JP2657801B2 - Glycosyl transfer reaction method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a glycosyltransfer reaction method, and more particularly, to a polypeptide having a specific amino acid sequence and having cyclomaltodextrin glucanotransferase activity. A sugar transfer reaction characterized by acting on starch.

(Prior Art) Cyclomaltodextrin glucanotransferase (hereinafter abbreviated as CGT-ase in the present specification) is
It is also called macerans amylase, and has long been known as an enzyme produced by Bacillus macerans. In recent years, CGT-ase has been used in addition to Bacillus macerans as well as Bacillus stearothermophilus, Bacillus circulans, Bacillus megaterium, Bacillus polymyxia, Klebsiella emmonia pneumoniae ) Are also known to be produced by microorganisms.

However, CGT-ase produced from these microorganisms
Is known for only a part of its enzymatic properties and is still insufficient for widespread industrial use with peace of mind.In particular, when used for the production of food and drink, CGT- It is desired to use ase.

(Problems to be Solved by the Invention) In order to use CGT-ase with a sense of security in industry, the present inventors have studied CGT-ase which has been elucidated in more detail, especially CGT-ase which has been elucidated even to the amino acid sequence. The present inventors have intensively studied the use of a polypeptide having activity (hereinafter, simply referred to as a polypeptide in the present specification) in a method for producing a food or drink.

As a result, the polypeptide is expressed as a partial amino acid sequence: (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Th
r, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Ph
e-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Me
t-Asp-Arg-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Me
has at least one sequence selected from t-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly More specifically, the partial sequence has the partial sequences (a), (b), (c), (d), and (e) in order from the N-terminal side. There was found.

And its characteristic property is a polypeptide which produces cyclodextrin from soluble starch and has a molecular weight of 70,000 ± 10,000 by SDS-polyacrylamide gel electrophoresis, and has a specific activity of 200 ± 30 units / mg protein. .

In addition, it was found that the polypeptide derived from Bacillus stearothermophilus had the amino acid sequence shown in Table 2-1 as described later.

Furthermore, a polypeptide derived from Bacillus macerans
As will be described later, it was found to have the amino acid sequence shown in Table 5-1.

Furthermore, the polypeptide of the present invention whose amino acid sequence has been elucidated can act not only on starch and used in a glycosyltransfer reaction, but can also be incorporated into foods and drinks together with the glycosyltransferred product and used as it is without losing flavor. It turned out to be gained.

Hereinafter, the contents of the present invention will be described in detail, and the effects of the present invention will be described together. When abbreviations are used for amino acids, peptides, and the like in the description of the present specification, they are based on commonly used abbreviations in the art. Examples are listed below. In the case where an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

DNA: deoxyribonucleic acid RNA: liponucleic acid A: adenine T: thymine G: guanine C: cytosine dNTP: deoxynucleotide triphosphate ddNTP: dideoxynucleotide triphosphate dCTP: deoxycytidine triphosphate SDS: sodium dodecyl sulfate Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Gln: Glutamine Glu: Glutamic acid Gly: Glycine His: Histidine Ile: Isoleucine Leu: Leucine Lys; Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: tryptophan Tyr: tyrosine Val: valine In the present invention, the amino acid sequence of the polypeptide is CG
After cloning a polypeptide gene from a Tase-producing bacterium, its nucleotide sequence was decoded and determined.

On the other hand, the amino acid sequence of the portion containing the N-terminus of the polypeptide was examined using a gas-phase protein sequencer after purifying the polypeptide to high purity.

Cloning of polypeptide gene After separating and purifying the DNA of the microorganism from a donor microorganism capable of producing a polypeptide, for example, sonication, cutting with a restriction enzyme, etc., and cutting the vector in the same manner as the obtained DNA fragment The vector fragment obtained by
A ligase is used to form a recombinant DNA containing the polypeptide gene.

At this time, as the donor microorganism, a microorganism having a polypeptide producing ability, for example, JP-A-47-20373, JP-A-50-63189, JP-A-50-88290, and Hans Bender (Hans Bander), Archives of Microbiolog
y), Vol. 111, 271-282 (1977) and other Bacillus genus such as Bacillus macerans, Bacillus megaterium, Bacillus circulans, Bacillus polymixer, Bacillus stearothermophilus, and Clavesea genus such as Clavesea pneumoniae. Such as recent is appropriately selected.

Still, a transformed microorganism having a polypeptide-producing ability introduced by genetic recombination can also be used as a donor microorganism.

The DNA derived from the donor microorganism is prepared by, for example, culturing the donor microorganism in a liquid medium with aeration and stirring for about 1 to 3 days, collecting the resulting culture by centrifugation, and then lysing it. be able to. As the lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or ultrasonic treatment is used. If necessary, other enzyme agents such as protease, a surfactant such as sodium lauryl sulfate, and the like, and freezing and thawing treatment can be freely used.

In order to separate and purify DNA from the lysate thus obtained, conventional methods such as phenol extraction, protein removal, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation are appropriately combined. It can be carried out.

The method of cutting DNA can be carried out, for example, by sonication, treatment with a restriction enzyme, and the like.However, in order to facilitate binding of the obtained DNA fragment to a vector fragment, a restriction enzyme, particularly a specific nucleotide sequence, is used. Act, for example, EcoR I, Hind II, BamH I, Sal I, Sla I, Xma I,
Type II restriction enzymes such as Mbo I, Xba I, Sac I, Pat I are suitable.

