JPH10337184A - Beta-1,3 glucanase gene and production of beta-1,3 glucanase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject gene for the production, etc., of an enzyme for producing a laminary oligosaccharide useful in the field of medicinal and agrochemical agents, cosmetics and foods by bringing the gene to consist of a DNA capable of coding protein expressing β-1,3-glucanase activity of a specific amino acid sequence. SOLUTION: This β-1,3 glucanase gene is a new DNA capable of coding a protein having 1-277 amino acid sequence in the amino acid arrangement of the formula, or a protein having 1-277 amino acid sequence in the amino acid arrangement of the formula having an amino acid sequence containing substitutions, deficiencies, insertions or transpositions of one or several amino acid residues and having β-1,3 glucanase activity and is used for a production, etc., of the above mentioned enzyme useful for a production of laminary oligosaccharides and a synthesis, etc., of glycoside compounds. This gene is obtained by screening a cDNA library derived from Streptomyces sp. DIC-108 strain (FERM BP-253).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a β-1,3 glucanase (hereinafter referred to as “β-1,3 glucanase”) involved in the production of laminarioligosaccharides, which are important oligosaccharides in the fields of medicine, agricultural chemicals, cosmetics and foods, and in the enzymatic synthesis of glycosyl compounds. , "SGTas
e), a recombinant vector containing the DNA,
Introduced microorganisms, and β-
The present invention relates to a method for producing 1,3 glucanase.

[0002]

2. Description of the Related Art SGTas by Streptomyces sp.
e, and a technique for producing a glycoside compound using the glycosyltransferase activity of the enzyme, has already been described in Japanese Patent Publication No. 4-37.
No. 719, Japanese Patent Application No. 7-10602, Japanese Patent Application No. 8-110
No. 789. Glycosyl compounds, for example, alkyl glycosides, have attracted attention as low-irritant surfactants, and paranitrophenyl laminarioligosaccharides can be used as β-1,3 glucanase measuring reagents. In addition, the application of glycosyl compounds is expected in a wide range of fields such as agrochemicals, foods, cosmetics, and test drugs, and there is an urgent need to develop a simple mass production method.

[0003] SGTase is useful for easily producing a glycosyl compound, but it has been known by conventional methods to use SGTase.
The productivity of e was very low, and an enzyme amount sufficient for mass production of glycosyl compounds could not be obtained. As a result, it was difficult to meet the needs of the industry as described above. In addition, during the production of SGTase, other glucanases are mixed as by-products, causing a reduction in the yield in the glycosyl compound production reaction.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to solve such a problem by preparing a base sequence encoding said enzyme for the purpose of mass production of SGTase, preferably in a system free of other enzymes. And a recombinant vector containing the DNA, a microorganism into which the DNA has been introduced, and a method for producing β-1,3 glucanase using the microorganism.

[0005]

Means for Solving the Problems The present inventors have proposed SGTa.
As a means of increasing the production amount and purity of se, the method by genetic engineering is considered to be advantageous, and the present inventors have worked diligently to obtain DNA containing a base sequence encoding SGTase and to produce SGTase by a recombinant microorganism, thereby completing the present invention. Reached.

That is, the present invention relates to (1) a DNA encoding a protein represented by the following (A) or (B): (A) an amino acid sequence represented by SEQ ID NO: 1 to 277 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; A protein having the sequence to be expressed. (B) having the sequence represented by amino acid numbers 1 to 277 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing,
Or a protein having an amino acid sequence containing substitution, deletion, insertion or transposition of several amino acid residues and having β-1,3 glucanase activity; (2) a protein having an amino acid sequence represented by SEQ ID NO: 1 Of which, amino acid numbers -64 to
(1) a DNA comprising the nucleotide sequence encoding the amino acid sequence represented by -1; (3) having the sequence represented by base numbers 367 to 1197 among the nucleotide sequence represented by SEQ ID NO: 1 (1) (4) The DNA described in (2) having the sequence represented by base numbers 175 to 1197 among the nucleotide sequence shown in SEQ ID NO: 1, (5) having the nucleotide sequence shown in SEQ ID NO: 1 And (6) (1).
Recombinant DNA obtained by linking the DNA according to any one of (1) to (5) with a vector autonomously replicable in a host cell.
NA, (7) D according to any one of (1) to (5).
A microorganism transformed by introducing NA into cells,
(8) The microorganism according to (7) and the microorganism according to (9), (7) or (8), which have been transformed with the recombinant DNA according to (6), are cultured in a suitable medium. −
Β-characterized by collecting 1,3 glucanase
And a method for producing 1,3 glucanase.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

