JP2000279180A - Gene coding for branching enzyme and microorganism having the gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new branching enzyme which is derived from Aspergillus nidulans, has a specific amino acid sequence, forms a branch via α-1,6 bond, and is useful for preventing the aging of starch in food industry and so on. SOLUTION: This is a new branching enzyme which is derived from Aspergillus nidulans, has the amino acid sequence shown by the formula or an amino acid sequence obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into the amino acid sequence, cleaves α-1,4 bond of α-glucan such a amylose and amylopectin to form a branch via α-1,6 bond, and is useful for preventing the aging of starch in food industry and so on. This enzyme is obtained by carrying out PCR of chromosomal DNA of A.nidulans using primers having its partial sequences, screening a genomic library of A.nidulans using the obtained DNA as a probe, followed by expressing the obtained gene in A.nidulans.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a filamentous fungus, Aspergillus nidulan.
lans), a DNA encoding a branching enzyme gene, and a transformant into which the gene is introduced.

[0002]

2. Description of the Related Art A branching enzyme was first discovered in rabbit liver tissue by Cori et al.
Its existence has been confirmed in a wide variety of organisms such as animals, plants, and microorganisms. A branching enzyme is an enzyme that acts on α-glucan such as amylose or amylopectin, cuts α-1,4 bonds, and forms branches by α-1,6 bonds. At present, starch aging is a major problem in the processing of substances containing starch. For example, it is known that aging of starch in foods leads to a decrease in storage stability and a decrease in digestibility. This aging is due to the association of the amylose molecules contained in the starch, followed by insolubilization. However, when a branching enzyme is acted on in a starch solution, the amylose and amylopectin molecules in the starch can be increased in the branched structure by α-1,6-glucosidic bonds, thereby suppressing the aging of starch. (Starch Chemistry, No. 30
Vol. 2, No. 2, p. 223).

Hitherto, the existence of branching enzymes has been confirmed in a wide variety of organisms such as animals, plants, microorganisms, etc., but those derived from mold have not been confirmed so far, and their amino acid sequences have been clarified. No report has been made of any successful cloning of the gene encoding it.

[0004]

In a method of obtaining a branching enzyme from a producing organism, which is known at present, a method of extracting from a microorganism or an animal or a plant is used. It is far from satisfying the required amount.

[0005]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors have studied from various aspects. As a result, a method using a genetic engineering technique is most suitable for achieving efficient production of a branching enzyme. I got the knowledge. As a result of intensive studies, the present inventors have clarified the amino acid sequence of the branching enzyme,
By determining a base sequence corresponding to the amino acid sequence and preparing a transformant into which the base sequence has been introduced, genetic engineering techniques have been applied and mass production of a branching enzyme has been successfully achieved for the first time. Was completed. Hereinafter, the present invention will be described in detail.

In order to clone a gene encoding a branching enzyme according to the present invention, first, the filamentous fungus Aspergillus nidulans (Aspergillus) is used.
maltose-binding protein (72 kDa protein: p7)
7) is separated. The isolated branching enzyme is subjected to protease treatment to obtain a peptide fragment, and its partial amino acid sequence is determined.

Since this partial amino acid sequence contains a part of the highly conserved region of the α-amylase family to which the branching enzyme belongs, necessary primers are designed and synthesized based on this partial amino acid sequence. nidulans
PCR using the chromosomal DNA of
An R. product R-branching enzyme gene fragment was obtained. The colony hybridization of the genomic library of Nidulans was carried out, a positive colony was successfully obtained, and the entire nucleotide sequence was determined (SEQ ID NO: 2 in the sequence listing, FIGS. 6 to 1).
0). In addition, cDNA library plaque hybridization was performed using the upstream side of the obtained gene as a probe, and the cDNA was successfully obtained.
Was determined (FIGS. 11 to 13).

As a result, it was found that the branching enzyme gene (p72 gene) was composed of 2524 bp (start codon ATG to TAA: stop codon was TAA) and that seven introns were present. And cDNA without intron
Consists of 2061 bp, comprising 686 amino acids,
It was found to encode a protein of about 78 kDa.

Furthermore, in the present invention, not only the gene corresponding to the branching enzyme was isolated in this way, but also the gene corresponding to Aspergillus nidulans G1 was isolated using a genetic recombination technique.
By introducing the gene into 91 strains, the gene encoding a branching enzyme was multiply copied on the chromosome, and the expression of the enzyme gene was successfully enhanced. The isolation of the branching enzyme gene of the present invention, the determination of the nucleotide sequence thereof, and the corresponding amino acid sequence are shown below by examples.

