JPH104972A - Gene coding heat-resistant beta-galactosidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene having a specific base sequence, coding a heat-resistant β-galactosidase originated from a bacterium belonging to the genus Thermo and useful for the genetic engineering production of the heat- resistant β-galactosidase used as an enzyme for food industry, etc. SOLUTION: This new β-galactosidase gene is originated from a bacterium belonging to the genus Thermus, codes heat-resistant β-galactosidase and has a base sequence of the formula or the substantially same function as the base sequence. The new β-galactosidase gene is useful for producing the heat-resistant β-galactosidase catalyzing reactions for hydrolyzing β-(D)1,4-galactoside bonds and used as an enzyme for food industry, etc. The β-galactosidase gene is obtained by separating the heat-resistant galactosidase from a highly thermopile bacterium belonging to the genus Thermus [e.g. Thermus S.P. A4 strain (FERMP-15236)], determining the N-terminal amino acid sequence of the enzyme, and subsequently screening the chromosomal DNA of the A4 strain with a synthesized probe having a base sequence corresponding to the determined N-terminal amino acid sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene encoding a thermostable β-galactosidase, and more particularly, to a gene derived from a bacterium belonging to the genus Thermus, which is a highly thermophilic bacterium.
The present invention relates to a gene encoding galactosidase, a transformant into which a DNA fragment containing the gene has been incorporated, and a method for producing β-galactosidase having high thermostability using the transformant.

[0002]

2. Description of the Related Art β-galactosidase according to the present invention is an enzyme that catalyzes a reaction for hydrolyzing a β- (D) 1,4-galactoside bond. The enzyme also catalyzes a reaction that generates a galactooligosaccharide by transferring a galactosyl group generated by hydrolysis to a sugar molecule serving as an acceptor depending on the substrate concentration. Industrially, this enzyme is used for producing glucose and galactose using lactose as a substrate, or for producing galactooligosaccharides. β-galactosidase is present in various microorganisms, and its gene has been well studied. As for thermostable β-galactosidase, Bacillus stearothermophilus which is a moderately thermophilic bacterium and Sulfolobus sulphatarix which is a hyperthermophilic bacterium are used.
olfataricus) and Thermotoga maritima (Thermotoga m
aritima) has been investigated for its gene structure. However, the gene sequence of β-galactosidase of the genus Thermus has not been clarified.

[0003]

Since β-galactosidase hydrolyzes lactose present in milk into glucose and galactose, it is used for producing low-lactose milk and syrup from lactose in cheese whey. Since thermostable β-galactosidase can be operated at a high temperature when the above reaction is carried out by immobilization, there is an advantage that the risk of contamination by other microorganisms can be prevented. Therefore, the problem to be solved by the present invention is to clarify the nucleotide sequence of a gene encoding β-galactosidase having excellent heat resistance, and to clarify this gene with other microorganisms such as Escherichia coli.
(E. coli, Escherichia coli) and the like to easily establish a method for producing thermostable β-galactosidase.

[0004]

Means for Solving the Problems The present inventors have determined that the Thermus sp. A4 strain (FERM) previously isolated from hot spring soil was used.
P-15236), β-galactosidase was purified, its physicochemical properties were clarified, and the N-terminal amino acid sequence was determined (Japanese Patent Application No. 7-305249). The β-galactosidase is excellent in heat resistance and is expected to be used as an industrial enzyme. Therefore, the present inventors first performed cloning of a gene encoding the enzyme and elucidated the nucleotide sequence thereof, aiming at mass production of the enzyme. In the present invention, an oligonucleotide is synthesized from the N-terminal amino acid sequence of β-galactosidase described above, and using this as a probe, a fragment containing a region encoding this β-galactosidase is obtained from chromosomal DNA of Thermus sp. A4 strain. Obtained and analyzed its base sequence. The thermostable β-galactosidase gene derived from a bacterium of the genus Thermus of the present invention has low homology to other β-galactosidase genes reported so far, and it has been revealed that the gene is a novel gene. Further, by determining the nucleotide sequence of the present gene, it becomes possible to clone a thermostable β-galactosidase gene of a bacterium of the genus Thermus or another genus based on this sequence.

