JPS61135581A - Polypeptide having cyclomaltodextrin glucanotransferase activity - Google Patents

Abstract

PURPOSE:To provide a polypeptide containing a specific partial amino acid sequence, having the activity of cyclomaltodextrin glucanotransferase and useful for the production of cyclodextrin and glycosylsucrose from starch, etc. CONSTITUTION:The amino acid sequence of a polypeptide having the activity of cyclomaltodextrin glucanotransferase and known as macerans amylase pro duced by Bacillus macerans was investigated and it has been found that the enzyme contains one or more sequences selected from 7 kinds of amino acid sequences of formulas. It has also been found that the partial sequences are arranged in the order of (a), (b), (c), (d) and (e) from the side close to the N- terminal. The polypeptide has a molecular weight of 70,000+ or -10,000 by SDS- polyacrylamide gel electrophoresis, and a specific activity of 200+ or -30 unit/mg- protein.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to polypeptides, particularly polypeptides having cyclomaltodextrin glucanotransferase activity.

More specifically, a microorganism capable of producing cyclomaltodextrin glucanotransferase, or a transformed microorganism into which a cyclomaltodextrin/glucanotransferase gene is introduced using recombinant DNA technology, is cultured in a nutrient medium. The polypeptide produced by 6 has the following partial amino acid sequence: (a) Asn-LJys-11s-Asn-Asp
-Gl)'-T)'r-Leu-Thr, (b) P
ro-Val-Phe-Thr-Phe-Gly-Gl
u-Trp-Phe-Leu.

(c) Val-Thr-Phe-1ie-Asp-
Asn-His-Asp-Met-ASP-Arg-p
He.

(d) l1e-Tyr-Tyr-Gly-Thr-
Qlu-Qln-Tlyr+fet-Thr-Gly-
Asn-Gly-Asp-Pro-Asn-Asn-A
rg, and (e) Asn-Pro-Ala-L
The present invention relates to a polypeptide having cyclomaltodextrin glucanotransferase activity, characterized in that it has one or more sequences selected from eu-Ala-'rxr-cty.

(Prior art) /Chromaltodextrin glucanotransferase (hereinafter abbreviated as CGTase in this specification)
is also called Bacillus macerans amylase, and is known as Carabacillus macerans (Bac (1) u8 macerans).
) is known as an enzyme that produces

In recent years, CGTase has been used not for Bacillus maceranstake but for Bacillus stearothermophilus (Bac(1)).
us stearothermophilus ), Bacillus circulans (13ac(1)ua cir
It is also known to be produced by microorganisms such as S. culans), and its transglycosylation activity has led to its widespread industrial use.

For example, cyclodextrin is produced by applying CGTaSe to a gelatinized starch solution, and glycodyl sucrose is produced by applying CGTase f to a mixed solution of liquefied starch and sucrose to utilize the sugar transfer reaction from starch to sucrose. are doing. Recently, cyclodextrin has been in increasing demand as a host for forming stable clathrates, e.g., by including organic compounds that are easily oxidized or volatile, and glycodylsucrose has been used to provide elegant sweetness. The demand for low caries sweeteners (manufactured by Hayashibara Co., Ltd., registered trademark coupling sugar) is increasing.

A stable supply of refined CGTase to meet this increasing demand has become an urgent need. Therefore, in order to stably supply CGTasst, it has become necessary to elucidate the entire amino acid sequence of a polypeptide (hereinafter simply referred to as polypeptide) having CGTase activity.

However, the amino acid sequence of the polypeptide is still unknown.

(Problems to be Solved by the Invention) The present inventors have clarified the amino acid sequences of polypeptides, secured a wide range of sources of polypeptides using recombinant DNA technology, and improved the production amount of polypeptides. Aiming for a series of diligent studies is a friend.

As a result, the polypeptide has the following partial amino acid sequence: (a) Asn-Lys-Ile-Asn=Asp-
Gly-T)rr-Leu-Thr.

(b) Pro-Val-Phe-Thr-Phe-
Gly-Glu-Trp-Phe-(,,eul(c)
Val-Thr-Phe-Ile-Asp-Asn
-His-Asp-Met-Asp-Arg-Phe
.

(d) Ile-Tyr-Tyr-Gly-Thr7
Glu-Gln-Tyr-Met-Thr-Gly-A
sn-GL7-Asp-Pro-Asn-Asn-Ar
g, and (e) Asn-Pro-Ala-Le
It was found that the partial sequences have one or more types of sequences selected from u-Ala-'pyr-Gly, and more specifically, the partial sequences are (a), (b) in order of proximity from the N-terminus. ,(
It was found that it had the partial sequences e), (d), and (e).

Its characteristic property is that it produces cyclodextrin from soluble starch, has a molecular weight of 70,000±10,000 by SDS-polyacrylamide gel electrophoresis, and has a specific activity of 200±30 units. /q
It is a protein.

It was found that a polypeptide derived from Bacillus stearothermophilus has the amino acid sequence shown in Table 2-1, as explained below.