(1) DNA of the Present Invention and Recombinant DNA Containing the Same The DNA of the present invention contains a base sequence encoding SGTase. SGTase (mature type) has the amino acid sequence shown in SEQ ID NO: 1;
It is a protein having the amino acid sequence represented by 277. The SGTase is composed of 277 amino acids and has a molecular weight of about 30,000, and has the ability to hydrolyze β-1,3 glycosyl sugar compounds to produce β-1,3 oligosaccharides and glucose, and also to transfer sugars to other compounds. To produce glycosyl compounds. Streptomyces
SGTase derived from SP (Streptomyces sp.) DIC-108 strain is once a secretory sequence (signal peptide).
It has been shown by the present invention that after being produced as a precursor containing, the peptide undergoes cleavage of the signal peptide to become a mature form.

[0009] The DNA of the present invention may be any of the amino acid sequences represented by SEQ ID NO: 1 as long as it comprises a base sequence encoding SGTase having the amino acid sequence represented by amino acids 1 to 277. There may be.
That is, although there are a plurality of base sequences that determine one amino acid sequence, any base sequence that encodes the amino acid sequence represented by amino acid numbers 1-277 among the amino acid sequences represented by SEQ ID NO: 1 It may be. As an example of such a DNA, specifically, a DNA having a base sequence represented by base numbers 367 to 1197 in the base sequence shown in SEQ ID NO: 1 can be mentioned.

[0010] The DNA of the present invention has one or several amino acid residue substitutions, deletions, insertions or transpositions as long as the β-1,3 glucanase activity of SGTase encoded thereby is not substantially impaired. Includes what you do.
DNA encoding such a protein substantially equivalent to SGTase can be obtained, for example, by subjecting DNA encoding SGTase or a microorganism having the same to mutation treatment,
A DNA which hybridizes from the DNA or the microorganism under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 1 or a part thereof, wherein SGTase
It can be obtained by selecting one that encodes a protein having activity. Here, “stringent conditions” means that a so-called specific hybrid is formed,
This refers to conditions under which non-specific hybrids are not formed. Although it is difficult to quantify these conditions clearly, as an example, nucleic acids having high homology, for example, 50% or more,
Preferably, DNAs having a homology of 70% or more hybridize with each other, and nucleic acids with lower homology do not hybridize with each other.

[0011] DNA encoding a protein substantially equivalent to the above-mentioned SGTase is SGTas.
It can also be obtained by directly introducing a mutation into a DNA encoding e, for example, a DNA having the base sequence shown in SEQ ID NO: 1 by a method such as site-specific mutation.

The number of substitutions, deletions, insertions or transpositions of amino acid residues is not particularly limited as long as it does not substantially affect SGTase activity. In addition, the DNA of the present invention comprises the above SGT
In the amino acid sequence shown in SEQ ID NO: 1 upstream of the sequence encoding the ase (mature form), amino acid numbers -64 to-
Amino acid sequence represented by 1 (including signal peptide)
May further be included. Specific examples of such DNA include a DNA having a sequence represented by base numbers 175 to 1197 in the base sequence shown in SEQ ID NO: 1.

Further, the DNA of the present invention may contain one or more of 5 ′ non-coding region, 3 ′ non-coding region, promoter and terminator. Specific examples of such DNA include a DNA having the base sequence shown in SEQ ID NO: 1. Hereinafter, mature SGTas
DNA encoding e, SGT containing signal peptide
The DNA encoding the ase precursor and the DNA containing the non-coding region and the like may be abbreviated as “SGTase gene” in some cases.

The recombinant DNA of the present invention comprises the above-mentioned SGTa
It is a recombinant DNA obtained by ligating a DNA fragment containing the se gene and a vector. The vector used for preparing the recombinant DNA may be appropriately selected depending on the host microorganism into which the SGTase gene is to be introduced, and a phage or plasmid capable of growing in the host microorganism is suitable. As the phage, a known phage can be appropriately used. For example, when Escherichia coli is used as a host microorganism, λ phage and M13 phage can be used. When the host microorganism is Escherichia coli, for example, pBR32
2, pUC118, pUC119, pZErO ^™ -1
(Invitrogen) can be used. Further, for example, Escherichia coli, Bacillus subtilis (Bacillus subti)
lis) as shuttle vectors capable of autonomous propagation in two or more host microorganisms such as pHY300PLK,
In addition to vectors such as pHV14 and pUR32, various known shuttle vectors can be used.

The vector used in the present invention may be a vector that can be integrated into chromosomal DNA of a host cell. That is, the DNA of the present invention may be retained outside the chromosome as a plasmid when introduced into the host cell, or may be retained by being integrated into the chromosomal DNA. Among these vectors, usually, vectors capable of autonomous replication in a host cell are preferable.