[0005] Next, a transformant containing the nucleotide sequence was prepared, and production of the enzyme was attempted. The DNA fragment having the above-described base sequence encoding β-galactosidase was introduced into Escherichia coli, and β-galactosidase was successfully expressed. When the β-galactosidase activity of the enzyme expressed in Escherichia coli was measured, it was confirmed that the enzyme had the same heat resistance as the enzyme purified from the wild strain. The present invention has been completed based on these findings.

That is, the present invention according to claim 1 has the nucleotide sequence of SEQ ID NO: 1 derived from a bacterium belonging to the genus Thermus and encoding a thermostable β-galactosidase or a substantially identical function thereto. Β-galactosidase gene. The present invention according to claim 2 is a transformant into which a DNA fragment containing the β-galactosidase gene according to claim 1 has been incorporated. The present invention according to claim 3 provides:
A method for producing thermostable β-galactosidase, comprising culturing the transformant according to claim 2, and collecting thermostable β-galactosidase from the culture. In addition,
The present invention also includes the following embodiments. (1) The β according to claim 1, wherein the bacterium belonging to the genus Thermus is Thermus SP A4 strain (FERM P-15236).
-The galactosidase gene. (2) A β-galactosidase gene encoding an amino acid sequence deduced from the nucleotide sequence according to claim 1, or an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted with respect to the amino acid sequence.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The Thermus bacterium used in the present invention may be any one having the desired thermostable β-galactosidase gene, and the Sirmas sp. A4 strain is particularly suitable. This strain is stored under the deposit number FERM P-15236 at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry, and is available to anyone. The genetic engineering techniques used in the present invention are known, for example,
Molecular Cloning (T. Maniatis et al., Cold Spring Har
bor Laboratory Press, 1989) and so on. Furthermore, the strains, vector DNAs, enzymes, reagents and the like used in the present invention are all commercially available and can be easily obtained.

[0008] Obtaining a gene encoding β-galactosidase having excellent thermostability and determining its base sequence can be carried out as follows. First, the bacterium of the genus Thermus is cultured by a conventional method, and the heat-resistant β purified from the culture is used.
-Prepare galactosidase. For example, β-galactosidase (hereinafter referred to as “thermus sp.
Abbreviated as A4-β-Gal. )), The method described in Japanese Patent Application No. 7-305249 is suitable.
That is, cells were collected from the culture of the present bacterium by solid-liquid separation means, suspended in a sodium phosphate buffer or the like,
The crude enzyme solution is obtained by crushing by a suitable means. For this crude enzyme solution, β-galactosidase is purified using the hydrolysis activity for ONPG as an index. First, this crude enzyme solution was subjected to ammonium sulfate salting-out with 80% saturated ammonium sulfate or the like, and the precipitate was recovered. The precipitate was dispersed in a Tris-HCl buffer or the like, and then dialyzed against the same buffer. Then, fractionation and purification are performed using various types of chromatography such as ion exchange chromatography and gel filtration chromatography. Next, the N-terminal amino acid sequence of A4-β-Gal, which is the obtained thermostable β-galactosidase, is determined using a protein sequencer or the like, and an oligonucleotide having a base sequence deduced from the amino acid sequence is obtained by a conventional method. With a DNA synthesizer. Next, the nucleotide is labeled by a conventional method to prepare a probe.

On the other hand, a bacterium of the genus Thermus is cultured, and a chromosomal DNA solution is prepared from the obtained cells according to a conventional method. This chromosomal DNA is digested with various restriction enzymes to prepare DNA fragments. Next, using the DNA fragment as a target,
Southern hybridization using the nucleotide probe described above is performed to specify the size of each restriction enzyme digest of a DNA fragment containing all or a part of the gene encoding thermostable β-galactosidase. In order to isolate only a DNA fragment containing a gene encoding the desired thermostable β-galactosidase from these, for example, colony hybridization can be performed.