[0010]

Examples of the present invention will be described below, but these examples are for the purpose of illustration only, and the present invention is not limited to these examples.

1. Purification of Branching Enzyme and Determination of Partial Amino Acid Sequence Aspergillus nidulans strain G191 (Univer
situation of Glasgow, Glasgow G
116 NU, Scotland, U.S.A. K. Or Fu
ngal Genetics Stock Center
10 spores purchased from r) to the DP-S medium 500ml ⁶
The cells were inoculated so as to obtain the number of cells / ml, and cultured with shaking at 37 ° C. overnight. After the cultured cells were collected by filtration, they were frozen with liquid nitrogen and crushed in a mortar. Thereafter, the disrupted cells were disrupted in 80 ml of a disruption buffer (5 mM EDTA · 2N).
a, 2 mM PMSF, 10 mM Tris-HCl,
5 mM mercaptoethanol, pH 7.5), and further crushed with a homogenizer.
The supernatant was recovered by centrifugation to obtain a cell extract. After applying this cell extract to an amylose column, 10 ml of a washing buffer (1 M NaCl, 10 mM Tris-H) was used.
Cl, 5 mM EDTA · 2Na, 2 mM PMSF,
The amylose column was washed with 5 mM mercaptoethanol, pH 7.5).

[0012] The elution of the branching enzyme is carried out using a maltose solution 10
ml (0.5 M maltose, 10 mM Tris-H
Cl, 5 mM mercaptoethanol, pH 7.5). Thereafter, the degree of purification was confirmed by SDS-PAGE. By this operation, the branching enzyme is confirmed by SDS-PAGE as a substantially single band. The branching enzyme was purified from Aspergillus nidulans strain G191, and the purified branching enzyme was treated with V8 protease and fragmented. After subjecting it to SDS-PAGE gel, PVDF
It was transferred to a membrane (manufactured by BioRad). After cutting out a fragment corresponding to 26 kDa from the membrane, the N-terminal amino acid sequence of this peptide was determined using a protein sequencer (Perkin Elmer Japan 610A) using the Edman degradation method. The determined N-terminal amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing.

2. Isolation of this enzyme gene To isolate the branching enzyme gene, a part of the amino acid sequence of the conserved region found when comparing the amino acid sequence obtained by the previous amino acid sequence determination with the branching enzymes of different origins The primers were designed, and an attempt was made to obtain a branching enzyme gene fragment by PCR. First, Rae
der and Broda's method (Lett. Appl. Mi.
crob. Chromosomal DNA from the cells of Aspergillus nidulans G191 strain according to
Extracted. Next, using this chromosomal DNA as a template, the primer shown in SEQ ID NO: 4 of the Sequence Listing and the primer shown in SEQ ID NO: 5 in the Sequence Listing are mixed, and a PCR reaction is performed using AmpliTaq DNA polymerase (Perkin Elmer Japan). Was. Furthermore, DN amplified by PCR
Using the A fragment as a template, a primer shown in SEQ ID NO: 6 in the sequence listing and a primer shown in SEQ ID NO: 7 in the sequence listing were mixed,
When the PCR reaction was performed again, an about 460 bp branching enzyme gene fragment was obtained.

In the sequence numbers 4 to 7, m, n,
r, y, and w mean the following, respectively. m: a, cn: a, c, g, tr: a, gy: c, tw: a, t

Article 19 of the PCR is as follows.

[0016] Fungal Genetics Sto
The chromosomal DNA library of Aspergillus nidulans obtained from ck Center was copied to a nylon membrane, and about 460 b
By performing colony hybridization using the DNA fragment of p as a probe, Escherichia coli containing a cloned cosmid of a DNA fragment containing a branching enzyme gene can be selected. By the above method, a cosmid containing a chromosome-derived branching enzyme gene was obtained and named pBE-G.

