nm decreased only for the crude enzyme solution prepared from ECMI113, and the maleate isomerase activity could be detected.

Example 5 Maleic Acid Isomerization Reaction by Transformant Containing Maleate Isomerase The ECMI113 prepared in Example 2 in the same manner as in Example 4.
The strain was inoculated into the above LB medium containing 50 μg / ml of ampicillin, and cultured under aerobic shaking at 30 ° C. for 15 hours.
The obtained culture is centrifuged (3,000 × g, 4 ° C., 2
0%) to collect the cells, and then add 0.9% (w / v) NaC
l.

Next, 0.1 g (wet weight) of the washed cells was suspended in 1 ml of 0.9% (w / v) NaCl, and the reaction solution [composition: sodium maleate 84 g / L, Trit
onX-100 1 g / l], and reacted by shaking at 45 ° C for 1 hour. The reaction solution after the reaction was centrifuged (3,000 × g, 4 ° C., 20 minutes), and the obtained supernatant was subjected to an organic acid column (SCR-10 manufactured by Shimadzu Corporation).
1H column), high performance liquid chromatography analysis using a UV detector (210 nm) (manufactured by Shimadzu Corporation, LC-5A)
Was served. As a result, a fumaric acid peak could be detected in addition to the raw material maleic acid peak. Also, by the above reaction,
It was also confirmed that the amount of maleic acid in the raw material was reduced. From the above results, it was confirmed that maleic acid was isomerized to fumaric acid by the action of the maleate isomerase contained in the transformant.

[0086]

According to the present invention, there are provided a DNA encoding a maleic acid isomerase derived from Bacillus circulans, a recombinant vector having the DNA, and a transformant containing the recombinant vector. By the transformant containing the maleate isomerase gene of the present invention, it is possible to produce a significant amount of maleate isomerase that stably retains its activity under normal temperature conditions as well as high temperature conditions, By using the present cells or the maleic acid isomerase prepared from the present cells, fumaric acid can be efficiently produced from maleic acid.

[0087]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 756 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism: Bacillus circulans Strain: MI-113 Sequence characteristics Symbol indicating characteristics: CDS Location: 1-753 Method for determining characteristics: E sequence ATG AGT AAG AAT TAT CGA GTA GGC CTT ATC GTA CCA AGC TCT AAC ACA 48 Met Ser Lys Asn Tyr Arg Val Gly Leu Ile Val Pro Ser Ser Asn Thr 1 5 10 15 ACG ATG GAA ACA GAA ATT CCG GCT ATG CTT CAT TCC AGA ATG CAG ACT 96 Thr Met Glu Thr Glu Ile Pro Ala Met Leu His Ser Arg Met Gln Thr 20 25 30 AAA CCA GAA GAA ACC TTT ACA TTC CAT TCT GCA CGT ATG CGG ATG ATG 144 Lys Pro Glu Glu Thr Phe Thr Phe His Ser Ala Arg Met Arg Met Met 35 40 45 CAT GTA AAT CCG GAT GAA TTA AAG AAA ATG GAT GTA GAT AGT GAC CGT 192 His Val Asn Pro Asp Glu Leu Lys Lys Met Asp Val Asp Ser Asp Arg 50 55 60 TGT GCA GTG GAG CTT TCC GAT GCA CGA TGT GAT GTG CTG GCT TAT GCA 240 Cys Ala Val Gl u Leu Ser Asp Ala Arg Cys Asp Val Leu Ala Tyr Ala 65 70 75 80 TGT CTA GTT GCC ATT ATG TGT CAG GGT CCG GGA TAT CAT CAC ATT TCT 288 Cys Leu Val Ala Ile Met Cys Gln Gly Pro Gly Tyr His His Ile Ser 85 90 95 GAA GAA CGC TTA GGA AAG GTT ACA GTC AGC AAT GGT GGT CCA GCT CCA 336 Glu Glu Arg Leu Gly Lys Val Thr Val Ser Asn Gly Gly Pro Ala Pro 100 105 110 ATT GTC AGC AGT GCA GGA GCA TTA ATT GAG GGC CTT GAA ACG ATT GGG 384 Ile Val Ser Ser Ala Gly Ala Leu Ile Glu Gly Leu Glu Thr Ile Gly 115 120 125 GCG AAA AAG GTT TCC ATT ATT ACA CCA TAT ATG AAG CCA TTG ACT AAA 432 Ala Lys Lys Val Ser Ile Ile Thr Pro Tyr Met Lys Pro Leu Thr Lys 130 135 140 ACG GTA ATT GAA TAT TTA AAT GCT GCT GGT ATT GAA GTC ATT GAT TCA 480 Thr Val Ile Glu Tyr Leu Asn Ala Ala Gly Ile Glu Val Ile Asp Ser 145 150 155 160 ATC AGC CTC GAA GTT GCG GAT AAC CTA GAG GTT GGC CGT TTG GAT CCG 528 Ile Ser Leu Glu Val Ala Asp Asn Leu Glu Val Gly Arg Leu Asp Pro 165 170 175 GAA AAC CTA ATA GGC CAT GCA GAC CGT TTG AAT ATT AAA GGA GCA GAT 576 Glu A sn Leu Ile Gly His Ala Asp Arg Leu Asn Ile Lys Gly Ala Asp 180 185 190 GCA GTT GTC CTA TCT GCA TGC GTT CAG ATG CCT TCT CTT CCA GCT ATT 624 Ala Val Val Leu Ser Ala Cys Val Gln Met Pro Ser Leu Pro Ala Ile 195 200 205 CAG GCT GTT CAA GAC CGT TTG GGA ATT CCA GTC TTG TCT GCC GGA GTT 672 Gln Ala Val Gln Asp Arg Leu Gly Ile Pro Val Leu Ser Ala Gly Val 210 215 220 GCA ACA GTT TTT AAA ATt CTC AAG GAA TTA AAC CTT TCA ACA GAG GTA 720 Ala Thr Val Phe Lys Ile Leu Lys Glu Leu Asn Leu Ser Thr Glu Val 225 230 235 240 CCA AAT GCA GGA CAT TTA CTA TCC GGT AAA TTT TAG 756 Pro Asn Ala Gly His Leu Leu Ser Gly Lys Phe 245 250