[0010] First, a recombinant plasmid is prepared by ligating a DNA fragment containing a target gene to a vector, and then introduced into Escherichia coli. Thereafter, colony hybridization is carried out according to a conventional method using the above-described nucleotide probe, and a recombinant Escherichia coli having a recombinant plasmid into which a DNA fragment containing all or a part of the gene encoding heat-resistant β-galactosidase has been inserted. Is separated. A DNA fragment is extracted from the separated E. coli by a conventional method, and the DNA fragment thus obtained is digested with a restriction enzyme to prepare a restriction enzyme map. The base sequence is determined using, for example, a DNA sequencer. This sequence is shown in SEQ ID NO: 1 in the sequence listing. Heat resistance β of the present invention
-The gene encoding galactosidase has been obtained by such a method.

Further, a recombinant plasmid into which a DNA fragment containing the entire gene encoding heat-resistant β-galactosidase has been inserted is constructed, and Escherichia coli transformed with the plasmid is cultured. β-galactosidase can be obtained. D used for transformation
The NA fragment is prepared by a conventional method. For example, a BamHI fragment or a BamHI-PstI fragment can be used. The plasmid used for the transformation is not particularly limited.
C118, pUC119 and the like can be used. The method for culturing the transformant is not particularly limited. However, since expression is expected to be regulated by the lac promoter, it is preferable to add IPTG for culturing. After the culture, when heat-resistant β-galactosidase is obtained from the cells, heat treatment can be performed to increase the specific activity.

[0012]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 Cloning of DNA Fragment Containing A4-β-Gal Gene According to the method described in Japanese Patent Application No. 7-305249, the Thermus SP A4 strain (FERM P-
15236) was used to purify thermostable β-galactosidase (hereinafter abbreviated as A4-β-Gal). Next, the N-terminal amino acid sequence of the obtained A4-β-Gal was determined using a protein sequencer. This A4-
The 44mer base sequence was estimated from the N-terminal amino acid sequence of β-Gal. This nucleotide sequence is shown in FIG. This 44
Oligonucleotides having a mer base sequence were synthesized using a DNA Synthesizer (Applied Biosystems) according to a conventional method. In addition, this oligonucleotide was ECL 3'-
A probe (hereinafter referred to as A4B) which is labeled by an oligo labeling system (Amersham) and used in the following experiments.
Abbreviated as GN. ).

The chromosomal DNA of the A4 strain was extracted according to a conventional method. A4 strain was transformed into 500m liquid medium for bacteria of the genus Thermos
1 and inoculated aerobically at 70 ° C. for 2 days. DNA obtained from cells recovered from this culture by centrifugation
Was dissolved in 2 ml of TE buffer and used as an A4 strain chromosomal DNA solution in the following experiments. A4 strain chromosomal DNA about 5
0 μg was completely digested with about 50 units of restriction enzyme to prepare a chromosomal DNA fragment. BamHI, Ec as restriction enzymes
oRI, HincII, HindIII, KpnI, PstI, SacI, SalI, Sp
hI and XhoI (both manufactured by Boehringer Mannheim) were used. The obtained chromosomal DNA fragment was subjected to electrophoresis in a 0.8% agarose gel, and after completion of the electrophoresis, was transferred to a nylon membrane (Hybond-N ⁺ , manufactured by Amersham) according to a conventional method.
The size of a chromosomal DNA fragment containing the whole or a part of the A4-β-Gal gene was determined by Southern hybridization using this membrane and the probe A4BGN. FIG. 2 shows the obtained results. Probe A4
Among several DNA fragments confirmed to hybridize with BGN, the total length of the A4-β-Gal gene is estimated to be about 2.2 kbp, so that 5.5 kbp
BamHI fragment, 2.1 kbp SacI fragment and 3.4 kb
The following experiment was conducted for the purpose of obtaining the SphI fragment of p.

The chromosomal DNA fragment obtained by the above-mentioned method is
Electrophoresis was carried out in an 8% low-melting point agarose gel, a gel containing a DNA fragment of the desired size was cut out, and phenol treatment, chloroform extraction, and ethanol precipitation were performed to extract a chromosomal DNA fragment. Vector
pUC118 or pUC119 was previously digested with a restriction enzyme generating the same end as the chromosomal DNA fragment, and treated with alkaline phosphatase to prevent self-ligation. About 0.1 μg of this vector DNA and about 1 μg of a chromosomal DNA fragment were mixed, T4 DNA ligase was added, and the mixture was reacted at 16 ° C. for 1 hour to ligate the DNA. DNA ligation kit ver.1 (Takara Shuzo) was used for T4 DNA ligase and its reaction solution. After completion of the ligation reaction, the resulting recombinant plasmid was recovered by ethanol precipitation, dissolved in 3 μl of sterilized water, and used for transformation.

