JP3044325B2 - Glucosyltransferase gene and method for producing glucosyltransferase using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to glucosyltransferase (hereinafter referred to as GTas
e. The present invention relates to a method for producing GTase by a microorganism into which a recombinant DNA incorporating a gene and a DNA containing the gene has been introduced.

[Prior art] Conventionally, Aspergillus nige
The structure of the GTase gene derived from r) is not known at all, and the gene has not been isolated. Furthermore, a method for producing GTase using a microorganism into which the gene has been introduced has not been performed.

GTase is an enzyme that catalyzes the action of transferring the glucosyl group of maltose to position 6 or 3 of glucose or an oligosaccharide composed of glucose, and has an enzyme number of EC 2.4.1.24.
are categorized. This enzyme is used in the starch industry.

[Problems of the prior art] The present inventors aimed at providing a method for producing GTase by genetic manipulation, and as a result of intensive studies on the GTase gene derived from Aspergillus niger, the inventors found that the GTase gene derived from this strain was the first to be obtained. The present inventors have succeeded in producing GTase by isolating, analyzing and incorporating it into other microorganisms, and completed the present invention.

[Means and Actions for Solving the Problems] The present invention is derived from a microorganism belonging to the genus Aspergillus, and is defined by the restriction map in FIG.
This is a method for producing GTase using a microorganism into which a GTase gene and a recombinant DNA incorporating the gene have been introduced.

A part of the GTase gene used in the present invention has the DNA sequence shown in FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail as a GTase-producing bacterium such as Aspergillus or Penicillium. As a source of the chromosomal DNA containing the GTase gene of the present invention, the above-mentioned GTase-producing bacteria are used. For example, Aspergillus niger
rgillus niger) No. 499.

 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. The basal mycelium is relatively dense. The colony surface is velvety. The color of the colony is initially white and becomes brown to black when a large number of conidia are formed. The back of the colony is initially colorless and later pale yellow.

(B) Tuapec agar medium Growth is good at 37 ° C. The basal hypha layer is relatively thin and flat. The colony surface is velvety to wool-like. The color of the colony is initially white and becomes 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: 4-7 Temperature: 25-35 ° C (b) Growth range (using malt extract medium) pH: 3-8 Temperature: 10-45 ° C (3) Morphological properties Conidial head: 200-500μ, black.

Conidiophores: length 500μ ~ 3mm, diameter 15 ~ 2μ.

Branches and rises from basal or aerial hyphae. Smooth colorless.

Capsule: 50-70μ in diameter, spherical.

Metre: about 25 × 5.3μ Friedride: about 11 × 3μ.

Conidia: 3.0-4.5μ in diameter, spherical, rough surface, agglomerated black.

Based on the above mycological properties, this strain belongs to the genus Aspergillus. In addition, conidial heads with metres are mixed, conidial heads are spherical, torn when old, conidiophores do not constrict just below the top, conidial heads are black, conidiophores are smooth, vertical The strain was identified as Aspergillus niger by splitting. This bacterium has been deposited with the National Institute of Microbial Industry and Technology as National Institute of Microorganisms No. 11316.

The above strain is cultured by an ordinary culture method to obtain a culture, and a culture solution is obtained from the culture by a conventional method, for example, treatment such as filtration or centrifugation.

GTase is purified from the culture solution of Aspergillus niger obtained above by a conventional method, for example, ammonium sulfate salting out, centrifugation, desalting and various types of chromatography.

Purified GTase is limitedly degraded with trypsin or the like, and the limitedly degraded peptide is separated by HPLC or the like.
The terminal amino acid sequence can be determined by, for example, a known literature [Eur. J. Biochem.,
1, 80-91 (1967)], using an automatic amino acid sequencer. A DNA having a base sequence corresponding to this amino acid sequence is synthesized. Synthetic D
NA can be made using, for example, an automatic DNA synthesizer. Labeling of synthetic DNA can be performed, for example, according to a known literature [Proc. Natl. Aca
USA, 74, 560-564 (1977)], and phosphorylating the 5 'end with γ- ³² P-ATP using T4 polynucleotide kinase. Thus, a probe is prepared.

Separately, Aspergillus niger is cultured, and the cells are disrupted 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), Cold Spring Harbor Laboratory Press
According to the method described in 1989, 9.34 to 9.58], the chromosomal DN
A 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. That is, DNA is adsorbed to a nitrocellulose filter and labeled
The NA probe is hybridized and an autoradiogram is taken. The restriction enzymes used include SphI and the like.