In order to further obtain the cDNA of the branching enzyme,
Fungal GeneticsStock Center
cdn obtained from Aspergillus nidulans
CDNA of an enzyme that branches from A library (using λZAPII (Stratagene) as a vector)
Tried to get. That is, the cDNA library was appropriately diluted, and E. coli XL1-Blue MRF '(St
ratagene) and an appropriate amount of NZY agar medium (1% NZ amine, 0.5% yeast extra, 0.5% NaCl, 0.2% MgSO ₄₎
・ 7H ₂ O, 1.5% agar; pH 7.5) was spread by an overlay method and kept at 37 ° C. overnight to obtain a plate on which plaques due to λ phage were scattered. Transfer the plaque from this plate to a nylon membrane,
Using a DNA fragment of about 460 bp obtained by the R reaction as a probe, λ phage cloned from a DNA fragment containing the cDNA of the present enzyme was selected by performing plaque hybridization. A recombinant phage having the restriction map shown in FIG. 1 was obtained and named λBE-1.

3. Determination of the present enzyme gene on the recombinant cosmid pBE-G and the nucleotide sequence on the recombinant phage λBE-1 Determination of the branching enzyme gene on the recombinant cosmid pBE-G and the nucleotide sequence on the recombinant phage λBE-1 And Sanger's dideoxy chain termination method (J. Mol. Biol., 94 , 441 (19).
75); Proc. Natl. Acad. Sci. US
A, 74 , 5463 (1977)). That is, the cosmid recombinant cosmid pBE
After subcloning the branching gene on -G into pUC118 (Takara Shuzo Co., Ltd.), the nucleotide sequence of the branching gene on chromosomal DNA was determined. Recombinant λ
The phage λBE-1 was prepared by pBluescriptSK containing the cDNA for branching enzyme by the in vitro excision method (in vitro Excision method).
After conversion to (-), the base sequence of the cDNA of the branching enzyme gene was determined. The determined total nucleotide sequence of the cDNA is shown in FIGS. In addition, as for the cDNA and the whole branching enzyme gene including introns, the entire base sequence determined as described above is determined by using SEQ ID NO: 2 in the sequence listing (and FIG. 6).
To FIG. 10).

As a result of comparing the genomic DNA and cDNA of the branching enzyme gene obtained by the above determination of the base sequence (ie, the chromosome and cDNA base sequences of the branching enzyme gene), the branching enzyme gene (p72 gene ) Is composed of 2524 bp including cDNA (SEQ ID NO: 2, FIGS. 6 to 10), and the presence of seven introns of 46 bp to 96 bp was confirmed (FIGS. 11 to 13, arrows). In the figure, light shaded portions indicate partial amino acid sequences determined from p72, and dark shaded portions indicate four conserved regions.

[0020] In the base sequence of the cDNA, DN
A is an open reading frame (OR) where the genetic information of A is transcribed into mRNA and translated into protein.
The search for F) indicated that the branching enzyme had a 2061 bp O starting at the start codon ATG and ending at the stop codon TAA.
RF was found (FIGS. 1-13) and was found to encode a protein of 686 amino acids, approximately 78 kDa. The amino acid sequence corresponding to this ORF is shown in SEQ ID NO: 1 (and FIGS. 3 to 5) in the sequence listing. Hereinafter, an example of a method for producing a branching enzyme gene, a plasmid, and a branching enzyme will be described.

5. Introduction of this enzyme gene into Aspergillus nidulans In order to efficiently introduce the branching enzyme gene into Aspergillus nidulans strain G191, the recombinant plasmid pT
G1-BE was produced. That is, pBE-G is converted to Xba
I, the 4147 bp band was recovered, and XbaI
Plasmid pTG1 (Mol. Gen. Ge)
net. , 254, 119 (1997)) and T4 DNA
Ligation was performed using ligase. Recombinant plasmid pTG1
-BE was added to Aspergillus nidulans strain G191 by Ba
Lance and Turner's method (Gene, 36,
321 (1985)). As a result, about 1
00 transformants were obtained. Ten strains were selected from among them, and a branching enzyme was extracted by the method shown in 1 to obtain SDS-P.
AGE was performed. As a result, the branching enzyme was replaced with SDS-PA
When subjected to GE, it was possible to obtain a transformant in which the observed 72 kD band was increased 5-fold as compared to Aspergillus nidulans G191 strain. This transformant was named Aspergillus nidulans BE strain. In order to confirm that the 72 kD protein produced in large quantities by Aspergillus nidulans strain BE was a branching enzyme gene product, this protein was digested with V8 protease in the same manner as in method 1, and then subjected to SDS-PAGE.