Escherichia coli (strain MV1184,
Hereinafter, it is abbreviated as MV1184 strain. ) Was in a state suitable for transformation by an electric pulse (electroporation method) according to a conventional method. The transformation was performed using the MV1184 strain cell suspension 40.
To 3 μl, 3 μl of the recombinant plasmid solution obtained by the above method was added, and pulsed with Gene-Pulser (BIO-RAD). Immediately after applying pulse, 1ml
SOC medium (2% bactotryptone, 0.5% yeast extract, 0.05% NaCl, 0.019% KCl, 10 mM
After suspending in MgCl ₂ , 20 mM glucose) and keeping at 37 ° C. for 1 hour, ampicillin (0.125 g /
L), X-gal (0.005 g / L) and IPTG (0.0
LB plate medium (1% bactotryptone, 5 mM),
A 0.5% yeast extract, 0.5% NaCl, 1.5% agar) was spread on Hybond-N ⁺ membrane and cultured overnight at 37 ° C to obtain a transformant having ampicillin resistance. Was.

A recombinant plasmid into which a chromosomal DNA fragment containing the whole or a part of the A4-β-Gal gene has been inserted by a colony hybridization method using a membrane on which the DNA of the colony of the transformant has been fixed and the probe A4BGN. Were identified and separated. In addition, the procedure of colony hybridization followed the protocol by the manufacturer of the ECL 3'-oligolabeling system. As a result, the chromosomal DNA Sa
At first, it was confirmed that three colonies out of about 1200 recombinants into which the cI fragment was inserted hybridized with the probe A4BGN. The nucleotide sequence was determined by the method described below, and the results were compared with the nucleotide sequences of β-galactosidase genes derived from other bacteria. As a result, the recombinant plasmids held by these three strains were all 5′-side of A4-β-Gal gene. It was found that an approximately 1.7 kbp SacI fragment containing a part of was inserted. This recombinant plasmid was named pBGS2.

FIG. 3 (A) shows a restriction map of the SacI fragment prepared by a conventional method. The arrow in the figure is A4-β-G
Indicates the position and orientation of the al gene. To obtain a plasmid containing the full length of the A4-β-Gal gene, a longer Ba
Colony hybridization was also performed on the recombinant into which the mHI fragment or the SphI fragment had been inserted.
In order to simplify the experimental procedure, the above SacI fragment was recovered by low-melting point agarose gel electrophoresis, phenol treatment, chloroform extraction, and ethanol precipitation.
Labeling was performed using a direct labeling system (manufactured by Amersham) and used as a probe. The colony hybridization procedure followed the protocol provided by the manufacturer of the ECL direct labeling system. As a result, it was found that one out of about 2,000 recombinant strains into which the BamHI fragment of the chromosomal DNA had been inserted contained all or a part of the A4-β-Gal gene. This recombinant plasmid was named pBGB1. When a restriction map was created (FIG. 3 (B)), pBGB1 was estimated to contain the full length of the A4-β-Gal gene.

Example 2 Determination of the Complete Nucleotide Sequence of the A4-β-Gal Gene The nucleotide sequence of the inserted fragment of plasmid pBGB1 extracted from the recombinant Escherichia coli isolated in Example 1 above was determined using the 373A DNA
It was determined by Sequencer (Applied Biosystems).
SEQ ID NO: 1 in the sequence listing contains heat-stable β-galactosidase A4
1 shows the DNA sequence of the structural gene of -β-Gal and the amino acid sequence deduced therefrom. A4-β-Gal gene is 215
Consisting of 7 base pairs, the start codon was ATG and the stop codon was TGA. The amino acid sequence deduced from the base sequence consists of 719 amino acid residues, and the deduced molecular weight is 7956.
It was 9 Da. As a result of examining the homology with the amino acid sequence of β-galactosidase of other bacteria, the most homologous Bacillus stearothermophilus (Bacillus stearothil) was found.
ermophilus), the homology is as low as about 30%.
-Β-Gal gene can be determined as a novel β-galactosidase gene.