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

Next, in order to perform colony hybridization, the recovered DNA was incorporated into
Is prepared. Integration of chromosomal DNA into vector DNA
Known literature [J. Mol. Biol., 96, 171-184 (1975)]
The method can be carried out by cleaving the chromosomal DNA and the vector DNA with a restriction enzyme according to the method described in (1) and then ligating them with ligase. As the vector DNA, for example, a plasmid DNA is mentioned, and particularly, pBR322 and pUC19 are preferable. Examples of the ligase include T4 DNA ligase.

Introduction of recombinant DNA into Escherichia coli can be performed, for example, according to the known literature [Mol
ecular Cloning (2nd Edition), Cold Spring H
arbor Laboratory Press 1989, 1.82 to 1.84]. The E. coli used was Escherichia coli HB101.
Strains are preferred. Selection of a strain containing a recombinant DNA containing the GTase gene can be performed, for example, by the method described in the known literature [Molecular Cloning].
(2nd Edition), publishing house Cold Spring Harbor Laborato
ry Press 1989, 1.90-1.104].
It can be performed by colony hybridization using the synthetic DNA as a probe. That is, recombinant DNA
Was spread on an L-broth agar medium containing ampicillin, cultured overnight, and then replicated on a nitrocellulose filter, followed by further addition of L-broth containing ampicillin for 2 to 3 hours.
Culturing on a broth agar medium, lysing and fixing the DNA to detect positive colonies to which the synthetic DNA hybridizes;

Next, from a positive strain, for example, a known literature [Molecular Cl
oning (2nd Edition), Cold Spring Harbor Lab
oratory Press 1989, 1.25 to 1.28], extracting and purifying the plasmid, decomposing it with various restriction enzymes, preparing a restriction enzyme map, and again examining that the hybridized DNA probe hybridizes by Southern hybridization. Confirm.

Next, a part of the base sequence to be hybridized with the synthetic DNA probe is determined.

Furthermore, a large-scale expression system for GTase will be established. For example, a commonly used expression system can be used for the transformation system. In particular, a transformation system has been established, and S-similar to Aspergillus niger
Aspergillus nidulan, which can be expected to plicing and adding and secreting sugar
s is preferred.

PSa123 can be used as a transformation vector. this is
Aspergillus nidulans arginine-requiring complement gene ar
Have gB. A plasmid in which a gene fragment is ligated is constructed. This is introduced into an arginine-requiring mutant of Aspergillus nidulans, and transformation is performed. The transformed strain is cultured to produce GTase, and GTase is collected from the culture.

Examples (1) Purification of GTase and determination of partial amino acid sequence Aspergillus niger No. 49
1 (1/3) of spore slant of 9 (Microtechnical Laboratory No. 11316)
Of CM-CSL medium (corn meal is liquefied with amylase, and corn steep liquor is added to a 2% solution thereof at a final concentration of 5%).
% And sterilized by autoclaving at 121 ° C. for 30 minutes) and shaking culture at 30 ° C. for 5 to 6 days. The cells are removed by filtration using 3MM filter paper (manufactured by Whatman), and ammonium sulfate is added to the culture solution so as to be 50% saturated. The resulting precipitate is then reduced to 10,000 rp.
The mixture is removed by centrifugation at m for 30 minutes, and ammonium sulfate is further added to the supernatant so as to be 90% saturated. Next, the precipitated fraction was
Collect by centrifugation at 000 rpm for 30 minutes and dissolve in 10 mM acetate buffer (pH 6.0). After insoluble matter was removed by centrifugation at 15,000 rpm for 30 minutes, the supernatant was filtered through a 0.45 μm filter, desalted and gel-filtered with Toyopearl HW-40C, and 10 mM acetate buffer (pH 6.0). Was carried out.

The thus obtained crude enzyme solution was separated using an FPLC system (manufactured by Pharmacia) and anion exchange chromatography using a Mono Q HR 10/10 column. Elution was performed with 0-500 mM NaCl in 10 mM acetate buffer (pH 6.0).
This was done by forming a linear gradient. Furthermore,
Gel filtration chromatography using a Superose 12 column from a fraction having GTase activity [100 mM acetate buffer (pH
6.0), 200 mM NaCl] to purify GTase.

