JPH09262090A - Laminaripentaose synthase gene - Google Patents

Abstract

(57) Abstract: DNA containing a nucleotide sequence encoding a laminaripentaose synthase, a recombinant vector containing this DNA, a microorganism into which the DNA has been introduced, and laminaripentaose utilizing this microorganism A method for producing a productive enzyme is provided. SOLUTION: A recombinant DNA obtained by ligating a DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 with a vector capable of autonomous replication in a host cell or a vector capable of being integrated into host cell chromosomal DNA. The microorganism transformed with the strain is cultured in a suitable medium, and the laminaripentaose-forming enzyme is collected from the culture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the production of laminaripentaose (hereinafter sometimes abbreviated as “LP”), which is an important oligosaccharide in the fields of medical and agricultural chemicals, cosmetics and foods. Pentaose synthase (hereinafter,
A DNA containing a base sequence encoding “LPHase”), a recombinant vector containing this DNA,
The present invention relates to a microorganism into which the DNA has been introduced, and a method for producing a laminaripentaose-forming enzyme using the microorganism.

[0002]

2. Description of the Related Art LPHas of the genus Streptomyces
Regarding the production method of e and the technique for producing LP from β-1,3 glycosyl sugar compound using the enzyme, Japanese Patent Publication No.
No. 37719. LP is not only important as a sugar raw material for anti-AIDS drugs, but also has a very wide range of applications such as in the fields of agrochemicals, foods, cosmetics, and in the field of test drugs, and establishment of a mass production method thereof is earnestly desired.

Although LPHase is useful for producing LP, the productivity of LPHase is low in the conventional method,
The amount of enzyme sufficient for mass production of LP was not obtained. Therefore, the reality is that it is not possible to meet the needs of the industry as described above. Further, during the production of LPHase, other glucanases were mixed as a by-product, which caused a decrease in LP production efficiency.

[0004]

The object of the present invention is to solve the above problems, for the large-scale production of LPHase, preferably for the production in a system free from contamination with other enzymes, the nucleotide sequence encoding the enzyme. It is intended to provide a DNA containing DNA, a recombinant vector containing the DNA, a microorganism into which the DNA has been introduced, and a method for producing a laminaripentaose-forming enzyme using the microorganism.

[0005]

The present inventors have found that LPHa
As a means for increasing the production amount and purity of se, the method by genetic engineering is considered to be a good idea, and the present invention was completed by earnest efforts to obtain a DNA containing a nucleotide sequence encoding LPHase.

That is, the present invention provides (1) an amino acid residue having the sequence represented by amino acid Nos. 36 to 401 among the amino acid sequences shown in SEQ ID NO: 1 and which does not substantially impair the laminaripentaose synthase activity. DNA containing a nucleotide sequence encoding a laminaripentaose synthase which may have a group substitution, deletion, insertion or transfer, (2) Furthermore, in the amino acid sequence shown in SEQ ID NO: 1, amino acid numbers 1 to 1 (1) DNA containing a nucleotide sequence encoding the amino acid sequence represented by 35, (3) DNA having the sequence represented by nucleotide numbers 634 to 1731 in the nucleotide sequence shown in SEQ ID NO: 1 (4) Of the base sequence shown in SEQ ID NO: 1, the DNA of (2) having the sequence represented by base numbers 529 to 1731, (5) The DNA of (2) having the base sequence shown in SEQ ID NO: 1, 6) Recombinant DNA obtained by ligating the DNA according to any one of the above (1) to (5) and a vector capable of autonomously replicating in a host cell or a vector capable of integrating into a host cell chromosomal DNA, (7) (1) to (5) above
(8) A microorganism transformed by introducing any of the DNAs into cells, (8) a microorganism transformed with the recombinant DNA according to (6) above, (9), (7) or (8) above. The method for producing a laminaripentaose synthase, comprising culturing the microorganism of (1) in a suitable medium and collecting the laminaripentaose synthase from the culture.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. <1> DNA of the present invention and recombinant DNA containing the same The DNA of the present invention contains a nucleotide sequence encoding LPHase.

LPHase (mature type) is a protein having an amino acid sequence represented by amino acid numbers 36 to 401 among the amino acid sequences shown in SEQ ID NO: 1. The L
PHase has a molecular weight of about 370 consisting of 366 amino acids.
00 protein and has the ability to produce LP from β-1,3 glycosyl sugar compounds. LPHase derived from Streptomyces sp. DIC-108 strain can be expressed as a precursor having a molecular weight of about 42900 consisting of 401 amino acids including a secretory sequence (signal peptide) consisting of 35 amino acids. , Demonstrated by the present invention.

