JPH09173083A - End-beta-n-acetylglucosaminidase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject gene necessary for the production of a polypeptide having an end-β-N-acetylglucosaminidase A activity useful for the analysis of the sugar chain structure of a glucoprotein, the modification of the sugar chain of a composite saccharide, the preparation of neoglycoprotein, etc. SOLUTION: A DNA containing a base sequence coding a polypeptide having an end-β-N-acetylglucosaminidase-A activity, such as an amino acid sequence of the formula. The DNA can be obtained from the genome DNA of Arthrobacter protophormiae AKU 0647 (FERM BP-4948), etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to endo-β-N-
DNA encoding a polypeptide having acetylglucosaminidase A activity, and endo-using the DNA
The present invention relates to a method for producing a polypeptide having β-N-acetylglucosaminidase A activity.

[0002]

2. Description of the Related Art In recent years, various physiological functions of sugar chain portions of molecules called so-called glycoconjugates such as glycoproteins and glycolipids have attracted attention. Glycolytic enzymes have become a very useful research tool for elucidating the structure of sugar chains and their biological activities. Above all, Endo-β-N-acetylglucosaminidase
Acts on the N-linked sugar chain of glycoprotein and is present at the reducing end of di-N-acetylchitobiose (di-N
-acetylchitobiose) GlcNAcβ1-4GlcNAc bonds are cleaved, and N-acetylglucosamine (N-
Acetylglucosamine) is left behind to catalyze the reaction of cleaving sugar chains from proteins, and it has been used for structural or functional analysis of glycoproteins.

Further, some endo-β-N-acetylglucosaminidases have already catalyzed the transfer reaction of sugar chains, and particularly endo-β derived from Arthrobacter protoformiae AKU 0647 strain. -N-acetylglucosaminidase A
[Hereinafter, it may be abbreviated as Endo-A in the present specification] has already been reported to have a very strong transglycosylation activity (JP-A-5-64594). . That is, endo-A acts on the N-linked oligomannose type sugar chain of glycoprotein to cut out the sugar chain,
It efficiently catalyzes the reaction of transferring the sugar chain to the acceptor sugar or glycoconjugate. Therefore, Endo-A is an enzyme that is extremely useful not only for analyzing the sugar chain structure of glycoproteins, but also for modifying glycoconjugate sugar chains and preparing neoglycoproteins. For End-A,
From Arthrobacter Protoformier [Applied and Environmental Microbiolog
y), 55, 3107-3112 (198).
9)] is known.

[0004]

However, in the case of the method of culturing Arthrobacter protoformier to obtain endo-A, protease and other glycosidases are produced at the same time, and these enzymes and endo-A Was difficult to separate and purify. Further, in order to induce endo-A enzyme production, it is necessary to add ovalbumin or a glycopeptide of ovalbumin to the medium at the time of culturing. Therefore, there has been a demand for a method of manufacturing high-purity endo-A at a lower cost.

Conventionally, a method for purifying Endo-A from Arthrobacter protoformier is known [Applied and Environmental Microbiology, Vol. 55, pp. 3107-3112 (198).
9)], but the amino acid sequence and gene structure of endo-A are not known at all, and the method for producing endo-A by genetic engineering is not known.

Therefore, the object of the present invention is to provide a D having a nucleotide sequence encoding a polypeptide having endo-A activity.
To provide NA. Another object of the present invention is to provide the D
It is to provide a recombinant DNA containing NA. Another object of the present invention is to provide an expression vector having the recombinant DNA inserted therein. Another object of the present invention is to provide a transformant transformed with the expression vector. Still another object of the present invention is to provide the DN
It is to provide a method for industrially producing a polypeptide having endo-A activity using A.

[0007]

The present inventors have found that
In order to clarify the amino acid sequence and base sequence of A, as a result of earnest research on the endo-A gene of a bacterium (Arthrobacter protoformier) that produces endo-A, the whole base sequence was finally determined. , Succeeded in clarifying the amino acid sequence of endo-A for the first time. Furthermore, we have succeeded in developing a method for industrially producing End-A using the End-A gene.
The present invention has been completed based on these facts.

That is, the gist of the present invention is (1) End-
An isolated DNA containing a nucleotide sequence encoding a polypeptide having β-N-acetylglucosaminidase A activity, (2) The polypeptide is derived from a strain belonging to the genus Arthrobacter. DN
A, (3) The DNA according to (1) or (2) above, wherein the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, (4)
The DNA according to (1) or (3) above, wherein the base sequence is the DNA sequence set forth in SEQ ID NO: 2.
(5) Endo-β-N-acetylglucosaminidase A containing a part of the amino acid sequence shown in SEQ ID NO: 1.
A DNA encoding a polypeptide having an activity or its functionally equivalent activity, (6) a DNA containing a part of the DNA sequence set forth in SEQ ID NO: 2, and
D encoding a polypeptide having β-N-acetylglucosaminidase A activity or its functionally equivalent activity
NA, (7) D according to any of (1) to (6) above
Endo-β-, which can hybridize with NA
DN encoding a polypeptide having N-acetylglucosaminidase A activity or its functionally equivalent activity
A, (8) One or more amino acid residues are deleted, added, inserted or substituted in the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, and endo-β-N
-A DNA comprising a nucleotide sequence encoding an amino acid sequence of a polypeptide having acetylglucosaminidase A activity or its functionally equivalent activity, (9) including the DNA according to any one of (1) to (8) above (10) The recombinant DNA according to the above (9)
(11) A transformant obtained by transforming the expression vector according to (10) above, which comprises a microorganism, an animal cell, or a plant cell as a host cell, and (12) above (1) To (9) A transformant obtained by introducing the expression vector into which the polynucleotide containing the DNA according to any one of (9) is inserted is cultured, and the polypeptide having endo-β-N-acetylglucosaminidase A activity is obtained from the culture. Is collected,
The present invention relates to a method for producing a polypeptide having endo-β-N-acetylglucosaminidase A activity.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, endo-β-
N-acetylglucosaminidase A is applied
And environmental microbiology, 55, 3107 to 3112 (1989), having the following physicochemical properties. 1. Action GlcNAc of di-N-acetylchitobiose which acts on the N-linked sugar chain of glycoprotein and is present in the reducing end portion thereof
It has the action of cleaving the bond between β1-4GlcNAc. 2. Substrate specificity It acts on oligomannose type sugar chains, glycopeptides and glycoproteins, but does not act on complex type (complex type) sugar chains. 3. Optimum pH and stable pH Optimum pH is pH 5.0-11.0, stable pH is pH
5.0-7.0. 4. Optimum temperature and stable temperature The optimum temperature is 60 ° C, and the stable temperature is stable up to 60 ° C.

Further, the polypeptide having endo-β-N-acetylglucosaminidase A activity means not only a natural type of endo-β-N-acetylglucosaminidase A but also endo-β-N-acetylglucosaminidase A.
As long as it has activity, the present invention also includes a polypeptide in which the amino acid sequence of the natural amino acid sequence is modified by deletion, addition, insertion, substitution, etc. of amino acid residues. Examples of the natural endo-β-N-acetylglucosaminidase A referred to here include those derived from strains belonging to the genus Arthrobacter, but the present invention is not limited thereto and includes bacteria. , Those derived from microbial cells such as yeasts, actinomycetes, filamentous fungi, ascomycetes and basidiomycetes, or those derived from organisms such as plants and animals.

In the present specification, the polypeptide having the functionally equivalent activity is as follows. Naturally occurring proteins have amino acid sequence changes caused by polymorphisms or mutations in the gene encoding the protein, as well as in the amino acid sequence due to modification reactions of the produced protein in vivo and during purification. Include those with mutations such as amino acid deletions, additions, insertions, substitutions, etc., but nonetheless, some have substantially the same physiological and biological activities as proteins without mutations. It has been known. Even if there are structural differences like this,
Those which are recognized to share the main functions are called polypeptides having functionally equivalent activity.

The same is true when artificially introducing the above-mentioned mutation into the amino acid sequence of the protein. In this case, it is possible to prepare a wider variety of mutants, but it is assumed that they have no mutation. As long as they show substantially equivalent physiological activity, these variants are considered to be polypeptides having functionally equivalent activity. For example, the methionine residue present at the N-terminus of a protein expressed in E. coli is
In most cases, methionine aminopeptidase is said to remove it, but depending on the type of protein, both those with and without methionine residue are produced. However, the presence or absence of this methionine residue often does not affect the activity of the protein. Further, it is known that a polypeptide in which a certain cysteine residue in the amino acid sequence of human interleukin 2 (IL-2) is replaced with serine retains the interleukin 2 activity [Science, 224]. Volume 14
31 (1984)].

Further, when a protein is produced by genetic engineering, it is often expressed as a fusion protein. For example, in order to increase the expression level of the target protein, an N-terminal peptide chain derived from another protein is added to the N-terminal of the target protein, or an appropriate peptide chain is added to the N-terminal or C-terminal of the target protein. Is added for expression and a carrier having an affinity for the added peptide chain is used to facilitate purification of the target protein.