A phage or plasmid capable of autonomous propagation in a host microorganism is suitable as a vector.

Examples of the phage include Escherichia coli (Ea
cherichia coli) is used as the host microorganism.
λC, λgt and λB are Bacillus subtilis (Baci
llus subtilis) as the host microorganism, ρ11,
φ1, φ105, etc. can be used.

Examples of the plasmid include pBR322 and pBR325 when Escherichia coli is used as a host microorganism, and pUP110, pTZ4 (pTP4) and pC194 when Bacillus subtilis is used as a host microorganism. For example, vectors capable of autonomous growth in two or more host microorganisms such as Escherichia coli and Bacillus subtilis can be used, for example, vectors such as pHV14, TRp7, YEp7, and pBS7. Such a vector,
Similar to the DNA described above, the vector is cleaved with a restriction enzyme or the like to obtain a vector fragment.

The method of binding the DNA fragment and the vector fragment may be a method using a known DNA ligase, for example, DNA
After annealing the fragment and the vector fragment, a recombinant DNA is prepared in vitro by the action of an appropriate DNA ligase. If necessary, after annealing, introduce into the host microorganism,
Recombinant DNA can also be obtained by using in vivo DNA ligase.

Any host microorganism may be used as long as the recombinant DNA is capable of stable and autonomous growth and capable of expressing its trait. Among them, microorganisms lacking the ability to produce α-amylase (EC 3.2.1.1) are not only easy to measure the amount of polypeptide produced, but also easy to separate and purify the produced polypeptide, which is advantageous. Available to

A method for introducing a recombinant DNA into a host microorganism is a known method, for example, in the case where the host microorganism is a microorganism belonging to the genus Escherichia, in the presence of calcium ions, and in the case of a microorganism belonging to the genus Bacillus, the competent cell method. Alternatively, a protoplast method or the like can be employed.

As a method for selecting a transformed microorganism into which the recombinant DNA has been introduced, a microorganism that grows on a plate medium containing starch and that generates cyclodextrin from the starch may be selected.

It has been found that the recombinant DNA containing the polypeptide gene once selected in this manner can be easily removed from the transformed microorganism and introduced into another host microorganism. In addition, the recombinant DNA containing the polypeptide gene is cleaved with a restriction enzyme or the like to contain the polypeptide gene.
It has been found that a DNA fragment can be easily combined with a vector fragment obtained by cutting a vector such as a plasmid in the same manner.

In addition, the recombinant DNA containing the polypeptide gene has a cleavage site by the restriction enzyme Pvu II (manufactured by Toyobo Co., Ltd.), and the polypeptide gene is cleaved by the action of the restriction enzyme Pvu II, and its expression It turned out to lose ability.

Nucleotide sequence of polypeptide gene The nucleotide sequence of the gene of the polypeptide is Gene,
Vol. 9,259-268 (1982)
What is necessary is just to decode by the chain terminator method.

In this method, a DNA fragment containing a polypeptide gene obtained by cloning is inserted into a cloning site of a plasmid such as plasmid pUC18 by using a restriction enzyme. The obtained recombinant plasmid is transferred to Escherichia coli JM83 or the like by transformation, and then a microorganism having the recombinant plasmid is selected.

A recombinant plasmid is prepared using the product obtained by growing this microorganism.

The obtained recombinant plasmid is annealed with a synthetic primer, and a Klenow fragment is acted on to anneal the primer to generate a complementary DNA.

After subjecting this reaction product to polyacrylamide gel electrophoresis and then to radioautography, the nucleotide sequence of the polypeptide gene is determined.

In addition, the nucleotide sequence of the signal peptide gene that secretes the polypeptide out of the cells is determined in the same manner.

Amino acid sequence of polypeptide The amino acid sequence of the polypeptide is determined from the base sequence.

In addition, the amino acid sequence of a signal peptide that causes the polypeptide to be secreted out of the cells is determined in the same manner.

A partial amino acid sequence containing the N-terminus of a polypeptide A microorganism belonging to the genus Bacillus capable of producing a polypeptide is cultured in a nutrient medium to produce the polypeptide. After completion of the culture, the supernatant is collected by centrifugation, and the supernatant is purified by ammonium sulfate fractionation, ion exchange chromatography, and high performance liquid chromatography to obtain a high-purity polypeptide. Using this sample, Journal of Biological Chemistry, Vol.
According to the description of 256,7990-7997 (1981), the partial amino acid sequence containing the N-terminus of the polypeptide is determined by digestion with a gas phase protein sequencer and by high performance liquid chromatography.

Preparation of polypeptide by transformed microorganism It has been found that a large amount of polypeptide can be stably produced by culturing the transformed microorganism obtained as described above in a nutrient medium.

Nutrient media include, for example, carbon sources, nitrogen sources, minerals,
If necessary, organic trace nutrients such as amino acids and vitamins may be contained.