The DNA of the present invention may be any DNA as long as it contains the nucleotide sequence encoding LPHase having the sequence represented by amino acid Nos. 36 to 401 among the amino acid sequences shown in SEQ ID No. 1. Good. That is, although there are a plurality of base sequences that determine one amino acid sequence, among the amino acid sequences shown in SEQ ID NO: 1, amino acid number 3
If it encodes the sequence represented by 6 to 401,
Any base sequence may be used. Specifically, as such DNA, a DNA having a sequence represented by base numbers 634 to 1731 in the base sequence shown in SEQ ID NO: 1.
Is mentioned.

The DNA of the present invention has amino acid residue substitutions and deletions unless the ability of the laminaripentaose synthase encoded thereby to produce LP from the β-1,3 glycosyl sugar compound is substantially impaired. Those having loss, insertion or metastasis are also included.

Further, the DNA of the present invention is the above LPHas.
Among the amino acid sequences shown in SEQ ID NO: 1, amino acid numbers 1 to 35 are provided upstream of the sequence encoding e (mature protein).
It may further include a nucleotide sequence encoding the amino acid sequence represented by (signal peptide). Such a D
Specific examples of NA include DNA having a sequence represented by base numbers 529 to 1731 in the base sequence shown in SEQ ID NO: 1.

Further, the DNA of the present invention may contain one or more kinds of 5'non-coding region, 3'non-coding region, promoter, terminator and the like. Specific examples of such DNA include a DNA having the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 1 contains a promoter sequence (-35 signal and -10 signal).

Hereinafter, D encoding mature LPHase
LPHase (precursor) containing NA and signal peptide
DNA that encodes DNA and further includes a non-coding region, etc.
A may be abbreviated as "LPHase gene".

The recombinant DNA of the present invention is the above-mentioned LPHa.
It is a recombinant DNA obtained by ligating a DNA fragment containing the se gene and a vector capable of autonomously replicating in a host cell or a vector capable of integrating into a host cell chromosomal DNA. The vector used for producing the recombinant DNA of the present invention is LPHa.
It may be appropriately selected depending on the host microorganism into which the se gene is to be introduced. Suitable vectors are phages or plasmids that can grow in host microorganisms. Known phages can be appropriately used. For example, when Escherichia coli ( Escherichia coli ) is used as a host microorganism, λ phage, M13 phage and the like can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, for example, pBR3
22, pUC118, pUC119 and the like can be used. Furthermore, for example Escherichia coli, a shuttle vector autonomously replicable in more than one host microorganisms, such as Bacillus subtilis (Bacillus subtilis), for example, pHY
In addition to vectors such as 300PLK, pHV14 and pUR32, various known shuttle vectors can be used. Preferred is pUC119.

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.

LP used for producing the recombinant DNA of the present invention
The DNA fragment containing the Hase gene is
When the Hase gene contains a promoter that can be expressed in the host cell, it may be ligated to the vector as it is. When the Hase gene does not contain a promoter that can be expressed in the host cell, an appropriate promoter sequence is used as LPHase.
It may be linked upstream of the coding region of. Alternatively, the LPHase gene may be inserted into an appropriate site of a heterologous protein expression vector containing a promoter. As such a promoter, for example, when Escherichia coli is used as a host microorganism, for example, trp promoter, lac promoter or the like is used, and when P. subtilis is used as a host microorganism, a promoter of Bacillus amyloliquefaciens is used. Examples include promoters derived from the α-amylase gene.

Furthermore, if necessary, upstream of the LPHase gene coding region, a DNA fragment encoding a secretory sequence (signal peptide) suitable for the host microorganism, for example, amino acid number 1 in the amino acid sequence shown in SEQ ID NO: 1 is used.
It is expected that LPHase can be produced extracellularly by inserting a nucleotide sequence encoding the amino acid sequence represented by ~ 35.

Next, a method for preparing the DNA of the present invention and a recombinant DNA containing the same will be described. First, LPHas
The chromosomal DNA is collected and purified from a donor microorganism capable of producing e, and the chromosomal DNA is cleaved with an appropriate restriction enzyme or the like. As the donor microorganism, L including transformant was used.
PHase-producing microorganisms can be widely used. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces, preferably Streptomyces sp. DIC-108 strain. In addition, the DIC-108 strain was obtained from the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry.
(FERM BP-253).