The codon (combination of three bases) designating an amino acid on a gene is 1 for each type of amino acid.
~ It is known that there are 6 types each. Therefore, a large number of genes encoding an amino acid sequence can exist, depending on the amino acid sequence. Genes are not always stable in nature,
Mutations in the nucleic acid are not uncommon. In some cases, the mutation that occurs in the gene does not change the encoded amino acid sequence (called a silent mutation), and in this case, it can be said that different genes encoding the same amino acid sequence have occurred. Therefore, even if a gene that encodes a specific amino acid sequence is isolated, it is undeniable that multiple types of genes that encode the same amino acid sequence will be created as the organism containing it is passaged. .

Further, it is not difficult to artificially prepare various kinds of genes encoding the same amino acid sequence by using various genetic engineering techniques. For example,
In genetically engineered protein production, if the codon used in the original gene that encodes the target protein is rarely used in the host being used, the expression level of the protein is low. There is. In such cases, it is possible to achieve high expression of the target protein by artificially converting codons into those commonly used in the host without changing the encoded amino acid sequence. Has been done. Needless to say, it is possible to artificially create many kinds of genes that code for specific amino acid sequences. Therefore, even these artificially prepared different polynucleotides are included in the present invention as long as they encode the amino acid sequences disclosed in the present invention.

Furthermore, a polypeptide obtained by deleting, adding, inserting or substituting one or more amino acid residues in the amino acid sequence of the target protein rarely has the activity functionally equivalent to the target protein. No, but a DN containing a nucleotide sequence encoding such a polypeptide
A, whether natural or artificially made, is also included in the present invention.

In general, a polypeptide having functionally equivalent activity often has homology in the nucleotide sequences encoding it. Therefore, all DNAs that can hybridize with the DNA of the present invention and that encode a polypeptide having endo-A activity are included in the present invention.

In the following, Endo-A derived from Arthrobacter protoformier AKU 0647 strain is taken as an example.
The present invention will be specifically described. This strain is Arthrobacter
It has been designated as protophormiae AKU 0647 and has been deposited as FERM BP-4948 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

First, Applied and Environmental Microbiology, Volume 55, Volume 310
7-3112, (1989) according to the method described in Arthrobacter protoformier AKU 0647.
The strain is cultured, and then Endo-A is isolated and purified from the culture. Next, information on the partial amino acid sequence of the purified endo-A is obtained. To determine the partial amino acid sequence,
For example, purified Endo-A is directly analyzed by an Edman degradation method for amino acid sequence analysis (protein sequencer 47).
6A, manufactured by Applied Biosystems Co., Ltd.) to obtain 10-2 of the N-terminal amino acid sequence of endo-A.
Determine 0 residues. Alternatively, it is obtained by subjecting a highly specific protein hydrolase such as Achromobacter protease I, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) -trypsin to limited hydrolysis to effect Reversed phase HPL
Separate and purify using C. It is effective to perform amino acid sequence analysis on the purified peptide fragment.

The endo-A gene is cloned based on the thus obtained partial amino acid sequence information. For that purpose, generally used PCR method or hybridization method is used. a) A synthetic oligonucleotide is designed as a probe for Southern hybridization based on the information of the partial amino acid sequence. b) Meanwhile, Arthrobacter Protoformier AK
The genomic DNA of U 0647 strain is completely digested with an appropriate restriction enzyme, separated by agarose gel electrophoresis, and then blotted on a nylon membrane according to a conventional method. c) Hybridization between the separated DNA fragment and a synthetic oligonucleotide designed based on the information of the partial amino acid sequence is performed under generally used conditions. For example, the nylon membrane is blocked in a prehybridization solution containing salmon sperm DNA, each synthetic oligonucleotide labeled with ³² P is added, and the mixture is kept warm overnight. After washing the nylon membrane, autoradiography is performed to detect a DNA fragment that hybridizes with the synthetic oligonucleotide probe. A DNA fragment corresponding to the detected band is extracted from the gel and purified.

D) The DNA fragment thus obtained which hybridizes with the synthetic oligonucleotide probe is incorporated into a plasmid vector by a commonly used method. Examples of plasmid vectors include pUC18, pUC19, pU
C119, pTV118N and the like can be preferably used, but the invention is not particularly limited thereto. e) Next, the recombinant plasmid is introduced into a host to transform the host. When Escherichia coli is used as the host, any wild strain or mutant strain can be used as long as it has transformability. The method of introduction is a method usually used, for example, Molecular Cloning Laboratory Manual (Molecular Cloning, A Labor
atory Manual) [T. Maniatis et al., Cold Spring Harbor Laboratory 1
982] The method described on page 250 can be used.

F) Next, a transformant having the desired DNA fragment is selected. For that purpose, the characteristics of the plasmid vector are utilized. For example, in the case of pUC19, ampicillin-resistant colonies on a plate containing ampicillin, or ampicillin, 5-bromo-4-chloro-
3-indolyl-β-D-galactoside (X-Gal) and isopropyl-β-D-thiogalactopyranoside (IPT
On a plate containing G), colonies having the foreign gene introduced therein are selected by selecting colonies that show ampicillin resistance and appear white. g) Next, a colony having a vector containing the target DNA fragment is selected from the above population. The selection method is colony hybridization depending on the type of vector,
Plaque hybridization is used as appropriate. Also,
The PCR method can also be used.

H) If a vector containing the desired DNA fragment can be selected, the desired DNA inserted in this vector
The nucleotide sequence of the fragment is analyzed by a conventional method such as the dideoxy chain terminator method [Proceedings of the
National Academy of Sciences of the USA (Proceedings of the National Academy of Sc
iences of the USA), Vol. 74, p. 5463 (19)
77)]. By comparing the determined base sequence with the N-terminal analysis of Endo-A, partial amino acid sequence, molecular weight, etc., it is determined whether the target Endo-A gene is all or part thereof. From the DNA fragment containing the endo-A gene thus obtained, the structure of the endo-A gene and the entire amino acid sequence of endo-A are determined.

I) If the vector containing the desired DNA fragment does not contain the entire length of the endo-A gene, the obtained DN
Genome DN of Arthrobacter protoformier AKU 0647 strain using a part of A fragment as a probe
In the same manner, the target endo-A gene full length can be obtained by obtaining a deficient portion by a hybridization method or the like from a digested A with another restriction enzyme and then ligating it.

In cloning the Endo-A gene derived from Arthrobacter protoformier AKU 0647, PCR was carried out using a synthetic oligonucleotide primer designed according to a standard method based on the information on the partial amino acid sequence as described below. The reaction was carried out to obtain the target gene, but the target gene was not obtained. However, as a result of earnest studies by the present inventors, when performing PCR, a specific oligonucleotide designed from the internal partial amino acid sequence of endo-A and synthesized was used as a primer, and genomic DNA was used as a template for P.
It was discovered that a part of the target endo-A gene was amplified for the first time by performing CR.

In more detail, first, PCR reaction is carried out using a synthetic oligonucleotide primer designed according to a standard method based on the information of the partial amino acid sequence using the genomic DNA of Arthrobacter protoformier AKU 0647 strain as a template. Then, a fragment of the target gene is obtained. That is, oligonucleotide primer 1 (SEQ ID NO: 6) designed from N-terminal amino acid sequence A-23 (SEQ ID NO: 5), partial amino acid sequence A-46
Oligonucleotide primer 2 (SEQ ID NO: 8) designed from (SEQ ID NO: 7), partial amino acid sequence A-
Oligonucleotide primer 3 (SEQ ID NO: 10) designed from 20 (SEQ ID NO: 9) and oligonucleotide primer 4 (SEQ ID NO: 12) designed from partial amino acid sequence A-12 (SEQ ID NO: 11) are synthesized respectively. . In addition, a BamHI site is added to the 5'end side of primer 1 and an EcoRI site is added to the 5'end side of the other primers to facilitate determination of the base sequence of the amplified product.

PCR is a PCR technology
logy), edited by Erlich HA (Erlich), published by Stockton Press (published in 1989).
For example, Perkin Elmer Cetus Instruments, Gene AmpliAgent Kit (Gene Amp R
eagent Kit). Reaction is 94 ° C: 1 minute, 4
3 cycles of 9 ° C: 1 minute 30 seconds, 72 ° C: 1 minute 30 seconds
Perform 0 cycles. Using the genomic DNA of Arthrobacter protoformier AKU 0647 as a template, the first PCR reaction was performed using a combination of primer 1 and primer 2, and then a part of this reaction solution was used to prepare primer 1
When the reaction solution was analyzed by agarose gel electrophoresis after the second PCR reaction with the combination of DNA and primer 3 or the combination of primer 1 and primer 4, a clear band that seems to be amplified DNA I can't find it.
Further, in a single PCR reaction using the combination of primer 1 and primer 4, a specific band that seems to be amplified DNA is detected by agarose gel electrophoresis. Furthermore, when the base sequence of this amplified DNA fragment is determined by a method commonly used, for example, the dideoxy chain terminator method, a sequence corresponding to the partial amino acid sequence of endo-A is found in the determined sequence in addition to the sequence of the synthetic DNA. A part of the target Endo-A gene can be obtained. Of course, the gene encoding the full-length End-A can be cloned by further performing a hybridization method or the like using the obtained gene as a probe.

