JPH02286077A - Bacillus-s-p, dna fragment containing l-lactic acid dehydrogenase gene, gene recombinant plasmid containing the same gene, l-lactic acid dehydrogenase gene and gene recombinant plasmid containing the same gene - Google Patents

Abstract

PURPOSE:To provide a novel thermophile, Bacillus.S.P, which can grow at relatively high temperature, does not cause any gelatin liquefaction, does not hydrolyze starch and produces L-lactic acid dehydrogenase. CONSTITUTION:A thermophile, Bacillus.S.P TP626 (FERM 10450), which can grow at a relatively high temperature such as 50-70 deg.C, does not cause any gelatin liquifaction, does not hydrolyze starch and produces L-lactic acid dehydrogenase was found out as a result of an examination for finding out a L-lactic acid dehydrogenase originated from a thermophile. The isolation of a complex plasmid pTCl containing a L-lactic acid dehydrogenase gene originated from the bacterium stain and a genetic recombination using the gene permits to massively produce the L-lactic acid dehydrogenase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to Bacillus sp.
・Sp. ), D containing the L-lactate dehydrogenase gene
The present invention relates to an NA fragment, a recombinant plasmid containing the same, and the milk M dehydrogenase gene shown in FIG. 1, which will be described later, and a recombinant plasmid containing the same.

``Prior art'' L-lactate de*acidase is determined by the ``enzymatic measurement method'' (No. 101).
102, published by Igakushoin, 1982), it is widely used as a diagnostic agent for human liver, and is a highly useful enzyme.

Also, "Bioindustry" (Volume 3, No. 9,
As described in pages 5 to 13), enzymes derived from thermophilic bacteria generally not only have sufficient basic properties as an enzyme, but also have excellent heat resistance and storage stability. ing. In general, thermophilic bacteria are stable even when subjected to heat treatment to prevent contamination with contaminants, so by taking advantage of this property, it is easy to obtain highly pure enzymes, but the biggest drawback of enzymes is that they are usually unstable. It can be said that it is an enzyme with high practical value that covers the following.

On the other hand, the L-lactate dehydrogenase gene, which produces L-lactate dehydrogenase within the bacterial body, has been published in "Biological Chemistry Hop-Saylor" (Biol.Chem.Hopp).
e-Seyter) J (Vol. 368, No. 1167~
1177 (1987)},
Some are known. However, it has the following characteristics, for example, it can grow well at around 50-70'C, does not liquefy gelatin, does not hydrolyze starch, and is a thermophilic bacterium that produces L-lactate dehydrogenase. Novel Bacillus sp., specifically Bacillus sp.
acillus-sp. ) The TP262 strain and the L-lactate dehydrogenase gene derived from this strain are unknown.

[Problems to be Solved by the Invention] Under these circumstances, it is necessary to provide heat-stable monate dehydrogenase and its genes through genetic manipulation, and to improve heat resistance and storage stability through genetic manipulation. It has been desired to develop a method for producing excellent and highly pure L-milk M dehydrogenase in large quantities.

[Means for Solving the Problems] As a result of intensive studies to find L-lactate dehydrogenase derived from thermophilic bacteria, the present inventors discovered thermophilic bacteria Bacillus sp isolated from hot spring water, for example. We found Bacillus sp. TP262 strain.

Furthermore, in order to solve the above-mentioned problems, the present inventors isolated a composite plasmid pTC1 containing the perch milk M dehydrogenase gene derived from this strain, introduced it into a microorganism using gene recombination technology, and L The present inventors have discovered that by expressing the monolactate dehydrogenase gene and using this gene, it is possible to easily and maximally produce a useful enzyme, and have completed the present invention.

More specifically, the present inventors have found that L-lactate dehydrogenase can be grown well at around 50 to 70°C, does not liquefy gelatin, does not hydrolyze starch, and is stable to heat. Bacillus sp., a thermophilic bacterium, such as Bacillus nis bis TP262 strain, was discovered. In addition, a recombinant plasmid was obtained by ligating the monolactate dehydrogenase gene on the DNA of this strain or a DNA fragment containing it with a vector plasmid.

In general, the L-lactate dehydrogenase gene is expressed by introducing this recombinant plasmid into a host microorganism such as the genus Nizierisia, including ESCheriChia col +), and the L-lactate dehydrogenase gene is expressed. The present inventors have discovered that several types of lactate dehydrogenase can be easily produced in large quantities by using a host microorganism containing the following, and have completed the present invention.

In addition, it was confirmed that the structure of the monolactate dehydrogenase gene derived from Bacillus sp. TP262 strain is shown as DNA base sequence 148 to 1104 in FIG.