Furthermore, it has been found that the polypeptide derived from Bacillus cerans has the amino acid sequence shown in Table 5-1, as will be explained later.

Furthermore, the amino acid sequence of the signal peptide used to separate the polypeptide from the producing microorganism has been determined.

Hereinafter, the contents of the present invention will be explained in detail, and the present invention will also be explained in detail. In the description of this specification, amino acids, peptides,
When other abbreviations are used, they are based on the abbreviations commonly used in the field. Examples of these are listed below. Regarding amino acids, if there are possible optical isomers, 5 optical isomers are shown unless otherwise specified.

DNA: Deoxyribonucleic acid RNA: Ribonucleic acid A: Adenine T: Thymine G Nigeanin C: Cytosine dNTP: Deoxynucleotide tris 7M.

ddNTP: dideoxynucleotide triphosphate dCT
P: deoxycytidine trisunate SDS sodium nidodecyl sulfate Ala Ni-22-Arg: arginine Asn: asparagine As p: aspartic acid C7S nigistein Gln: glutamine Glu: glutamate Gly nigericin) (is: histidine Ile: inoleucine Leu: leucine L 7': Lysine Met: Methionine Phe: Phenylalanine pro: Gronin 3er: Serine Thr: Threonine'['rp Nitributophane Tyr: Tyrosine Val': Valine In the present invention, the amino acid sequence of the polypeptide is CG
After cloning the entire polypeptide gene from a Tase-producing bacterium, its nucleotide sequence was determined by decoding.On the other hand, the amino acid sequence of the portion containing the N-terminus of the polypeptide was determined after the polypeptide was purified to a high degree of purity. , was investigated using a gas-phase protein enhancer.

Cloning of polypeptide' gene The present invention involves separating and purifying the DNA of a donor microorganism capable of producing a polypeptide, and then cleaving it with, for example, ultrasound or restriction enzymes, and then using the resulting DNA fragments. , and a vector fragment obtained by completely cutting the vector in the same manner, for example, by using DNA IJ gauze or the like,
Forming recombinant DNA containing the polypeptide gene.

In this case, the donor microorganism is a microorganism capable of producing a polypeptide, such as JP-A-47-20373, JP-A-50-63189, JP-A-50-882.
No. 90 and Hans Ben
der), Archives of Microbiology
), Mo1.111.271-282 (1977
Bacillus species such as Bacillus macerans, Bacillus megaterium, Bacillus circulans, Bacillus zerimixa, and Bacillus stearothermophilus, and Klebscilla species such as Klefunula pneumoniae, as shown in 2010), are appropriately selected and identified.

Furthermore, a transformed microorganism into which polypeptide-producing ability has been introduced by genetic recombination can also be used as a donor microorganism.

DNA derived from the donor microorganism is prepared by, for example, culturing the donor microorganism in a liquid medium with aeration and agitation for about 1 to 3 days, centrifuging the resulting culture to collect the bacteria, and then completely lysing the culture. can do. As the bacteriolytic method, for example, treatment with a cell wall lytic enzyme such as lysozyme or β-glucanase, ultrasonic treatment, etc. are used. Also,
If necessary, other enzyme agents such as rainbow protease or surfactants such as sodium lauryl sulfate may be used in combination, and freeze-thaw treatment may also be performed.

To separate and purify DNAt from the lysate thus obtained, conventional methods such as phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment,
This can be done by appropriately combining methods such as alcohol precipitation and centrifugation.

DNAt-cleavage can be performed by, for example, ultrasonication, restriction enzyme treatment, etc.
In order to facilitate the ligation of the A fragment and the vector fragment,
Restriction enzymes, especially those that act on specific nucleotide sequences,
For example, EcoRI, [ind[, BamHI,
Sal l, Sla I, X+na f 1Mbo
Type I restriction enzymes such as I, XblL l, 3ac l, pst l are suitable.

As a vector, it can reproduce autonomously in host microorganisms7
age or plasmids are suitable.

Examples of phages include Escherichia coli (Es.
Cherichia coli) T-host microorganisms include Carbacillus subtilis (13ac(1)us 5ubti) such as λgt, λC1λgt, λB.
lis) When using a t-host microorganism, pH, ψ
1, ψ105, etc. can be used.

In addition, as a plasmid, for example, Etscherichia
In the case of coli whole host microorganisms, I) BR322, P
When BR325 and the like use Bacillus subtilis as one main microorganism, pUB 110. p'rz 4(
pTP4), pc194, etc. can be used, and furthermore, for example, microorganisms that can autonomously reproduce in two or more types of host microorganisms such as E. coli, Bacillus subtilis, etc.
It is also possible to utilize vectors such as pHV14, TRp7, YEp7, PBS 7, etc. Such a vector is cleaved with a restriction enzyme or the like in the same manner as the DNA described above to obtain a vector fragment.

The method for joining the DNA fragment and the vector fragment may be any method using known DNA I7 gauze.
For example, after annealing a DNA fragment and a vector fragment, recombinant DNlt is generated in vitro by the action of an appropriate NA ligase. If necessary, after annealing, the DNA IJ gauze can be introduced into a host microorganism and in vivo. It can also be used to make recombinant DNA.