The entire nucleotide sequence of the gene encoding endo-A produced by the Arthrobacter protoformier strain AKU0647 obtained as described above is the one described in SEQ ID NO: 2, and is thus deduced. The entire amino acid sequence was determined to be that set forth in SEQ ID NO: 1. It should be noted that there are numerous nucleotide sequences corresponding to the amino acid sequences described in SEQ ID NO: 1 except those described in SEQ ID NO: 2, but all of them are included in the scope of the present invention. The DNA of the present invention is a DNA encoding a polypeptide having an endo-A activity containing a part of the amino acid sequence shown in SEQ ID NO: 1 or a functionally equivalent activity thereof, and is shown in SEQ ID NO: 2. Endo-A activity which is a DNA containing a part of a DNA sequence and which encodes a polypeptide having endo-A activity or its functionally equivalent activity, and which is further capable of hybridizing with these DNAs Alternatively, it also includes a DNA encoding a polypeptide having the functionally equivalent activity.

Genomic DNs obtained from organisms other than Arthrobacter protoformier using the whole or part of the endo-A gene whose whole nucleotide sequence has been elucidated as described above as a probe for hybridization.
From the A or cDNA library, DNA encoding a polypeptide having endo-A activity and having high homology with the endo-A gene can be selected. Hybridization is carried out under generally used conditions, for example, a nylon membrane having a genomic DNA or cDNA library obtained from an organism other than Arthrobacter protoformier immobilized thereon is prepared, and 6xSSC, 0.5%
SDS, 5x Denhardt, 100 μg / ml salmon sperm DN
After blocking the nylon membrane in a prehybridization solution containing A at 65 ° C, each probe labeled with ³² P is added and incubated at 65 ° C overnight. This nylon membrane was washed in 6xSSC at room temperature for 10 minutes, 2xSSC containing 0.1% SDS at room temperature for 10 minutes, 0.2xSSC containing 0.1% SDS at 45 ° C for 30 minutes, and then autoradio Although DNA that hybridizes with the probe can be detected by taking a graph, genes showing various homologies can be obtained by changing conditions such as washing.

On the other hand, from the nucleotide sequence of the gene of the present invention to PC
Primers for R reactions can be designed. By carrying out a PCR reaction using this primer, a gene fragment highly homologous to the gene of the present invention can be detected,
Furthermore, the whole gene can be obtained.

The following method is convenient for producing a polypeptide having endo-A activity using the endo-A gene of the present invention. First, a host is transformed with a vector containing the target Endo-A gene, and then the transformant is cultured under conditions normally used to produce a polypeptide having Endo-A activity. You can In some cases, the polypeptide may be produced in the form of an inclusion body. In addition, microorganisms, animal cells, plant cells and the like can be used as the host.

It is convenient to confirm the expression by, for example, measuring the endo-A activity. For the activity measurement, for example, using a cell extract of recombinant Escherichia coli as an enzyme solution, Applied and Environmental Microbiology, Vol. 55, pp. 3107-3112, (198).
It can be performed by the method described in 9). End of purpose-
If expression of A is observed, for example, if the transformant is Escherichia coli, optimal conditions for endo-A expression such as medium composition, medium pH, culture temperature, use amount of inducer, use time, and culture time are considered. It is possible to efficiently produce End-A by determining

Conventional methods are used to purify Endo-A from the transformant culture. When Endo-A accumulates intracellularly, as in the case where the transformant is E. coli,
After completion of the culture, the transformants are collected by centrifugation, disrupted by sonication or the like, and then centrifuged or the like to obtain a cell-free extract. This can be purified by a general protein purification method such as salting out, ion exchange, gel filtration, hydrophobicity, various chromatography such as affinity, and the like. The expression product may be secreted out of the transformant depending on the transformant used, and in such a case, it may be purified similarly from the culture supernatant.

When the endo-A produced by the transformant is produced in the microbial cells, various enzymes in the microbial cells coexist, but since these are only a trace amount compared to the amount of endo-A, Purification of endo-A is extremely easy. In addition, when Endo-A is secreted outside the cells, medium components and the like coexist, but since these usually contain almost no protein components that hinder the purification of Endo-A, Arthrobacter protoplasts are used. There is an advantage that the complicated separation and purification operation necessary for the purification of Endo-A from the culture of Formier AKU 0647 strain is not required.

When the host is Escherichia coli, the expression product may be formed as an insoluble inclusion body. In this case, after the completion of the culture, the bacterial cells are collected by centrifugation, crushed by ultrasonic treatment or the like, and then the insoluble fraction containing inclusion bodies is collected by centrifugation or the like. After washing the inclusion body, solubilize it with a commonly used protein solubilizer such as urea or guanidine hydrochloride, and perform various chromatography such as ion exchange, gel filtration, hydrophobicity, affinity, etc., if necessary. After purification by
End-A, which is the target of which the activity was retained, by performing a reholding operation using a dialysis method or a dilution method
A polypeptide having activity can be obtained. If necessary, this preparation can be further purified by various kinds of chromatography to obtain a highly pure polypeptide having endo-A activity.

Thus, the present invention provides the primary structure and gene structure of endo-A derived from Arthrobacter protoformier strain AKU 0647. Also,
It enables genetic engineering production of a polypeptide having endo-A activity. By using the genetic engineering production method of the present invention, a highly pure polypeptide having endo-A activity can be obtained at low cost.

[0037]

The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Embodiment 1 Cloning of Endo-A structural gene (1) Extraction and purification of genomic DNA Endo-A producing strain Arthrobacter protoformier AKU0647 (FERM BP-4948) was prepared with yeast extract 0.5%, peptone 0.5%, NaCl 0.5%,
Inoculate 10 ml of medium consisting of pH 7.5 and
After preculturing for 8 hours, 1 ml of the culture solution was inoculated into each of 5 Erlenmeyer flasks containing 100 ml of the same medium, and shake-cultured for 24 hours. After culturing, the culture solution is centrifuged to collect the cells, and the obtained cells are treated with Saline-EDTA solution (0.1
After washing twice with 5M NaCl, 0.1M EDTA, pH 8.0 and suspending in 20 ml of Saline-EDTA solution, lysozyme solution [Saline-TE solution (0.1M NaCl, 10 mM EDTA at a concentration of 20 mg / ml) was added. , 0.1M
Dissolved in Tris-HCl, pH 8.0)], and shaken at 37 ° C. for 10 minutes. Next, 5 ml of 5% SDS solution (dissolved in Saline-TE solution) was added, and the mixture was shaken at 60 ° C for 20 minutes, and then proteinase K was added.
130 μl (final concentration 50 μg / ml) of [proteinase K (10 mg / ml)] was added and incubated at 37 ° C. for 3 hours. After this, the temperature is returned to room temperature, and an equal volume of TE buffer solution (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0) Saturated phenol was added, the mixture was gently stirred, and centrifuged at 8000 rpm for 20 minutes, and then the upper layer was collected (hereinafter, abbreviated as phenol extraction). To the aqueous layer was slowly added twice the amount of cold ethanol to precipitate DNA, which was wound on a glass rod, washed with 70%, 80%, and 90% cold ethanol solutions and air-dried lightly (hereinafter , Abbreviated as ethanol precipitation). 0.1xSSC [20xSSC (3M
NaCI, 0.3M sodium citrate was used after dilution] 18 ml was added to dissolve, 10 ml of 10xSSC and 10 μl of RNase A (10 mg / ml) (final concentration 50 μg / ml) were added, and the mixture was incubated at 37 ° C. It was kept warm for 1 hour. After the reaction, deproteinization was performed and ethanol precipitation was performed. This was dissolved in 2 ml of 0.1 × SSC, dialyzed against TE buffer for 24 hours, extracted with phenol / chloroform, extracted with chloroform, and precipitated with ethanol, and then DNA was recovered by centrifugation to remove TE. It was dissolved in a buffer solution to obtain a genomic DNA solution. The concentration of genomic DNA obtained by this operation was 509 μg / m from its absorbance.
It was calculated as 1. Furthermore, by agarose electrophoresis,
It was confirmed that the size was 24 kb or more.

(2) Determination of partial amino acid sequence of endo-A Applied and Environmental Microbiology, 55, 3107-3112 (1
The endo-A purified by the method described in 989) was directly subjected to the amino acid sequence analysis by the vapor phase Edman degradation method according to the conventional method to determine the N-terminal amino acid sequence A-23 (SEQ ID NO: 5). Furthermore, the enzyme protein was pyridylethylated [1 nmol of endo-A protein was 450 mM, N-
Apply to a desalting column (first desorting column PC3.2 / 10, manufactured by Pharmacia) equilibrated with ethylmorpholine / formate buffer pH 8.5 salt,
Elution was performed with the same buffer. The eluate was collected in a glass vial and concentrated to dryness. 10 μl of pyridine, 2 μl of 4-vinylpyridine, 2 μl of tri-N-butylphosphine, and 10 μl of water were placed in a glass test tube having a size larger than that of the other. A glass vial containing the sample was placed in the glass test tube, the glass test tube was sealed, and then reacted at 100 ° C. for 5 minutes. After completion of the reaction, the glass vial was taken out and thoroughly dried. The pyridylethylated product thus obtained is used for digestion with lysyl endopeptidase. ], And digested with lysyl endopeptidase [40 mM 10 mM Tris-HCl buffer (pH 7.5) containing 4 M urea in a glass vial.
μl, 10 mM Tris-HCl buffer (pH 7.5) 5
0 μl, 10 μl of 0.1 M calcium chloride, and 2 pmol of lysyl endopeptidase were added, and the reaction was carried out at 37 ° C. overnight. ], The peptide fragment is
PLC (Smart System, Pharmacia, column: mRPC C2 / C18, SC2.1 / 10, flow rate: 1 ml / min, eluent A: 0.1% trifluoroacetic acid solution, eluent B: 0.1% tri Acetonitrile containing fluoroacetic acid, elution: Eluent B at sample application
To 0%, the eluent B ratio to 10% at the end of sample application, and the eluent B ratio to 6% in 85 minutes.
Raise it to 0%. ], And separated and purified. Amino acid sequence analysis was performed on each peptide fraction, and the partial amino acid sequence A
-46 (SEQ ID NO: 7), A-20 (SEQ ID NO: 9),
A-12 (SEQ ID NO: 11) was determined.