At this time, as the carbon source, starch, starch partially hydrolysate, saccharides such as glucose, fructose, and sucrose are advantageously used. As the nitrogen source, an inorganic nitrogen source such as ammonia gas, aqueous ammonia, ammonium salt, and nitrate, and an organic nitrogen source such as peptone, yeast extract, defatted soybean, corn steep liquor, and meat extract are appropriately used.

The culture method is, for example, a liquid medium at pH 4 to 10 and a temperature of 25 to 6
The polypeptide may be cultured for about 1 to 4 days under aerobic conditions such as aeration and stirring while maintaining the temperature in the range of 5 ° C. to produce and accumulate the polypeptide.

The polypeptide in the culture can be collected and used as it is, but is generally used after being separated into a polypeptide solution and microbial cells by filtration, centrifugation or the like according to a conventional method.

When the polypeptide is present in the cells, the cells are treated with ultrasonic waves, a surfactant, a cell wall lysing enzyme and the like, and then the polypeptide solution is collected by filtration and centrifugation.

The polypeptide solution thus obtained can be used as it is, or, for example, concentrated under reduced pressure, concentrated by membrane,
Starch adsorption, elution, further purification by appropriately combining salt precipitation with ammonium sulfate, sodium sulfate, etc., fractional precipitation with methanol, ethanol, acetone, etc., and collecting and using a polypeptide of higher purity, Also, after utilizing this polypeptide for the reaction, it is recovered by membrane separation,
Repetition and use, furthermore, this polypeptide,
For example, JP-A-58-19276 and JP-B-60-1879
It can be immobilized by the immobilization method described in Japanese Patent Application Laid-Open Publication No. H10-15095 and used as an immobilization enzyme.

The polypeptide used in the present invention is not limited to those derived from the above-described transgenic microorganism by genetic recombination, as long as the amino acid sequence to be specified is elucidated and can be used with a sense of security. .

The polypeptide having the amino acid sequence specified in the present invention can be advantageously used in the glycosyltransfer reaction method of the present invention.

That is, the glycosyltransfer reaction method of the present invention comprises causing the polypeptide to act on starches such as gelatinized starch, liquefied starch and amylose to cause an intramolecular transfer reaction, for example, α-cyclodextrin, β-cyclodextrin, γ To produce a cyclodextrin or the like, or to act on a mixture of a sugar donor such as starch and a sugar acceptor other than starch or a sugar acceptor such as a glycoside to cause an intermolecular transfer reaction to cause various sugars. This is a method for generating a transfer reaction product. Various cyclodextrins obtained by an intramolecular reaction can be advantageously used in foods and drinks, cosmetics, pharmaceuticals, daily necessities, and the like by forming inclusion compounds with fragrances, pigments, seasonings, oil-soluble substances, and the like.

When used for the intermolecular transfer reaction, for example, starch,
Liquefied starch, dextrin, cyclodextrin, amylose and other starches as sugar donors, for example, xylose, sorbose, sugars selected from monosaccharides such as fructose, sucrose, maltulose, and disaccharides such as isomaltulose Using a sugar as a sugar acceptor, a polypeptide is allowed to act on a mixture of these sugar donors and sugar acceptors, and an α-glycosylated saccharide sweetener such as α-glucosyl, α-maltosyl, α-maltotriosyl is obtained. The preparation can be carried out very advantageously. The obtained α-glycosylated saccharide sweetener has a mildly improved taste as compared with the saccharide sweetener of the raw material, its water solubility is increased, crystal precipitation is prevented, and the saccharide The use of sweeteners is significantly expanded and can be advantageously used for various foods and drinks.

Further, in the intermolecular transfer reaction, as a sugar acceptor, for example, steviol glycosides such as stevioside, rebaudioside, and rubusoside, glycyrrhizin, soyasaponin, tea saponin, ginsenoside, rutin, when using a glycoside such as esculin By reacting a polypeptide on a mixture of a sugar donor such as starch and a glycoside, α-glucosyl, α-maltosyl, α-glucosyl
The preparation of α-glycosylated glycosides such as maltotriosyl can also be advantageously carried out.

The resulting α-glycosylated glycoside has improved properties such as bitterness, astringency and other unpleasant tastes and increased water solubility, as compared with the glycoside of the raw material, Can be significantly expanded. In particular, in the case of α-glycosylated steviol glycoside or α-glycosylated glycyrrhizin, the taste is remarkably improved and the sweetness is close to that of sugar. It can be used to advantage. Further, the present invention is a method for producing a food or drink using the above-mentioned glycosyltransfer reaction method.

That is, in the production of foods and drinks, a polypeptide is allowed to act on starch, and the resulting transglycosylation reaction is contained to produce foods and drinks of improved quality.

The starch used in the present invention may be any starch capable of causing an intramolecular or intermolecular transfer reaction by the action of a polypeptide, for example, starch, amylose, amylopectin, or partially hydrolyzing these with an acid or an enzyme. Starch such as decomposed dextrin, and cereals such as rice, barley, wheat, corn, adzuki beans, cowpea, etc., potatoes such as sweet potato, potato, or chips, grits, powder thereof And so on.

In the production of foods and drinks, a method in which a polypeptide is allowed to act on starch and the resulting glycosyltransferase is contained may be any method as long as the object of the present invention can be achieved.For example, a method for preliminarily gelatinizing and dispersing a starch solution may be used. After the polypeptide is allowed to act and concentrated or dried as it is or as necessary,
You may mix it with food and drink.