In preparing the recombinant DNA of the present invention, if the DNA fragment containing the SGTase gene to be used contains a promoter that can be expressed in a host cell, it may be directly ligated to a vector, but if it is not contained, May be added with a promoter upstream of the SGTase gene and then ligated to a vector. Alternatively, the SGTase gene may be inserted into an appropriate site of a heterologous protein expression vector including a promoter. As a promoter, for example, when Escherichia coli is used as a host microorganism, for example, the trp promoter, lac promoter and the like are used. When Bacillus subtilis is used as a host microorganism, Bacillus amyloliquefaciens (Bacillus a) is used.
and a promoter derived from the α-amylase gene of C. myloliquefaciens).

Further, by inserting a DNA fragment encoding a signal peptide suitable for a host microorganism into the upstream or downstream of the SGTase coding region, SGTas can be obtained.
It can be expected that extracellular production of e will be possible. The signal peptide includes, for example, a signal peptide included in a sequence N-terminal to mature SGTase in the amino acid sequence shown in SEQ ID NO: 1.

Next, a method for preparing the DNA of the present invention and a recombinant DNA containing the same will be described. First, SGTas
e The chromosomal DNA is collected and purified from a donor microorganism capable of producing e, and the chromosomal DNA is cleaved. Donor microorganisms include Streptomyces sp.
es sp.) DIC-108 strain. The strain has been deposited with the Ministry of International Trade and Industry at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, as a microfabrication laboratory deposit No. 253 (FERM BP-253). The cleaved DNA is separated by agarose gel electrophoresis and transferred to a nylon membrane or the like.

On the other hand, SGTa collected from a donor microorganism
A DNA probe is synthesized based on the nucleotide sequence deduced from the known N-terminal amino acid sequence of se. Using this membrane and a labeled DNA probe, Southern hybridization was performed, and a chromosomal DNA restriction enzyme cleavage fragment having a molecular weight corresponding to the positive band expected to contain the SGTase gene was separately determined with reference to the obtained positive band molecular weight. Get.

The DNA fragment and the vector are chromosomal DNA
The vector fragment obtained by cleavage in the same manner as the cleavage of
Form a library. Next, a host microorganism such as Escherichia coli is transformed (transformed) with the library, and a recombinant DNA containing the SGTase gene is identified and singly identified by PCR using two primers corresponding to the known N-terminal amino acid sequence. Let go. When the DNA does not contain the full-length SGTase gene, when the chromosomal DNA is cut, the above operation is repeatedly performed in combination with adjustment of the degree of cleavage, use of a different restriction enzyme, and the like, to thereby obtain the full-length SGTase gene. A DNA containing the SGTase gene is obtained.

The nucleotide sequence of the isolated DNA is determined by using an auto sequencer or the like according to a conventional method.
The chromosomal DNA of the donor microorganism used in the present invention is prepared by culturing the donor microorganism in a liquid medium, for example, with aeration and stirring for 1 to 3 days, collecting the resulting culture by centrifugation, and then lysing this. can do. As the lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or ultrasonic treatment is used. If necessary, other enzyme agents such as protease and ribonuclease and sodium lauryl sulfate (SD
A surfactant such as S) is used in combination. In some cases, freeze-thaw treatment is used. In order to separate and purify DNA from the lysate thus obtained, phenol extraction, chloroform extraction, ethanol precipitation, ribonuclease treatment, protease treatment and the like are appropriately combined in a conventional manner.

The DNA may be cut by, for example, ultrasonic treatment or restriction enzyme treatment. After cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used as necessary. Disconnected DN
From the A fragment, a fragment having an optimal length can be obtained by a sucrose density gradient centrifugation method or an extraction method from an electrophoresed gel.

As a vector, a phage or a plasmid capable of growing in a host microorganism is suitable. As the phage, a known phage can be appropriately used. For example, when Escherichia coli is used as a host microorganism, λ phage and M13 phage can be used.
When the host microorganism is Escherichia coli, for example, pBR322, pUC
118, pUC119, pZErO ^™ -1 and the like can be used. Further, as a shuttle vector capable of autonomous propagation in two or more host microorganisms such as Escherichia coli and Bacillus subtilis, for example, pHY300PLK,
In addition to vectors such as pHV14 and pUR32, various known shuttle vectors can be used.

The method for binding the DNA fragment to the vector fragment is preferably a method using a known ligase. For example, a DNA purified by phenol extraction and ethanol precipitation is used.
The fragment and the vector fragment are mixed at an appropriate ratio, and an appropriate D
A recombinant DNA is prepared by the action of NA ligase.