(3) Preparation of a gene library of Arthrobacter protoformier AKU 0647 strain Genomic DNA (509 μg / ml) 1 prepared in (1)
After adding 8 units of restriction enzyme Sau3AI (manufactured by Takara Shuzo Co., Ltd.) to 0 μl to make a total volume of 50 μl, the mixture was incubated at 37 ° C. for 20, 3
Digested for 0, 40, 60 seconds. 100 each time
Add 15 μl of mM EDTA (pH 8.0), and add 60
The reaction was stopped by heating at 0 ° C for 20 minutes. This genomic DNA is approximately 24 kb as the reaction time elapses.
It was confirmed by agarose gel electrophoresis that partial digestion from about 1 kb to about 1 kb was carried out.

The above partial digestion solutions were combined and subjected to agarose gel electrophoresis to obtain a DNA having a size of about 4 to 23 kb.
The fragment was cut out, the DNA was recovered using EASY TRAP (manufactured by Takara Shuzo), ethanol precipitated, and dissolved in 10 μl of TE buffer. λEMBL3
Arm [Stratagene] and the obtained DNA fragment of about 4 to 23 kb were ligated with a ligation kit (Takara Shuzo) at the composition shown in Table 1 at 16 ° C.
The reaction was carried out for 0 minutes to prepare a recombinant vector.

[0042]

[Table 1]

The reaction solution was precipitated with ethanol and dissolved in 4 μl of TE buffer, which was used as a ligation DNA solution. Ligation DNA solution with Gigapack II
Gold Packaging Extract (Gigapack
II Gold packaging Extract, manufactured by Stratagene) was used for in vitro packaging. E. coli P2392 (E. coli P2392) suspension [10mM Mg]
TB medium containing SO ₄ and 0.2% maltose (NaCl
0.5%, peptone 1.0%, pH 7.4) 50m
After culturing at 28 ° C for 10 hours at 28 ° C, cells were collected, and the obtained cells were suspended in 10 mM MgSO _{4 so} that the absorbance (600 nm) was 0.5. ] 600 μl was added and the mixture was incubated at 37 ° C. for 15 minutes to infect the phage. Next, top agar [NZ
Y medium (NaCl 0.5%, yeast extract 0.5%,
_{MgSO 4 · 7H 2 O 0.2%} , NZ amine 1.0
%) 0.7% agarose] 3 ml of the above-mentioned solution infected with the phage solution was added, and mixed quickly,
The mixture was poured onto bottom agar (NZY medium, 3% agar) kept at 7 ° C, and incubated at 37 ° C for 8 hours. After that, the plaque on the plate is 0.5-1.0 m
After confirming that the size became m, it was stored in a refrigerator (4 ° C.). The plaques that appeared in this way were used as a gene library. When 6 inserted plaques were arbitrarily collected and the inserted DNA fragment was confirmed, an inserted DNA fragment of about 10 kb was confirmed in 4 out of 6 clones. Further, the same operation was performed to prepare a gene library of about 10,000.

(4) Cloning of DNA fragment containing endo-A gene Primer 1 (SEQ ID NO: 5) designed from the N-terminal amino acid sequence A-23 (SEQ ID NO: 5) determined in (2)
6), primer 2 (SEQ ID NO: 8) designed from partial amino acid sequence A-46 (SEQ ID NO: 7), primer 3 (SEQ ID NO: 10) designed from partial amino acid sequence A-20 (SEQ ID NO: 9) , Partial amino acid sequence A-
Primer 4 designed from 12 (SEQ ID NO: 11)
(SEQ ID NO: 12) was synthesized. A BamHI site was added to the 5'end of primer 1 and an EcoRI site was added to the 5'end of the other primers to facilitate determination of the base sequence of the amplified product. Using these primers, the genomic DNA of Arthrobacter protoformier AKU0647 strain was used as a template, and P
CR was performed. PCR was performed using the Gene Amp Reagent Kit (Perkin Elmer Cetus Instruments, Inc.) according to the method described in PCR Technology, edited by Erlich HA, Stockton Press, Inc. (published in 1989). It was Reaction: 94 ° C: 1 minute, 49 ° C: 1 minute 30 seconds, 72
C .: 1 minute and 30 seconds were cycled 30 times. As a result of this PCR reaction, one P
It was specifically amplified by CR reaction. A DNA fragment (about 1.2 kb) amplified by the combination of primer 1 and primer 4 was cut out, and the DNA was recovered using an Easy Trap (Takara Shuzo). Use this DNA fragment with the restriction enzyme EcoRI
Digested with BamHI (both manufactured by Takara Shuzo) and plasmid pBlue
The EcoRI and BamHI sites of script (Stratagene) were linked using a ligation kit (Takara Shuzo).

Furthermore, the amplified DNA fragment (about 1.2 k
To create the restriction map of b), the restriction enzyme Hind III
When digested with (Takara Shuzo), it was found that there was one HindIII site near the center of this DNA fragment (Fig. 1). Next, the nucleotide sequences of the amplified DNA fragment from the Bam HI site side and the Eco RI site side were determined by the dideoxy chain terminator method. Furthermore, the nucleotide sequences of both sides of the Hind III site, which exists only at one site in the center of this amplified DNA fragment, were determined by the dideoxy chain terminator method. Determined B
am HI site side nucleotide sequence SEQ ID NO: 13
The nucleotide sequence of Eco RI site is shown in SEQ ID NO:
14, the nucleotide sequence on the Bam HI site side of the Hind III site is shown in SEQ ID NO: 15 in the sequence listing, and the nucleotide sequence on the Eco RI site side of the Hind III site is shown in SEQ ID NO: 16 of the sequence listing. As a result, in addition to the sequences of primer 1 and primer 4 in the determined sequence, a sequence corresponding to the partial amino acid sequence of endo-A was found, and a part of the target endo-A gene was successfully obtained. .

(5) Cloning of Endo-A Gene Next, the gene library prepared in (3) was screened using the DNA fragment (about 1.2 kb) obtained in (4) as a probe. First, amplified DNA
Fragment (about 1.2 kb) 480 μg was transferred to ECL random prime labeling system (ECL random prime l
Labeling was carried out using an abelling system manufactured by Amersham Co., Ltd. according to the protocol of the system. Using the labeled DNA fragment as a probe, plaque hybridization was carried out with the gene library prepared in (3). Plaque hybridization is ECL
Random Prime Labeling System Described Method and Molecular Cloning Laboratory
Manual, Second Edition, Mantis et al., Chapter 2, Chapter 10
8 to 122, Cold Spring Harbor Laboratory Co., Ltd., 1989. In other words, Amersham Nylon membrane [Product name Hybond N + (Hy
bond-N +)] was cut into a plate shape, and a notch of about 1 mm was placed on the plate of the gene library prepared in (3) so that the orientation of the nylon film could be seen. It was left as it was for 5 minutes, then the nylon membrane was slowly peeled off from the plate, placed on a filter paper moistened with 0.5 M NaOH with the surface in contact with the plate facing upward, and left for 5 minutes. Next, this nylon membrane was transferred onto a dry filter paper to remove water. FUNA-UV-LINKE
DNA was immobilized on a nylon membrane with RFS-800 (Funakoshi). In this way, a plaque hybridization filter was prepared.

Prepare the prepared filter with 5xSSC (1xS
SC is 8.77 g of NaCl, 4.41 g of sodium citrate dissolved in 1 l of water), 0.5% SD
Prehybridization was performed at 60 ° C. for 1 hour in a solution containing S, 100 μg / ml salmon sperm DNA, 5 × Denhaldt's [containing bovine serum albumin, polyvinylpyrrolidone, and ficoll at 0.1% concentrations respectively]. Then, the DNA fragment labeled above is added as a labeled probe to a concentration of 5 ng / ml (the labeled probe is previously heated in boiling water for 5 minutes and then rapidly cooled in ice), 60 ° C., Hybridization was performed for 8 hours and 50 minutes.

Next, 6 in 1 × SSC, 0.1% SDS
15 minutes at 0 ° C. in 0.5 × SSC, 0.1% SDS,
60 ° C, 15 minutes, buffer A (0.1M Tris
-HCl, pH 7.5, 0.6 M NaCl), 25
Washed at ℃ for 1 minute. Next, in order to further lower the hybridization background, the liquid block attached to the system was added to buffer A.
It was washed for 30 minutes at 25 ° C. in a solution diluted 20 times with.