The purpose of the present invention is to develop Bacillus S., a thermophilic bacterium that can grow well at around 50 to 70°C, does not liquefy gelatin, does not hydrolyze starch, and produces L-lactate dehydrogenase. An object of the present invention is to provide a DNA fragment containing the L-lactate dehydrogenase gene and a recombinant plasmid containing the L-lactate dehydrogenase gene, as well as a recombinant plasmid containing the L-lactate dehydrogenase gene shown in FIG. 1 described below. .

By using host microorganisms into which these recombinant plasmids have been introduced, monolactate dehydrogenase derived from the thermophilic bacterium Bacillus sp. can be easily produced in large quantities.

In addition, the Bacillus sp. 18262 strain, which is one of the objects of the present invention, produces several types of monolactate dehydrogenase isozymes, as shown in Figure 5-8. It is a useful strain that can produce several types of lactate dehydrogenase at once.

The present invention will be explained in detail below.

FIG. 1 shows a region containing the L-lactate dehydrogenase gene among the DNA fragments derived from the thermophilic bacterium Bacillus sp inserted into the composite plasmid pTC1 containing the L-lactate dehydrogenase gene of the present invention, which will be described later. The base sequence of The structural gene of this base sequence is 148 to 1104 of the base sequence in FIG.
is shown. That is, the base sequence in Figure 1 contains 148 to
Starting at 150 (ATG start codon), starting at 1105
An open reading frame consisting of 957 base pairs ending at 1107 [Oker (TAA stop codon)]
These encode a protein consisting of 319 amino acids). Although there are several ATGs other than 148-150, (1) this is the only base sequence that encodes the confirmed amino-terminal amino acid sequence, that is, methionine-lysine-arginine;
(2) Shine-Dalgarno (Shine-DalQarnO) sequence, which is a liposome binding site, exists at 129-135, and (3) purified from Bacillus sp. 18262 strain. Molecular weight of monolactate dehydrogenase subunit 3
Molecular weight 35. which approximately corresponds to 7.000-39,000.
Since it encodes 894 proteins, 148
ATG present at ~150 is considered to be the translation initiation codon of the L-lactate dehydrogenase gene.

Also, 101 to 106 upstream of 148 (TTGTGA>
and 121-126 (AATTTT>, the "-35" region and "-1" region of the promoter structure, respectively)
There is a @ group sequence corresponding to the 0'' region.

The monolactate dehydrogenase gene of the present invention is derived from Bacillus sp., for example, Bacillus nis bis strain TP262, and is present on the chromosomal DNA of the bacterium. Bacillus sp. strain 18262 is a thermophilic bacterium isolated from hot spring water, and has the mycological properties shown in Table 1 below.

/ / / / Table-1 Table-1 (Continued) The above properties are summarized in Berc+ey's Manual of Systematic Bacteriology (Berc+ey's Manual of Systematic Bacteriology).
) ranual of Systematic Bac
terioloc+y) Volume 1 (1984), the characteristics of this strain were found to be similar to that of Bacillus (BaCi).
This strain closely resembles the characteristics of the genus Ius.
It was identified as a bacterium belonging to the genus Bacillus. In general, this strain can grow well at around 50-70'C, does not liquefy gelatin in broth gelatin puncture medium, does not hydrolyze starch, and produces heat-stable L-lactate dehydrogenase. Since it is a thermophilic bacterium, it is a new bacterium.
This bacterial strain was named Bacillus sp. TP262 strain (Feikoken Bacteria No. 10450).

Note that this strain is not pathogenic to animals and plants.

In the present invention, in addition to the above-mentioned bacterial strains, all of their natural mutant strains and artificial mutant strains can be used.

Next, the novel method for culturing Bacillus varnish and pea of the present invention will be explained.

The novel Bacillus sp of the present invention can be obtained using conventional methods for culturing microorganisms belonging to the genus Bacillus, such as meat juice, yeast extract, peptone, bovine brain extract powder, heart extract powder, etc. It can be cultured under aerobic conditions in a medium containing nutrient sources. Also,
If necessary, an inorganic compound selected from common salt, phosphate, etc. can also be added to the above medium.

This strain may be cultured by directly inoculating the cells into the medium as described above.

The culture temperature is appropriately set within a range where microorganisms can grow, but is usually 37 to 70°C, preferably 50 to 70°C.
It is.

Further, the pH of the medium is usually adjusted to pH 6.0 to 8.5.

The culture period varies depending on the type of medium used, but is usually about 10 to 20 hours.

Next, Bacillus sp., e.g.
A method for cloning the monolactate dehydrogenase gene or a DNA fragment containing it on the chromosomal DNA of the S.P. TP262 strain will be described.