In addition, in the case of a thermophilic substance such as a polypeptide derived from Bacillus stearothermophilus, starch and gelatin are preliminarily coexisted and gelatinization of the starch, liquefaction by the polypeptide, and a sugar transfer reaction are performed. May be performed at the same time to produce food and drink. In addition, other enzymes such as amylase, protease, lipase and the like can be used in combination with the polypeptide. The food and beverage thus obtained suppresses starch aging by the action of the polypeptide, and not only improves the mouthfeel, taste, and flavor of the food, but also prevents crystal precipitation, emulsifying properties, moisturizing properties, and the like. Since the properties are improved, these properties are hardly changed, and high quality can be maintained for a long period of time, the commercial value is extremely high.

As foods and drinks to which the present invention can be applied, foods containing starchy material or foods made from starchy material are suitable, for example, cookies, biscuits, oysters, sponge cake, uiro, fertilizer, Cakes such as mochi, buns such as sweet bread, bread, noodles such as udon, buckwheat, ramen, macaroni, fish meat products such as kamaboko, fish sausage, grilled meat sauce, curry roux, stew ingredients, soup ingredients, α Α-glycosyl steviol glycosides such as glycosyl stevioside, α-glycosyl rebaudioside, α-glycosyl rubusoside, α
-Glycosyl, seasonings such as glycyrrhizin, amazake, hard drinks, drinks such as shiruko, sake, sake, beer, vinegar,
Fermented foods and drinks such as soy sauce and miso are suitable.

 Hereinafter, the present invention will be described in detail by experiments.

Experiment 1 Cloning of Bacillus stearothermophilus polypeptide gene into Escherichia coli (1) Preparation of chromosomal DNA containing Bacillus stearothermophilus heat-resistant polypeptide gene Bacillus stearothermophilus heat-resistant polypeptide gene Chromosomal DNA containing DNA can be prepared by the method of Saito, Miura, etc. [Biochimica et Biophisica acta (Biochimica et Biophisica
Acta), Vol. 72, 619-629 (1963)]. That is, Bacillus stearothermophilus FERM-
P No. 2225 was cultured in a Brain Heart Infusion medium at 50 ° C. overnight with aeration and agitation. The culture solution is collected by centrifugation, and the obtained cells are suspended in a TES buffer (pH 8.0, containing trisaminomethane, hydrochloric acid, EDTA, and sodium chloride), and then lysozyme is added at a rate of 2 mg / ml. At 37 ° C
Hold for 0 minutes. After freezing overnight at -20 ℃, TSS
After adding a buffer solution (pH 9.0, containing trisaminomethane, HCl, sodium lauryl sulfate, and sodium chloride) and heating to 60 ° C., a TES phenol mixture (TES buffer solution (pH 7.5) 1
The mixture was cooled in ice water and centrifuged to obtain a supernatant. To the supernatant was added two volumes of cold ethanol to recover the crude chromosomal DNA, which was dissolved in SSC buffer (pH 7.1, containing sodium chloride and trisodium citrate), and liponuclease (manufactured by Sigma). RNase A) and protease (Pronase E, manufactured by Kaken Pharmaceutical Co., Ltd.), then add a TES phenol mixture, cool and centrifuge, and add 2 volumes of cold ethanol to the resulting supernatant. The purified chromosomal DNA was recovered, dissolved in a buffer (pH 7.5, containing trisaminomethane, HCl, and EDTA) and stored at -20 ° C.

(2) Preparation of plasmid pBR322 Plasmid pBR322 (ATCC 37017) was prepared by the method of J. Meyers (Journal of Bacteriology, Vol. 127, 1).
524-1537 (1976)] and prepared from Escherichia coli.

(3) Preparation of Recombinant DNA Containing Polypeptide Gene The purified chromosomal DNA containing the heat-resistant polypeptide gene prepared in Experiment 1- (1) was allowed to act on the restriction enzyme Mbo I (manufactured by Nippon Gene Co., Ltd.) DNA fragment is 1-20K
The chromosomal DNA was partially cut to bp. On the other hand, a restriction enzyme BamHI (manufactured by Nippon Gene Co., Ltd.) was allowed to act on the plasmid pBR322 prepared in Experiment 1- (2) to completely cut the plasmid, and then Escherichia coli was used to prevent self-ligation of the plasmid fragment. Alkaline phosphatase derived from Takara Shuzo Co., Ltd.
To dephosphorylate the 5 'end of the pBR322 fragment.

Binding of both fragments was carried out by reacting T ₄ DNA ligase (manufactured by Nippon Gene Co., Ltd.) at 4 ° C. overnight, and the recombinant DN was
A was prepared.

(4) Introduction of Recombinant DNA into Escherichia coli As a host microorganism, Escherichia coli HB101 (ATCC33694) lacking amylase-producing ability was used.