The host microorganism may be one in which the recombinant DNA is stable, capable of autonomous growth, and capable of expressing its characteristics. Among these, microorganisms lacking the ability to produce α-amylase (EC3,2,1,1) not only make it easy to measure the amount of polypeptide produced, but also to isolate the polypeptide produced.
It is easy to purify and can be used advantageously.

Recombinant DNAt can be introduced into the host microorganism using known methods, for example, in the case of the host microorganism belonging to the genus E./Elichia, it is carried out in the presence of carunium ions, and in the case of the microorganism belonging to the genus Bacillus, it is carried out in the presence of competent ions. A cell method, a protoplast method, etc. can be adopted.

A method for selecting a transformed microorganism into which recombinant DNA has been introduced is to select one that grows on a plate medium containing starch and that produces ncrodextrin from starch.

It has been found that the recombinant DNA containing the polypeptide gene once selected in this manner can be easily extracted from the transformed microorganism and introduced into other host microorganisms. Also, recombinant DNA containing polypeptide genes
? It has now been discovered that it is also possible to easily and efficiently cleave a DNA fragment containing a polypeptide gene with a restriction enzyme, etc., and then ligate it to a vector fragment obtained by similarly cleaving a vector such as a plasmid.

Mayu, recombinant DNA containing the polypeptide gene of the present invention
is restriction enzyme pvu I+ (manufactured by Toyobo Co., Ltd.)
It has been found that the polypeptide gene has a cleavage site of L, and when subjected to the action of the restriction enzyme pvu 11, the polypeptide gene is cleaved and loses its ability to express its expression.

Base sequence of polypeptide gene The gene base sequence of polypeptide is Qene
), Vol. 9.259-268 (1982).

In this method, a DNA fragment containing a polypeptide gene obtained by cloning is inserted into a plasmid such as Gold or Grasmid pUc18 at its cloning site using restriction enzymes. The obtained recombinant plasmid is transferred to Escherichia coli JM83 etc. by transformation, and then microorganisms having the recombinant plasmid are selected.

Recombinant grasmid is prepared using this microorganism that is grown completely.

The resulting recombinant plasmid is annealed with a synthetic primer, and a Klenow fragment is added thereto to fully extend the primer and generate complementary DNA At'-.

This reaction product is subjected to polyacrylamide gel electrophoresis, followed by radioautography, and then the base sequence of the polypeptide gene is determined.

The nucleotide sequence of the signal peptide gene that secretes the polypeptide outside the bacterial cell is also determined in the same manner.

1.1 Peptide 8-1 The amino acid sequence of the polypeptide is determined by base sequencing.

Furthermore, the amino acid sequence of a signal peptide that causes the polypeptide to be secreted outside the bacterial cell is determined in the same manner.

A microorganism belonging to the genus Bacillus having the ability to produce a polypeptide is cultured in a total nutrient medium to produce a polypeptide. After completion of the culture, the supernatant is collected by centrifugation and purified by ammonium sulfate fractionation, ion exchange atomography, and high performance liquid chromatography to obtain a highly pure polypeptide. This sample was used in the Journal of Biological Gummistry.
Chemistry), Vol, 256°799
0-7997 (198)), the amino acid sequence containing the N-terminus of the polypeptide is determined by elilysis into gas-phase protein/-quencer and fixation with high performance liquid chroma dog 2-fi. .

Preparation of polypeptides using transformed microorganisms We have also found that scales can stably produce large amounts of polypeptides by culturing the transformed microorganisms obtained as described above in a nutrient medium.

The nutrient medium may contain, for example, a carbon source, a nitrogen source, minerals, and, if necessary, organic micronutrients such as amino acids and pitami.

At this time, the carbon source is starch, starch partial hydrolyzate,
Carbohydrates such as glucose, fructose and sucrose are advantageously used. Nitrogen sources include ammonia gas, ammonia water, and ammonium salts.

Inorganic nitrogen sources such as nitrates, organic nitrogen sources such as peptone, yeast extract, defatted soybean, go/stay liquor, meat extract, etc. are used as appropriate.

The culture method is, for example, maintaining a liquid medium in the range of pH 4 to 1O1 @ 25 to 65 degrees Celsius, culturing it under aerobic conditions such as aeration and stirring for about 1 to 4 days, and producing and accumulating polypeptides. If you force it, it will work.

Although the polypeptide in the culture can be collected and used in its entirety as it is, it is generally used after being separated into a polypeptide solution and microorganism cells by filtration, centrifugation, etc., according to a conventional method.

If the polypeptide is present in the bacterial cells, the cells are treated with ultrasound, a surfactant, a cell wall lytic enzyme, etc., and then the polypeptide solution is collected by filtration, centrifugation, etc.

The polypeptide solution obtained in this way is, for example,
Vacuum concentration, membrane concentration, and further ammonium sulfate, sulfuric acid, etc.:
The purified polypeptide can be purified by appropriately combining salting-out methods such as salting out, fractional precipitation using methanol, ethanol, acetone, etc., to collect all the polypeptides of higher purity, which can be advantageously used as an industrial polypeptide agent.