The replicable expression vector and host microorganism that can be expressed in a suitable host microorganism used in the present invention include replicable expression vectors and host microorganisms commonly used in the field of genetic engineering.

Expression vectors include, for example, pUC-based vectors such as pU C19 for N. coli, pB
pBR-based vectors such as R322, I) ACY018
plasmid vectors such as 4, phage vectors such as λ and M2S; for Bacillus subtilis pUBllo, pc
194 and pE194.

In addition, host microorganisms include, for example,
JM-type hosts such as E. coli strains JM103 and JM109, as well as microorganisms of the genus Nigelysia, such as HBLol and caoo, and Bacillus subtilis.
lus 5ubtilis) RM125 strain and R
Examples include Bacillus subtilis such as M141 strain.

First, Bacillus ◆ Nis P, for example, Bacillus sp. TP2B2 strain, was cultured in a conventional manner, and then the bacteria were collected.
A chromosomal DNA specimen is collected by processing in a conventional manner. Cut the chromosomal DNA preparation with old ndl [. On the other hand, pUc19 as an expression vector was cut with HindI and D
The NA fragments are ligated with pUC19 cut with heavy dlIl and gauze to obtain a composite plasmid.

In order to select and collect a composite plasmid containing a heat-stable L-lactate dehydrogenase gene from the obtained composite plasmid, this is transformed into, for example, N. coli strain JM103 [Pharmacia Co., Ltd. ] [Reference: Nucleitsk Acid Research (N
9, pp. 309-321 (1981)] to obtain a transformed strain that is resistant to ampicillin and cannot degrade lactose.

The obtained transformed strain is cultured in a conventional manner, and a strain exhibiting L-lactate dehydrogenase activity at 60°C is isolated in a conventional manner.CTCI
(referred to as stocks). The obtained strain 701 was cultured in a conventional manner, and then a complex plasmid (1
) TCl) is isolated by a conventional method such as the Boiling Method or the Triton x-ioo method described below.

The molecular weight of the composite plasmid pTC1 obtained as described above was determined by agarose gel electrophoresis analysis, and was approximately 4.3 megadaltons (6.6 Kb), which contained the lactate dehydrogenase gene. The molecular weight of the DNA fragment derived from Bacillus sp. TP262 strain was 2.5 megadaltons (3,9 b).

The restriction enzyme map of pTCI is shown in FIG.

In FIG. 2, the part indicated by the bold line is the DNA part containing the lactate dehydrogenase gene, and its restriction enzyme map is shown in FIG.

On the other hand, we separately prepared plasmids in which various parts of the inserted DNA part of pTCl were deleted, and using these plasmids, we obtained transformed strains of Nijierisia temple coli strain JM103 or JM109, and examined the presence or absence of L-lactate dehydrogenase activity. As a result, it was found that the L-lactate dehydrogenase gene was present in the range including the cleavage sites by Xba I, Ava engineering, and old ncl shown in FIG. Therefore, the nucleotide sequence of the DNA fragment including the cleavage site by XbaLAva I and old nclJ was
fdeoxy) method. The results are shown in FIG. The nucleotides shown as FA group sequences 1 to 1197 in FIG. 1 contain the lactate dehydrogenase gene of the present invention.

After culturing the microorganism transformed by the above method and obtaining bacterial cells, the thermophilic bacterium Bacillus sp.
Several L-lactate dehydrogenase isozymes derived from strain TP262 can be purified and isolated.

[Effect of the invention] The Bacillus sp of the present invention, for example, Bacillus sp.
S.P. strain TP262 is a new and useful bacterium that can produce useful L-lactate dehydrogenase. In general, Bacillus sp. TP262 strain is shown in Figure 5-A.
As shown in Figure 1, since several types of L-lactate monolactate dehydrogenase isozymes are produced, several types of L-lactate monolactate dehydrogenase can be produced at once.

Furthermore, by genetic manipulation using a complex plasmid containing the monolactate dehydrogenase gene of the present invention, Bacillus sp., for example, Bacillus sp. TP26
Several types of L-lactate dehydrogenase isozymes of two strains can be produced in host microorganisms such as the genus Cielysia, and the productivity is similar to that of the production of monolactate dehydrogenase isozyme by Bacillus sp. better than sex.

In general, by utilizing the heat resistance of the L-lactate dehydrogenase of the present invention, it is easy to separate and purify the protein from host microorganism proteins such as heat-labile host microorganisms such as Elysia sp.