The DNA fragments cleaved with the restriction enzymes as described above are separated by agarose gel electrophoresis and transferred to a nylon membrane or the like. On the other hand, a DNA probe is synthesized based on the base sequence deduced from the N-terminal amino acid sequence of LPHase collected from a donor microorganism. Southern hybridization was carried out using this membrane and a RI-labeled DNA probe, and the molecular weight of the positive band obtained was used as a reference to prepare LPHa separately.
A chromosomal DNA restriction enzyme cleavage fragment having a molecular weight corresponding to the positive band expected to contain the se gene is obtained. The DNA fragment and the vector fragment obtained by cleaving the vector in the same manner as the chromosomal DNA are ligated with, for example, DNA ligase to form a chromosomal DNA library. Then, a host microorganism such as Escherichia coli is transformed (transformed) with the library, and a clone containing the LPHase gene is identified and isolated by colony hybridization using the above-mentioned labeled probe. When the DNA does not contain the full-length LPHase gene, when the chromosomal DNA is cut, the above operations are repeated by combining the degree of cutting, the use of different restriction enzymes, etc. LPHa
A DNA containing the se gene is obtained.

The base sequence of the isolated DNA is determined by using an autosequencer 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 a lysing method, for example, treatment with a cell wall lysing enzyme agent 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 according to a conventional method.

The DNA can be cleaved by, for example, ultrasonic treatment or restriction enzyme treatment. After cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used if 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 used for preparing a chromosomal DNA library, a phage or a plasmid capable of growing in a host microorganism is suitable. As the phage, known ones can be appropriately used. For example, when Escherichia coli is used as the host microorganism, λ phage, M13 phage, etc. can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, for example, p
BR322, pUC118, pUC119, etc. can be used. Furthermore, as a shuttle vector capable of autonomously replicating in two or more host microorganisms such as Escherichia coli and Bacillus subtilis, for example, pHY300PLK, p
In addition to vectors such as HV14 and pUR32, various known shuttle vectors can be used.

The method of ligating the DNA fragment and the vector fragment is preferably a method using a known ligase, for example, DNA purified by phenol extraction and ethanol precipitation.
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 into which the chromosomal DNA library is introduced may be any one if the recombinant vector is stable and capable of autonomous replication. For example, Escherichia coli, Bacillus sp.

The method for introducing the recombinant vector into the host microorganism is a known method, for example, when the host microorganism is Escherichia coli, the calcium method (Lederberg, EM and
Cohen, SM, J. Bacteriol., 119, 1072 (1974)) and electroporation method (Hiroto Okayama, Masami Matsumura, edited by Experimental Medicine, Gene Handbook, Yodosha (1991)) can be used. In case of λ phage vector, D
Incorporating NA into phage particles in vitro and infecting bacteria as phage (B.Horn: Methods in Enzym
ology, 68, 299 (1979)) is used.

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
A restriction enzyme fragment map of a DNA fragment containing the LPHase gene obtained from Streptomyces sp. DIC-108 strain is shown in Fig. 1. Further, in this DNA fragment, the base sequence of the coding region of the LPHase gene and its adjacent region and the amino acid sequence predicted from the base sequence are shown in SEQ ID NO: 1.

In completing the present invention, the D
NA was obtained as described above, but since the amino acid sequence of LPHase and the nucleotide sequence encoding the same were clarified by the present invention, the DNA of the present invention was obtained.
Can be synthesized based on the sequence described in the sequence listing, and the donor can be obtained by PCR (polymerase chain reaction) using an oligonucleotide primer synthesized based on this base sequence. It can also be easily obtained by amplifying the LPHase gene from the chromosomal DNA of.

The vector containing the LPHase gene obtained as described above is used as it is as the recombinant DNA of the present invention when the promoter specific to the LPHase gene functions in the host cell into which the LPHase gene is to be introduced. can do. If the LPHase gene does not contain a promoter, or LPH
When the promoter specific to the ase gene does not function efficiently in the host cell, an appropriate promoter sequence is
The recombinant DNA of the present invention can be obtained by ligating upstream of the coding region of Hase or by inserting the LPHase 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 LPHase gene into cells. 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 LPH
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 LPHase gene is introduced into the above-mentioned microorganisms, it is necessary to introduce a recombinant DNA using a plasmid vector of another copy number so that the gene amplification effect (gene d
osage effect), but the host chromosomal DNA
Can be incorporated into

By introducing the DNA of the present invention into a microorganism that does not produce LPHase, it is possible to impart the ability to produce LPHase. Further, the amount of LPHase produced can be increased by introducing the DNA of the present invention into a microorganism that originally produces LPHase.

