JPH05268953A - Production of glucoamylase with low glucosyltransferase activity - Google Patents

Abstract

PURPOSE:To produce a glucoamylase having a low glucosyltransferase activity by using a gene recombination technique. CONSTITUTION:The objective method for producing a glucoamylase having a low glucosyltransferase activity is to culture in a nutrient culture medium Aspergillusniger having the productivity of glucoamylase in which a recombinant DNA prepared by integrating a DNA containing a glucosyltransferase structural gene deficient in N- and C-terminals thereinto is transduced, produce the glucoamylase in the resultant culture and then collect the obtained glucoamylase from the culture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a glucosyltransferase (hereinafter referred to as GTase) having N-terminal and C-terminal deletions.
Say. ) Aspergillus niger (As) into which a recombinant DNA incorporating a DNA containing a structural gene has been introduced
The present invention relates to a method for producing glucoamylase (hereinafter referred to as GAase) by pergillus niger.

[0002]

2. Description of the Related Art GAase is an enzyme that catalyzes the action of hydrolyzing the α-1,4 glucoside chain of starch sugar from the non-reducing end to produce glucose.
It is classified into 3.2.1.3. This enzyme is used in the starch industry. Conventionally, GAase has been produced by culturing microorganisms such as Aspergillus niger.

[0003]

When GAase is produced by Aspergillus niger, the GTa is simultaneously produced.
GTase that has been produced and has been generated
Is known to be difficult to remove even in the purification step. Therefore, in the starch industry Aspergillus
When glucose is produced from starch using Niger's GAase, there is a problem that isomerized sugar is produced by GTase mixed with by-produced oligosaccharide, which reduces the yield and purity of glucose. Was. In addition, conventionally-improved methods for reducing GTase by various artificial mutagenesis means that mutations occur randomly, and thus it is not always possible to obtain a highly targeted strain. Had a problem that the growth and the conidia attachment performance were deteriorated by the mutation treatment.

[0004]

[Means for Solving the Problems] Therefore, the present inventors have studied to obtain an improved strain in which only the GTase activity is reduced while maintaining high GAase productivity by means of gene recombination. .. Then, a plasmid containing a gene in which the N-terminal and the C-terminal of the GTase structural gene were deleted was prepared, cleaved at the SnaBI site located almost in the center of this gene, and then introduced into protoplasts for homologous recombination. High GAa by raising
The present invention succeeded in obtaining a transformant having an ability to produce a se activity and a reduced GTase activity.

That is, the present invention is derived from a microorganism belonging to the genus Aspergillus, and is defined by the restriction enzyme map of FIG. 1 (b), the GTase structural gene having the N-terminal and C-terminal deleted, and the GTase structural gene. A recombinant Aspergillus niger introduced with a recombinant DNA containing DNA containing GA is cultivated in a nutrient medium,
A method for producing GAase having a low GTase activity, which comprises producing GAase in a culture and then collecting the GAase from the culture.

The GA of the present invention will be described in detail below.
Examples of the ase-producing bacterium include Aspergillus genus and Rhizopus genus. As a source of the chromosomal DNA containing the GTase gene of the present invention, the GAase-producing bacterium as described above is used. For example, Aspergillus niger No. 499 etc. are mentioned.

The mycological properties of this strain are as follows. (1) Growth state in each medium (a) Malt extract agar medium Growth is good at 37 ° C. Basal hyphae are relatively dense. The surface of the colony is velvety. The color of the colony is initially white and turns brown to black when a large number of conidia are formed. The back of the colony is initially colorless and later becomes pale yellow. (B) Tuapec agar medium Growth is good at 37 ° C. Basal hyphae are relatively thin and flat. The surface of the colony is velvety to wool. The color of the colony is initially white and turns brown to black when a large number of conidia are formed.
The back of the colony is initially colorless and later yellow.

(2) Physiological and ecological properties (a) Optimal growth conditions (using malt extract medium) pH: 4 to 7 Temperature: 25 to 35 ° C (b) Range of growth (using malt extract medium) pH: 3-8 Temperature: 10-45 ° C

(3) Morphological properties Conidial head: 200 to 500 μm, black. Conidia peduncle: length 500μ-3mm, diameter 15-20
μ. It branches from basal hyphae or aerial hyphae and rises.
Smooth surface colorless. Apex: 50-70μ in diameter, spherical. Metre: about 25 × 5.3μ Fluoride: about 11 × 3μ. Conidia: diameter 3.0-4.5μ, spherical, rough surface, agglomerate black.

From the above-mentioned mycological properties, this strain belongs to the genus Aspergillus. Also, conidia heads with meteor are mixed, conidia head is spherical, tears as it gets old, conidia peduncle cannot constrict directly under the apex, conidia head is black, conidia stalk is smooth, vertical This strain was identified as Aspergillus niger by tearing. This strain has been deposited with the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Micromachine Research Institute No. 11316 (FERM P-11316).

The above-mentioned strain is cultivated by an ordinary culturing method to obtain a culture, and the culture is obtained from the culture by a conventional method such as filtration and centrifugation. From the Aspergillus niger culture broth obtained above, GT was subjected to conventional methods such as ammonium sulfate salting-out, centrifugation, desalting and various chromatographies.
purify the ase.