[Examples] Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

In the specification and drawings of this application, when bases, etc. are indicated by abbreviations, the International Union of Pure and Applied Chemistry (1nt
ernat 1onalυn1on of Pure
andApplied chen++5try (abbreviated as IUPAC): and International Union of Biochemistry (Interna
tional Union ofB+ochem+5t
ry ; abbreviated as IUB) Biochemical Nomenclature Committee (Commi
sion on Biochemical Nomen
clature: Based on the abbreviation CBN> or a profanity used in the field, examples of which are given below.

DNA: Deoxyribonucleic acid A: Adenine T: Thymine G Nigeanine C: Cytosine DEAE Nidiethylaminoethyl SDS Sodium nidodecyl sulfate EDTA: Ethylenediaminetetraacetic acid NADH, Reduced oral codinamide adenine dinucleotide Example 1 Bacillus sp. TP262 strain Purification of monolactate dehydrogenase produced (1) Bacillus φ Nis P strain TP262 is the fifth
As shown in Figure A, several types of L-lactate dehydrogenase (
Hereinafter, the band of monolactate dehydrogenase shown in FIG. 5-A will be referred to as Band-E-Vl. ), or among these,
A method for purifying L-lactic acid monohydrogenase, which is particularly active in band IVb, is shown.

Bacillus sp. TP262 strain was grown on L-agar plate medium (1% polypeptone, 0.5% yeast extract, 0.1
% glucose, 0.5% salt, 0.3% Shaten, p, H7,
After inoculating O) and culturing at 60°C for 115 to 18 hours,
If the bacteria are collected and washed with phosphate buffer (pH 7.0) for 0.1 day, a wet weight of 959 bacterial cells will be obtained. The obtained bacterial cells were added to 0, 1M phosphate buffer (pH 7,0 > 40017
+72, and the resulting suspension was subjected to ultrasonic disruption [The L-lactate dehydrogenase activity of the ultrasonic-disrupted microbial cells was 78 units for 19 of the disrupted microbial cells. . After 1 hour, centrifugation (10,000 rpm, 20
for 1 minute) to prepare a cell extract. Add ammonium sulfate to the obtained cell extract to a saturation of 40%, and collect the supernatant. The obtained supernatant liquid was reduced to 40% in advance.
Saturated ammonium sulfate - E phosphate buffer (pH 7,
Butyl Toyopearl 650S [
Tosoh Co., Ltd.] column (bed volume 8 (W)), and then 20% saturated ammonium sulfate-0,1
After washing with M phosphate buffer (pH 7,0), 15% saturated ammonium sulfate-0,18 phosphate buffer (pH 7,
Both fractions eluted in step 0) are collected, and both fractions of 1q are desalted by ultrafiltration. Both portions containing the desalted crude lactate dehydrogenase were added in advance to 50mM Phosphorus Ill buffer (pH 7,
DEAE-cellulose DE5 equilibrated with 0)
2 [Manufactured by Whatman] Column (bed volume 50), and then 50mM phosphate buffer (pH 7, O
)) Separate both volumes. The obtained eluate was subjected to ultrafiltration, desalted and diluted with 50mM phosphate buffer (pH 7).
, O). This was adsorbed onto a DEAE-cellulose DE52 column equilibrated with 50 nIH phosphate buffer (pH 7,0), washed with 150 mM phosphate buffer (pH 7,0), and then washed with 200 mN phosphate buffer (pH 7,0). Elute at pH 7.0). This eluate was added in advance to
Adsorb onto an AGNAD (manufactured by P-L Biochemicals) affinity column equilibrated with 0mM phosphate buffer (pH 7,0), and then 2) 1M potassium-50mM phosphate buffer (pH 7,0). Collect the eluted fraction. The resulting eluate was precipitated with ammonium sulfate, and then the precipitate was dissolved in 50 IN phosphate buffer (pH
7,0)), and the resulting solution was subjected to high performance liquid chromatography [TSK Glue G3000 SR (manufactured by Tosoh Corporation)] to obtain Ck, 571Q.
Obtain purified enzyme. The obtained purified enzyme was subjected to polyacrylamide electrophoresis, silver staining and activity staining, and as a result, a single band was detected (Fig. 5-B). Its specific activity is 135 units/myN compared to white and crude enzyme.
It was purified 6 times. In addition, in the measurement of enzyme activity, 1 μH of L from pyruvonic acid was added for 1 minute at 60°C.
- The amount of enzyme that produces lactic acid was measured as 1 unit.

Example 2 (1) Collection of chromosomal DNA specimens of Bacillus sp. TP262 strain Chromosomal DNA of Bacillus sp.
a) Vol. 72, pp. 619-629 (1963)]. Bacillus sp. TP262 strain is cultured in the same manner as described in Example 1, collected, sequentially subjected to lysozyme treatment and freeze-thaw SDS treatment, and then lysed to extract DNA. The obtained DNA was sequentially subjected to phenol treatment and phenol/chloroform treatment to remove protein.
Precipitate the DNA with ethanol. This was mixed with TE buffer [
10m)l Tris-HCl (pH 8,0) and 1m)i
EDTA], the resulting solution is subjected to ribonuclease treatment, further subjected to phenol/chloroform treatment, and then precipitated with ethanol, and the resulting precipitate is dissolved in TE buffer. This solution is used as a chromosomal DNA standard.