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 carried out in the same manner as described in <1> above.

<3> Method for Producing LPHase of the Present Invention LPHase can be produced by culturing the microorganism of the present invention in a suitable medium and collecting LPHase from the culture.

Any medium can be used as long as it is a medium commonly used for culturing host microorganisms, and either a natural medium or a synthetic medium can be used. Examples of carbon sources include curdlan, laminaran, pakiman, paramylon,
Β-1,3 glycosyl sugar compounds such as yeast cell wall and glucose, glycerin, ethanol and the like can be used, but the presence of β-1,3 glycosyl sugar compound is more preferable for the production of LPHase. As the nitrogen source, ammonium sulfate, ammonium chloride, urea, ammonia, yeast extract, polypeptone, meat extract and the like are used. As the inorganic salt, potassium phosphate, sodium phosphate, ammonium phosphate, magnesium sulfate and the like can be used. Also, vitamins, amino acids, etc. may be added as a trace nutrient source, and iron, cobalt, manganese, aene, etc. may be added in the form of salt as trace elements.

Culturing is usually preferably carried out under aerobic conditions such as aeration and stirring, but it can also be carried out under static conditions. As a typical medium composition, for example, 0.5%
The medium of glucose, 0.5% curdlan, 0.2% yeast extract, 0.2% dipotassium phosphate, 0.1% magnesium sulfate was inoculated with the microorganism of the present invention, and the culture temperature was from 10 ° C. In the range of 50 ° C, preferably 30 ° C to 40
In the range of 0 ° C, the culture pH can be 3 to 9, but preferably about 6 to 8. Under the above conditions, aeration and stirring culture are usually performed for 1 to 3 days.

After the culturing, when the enzyme is present in the microbial cell, the microbial cell is first collected by centrifugation or the like and then crushed to collect the enzyme from the microbial cell. As a crushing method, a microbial cell suspended in an appropriate buffer solution or the like is ultrasonically crushed, a dynomill method,
A physical method such as a freeze-thaw method or a chemical method such as a solvent method or an enzyme method can be used, and of course, two or more of them may be combined. The extraction conditions can be carried out at room temperature in a water suspension, but in order to prevent the inactivation of the enzyme to a minimum, it is carried out at 10 ° C or lower, preferably 2 ° C to 10 ° C, and in the presence of an antioxidant. Is preferred.

On the other hand, when the enzyme is present outside the bacterial cells (in the medium), the bacterial cells are removed from the culture solution by centrifugation or the like, and then the following operation is performed. From the obtained crude enzyme solution, LPHase is recovered and purified by a suitable method, for example, salting out such as ammonium sulfate fractionation method, dialysis, ion chromatography method, gel filtration chromatography method, or a combination thereof.
The molecular weight of the purified LPHase thus obtained is about 37,000 (from SDS-PAGE under reducing conditions), the optimum pH in the reaction is around 5.5, and the optimum temperature is about 55 ° C.

[0040]

EXAMPLES The present invention will be described below in greater detail by giving Examples, but the present invention is not limited thereto.

[0041]

[Example 1] [Streptomyces SP DIC-
Preparation of chromosomal DNA from 108 bacteria] 500 ml of 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 EDT was used.
A) Suspended with 5 ml. This was frozen and thawed and crushed appropriately with a homogenizer.

15 ml of 33% sucrose solution (pH 8.0) and 0.25 M EDTA (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.

[0043]

[Example 2] [Preparation of labeled probe based on N-terminal amino acid sequence of LPHase] LPHase recovered from Streptomyces sp. DIC-108 strain was subjected to ion exchange column chromatography to obtain partially purified LPHase. The obtained purified LPHase was separated by SDS polyacrylamide gel electrophoresis, transferred to a nylon membrane, and the N-terminal amino acid sequence of LPHase was determined by a protein sequencer. This is shown in SEQ ID NO: 2.

The nucleotide sequence corresponding to the 17th to 25th amino acids in the sequence was deduced, and the oligonucleotide shown in SEQ ID NO: 3 was synthesized by a DNA synthesizer. The third base (K) is a mixture of T and G, 25
The second base (Y) was a mixture of T and C. Using polynucleotide kinase, the 5'end of synthetic DNA was
- ^{3 2} P] labeled with ATP, which was used as a labeled probe.