The purified GTase is subjected to limited digestion with trypsin and the like, the limited digested peptide is separated by HPLC, and the N-terminal amino acid sequence of the separated peptide is described, for example, in a known document [Eur. J. Biochem., Vol. 1, 80-91. Page (1967)] is applied using an automated amino acid sequencer. A DNA having a base sequence corresponding to this amino acid sequence is synthesized. Synthetic DNA can be produced, for example, by using an automatic DNA synthesizer. Labeling of synthetic DNA is carried out, for example, by publicly known literature [Proc. Natl. Acad. Sci. US
A., 74, 560-564 (1977)].
The 5'end was γ- ³² using 4 polynucleotide kinase.
It can be performed by phosphorylation with P-ATP. The probe is prepared in this manner.

Separately, Aspergillus niger (Aspe
rgillus niger) is cultured, and the cells are crushed with a homogenizer or the like, and then chromosomal DNA is obtained according to a conventional method. Then, a known document [Molecular Cloning (2nd Edi
tion), Publisher Cold Spring Harbor Laboratory Press
(1989) 9.34-9.58], the chromosomal DNA is cleaved with a restriction enzyme, separated by agarose gel electrophoresis according to the fragment length, and then subjected to Southern hybridization using the above probe. To do. That is,
DNA is adsorbed to a nitrocellulose filter and hybridized with a labeled synthetic DNA probe, and an autoradiogram is taken. The restriction enzyme used is SphI
And so on.

Then, an agarose gel described in, for example, a known document [Anal. Biochem., 101, 339-341 (1980)] was used to collect a DNA fragment assembly having a certain length containing a chromosomal DNA fragment to which the probe hybridizes. It can be recovered according to the DNA extraction method from.

Next, in order to perform colony hybridization, the above-mentioned recovered DNA is incorporated into vector DNA to prepare a recombinant DNA. Incorporation of chromosomal DNA into vector DNA is well known [J. Mol. Biol., 96].
Vol. 17, pp. 171-184 (1975)].
It can be carried out by cutting NA and vector DNA with a restriction enzyme, and then ligating with ligase.
Examples of the vector DNA include plasmid DNA, and particularly pBR322 and pUC19 are preferable.
Examples of the ligase include T4 DNA ligase.

Introduction of recombinant DNA into Escherichia coli can be carried out, for example, by publicly known literature [Molecular Cloning (2nd Edition), Publisher C.
old Spring Harbor Laboratory Press (1989) 1.82-1.
84]. Escherichia coli to be used is Escherichia coli (Escher)
ichia coli) HB101 strain is preferable. GT
The selection of the strain containing the recombinant DNA containing the ase gene can be carried out, for example, by publicly known literature [Molecular Cloning (2nd Editi
on), Publisher Cold Spring Harbor Laboratory Press (1
989) 1.90 to 1.104], and the synthetic D
It can be performed by colony hybridization using NA as a probe. That is, Escherichia coli introduced with recombinant DNA was spread on L-broth agar medium containing ampicillin, and after overnight culture, it was replicated on a nitrocellulose filter and further cultured for 2-3 hours on L-broth agar medium containing ampicillin. Then, lysis and DNA immobilization are performed to detect positive colonies to which the synthetic DNA hybridizes.

Next, from the positive strains, for example, known documents [Mo
lecular Cloning (2nd Edition), Publisher Cold Spring
Harbor Laboratory Press (1989) 1.25 to 1.28], the plasmid is extracted and purified by various methods, and digested with various restriction enzymes to prepare a restriction enzyme map, and again synthetic DNA by Southern hybridization.
Make sure the probe hybridizes.

Next, the GTase gene is confirmed by determining the base sequence of GTase and expressing it. As a transformant, Aspergillus niger, which is a high-producing strain of GAase, is preferable.

PDH25 can be used as a transformation vector. pDH25 carries the hygromycin B phosphotransferase gene hph expressed in Aspergillus niger. This and a plasmid into which the GTase structural gene having N-terminal and C-terminal deletions were inserted are simultaneously introduced into Aspergillus niger by the co-transfomation method to obtain a hygromycin-resistant transformant. This transformant strain is cultivated to produce GAase with low GTase activity in the culture, and GAa is produced from the culture.
se.

[0020]

【Example】

Example (1) Purification of GTase and determination of partial amino acid sequence Aspergillus niger No. 499 (Microtech Lab.
Spore slant ⅓) of CM-CSL medium (cornmeal was liquefied with amylase, and corn steep liquor was added to the 2% solution so that the final concentration was 5%, and the temperature was 121 ° C for 30 minutes. Cell sterilized by the autoclave of 1.) and cultured with shaking at 30 ° C. for 5 to 6 days. 3 cells
Removed by filtration using MM filter paper (manufactured by Whatman),
Ammonium sulphate is added to the culture medium to 50% saturation. Next, the resulting precipitate is removed by centrifugation at 10,000 rpm for 30 minutes, and ammonium sulfate is added to the supernatant so that it becomes 90% saturated. Next, the precipitated fraction is collected by centrifugation at 10,000 rpm for 30 minutes and dissolved in 10 mM acetate buffer (pH 6.0). The insoluble matter was removed by centrifugation at 15,000 rpm for 30 minutes, the supernatant was filtered through a 0.45 μm filter, and desalted and 10 mM acetate buffer (pH 6.0) by gel filtration using Toyopearl HW-40C. Was equilibrated to.

The crude enzyme solution thus obtained was separated by anion exchange chromatography using a FPLC system (Pharmacia) and a Mono Q HR 10/10 column. Elution is 0 to 5 in 10 mM acetate buffer (pH 6.0).
This was done by forming a 00 mM NaCl linear gradient. Furthermore, from the fraction having GTase activity, Superose12
Gel filtration chromatography using a column [100 mM
GT with acetate buffer (pH 6.0), 200 mM NaCl]
The ase was purified.