Example 3 Expression of A4-β-Gal Gene in E. coli and Confirmation Thereof A new plasmid pBGB3 was constructed for the purpose of expressing the A4-β-Gal gene in E. coli (FIG. 3).
(C)). This was done by removing about 1 kbp of the BamHI-PstI fragment upstream of the A4-β-Gal gene from the inserted fragment of pBGB1.
-It is linked so that the orientation of the β-Gal gene and the lac promoter in the pUC119 vector are aligned. Also,
Similarly, plasmid pBGB2 was constructed. This is one in which the orientation of the lac promoter and the A4-β-Gal gene in the pUC118 vector are aligned with the orientation of the insert fragment of pBGB1 reversed. E. coli transformed with these plasmids were transformed with ampicillin (80 μg / ml) and IPTG (0.0
3 mM L-broth (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, pH 7.0) containing 6 mM) and shaken at 37 ° C. overnight. Cultured. As a control, Escherichia coli transformed only with the vector DNA was similarly cultured.

The cells were collected by centrifugation, washed once with 50 mM sodium phosphate buffer (pH 6.5), dispersed in 3 ml of the same buffer, and sonicated. The supernatant obtained by centrifuging the crushed product (15000 rpm, 5 minutes) was measured for protein concentration and β-galactosidase activity at 70 ° C. using ONPG as a substrate as follows. Reaction mixture in cuvette (50 mM sodium phosphate buffer (pH 6.5), 1
mM MgCl ₂ , 2.8 mM ONPG, 2.9 ml in total) was kept at 70 ° C., and 100 μl of the enzyme solution was added, and after 30 seconds, the change in absorbance at 405 nm for 30 minutes was measured.
The molar extinction coefficient of o-nitrophenol at 405 nm was calculated as 3.1 × 10 ³ M ⁻¹ cm ⁻¹ . Enzyme 1
Unit was defined as the amount of enzyme that produced 1 μmol of o-nitrophenol per minute. The protein concentration was measured using a Bio-Rad Protein assay (manufactured by Bio-Rad). Bovine serum albumin was used as a standard.

As a result, both transformants produced β-galactosidase, but the E. coli crushed cells transformed with pBGB3 had higher activity, and the specific activity was 2.5.
It was 2 units / mg. Furthermore, the specific activity after heat treatment at 75 ° C. for 15 minutes further increased to 10.9 uni.
t / mg. That is, it was revealed that E. coli transformed with pBGB3 produced heat-resistant β-galactosidase. On the other hand, in the case of Escherichia coli transformed with the control vector DNA alone, the activity was almost 0.

[0022]

Industrial Applicability According to the present invention, there are provided a nucleotide sequence of a gene encoding a thermostable β-galactosidase possessed by a bacterium of the genus Thermus, for example, a Thermus sp. A4 strain, and an amino acid sequence deduced therefrom. In addition, the disrupted E. coli cells transformed with the plasmid containing the nucleotide sequence have a high β-galactosidase activity at 70 ° C., similar to that of the Thermus sp. A4 strain. Therefore, according to the present invention, heat-resistant β-galactosidase useful for the food industry can be mass-produced using a genetic recombination technique. Further, the DNA of the present invention
Using the sequence, it is also possible to clone the β-galactosidase gene of another bacterium of the genus Thermus or a bacterium of another genus.