The host microorganism may be any microorganism as long as the recombinant DNA is stable, capable of autonomous growth, and capable of expressing its trait. For example, Escherichia coli, Bacillus sp. A method for introducing a recombinant vector into a host microorganism is a known method. For example, when the host microorganism is Escherichia coli, a calcium method (Lederb
erg, EM and Cohen, SM, J. Bacteriol., 119, 1072 (197
4)) and the electroporation method (Hirato Okayama, Masami Matsumura, edited by Experimental Medicine, Gene Handbook, Yodosha (19)
91)) can be used. In the case of a λ phage vector, a method of incorporating DNA into phage particles in vitro and infecting bacteria as phage (B. Horn: Met
hodsin Enzymology, 68, 299 (1979)).

The transformed microorganism into which the recombinant vector has been introduced can be selected by a method using an antibiotic, or by inoculating the transformed strain on an agar plate mixed with a β-1,3 glycosyl sugar compound such as Pakiman or curdlan. The dissolution zone due to the decomposition of the β-1,3 glucosyl sugar compound can be detected by a method such as detection by dye staining.

As described above, Streptomyces
FIG. 1 shows a restriction enzyme map of a DNA fragment containing the SGTase gene obtained from SP (Streptomyces sp.) DIC-108 strain. Also, of this DNA fragment, S
SEQ ID NO: 1 shows the nucleotide sequence of the coding region of the GTase gene and its adjacent region, and the amino acid sequence predicted from the nucleotide sequence.

Although the DNA of the present invention has been obtained as described above, the amino acid sequence of SGTase and the nucleotide sequence encoding it have been clarified by the present invention. Can be synthesized from the donor chromosomal DNA by PCR (polymerase chain reaction) using oligonucleotide primers synthesized based on this base sequence. It can also be easily obtained by amplifying the SGTase gene.

The vector containing the SGTase gene obtained as described above can be directly used as the recombinant DNA of the present invention when the promoter specific to the SGTase gene functions in a host cell into which the SGTase gene is to be introduced. can do. When the SGTase gene does not contain a promoter, or when SGT
If the promoter specific to the ase gene does not function efficiently in the host cell, an appropriate promoter
The recombinant DNA of the present invention can be obtained by ligating upstream of the coding region of Tase or inserting the SGTase gene into an appropriate site of an expression vector containing a promoter.

(2) Microorganism of the Present Invention The microorganism of the present invention is a microorganism transformed by introducing the SGTase gene into a cell. Such a microorganism can be obtained, for example, by transforming the above-described recombinant DNA of the present invention.

The microorganism used as a host is SGT
There is no particular limitation as long as the ase gene can be stably expressed, but any microorganism that is usually used for producing a heterologous protein can be used. Specifically, Escherichia bacteria such as Escherichia coli, Bacillus
Bacillus bacteria such as subtilis, Streptomyces bacteria, Saccharomyces cerevisiae (Saccharomyces)
cerevisiae).

When the SGTase gene is introduced into the above microorganism, it is important to introduce a recombinant DNA using a large number of plasmid vectors to achieve a gene amplification effect (gene d).
osage effect), but the host chromosomal DNA
Can be incorporated into If the recombinant DNA of the present invention is introduced into a microorganism that does not produce SGTase,
ase production ability can be imparted. In addition, when the DNA of the present invention is introduced into a microorganism that originally produces SGTase, the amount of SGTase production can be increased.

The method of introducing the recombinant DNA into the host microorganism and the selection of the transformed microorganism into which the recombinant vector has been introduced may be performed in the same manner as described in (1) above.

(3) Method for Producing SGTase of the Present Invention SGTase can be produced by culturing the microorganism of the present invention in a suitable medium and collecting SGTase from the culture. Any medium can be used as long as it is generally used for culturing host microorganisms, and a natural medium or a synthetic medium can be used. For example, L
B medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride) and 2 × YT medium (1.6% tryptone,
1% yeast extract, 0.5% sodium chloride), BY medium (0.5% meat extract, 0.2% yeast extract, 0.2% sodium chloride, 1% polypeptone), YS medium (1% yeast extract, 1% sucrose, 0.25% sodium chloride) and the like. Also, if necessary, antibiotics such as ampicillin, chloramphenicol, Zeoci
^nTM or the like may be added.

The cultivation is usually preferably carried out under aerobic conditions such as aeration stirring and shaking, but can also be carried out under static conditions. The culture temperature is in the range of 10 ° C to 50 ° C, preferably in the range of 20 ° C to 45 ° C.
To 9 are possible, but preferably performed in the vicinity of 5 to 8. Under the above conditions, aeration and stirring culture are usually performed for half a day to 3 days.