(2) Insertion of chromosomal DNA into pUC19 Approximately 5 g of the chromosomal DNA preparation obtained in (1) was inserted into 100 units of old n
Using dlO, react at 37°C for 2 hours. on the other hand,
1 gram of plasmid 1c19 (manufactured by Pharmacia Co., Ltd.)
is completely cleaved with old ndl in the same manner as above, and then treated with alkaline phosphatase derived from N. coli. Chromosomal DNA cut with old ndl [I and 1) U
Mix Cl3, add 5 units of T4 DNA ligase, and react the resulting mixture at 15°C for 1 hour to prepare a composite plasmid.

(3) Introduction of the complex plasmid into N. coli JM103 strain (preparation of transformed strain) Introduce the complex plasmid prepared in (2) into N. coli JM103 strain (manufactured by Pharmacia Co., Ltd.) using the usual transformation method. did. A competent cell suspension of N. coli JM103 strain was added to 50 ρ of the complex plasmid solution.
．． 2d, and the resulting mixture was kept at O'C for 30 minutes, then subjected to heat shock at 42°C for 2 minutes, and immediately added with 10 g of Batatobeptone and 5 g of yeast extract.
, Shiichi broth (i) with a composition of 59 sodium chloride, 1 g of glucose, and 11 g of water was added to 117.0>1 precipitate and shaken at 37° C. for 1 hour. This was then mixed with ampicillin 50 g/w, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (x-qal) 100 g/w.
1R/mfl and isopropyl-β-D-thiogalactopyranoside (l PTG) 100 n/rrd! and cultured overnight at 37°C,
The white colonies that appear are obtained as transformed strains.

(4) Selection of monolactate dehydrogenase-producing strains from transformed strains The transformed strains obtained in (3) were cultured at 1 d per strain, and after being treated at 60°C for 20 minutes, 10 strains were cultured. Collect bacteria. 2d of 0.1M potassium phosphate buffer (pH 7.0) was added to the obtained bacterial cells, the resulting mixture was disrupted by ultrasonic waves, and the mixture was crushed into 0.1 m! sample, substrate solution and final concentration 0.
1M potassium phosphate buffer (pH 7.0).

0.85 mH sodium pyruvate and 0.1511
18N ADH]. This mixture was heated to 60℃ for 3
After standing still for 0 minutes, the absorbance at 3401 m was measured, and one sample in which a significant decrease was observed was obtained. We cultured 10 strains of this specimen again, prepared an enzyme solution for each strain from each culture solution, and measured the L-lactate dehydrogenase activity. A strain exhibiting enzymatic activity was obtained. This strain is Nijierisia coli Te
The strain was named 3 (Feikoken Bacteria No. 10449).

(5) Isolation of a complex plasmid containing the L-lactate dehydrogenase gene The Te3 strain obtained in (4) is cultured in L-broth 1rd containing Anji 9250 stable, and then harvested.

Isolation of plasmids is performed using molecular cloning (
Molecular C1onina) No. 366-36
Page 7,] - Rude Spring Harbor Laboratory
(Cold Spring Harbor Labor
boiling method (f3
oiling method).

That is, 0.5% Triton X-10 was added to the obtained bacterial cells.
Add 0.35! nl of a solution of 0 [8% sucrose, 50 m HEDTA and 10 mM Tris-HCl (pH B. The resulting mixture is boiled in boiling water for 40 seconds to lyse the bacteria, and centrifuged (10,000 rpm+, 10 minutes) to remove insoluble materials. Add 40 lJI of 3H sodium acetate and 420 lJI of isopropanol;
The obtained solution is allowed to stand at -70°C for 15 minutes, and then centrifuged (10.00 rpm, 15 minutes) to obtain a precipitate (DNA fraction). The obtained precipitate is dissolved in 20 ρ of TE buffer. This precipitate (complex plasmid)
As a result of measuring the molecular weight by agarose gel electrophoresis, it was approximately 4.3 megadaltons (approximately 6.8 Kb), of which the molecular weight of the DNA fragment containing the L-lactate dehydration enzyme gene was 2.5 megadaltons (3 , 9Kb). This plasmid was named pTC1. In order to create a restriction enzyme map, [R New Gene Manipulation Practical Handbook, No. 146]
Large-scale preparation was carried out according to the Triton X-100 method described in Jacchik Publishing (1985), p. 147. That is, the bacterial cells were suspended in 50mM Tris-HCl (1)88,0) containing 25% sucrose, treated with lysozyme, and treated with RNAse (RNase)51! The mixture was treated with Triton X100, 5H sodium chloride was added to a final concentration of 1 to 1, mixed, centrifuged (14,000 rpm, 40 minutes), and the supernatant liquid was collected.