Next, trypsin is added to 100 mg of purified GTase
μg was added, and the mixture was allowed to act at 37 ° C. for 5 hours in 100 mM Tris-HCl (pH 8.5) and 0.001% sodium dodecyl sulfate (SDS) to perform limited degradation of GTase. Further, the reaction solution was
It was applied to 8 columns (VP-318-2251, manufactured by Senshu Kagaku KK), and the limited peptide of GTase was fractionated by a 0-80% acetonitrile linear gradient by high performance liquid chromatography in the presence of 0.1% trifluoroacetic acid. Some N-termini of the fractionated peptides were determined by Edman degradation using a model 470A type (manufactured by Applied Biosystems) amino acid sequencer. The determined amino acid sequence is shown below.

(Note that X indicates an amino acid that could not be determined.) (2) Identification of GTase gene A portion of the amino acid sequence of GTase which is considered to have high specificity when converted to a DNA base sequence was searched for. Two synthetic DNAs having sequences corresponding to the A part and the B part of the amino acid sequence were synthesized using a 380B type (manufactured by Applied Biosystems) DNA synthesizer. The synthesized sequence is shown below.

(However, R indicates A or G, Y indicates C or T, M indicates A, C or T, and N indicates A, C, G or T.) The synthetic DNA solution is treated at 65 ° C. for 12 hours to remove the protecting group. After that, the mixture was concentrated to about 100 μl using an N-1 type rotary evaporator (manufactured by Tokyo Rika Co., Ltd.), 10 μl of 3M sodium acetate (pH 4.8) and 250 μl of ethanol were added, and the mixture was allowed to stand at −80 ° C. for 30 minutes, and then centrifuged. Then, ethanol precipitation was performed. 0.5 μg of the DNA thus obtained
In a solution containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 10 units of T4 DNA kinase, 1.85 MBq of (γ- ³² P-) ATP (PB1021 manufactured by Amersham)
8) was incubated at 37 ° C. for 30 minutes to label the 5 ′ end to obtain a probe.

Next, Aspergillus niger N
o.499 in YPD medium (2% glucose, 1% peptone, 0.5%
% Yeast extract) at 30 ° C. for 30 hours, followed by filtration with gauze to collect the cells, and the cells were frozen with liquid nitrogen. The frozen cells are homogenizer (AM-8, Nippon Seiki Seisakusho)
At 18,000 rpm for 15 minutes. Then 5
0 mM EDTA (pH 8.0), 0.5% SDS, and 0.1 mg / ml proteinase K (final concentration) were added, and the mixture was incubated at 50 ° C. for 4 hours. Further, TE (10 mM Tris-HCl, 1 mM
(EDTA, pH 8.0) An equivalent amount of saturated phenol was added, and the mixture was stirred gently and centrifuged at 15,000 rpm for 30 minutes to separate the aqueous phase. After performing the same treatment once again, instead of TE-saturated phenol, TE-saturated phenol and chloroform were mixed at a ratio of 1: 1 and then the same treatment was performed twice using chloroform. 50% of the nucleic acid crude extract thus obtained
Add 1/4 volume of polyethylene glycol # 6000, 0 ℃, 2
Incubated for hours. After collecting the precipitated fraction by centrifugation at 3,000 rpm for 3 minutes, 8 ml of TES buffer (20 mM Tris-H
Cl, 5 mM EDTA, 100 mM NaCl, pH 7.5)
Add CsCl, add another 5 mg of ethidium bromide to 44,500 rp
m, 16 h CsCl-ethidium bromide equilibrium density gradient centrifugation. The DNA band formed by this centrifugation was collected, and dialyzed against TE except for ethidium bromide using 1-butanol. With this method, 100 g of wet cells
4.5 mg of chromosomal DNA was obtained.

Complete degradation was carried out by allowing 30 units of the restriction enzyme SphI to act at 37 ° C. for 3 hours on 10 μg of the chromosomal DNA of Aspergillus niger No.499 obtained as described above. 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 trisodium cit
rate), 0.1% SDS, 0.2% bovine serum albumin, 0.1%
Pelle hybridization was carried out by incubating the filter at 65 ° C. for 8 hours in 2% Ficoll 400 and 0.2% polyvinylpyrrolidone.
Hybridization was carried out by allowing the mixture to stand at 0 ° C. overnight. The filters were then washed in 6 × SSC, 0.1% SDS at 52 ° C. for 30 minutes and analyzed by autoradiography. As a result, it was observed that both types of probes hybridized to the 4.3 Kb position.