SEQ ID NO: 2 Sequence length: 24 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence Ser Lys Asn Tyr Arg Val Gly Leu Ile Val Pro Ser Ser Asn Thr Thr 1 5 10 15 Met Glu Thr Glu Ile Pro Ala Met 20 24

SEQ ID NO: 3 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic oligonucleotide) Sequence characteristics: Characteristic symbols : Location: Method of determining the characteristics: Other information: N represents inosine Sequence AARAAYTAYC GYCTNGGNCT NATYGTNCC 29

SEQ ID NO: 4 Sequence length: 33 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic oligonucleotide) Sequence Sequence characteristics: Characteristic Symbol: Location: Method for determining the characteristics: Other information: N represents inosine. AARACNACNA TGGARACNGA RATNCCNGCN ATG 33

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location // (C12N 15/09 ZNA C12R 1:09) (C12N 1/21 C12R 1:19) (C12N 9/90 C12R 1:19) (72) Inventor Miki Kobayashi 3-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Prefecture Inside the Tsukuba Research Laboratory, Mitsubishi Chemical Corporation (72) Inventor Hideaki Yukawa Chuo-Hachi, Ami-cho, Inashiki-gun, Ibaraki Prefecture Chome 3-1 Tsukuba Research Laboratory, Mitsubishi Chemical Corporation

Claims (6)

[Claims] 1. It has the amino acid sequence shown in SEQ ID NO: 1,
A DNA encoding a maleic acid isomerase which may have substitution, deletion or insertion of an amino acid residue which does not substantially impair the activity of isomerizing maleic acid to produce fumaric acid. 2. The DNA according to claim 1, which is represented by the nucleotide sequence shown in SEQ ID NO: 1. 3. A recombinant vector comprising the DNA according to claim 1 ligated to a vector. 4. A transformant transformed by introducing the DNA according to claim 1 or 2. 5. A method for producing a maleate isomerase, comprising culturing the transformant according to claim 4 in a medium, and collecting the maleate isomerase from the culture. 6. A method comprising adding the transformant according to claim 4 or a partially purified maleate isomerase or a purified enzyme obtained from a culture of the transformant to an aqueous solution containing maleic acid. A process for producing fumaric acid, comprising isomerizing an acid to produce fumaric acid.