After culturing, if the enzyme is present in the cells, the cells are first collected by centrifugation or the like, and then crushed to collect the enzyme from the cells. (Enzymes eluted partially into the culture medium by lysis can be recovered according to the method described below when they are present outside the cells.) As the disruption method, cells suspended in an appropriate buffer or the like are used. The ultrasonic crushing method,
A physical method such as a dynomill method or a freeze-thaw method, or a chemical method such as a solvent method or an enzymatic method can be used. The extraction can be carried out at room temperature in an aqueous suspension, but in order to minimize the inactivation of the enzyme, the extraction is performed at a temperature of 10 ° C. or lower, preferably 2 ° C.
It is preferable to carry out at 10 ° C. and in the presence of an antioxidant or a protease inhibitor.

On the other hand, when the enzyme is present outside the cells (in the medium), the cells are removed from the culture solution by centrifugation or the like, and then the following operation is performed. SGTase is recovered and purified from the obtained crude enzyme solution by an appropriate method, for example, salting out such as ammonium sulfate fractionation, dialysis, ion chromatography, gel filtration chromatography or the like, alone or in combination.

[0038]

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

(Example 1) Preparation of chromosomal DNA from Streptomyces sp. DIC-108 bacteria 500 ml of a liquid medium (0.5% glucose, 0.5%
Streptomyces sp. DI cultured on curdlan, 0.2% yeast extract, 0.2% dipotassium phosphate, 0.1% magnesium sulfate) at 30 ° C for 2 days
About 1 g of C-108 bacteria was collected by filtration, and TE buffer (50 mM Tris-HCl pH 8.0, 50 mM E
(DTA) 5 ml. This was frozen and thawed and crushed appropriately with a homogenizer.

15 ml of a 33% sucrose solution (pH 8.0) and 0.25 MEDTA (pH
8.0) 4 ml of 8 ml and 10 mg / ml lysozyme solutions
After incubating at 37 ° C. for 1 hour,
10 ml of M sodium perchlorate and 4 ml of 10% SDS were added to completely lyse the cells. Nucleic acid is recovered from the lysate by chloroform extraction and ethanol precipitation,
e, protease treatment, followed by phenol extraction and ethanol precipitation to give purified chromosome DN.
A was obtained.

Example 2 Preparation of Labeled Probe Based on N-Terminal Amino Acid Sequence of SGTase SGTase recovered from Streptomyces sp. DIC-108 strain is subjected to ion exchange column chromatography to obtain partially purified SGTase. The resulting partially purified SGTase was separated by SDS polyacrylamide gel electrophoresis, and then transferred to a nylon membrane.
The N-terminal amino acid sequence of SGTase was determined using a protein sequencer. This is shown in SEQ ID NO: 2.

The base sequence corresponding to the 8th to 22nd amino acids in the sequence was estimated, and
The oligonucleotide shown in SEQ ID NO: 3 was synthesized.
The symbol (S) in the base sequence indicates that the base is G or C. Amersham ECL3'-oligolabelling and d
The 3 'end of the synthetic oligonucleotide was labeled using etection systems, and this was used as a labeled probe.

Example 3 Preparation of Gene Library of Streptomyces sp. DIC-108 Bacteria 12 μg of the chromosomal DNA obtained in Example 1 was
After complete digestion with mal, this was subjected to agarose gel electrophoresis and subsequently transferred to a nylon membrane by the capillary method. This membrane and Example 2
Southern hybridization using the labeled probe obtained in Amersham ECL 3'-oligolabelling and
The hybridization was carried out according to the method of detection systems, and a band that hybridized was detected by exposure to an X-ray film, and the size thereof was confirmed (about 1.7 kb).

On the other hand, chromosome D digested with SmaI
NA was separated by agarose gel electrophoresis, and a fraction containing a fragment having a size hybridized with the probe was extracted and collected. Using a ligation kit (Takara Shuzo Co., Ltd.), these fragments were ligated to pZErO ^™ -1 digested with EcoRV to prepare a DNA library used for clone identification by the following PCR.

Example 4 SGTase by PCR
Identification of Gene Clones and Preparation of Plasmid Using a DNA library, 25 E. coli DH5α strains were transformed by the competent cell method, and LB-Z
eocin ^™ agar medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, 50%
(μg / ml Zeocin ^™ , 1 mM IPTG).