Next, 100 μg of trypsin was added to 5 mg of the obtained purified GTase, and 100 mM Tris-HCl (pH 8.
5), 37 in 0.001% sodium dodecyl sulfate (SDS)
It was allowed to act at 5 ° C. for 5 hours to perform limited decomposition of GTase.
Further, the reaction solution was applied to a C-18 column (VP-318-2251, Senshu Kagaku Co., Ltd.), and the GTase limited decomposition peptide was subjected to high performance liquid chromatography in the presence of 0.1% trifluoroacetic acid to give a linear gradient of 0 to 80% acetonitrile. It was collected by. Some N of the fractionated peptides
The ends were determined by the Edman degradation method using a model 470A type (manufactured by Applied Biosystems) amino acid sequencer. The determined amino acid sequence is SEQ ID NO: 1.
And SEQ ID NO: 2.

(2) Identification of GTase gene The amino acid sequence of GTase is searched for a part which seems to have high specificity when converted into a DNA base sequence, and corresponds to parts A and B of SEQ ID NO: 1. Two synthetic DNAs having the sequence shown below were synthesized using a 380B type (Applied Biosystems) DNA synthesizer. The synthesized sequences are shown below. A: GARTAYATMTGGACNGA B: GAYTAYATGCAYGGNTA (where R is A or G, Y is C or T, M is A, C or T, N is A, C, G or T)

Synthetic DNA solution is used to remove the protecting group.
After treatment at ℃ for 12 hours, concentrated to about 100 μl with N-1 type rotary evaporator (manufactured by Tokyo Rika Co., Ltd.), added 10 μl of 3M sodium acetate (pH 4.8) and 250 μl of ethanol, and kept still at -80 ° C. for 30 minutes. After standing, centrifugation was performed to precipitate ethanol. 0.5 μg of the DNA thus obtained was added to 50 mM Tris-HCl (pH 7.6), 10 mM
10 units of T4 DNA kinase, (γ- ³² P-) ATP1.85 in a solution containing MgCl ₂ , 10 mM 2-mercaptoethanol
MBq (PB10218 from Amersham) and 37 ℃, 30
Label the 5'end by incubating
It was used as a probe.

Next, Aspergillus niger No. 499
YPD medium (2% glucose, 1% peptone, 0.5%
After culturing in yeast extract) at 30 ° C. for 30 hours, the cells were collected by filtration with gauze and frozen with liquid nitrogen. Use a homogenizer (AM-8 type, manufactured by Nippon Seiki Seisakusho) to freeze cells.
It was crushed by treating at 8,000 rpm for 15 minutes. Then 50 mM
EDTA (pH 8.0), 0.5% SDS, 0.1 mg / ml proteinase K (each final concentration) was added, and the mixture was incubated at 50 ° C for 4 hours. Furthermore, TE (10 mM Tris-HCl, 1
mM EDTA, pH8.0) an equal amount of saturated phenol was added and gently stirred, and then centrifuged at 15,000 rpm for 30 minutes to separate the aqueous phase.

After the same treatment was carried out once again, a mixture of TE saturated phenol and chloroform at a ratio of 1: 1 instead of TE saturated phenol was used, and then the same treatment was repeated twice using chloroform. A 1/4 amount of 50% polyethylene glycol # 6000 was added to the thus obtained crude nucleic acid extract, and the mixture was incubated at 0 ° C. for 2 hours. 3,00
After collecting the precipitated fraction by centrifugation at 0 rpm for 3 minutes, 8 ml of TES buffer (20 mM Tris-HCl, 5 mM EDTA, 100 mM NaC was added.
redissolved in pH 7.5), added 8.7g of CsCl, and added 5mg
Ethidium bromide was added and subjected to CsCl-ethidium bromide equilibrium density gradient centrifugation at 44,500 rpm for 16 hours. The DNA band formed by this centrifugation was recovered, and dialyzed against TE after removing ethidium bromide with 1-butanol. By this method, 4.5 mg of chromosome D from 100 g of wet cells
Obtained NA.

Aspergillus niger No. obtained as described above. Complete decomposition was carried out by reacting 10 units of 499 chromosomal DNA with 30 units of the restriction enzyme SphI at 37 ° C. for 3 hours. The reaction solution was subjected to 1% agarose gel electrophoresis, and Southern hybridization was performed using the above probe.

6 × SSC (1 × SSC is 150 mM NaCl, 15 mM t
(including risodium citrate), 0.1% SDS, 0.2% bovine serum albumin, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone at 65 ° C for 8 hours to perform prehybridization, and then add the above probe. Hybridization was carried out at 40 ° C. overnight. The filters were then washed in 6X SSC, 0.1% SDS for 30 minutes at 52 ° C and analyzed by autoradiography. As a result, it was observed that the two kinds of probes both hybridized to the position of 4.3 Kb.

(3) Cloning of GTase gene Aspergillus niger No. 499 chromosome DNA 5μ
g was completely decomposed with 10 units of SphI, then subjected to 1% agarose gel electrophoresis, stained with ethidium bromide and stained with about 4.3
Cut off the gel at the position that hits Kb, and cut the gel piece into 3 pieces.
Double volume of 8N NaClO ₄ is added and incubated at 37 ° C. for 10 minutes to dissolve agarose. Then the DNA in the solution
Was adsorbed on a GF / C (manufactured by Whatman) glass filter having a diameter of 6 mm, and the filter was dissolved in 1 ml of TE to give 6N.
It was washed with a solution of NaClO ₄ followed by 1 ml of 95% ethanol and dried.