This microorganism is cultured in L-broth at 37 ° C. for 4 hours and collected, and then contains 10 mM sodium chloride and 50 mM manganese chloride.
The cells were suspended in 10 mM acetate buffer (pH 5.6), collected by centrifugation, and then suspended in 10 mM acetate buffer (pH 5.6) containing 25 mM calcium chloride and 125 mM manganese chloride. , The recombinant DNA obtained in Experiment 1- (3) was added, and
Let stand for 0 minutes. Further, the mixture was heated to 37 ° C, L-broth was added, and the mixture was kept at 37 ° C for 30 minutes.
g / ml and spread on L-Agar plate containing 2 mg / ml starch and kept at 37 ° C for 24 hours to allow colonies to form.

From this plate, the starch was decomposed by the iodine coloration method, and colonies producing cyclodextrin were selected.
Transformed microorganisms into which the recombinant DNA containing the polypeptide gene had been introduced were selected. This microorganism is grown, and recombinant DNA is prepared according to the plasmid preparation method of Experiment 1- (2).
Was taken out, various restriction enzymes were allowed to act thereon, the cleavage site was determined, and then the recombinant DNA and the plasmid pBR322 were completely cleaved with the restriction enzyme EcoRI (manufactured by Nippon Gene Co., Ltd.). ) and reacted with similarly T ₄ DNA ligase to prepare a recombinant DNA, a small recombinant DN comprising a polypeptide gene according to the method in experiment 1- (4)
Transformed microorganisms carrying A were selected.

Further, this small recombinant DNA and plasmid pBR322 were completely digested with the restriction enzyme SalI (manufactured by Nippon Gene Co., Ltd.), and then treated in the same manner as in the case of EcoRI described above to obtain a further smaller plasmid containing the polypeptide gene. Transformed microorganisms carrying the recombinant DNA were selected.

Escherichia coli TCH201
(FERM P-7924) and the recombinant DNA it possesses
Was named pTCH201.

FIG. 1 shows a restriction map of the DNA portion derived from Bacillus stearothermophilus for the recombinant DNA pTCH201.

As is clear from FIG. 1, it was found that cleavage was caused by restriction enzymes Pvu II (manufactured by Toyobo Co., Ltd.), Kpn I, Hind III (manufactured by Nippon Gene Co., Ltd.), and Xba I (manufactured by Takara Shuzo Co., Ltd.).

On the other hand, restriction enzymes EcoR I, BamH I, Pst I, Xho I, Bgl
II, Acc I (above, manufactured by Nippon Gene Co., Ltd.) did not cut.

Experiment 2 Cloning of Bacillus stearothermophilus polypeptide gene into Bacillus subtilis (1) Preparation of recombinant DNA pTCH201 Recombinant DNA pTCH201 was prepared according to the method of Experiment 1- (2), Escherichia coli TCH201 (FERM P-7924). ).

(2) Preparation of plasmid pUB110 Plasmid pUB110 (ATCC 37015) was prepared according to the method of Gryczan et al. [Journal of Bacterioriology, Vol.
329 (1978)] and prepared from Bacillus subtilis.

(3) Preparation of Recombinant DNA Containing Polypeptide Gene Restriction enzymes EcoR I and Xba I were allowed to act simultaneously on recombinant DNA pTCH201 containing the heat-resistant polypeptide gene prepared in Experiment 2- (1) and completely. Cut.

On the other hand, restriction enzymes EcoRI and XbaI acted similarly on plasmid pUB110 prepared in Experiment 2- (2) to completely cut it.

Binding of both fragments using T ₄ DNA ligase, experiment 1
A recombinant DNA was prepared by the same action as in (3).

(4) Introduction of recombinant DNA into Bacillus subtilis Bacillus subtilis 715A lacking amylase-producing ability was used as a host microorganism. After culturing the microorganism in a Brain Heart Infusion medium at 28 ° C. for 5 hours and collecting cells, a method such as Schacffer is used [Proceeding of the National Academy of Sciences of the United States of America (Proceeding of the National States).
of Academy of Sciences of the United States of Am
erica), Vol. 73, 2151-2155 (1976)] to prepare a protoplast suspension.

To this solution, the recombinant DNA prepared in Experiment 2- (3) was added, and the method of Sekiguchi et al. [Agricultural and Biological chemistry (Agricultural and Biologi
cal Chemistry), Vol. 46, 1617-1621 (1982)], and an agar plate medium containing the antibiotic kanamycin 250 μg / ml in the HCP medium and starch 10 mg / ml in the HCP medium. The cells were spread on the top and kept at 28 ° C. for 72 hours to form colonies.

Thereafter, a transformed microorganism into which a recombinant DNA containing a thermostable polypeptide gene had been introduced was selected according to the method of Experiment 1- (4). One strain of the present microorganism was designated as Bacillus subtilis TCU211 (FERM P-7927), and the recombinant DNA contained in the strain was designated as pTCU211.

FIG. 2 shows a restriction map of the DNA portion derived from Bacillus stearothermophilus of the recombinant DNA pTCU211. As is clear from FIG. 2, the restriction enzymes Pvu II,
Kpn I and Hind III were found to undergo cleavage.

On the other hand, restriction enzymes EcoR I, BamH I, Pst I, Xho I, Bgl
It was not cleaved by II, Acc I and Xba I.