[0045]

[Embodiment 3] [Streptomyces SP DIC-
Preparation of Gene Library of 108 Bacteria] 1 μg of chromosomal DNA obtained in Example 1 was treated with restriction enzyme Sa
After being completely decomposed with II, it was subjected to agarose gel electrophoresis and subsequently transferred to a nylon membrane by the capillary method. Southern hybridization using this membrane and the labeled probe obtained in Example 2 was performed according to a conventional method, and a hybridizing band was detected by autoradiography to confirm its size (molecular weight: about 1.4 kb. ).

On the other hand, chromosome D digested with SalI
NA was separated by agarose gel electrophoresis, and a fraction containing a fragment having a size hybridized with the probe was extracted and collected. Ligation kit (Takara Shuzo Co., Ltd.)
These fragments were ligated to pUC119 similarly digested with SalI to prepare a DNA library used for the following colony hybridization.

[0047]

[Example 4] [Identification of LPHase gene clone by colony hybridization method and preparation of plasmid] Escherichia coli DH5α strain of about 3000 cells was transformed by the competent cell method using a DNA library, and L
A agar medium (1% polypeptone, 0.5% yeast extract,
0.5% sodium chloride, 1.5% agar, 50 μg / m
1 ampicillin). The colonies were transferred onto a nylon membrane, transferred to a new agar medium, and then cultured at 37 ° C. for about 3 hours. Peel off the membrane and denature solution (0.5N NaOH, 1.5M
NaCl) moistened filter paper for 10 minutes, then neutralized (0.5 M Tris-HCl buffer pH 7.5, 1.
It was left still for 10 minutes on a filter paper soaked with 5M NaCl). After air-drying, DNA was immobilized by UV irradiation.

Using the membrane and the labeled probe obtained in Example 2, colony hybridization was performed according to a conventional method. However, the hybridization temperature is 37 ° C., the washing conditions are 2 × SSC, 0.1% SDS,
It was 37 ° C. As a result, a positive clone was obtained, and the insert was excised with SphI and SalI and subcloned into pUC119. This plasmid was named pLA01.

In the next step, the full length of the target gene was obtained. First, 1 μg of chromosomal DNA is treated with restriction enzyme S
It was completely digested with acI, subjected to agarose gel electrophoresis, and transferred to a membrane. On the other hand, the pLA01 plasmid was cleaved with restriction enzymes SphI and SalI, subjected to agarose gel electrophoresis, and the insert DNA was extracted and labeled with [α- ³² P] dCTP by the random prime method. Using this as a labeled probe, Southern hybridization, ligation, transformation, and colony hybridization were performed in the same manner as above. Hybridization was carried out at 37 ° C. and 50% formamide, and washing conditions were 0.2 × SSC, 0.1%.
SDS, 65 ° C. As a result, a positive clone having pUC119 in the vector portion was obtained, which was named pLE01. The plasmid contained the full length LPHase gene. A restriction enzyme cleavage map of the insert fragment contained in pLE01 is shown in FIG.

[0050]

[Example 5] [Determination of nucleotide sequence of LPHase gene and estimation of LPHase amino acid sequence] Regarding the portion derived from Streptomyces sp. DIC-108 chromosomal DNA in the plasmid pLE01, AutoRead ALFred, Sequence.
The sequence was performed using ing Kit (Pharmacia). As a result, LPHase shown in SEQ ID NO: 1
The base sequence of the gene was determined. In addition, an open reading frame (base numbers 529 to 17) is included in the base sequence.
31) was found and the corresponding amino acid sequence was deduced.

Amino acid Nos. 36 to 36 in the above amino acid sequence
69 is LP determined by protein sequencer
It completely matched the N-terminal amino acid sequence of Hase. From this, the gene contained in the pLE01 insertion fragment was
It was confirmed that it encodes LPHase. Also, L
It was revealed that PHase is encoded as a precursor protein having a signal peptide consisting of 35 amino acids.

[0052]

INDUSTRIAL APPLICABILITY The present invention provides a DNA encoding LPHase. The DNA of the present invention has been isolated for the first time, and enables the production of LPHase simply by recombining the vector (promoter if necessary) portion depending on the host. Thus, not only can the enzyme be mass-produced, but it can also be produced in a state where other glucanases that reduce the LP production efficiency are not mixed.

The recombinant DNA containing the DNA of the present invention and the microorganism into which the DNA of the present invention has been introduced can be suitably used for the production of LPHase using the LPHase gene.