(3) Cloning of GTase gene Aspergillus niger No.49
After 5 μg of 9 chromosomal DNA was completely digested with 10 units of SphI, it was subjected to 1% agarose gel electrophoresis, stained with ethidium bromide, cut off at a position corresponding to about 4.3 Kb, and 3 times the volume of 8N NaClO Add ₄ and 37 ℃, 10
Incubate for a minute to dissolve the agarose. The DNA in the solution was then adsorbed to a 6 mm diameter GF / C (Whatman) glass filter, and the filter was washed with a solution of 6N NaClO ₄ in 1 ml of TE, followed by 1 ml of 95% ethanol, Let dry. Add 30 μl of TE to this filter and add
Incubated at 30 ° C for 30 minutes. Then 15,000rpm, 2
The aqueous phase was collected by centrifugation for 1 minute. In this way, the agarose gel is converted to Aspergillus niger.
ger) A SphI fragment near 4.3 Kb derived from No.499 chromosomal DNA was recovered. Separately, 0.1 μg of vector plasmid pBR322
The mixture was treated with 5 units of SphI at 37 ° C. for 2 hours, and further treated with 1M Tris-HCl (p
H9.0) was added to the reaction solution at a 1/5 volume, and 0.5 unit of alkaline phosphatase was added thereto, followed by incubation at 65 ° C for 30 minutes. The reaction mixture was subjected to agarose gel electrophoresis and brominated.
After ethidium staining, a band corresponding to the molecular weight of pBR322 was cut out and collected. The above two DNAs were mixed and 66 mM Tr
Ligation reaction was performed by adding 300 units of T4 DNA ligase with is-HCl (pH 7.6), 6.6 mM MgCl ₂ , 10 mM dithiothreitol, and 1 mM ATP (final concentration), and incubating at 4 ° C. overnight. Next, competent cells of Escherichia coli HB101 were prepared and transformed using the above ligation reaction product. The transformed strain was aspicillin 50 μg / ml, 1.2
Probe on a nitrocellulose filter after selection on an LB medium containing 1% agar (1% Bacto Tryptone (Difco), 0.5% yeast extract, 0.5% NaCl).
Was used for colony hybridization. However, hybridization and washing conditions were the same as in the case of Southern hybridization. As a result of analysis by autoradiography, 24 positive clones out of about 1,000 transformants were obtained. Plasmid DNA was prepared from these transformants and 0.1 μg each.
5 units of each of the restriction enzymes shown in Table 1 were allowed to act for 2 hours at 37 ° C., and analyzed by agarose gel electrophoresis. As a result, all of the plasmids were converted to the 4.3 Kb DNA fragment shown in FIG. At the SphI site. This plasmid
It was named pGTY02.

(4) Determination of partial nucleotide sequence of GTase gene Plasmid pGTY02 was digested with various restriction enzymes, agarose gel electrophoresis was performed, and Southern hybridization using probes A and B was performed. The results showed that both probes hybridized in the 0.5 Kb EcoRV fragment. Therefore, a DNA fragment of about 1.6 Kb obtained by treating pGTY02 with PvuII and EcoRI was excised and recovered from an agarose gel, and subjected to a ligation reaction with a separately treated plasmid pUC118 treated with HincII and EcoRI. In addition, pGTY02 is replaced with SalI, EcoRI
A DNA fragment of about 2.5 Kb obtained by the treatment was excised and recovered from the agarose gel, and separately treated with SalI and EcoRI.
A ligation reaction was performed with UC118. Escherichia coli MV1184 strain was transformed using these reaction solutions, and a plasmid containing the correct insert fragment in the vector was selected by digestion with various restriction enzymes. The plasmid thus obtained was subjected to a kilosequence deletion kit (Takara Shuzo Co., Ltd.) to transform E. coli MV1184,
Clones containing plasmids deleted at various lengths of about 200 bases each were selected. Single-stranded DNA was prepared from Escherichia coli MV1184 carrying these plasmids, and a part of the nucleotide sequence was determined using a DNA sequencing kit (Takara Shuzo). The result is shown in FIG. The thick underlined portion in FIG. 2 is a portion that matches the nucleotide sequence of the probe, and the enclosed amino acid sequence is a portion that matches the determined amino acid sequence. SphI DNA cloned from the above results
Fragment is Aspergillus niger N
It was shown to contain the GTase gene from o.499. In addition, it was shown that an intron consisting of 51 bases (a part of the base sequence indicated by the underlined dashed line) exists in the hybridizing part of the probe A in view of the amino acid sequence and the nucleic acid sequence.