Experiment 3 Partial amino acid sequence containing N-terminal of polypeptide derived from Bacillus stearothermophilus (1) Preparation of polypeptide Bacillus stearothermophilus FERM-P No. 222
5 was subjected to liquid culture in the same manner as in Experiment 5, to produce a polypeptide in the medium. The supernatant was collected by centrifugation, the polypeptide fraction was obtained by sulfate precipitation, and then anion exchanger column chromatography (manufactured by Toyo Soda Co., Ltd., using trade name DEAE / Toyopearl 650), chromatofocusing method (Manufactured by Pharmacia, using trade name Mono P) to collect highly purified polypeptide.

This product is available from K. Weber and M. Osborn, Journal of Biological Chemistry
mistry), Vol. 244, 4406 (1969), 70,000 ± 1 by SDS-polyacrylamide gel electrophoresis.
It had a molecular weight of 0,000.

 The specific activity was 200 ± 30 units / mg protein.

One unit of the CGTase activity referred to in the present invention is pH w / 0.3 w / 0.02 M acetate buffer and 2 × 10 ⁻³ M calcium chloride.
0.2 ml of an appropriately diluted enzyme solution was added to 5 ml of w% soluble starch solution and reacted at 40 ° C. for 10 minutes.
Then, the reaction was stopped by mixing with 15 ml of a 0.02 N-sulfuric acid aqueous solution, and 0.2 ml of a 0.1 N potassium iodide iodide solution was added to the reaction stop solution to cause color development. The amount of enzyme that completely eliminates the color of iodine in 15 mg of soluble starch by reacting at 10 ° C. for 10 minutes.

(2) Partial amino acid sequence containing N-terminus The polypeptide prepared by the method of Experiment 3- (1) was applied to a gas-phase protein sequencer (manufactured by Applied Biosystems, trade name: Model 470A), and then subjected to high performance liquid chromatography. And the partial amino acid sequence containing the N-terminus was determined.

The result was Ala-Gly-Asn-Leu-Asn-Lys-Val-Asn-
It was found to have a Phe-Thr sequence.

Experiment 4 Base sequence of polypeptide gene derived from Bacillus stearothermophilus and amino acid sequence of polypeptide (1) Preparation of plasmid pUC18 Plasmid pUC18 was prepared from Escherichia coli JM83 (ATCC 35607) into which the plasmid pUC18 was introduced, using Experiment 1- ( 2)
It was prepared according to the method described above.

(2) Preparation of Recombinant DNA Containing Polypeptide Gene Recombinant DNA was prepared according to the method of Experiment 1- (3).

That is, the fragment containing the polypeptide gene prepared by the method of Experiment 2- (3) is further cleaved by the action of various restriction enzymes, and the plasmid pUC18 prepared by the method of Experiment 4- (1) is ligated. similarly cleaved with various restriction enzymes to prepare a recombinant DNA by the action of T ₄ DNA ligase in these two fragments.

(3) Introduction of recombinant DNA into Escherichia core Escherichia coli JM83 was used as a host microorganism.

Recombinant DNA was introduced into this microorganism and transformed according to the method of Experiment 1- (4).

Then, 5-bromo-4-chloro-3-indolyl-
β-galactoside (5-bromo-4-chloro-3-indol
A microorganism that grew on a medium containing (y-β-galactoside or Xgal) and formed a colorless plaque was selected as a transformed microorganism.

(4) Preparation of Recombinant DNA from Transformed Microorganism The transformed microorganism was cultured in L-broth containing 50 μg / ml of the antibiotic ampicillin, and recombinant DNA was prepared from the obtained cells by an alkaline lysis method.

(5) Nucleotide sequence of recombinant DNA Decoded according to the dideoxy chain terminator method.

That is, after adding the recombinant DNA prepared in Experiment 4- (4) and a synthetic primer (17 bases) and annealing at 60 ° C. for 20 minutes, dNTP, ddNTP, [α- ³² P] dCTP and Klenow fragment were added. The reaction was carried out at 37 ° C. for 30 minutes, and the primer was extended from the 5 ′ side to the 3 ′ direction to generate complementary DNA. An excess of dNTP was added thereto, and the mixture was further reacted at 37 ° C. for 30 minutes. Then, a formamide dye solution was added to stop the reaction. Then, the mixture was boiled for 3 minutes and electrophoresed on a 6% polyacrylamide gel at about 25 mA to separate the elongated complementary DNA. After electrophoresis, the gel was fixed and dried.

Perform autoradiography using this gel,
The nucleotide sequence of the polypeptide gene was determined by performing sequence analysis on the base fragment on the autoradiogram.

 The results are shown in Table 1-1.

In addition, the nucleotide sequence of the signal peptide gene following the 5 'upstream thereof was examined in the same manner.

 The results are shown in Table 1-2.

(6) Amino acid sequence of polypeptide The amino acid sequence of the polypeptide was determined using the nucleotide sequences in Table 1-1, and the results are shown in Table 2-1.

In addition, the amino acid sequences of the signal peptides following the N-terminal upstream thereof were determined, and the results are shown in Table 2-2.

From the above results, it was revealed that the amino acid sequence of the polypeptide derived from Bacillus stearothermophilus has the sequence shown in Table 2-1.

Experiment 5 Preparation of polypeptide by transformed microorganism (1) Preparation of polypeptide and measurement of activity Transformed microorganism Escherichia coli TCH201 (FERM P) into which a recombinant DNA containing a thermostable polypeptide gene derived from Bacillus stearothermophilus was introduced. -7924) and Bacillus subtilis TCU211 (FERM P-7927) to prepare polypeptides.