PCR reaction solution (PCR buffer, 0.2 mM
dNTPmix, 5 pmole primer No. 1,
5 pmole primer no. The microbial cells were suspended in 10 µl of 2, 0.3 u Taq polymerase) with a small amount of toothpick and picked up with a thermal cycler DNA E from MJ Research.
PCR was performed using ngine ^™ PTC-200. Primer No. The base sequence of SEQ ID NO.
The nucleotide sequence of No. 2 is shown in SEQ ID NO: 5. Primer No.
1 is the base number 388 to 4 of the base sequence shown in SEQ ID NO: 1.
07, primer No. 2 corresponds to a complementary strand of base numbers 409 to 428 of the base sequence shown in SEQ ID NO: 1. The symbol (S) in the base sequence indicates that the base is G or C, and (W) indicates that the base is A or T. The reaction conditions were “96 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C.
One minute cycle was repeated 30 times. After completion of the reaction, the SGTase gene clone was identified by detecting the amplified DNA fragment by polyacrylamide gel electrophoresis.

As a result, a positive clone having pZErO ^™ -1 in the vector portion was obtained.
-1. The plasmid contained the full length SGTase gene. FIG. 1 shows a restriction map of the inserted fragment of pSGT-1.

Example 5 Determination of SGTase Gene Base Sequence and Estimation of SGTase Amino Acid Sequence ABI PRISM ^™ Dye Terminator Cycle for plasmid pSGT-1 derived from the chromosomal DNA of Streptomyces sp. DIC-108 was used. Sequencing Co
Sequence was performed using re Kit (PERKIN ELMER).
As a result, the base sequence of the SGTase gene shown in SEQ ID NO: 1 was determined. In addition, an open reading frame (base numbers 175 to 1197) was found in the base sequence, and the corresponding amino acid sequence was estimated.

The amino acid numbers 1-2 in the above amino acid sequence
2 is the SGT determined by the protein sequencer
It completely matched the N-terminal amino acid sequence of ase. From this, the gene contained in the inserted fragment of pSGT-1 was S
It was confirmed to encode GTase. Also, SG
Tase was found to be encoded as a precursor protein having a signal peptide.

(Example 6) SGTa by recombinant Escherichia coli
Production of se First, in order to synthesize a gene portion required for production of mature SGTase in pSGT-1, PCR using primers N and C was performed. The nucleotide sequence of primer N is shown in SEQ ID NO: 6, and the nucleotide sequence of primer C is shown in SEQ ID NO: 7. Primer N corresponds to base numbers 367 to 385 of the base sequence shown in SEQ ID NO: 1, and primer C
Represents base numbers 1601-1 of the base sequence shown in SEQ ID NO: 1.
620 complementary strands. The reaction conditions are “96 ° C. 1
Minute, 72 ° C., 5 minutes ”cycle 30 times.

Next, the amplified fragment recovered by extraction from agarose gel electrophoresis was once subjected to plasmid pT7Bl
ue (Novagen), a fragment was cut out therefrom with the restriction enzymes EcoRI and HindIII, and the plasmid pUC also cut with EcoRI and HindIII.
It was subcloned into 118. The clone thus obtained was designated as pSGTEX-1.

Escherichia coli J transformed with pSGTEX-1
M105 was shake-cultured in a test tube containing 5 ml of a medium at 37 ° C. for 20 hours. The medium components were 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 5%
0 μg / ml ampicillin, 0.2 mM IPTG.

The supernatant obtained by centrifuging the above culture solution was concentrated and used as a crude enzyme solution in the following sugar transfer reaction. PNPG
(Paranitrophenyl glucoside) 50mg, Laminaripentaose 50mg, 0.5M acetate buffer 1m
1 and 4 ml of the crude enzyme solution were mixed and shaken at 55 ° C. for 5 hours. After completion of the reaction, the reaction product was detected by HPLC (using an ODS column).
The formation of PNP oligoglycosides having (para-nitrophenyl laminaritetraoside) as a main component was observed. Thereby, production of SGTase by the recombinant E. coli was confirmed.

[0054]