Add pronase to the iq Radar supernatant, incubate at 37°C for 30 minutes, add 40% polyethylene glycol 6000 (manufactured by Wako Pure Chemical Industries, Ltd.) to the final temperature of 10%, and mix the resulting mixture. After standing at O'C for 2 hours, centrifugation at 4°C (3000 ppm
, 20 minutes) to obtain a precipitate. This precipitate is T
The plasmid was isolated and purified by dissolving it in E-milk solution and performing cesium chloride equilibrium density gradient centrifugation.

The restriction enzyme map of pTCl is shown in FIG.

FIG. 3 shows a restriction enzyme map of the inserted DNA portion (indicated by a bold line) in FIG. 2, that is, a DNA fragment containing the L-lactate dehydrogenase gene.

(6) Confirmation of location of L-lactate dehydrogenase gene (5)
D inserted into the composite plasmid pT C 1 obtained in
A plasmid prepared by shortening the NA fragment was used to transform N. coli, and the inserted DNA was determined by examining whether the transformed strain produced dehydrogenase.
The location of the L-lactate dehydrogenation gene on the A fragment was confirmed. First of all, I) TCI! -old ncI[
After cleaving with 4
A plasmid pTC5 is prepared in which two DNA fragments of 1 kb and 2.5 kb generated by CHI digestion are deleted (Fig. 4). Second, pTCl was cut with Ava technology and ligated with T4 DNA ligase to create a 2.4 kb 1
A plasmid pTC6 is constructed in which the DNA fragment generated by Ava I digestion is deleted (Fig. 4 ii).

Third, after pTCl is cut with old n+4, agarose gel electrophoresis is performed, and a gel piece containing a 2.5 kb DNA fragment band is excised.

From this gel piece, we use the DNA recovery kit GENE CLEAN [BIO 101].
101) [Reference: Proceedings of the National Academy of Sciences]
of the United States of America (Proc. Natl. Acad. Sci. U.S.
．． A. ) Vol. 76, p. 615 (1974)], a 2.5 kb old nc[-DNA fragment is recovered. On the other hand, plasmid vector p, Uc19 if i
nc n and ligated with the 2.5 kb old ncI[-DNA fragment using T4 DNA ligase to create pTclo (Fig. 4 iii). Fourth, pTCl was
After cutting with aI, ligating with T4 DNA ligase, 2.
A plasmid pTC7 is created in which a single 1 kb DNA fragment generated by Xba I digestion is deleted (Fig. 4i).
v). Fifth, after cutting pTC1 with Xba I, agarose gel electrophoresis is performed, and a 2.1 kb XbaI-DNA fragment is recovered by scene clean. On the other hand, plasmid vector J)LJC19 was cut with xbaI, and a 2.1kb XbaI-DNA fragment, T4D
Ligate with NA ligase to create pTC9 (Figure 4 V
). Sixth, pTCl was cut with HindIII, agarose gel electrophoresis was performed, and in step 3.9, the HindIII of b
Cut out the gel piece where the ndll fragment band is located.

From this gel piece, the old ndII-DNA fragment of 3 and 9 b is recovered by Sheen Clean. 3.9 old n of b
The dl[l-DNA fragment is partially digested with 5au3AI, subjected to agarose gel electrophoresis, and a DNA fragment of approximately 2 to 6 lengths is recovered from the gel piece using Scene Clean. On the other hand, plasmid vector pUc19 was
After cutting with HI, the above 21 (about 6 length DNA)
The fragments are ligated with T4 DNA ligase to create pTC17.

The procedure for producing these plasmids is shown in FIG.

Plasmid pTC5, pTC6, pTC7, pTC9 or I) N. coli JM1 by TCIO
03 strain (manufactured by Pharmacia Co., Ltd.), and pT C
17 by N. coli 0M109 strain [STRATAGENE @! ), and whether or not the obtained transformed strain exhibits L-lactate dehydrogenase activity derived from the thermophilic bacterium Bacillus sp. TP262 was determined using the same method as described in (4) above. I investigated it.

Figure 3 shows the range of DNA fragments derived from the Bacillus sp. strain TP262 chromosome contained in the plasmids used for transformation and the presence or absence of L-lactate dehydrogenase activity in the transformants obtained by introducing these plasmids. . pT
The TC17 strain containing Cl7 showed L-lactate dehydrogenase activity, but the other strains did not show [-lactate dehydrogenase activity.