FIG. 2 shows the migration pattern. In the figure,
Lane 1 shows a molecular weight marker, lane 2 shows a branching enzyme produced by Aspergillus nidulans BE strain, and lane 3 shows a branching enzyme after V8 protease treatment.
As is apparent from the results, the migration pattern almost coincided with the pattern obtained when the branching enzyme was treated with V8 protease, suggesting that the p72 protein is a branching enzyme. Further, the amino acid sequence of the band of the same molecular weight (peptide 1 in FIG. 2) as the 26 kDa band whose amino acid sequence was determined in 1 was determined. The determined N-terminal amino acid sequence is Ser-His-Asp-Gln-
Ala-Leu-Val-Gly, the sequence of which is:
It corresponded to the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing, and corresponded to a part of the amino acid sequence found from the branching enzyme gene. From this, Aspergillus nidulans B
The 78 kD protein of the E strain was confirmed to be the gene product according to the present invention.

[0023]

According to the present invention, the branching enzyme gene and the amino acid sequence of the branching enzyme have been clarified, and the expression of the gene in a transformant into which the gene has been introduced has also been confirmed. As a result, the enzyme can be industrially produced, and is expected to be used for a wide range of uses in the food industry and the like, such as suppression of starch aging.

[0024]

[Sequence List] SEQUENCE LISTING <110> Higeta Shoyu Co., Ltd. <120> Gene Encoding Glycogen Branching Enzyme and Microorganism Gontaining there <130> 6149 <141> 1999-4-1 <160> 7 <210> 1 <211 > 686 <212> PRT <213> Aspergillus nidulans <400> 1 Met Thr Ser Thr Ala Pro Ser Asp Gly Thr Gly Ile Ile Asp Leu 1 5 10 15 Asp Pro Trp Leu Glu Pro Phe Arg Glu Ala Ile Lys Arg Arg Phe 20 25 30 Asp Tyr Val Glu Ser Trp Ile Lys Thr Val Asp Glu Val Glu Gly 35 40 45 Gly Leu Asp Lys Phe Ser Lys Gly Tyr Glu Lys Phe Gly Phe Asn 50 55 60 Val Ser Glu Thr Gly Asp Ile Thr Tyr Arg Glu Trp Ala Pro Asn 65 70 75 Ala Ile Glu Ala Ala Leu Val Gly Asp Phe Asn Asn Trp Asp Thr 80 85 90 Lys Ala Asn Pro Met Thr Arg Asp Asn Phe Gly Val Trp Glu Ile 95 100 105 Ala Leu Pro Ala Lys Asn Gly Thr Pro Val Ile Pro His Asp Ser 110 115 120 Lys Val Lys Val Lys Ile Thr Met Val Thr Arg Ser Gly Glu Arg 125 130 135 Ile Tyr Arg Ile Pro Ala Trp Ile Lys Arg Val Val Gln Asp Leu 140 145 150 Asn Val Ser Pro Ile Tyr Glu Ser Val Phe Trp Asn Pro Pro Lys 155 160 165 Ala Glu Arg Tyr Asn Phe Gln His Ala Arg Pro Lys Lys Pro Glu 170 175 180 Ser Leu Arg Ile Tyr Glu Ala His Val Gly Ile Ser Ser Pro Asp 185 190 195 Thr Arg Val Ala Thr Tyr Lys Glu Phe Thr Ala Asn Met Leu Pro 200 205 210 Arg Ile Lys Tyr Leu Gly Tyr Asn Ala Ile Gln Leu Met Ala Ile 215 220 225 Met Glu His Ala Tyr Tyr Ala Ser Phe Gly Tyr Gln Val Asn Asn 230 235 240 Phe Phe Ala Ala Ser Ser Arg Tyr Gly Lys Pro Glu Asp Leu Lys 245 250 255 Glu Leu Val Asp Thr Ala His Ser Met Gly Leu Val Val Leu Leu 260 265 270 Asp Val Val His Ser His Ala Ser Lys Asn Val Asp Asp Gly Leu 275 280 285 Asn Met Phe Asp Gly Ser Asp His Leu Tyr Phe His Ser Gly Ser 290 295 300 Lys Gly Gln His Glu Leu Trp Asp Ser Arg Leu Phe Asn Tyr Gly 305 310 315 Asn His Glu Val Leu Arg Phe Leu Leu Ser Asn Leu Arg Phe Trp 320 325 330 Met Glu Glu Tyr Gly Phe Asp Gly Phe Arg Phe Asp Gly Val Thr 335 340 345 Ser Met Leu Tyr Thr His His Gly Ile Gly Thr Gly Phe Ser Gly 350 355 360 Gly Tyr His Glu Tyr Phe Gly Pro Ala Val Asp AspAsp