Next, 1/1000 volume of the buffer A (containing 0.5% BSA) HRP attached to the system.
An antibody reaction was carried out at 25 ° C. for 30 minutes in a solution containing a labeled anti-fluorescein antibody (anti-fluorescein-HRP). Then, in buffer A, 0.5% BSA, 25 ° C. for 30 minutes, in buffer A, 0.1% Tween 20, 25
It was washed at 10 ° C. for 10 minutes, and this operation was repeated 3 times.

Next, a detection reaction was carried out for 1 minute at 25 ° C. in a solution in which equal amounts of the detection reagents 1 and 2 attached to the same system were combined, and then this filter was used for 20 minutes in the same manner as in autoradiography. Exposed. As a result, 1
Three positive plaques were obtained. Each of these 13 plaques was treated with SM buffer (0.58% NaC).
_{l, 0.2% MgSO 4 · 7H} 2 O, 50mM Tri
s-HCl, pH 7.5, 0.01% gelatin) 500
After suspending in μl, the mixture was left at room temperature for 1 hour. Then, the mixture was centrifuged and the supernatant was recovered as a phage solution, and one drop of chloroform was added for storage and stored at 4 ° C.

Phage DNA was recovered from the phage solution thus recovered, and PCR was performed using the phage DNA as a template and the primers 1 (SEQ ID NO: 6) and 4 (SEQ ID NO: 12) to carry out PCR (4). As a result of carrying out under the conditions described in 1., about 1.2 kb DNA fragment expected in 2 out of 13 DNA clones was confirmed by agarose electrophoresis.

In order to unify the obtained two phage DNAs, 1, 5, and 10 μl each of the phage solution prepared above corresponding to this phage DNA was added to Escherichia coli P.
2392 suspension [10 mM MgSO4, TB medium containing 0.2% maltose (NaCl 0.5%, peptone 1.0%, pH 7.4) 50 ml, cultured at 28 ° C for 10 hours, and then harvested. The obtained bacterial cells are treated with 10 mM Mg
A solution suspended in SO _{4 so} that the absorbance (600 nm) becomes 0.5. ] 600 μl was added and the phage solution was infected by incubating at 37 ° C. for 15 minutes.

Next, top agar [N
ZY medium (NaCl 0.5%, yeast extract 0.5
_{%, MgSO 4 · 7H 2 O} 0.2%, NZ amine
1.0%) 0.7% agarose] 3 ml of the above solution infected with the phage solution was added and mixed quickly, and bottom agar (NZY medium,
3% agar) and incubated at 37 ° C. for 8 hours.

From the plates on which plaque appeared, one plate having a single plaque was selected and subjected to plaque hybridization. The conditions are the same as above. As a result, two plates were selected from each of the obtained positive plaques, a phage solution was prepared in the same manner, and a phage DNA was prepared. Using the obtained phage DNA as a template, PCR was similarly performed using primer 1 (SEQ ID NO: 6) and primer 4 (SEQ ID NO: 12) under the conditions described in (4), and 3 out of 4 The expected 1.2 kb DNA fragment in the phage DNA of E. coli was confirmed by agarose electrophoresis.

In order to confirm whether or not the obtained phage DNAs are the same, restriction enzymes Bam HI and Hind III are used.
Upon digestion with (Takara Shuzo Co., Ltd.), two electrophoretic patterns showed the same electrophoretic pattern, and the other one showed a pattern different from this electrophoretic pattern. Thus, we succeeded in cloning a part of the target Endo-A gene. Obtained phage DNA 1 and phage DNA
Of the two types of 10, the subsequent experiments were carried out on the phage DNA1 because of its easy handling.

(6) Subcloning of Endo-A Gene The DNA clone 1 obtained in (5) was cloned into the restriction enzymes Cla I and Hi.
Digested with nd III, Pst I, and Sal I (all manufactured by Takara Shuzo),
DNA subjected to agarose electrophoresis and labeled with (5)
Using the fragment (about 1.2 kb) as a probe, Molecular Cloning Laboratory Manual, 2nd
Hybridization was carried out at 60 ° C. for 12 hours according to the method described in ed., Manitis et al., Ed., Chapter 9, pages 31-58, Cold Spring Harbor Laboratory, 1989.

As a result, the DNA fragment of about 3 kb obtained by digesting with the restriction enzyme Cla I was the DNA labeled with (5).
It hybridized with the fragment (about 1.2 kb). This hybridized DNA fragment of about 3 kb was recovered and pBlues
It was ligated to the Cla I site of cripte SK (-) (manufactured by Stratagene). This plasmid was named Cla I-3kb. A restriction enzyme map of the Cla I-3 kb insert is shown in FIG. When the nucleotide sequence of the insert fragment of this plasmid was determined by the dideoxy chain terminator method, the sequences of N-terminal primer 1 and primer 4 were found in the determined sequence. Only a portion of about 0.3 kb was included.

To obtain the C-terminal side, Cla I-3kb
The Hind III-Cla I fragment (about 0.9 kb), which is the most C-terminal side of the inserted fragment, was labeled by the same method as described in (5), and this was used as a probe to obtain the DNA obtained in (5). Clone 1 is a restriction enzyme Hind III, Kpn I, Pst I, Pvu I
I, then Hind III-Kpn I, Hind III-Pst I, Hind III-
Digest with Pvu II, perform agarose gel electrophoresis, Molecular Cloning Laboratory Manual,
Hybridization was carried out at 60 ° C. for 12 hours according to the method described in Second Edition, Manitish et al., Chapter 9, pp. 31-58, Cold Spring Harbor Laboratory, 1989.

As a result, an approximately 2.5 kb DNA fragment upon digestion with Hind III-Pst I hybridized with this probe. This hybridized DNA of about 2.5 kb
The fragment was recovered and ligated to the Hind III / Pst I site of pBluescripte SK (-) (manufactured by Stratagene).
This plasmid was named Hind III / Pst I-2.5kb.
The restriction enzyme map of the HindIII / PstI-2.5 kb insert is shown in FIG. When the nucleotide sequence of the insert fragment of this plasmid was determined by the dideoxy chain terminator method, it was found that the Hind II fragment of the insert fragment in Cla I-3kb was found.
It contains the I-Cla I fragment (about 0.9 kb) and further contains C
The termination codon on the terminal side was recognized.

By combining the plasmid Hind III / Pst I-2.5 kb with the plasmid Cla I-3 kb, the full length of the endo-A gene can be known. An example of the base sequence of the open reading frame (ORF) of endo-A is shown in SEQ ID NO: 4 in the sequence listing, and the amino acid sequence encoded by this base sequence is shown in SEQ ID NO: 3 in the sequence listing. In addition, from the knowledge of the N-terminal amino acid sequence A-23 (SEQ ID NO: 5) of endo-A obtained in (2), an example of a base sequence encoding endo-A is shown in SEQ ID NO: 2 in the sequence listing, The amino acid sequence encoded by this base sequence is shown in SEQ ID NO: 1 in the sequence listing.

Embodiment 2 FIG. Construction of endo-A expression plasmid (1) Construction of plasmid containing the full-length endo-A gene The Hind III-Cla I fragment was removed from the plasmid Hind III / Pst I-2.5 kb and the insert fragment in the plasmid Cla I-3 kb was removed. Inserted. As a result, a Cla I-Pst I plasmid containing a gene encoding the entire length of endo-A was prepared. The plasmid containing the full length of the endo-A gene thus obtained was
It was named pEACP. The E. coli XL1-Blue strain into which pEACP was introduced is designated as Escherichia coli XL1-Blue / pEACP. Escherichia coli XL1-Blue strain introduced with pEACP was Escherich
Designated as ia coli XL1-Blue / pEACP, it has been deposited as FERM BP-5581 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(2) Measurement of endo-A activity 100 μg of Escherichia coli XL1-Blue / pEACP
In 5 ml of 2xTY medium containing 1 / ml of ampicillin,
It was cultured at 37 ° C. for about 10 hours. A part of this culture solution was centrifuged, and the supernatant was used as a crude enzyme solution and applied and
Environmental Microbiology, 55th
Enzyme activity was measured by the method described in Vol. 3, pp. 3107-3112 (1989). That is, as a result of reacting with the composition shown in Table 2 at 37 ° C. for 1 hour, about 9 milliunits /
ml endo-A activity was observed.

[0063]

[Table 2]

(3) Western Blotting of Endo-A Endo-A in the crude enzyme solution prepared in (2) above was derived from Arthrobacter protoformier AKU 0647 strain by Escherichia coli XL1-Blue / pEACP. -To see if it is the same as A,
Molecular Cloning Laboratory Manual, 2nd Edition, Manitis et al., Chapter 18, Chapters 60-7
Page 4, Cold Spring Harbor Laboratory, 1
Western blotting was performed according to the method described in 989. The antibody of Endo-A used was Applied and Environmental Microbiology, 55, 3107-3112 (1989).
According to the method described in Molecular Cloning Laboratory Manual, 2nd Edition, Manitish et al., Chapter 18, pp. 3-17, Cold Spring Harbor Laboratory, 1989, using Endo-A purified by the method described in 1. Prepared. The result is shown in FIG.
In FIG. 3, lane 1 shows about 15 endo-A prepared from Arthrobacter protoformier AKU0647 strain.
lane is used, and lane 2 is Escherichia coli XL1
-By Blue / pEACP, a protein amount of about 2 µg of the crude enzyme solution prepared in (2) above was used. As can be seen from FIG. 3, Endo-A of Escherichia coli XL1-Blue / pEACP is Arthrobacter protoformier AK.
It was confirmed that it was the same as End-A derived from U 0647 strain.