[0023]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2157 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Thermus sp. : Characteristic of A4 sequence Symbol indicating characteristic: CDS Location: 1. . 2157 Characterization method: S sequence ATG CTC GGC GTT TGC TAT TAC CCC GAA CAC TGG CCC AAG GAG CGC TGG 48 Met Leu Gly Val Cys Tyr Tyr Pro Glu His Trp Pro Lys Glu Arg Trp 1 5 10 15 AAG GAG GAC GCC CGG CGC ATG CGG GAA GCG GGG CTT TCC CAT GTA CGT 96 Lys Glu Asp Ala Arg Arg Met Arg Glu Ala Gly Leu Ser His Val Arg 20 25 30 ATA GGG GAG TTC GCC TGG GCC TTA TTG GAA CCG GAG CCT GGA AGG CTG 144 Ile Gly Glu Phe Ala Trp Ala Leu Leu Glu Pro Glu Pro Gly Arg Leu 35 40 45 GAG TGG GGT TGG TTG GAC GAG GCC ATC GCC ACC CTG GCC GCC GAG GGG 192 Glu Trp Gly Trp Leu Asp Glu Ala Ile Ala Thr Leu Ala Ala Glu Gly 50 55 60 CTT AAG GTG GTC CTG GGC ACG CCC ACG GCC ACG CCG CCC AAG TGG CTC 240 Leu Lys Val Val Leu Gly Thr Pro Thr Ala Thr Pro Pro Lys Trp Leu 65 70 75 80 GTA GAC CGT TAT CCG GAG ATC CTC CCC GTG GAC CGG GAA GGG CGG AGG 288 Val Asp Arg Tyr Pro Glu Ile Leu Pro Val Asp Arg Glu Gly Arg Arg 85 90 95 CGG CGG TTT GGG GGG CGG CGG CAC TAC TGC TTT TCC AGC CCC GTT TAC 336 Arg Arg Phe Gly Gly Arg Arg H is Tyr Cys Phe Ser Ser Pro Val Tyr 100 105 110 CGG GAA GAG GCC CGG CGC ATC GTG ACG CTA CTC GCC GAG CGC TAC GGG 384 Arg Glu Glu Ala Arg Arg Ile Val Thr Leu Leu Ala Glu Arg Tyr Gly 115 120 125 GGC CTC GAG GCC GTG GCG GGC TTC CAG ACC GAC AAC GAG TAC GGC TGC 432 Gly Leu Glu Ala Val Ala Gly Phe Gln Thr Asp Asn Glu Tyr Gly Cys 130 135 140 CAC GAC ACC GTG CGC TGC TAC TGC CCC CGC TGC CAA GAG GCC TTC CGG 480 His Asp Thr Val Arg Cys Tyr Cys Pro Arg Cys Gln Glu Ala Phe Arg 145 150 155 160 GGG TGG CTC GAG GCC CGG TAC GGC ACC ATT GAA GCC CTG AAC GAG GCC 528 Gly Trp Leu Glu Ala Arg Tyr Gly Thr Ile Glu Ala Leu Asn Glu Ala 165 170 175 TGG GGG ACG GCC TTC TGG AGC CAG CGT TAT CGG AGC TTT GCC GAG GTG 576 Trp Gly Thr Ala Phe Trp Ser Gln Arg Tyr Arg Ser Phe Ala Glu Val 180 185 190 GAG CTC CCC CAC CTC ACC GTG GCC GAG CCT AAC CCG AGC CAC CTC CTG 624 Glu Leu Pro His Leu Thr Val Ala Glu Pro Asn Pro Ser His Leu Leu 195 200 205 GAC TAC TAC CGC TTC GCC TCG GAC CAG GTG AGG GCC TTT AAC CGC CTC 672 Asp Tyr Tyr Arg P he Ala Ser Asp Gln Val Arg Ala Phe Asn Arg Leu 210 215 220 CAG GTG GAG ATC CTG AGG GCC CAT GCC CCC GGG AAG TTC GTC ACC CAC 720 Gln Val Glu Ile Leu Arg Ala His Ala Pro Gly Lys Phe Val Thr His 225 230 235 240 AAC TTC ATG GGC TTC TTC ACC GAC TTG GAC GCT TTC GCC TTG GCC CAG 768 Asn Phe Met Gly Phe Phe Thr Asp Leu Asp Ala Phe Ala Leu Ala Gln 245 250 255 GAC CTG GAC TTC GCC AGC TGG GAC AGC TAC CCT CTG GGC TTC ACC GAC 