Add 30 μl of TE to this filter, and add 37
Incubated at 30 ° C for 30 minutes. Then 15,000 rpm, 2
The aqueous phase was recovered by centrifugation for minutes. In this way, the Aspergillus niger No. A SphI fragment around 4.3 Kb derived from 499 chromosomal DNA was recovered.

Separately, the vector plasmid pBR322
Treat 0.1 μg with 5 units of SphI for 2 hours at 37 ℃
1/5 M Tris-HCl (pH 9.0) to the reaction solution
Amount, add 0.5 units of alkaline phosphatase, 65 ℃, 3
Incubated for 0 minutes. This reaction solution was subjected to agarose gel electrophoresis and stained with ethidium bromide, and then a band corresponding to the molecular weight of pBR322 was cut out and collected.

66 mM Tris-H was prepared by mixing the above two kinds of DNA.
Cl (pH 7.6), 6.6 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP (each final concentration) 300 units of T4DN
Ligation reaction was performed by adding A ligase and incubating at 4 ° C. overnight. Next, competent cells of Escherichia coli HB101 strain were prepared and transformed with the above ligation reaction product. Transformant is ampicillin 50 μg
/ Ml, LB medium containing 1.2% agar [1% Bacto Trypton
e (manufactured by Difco), 0.5% yeast extract, 0.5% NaCl]
After selecting above, the cells were transferred onto a nitrocellulose filter and subjected to colony hybridization using probe B.

However, the hybridization and washing conditions were the same as those for Southern hybridization. As a result of analysis by autoradiography, 24 positive clones were obtained out of about 1000 transformants. Plasmid DNA was prepared from these transformants, and BamHI, EcoRI, PvuII, Sn were added to 0.1 μg of each.
When 5 units each of restriction enzymes such as aBI and SphI were allowed to act at 37 ° C. for 2 hours and analyzed by agarose gel electrophoresis, a 4.3 Kb DNA fragment was pBR3 for all plasmids.
It was included in 22 SphI sites. This plasmid is called pGT
It was named Y02.

(4) Determination of nucleotide sequence of 4.3 kb fragment The DNA fragment obtained by digesting the plasmid pGTY02 with various restriction enzymes was subcloned into pUC118 and pUC119. The thus obtained plasmid was purified by CsCl-ethidium bromide density gradient centrifugation, and then subjected to the method of Henikoff et al. [Gene, 28, 351-359 (198
4)] and the deletion series was prepared. E. coli MW1184 strain was transformed with the prepared deletion series to obtain transformants carrying plasmids having DNAs of various lengths.

Next, the obtained transformant was cultured in 2 × YT medium and infected with helper phage M13K07 to obtain single-stranded DNA, which was purified and used for determining the nucleotide sequence.

The nucleotide sequence is determined by the Sanger Rano dideoxy method [P
roc. Nat. Acad. Sci. USA., Vol. 74, 5463-5467 (197
7)]. Ampli Taq is mainly used for the extension reaction.
(Takara Shuzo) was used. If partial degeneracy occurs,
This was resolved by using Sequenase (Toyobo) and substituting dITP for dGTP. Regarding the amino acid sequence encoded by the GTase structural gene, the entire amino acid sequence of the GTase protein has been reported by Chiba et al. [Journal of the Japanese Society of Agricultural Chemistry, 64, 510 (1990) and Biochemistry, 63, 787]. Page (1991)] It was decided with reference to it. The results are shown in SEQ ID NO: 3. Sequence number:
The underlined part in 3 indicates the presence of an intron,
The amino acid sequence portion shows the primary structure of GTase protein.

(5) Method for Obtaining GTase Activity-Lowing Strain Using High GAase Activity Aspergillus niger Producing Strain First, GTas was used as a plasmid for transformation.
e PvuII-EcoR with deletion of N-terminal and C-terminal of structural gene
The I fragment was inserted into the HincII-EcoRI site of pUC19, and this plasmid was named pGTID1 and shown in FIG.

Next, using pGDID1 and a control plasmid pUC19 (20 μg each) and a plasmid pDH25 (10 μg) having a hygromycin B phosphotransferase-required complementary gene hph, co-transformati was used.
It was introduced into Aspergillus niger by the on method and transformed. As a result, hygromycin B 100 μg / ml
A transformant strain that grew well even in a medium containing spores, that is, a pGTID1-derived TRD1-24 (target strain) and a control pUC19-derived TR19-1 strain were obtained.

The time-dependent changes in GAase and GTase production (that is, changes in GAase and GTase activity per ml of culture supernatant) are shown in FIGS. 1 and 2. As is clear from FIGS. 1 and 2, the target strain among the transformants obtained had the same GAase activity as the control strain, but only the GTase activity decreased to 1/3. all right.

The GAase and GTase activities were measured by the following method. (1) Preparation of enzyme solution 10 ml of culture solution was sampled, centrifuged at 15000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter. 2 ml of this filtrate was concentrated to 100 μl or less, and this was concentrated with 50 mM sodium acetate. It was diluted to a total of 500 μl and used as the enzyme solution.