In addition, the amounts of polypeptides produced by these transformed microorganisms, the host microorganisms and the donor microorganism Bacillus stearothermophilus were compared by their activities.

The liquid medium is corn steep liquor 1.0 w / v%, ammonium sulfate 0.1 w / v%, calcium carbonate 1.0 w / v%, starch
It was made up of 1 w / v% and water, adjusted to pH 7.2, sterilized at 120 ° C. for 20 minutes, and cooled. Escherichia coli TC
In the case of H201, the antibiotic ampicillin is added to this medium.
Inoculate at a rate of 50 μg per ml, and in the case of Escherichia coli HB101, inoculate without adding antibiotics.
Each was cultured at 37 ° C. for 48 hours with aeration and stirring.

In the case of Bacillus subtilis TCU211, the antibiotic kanamycin was added to the liquid medium at a rate of 5 μg per ml, and the mixture was inoculated with Bacillus subtilis 715A.
In the case of, inoculate without adding antibiotics,
The culture was performed for 72 hours.

Also, Bacillus stearothermophilus FERM-PN
In the case of o.2225, the liquid medium was cultured at 50 ° C. for 48 hours without adding antibiotics. The horn culture solution is centrifuged, separated into a supernatant and cells, the supernatant is measured for activity as it is, the cells are disrupted by sonication, and the activity is measured. I asked. The results are shown in Table 3.

As is clear from the results in Table 3, the amount of polypeptide produced from the transformed microorganism is improved, which is advantageous as an industrial production method.

(2) Physicochemical properties of polypeptide The supernatant of Escherichia coli TCH201 obtained in Experiment 5 (1) was salted out with ammonium sulfate 0.5 saturation to prepare and collect a crude polypeptide agent. Using this polypeptide agent, the method of N. Catsimpoolas [Analytical Biochemistr
y), Vol. 26, pp. 480-482 (1968), and the isoelectric point of the polypeptide was examined, specifically, a gel for isoelectric focusing (LKB, trade name " Isoelectric focusing was carried out using a glass column having a length of 65 mm packed with “ampholine”) under a normal condition of a constant voltage of 200 V for 16 hours and further a constant voltage of 400 V for 1 hour. After the power was turned off, the gel was removed from the glass column, cut at 5 mm intervals, extracted with water, and its pH was measured.
The gradient was determined. Separately, the gel removed from the glass column was stained with Coomassie Blue, decolorized, and then applied to a chromatoscanner (Shimadzu Corp., trade name "Model CS-930") at a wavelength of 600 nm. Is
As shown in FIG.

From the results of this experiment, it was found that the isoelectric point of the polypeptide from the transformed microorganism was 5.0 ± 0.1 (± SD, n = 8). Similarly, Bacillus subtilis
When the isoelectric point of TCU211 was examined, the same result as that of Escherichia coli TCH201 was obtained.

In addition, Escherichia coli TCH2 obtained in Experiment 5 (1)
The thermal stability was examined using a crude polypeptide agent obtained by salting out the supernatant of 01 with ammonium sulfate 0.6 saturation.

50mM Tris containing CaCl ₂ of 5 mM - maleate buffer C. in (pH 7.0), the polypeptide agent at different temperatures 30
Incubated for minutes. Thereafter, the remaining CGTase activity was measured, and the remaining activity was expressed as a relative activity. Result is,
The results are shown in Table 3-2.

From the results in Table 3-2, it was found that the heat stability of the polypeptide from the transformed microorganism was 70 ° C or higher.
Similarly, when the thermal stability of the polypeptide from Bacillus subtilis TCU211 was examined, the same results as those obtained from the polypeptide from Escherichia coli TCH201 were obtained.

Furthermore, Escherichia coli TC obtained in Experiment 5 (1)
The supernatant of H201 and Bacillus subtilis TCU211 was added to ammonium sulfate.
Using a crude polypeptide agent prepared and collected by salting out at 6 saturation, the amount of sugar transfer from starch to sucrose, the amount of cyclodextrin produced from starch, the production ratio of cyclodextrin α, β and γ, the optimal temperature, Examination of the enzyme properties such as optimum pH and stable pH revealed that the polypeptide of the transformed microorganism showed properties similar to those of the donor microorganism Bacillus stearothermophilus.

 Hereinafter, a few examples will be described.

Example 1 Bread Polypeptide prepared from Bacillus stearothermophilus in 650 g of wheat flour according to the method of Experiment 3- (1)
Add 3,000 units, then dissolve 20 g of sugar and 11 g of salt in a small amount of water, stir well, and press squeeze yeast 13
g, 13g of short nig oil, 2g of yeast food, add an appropriate amount of water and knead well to make a dough,
After that, ferment at 26 ° C for 2 hours according to the usual method.
Minutes, a bench time of 15 minutes, and then baked at an open temperature of about 200 ° C. for about 40 minutes to produce bread.

This product had good flavor, moderate elasticity, and little change over time.

Example 2 Amazake The polypeptide prepared by the method of Experiment 5 was added to 1 kg of cooked rice.
After adding 2,000 units and reacting at 70 ° C. for 3 hours, the temperature was lowered to 55 ° C., and 300 g of commercially available noodles were added and reacted for 24 hours to produce amazake.