One unit of CGTase activity in the present invention means pH 5,
Appropriately diluted in a 0.3 w/w % soluble starch solution containing 5,0,02 M acetate buffer and 2 x 10-3 M carunium chloride, the following enzyme solution was prepared at 0.2 fRt'.
Add i and react at 40℃ for 10 minutes, then add 0.5 of the reaction solution.
d i and ci, 0.02 N-sulfuric acid aqueous solution 15 + dlC
! to stop the reaction, and further add O, L to this reaction stop solution.
Add 0.2 mt of N-iodine potassium iodide solution to develop color, then measure the total absorbance at 660 nm to obtain 40
The amount of enzyme that completely eliminates the iodine coloration of 15 cm of Norupur starch by reacting at ℃ for 10 minutes.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Cloning of the Bacillus stearothermophilus polypeptide gene into Eno/Erichia coli +11 Preparation of chromosomal DNA containing the heat-resistant polypeptide gene of Bacillus stearothermophilus Chromosomal DNA containing peptide genes was obtained using the method of Saito, Miura et al.
ca et Biophysica Acta),
Vol. 72.619-629 (1963)]. That is, Bacillus stearothermophilus FERM-P FERM-P 2225 t-Plain Heart Ia 7 Eugene medium was cultured at 50° C. with agitation and aeration overnight. The culture solution was collected by centrifugation, and the obtained bacterial cells t-T
ESI! The mixture was suspended in a buffer solution (pH 8.0, containing trisaminomethane, hydrochloric acid, EDTA, and sodium chloride), and then lysozyme was added at a rate of 2 m/d and maintained at 37° C. for 30 minutes. This was frozen at t-1-20℃ overnight, and then TSS
Mild lI solution (pH 9.0, trisaminomethane, HCI,
Sodium lauryl sulfate, sodium chloride l) Contains rum) t-
After heating to 60°C, add TES-phenol mixture (T
A mixed solution of 1 volume of ES buffer (pH 7.5) and 4 volumes of 7enol) was added, and after cooling in ice water, centrifugation was performed to obtain an upper groove.

Add 2 volumes of cold ethanol to this supernatant and extract the crude chromosomal DNA.
was collected, dissolved in t-3SC buffer (pH 7.1, containing sodium chloride, trisodium citrate), and treated with ribonuclease (Ngumasha Rizou, trade name 1Nase A).
) and protease (manufactured by Kaken Pharmaceutical Co., Ltd., trade name pronase, E). Then, a TES-phenol mixture was added, cooled and centrifuged, and the resulting supernatant was diluted with twice the volume of cold ethanol. Purified chromosomal DNA was collected from the chin, dissolved in a buffer solution (pf (7,5, containing trisaminomethane, HCI, and EDTA), and stored at -20°C.

(2) Preparation of plasmid pBR322 Plasmid PBR322 (ATCC37017) was prepared by the method of J3Mayers et al.
Op Bacterio a) - (Journalof B
acteriolog)r), Vol, 127
．． 1524-1537 (1976)] was isolated and prepared from E. coli.

(3) Recombinant DNA containing a polypeptide gene
Preparation of Restriction enzyme [bol
(manufactured by Nopon Gene Co., Ltd.), DN
Fragment A becomes 1-20 Kbp: Ratio of partially cleaved chromosomal DNA. On the other hand, t prepared in Example 1-+2)
Plasmid pBR322 was treated with the restriction enzyme BamHI (Nippo/Gene Manufacturing Co., Ltd.) to completely cleave it, and then alkaline phosphatase derived from Escherichia coli (Takara Shuzo Co., Ltd.) was added to completely prevent self-ligation of the plasmid fragment. (manufactured) was applied to remove/oxidize the less than 5' of the PBR322 fragment.

The two fragments were joined together by reacting overnight at 4°C with DNAIJ gauze (manufactured by Nippo/Gene Co., Ltd.) to prepare recombinant DNA. , E. coli I (Blot (ATCC336
94) was used.

After culturing and collecting bacteria in this machine's biological 'tL medium at 37°C for 4 hours, they were suspended in 10 mM acetate buffer (pi(5,6)) containing 10 mM sodium chloride and 50 mM manganese chloride, and centrifuged. Bacteria were collected using 10 mM chloride containing 25 mM calcium/umium chloride and 125 mM manganese chloride.
The nine recombinant DNA obtained in Example 1-+31 was added to the suspension in acetate buffer (PI(5,6)), and the suspension was incubated in ice water for 30 minutes.
Let it sit for a minute. Furthermore, warm to 37°C, add L medium,
After keeping at 7℃ for 30 minutes, add antibiotic ampicillin 50
It was spread on an L-agar plate medium containing μf/mlf and 2 q/at of starch and kept at 37° C. for 24 hours to form colonies.