[0054]

[Sequence listing] SEQ ID NO: 1 Sequence length: 2776 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 features Feature symbol: -35 signal Location: 445..450 Feature determination method: S Feature signature: -10 signal Location: 468..473 Feature determination method: S Feature Symbol: RBS Location: 519..524 Characteristic determination method: S Characteristic symbol: CDS Location: 529..1731 Characteristic determination method: P Characteristic symbol: sig peptide Location: 529 .. 633 methods to determine the characteristics: E SEQ GAGCTCGAAC AGGGCGACCT CTACGAGCAC TTCCGCGACC GGCACGGGGT GCGGGTCAGC 60 GAGGCCGTGC AGTCCGTCGA GCCGACCGTG GTGACCCGTG CGGAGGCGGA GCTGCTGGAC 120 GTGCCCGAGC TGTCGCCGGC CCTCCTCTTC GAGCGCCTCA CCACCGACAC GGCCGGGCGT 180 CCGGTGGAGT ACGTCCACTC CGTCTACCGG GGCGAACCGC TACCGCATCG TCTCCCGGCT 240 CGCCCTGGGG CCCCCGGTCT CGCGTGCGGA CGGGACGGGA GAGGGCCGAG GGGATCACCA 300 TCCGGGCTTC CCGCCCGGCG ACCTCACCTC CCGCGGCCTG GTCAGCTTCT CGGCGCACGG 360 CGTCGTCCAG CACGACTGAT CCACCCCCGG AGCGGCGCGC ACAGGCATGC GGAACCATCG 420 CCTCCTCCAA TTTCGCTTGA ACCGTTGACA GGTCCATACC AGGGCGGGAT TCTCTTTCTG 480 AGAGCGCTCT CACAGCAGTT CCCGGCTACT CGTCGAACGG AGAACGAC ATG CTG CGC 537 Met Leu Arg 1 ACT CTC AGA CGC CGG GTC ACC GCC GTG GCG CTG GGC CTC GCC ACC GCG 585 Thr Leu Arg Arg Arg Val Thr Ala Val Ala Leu Gly Leu Ala Thr Ala 5 10 15 CTG GGC GGC GGC TGG CTC GCC GCC GGG GTC CCG TCC CCG GCG CAC GCC 633 Leu Gly Gly Gly Trp Leu Ala Ala Gly Val Pro Ser Pro Ala His Ala 20 25 30 35 GCG GTC CCG GCC ACC ATC CCG CTG ACC ATC ACC AAC AAC TCG GGC CGC 681 Ala Val Pro Ala Thr Ile Pro Leu Thr Ile Thr Asn Asn Ser Gly Arg 40 45 50 GCC GAA CAG ATC CAC ATC TAC AAC CTC GGC ACC GAG CTG TCG TCC GGC 729 Ala Glu Gln Ile His Ile Tyr Asn Leu Gly Thr Glu Leu Ser Ser Gly 55 60 65 CGG CAG GGC TGG GCC GAC GCG AGC GGC GCC TTC CAC CCC TGG CCC GCG 777 Arg Gln Gly Trp Ala Asp Ala Ser Gly Ala Phe Hi s Pro Trp Pro Ala 70 75 80 GGC GGC AAT CCC CCC ACC CCC GCA CCC GAC GCC TCC ATC CCG GGC CCG 825 Gly Gly Asn Pro Pro Thr Pro Ala Pro Asp Ala Ser Ile Pro Gly Pro 85 90 95 GCC CCC GGC CGG TCC ACC ACC ATC CAG ATC CCC AAG TTC TCC GGC CGC 873 Ala Pro Gly Arg Ser Thr Thr Ile Gln Ile Pro Lys Phe Ser Gly Arg 100 105 110 115 ATC TAC TTC TCC TAC GGC CGC AAG ATG GAG TTC CGG CTC ACC ACC GGC 921 Ile Tyr Phe Ser Tyr Gly Arg Lys Met Glu Phe Arg Leu Thr Thr Gly 120 125 130 GGC CTG GTG CAG CCC GCC GTG CAG AAC CCC ACC GAC CCC AAC CGC GAC 969 Gly Leu Val Gln Pro Ala Val Gln Asn Pro Thr Asp Pro Asn Arg Asp 135 140 145 ATC CTC TTC AAC TGG TCC GAG TAC ACG CTC AAC GAC TCG GGG CTG TGG 1017 Ile Leu Phe Asn Trp Ser Glu Tyr Thr Leu Asn Asp Ser Gly Leu Trp 150 155 160 ATC AAC AGC ACC CAG GTC GAC ATG TTC TCC GCG CCC TAC ACG GTG GGC 1065 Ile Asn Ser Thr Gln Val Asp Met Phe Ser Ala Pro Tyr Thr Val Gly 165 170 175 GTG CGG CGC GGC GAC GGC ACC ACA CTG AGC ACC GGC AAG CTG CGC CCC 1113 Val Arg Arg Gly Asp Gly Thr Thr Leu S er Thr Gly Lys Leu Arg Pro 180 185 190 195 GGC GGC TAC AAC GGC GTG TTC AAC GCC CTC AGG GGA CAG TCC GGC GGC 1161 Gly Gly Tyr Asn Gly Val Phe Asn