(2) GAase activity measurement 5% starch 0.1 ml, 0.25M sodium acetate buffer (pH 4.5)
A substrate consisting of 0.1 ml and 0.25 ml of water is preheated at 37 ° C for 5 minutes, 0.05 ml of the enzyme solution of (1) is added, and after reacting at 37 ° C for 30 minutes, 0.02 ml of the reaction solution is taken and glucose CII-test is performed. Add Wako (Wako Pure Chemical Industries, Ltd. product) coloring reagent. 37
The mixture was heated at ℃ for 5 minutes, and the absorbance at a wavelength of 505 nm was measured. The amount of glucose produced by the reaction was defined as the enzyme activity. As the unit of enzyme activity, the amount of enzyme that liberates 1 μmol glucose per minute was defined as 1 unit.

(3) Measurement of GTase activity It was carried out based on the method of Iwano et al. [J. Brew. Soc. Japa
n, 72, 517-520 (1977)]. That is, 0.5 ml of 4% methyl-α-glucoside dissolved in 50 mM sodium acetate buffer (pH 5.0) is preheated at 40 ° C. for 5 minutes, and 0.1 ml of the enzyme solution (1) is added. After reacting at 40 ℃ for 30 minutes, DN
After adding 1 ml of S solution and boiling at 100 ° C. for 5 minutes, it was rapidly cooled in ice water, diluted with 4.5 ml of water, and the absorbance was measured at a wavelength of 525 nm. The amount of glucose produced by the reaction was taken as the enzyme activity amount. As the unit of enzyme activity, the amount of enzyme that liberates 1 μmol glucose per minute was defined as 1 unit.

[0043]

According to the present invention, the N-terminal and C of the present invention are
GAase with low GTase activity was obtained by culturing Aspergillus niger containing recombinant DNA in which a GTase structural gene having a terminal deletion was incorporated in a medium.
Can contribute to the starch industry.

[0044]

[Sequence list]

SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid sequence Phe Glu Ile Pro Leu Glu Tyr Ile Trp Thr Asp Ile Asp Tyr Met 15 AB His Gly Tyr Arg 19

SEQ ID NO: 2 Sequence length: 20 Sequence type: Amino acid sequence Leu X Glu Ser Gly Arg Tyr Tyr Val Pro Ile Val Asp Ala Ala 15 Leu Tyr Ile Pro Asn 20 (X cannot be determined) Show.)