816 Asp Leu Asp Phe Ala Ser Trp Asp Ser Tyr Pro Leu Gly Phe Thr Asp 260 265 270 CTC ATG CCC CTA CCT CCG GAG GAG AAG CTC CGC TAC GCC CGC ACC GGC 864 Leu Met Pro Leu Pro Pro Glu Glu Lys Leu Arg Tyr Ala Arg Thr Gly 275 280 285 285 CAC CCC GAC GTG GCC GCC TTC CAC CAC GAC CTC TAC CGG GGG GTG GGA 912 His Pro Asp Val Ala Ala Pla His His Asp Leu Tyr Arg Gly Val Gly 290 295 300 CGG GGG AGG TTT TGG GTG ATG GAG CAA CAG CCG GGC CCT GTG AAC TGG 960 Arg Gly Arg Phe Trp Val Met Glu Gln Gln Pro Gly Pro Val Asn Trp 305 310 315 320 GCC CCT CAC AAC CCG AGC CCC GCT CCC GGG ATG GTG CGG CTT TGG ACC 1008 Ala Pro His Asn Pro Ser Pro Ala Pro Gly Met Val Arg Leu Trp Thr 325 330 335 TGG GAG GCC CTG GCC CAC GGT GCG GAG GTG GTT TCC TAC TTC CGC TGG 1056 Trp Glu Ala Leu Ala His Gly Ala Glu Val Val Ser Tyr Phe Arg Trp 340 345 350 CGC CAG GCG CCT TTT GCC CAG GAG CAG ATG CAC GCC GGG CTC CAC CGA 1104 Arg Gln Ala Pro Phe Ala Gln Glu Gln Met His Ala Gly Leu His Arg 355 360 365 CCG GAT TCT GCC CCC GAC CAA GGC TTC TTT GAG GCG AAG CGC GTG GCC 1152 Pro Asp Ser Ala Pro Asp Gln Gly Phe Phe Glu Ala Lys Arg Val Ala 370 375 380 GAG GAG CTC GCC GCC CTG GCC CTG CCT CCC GTG GCC CAG GCC CCT GTG 1200 Glu Glu Leu Ala Ala Leu Ala Leu Pro Pro Val Ala Gln Ala Pro Val 385 390 395 400 GCC CTG GTT TTT GAC TAC GAG GCC GCC TGG ATT TAC GAG GTC CAG CCC 1248 Ala Leu Val Phe Asp Tyr Glu Ala Ala Trp Ile Tyr Glu Val Gln Pro 405 410 415 CAA GGG GCA GAG TGG AGC TAT CTG GGC CTC GTC TAT CTC TTT TAC AGC 1296 Gln Gly Ala Glu Trp Ser Tyr Leu Gly Leu Val Tyr Leu Phe Tyr Ser 420 425 430 GCC CTC CGG CGG CTG GGA CTG GAT GTG GAT GTG GTT C CC CCG GGG GCT 1344 Ala Leu Arg Arg Leu Gly Leu Asp Val Asp Val Val Pro Pro Gly Ala 435 440 445 TCT TTA AGG GGC TAC GCC TTC GCC GTG GTC CCG AGC CTC CCC ATC GTG 1392 Ser Leu Arg Gly Tyr Ala Phe Ala Val Val Pro Ser Leu Pro Ile Val 450 455 460 CGG GAG GAG GCC TTG GAA GCC TTC CGG GAA GCC GAG GGG CCT GTC CTC 1440 Arg Glu Glu Ala Leu Glu Ala Phe Arg Glu Ala Glu Gly Pro Val Leu 465 470 475 475 480 TTC GGT CCC CGC TCG GGG AGC AAG ACG GAA ACT TTT CAG ATT CCC AAG 1488 Phe Gly Pro Arg Ser Gly Ser Lys Thr Glu Thr Phe Gln Ile Pro Lys 485 490 495 GAG CTT CCT CCC GGC CCC CTC CAG GCC CTC CTT CCC CTT AAG GTG GTT 1536 Glu Leu Pro Pro Gly Pro Leu Gln Ala Leu Leu Pro Leu Lys Val Val 500 505 510 CGG GTG GAA AGC CTT CCC CCG GGT CTT CTA GAG GTG GCG GAG GGG GCG 1584 Arg Val Glu Ser Leu Pro Pro Gly Leu Leu Glu Val Ala Glu Gly Ala 515 520 525 CTC GGC CGC TTC CCT CTG GGT CTG TGG CGG GAA TGG GTG GAG GCT CCC 1632 Leu Gly Arg Phe Pro Leu Gly Leu Trp Arg Glu Trp Val Glu Ala Pro 530 535 540 CTA AAG CCC CTC CTT ACC TTC CA G GAC GGG AAG GGA GCC CTC