[0065]

INDUSTRIAL APPLICABILITY According to the present invention, the amino acid sequence and the base sequence of endo-A are clarified for the first time, which provides an industrially advantageous method for genetically producing a polypeptide having endo-A activity.

[0066]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 621 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence: Ser Thr Tyr Asn Gly Pro Leu Ser Ser His Trp Phe Pro Glu Glu 1 5 10 15 Leu Ala Gln Trp Glu Pro Asp Ser Asp Pro Asp Ala Pro Phe Asn 20 25 30 Arg Ser His Val Pro Leu Glu Pro Gly Arg Val Ala Asn Arg Val 35 40 45 Asn Ala Asn Ala Asp Lys Asp Ala His Leu Val Ser Leu Ser Ala 50 55 60 Leu Asn Arg His Thr Ser Gly Val Pro Ser Gln Gly Ala Pro Val 65 70 75 Phe Tyr Glu Asn Thr Phe Ser Tyr Trp His Tyr Thr Asp Leu Met 80 85 90 Val Tyr Trp Ala Gly Ser Ala Gly Glu Gly Ile Ile Val Pro Pro 95 100 105 Ser Ala Asp Val Ile Asp Ala Ser His Arg Asn Gly Val Pro Ile 110 115 120 Leu Gly Asn Val Phe Phe Pro Pro Thr Val Tyr Gly Gly Gln Leu 125 130 135 Glu Trp Leu Glu Gln Met Leu Glu Gln Glu Glu Asp Gly Ser Phe 140 145 150 Pro Leu Ala Asp Lys Leu Leu Glu Val Ala Asp Tyr Tyr Gly Phe 155 160 165 Asp Gly Trp Phe Ile Asn Gln Glu Thr Glu Gly Ala Asp Glu Gly 170 175180 Thr Ala Glu Ala Met Gln Ala Phe Leu Val Tyr Leu Gln Glu Gln 185 190 195 Lys Pro Glu Gly Met His Ile Met Trp Tyr Asp Ser Met Ile Asp 200 205 210 Thr Gly Ala Ile Ala Trp Gln Asn His Leu Thr Asp Arg Asn Lys 215 220 225 Met Tyr Leu Gln Asn Gly Ser Thr Arg Val Ala Asp Ser Met Phe 230 235 240 Leu Asn Phe Trp Trp Arg Asp Gln Arg Gln Ser Asn Glu Leu Ala 245 250 255 Gln Ala Leu Gly Arg Ser Pro Tyr Asp Leu Tyr Ala Gly Val Asp 260 265 270 Val Glu Ala Arg Gly Thr Ser Thr Pro Val Gln Trp Glu Gly Leu 275 280 285 Phe Pro Glu Gly Glu Lys Ala His Thr Ser Leu Gly Leu Tyr Arg 290 295 300 Pro Asp Trp Ala Phe Gln Ser Ser Glu Thr Met Glu Ala Phe Tyr 305 310 315 Glu Lys Glu Leu Gln Phe Trp Val Gly Ser Thr Gly Asn Pro Ala 320 325 330 Glu Thr Asp Gly Gln Ser Asn Trp Pro Gly Met Ala His Trp Phe 335 340 345 Pro Ala Lys Ser Thr Ala Thr Ser Val Pro Phe Val Thr His Phe 350 355 360 Asn Thr Gly Ser Gly Ala Gln Phe Ser Ala Glu Gly Lys Thr Val 365 370 375 Ser Glu Gln Glu Trp Asn Asn Arg Ser Leu Gln Asp Val Leu Pro380 385 390 Thr Trp Arg Trp Ile Gln His Gly Gly Asp Leu Glu Ala Thr Phe 395 400 405 Ser Trp Glu Glu Ala Phe Glu Gly Gly Ser Ser Leu Gln Trp His 410 415 420 Gly Ser Leu Ala Glu Gly Glu His Ala Gln Ile Glu Leu Tyr Gln 425 430 435 Thr Glu Leu Pro Ile Ser Glu Gly Thr Ser Leu Thr Trp Thr Phe 440 445 450 Lys Ser Glu His Gly Asn Asp Leu Asn Val Gly Phe Arg Leu Asp 455 460 465 Gly Glu Glu Asp Phe Arg Tyr Val Glu Gly Glu Gln Arg Glu Ser 470 475 480 Ile Asn Gly Trp Thr Gln Trp Thr Leu Pro Leu Asp Ala Phe Ala 485 490 495 Gly Gln Thr Ile Thr Gly Leu Ala Phe Ala Ala Glu Gly Asn Glu 500 505 510 Thr Gly Leu Ala Glu Phe Tyr Ile Gly Gln Leu Ala Val Gly Ala 515 520 525 Asp Ser Glu Lys Pro Ala Ala Pro Asn Val Asn Val Arg Gln Tyr 530 535 540 540 Asp Pro Asp Pro Ser Gly Ile Gln Leu Val Trp Glu Lys Gln Ser 545 550 555 Asn Val His His Tyr Arg Val Tyr Lys Glu Thr Lys His Gly Lys 560 565 570 Glu Leu Ile Gly Thr Ser Ala Gly Asp Arg Ile Tyr Leu Glu Gly 575 580 585 Leu Val Glu Glu Ser Lys Gln Asn Asp Val Arg Leu HisIle Glu 590 595 600 Ala Leu Ser Glu Thr Phe Val Pro Ser Asp Ala Arg Met Ile Asp 605 610 615 Ile Lys Ser Gly Ser Phe 620

SEQ ID NO: 2 Sequence Length: 1863 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Sequence: TCTACGTACA ACGGCCCGCT GTCCTCCCAT TGGTTTCCAG AGGAACTTGC CCAATGGGAA 60 CCAGACTGTG ATCCAGACGC ACCCTTTAAC AGCCGCAGGA ACCAGGCCGC 120 GTTGCGAATA GGGTAAATGC TAATGCAGAC AAGGACGCAC ACCTTGTTTC GTTGTCCGCG 180 CTAAACAGGC ATACATCAGG TGTTCCATCG CAAGGAGCGC CAGTTTTCTA TGAAAATACG 240 TTCAGCTATT GGCATTATAC AGATTTGATG GTTTATTGGG CTGGTTCAGC TGGCGAAGGC 300 ATTATCGTTC CGCCAAGTGC CGATGTCATT GATGCATCGC ACCGAAATGG GGTGCCGATT 360 TTAGGAAATG TGTTCTTCCC GCCGACGGTT TATGGAGGGC AGCTAGAGTG GCTAGAACAA 420 ATGTTAGAGC AAGAGGAGGA CGGTTCATTC CCCCTTGCTG ACAAATTGCT AGAAGTCGCA 480 GACTATTATG GGTTTGACGG CTGGTTTATT AACCAAGAAA CAGAAGGGGC AGACGAAGGA 540 ACAGCCGAAG CCATGCAAGC TTTTCTCGTT TATTTGCAGG AACAAAAAAGCC AGAAGGCATG 600 CACATCATGT GGTATGACTC GATGATTGAT ACAGGGGCGA TCGCCTGGCA AAACCATTTA 660 ACGGATCGAA ATAAAATGTA CTTGCAAAAT GGCTCGACCC GCGTCGCTGA CAGCAT GTTT 720 TTGAACTTTT GGTGGCGTGA CCAGCGCCAA TCGAACGAAT TGGCACAAGC ACTTGGCAGG 780 TCTCCGTATG ACCTCTATGC CGGAGTGGAT GTGGAAGCAC GAGGGACAAG TACCCCTGTT 840 CAGTGGGAAG GCCTGTTTCC TGAAGGAGAA AAGGCGCATA CATCACTCGG GTTATACCGT 900 CCAGATTGGG CATTTCAGTC AAGTGAAACA ATGGAAGCGT TTTATGAAAA AGAACTACAA 960 TTTTGGGTTG GCTCGACAGG AAATCCAGCC GAAACAGACG GCCAGTCAAA TTGGCCTGGC 1020 ATGGCGCACT GGTTTCCCGC GAAAAGCACC GCTACTTCGG TACCCTTTGT GACTCACTTT 1080 AATACGGGCA GCGGCGCTCA GTTTTCGGCA GAAGGCAAAA CTGTGTCGGA ACAGGAATGG 1140 AATAACCGCA GCCTTCAAGA TGTGCTGCCG ACATGGCGCT GGATTCAGCA TGGCGGCGAT 1200 TTAGAGGCAA CATTTTCTTG GGAAGAAGCG TTTGAAGGGG GAAGCTCGTT ACAATGGCAT 1260 GGCTCATTAG CGGAAGGAGA ACACGCCCAA ATCGAGCTCT ATCAAACAGA GTTGCCGATA 1320 AGCGAAGGCA CTTCGCTAAC GTGGACATTT AAAAGCGAGC ACGGCAACGA TTTAAATGTG 1380 GGCTTCCGTT TAGATGGGGA AGAGGACTTC CGTTATGTGG AAGGAGAACA GCGTGAATCG 1440 ATAAATGGTT GGACGCAGTG GACGTTGCCG CTGGATGCGT TTGCTGGTCA GACGATAACA 1500 GGGCTGGCAT TTGCAGCGGA AGGGAATGAG ACTGGGCTGG CAGAATTCTA TATTGGACAA 1560 C TGGCCGTAG GTGCTGATAG CGAAAAGCCT GCCGCTCCAA ACGTGAACGT ACGCCAGTAC 1620 GACCCAGACC CGAGTGGCAT TCAGCTCGTA TGGGAAAAAC AAAGCAACGT CCACCATTAC 1680 CGCGTTTATA AAGAAACAAA GCACGGCAAA GAGCTAATTG GCACATCTGC TGGAGATCGA 1740 ATTTACCTAG AAGGCCTAGT CGAGGAAAGC AAACAAAACG ACGTGCGTCT GCATATAGAA 1800 GCACTAAGTG AAACATTTGT GCCAAGTGAT GCTCGCATGA TCGACATAAA AAGCGGCTCG 1860 TTT 1863