From this plate, colonies that had completely degraded starch and produced cyclodextrin were selected using the iodine coloring method, and colonies that had completely degraded starch and produced cyclodextrin were selected. A transformed microorganism into which recombinant DNA containing Choose your friend. This microorganism was grown, and recombinant D
After extracting the NA and exposing it to the action of all the various restriction enzymes and determining all the cleavage sites, complete cleavage was performed with the restriction enzyme EcoRI (Nutatsubo Co., Ltd./Gene Manufacturing), and then Example 1
- Apply T4 DNA ligase in the same way as for +31, and make recombinant DNA? A transformed microorganism containing a small recombinant DNA Th containing a polypeptide gene was selected according to the method of Example 1-(4).

Furthermore, this small recombinant DNA t-restriction enzyme 5all
(Nippo Co., Ltd./Gene Manufacturing) to completely cut the
The same treatment as in the case of EcoR,I was performed to select a transformed microorganism carrying a smaller recombinant DNA containing the polypeptide gene gold.

One strain of this microorganism is E. coli TCH201.
(FEBM P-7924), and the recombinant DNA it possesses was named pTCH201 and recombinant D
For NA pTcH201, the restriction enzyme cleavage map of the Bacillus stearothermophilus white rice DNA portion was
As shown in the figure.

As is clear from FIG. 1, the restriction enzymes pvul [(manufactured by Toyobo Co., Ltd.), Kpn I, Hind 1
(manufactured by Nipponji-7 Co., Ltd.) and Xbal (manufactured by Takara Shuzo Co., Ltd.) were found to undergo cleavage.

On the other hand, the restriction enzyme EcoRI %B, amH[,, pst
It was not cleaved by I, Xho I, Bgl II, and Ace I (manufactured by Ninobon Gene Co., Ltd.).

Example 2 Cloning of Bacillus stearothermophilus polypeptide gene into Bacillus subtilis (1) Preparation of recombinant DNA pTCH201 Recombinant D
NA pTcfj201 was obtained from Ezoelichia coli TCH201 (FEB) according to the method of Example 1-+2).
MP-7924).

(2) Preparation of plasmid pUB110 Plasmid pUBIIO (ATCC37015) was prepared by the method of Gryczan et al.
iology).

Vol. 134, 318-329 (1978)].

(3) Preparation of recombinant DNA containing a polypeptide gene Example 2 - (9 thermostable IJ prepared in step 11, restriction enzyme for the recombinant DNA pTCH201 containing the petid gene): coRI-produced Xba I act at the same time and completely cut it off.

On the other hand, Example 2-(2+ prepared plasmid pUB1
10 was treated with the restriction enzyme ECORI and Xba+ in the same manner to completely cleave it.

Both fragments were joined together using T, DNA IJ gauze in the same manner as in Example 1-+31 to prepare recombinant DNAt.

(4) Introduction of recombinant DNA into Bacillus subtilis.
A friend using Chillus subtilis 715A.

After culturing this microorganism in plain/・-t infusion medium at 28°C for 5 hours and collecting the bacteria, Zoe-77-(
5chaffer), etc.
Op the Su/Yonal Academy of Sciences of the Enlightened States Op America (
proceeding of the National
of Academy of 5 Sciences o
f the United States of America
rica), Vol, 73.2)51-2)5
5 (197, 6)].

The recombinant DNA prepared in Example 2-J31 was added to the aqueous solution, and the method of Sekiguchi et al.
and Biological Chemistry)
.

Vol, 46.1617-162) (1982)]
After transformation according to the method, spread on an agar plate medium containing HCP medium containing the antibiotic kanainone 250μSF/mj t- and starch 10~/-, and incubate at 28°C.
, keep for 72 hours to ensure colony formation.

Thereafter, the method of Example 1-+4) was carried out to select transformed microorganisms into which the recombinant DNA containing the heat-resistant polypeptide gene had been introduced. One strain of this microorganism is Bacillus spp. TCU2) 1 (FEBM P-7927).
The group envy DNA it possesses is named pTCU2)
I named it 1.

Recombinant DNA pTcU2)1 is cut with the restriction enzymes Pvu I[, Kpn I, Hind 1? It turned out that I would receive it.

On the other hand, the restriction enzyme ECOR[, B&mf(I, pst
l, XhOI, Bgi If, Acc [, Xb
There was no cleavage in aI.

Example 3 Preparation of N-terminal gold-containing partial amino acid sequence fl+ polypeptide of polypeptide derived from Bacillus stearothermophilus Bacillus stearothermophilus yx FERMP-
2225t-1 was cultured in liquid in the same manner as in Example 5 to produce polypeptide in the medium. The supernatant was collected by centrifugation, subjected to ammonium sulfate salting out to obtain 9 polypeptide fractions, and then subjected to anion exchange column chromatography (
The highly purified polypeptide was purified by the Talomad 7-Okano 7 method (manufactured by Toyo Ichita Co., Ltd., using the trade name DEAE/Toyopearl 650) and the Talomad 7-Organization method (manufactured by Farman Co., Ltd., using the trade name Mow P).