 This product had good flavor and mouthfeel.

Example 3 Steamed Kankan Wheat Flour 600g, Potato Flour 250g, Salt 20g, Bleached Bean 400g, Sugar 1Kg, Maltose 1.4Kg and Experiment 3
After well kneading with water 2.4 containing 1,000 units of the polypeptide produced by the method (1), it was molded and steamed for about 40 minutes according to a conventional method.

This product is resistant to aging, has good mouthfeel, good flavor,
Maintained high quality for a long time.

Example 4 Cyclodextrin-containing starch syrup 10% w / w potato starch milk was subjected to Bacillus by the method of Experiment 5.
The polypeptide prepared from T.subtilis TCU211 was added to give 2 units per gram of starch, liquefied at pH 6.5 and a temperature of 85 ° C, cooled to 70 ° C, added with the same amount of polypeptide, and allowed to react for 40 hours. According to the method, decolorization with activated carbon, desalting with an ion exchange resin, purification, and concentration were performed to obtain a syrup containing cyclodextrin in a yield of 92% based on solids. This syrup is a syrup with good fragrance retention and can be advantageously used as a raw material for foods and drinks with good flavor, and as a compounding material for flavors and cosmetics that value fragrance.

In addition, this starch syrup, α- cyclodextrin, by a method using an organic precipitant such as toluene and trichloroethane, a known method such as column chromatography,
It is also advantageous to separate and collect β-cyclodextrin and γ-cyclodextrin.

In the production process of the cyclodextrin-containing starch syrup, after the polypeptide reaction, β-amylase and isoamylase (EC3.2.1.68) are allowed to act to obtain a high content of cyclodextrin and maltose. .41), purified and concentrated to produce maltocyclodextrin-rich starch syrup.
From this, high purity maltocyclodextrin is separated,
Harvesting can also be performed advantageously.

Example 5 200 g of α-glycosyl stevioside stevioside and 600 g of dextrin (DE8) were dissolved by heating in water 3 and then cooled to 70 ° C., and the polypeptide prepared from Bacillus subtilis TCU211 by the method of Experiment 5 was added to the dextrin trauma. Add 5 units
The reaction was carried out at pH 6.0 and a temperature of 65 ° C. for 35 hours. After the end of the reaction, 95
Heated to 15 ° C for 15 minutes, overpurified, concentrated, dried and powdered to give a powdered sweetener containing α-glycosyl stevioside in 92% yield per solid.

This sweetener had a sweetness similar to that of sugar in which stevioside was eliminated, and the degree of sweetness was about 100 times that of sugar.

Since the present sweetener has no fear of tooth decay and has almost no calories, it can be freely used as a diet sweetener or in combination with other sweeteners for seasoning various foods and drinks.

(Effects of the Invention) As is clear from the above, the present invention is a glycosyltransfer reaction using the elucidated polypeptide, elucidating the amino acid sequence of the polypeptide, and a method for producing food and drink. is there.

In the present invention, not only can a glycosyltransfer reaction be performed with confidence by using a polypeptide having a specific amino acid sequence, but this glycosyltransfer reaction can be used as it is in a method for producing a flavorful food or drink. It turns out, its industrial consciousness is very large.

Further, when producing foods and drinks, it is also a great feature that the polypeptide contained in the starch can be used for food as it is without any loss of the flavor of the foods and drinks.

[Brief description of the drawings]

FIG. 1 shows a restriction map of a DNA portion derived from Bacillus stearothermophilus for recombinant DNA pTCH201. FIG. 2 shows a restriction map of a DNA portion derived from Bacillus stearothermophilus for the recombinant DNA pTCU211. FIG. 3 shows that the transformed microorganism Escherichia coli TCH2
FIG. 3 shows the isoelectric point of the polypeptide from 01.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12N 15/09 ZNA C12R 1:07)

Claims (5)

(57) [Claims] (1) As partial amino acid sequences, in order from the N-terminal side, (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Th
r, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe
-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Met
-Asp-Arg-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met
-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) having the sequence of Asn-Pro-Ala-Leu-Ala-Tyr-Gly and having a molecular weight of SDS-polyacrylamide gel. A microorganism capable of producing a polypeptide derived from Bacillus stearothermophilus having a thermostability of 70,000 ± 10,000, thermostability of 70 ° C. or higher, and cyclomaltodextrin glucanotransferase activity is cultured in a nutrient medium to obtain A glycosyltransfer reaction method comprising causing the polypeptide to act on starch. 2. The polypeptide according to claim 1, wherein the partial amino acid sequence having an N-terminus is Ala-Gly-Asn-Leu-Asn-Lys-
2. The method according to claim 1, wherein the method has a Val-Asn-Phe-Thr sequence. (3) the polypeptide has an amino acid sequence: The method according to claim 1 or 2, wherein the method comprises the steps of: 4. The method according to claim 1, wherein the starch is selected from starch, amylose, cyclodextrin, and dextrin. 5. The method according to claim 1, wherein the glycosyl transfer reaction is an intramolecular transfer reaction or an intermolecular transfer reaction.
Item 5. The method for transglycosylation according to Item 2, 2, 3 or 4.