From this result, the L-lactate dehydrogenase gene is located at a site that completely encompasses at least a 0.4 kb range from the Estimated to exist.

(7) Confirmation of the amino-terminal amino acid sequence of L-lactate dehydrogenase The amino-terminal amino acid sequence of L-lactate dehydrogenase obtained by the method (1) was directly analyzed using the Edman method [phenylinthiocyanate (PITC) method]. ] [Reference: Biochemistry Experiment Lecture 2, Protein Chemistry, Japanese Biochemistry, Volume 31
3-317 (Tokyo Kagaku Dojin) (1987)].

As a result, it was confirmed that the three amino acids from the amino terminal were arranged in the order of methionine, lysine, and arginine.

(8) Determination of base sequence of L-lactate dehydrogenase gene The base sequence of 1197 bases to the left from the vicinity of the HpaI cleavage site of pTcl shown in FIG. 3 was determined by the dideoxy method. The results are shown in FIG.

In Lai, (6) 2.1kb of Xba from the result in 2
When we searched for an open reading frame between the I''1 site and the 2.5kb Hinc n site, we found an open reading frame that starts with the ATG start codon at positions 148-150.
There are several ATGs other than 150, but (1) this is the only nucleotide sequence that encodes the amine-terminal amino acid sequence, methionine-lysine-arginine, and (2) Shine-Dalgarno (ribosome binding site)
rno) sequence, i.e. GAAAGGA is 129-1
35, with a molecular weight of 35.894, which almost matches the molecular weight of the subunit of L-lactate dehydrogenase produced from Bacillus sp. TP262 strain 37.000 to 39,000. Since it encodes a protein, 148-150 (ATG) is considered to be the translation initiation codon of the L-lactate dehydrogenase gene. The structural gene of the base sequence is from 148 to 148 of the base sequence in Figure 1.
Shown at 1104. That is, the base sequence in Figure 1 begins with 148-150 (ATG start codon) and begins with 1
105-1107 [Ocher (TAA stop codon)]
There is an open reading frame consisting of 957 salt pairs ending in , which encodes a protein consisting of 31911t amino acids.

(9) Purification of L-lactate dehydrogenase and its properties Purification of L-lactate dehydrogenase produced by N. coli Te3 strain Bacillus coli
Similar to the S.P. TP262 strain, it produces several types of L-lactate dehydrogenase (Bandoe-Vl) (Photograph 6).
A) shows a method for purifying monolactate dehydrogenase, which is represented by band IVb, which has particularly strong activity.

After culturing N. coli Te3 strain at 37° C. in L-broth 51 containing 50 g/m of ampicillin, the cells were collected to obtain 409 bacterial cells as a wet weight. The obtained bacterial cells were added with 100mH of 0.1M phosphate buffer (pH 7.0)2.
80 d was added to make a suspension, and after incubation at 60°C for 15 minutes, it was ultrasonically disrupted (the L-lactate dehydrogenase activity of the ultrasonically disrupted microbial cells was 574 units per gram of the disrupted microbial cells). (Jnit)) and again 6
After incubation at 0'C for 15 minutes, centrifugation was performed to separate the supernatant and precipitate. Add the above buffer solution 1 to the precipitate.
20ml was added and re-extracted.

Combine the two supernatants, add streptomycin sulfate to remove nucleic acids, and add saturated ammonium sulfate aqueous solution (t).
Add 380 ml of H7,5) to achieve 50% saturation and remove the precipitate. 730 d of this supernatant was heated at 70'C for 1
After incubating for 5 minutes, remove the precipitate again.

This supernatant was adsorbed onto a Butyl Toyopearl 6503 column (bed volume 100 d) that had been equilibrated with a 25% saturated ammonium sulfate aqueous solution, washed with a 20% saturated ammonium sulfate aqueous solution, and then eluted with a 15% saturated ammonium sulfate aqueous solution. Separate both portions. After concentrating the obtained eluate by ultrafiltration, it was diluted with 10 mM phosphate buffer (
pH 7.0), and then this solution was preliminarily diluted with 50 m
D equilibrated with M phosphate buffer (pH 7,0)
Adsorbed onto EAE-Sef1dex A-25 (manufactured by Pharmacia Co., Ltd.) (bed volume 130), 100mM.
After washing with phosphate buffer, both fractions eluted with 200m) I phosphate buffer (pH 7,0) are separated. The eluate was subjected to gel filtration using Sephadex G100 (manufactured by Pharmacia Co., Ltd.) and high performance liquid chromatography [
TSK gel G3000 SW (manufactured by Tosoh Corporation)]
After sequential gel filtration, the obtained purified # element was subjected to polyacrylamide gel electrophoresis, silver staining and activity staining, and as a result, a single band was detected (Figure 6-B).