The crystal is K Waper & M Osbo-7 (
K, Weber and M, Qsborn), Journal of Biological Chemistry (Jo
urnal of biological ('hem
istry ) + Vol, 244°4406 (+9
A molecular weight of 70,000±10,000 was obtained by SDS-polyacrylamide gel electrophoresis performed according to the method described in 1969).

Is the specific activity 200±30 units/ki protein? (2) Partial amino acid sequence containing N-terminus Example 3-(
Complete polypeptide prepared by the tl method, gas phase protein sequencer (manufactured by Applied Biosystems,
470A) and then analyzed by high performance liquid chromatography to determine the partial amino acid sequence containing the N-terminus.

The result is Ala-Gly-Asn-Lau-Asn-L
It was found that it had the sequence Iys-Val-Asn-Phe-Thr.

Practical Example 4 Base sequence of polypeptide gene derived from Bacillus stearothermophilus: and amino acid sequence of polypeptide il+ Plasmid Preparation of pUc18 Plasmid
ptrc ts was prepared from E. coli JM 83 (A, TCC35607) according to the method of Example 1-+2).

(2) Preparation of recombinant DNA containing polypeptide gene Recombinant DNAt was prepared according to the method of Example 1-+31.

That is, the fragment containing the polypeptide gene gold prepared by the method of Example 2-+31 was further cut by the action of various restriction enzymes, and then the plasmid pUc18 prepared by the method of Example 4-+11 was similarly digested. Cut with various restriction enzymes and apply T4DA N IJ gauze to both fragments to create recombinant DNA'.
``Friend.

The recombinant DNA was introduced into this microorganism using the method described in Example 1-4), and the microorganism was transformed.

5-bromo-4-chloro-3-indolyl-β-galactoside (5-bromo-4-ch
loro-3-1ndoly-β-galactos
The microorganisms that formed colorless plaques were selected as transformed microorganisms.

(4) Preparation of recombinant DNA from transformed microorganisms Transformed microorganisms, antibiotic ampirin 50μf/df
Recombinant DNAt was prepared from the resulting bacterial cells using the alkaline lysis method.

(5) The base sequence of the recombinant DNA was decoded according to the dideoquine chain/terminator method.

That is, after adding the recombinant DNA prepared in Example 4-+4) and a synthetic primer (17 bases) and annealing at 60°C for 20 minutes, dNTP, ddNTP[αT
”P) Add dCTP-treated Klenow fragment and heat at 37°C.
The primer was reacted for 30 minutes, and the primer was extended from the 5' side to the 3' direction to generate complementary DNA.

To this, excess dNTP t- was added, and further 3
After reacting for 0 minutes, a formamide dye solution was added to stop the reaction and the ratio was adjusted. Next, the mixture was boiled for 3 minutes, and electrophoresed on a 6-thick polyacrylamide gel at about 2.0 OOV and about 25 mA to separate the elongated complementary DNA. After electrophoresis, the gel was fixed and dried.

Autoradiograph 2 fee using this gel? The nucleotide sequence of the polypeptide gene was determined by conducting /-fence analysis of the nucleotide fragments on the autoradiogram.

The results are shown in Table 1-1.

In addition, the nucleotide sequence of the Gunnar peptide gene that follows upstream on the 5' side was also investigated in the same manner.

The results are shown in Table 1-2.

(6) Amino acid sequence of polypeptide Using the base sequence in Table 1-1, the entire amino acid sequence of the polypeptide was determined, and the results are shown in Table 2-1.

The amino acid sequence of the N-terminus upstream peptide was determined, and the results are shown in Table 2-2.

From the above results, it is clear that the amino acid sequence of the Bacillus stearothermophilus-derived polypeptide contains the sequence shown in Table 2-1.

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(0-* -Co-e-e -40-'s -1''ψto's = YuQ:a:e:; t;4 z き宮=Lu;=;Tsubasa-c&->alooc-tceo , Knee U-L-L Knee〇--L+-1-Ikui≦--Tame2υ-←口φlj −
(L L U :><((> )-←Ko-shu-GourdΦI
-I'1l-N-4-low pU4J bow to one bird V2H:A
7t4G==2-22sha2;=2go=go270:gi,
, Go 27 group ``Mi pasting work 7 miro ζψ fake-ri cpa octopus-kuku ←-← Otori Kazutochi 8 words 1 go 1 woman 72 Daig=; Goba'('I L (＞ -B〉!+Z biased tts<>
ai ← 1: I knee 14-2 > O-)% :ll
+ -+ 1-1-1- :: HOG +-; 2; Mimo to G;; yo = ρ to Zahei 1t Σ; Engineering 5 is seven discharges 2■l yo =; Apology is -<->-u <>>mouth←octopus-ni-C:J <:>φ
← くφ龜ら-え一≧战れ＊-hat-〇-山一一! -b
ll'l IJ n (1-1-nitsu2←* +6-a
> I:4) ψλ− taste (← Kurikuri (=<←>ζ
〉ku←OtsucQ＜←(Tallacle ξ cruel story city book 5 north mE book ξ 1- H>I:1--201-4) U &-a mouth α mouth 1 et al., x; l: G2 ko; the = = 2 = t;; and 22 =; Example 5
Preparation of polypeptide using a transformed microorganism A recombinant DNA containing a heat-resistant polypeptide gene derived from Bacillus stearothermophilus was introduced into a transformed microorganism called Enschierichia;
P-7924) and Bacillus subtilis TCU
2) 1(FEBM P-7927')r was used to determine the amount of polypeptide produced by these transformed microorganisms, the host microorganism, and the donor microorganism Bacillus stearothermophilus. Compare with friends.