Ala Leu Arg Gly Gln Ser Gly Gly 200 205 210 TGG GCG AAC CTC ATC CAG ACC CGG TCC GAC GGC ACC GTG CTG CGC GCC 1209 Trp Ala Asn Leu Ile Gln Thr Arg Ser Asp Gly Thr Val Leu Arg Ala 215 220 225 CTG TCC CCG CTC TAC GGG GTG GAG ACC GGC GCC CTC CCG GCG TCG GTC 1257 Leu Ser Pro Leu Tyr Gly Val Glu Thr Gly Ala Leu Pro Ala Ser Val 230 235 240 ATG GAC GAC TAC ATC AAC CGG GTC TGG AAC AAG TAC ACA GGC ACG GAC 1305 Met Asp Asp Tyr Ile Asn Arg Val Trp Asn Lys Tyr Thr Gly Thr Asp 245 250 255 CTG ATC GTC ACG CCC TTC GCC GAC CGT CCC GAC GTC CGG TAC ACC GGC 1353 Leu Ile Val Thr Pro Phe Ala Asp Arg Pro Asp Val Arg Tyr Thr Gly 260 265 270 275 CGG GTC TCG GGC GGC GTG CTG CGG TTC ACC GAC GGC TCC GGC GCC GTG 1401 Arg Val Ser Gly Gly Val Leu Arg Phe Thr Asp Gly Ser Gly Ala Val 280 285 290 GTC ACC ACC TTC CAG AAG CCG GAC GCC TCG TCC GTC TTC GGC TGC CAC 1449 Val Thr Thr Ph e Gln Lys Pro Asp Ala Ser Ser Val Phe Gly Cys His 295 300 305 CGC CTC CTC GAC GCG CCC AAC GAC CAG GTG CGC GGC CCC ATC TCG CGC 1497 Arg Leu Leu Asp Ala Pro Asn Asp Gln Val Arg Gly Pro Ile Ser Arg 310 315 320 ACC CTG TGC GCG GGC TTC AAC CGC ACC ACC CTG CTC GCC AAC CCC CAC 1545 Thr Leu Cys Ala Gly Phe Asn Arg Thr Thr Leu Leu Ala Asn Pro His 325 330 335 CAG CCC GAC CGG AGC GCG GCC GGC TTC TAC CAG GAG CCG GTC ACC AAC 1593 Gln Pro Asp Arg Ser Ala Ala Gly Phe Tyr Gln Glu Pro Val Thr Asn 340 345 350 355 CAC TAC GCG CGG ATC ATC CAC GCG CAC ATG GCC GAC GGG AAG GCG TAC 1641 His Tyr Ala Arg Ile Ile His Ala His Met Ala Asp Gly Lys Ala Tyr 360 365 370 GGC TTC GCC TTC GAC GAC GTC GGC CAC CAC GAG TCG CTC GTC CAC GAC 1689 Gly Phe Ala Phe Asp Asp Val Gly His His Glu Ser Leu Val His Asp 375 380 385 GGC GAC CCG CGC GGG GCG TCC CTG ACG CTC GAC CCG TTC GAC 1731 Gly Asp Pro Arg Gly Ala Ser Leu Thr Leu Asp Pro Phe Asp 390 395 400 TGACCCCGCC CCCCGCGCGCCG TGTCCGGCCG GGCCTCCGGC CGGGCGCGGG GCACCTGACG 1791 CACGTCGG CA CACGTCTCCT GTGTCCCGCG ATCACCGCAC ACTCGCGGGC ACCATAGGGT 1851 GCCACCATGT CTGCCACGTT GAACGCCGGT AAAACGCGCG CCGCCGTGCG CACCTCGGCA 1911 CGCATCTCGG GGGAACTGCT GCTGGTCCTG GTGGCGACCG GGGTGGCCCT CTGGCTGCTG 1971 GGCCGGATGT GGTCGGTGGT CTGGCCCCTC ATAGTCGGCC TGCTGCTCAC CACGCTCACC 2031 TGGCCTCCGA CCCGCTGGCT GCTCCGGCGC GGACTGCGCC CCGCGGTCGC CGCCTCCCTG 2091 GTGACCGTGC TGTTCCTGCT GGTCGGCGCG GGCACCGTGG CGCTCATCGC GGTGCCCGTC 2151 GCGTCGGAGT CCGGTGAACT CGGCGACGGC GTCGTCGAGG GCGTGCAGAA GCTGCGCGAG 2211 TGGGCCGAGG GCCGCCCGCT GAACATCGAC GACGCCCAGA TCACTTGCGC CTTCGACGCC 2271 GCGACCGACC GCATCCAGGA CAGCGCCGGG TCGATCGCCA CCACCGCCGT CACCGGGGTG 2331 AGCACCGTCT TCAGCGGTGT CGTCACCGCC GTCCTGGCGC TCTTCCTGAT GTTCTTCTTC 2391 CTCAAGGACG GGCCGCGTTT CCTGCCGTGG CTGCGCCGCC AGCTCCCCGG CCGGCTCGCC 2451 ACCGACGTGC CGGTCGTCGC CGCGCGCTGC TGGGACACCC TCGGGGCGTT CGTGCGGTCG 2511 CAGGCGCTCG TGGGCCTCAT CGACGCCGTG CTGATCGGCC TCGGCCTGTG GGTCCTCGAC 2571 GTGCCGCTGG TCCTGCCGCT CGCGGTGCTG ACCTTCGTGT CCGCGTTTCG TGCCCATCGT 2631 CGGCGCCCTG TTC GCCGGCC TCGTCGCCGT GCTGATCGCG CTGGTGTCCA ACGGGTTCAT 2691 GGACGCGGTG CTCGTGCTCG GTTCTCATCG TGGTCGTGCA GCAGCTCGAG GGGAACGTGT 2751 TCCAGCCGAT GATCCAGAGC CGCGG 2776