TAC CGG 1680 Leu Lys Pro Leu Leu Thr Phe Gln Asp Gly Lys Gly Ala Leu Tyr Arg 545 550 555 560 GAG GGG CGA TAC CTC TAC CTT GCG GCC TGG CCC TCG CCC GAA CTC GCG 1728 Glu Gly Arg Tyr Leu Tyr Leu Ala Ala Trp Pro Ser Pro Glu Leu Ala 565 570 575 GGG AGG CTC CTC TCC GCT CTC GCC GCC GAG GCG GGC CTA AAG GTC CTT 1776 Gly Arg Leu Leu Ser Ala Leu Ala Ala Glu Ala Gly Leu Lys Val Leu 580 585 590 TCC CTG CCC GAG GGC CTA AGG CTC AGG CGG CGG GGG ACC TGG GTC TTT 1824 Ser Leu Pro Glu Gly Leu Arg Leu Arg Arg Arg Gly Thr Trp Val Phe 595 600 605 GCC TTC AAC TAC GGG CCG GAG GCG GTG GAG GCC CCC GCC TCA GAG GGG 1872 Ala Phe Asn Tyr Gly Pro Glu Ala Val Glu Ala Pro Ala Ser Glu Gly 610 615 620 GCC CGG TTC CTC CTG GGG AGT AGG CGG GTG GGC CTT ATG ACC TCG CCG 1920 Ala Arg Phe Leu Leu Gly Ser Arg Arg Val Gly Leu Met Thr Ser Pro 625 630 635 640 TCT GGG AGG AGG CAT GAG GCT GAG GTT GGG GGA ACT GGA GGT TTT CGT 1968 Ser Gly Arg Arg His Glu Ala Glu Val Gly Gly Thr Gly Gly Phe Arg 645 650 655 GGA GG C GGA GGG CCT GGA GGA GGC CCC GGG GGG AGT GCG CCT TTG GGG 2016 Gly Gly Gly Gly Gly Gly Gly Gly Pro Gly Gly Ser Ala Pro Leu Gly 660 665 670 CAG GGA GGT GCG GGT CTT TCC CCC TTT CCC CGC CAA GGG GTT CTT CCG 2064 Gln Gly Gly Ala Gly Leu Ser Pro Phe Pro Arg Gln Gly Val Leu Pro 675 680 685 CCA CGG CTG GCA GAG CTG GAG CCT CGC CGC CTG GGT GGA CCC CGC TCA 2112 Pro Arg Leu Ala Glu Leu Glu Pro Arg Arg Leu Gly Gly Pro Arg Ser 690 695 700 GGC CCC CAC GCC CCT CCT CCC CGA GGC GCG CCG CCC CCA GGC GGA 2157 Gly Pro His Ala Pro Pro Pro Arg Gly Ala Pro Pro Pro Gly Gly 705 710 715 715 719

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of an oligonucleotide deduced from the N-terminal amino acid sequence of thermostable β-galactosidase A4-β-Gal.

FIG. 2 is a diagram showing the results of Southern hybridization targeting a restriction enzyme digest of chromosomal DNA of Thermus SP A4 strain.

FIG. 3 is a diagram showing a restriction enzyme digestion map, (A)
pBGS2, (B) shows pBGB1 and (C) shows restriction enzyme digestion maps of the inserted fragment of pBGB3.

Claims (3)

[Claims] 1. A β-galactosidase gene which is derived from a bacterium belonging to the genus Thermus and encodes a thermostable β-galactosidase, having the nucleotide sequence of SEQ ID NO: 1 or having substantially the same function as the nucleotide sequence. 2. A transformant into which a DNA fragment containing the β-galactosidase gene according to claim 1 has been incorporated. 3. A method for producing thermostable β-galactosidase, which comprises culturing the transformant according to claim 2, and collecting thermostable β-galactosidase from the culture.