The liquid medium is cornstap liquor 1. OW/vchi,
Ammonium sulfate Q, 1w/v%, Cal/Rum carbonate 1
, Qw/v, starch 1w/v, water, pH
7.2, sterilize at 120°C for 20 minutes, cool and prepare. In the case of E. coli TCH201, the antibiotic ampicillin/d was added to this medium at 50 μl/d.
? In addition, Eno7 Erychia coli was added at a ratio of
In the case of HB 101, the cells were inoculated without antibiotics and cultured with aeration at 37°C for 48 hours.

Mata, Bacillus subtilis TCU 2) 16! In
In this case, the liquid medium should be supplemented with antibiotics Kanamaine 7 &:W.
4) In the case of Bacillus subtilis 715A, inoculate without adding antibiotics and culture at 28'C for 72 hours.

In addition, Bacillus steamythermophilus FERM-P
&2225, the liquid medium was cultured for 48 hours at 50 °C without adding antibiotics. Each culture solution
Centrifugation was performed to separate the supernatant and bacterial cells, the supernatant was directly determined for its activity (11111), the bacterial cells were destroyed by ultrasonication, the specific activity was measured, and the activity was calculated by converting it into the amount of culture solution. . Result is,
It is shown in Table 3.

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

However, these supernatants were salted out with 0.6 saturated ammonium sulfate to prepare and collect a crude polypeptide agent. Using this polypeptide agent, the amount of sugar transfer from starch to sucrose, the amount of cyclodextrin produced from starch, cyclodextrin α, β, r
Production ratio, optimum temperature, optimum pH1 stable temperature, stable pH
A comparison of the enzymatic properties of the transformed microorganisms showed that the polypeptides of the transformed microorganisms were identical to those of the donor microorganism (Tillus stearothermophilus).

Example 6 Cloning of Bacillus macerans polypeptide gene into Erychia coli +11 Preparation of chromosomal DNA containing Bacillus macellans polypeptide gene Chromosomal DNA containing Bacillus macellans polypeptide gene was cultured at -28°C at Bacillus macellans 17At. Except for this, the sample was prepared simply by the method of Example i-tt+.

゛(2) Preparation of recombinant DNA containing a polypeptide gene Example 6 - Purified chromosomal DNA containing a polypeptide gene derived from Bacillus macerans was prepared using CI+ and was treated with a restriction enzyme [1ndl (Nippon Gene Manufacturing Co., Ltd.)]. was applied and partially cut in the same manner as in Example 1-(3).

On the other hand, the plasmid prepared by the method of Example 1-12)
For pBR322, restriction enzyme) (indl 'c
The 5′ end was then dephosphorylated by the method described in Example 1-(3), and the two fragments were ligated according to the method of Example 1-+31. ,
Next, create recombinant DNA.

(3) Introduction of recombinant DNA into En7 Erychia coli As a host microorganism, En7 Erychia coli HB 101 (ATCC336
94) according to the method of Example 1-+4) using
A transformed microorganism into which the recombinant DNA had been introduced was selected. Recombinant DNA was extracted from this microorganism, treated with various restriction enzymes, and its cleavage site was determined, and then partially cleaved with restriction enzyme 5AU3AI (manufactured by Nippon Gene Co., Ltd.).Meanwhile, the method of Example 1-12) After completely cleaving the tokutomo plasmid pBR322f with the restriction enzyme BamH■, the 5' end was dephosphorylated in the same manner as in Example 1-+31, and T4DNAIJ gauze was further applied in the same manner to prepare recombinant DNA At-. , a small recombinant DNA containing a polypeptide gene according to the method of Example 1-+4)
Select all the transformed microorganisms harboring T-.

One strain of this microorganism was used as En7 Erychia coli MAI (2(
FERM P-7925), and the recombinant DNA contained therein was named At-pMAH2.

For the recombinant DNA pMAI (2), the restriction enzyme cleavage map of the DNA portion derived from Bacillus macerans is shown in FIG.

It is clear from Figure 3: sea urchin, restriction enzyme pvu tl, S
al I, Ava l (Nippon Gene Manufacturing Co., Ltd.),
It turned out that my friend would undergo amputation due to PST L (Nippon Gene Manufacturing Co., Ltd.).

On the other hand, restriction enzyme EcORI%Hindl, Kpnl
.

It was not cleaved by Bamf(I, Xba I, Xho I, and Smal).

Example 7 Cloning of Bacillus macerans polypeptide gene into Bacillus subtilis Preparation of recombinant DNA pMA) (2) Recombinant DNA pMAH2 was prepared from Escherichia coli MAU2 (FEBMP-2) according to the method of Example 1-(2).
7925) and prepared as follows.