SEQ ID NO: 2 Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Val Pro Ala Thr Ile Pro Leu Thr Ile Thr Asn Asn Ser Gly Arg 1 5 10 15 Ala Glu Gln Ile His Ile Tyr Asn Leu Gly Thr Glu Leu Ser Ser Gly 20 25 30 Arg Gln

SEQ ID NO: 3 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GCKGAGCAGA TCCACATCTA CAACYTGGG 29

[Brief description of drawings]

FIG. 1 shows a restriction enzyme digestion map of the pLE01 insert portion.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Taishi Nishijima 7077-8 Sanadaso, 1 town, Igarashi, Niigata City, Niigata Prefecture (72) Inventor Takeshi Ehara 1-27-1, Osakidai, Sakura City, Chiba Prefecture Dai Nippon Inn Dormitory B-318 (72) Inventor Shuji Nishihashi 2-23-9 Osakidai, Sakura City, Chiba Prefecture (72) Inventor Mikio Oyama 3-13-13-403 Mitsuwadai, Wakaba-ku, Chiba City, Chiba Prefecture

Claims (9)

[Claims] 1. Among the amino acid sequences shown in SEQ ID NO: 1,
Laminaripentaose production which has a sequence represented by amino acid Nos. 36 to 401 and may have substitution, deletion, insertion or transfer of amino acid residues that do not substantially impair the activity of laminaripentaose synthase A DNA containing a nucleotide sequence encoding an enzyme. 2. The DNA according to claim 1, which further comprises a base sequence encoding the amino acid sequence represented by amino acid numbers 1 to 35 among the amino acid sequences shown in SEQ ID NO: 1. 3. The nucleotide sequence shown in SEQ ID NO: 1 having a sequence represented by nucleotide numbers 634 to 1731.
The described DNA. 4. The base sequence shown in SEQ ID NO: 1 having a sequence represented by base numbers 529 to 1731.
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,
Recombinant DNA obtained by ligating NA with a vector capable of autonomous replication in a host cell or a vector capable of integrating into a host cell chromosomal DNA. 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 a laminaripentaose producing enzyme, which comprises culturing the microorganism according to claim 7 or 8 in a suitable medium and collecting the laminaripentaose producing enzyme from the culture.