Furthermore, high performance liquid chromatography and polyacrylamide electrophoresis revealed that the molecular weight of this enzyme was approximately 120.
．． It was 000. According to the 5DS-polyacrylamide method, a single molecule with a molecule fi of 39.000 is obtained, so it is thought that this L-lactate dehydrogenase is composed of four subunits.

The specific activity of the purified enzyme was 103 units/mff protein, which was approximately 32 times more purified than the crude enzyme.

In addition, the Te1B strain and Te17 strain obtained by transforming N. coli JM109 strain with pT C1 and ρTC17 were cultured in the same manner as described in (4) above to prepare a crude enzyme solution. As a result of measuring the L-lactate dehydrogenase activity, the enzyme activity was found to be 2.38 and 2.05 times higher than that of the Te3 strain described above, respectively.

(10) Gene recombination pTcl into pBR322
After cutting the 3.9 Kb DNA fragment containing the L-lactate dehydrogenase gene with J and performing agarose gel electrophoresis, the former ndl of b
[Recover l-DNA fragment. On the other hand, plasmid BR
322 was cut with Hi nd m, and the old n of 3.9Kl)
dl [- DNA fragment and T4 DNA ligase,
Obtain pT020.

The obtained plasmid was introduced into Elysia coli to obtain a transformed strain, which was cultured in the same manner as in (4) above to prepare 1 ill of a crude enzyme solution, and the L-lactate dehydrogenase activity was measured. It was found that the strain had the same lactate dehydrogenase activity as the N. coli T01 strain.

[Brief explanation of the drawing]

Figure 1 shows the base sequence of the DNA fragment containing the monolactate dehydrogenase gene of plasmid pTC1, Figure 2 shows the restriction enzyme map of plasmid pTc1, and Figure 3 shows the plasmid pTC1, I) Te3. , pTC6
, pTC7, pTC9, p”rclo and 1) Te1
Figure 4 shows the restriction enzyme map of the inserted DNA fragment derived from thermophilic bacteria No. 7 and the presence or absence of monolactate dehydrogenase activity of the strains transformed with these plasmids.
A schematic diagram showing the production route of C5, DTC6, pTC7, pTc9, pTclo and pTc17 is shown. This is a photograph showing 5DS-polyacrylamide electrophoresis of elementary enzymes (bands ■ to Vl) and purified enzymes of band lVb from these enzymes, followed by activity staining. Figures 6-8 and 6-B show several types of L-lactate dehydrogenases (Bandoe to Vl) produced from recombinant N. coli Te3 strain and band IV from these enzymes.
This is a photograph in which only the enzyme b was subjected to 5DS-polyacrylamide electrophoresis and activity staining was performed.

Claims (9)

[Claims] (1) Bacillus sp. is a thermophilic bacterium that can grow at around 50 to 70°C, does not liquefy gelatin, does not hydrolyze starch, and produces L-lactate dehydrogenase. (2) Bacillus sp. Bacillus sp.
Claim No. 2, characterized in that it is P262 strain (
Bacillus sp. described in section 1). (3) Contains the L-lactate dehydrogenase gene derived from Bacillus sp. and is characterized by the restriction enzyme map shown in the figure below.The molecular weight is 2.
5 megadalton (3.9Kb) DNA fragment. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (4) Bacillus sp.
Claim No. 2, characterized in that it is P262 strain (
3) DNA fragment described in section 3). (5) Contains the L-lactate dehydrogenase gene derived from Bacillus sp. and is characterized by the restriction enzyme map shown in the figure below.The molecular weight is 2.
A recombinant plasmid in which a 5 megadalton (3.9 Kb) DNA fragment is ligated to a replicable expression vector that can be expressed in a host microorganism. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (6) Bacillus sp.
Claim No. 2, characterized in that it is P262 strain (
The recombinant plasmid described in section 5). (7) A base sequence in which at least one base is substituted or unsubstituted based on the degeneracy of the genetic code; L-lactate dehydrogenase gene indicated by [gene sequence available]. (8) Base sequence; [There is a gene sequence] The L-lactate dehydrogenase gene according to claim (7), which is linked to the 5' end of the base sequence; [There is a gene sequence]. (9) A claim consisting of a base sequence; [there is a gene sequence] and a base sequence; [there is a gene sequence] linked to the 5' and 3' ends of the base sequence; [there is a gene sequence], respectively. The L-lactate dehydrogenase gene according to item (7).