Gly Val 365 370 375 Met Tyr Leu Ala Leu Ala Asn Glu Met Leu His Arg Leu Tyr Pro 380 385 390 Asp Cys Ile Thr Val Ala Glu Asp Val Ser Gly Met Pro Ala Leu 395 400 405 Cys Leu Pro His Gly Leu Gly Gly Val Gly Phe Asp Tyr Arg Leu 410 415 420 Ala Met Ala Ile Pro Asp Met Tyr Ile Lys Leu Leu Lys Glu Lys 425 430 435 Ser Asp Asn Asp Trp Asp Ile Gly Asn Leu Ala Phe Thr Leu Thr 440 445 445 450 Asn Arg Arg His Gly Glu Lys Thr Ile Ala Tyr Ala Glu Ser His 455 460 465 Asp Gln Ala Leu Val Gly Asp Lys Ser Leu Met Met Trp Leu Cys 470 475 480 Asp Lys Glu Met Tyr Thr His Met Ser Val Leu Thr Glu Phe Thr 485 490 495 Pro Val Ile Glu Arg Gly Met Ala Leu His Lys Met Ile Arg Leu 500 505 510 Val Thr His Ala Leu Gly Gly Glu Gly Tyr Leu Asn Phe Glu Gly 515 520 525 Asn Glu Phe Gly His Pro Glu Trp Leu Asp Phe Pro Arg Ala Gly 530 535 540 Asn Asn Asn Ser Phe Trp Tyr Ala Arg Arg Gln Leu Asn Leu Thr 545 550 555 Glu Asp His Leu Leu Arg Tyr Arg Phe Leu Asn Glu Phe Asp Arg 560 565 570 570 Ala Met Gln Leu Thr Glu Ser Lys Tyr GlyTrp Leu His Ala Pro 575 580 585 Gln Ala Tyr Ile Ser Leu Lys His Glu Gly Asp Lys Val Leu Val 590 595 600 Phe Glu Arg Ala Asp Leu Leu Trp Ile Phe Asn Phe His Pro Thr 605 610 615 Glu Ser Phe Thr Asp Tyr Arg Val Gly Val Glu Gln Ala Gly Thr 620 625 630 Tyr Arg Val Val Leu Asp Thr Asp Asp Gln Ala Phe Gly Gly Leu 635 640 645 Gly Arg Ile Asp Gln Gly Thr Arg Phe Phe Thr Thr Asp Met Glu 650 655 660 Trp Asn Gly Arg Arg Asn Tyr Leu Gln Val Tyr Ile Pro Thr Arg 665 670 675 Thr Ala Leu Ala Leu Ala Leu Glu Glu Thr Leu 680 685 <210> 2 <211> 3037 <212> DNA <213> Aspergillus nidulans <400> 2 tctagaggga gagcgaggat cgcaacagag cctgtgatgt ccctgacaaa atagtggtgt -1020 gacgggcatt tagggctgcc tgtggtgctc acttcccaac gtcaccggcc ttgtgcacac -960 acaaagtcta tatatcatga cctgagcccg agtggtattc catgctcaga gcagaagatg -900 ggcttcttca gcagtctctg atatgatgga tcacatgttg ccataacgcg caaagtcgat -840 aggcggcggg atattgcttg acgcaacagt caaggaggga gactgatgct aattcgacta -780 actgagccta gatactctgc agttacttgc aattatggtc gtcatctagc cttttgcta c -720 agtcattgga ggttatgcta caggcacttg atttcttaca tctgtactgt ttctcccttc -660 tctgtcggcg cctgcaccta ggggtaaagc accccaactc gctgcacaac ataatgcgct -600 gacatcggcc tattactggt ttgaggaagg tccaaatcgt ttgatctggt aaaggaaaag -540 gttcgaaagt caggagaagg agtatatgat ttcgtagcac tatctgcttt tgattcaaag -480 ttctaggaac tttaggaact gctttttcct tgggttatgg tcctctaacc tccgggtaac -420 gggatacaga acaaatacat aaacagcacg cgcactgatg ttactcaaca agtggcccgt - 360 ttggccactt tgaatagaaa ccactaacta ccgccactat gcagacgatg atcgcagtgt -300 ccaatcagac cacggcacac ccttgaaacc cagccgatta aaggtgataa aaacccgtcg -240 atggctgttc caagaccaaa aagaggttgg gaaaagatag tgatgtcagc gatgtggaat -180 gttggcagga cacggacccc cgcaatcaac tcctcccagg aacaagagct ctgaaccgct -120 tctttcccgc atccggttca cttttttttt actctctaaa acttcgaaga ctcaatcaat -90 tcctactcta ttttggccct ggagtttatt cctgacgcga ctctcaacgc gtatcgagcc -30 tgagccagct tatctcgttg ctcaacggcc atgacttcca cagctccatc tgacggcact 30 ggtgggtcaa agaacgccga ttattcagtc aatgacattc taacgctgat ctacaggtat 90 catcg atctt gatccgtggt tagagccttt tcgggaggct atcaagcgcc gattcgatta 150 tgttgagagc tggatcaaga ccgttgatga ggtggaggga ggtctcgata agttcagcaa 210 ggtgagtaga ctatcaacta tccagttgtg ttccccattt tctctataac ttgtgtgcaa 270 cggctaattg gagagctttt tccatgggga cgatagggct atgagaaatt cggcttcaat 330 gtcagcgaga cgggcgacat cacctacagg gaatgggctc caaacgccat agaagcggcg 390 ctggtgggtg acttcagtac gtctgtctgg cacctggggt gggtaaaatc gccagttaaa 450 gcacgttact aagactggca gacaattggg ataccaaggc gaatccaatg acgagagaca 