SEQ ID NO: 3 Sequence length: 645 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence: Leu Arg Lys Ala Phe Leu Val Gly Leu Val Cys Thr Ala Cys Val 1 5 10 15 Leu Leu His Asp Asp Pro Val Ala Ala Ser Thr Tyr Asn Gly Pro 20 25 30 Leu Ser Ser His Trp Phe Pro Glu Glu Leu Ala Gln Trp Glu Pro 35 40 45 Asp Ser Asp Pro Asp Ala Pro Phe Asn Arg Ser His Val Pro Leu 50 55 60 Glu Pro Gly Arg Val Ala Asn Arg Val Asn Ala Asn Ala Asp Lys 65 70 75 Asp Ala His Leu Val Ser Leu Ser Ala Leu Asn Arg His Thr Ser 80 85 90 Gly Val Pro Ser Gln Gly Ala Pro Val Phe Tyr Glu Asn Thr Phe 95 100 105 Ser Tyr Trp His Tyr Thr Asp Leu Met Val Tyr Trp Ala Gly Ser 110 115 120 Ala Gly Glu Gly Ile Ile Val Pro Pro Ser Ala Asp Val Ile Asp 125 130 135 Ala Ser His Arg Asn Gly Val Pro Ile Leu Gly Asn Val Phe Phe 140 145 150 Pro Pro Thr Val Tyr Gly Gly Gln Leu Glu Trp Leu Glu Gln Met 155 160 165 Leu Glu Gln Glu Glu Asp Gly Ser Phe Pro Leu Ala As p Lys Leu 170 175 180 Leu Glu Val Ala Asp Tyr Tyr Gly Phe Asp Gly Trp Phe Ile Asn 185 190 195 Gln Glu Thr Glu Gly Ala Asp Glu Gly Thr Ala Glu Ala Met Gln 200 205 210 Ala Phe Leu Val Tyr Leu Gln Glu Gln Lys Pro Glu Gly Met His 215 220 225 Ile Met Trp Tyr Asp Ser Met Ile Asp Thr Gly Ala Ile Ala Trp 230 235 240 Gln Asn His Leu Thr Asp Arg Asn Lys Met Tyr Leu Gln Asn Gly 245 250 255 Ser Thr Arg Val Ala Asp Ser Met Phe Leu Asn Phe Trp Trp Arg 260 265 270 Asp Gln Arg Gln Ser Asn Glu Leu Ala Gln Ala Leu Gly Arg Ser 275 280 285 Pro Tyr Asp Leu Tyr Ala Gly Val Asp Val Glu Ala Arg Gly Thr 290 295 300 Ser Thr Pro Val Gln Trp Glu Gly Leu Phe Pro Glu Gly Glu Lys 305 310 315 Ala His Thr Ser Leu Gly Leu Tyr Arg Pro Asp Trp Ala Phe Gln 320 325 330 Ser Ser Glu Thr Met Glu Ala Phe Tyr Glu Lys Glu Leu Gln Phe 335 340 345 Trp Val Gly Ser Thr Gly Asn Pro Ala Glu Thr Asp Gly Gln Ser 350 355 360 Asn Trp Pro Gly Met Ala His Trp Phe Pro Ala Lys Ser Thr Ala 365 370 375 Thr Ser Val Pro Phe Val Thr His Phe Asn Th r Gly Ser Gly Ala 380 385 390 Gln Phe Ser Ala Glu Gly Lys Thr Val Ser Glu Gln Glu Trp Asn 395 400 405 Asn Arg Ser Leu Gln Asp Val Leu Pro Thr Trp Arg Trp Ile Gln 410 415 420 His Gly Gly Asp Leu Glu Ala Thr Phe Ser Trp Glu Glu Ala Phe 425 430 435 Glu Gly Gly Ser Ser Leu Gln Trp His Gly Ser Leu Ala Glu Gly 440 445 450 Glu His Ala Gln Ile Glu Leu Tyr Gln Thr Glu Leu Pro Ile Ser 455 460 465 Glu Gly Thr Ser Leu Thr Trp Thr Phe Lys Ser Glu His Gly Asn 470 475 480 Asp Leu Asn Val Gly Phe Arg Leu Asp Gly Glu Glu Asp Phe Arg 485 490 495 Tyr Val Glu Gly Glu Gln Arg Glu Ser Ile Asn Gly Trp Thr Gln 500 505 510 Trp Thr Leu Pro Leu Asp Ala Phe Ala Gly Gln Thr Ile Thr Gly 515 520 525 Leu Ala Phe Ala Ala Glu Gly Asn Glu Thr Gly Leu Ala Glu Phe 530 535 540 Tyr Ile Gly Gln Leu Ala Val Gly Ala Asp Ser Glu Lys Pro Ala 545 550 555 Ala Pro Asn Val Asn Val Arg Gln Tyr Asp Pro Asp Pro Ser Gly 560 565 570 Ile Gln Leu Val Trp Glu Lys Gln Ser Asn Val His His Tyr Arg 575 580 585 Val Tyr Lys Glu Thr Lys His Gly Ly s Glu Leu Ile Gly Thr Ser 590 595 600 Ala Gly Asp Arg Ile Tyr Leu Glu Gly Leu Val Glu Glu Ser Lys 605 610 615 Gln Asn Asp Val Arg Leu His Ile Glu Ala Leu Ser Glu Thr Phe 620 625 630 Val Pro Ser Asp Ala Arg Met Ile Asp Ile Lys Ser Gly Ser Phe 635 640 645

SEQ ID NO: 4 Sequence Length: 1935 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Sequence: TTGAGAAAAG CTTTTTTAGT CGGTCTTGTT TGCACAGCGT GTGTATTGCT CCATGATGAT 60 CCAGTTGCCG CATCTACGTA CAACCCCCGTCTTTCC AGAGGAACTT 120 GCCCAATGGG AACCAGACAG TGATCCAGAC GCACCCTTTA ACAGAAGCCA TGTTCCGCTG 180 GAACCAGGCC GCGTTGCGAA TAGGGTAAAT GCTAATGCAG ACAAGGACGC ACACCTTGTT 240 TCGTTGTCCG CGCTAAACAG GCATACATCA GGTGTTCCAT CGCAAGGAGC GCCAGTTTTC 300 TATGAAAATA CGTTCAGCTA TTGGCATTAT ACAGATTTGA TGGTTTATTG GGCTGGTTCA 360 GCTGGCGAAG GCATTATCGT TCCGCCAAGT GCCGATGTCA TTGATGCATC GCACCGAAAT 420 GGGGTGCCGA TTTTAGGAAA TGTGTTCTTC CCGCCGACGG TTTATGGAGG GCAGCTAGAG 480 TGGCTAGAAC AAATGTTAGA GCAAGAGGAG GACGGTTCAT TCCCCCTTGC TGACAAATTG 540 CTAGAAGTCG CAGACTATTA TGGGTTTGAC GGCTGGTTTA TTAACCAAGA AACAGAAGGG 600 GCAGACGAAG GAACAGCCGA AGCCATGCAA GCTTTTCTCG TTTATTTGCA GGAACAAAAG 660 CCAGAAGGCA TGCACATCAT GTGGTAGCACTCGATGATTG ATACAGGGGC GATCGC CTGG 720 CAAAACCATT TAACGGATCG AAATAAAATG TACTTGCAAA ATGGCTCGAC CCGCGTCGCT 780 GACAGCATGT TTTTGAACTT TTGGTGGCGT GACCAGCGCC AATCGAACGA ATTGGCACAA 840 GCACTTGGCA GGTCTCCGTA TGACCTCTAT GCCGGAGTGG ATGTGGAAGC ACGAGGGACA 900 AGTACCCCTG TTCAGTGGGA AGGCCTGTTT CCTGAAGGAG AAAAGGCGCA TACATCACTC 960 GGGTTATACC GTCCAGATTG GGCATTTCAG TCAAGTGAAA CAATGGAAGC GTTTTATGAA 1020 AAAGAACTAC AATTTTGGGT TGGCTCGACA GGAAATCCAG CCGAAACAGA CGGCCAGTCA 1080 AATTGGCCTG GCATGGCGCA CTGGTTTCCC GCGAAAAGCA CCGCTACTTC GGTACCCTTT 1140 GTGACTCACT TTAATACGGG CAGCGGCGCT CAGTTTTCGG CAGAAGGCAA AACTGTGTCG 1200 GAACAGGAAT GGAATAACCG CAGCCTTCAA GATGTGCTGC CGACATGGCG CTGGATTCAG 1260 CATGGCGGCG ATTTAGAGGC AACATTTTCT TGGGAAGAAG CGTTTGAAGG GGGAAGCTCG 1320 TTACAATGGC ATGGCTCATT AGCGGAAGGA GAACACGCCC AAATCGAGCT CTATCAAACA 1380 GAGTTGCCGA TAAGCGAAGG CACTTCGCTA ACGTGGACAT TTAAAAGCGA GCACGGCAAC 1440 GATTTAAATG TGGGCTTCCG TTTAGATGGG GAAGAGGACT TCCGTTATGT GGAAGGAGAA 1500 CAGCGTGAAT CGATAAATGG TTGGACGCAG TGGACGTTGC CGCTGGATGC GTTTGCTGGT 1560 C AGACGATAA CAGGGCTGGC ATTTGCAGCG GAAGGGAATG AGACTGGGCT GGCAGAATTC 1620 TATATTGGAC AACTGGCCGT AGGTGCTGAT AGCGAAAAGC CTGCCGCTCC AAACGTGAAC 1680 GTACGCCAGT ACGACCCAGA CCCGAGTGGC ATTCAGCTCG TATGGGAAAA ACAAAGCAAC 1740 GTCCACCATT ACCGCGTTTA TAAAGAAACA AAGCACGGCA AAGAGCTAAT TGGCACATCT 1800 GCTGGAGATC GAATTTACCT AGAAGGCCTA GTCGAGGAAA GCAAACAAAA CGACGTGCGT 1860 CTGCATATAG AAGCACTAAG TGAAACATTT GTGCCAAGTG ATGCTCGCAT GATCGACATA 1920 AAAAGCGGCT CGTTT 1935