[0046] SEQ ID NO: 3 Length of sequence: 4300 SEQ type: nucleic acid sequence GCATGCCATG AGTTGTTGGG CCGGCCTGAA GGATCCATCA TTGGGACCAA GGGCATCATC 60 CATGCGCTAC GGAGTACTTT CGGAGAATCA GCACCCCTGC ACAAAGCATT GTCAATGTGT 120 TTTCTTATGT CAAAAGCTGA CAGAGTCTGA GGCTCGCTGA CGATGGGATT CATGCTAATG 180 ACGGTCCGAA AGAGCTTTCA CGTAACACTG GTGAACATCC CACTCGGGAA GCCGAGACTT 240 GTGACCTACT TAGTCAAATG AGATGATTAT CAAAGCCATT AAATGCCTCG CTGTCAGGGG 300 CCCTGGTAAG TGTCTTCATT AATCGAAACC CATCTTCATT CGTCCCCGCC TTCAGTGCTC 360 ATCATTTTAG GTTTAGAAGC AAGATTGAGT GCCACCTGCT TTACAAACCA GCATGGGTAG 420 TCTGCTGTTG AAATTCTTCA CCGGGAGCAT TCTGGGGAAG GTGCAAAAGG CGGCGCGAAG 480 TGGTCGGGTC GCGATTGTAG TCTGGATTGG AGCACAAGAA TCGTCAGAGC CGAAGCCCGA 540 ACTGAGGGGG TCTCGGTCAT TTATCGGGAT GAGAGCCAAT CAGCGTGCGC TCATCATCTG 600 ATCGTCTGGC TGCCAGGCCC CTCAGGCATC AATACGGTAC TCGGCAGTAT CCACTCCCGT 660 TTCTCCGGTG CAACAAATCA TCGTTGGAGA ATCCCCAGCT CCCCCGCCAA CTGGGGTCGA 720 TGCTTCTCCA GTTGTCCTGG TTTCTCCCAT GAACTCGCTT ACGATAAGCT GCTGTACCA G 780 CCCACCAGCA CAACAATATC TTCAATCAGG TAGGTGCTTG TTCGTTACCT GCCCCATCCT 840 CTCCTCTTCT TCGGTCATTA TGAACTCAAT TCGGTCGCTA GCTTTGCCGA TTCTCCGCAG 900 TCCATAAAAA TATATCTGCA TTTGCCCCTT ACACGTCGGG AATTCACCGG CGCAATGAGC 960 CTTCGGGTAT GGTCGCACAG CGTCATGTCA ATAGGAGGCT GCTCCTAGTG GTGATCTACT 1020 AGTCGCCTCA ACACAGCAAT ATATAAATAA CAAGAGCATT CCTTGAGCAC ATCTGGGTAA 1080 TAGCTGTTCC ATTCTCATCA AGGATTACGC GACCGTGCCT CGAGCCTCCT TAAGCGAGCC 1140 ATG GTG AAG TTG ACG CAT CTC CTT GCC AGA GCA TGG CTT GTC CCT CTG 1188 Met Val Lys Leu Thr His Leu Leu Ala Arg Ala Trp Leu Val Pro Leu GCT TAT GGA GCG AGC CAG TCA CTC TTA TCC ACC ACT GCC CCT TCG CAG 1236 Ala Tyr Gly Ala Ser Gln Ser Leu Leu Ser Thr Thr Ala Pro Ser Gln CCG CAG TTT ACC ATT CCT GCT TCC GCA GAT GTC GGT GCG CAG CTG ATT 1284 Pro Gln Phe Thr Ile Pro Ala Ser Ala Asp Val Gly Ala Gln Leu Ile GCC AAC ATC GAT GAT CCT CAG GCT GCC GAC GCG CAG TCG GTT TGT CCG 1332 Ala Asn Ile Asp Asp Pro Gln Ala Ala Asp Ala Gln Ser Val Cys Pro GGC TAC AAG GCT TCA AAA GTG CAG CAC AAT TCA CGT GGA TTC ACT GCC 1380 Gly Tyr Lys Ala Ser Lys Val Gln His Asn Ser Arg Gly Phe Thr Ala AGT CTT CAG CTC GCG GGC AGG CCA TGT AAC GTA TAC GGC ACA GAT GTT 1428 Ser Leu Gln Leu Ala Gly Arg Pro Cys Asn Val Tyr Gly Thr Asp Val GAG TCC TTG ACA CTG TCT GTG GAG TAC CAG GAT TCG GAT CGA CTG AAT 1476 Glu Ser Leu Thr Leu Ser Val Glu Tyr Gln Asp Ser Asp Arg Leu Asn ATT CAG ATT CTC CCC ACT CAT GTT GAC TCC ACA AAC GCT TCT TGG TAC 1524 Ile Gln Ile Leu Pro Thr His Val Asp Ser Thr Asn Ala Ser Trp Tyr TTT CTT TCG GAA AAC CTG GTC CCC AGA CCC AAG GCT TCC CTC AAT GCA 1572 Phe Leu Ser Glu Asn Leu Val Pro Arg Pro Lys Ala Ser Leu Asn Ala TCT GTA TCC CAG AGC GAC CTT TTT GTG TCA TGG TCA AAT GAG CCG TCG 1620 Ser Val Ser Gln Ser Asp Leu Phe Val Ser Trp Ser Asn Glu Pro Ser TTC AAT TTC AAG GTG ATC CGA AAG GCT ACA GGC GAC GCG CTT TTC AGT 1668 Phe Asn Phe Lys Val Ile Arg Lys Ala Thr Gly Asp Ala Leu Phe Ser ACA GAA GGC ACT GTG CTC GTA TAT GAG AAT CAG TTC ATC GAA TTT GTG 1716 Thr Glu Gly Thr Val Leu Val Tyr Glu Asn Gln Phe Ile Glu Phe Val ACC GCG CTC CCT GAA GAA TAT AAC TTG TAT GGC CTT GGG GAG CAT ATC 1764 Thr Ala Leu Pro Glu Glu Tyr Asn Leu Tyr Gly Leu Gly Glu His Ile ACG CAA TTC CGC CTC CAG AGA AAT GCT AAT CTG ACC ATA TAT CCT TCG 1812 Thr Gln Phe Arg Leu Gln Arg Asn Ala Asn Leu Thr Ile Tyr Pro Ser GAT GAT GGA ACA CCT ATT GAC CA G TGAGTACTGA TATCCCGCCC GTATCTTCTG 1866 Asp Asp Gly Thr Pro Ile Asp Gln GTTCTACTCTTCTAGAACT GGC 1908 Asn Leu Tyr Gly CAA CAT CCC TTC TAT CTG GAT ACA AGA TAT TAC AAA GGA GAT AGG CAG 1956 Gln His Pro Phe Tyr Leu Asp Thr Arg Tyr Tyr Lys Gly Asp Arg Gln AAT GGG TCT TAT ATT CCC GTC AAA AGC AGC GAG GCT GAT GCC TCG CAA 2004 Asn Gly Ser Tyr Ile Pro Val Lys Ser Ser Glu Ala Asp Ala Ser Gln GAT TAT ATC TCC CTC TCT CAT GGC GTG TTT CTG AGG AAC TCT CAT GGA 2052 Asp Tyr Ile Ser Leu Ser His Gly Val Phe Leu Arg Asn Ser His Gly CTT GAG ATA CTC CTC CGG TCT CAA AAA TTG ATC TGG CGG ACC CTA GGT 2100 Leu Glu Ile Leu Leu Arg Ser Gln Lys Leu Ile Trp Arg Thr Leu Gly GGA GGA ATC GAT CTC ACC TTC TAC TCA GGC CCC GCC CCG GCC GAT GTT 2148 Gly Gly Ile Asp Leu Thr Phe Tyr Ser Gly Pro Ala Pro Ala Asp Val ACC AGG CAA TAT CTT ACC AGC ACT GTG GGA TTA CCG GCC ATG CAG CAA 2196 Thr Arg Gln Tyr Leu Thr Ser Thr Val Gly Leu Pro Ala Met Gln Gln TAC AAC ACT CTT GGA TTC CAC CAA TGT CGT TGG GGC TAC AAC AAC TGG 2244 Tyr Asn Thr Leu Gly Phe His Gln Cys Arg Trp Gly Tyr Asn Asn Trp TCG GAT CTG GCG GAC GTT GTT GCG AAC TTT GAG AAG TTT GAG ATC CCG 2292 Ser Asp Leu Ala Asp Val Val Ala Asn Phe Glu Lys Phe Glu Ile Pro TTG GAA TAT ATC TG G TGCGTATTGT ACTGGTTTAT GGTATCTCAA AACAGTCTAA 2347 Leu Glu Tyr Ile Trp CAGGCACTT AG G ACC GAT ATT GAC TAC ATG CAC GGA TAT CGC AAC TTT 2395 Thr Asp Ile Asp Tyr Met His Gly Tyr Arg Asn Phe GAC AAC GAT CAA CAT CGC TTT TCC TAC AGT GAG GGC GAT GAA TTT CTC 2443 Asp Asn Asp Gln His Arg Phe Ser Tyr Ser Glu Gly Asp Glu Phe Leu AGC AAG CTA CAT GAG AGT GGA CGC TAC TAT GTA CCC ATT GTT GAT GCG 2491 Ser Lys Leu His Glu Ser Gly