510 acttcggtgt ttgggagatt gcccttcctg cgaagaatgg cacgccggtc ataccgcatg 570 atagtaaggt taaggtatgt ttacggcatg cagcttcacc accgtggggg ttctaatgct 630 gacttcctac agataactat ggtcacccgc agcggagaac gtatatatcg tattcccgca 690 tggatcaagc gtgttgtgca agatctgaat gtatcgccta tctacgaatc tgtcttttgg 750 aacccaccaa aggcagagcg gtataacttt cagcatgcgc gccctaagaa acccgaaagc 810 ctacggatct atgaagctca cgtcggtatc tcgtccccgg ataccagagt agcaacatac 870 aaggagttca cagccaacat gctccctcgg attaagtacc taggctacaa cgcaatccag 930 ctcatggcca ttatggaaca tgc ctactat gccagcttcg ggtaccaggt taacaatttc 990 ttcgcagcga gtagtcgcta cggtaagccc gaggatctga aggaactggt tgacacggca 1050 catagtatgg gcttggtagt tctcctcgac gtggtgcaca gccacgcatc gaagaacgtc 1110 gatgacggac tgaacatgtt tgatggcagt gaccatttat atttccattc cgggtcaaaa 1170 ggtcagcacg agctatggga cagccgactg tttaactacg gaaaccatga agttctgcgg 1230 ttcctcctta gcaatcttcg cttttggatg gaggaatacg gctttgacgg ttttcgattt 1290 gatggtgtca ccagcatgtt atatacccat cacggtattg ggacgtgagt cacgataatc 1350 tcgatatttt ttacaagtgc tcaccagtgt cagtggcttt tcaggcggat accacgaata 1410 cttcggtcca gcagttgacg acgatggcgt tatgtaccta gccctagcaa acgaaatgct 1470 gcaccgtctt tacccagatt gcataaccgt ggcagaggat gtttcgggaa tgccagcgtt 1530 atgcatacca cacggccttg gtggagtcgg attcgactat cgtctcgcga tggccattcc 1590 agatatgtat atcaagcttc taaaagagaa gtcagacaac gattgggaca ttggcaatct 1650 cgccttcacg ttgacaaacc gacgccatgg cgagaagacc attgcatacg cagaaagcca 1710 cgaccaagcg taagtcctcc cgtcttctgt aacagaccca aatcataaca taacccagtc 1770 tcgtcggcga caaatctctc atgatgtggc tctgcgataa agaaatgtac acacacatgt 1830 ccgtcctgac agagttcacc cccgtcatcg aacgcggcat ggcactccac aaaatgatcc 1890 ggcttgtcac acacgccctt ggtggcgaag gctacctcaa tttcgaaggt aacgaattcg 1950 ggcacccgga atggctcgac tttccccgcg ccggcaacaa caactccttc tggtacgcac 2010 gtcgccaact aaacctgacc gaagatcact tcctctacgc tacagattcc ttaacgagtt 2070 cgaccgcgcc atgcagttga cagaatccaa gtacggctgg ctccatgcgc cccaggctta 2130 tatttctctc aagcacgaag gagataaagt gcttgtcttt gagcgggcgg atttgctatg 2190 gattttcaat ttccatccta cggagagttt taccgattat agggtcggtg tagaacaagc 2250 tgggacttat cgggttgtgc ttgacactga tgaccaggcg tttgggggct tgggaaggat 2310 cgatcagggg actagattct ttacaaccga tatggaatgg aatgggagga ggaattactt 2370 gcaggtttat attccgacta ggactgctct ggtgagtcta ctcgttcttg aacccttcat 2430 gcctttttct cttcgtggtt gtcatctgta tacgaaactc gatactaatc acttctgttg 2490 gatgtaggcc ctagcattgg aagagacgct gtaatactcc actccggagc ttttacgtgc 2550 gaatatattc gctattaaga tactctttct cgaaaactaa cgccagacca aactatatat 2610 ctatatgtac cgaccatata tccgtatcct agcat gttga gatagttcat agcataattt 2670 aatctttgtt aaccgcgcaa tttctcaccc cttccctttg ctgtacacta tgggtgattt 2730 tgttcatcat tggcctcgta catagtacat gagacgttaa acagccagga tgagttcaga 2790 tagcagggac ataacatcta ctgcgagagc catgctgcaa tctccatcat ttgatgaagc 2850 tgcacaagct ccagacaaca tactacagag gctatacgag aggtactaca atggagaaaa 2910 agaataaacc atattttcca atccaacaaa agaaatgata catgttgtcc ctgcccgagg 2970 gtataaggcc gggtgtggtc atttaattta cttacatgag cacaatatat tgtcacacga 3030 gtctaga 3037 <210 > 3 <211> 10 <212> PRT <213> Aspergillus nidulans <400> 3 Ser His Asp Gln Ala Leu Val Gly Asp Lys 1 5 10 <210> 4 <211> 26 <212> DNA <213> Artificial sequence < 400> 4 ttygayggnt tymgnttyga ygtnac 26 <210> 5 <211> 23 <212> DNA <213> Artificial sequence <400> 5 gtyttrtcnc cnacnarngc ytg 23 <210> 6 <211> 27 <212> DNA <213> Artificial sequence < 400> 6 ggnttymgnt tygaygtnac nwsnatg 27 <210> 7 <211> 29 <212> DNA <213> Artificial sequence <400> 7 gtyttrtcnc cnacnarngc ytgrtcrtg 29