SEQ ID NO: 5 Sequence length: 22 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence: Ser Thr Tyr Asn Gly Pro Leu Ser Ser His Xaa Phe Pro Glu Glu 1 5 10 15 Leu Ala Gln Xaa Glu Pro Asp 20

SEQ ID NO: 6 Sequence length: 30 Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTGGATCC TTYCCNGARG ARYTNGCNCA 30

SEQ ID NO: 7 Sequence length: 32 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Ala Ala His Leu Val Ser Leu Ser Ala Leu Asn Arg His Thr Ser 1 5 10 15 Gly Val Pro Ser Gln Gly Ala Pro Val Phe Tyr Glu Asn Thr Phe 20 25 30 Ser Tyr

SEQ ID NO: 8 Sequence length: 30 Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTGAATTC ANAGTRTTYT CRTARAANAC 30

SEQ ID NO: 9 Sequence length: 27 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Ala His Thr Ser Leu Gly Leu Tyr Arg Pro Asp Trp Ala Phe Gln 1 5 10 15 Ser Ser Glu Thr Met Glu Ala Phe Tyr Glu Ser Leu 20 25

SEQ ID NO: 10 Sequence length: 33 Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTGAATTC TCRTARAANG CYTCCATNGT YTC 33

SEQ ID NO: 11 Sequence length: 25 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Ser Thr Ala Thr Ser Val Pro Phe Val Thr His Phe Asn Thr Gly 1 5 10 15 Ser Gly Ala Gln Phe Ser Ala Glu Gly Lys 20 25

SEQ ID NO: 12 Sequence length: 30 Strand number: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTGAATTC TGRTTRAART GNGTNACRAA 30

SEQ ID NO: 13 Sequence length: 260 Number of chains: Double-strand topology: Linear Sequence type: Genomic DNA Sequence: GGATCCTTTC CCGGAGAGCT TGCGCAATGG GAACCAGACA GTGATCCAGA CGCACCCTTT 60 AACAGAAGCC ATGTTCCGCT GGAACCAGGC CGCGCATCCATCCACTCAGACTCAGACTAGGA 120AG GCGCTAAACA GGCATACATC ARGTGTTCCA 180 TCGCAAGGAG CGCCAGTTTT CTATGAAAAT ACGTTCAGCT ATTGGCATTA TACAGATTTG 240 ATGGTTTATT GGGCTGGTTC 260

SEQ ID NO: 14 Sequence length: 359 Number of chains: Double-stranded topology: Linear Sequence type: Genomic DNA Sequence: KGTGCGTGGA CCAGCGCCAA TCGAACGAAT TGCACAAGCA CTTTGGCAGG TCTCCGTATG 60 ACCTCTATGC CGGAGTGGAT GTGGAAGCAC GAGGACAAGT ACCCCKGT 120 CCACAGGTTC TCACTCGGGT TATACCGTCC AGATTGGGCA 180 TTTCAGTCAA GTGAAACAAT GGAAGCGTTT TATGAAAAAG AACTACAATT TGGGGTTGGC 240 TCGACAGGAA ATCCAGCCGA AACAGACGGC CAGTCAAATT GGCCTGGCAT GGCGCACTGG 300 TTTCCCGCGA AAAGCACCCTTTAATCAGTA

SEQ ID NO: 15 Sequence length: 322 Number of chains: Double-strand topology: Linear Sequence type: Genomic DNA sequence: GCTATTGGCA TTATACMAGA TTTGATGGTT TATTGGGCTG GTTCAGCTGG SCGAAGNCAT 60 TAATCGTTCC GVCCAAGTGCTT GCTCAATGCGTT ACCGAAATGG TGTGT TATGGAGGGC AGCTAGAGTG GCTAGAACAA 180 ATGTTAGAGC AAGAGGAGGA CGGTTCATTC CCCCTTGCTG ACAAATTGCT AGAAGTCGCA 240 GACTATTATG GGTTTGACGG CTGGTTTATT AACCAAGAAA CAGAAGGGGC AGACGAAGGA 300 ACAGCCGAAG CCATGCAAGC TT 322

SEQ ID NO: 16 Sequence length: 335 Number of chains: Double strand Topology: Linear Sequence type: Genomic DNA Sequence: AAGCTTTTCT CGTTTATTTG CAGGAACAAA AGCCAGAAGG CATGCACATC ATGTGGTATG 60 ACTCGATGAT TGATACAGGGAAGA CGCCTGGCAAAAT AGAGTCAAAAA CGAGTAAACA CGATTATACAGA CTGACAGCAT GTTTTTGAAC TTTTGGTGGC 180 GTGACCAGCG CCAATCGAAC GAATTGRCAC AARRCACTTG GCAGGTCTCC RTATGACCTC 240 TADTRCCGGA GTAGATGTGG AAGCACGAGG GACAAGTACC CCTGTTCAGT GGGAAGRCCT 300 GTTTCCTGAA GAGAAAGGNG CATACATVAC TC

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction map of a DNA fragment amplified by PCR.

FIG. 2 shows Cla I-3kb and Hind III / Pst I-2.5k.
It is a figure which shows the restriction enzyme map of the insert fragment of b.

FIG. 3 is an electrophoretic photograph showing the result of Western blotting of endo-A.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:06) (C12N 9/42 C12R 1:06)

Claims (12)

[Claims] 1. An isolated DNA containing a nucleotide sequence encoding a polypeptide having endo-β-N-acetylglucosaminidase A activity. 2. The DNA according to claim 1, wherein the polypeptide is derived from a strain belonging to the genus Arthrobacter. 3. The DNA according to claim 1 or 2, wherein the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 1 in Sequence Listing. 4. The nucleotide sequence represented by D shown in SEQ ID NO: 2.
An NA sequence, wherein the NA sequence is used.
The described DNA. 5. A DNA encoding a polypeptide having an endo-β-N-acetylglucosaminidase A activity or a functionally equivalent activity thereof, which comprises a part of the amino acid sequence set forth in SEQ ID NO: 1. 6. A DNA comprising a part of the DNA sequence set forth in SEQ ID NO: 2 and encoding a polypeptide having endo-β-N-acetylglucosaminidase A activity or its functionally equivalent activity. DNA. 7. An endo-capable of hybridizing with the DNA of any one of claims 1 to 6.
D encoding a polypeptide having β-N-acetylglucosaminidase A activity or its functionally equivalent activity
NA. 8. The amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing has one or more amino acid residues deleted, added, inserted or substituted, and endo-β-N.
-A DNA comprising a base sequence encoding the amino acid sequence of a polypeptide having acetylglucosaminidase A activity or its functionally equivalent activity. 9. A recombinant DNA comprising the DNA according to any one of claims 1 to 8. 10. An expression vector using the recombinant DNA according to claim 9 as a host cell, which is a microorganism, an animal cell or a plant cell. 11. A transformant obtained by transforming with the expression vector according to claim 10. 12. A transformant obtained by introducing the expression vector into which the polynucleotide containing the DNA according to any one of claims 1 to 9 is inserted, and the transformant is cultivated. Collecting a polypeptide having N-acetylglucosaminidase A activity,
A method for producing a polypeptide having endo-β-N-acetylglucosaminidase A activity.