The DNA of the present invention has been isolated for the first time, and the use of the recombinant DNA of the present invention containing the same makes it possible to easily produce a large amount of SGTase. In addition, SGTas was prepared in the absence of other glucanases that would cause a decrease in yield during the synthesis of glycosyl compounds.
e can be produced.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1665 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Streptomyces sp. Strain name: DIC-108 Sequence characteristics Features Symbol representing -35 signal Location: 118..123 Method that determined the feature: S Symbol representing feature: -10 signal Location: 141..146 Method that determined the feature: Symbol representing feature: mat_peptide Position: 367..1197 Method used to determine characteristics: S Symbol representing characteristics: CDS Location: 175..1200 Method used to determine characteristics: E sequence GGGCTACGGC CCCGAGCACC GCGCCCGTGT GCGCACCCCC CGGCCCGGTT TCCCCGGTTC 60 GCGCCCATTC GAGAGGTGAA CTCCCGCG ACCGGGTCGACC ACCGGGTCGACGAC CACATTGGCG GGACCGTGAG AGCGCTCTCA CGCG ATG 177 Met -64 TTC CGG GCT CTC AGG GCC TGT CCG CAC AAC CAT CAG CAC CGA GAG GTC 225 Phe Arg Ala Leu Arg Ala Cys Pro His Asn His Gln His Arg Glu Val -60 -55 -50 ATC C CAC ATG AAC GAC ACC TCC GGC ACC CCC CGA TCC CCC CAC GC A 273 Ile Pro His Met Asn Asp Thr Ser Gly Thr Pro Arg Ser Pro His Ala -45 -40 -35 CGG CGG CGG CTG AGG CGC ACC CTC GTC GCC CTC GCG GGC GCA CTG GCG 321 Arg Arg Arg Leu Arg Arg Thr Leu Val Ala Leu Ala Gly Ala Leu Ala -30 -25 -20 CTC GGG GCG GGC GCC CTC ACC CTC ACC GGC CCC ACC GCC AGC GCC TCC 369 Leu Gly Ala Gly Ala Leu Thr Leu Thr Gly Pro Thr Ala Ser Ala Ser -15 -10 -5 1 GTG CCG CCC CCG CCG TCG GGC TGG ACC CAG GTC TTC GCC GAC GAC TTC 417 Val Pro Pro Pro Pro Ser Gly Trp Thr Gln Val Phe Ala Asp Asp Phe 5 10 15 GAC GGC CCC AAG GGC AGC GGC GTC GAC ACC GGC GAC TGG CGG TAC GCC 465 Asp Gly Pro Lys Gly Ser Gly Val Asp Thr Gly Asp Trp Arg Tyr Ala 20 25 30 ACC GGC ACC GGC TAC CCC GGC GGC CCC TCC AAC TGG GGC ACC GGC GAG 513 Thr Gly Thr Gly Tyr Pro Gly Gly Pro Ser Asn Trp Gly Thr Gly Glu 35 40 45 ATC GAG ACG ATG ACG TCG AAC CCG GAG AAC GTC TCC CTC GAC GGC AAC 561 Ile Glu Thr Met Thr Ser Asn Pro Glu Asn Val Ser Leu Asp Gly Asn 50 55 60 65 GGC AAC CTG CGC ATC ACC CCG CGG CGC GAC GGC GCC GGC AAC TGG ACG 609 Gly Asn Leu Arg Ile Thr Pro Arg Arg Asp Gly Ala Gly Asn Trp Thr 70 75 80 TCG GGG CGC ATC GAG ACC GCC CGC GAC GAC TTC CAG CCC CCG GCC GGC 657 Ser Gly Arg Ile Glu Thr Ala Arg Asp Asp Phe Gln Pro Pro Ala Gly 85 90 95 GGC ACG CTG CGG GTC GAG GCC CGC ATC CAG GTC CCG AAC GTC ACC GGC 705 Gly Thr Leu Arg Val Glu Ala Arg Ile Gln Val Pro Asn Val Thr Gly 100 105 110 GAC GCG GCC AAG GGC TAC TGG CCC GCG TTC TGG ATG CTC GGC GCC CCG 753 Asp Ala Ala Lys Gly Tyr Trp Pro Ala Phe Trp Met Leu Gly Ala Pro 115 120 125 TAC CGG GGC GAC TAC TGG AAC TGG CCC GCC GTC GGC GAG CTG GAC ATC 801 Tyr Arg Gly Asp Tyr Trp Asn Trp Pro Ala Val Gly Glu Leu Asp Ile 130 135 140 145 ATG GAG AAC ACC CAG GGC ATG AAC ACG GTG TTC GCC ACG ATG CAC TGC 849 Met Glu Asn Thr Gln Gly Met Asn Thr Val Phe Ala Thr Met His Cys 150 155 160 GGC ACC TCG CCG GGC GGC CCG TGC AAC GAG ACC AGC GGC ATC GGC GGC 897 Gly Thr Ser Pro Gly Gly Pro Cys Asn Glu Thr Ser Gly Ile Gly Gly 165 170 175 CAG ACC ACC TGC CAG GGC ACG ACC TGT CAG GCC GGC TTC CAC A CC TAC 945 Gln Thr Thr Cys Gln Gly Thr Thr Cys Gln Ala Gly Phe His Thr Tyr 180 185 190 CGG ATG GAG TGG GAC CGC TCG TCG GAC GTG GAG GAG ATC CGC TTC TCC 993 Arg Met Glu Trp Asp Arg Ser Ser Asp Val Glu Glu Ile Arg Phe Ser 195 200 205 CTC GAC GAC CAC ACC TTC CAC ACC GTC CGG GAG AAC CAG GTC GAC GCG 1041 Leu Asp Asp His Thr Phe His Thr Val Arg Glu Asn Gln Val Asp Ala 210 215 220 225 ACG ACC TGG TCG AAC GCC ACC GAC CAC GGC TTC TTC GTC ATC CTC AAC 1089 Thr Thr Trp Ser Asn Ala Thr Asp His Gly Phe Phe Val Ile Leu Asn 230 235 240 GTG GCG ATG GGC GGC GGC TTC CCC GAC GCG TTC GGC GGC GGC CCC GAC 1137 Val Ala Met Gly Gly Gly Phe Pro Asp Ala Phe Gly Gly Gly Pro Asp 245 250 255 GCG GGC ACC CAG CCC GGC CAC TCG ATG CTC GTG GAC TAC GTG CAG GTG 1185 Ala Gly Thr Gln Pro Gly His Ser Met Leu Val Asp Tyr Val Gln Val 260 265 270 CTC ACC GCC TCC TGACCCGCCT GTCCCACCCC CCACACCCGG CTCATCCGGC 1237 Leu Thr Ala Ser 275 CGGTGACGAG GGCCCCGCGC ACCCCGCGCG GGGCCCTCGT CGGCGTACCG GCCCCGTCGT 1297 TCAGCGGCGT TCCGCGCGGA ACGCCC GCCG CGTCCGGGAA GCCGACCGCG GCCCCGCCCC 1357 ACGCCGCCGG CAGCTCGGTG TGCGCGAGGA GCCGCTTGGC CTCCAGCACA ACACGGGCCA 1417 GGGTGCGCAG GGAGCAGCCG AAGGCGTCCG CGGACGCGGT GGCGGGTGGC GAAGCCCCGC 1477 TCCACGAGCC GCAGCAGGGA CGCGGTCGAC GCGTGGCGCG GCACGGCCGT GTGCAGGCTC 1537 GGCGGCAGGG TGACGGCGTG CTCGTGCCAA CGGCGCAGAG ACGGCAGGAC GCGGGGCGGA 1597 GCGCGGCGAG CCGGGGCGGT CCGGGCCGGG CTGCATGGCG AGGACGGTTC CGCGGTACCC 1657 GGCGTCCC 1665