[Brief description of the drawings]

FIG. 1 shows a restriction map of the cDNA of the branching enzyme.

FIG. 2 shows an electrophoresis pattern of a protease degradation product of a branching enzyme.

FIG. 3 shows the amino acid sequence 1 of the branching enzyme.

FIG. 4 shows the same.

FIG. 5 shows the same.

FIG. 6 shows base sequence 1 of a branching enzyme gene.

FIG. 7 shows the same.

FIG. 8 shows the same.

FIG. 9 shows the same.

FIG. 10 shows the same.

FIG. 11 shows the nucleotide sequence 1 of the branching enzyme gene cDNA.

FIG. 12 shows the same.

FIG. 13 shows the same.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/15 C12R 1:66) (C12N 9/10 C12R 1:66) (72) Inventor Masato Kato 1-11, Kita Chikusa 1-chome, Chikusa-ku, Nagoya-shi, Aichi No. 8 Building 34, No. 34 (72) Inventor: Aya Matsuno GA11 4B050 CC04 LL02 4B065 AA60X AA60Y AB01 AC14 BA02 CA29 CA41

Claims (5)

[Claims] 1. A branching enzyme represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A DNA comprising a branching enzyme gene represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing and encoding the amino acid sequence according to claim 1. 3. A recombinant plasmid comprising at least a part of the DNA according to claim 2. 4. A transformant into which the recombinant plasmid according to claim 3 has been introduced, Aspergillus nidulans (A)
spergillus nidulans) BE. 5. The transformant Asper according to claim 4,
A method for producing a branching enzyme, which comprises using Gillus nidulans BE.