(5) Expression of GTase in Aspergillus nidulans 4.3 Kb using XbaI at EcoRI site of vector pSa123
The plasmid pSGTx01 ligated with the DNA fragment was constructed. It was introduced into an arginine auxotroph of Aspergillus nidulans and transformed. As a result, four transformed strains which grew well in an arginine-minus medium and formed spores were obtained.

SDS-PAGE was performed on the culture supernatant of this transformant, and it was confirmed that a protein having the same molecular weight as GTase of Aspergillus niger was secreted in the three strains.

 The GTase activity is shown in Table 2.

These three strains showed GTase activity of 4 to 5 times in total activity and about 3 times in specific activity as compared with the transformant using pSa123 alone.

Therefore, from the transformed strain into which the plasmid pSGTx01 was introduced, a strain secreting a protein having the same molecular weight as GTase of aspergillus niger was selected, and it was confirmed that the strain exhibited effective GTase activity as shown in Table 2. Under the confirmation, such a transformant can be cultured to collect GTase from the culture.

[Effects of the Invention] According to the present invention, GTase can be obtained extremely efficiently by culturing a microorganism containing a recombinant DNA into which the GTase gene of the present invention is incorporated, for example, by culturing the microorganism in a medium,
Further, the above gene can be used as a sample for protein engineering and greatly contributes to industry.

[Brief description of the drawings]

FIG. 1 shows a glucosyltransferase gene derived from a microorganism belonging to the genus Aspergillus. (Where B is BamHI, EI is EcoRI, Ev is EcoRV, C is C
laI, S is SacI, Pv is PvuII, Ps is PstI, SI is SalI, Sp is
Shows SphI. FIG. 2 shows the partial nucleotide sequence of the glucosyltransferase gene. The thick underlined portion in the figure is a portion corresponding to the nucleotide sequence of the probe, and the enclosed amino acid sequence is a portion corresponding to the determined amino acid sequence. The underlined base sequence indicates an intron.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Haruhiko Masaki 1-170 Yayoi-cho, Chiba City, Chiba Prefecture 2-105 Staff dormitory 2-105 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/54 C12N 9/10 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A probe set based on a partial amino acid sequence of glucosyltransferase produced by Aspergillus niger, among DNA fragments obtained by cutting chromosomal DNA of Aspergillus niger with a restriction enzyme, hybridizes. A glucosyltransferase gene contained in a DNA fragment selectively recovered by using as an indicator whether or not the DNA fragment has a 4.3 kb base pair defined by the restriction map in FIG. 1, and A glucosyltransferase gene comprising the DNA sequence shown in FIG. 2. A probe set based on a partial amino acid sequence of glucosyltransferase produced by Aspergillus niger, among DNA fragments obtained by cutting chromosomal DNA of Aspergillus niger with a restriction enzyme, hybridizes. A glucosyltransferase gene contained in a DNA fragment selectively recovered using as an indicator whether or not the glucosyltransferase gene is transformed by a transformed microorganism of the genus Aspergillus into which a plasmid ligated with the recovered DNA fragment is introduced. Glucosyltransferase whose expression has been confirmed and which is contained in a DNA fragment having a 4.3 kb base pair defined by the restriction map in FIG. 1 and which comprises the DNA sequence shown in FIG. 2 gene. 3. A probe set based on a partial amino acid sequence of glucosyltransferase produced by Aspergillus niger among DNA fragments obtained by cutting chromosomal DNA of Aspergillus niger with a restriction enzyme, and hybridizing. Whether or not the expression of the glucosyltransferase gene is confirmed by a transformed microorganism of the genus Aspergillus obtained by introducing a plasmid to which the recovered DNA fragment is ligated. 1. A DNA fragment having a 4.3 kb base pair defined by the restriction map shown in FIG. 1 and containing a glucosyltransferase gene comprising the DNA sequence shown in FIG. 4. A probe set based on a partial amino acid sequence of glucosyltransferase produced by Aspergillus niger among DNA fragments obtained by cutting chromosomal DNA of Aspergillus niger with a restriction enzyme, to hybridize. A glucosyltransferase gene contained in a DNA fragment selectively recovered by using as an indicator whether or not 4.3 kDa is defined by the restriction map shown in FIG.
A glucosyltransferase gene having the base pair of b and containing the DNA sequence shown in FIG. 2 is introduced into a microorganism of the genus Aspergillus together with the DNA fragment to prepare a transformed microorganism. And producing glucosyltransferase from the culture, and collecting glucosyltransferase from the culture.