Arg Tyr Tyr Val Pro Ile Val Asp Ala GCG CTC TAC ATT CCT AAT CCC GAA AAT GCC TCT GAT GC G TAAGTGTCTA 2540 Ala Leu Tyr Ile Pro Asn Pro Glu Asn Ala Ser Asp Ala GTGACAAATT ATATTACTGC CTGTATGCTA ATTAGCGATA CAG A TAC GCT ACG TAT 2596 Tyr Ala GGA GAC ACT GAC GTC TTC CTC AAG AAT CCC GAT GGT AGC 2644 Asp Arg Gly Ala Ala Asp Asp Val Phe Leu Lys Asn Pro Asp Gly Ser CTC TAT ATT GGA GCC GTT TGG CCA GGA TAT ACA GTC TTC CCC GAT TGG 2692 Leu Tyr Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Phe Pro Asp Trp CAT CAT CCC AAG GCA GTT GAC TTC TGG GCT AAC GAG CTT GTT ATC TGG 2740 His His Pro Lys Ala Val Asp Phe Trp Ala Asn Glu Leu Val Ile Trp TCG AAG AAA GTG GCG TTC GAT GGT GTG TGG TAC GAC ATG TCT GAA GTT 2788 Ser Lys Lys Val Ala Phe Asp Gly Val Trp Tyr Asp Met Ser Glu Val TCA TCC TTC TGT GTC GGG AGC TGT GGC ACA GGT AAC CTG ACT CTG AAC 2836 Ser Ser Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn CCG GCA CAC CCA TCG TTT CTT CTC CCC GGT GAG CCT GGT GAT ATC ATA 2884 Pro Ala His Pro Ser Phe Leu Leu Pro Gly Glu Pro Gly Asp I le Ile TAT GAT TAC CCA GAG GCT TTC AAT ATC ACC AAC GCT ACA GAG GCG GCG 2932 Tyr Asp Tyr Pro Glu Ala Phe Asn Ile Thr Asn Ala Thr Glu Ala Ala TCA GCT TCG GCG GGA GCT TCC AGT CAG GCT GCA GCA ACC GCG ACC ACC 2980 Ser Ala Ser Ala Gly Ala Ser Ser Gln Ala Ala Ala Thr Ala Thr Thr ACG TCG ACT TCG GTA TCA TAT CTG CGG ACA ACG CCC ACG CCT GGT GTC 3028 Thr Ser Thr Ser Val Ser Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val CGC AAT GTT GAG CAC CCA CCC TAT GTG ATC AAC CAT GAC CAA GAA GGC 3076 Arg Asn Val Glu His Pro Pro Tyr Val Ile Asn His Asp Gln Glu Gly CAT GAT CTC AGT GTC CAT GCG GTG TCG CCG AAT GCA ACG CAT GTT GAT 3124 His Asp Leu Ser Val His Ala Val Ser Pro Asn Ala Thr His Val Asp GGT GTT GAG GAG TAT GAT GTG CAC GGT CTC TAC GGA CAT CAA GGA TTG 3172 Gly Val Glu Glu Tyr Asp Val His Gly Leu Tyr Gly His Gln Gly Leu AAC GCT ACC TAC CAA GGT CTG CTT GAG GTC TGG TCT CAT AAG CGG CGG 3220 Asn Ala Thr Tyr Gln Gly Leu Leu Glu Val Trp Ser His Lys Arg Arg CCA TTT ATT ATT GGC CGC TCA ACC TTC GCT GGC TCT GGC AAA TGG GC A 3268 Pro Phe Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala GGC CAC TGG GGC GGC GAC AAC TAT TCC AAA TGG TGG TCC ATG TAC TAC 3316 Gly His Trp Gly Gly Asp Asn Tyr Ser Lys Trp Trp Ser Met Tyr Tyr TCC ATC TCG CAA GCC CTC TCC TTC TCA CTT TTC GGC ATT CCG ATG TTT 3364 Ser Ile Ser Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile Pro Met Phe GGT GCG GAC ACC TGT GGG TTT AAC GGA AAC TCC GAT GAG GAG CTC TGC 3412 Gly Ala Asp Thr Cys Gly Phe Asn Gly Asn Ser Asp Glu Glu Leu Cys AAC CGA TGG ATG CAA CTG TCC GCA TTC TTC CCA TTC TAC CGA AAC CAC 3460 Asn Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His AAT GAG CTC TCC ACA ATC CCA CAG GAG CCT TAT CGG TGG GCT TCT GTT 3508 Asn Glu Leu Ser Thr Ile Pro Gln Glu Pro Tyr Arg Trp Ala Ser Val ATT GAA GCA ACC AAG TCC GCC ATG AGA ATT CGG TAC GCC ATC CTA CCT 3556 Ile Glu Ala Thr Lys Ser Ala Met Arg Ile Arg Tyr Ala Ile Leu Pro TAC TTT TAT ACG TTG TTT GAC CTG GCC CAC ACC ACG GGC TCC ACT GTA 3604 Tyr Phe Tyr Thr Leu Phe Asp Leu Ala His Thr Thr Gly Ser Thr Va l ATG CGC GCA CTT TCC TGG GAA TTC CCT AAT GAC CCA ACA TTG GCT GCG 3652 Met Arg Ala Leu Ser Trp Glu Phe Pro Asn Asp Pro Thr Leu Ala Ala GTT GAG ACT CAA TTC ATG GTT GGG CCG GCC ATC ATG GTG GTC CCG GTA 3700 Val Glu Thr Gln Phe Met Val Gly Pro Ala Ile Met Val Val Pro Val TTG GAG CCT CTG GTC AAT ACG GTC AAG GGC GTA TTC CCA GGA GTT GGA 3748 Leu Glu Pro Leu Val Asn Thr Val Lys Gly Val Phe Pro Gly Val Gly CAT GGC GAA GTG TGG TAC GAT TGG TAC ACC CAG GCT GCA GTT GAT GCG 3796 His Gly Glu Val Trp Tyr Asp Trp Tyr Thr Gln Ala Ala Val Asp Ala AAG CCC GGG GTC AAC ACG ACC ATT TCG GCA CCA TTG GGC CAC ATC CCA 3844 Lys Pro Gly Val Asn Thr Thr Ile Ser Ala Pro Leu Gly His Ile Pro GTT TAT GTA CGA GGT GGA AAC ATC TTG CCG ATG CAA GAG CCG GCA TTG 3892 Val Tyr Val Arg Gly Gly Asn Ile Leu Pro Met Gln Glu Pro Ala Leu ACC ACT CGT GAA GCC CGG CAA ACC CCG TGG GCT TTG CTA GCT GCA CTA 3940 Thr Thr Arg Glu Ala Arg Gln Thr Pro Trp Ala Leu Leu Ala Ala Leu GGA AGC AAT GGA ACC GCG TCG GGG CAG CTC TAT CTC GAT GAT GGA GAG 398 8 Gly Ser Asn Gly Thr Ala Ser Gly Gln Leu Tyr Leu Asp Asp Gly Glu AGC ATC TAC CCC AAT GCC ACC CTC CAT GTG GAC TTC ACG GCA TCG CGG 4036 Ser Ile Tyr Pro Asn Ala Thr Leu His Val Asp Phe Thr Ala Ser Arg TCA AGC CTG CGC TCG TCG GCT CAA GGA AGA TGG AAA GAG AGG AAC CCG 4084 Ser Ser Leu Arg Ser Ser Ala Gln Gly Arg Trp Lys Glu Arg Asn Pro CTT GCT AAT GTG ACG GTG CTC GGA GTG AAC AAG GAG CCC TCT GCG GTG 4132 Leu Ala Asn Val Thr Val Leu Gly Val Asn Lys Glu Pro Ser Ala Val ACC CTG AAT GGA CAG GCC GTA TTT CCC GGG TCT GTC ACG TAC AAT TCT 4180 Thr Leu Asn Gly Gln Ala Val Phe Pro Gly Ser Val Thr Tyr Asn Ser ACG TCC CAG GTT CTC TTT GTT GGG GGG CTG CAA AAC TTG ACG AAG GGC 4228 Thr Ser Gln Val Leu Phe Val Gly Gly Leu Gln Asn Leu Thr Lys Gly GGC GCA TGG GCG GAA AAC TGG GTA TTG GAA TGG TAGTGTCAGC CACAAGCCAG 4281 Gly Aly Glu Asn Trp Val Leu Glu Trp * GTGTGCGCGTACAGCATGC 4300