SEQ ID NO: 2 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Val Pro Pro Pro Ser Gly Trp Thr Gln Val Phe Ala Asp Asp Asp 1 5 10 15 Phe Asp Gly Pro Lys Gly Ser Gly Val 20 25

SEQ ID NO: 3 Sequence length: 44 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGSTGGACSC AGGTSTTCGC SGACGACTTC GACGGSCCSA AGGG 44

SEQ ID NO: 4 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGSTGGACSC AGGTSTTCGC 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGWTGGACWC AAGTWTTTGC 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCCGTGCCGC CCCCGCCGTC 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGGCGAGCCG GGGCGGTCCG 20

[Brief description of the drawings]

FIG. 1 shows a restriction enzyme map of a pSGT-1 insert.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:19) (C12N 9/24 C12R 1:19) (72) Inventor Chitoshi Oka Wakaba, Chiba City, Chiba Prefecture 889 Kasori-cho, Ward Inside the Chiba Industrial Research Institute

Claims (9)

[Claims] 1. A DNA encoding a protein represented by the following (A) or (B): (A) a protein having a sequence represented by amino acids 1 to 277 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, (B) having the sequence represented by amino acid numbers 1 to 277 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing,
Alternatively, a protein having an amino acid sequence containing substitution, deletion, insertion or transposition of several amino acid residues, and having β-1,3 glucanase activity. 2. The DN according to claim 1, further comprising a base sequence encoding the amino acid sequence represented by amino acid numbers -64 to -1 in the amino acid sequence shown in SEQ ID NO: 1.
A. 3. The nucleotide sequence represented by SEQ ID NO: 1 having a sequence represented by nucleotide numbers 367 to 1197.
The described DNA. 4. The nucleotide sequence represented by SEQ ID NO: 1 having a sequence represented by nucleotide numbers 175 to 1197.
The described DNA. 5. The DNA according to claim 2, which has the nucleotide sequence shown in SEQ ID NO: 1. 6. D according to any one of claims 1 to 5,
A recombinant DNA obtained by linking NA and a vector capable of autonomous replication in a host cell. 7. D according to any one of claims 1 to 5,
A microorganism transformed by introducing NA into cells. 8. The microorganism according to claim 7, which has been transformed with the recombinant DNA according to claim 6. 9. A method for producing β-1,3 glucanase, comprising culturing the microorganism according to claim 7 or 8 in a suitable medium, and collecting β-1,3 glucanase from the culture.