[Brief description of drawings]

FIG. 1 shows the plasmid pGTID1. (A) in the figure shows the full length of the 4.3 kb SphI fragment of FIG. 1, and (b) in the figure shows the structure of pGTID1. In addition, Sp is SphI and B is
BamHI, Hc is HincII, Pv is PvuI
I, SB indicates SnaBI, E indicates EcoRI, and Sc indicates SacI.

FIG. 2 shows the time course of GTase production in transformants. In the figure, the black triangle indicates TRD1-24, and the square indicates TR19-1.

FIG. 3 shows the time course of GAase production of transformants. In the figure, the black triangle indicates TRD1-24, and the square indicates TR19-1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12N 15/56 C12R 1: 685) (72) Inventor Ikuko Nishimura 2-18 Nishikata, Bunkyo-ku, Tokyo −15 Maison de Istoire Room 302

Claims (3)

[Claims] 1. Aspergillus
s) A DNA fragment in which the N-terminal and C-terminal of the glucosyltransferase structural gene derived from a microorganism belonging to the genus are deleted in an arbitrary length. 2. Aspergillus
s) A glucosyltransferase structural gene derived from a microorganism belonging to the genus and defined by the restriction map of FIG. 1 (b) and having N-terminal and C-terminal deletions. 3. An Aspergillus niger having a glucoamylase-producing ability, into which a recombinant DNA incorporating the DNA containing the glucosyltransferase structural gene according to claim 1 or 2 has been introduced, is cultured in a nutrient medium and cultured. A method for producing glucoamylase having a low glucosyltransferase activity, which comprises collecting glucoamylase in a product and then collecting glucoamylase from the culture.