(2) Preparation of polypeptide gene Kanakuramu recombinant DNA The recombinant DNA pMAH2 prepared in Example 7-4) 1 and containing the polypeptide gene was treated with the restriction enzyme Ec.
oRI and Bamf (I'c) were applied simultaneously to completely cleave.

On the other hand, the next plasmid prepared by the method of Example 2-[2)
For pUBllo, use the restriction enzyme EcoRI Ba.
mHI k can be used at the same time to completely cut it off.

Both fragments were ligated using T4 DNA ligase in the same manner as in Example 1-+31 to produce recombinant DNAt&:.

(3) Introduction of recombinant DNA into Bacillus subtilis Example 2-+ Using Bacillus subtilis 715At, which lacks amylase-producing ability, as a host microorganism
According to method 4), a transformed microorganism into which a recombinant DNA containing a polypeptide gene derived from Bacillus macerans has been introduced is selected.

One strain of this microorganism is Bacillus subtilis MAU2)0
(FERM P-7926), and the recombinant DNA it possessed was named AtpMAU2)0.

FIG. 4 shows a restriction enzyme cleavage map of the Bacillus macerans-derived DNA portion of the recombinant DNA pMAU2)0. As is clear from FIG. 4, the restriction enzyme Pvu U
, Sal I, Ava I% Pst I.

On the other hand, restriction enzymes EcoRl, Hindl, K
pn I, BamH[, Xba l, Xho I
It was not cleaved by %SmaI.

Example 8 N of polypeptide derived from Bacillus macerans
Preparation of partial amino acid sequence containing terminal +11 polypeptide Bacillus subtilis MAU 2) 0 (FERMP
-7926)! -1 Polypeptide was produced in the medium by liquid culture in the same manner as in Example 10.

Next, the polypeptide was purified according to the method of Example 4-fil, and a very highly purified polypeptide was collected.

Crystal is 70.00 by SDS-polyacryl electrophoresis method.
Shows a molecular weight of 0±10,0OOCI and a specific activity of 200±3
It showed 0 units/Misaki protein.

(2) Partial amino acid sequence containing N-terminus Example 8-f
Example 3 using the polypeptide prepared by the method of il.
-(2+) The N-terminal gold-containing partial amino acid sequence was determined and compared.

The result is Ser-Pro-Asp-Thr-Ser
-Val-Asn-Asn-T, '1s-Lllu.

Example 9 Base sequence of polypeptide gene derived from Bacillus macerans and amino acid sequence of polypeptide (11 Preparation of recombinant DNA containing polypeptide gene Recombinant DNA was prepared according to the method of Example 4-131.

That is, prepared by the method of Example 7-+2),
<The fragment containing the peptide gene was further digested with various restriction enzymes, and also digested with restriction enzymes in the same manner as pUC18k prepared by the method of Example 4-+2), and both of these fragments were KT4DNA! Create recombinant DNA using gauze.

(2) Introduction of recombinant DNA into Escherichia coli Using Escherichia coli JM38i as a host microorganism, the entire recombinant DNA was simply introduced using the method of Example 4-(3+), and transformed microorganisms were selected.

(3) Preparation of recombinant DNA from transformed microorganisms Recombinant DNA was prepared according to the method of Example 4-+4).

(4) Base sequence of recombinant DNA Example 4-(T-determined base sequence of polypeptide gene according to method 51).

The results are shown in Table 4-1.

The sequence of the signal peptide gene following the 5' side was also examined in the same manner.

The results are shown in Table 4-2.

(5) Amino acid sequence of polypeptide Using the base sequence in Table 4-1, determine the amino acid sequence of the polypeptide. The results are shown in Table 5-1.

The amino acid sequence of the 7-terminal peptide following the N-terminus was determined, and the results are shown in Table 5-2. From over 90 results, the amino acid sequence of the polypeptide derived from Bacillus macerans is the 5th one. - It is clear that Natsuko has the sequence shown in Table 1.

Furthermore, from the results in Tables 2-1 and 5-1, the partial amino acid sequence common to all polypeptides is (a) Asn-L)rs-11a-Asn-Asp
-Gly-Tyr-Leu-Thr.

(b) Pro-val-Phe-Thr-phe-
Gly-Glu-Trp-Phe-Leu.

(c) Val-Thr-Phe-Ile-Asp-
zsn-His-Asp-Met-Asp-Arg-P
He.

(d) l1e-Tyr-Tlyr-Gl)'-Th
r-Qlu-Qln-Tyr+1et-Thr-Gly
-Asn-Gly-Asp-Pro-Asn-Asn-
Arg, and (e) Asn-Pro-A11L
-Leu-AI&-Tyr-Gly, and these partial amino acid sequences are N
In order from the terminal side, (a), (b), (e), (d),
A friend who was found to have the subsequence of (e).

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輻Mumu, Ukimui-tr+ a n 飄mul+I:i-
υ--Tameichi) 1 咄-1 飄Q Kojima Island (ri(-←LrJ
A<<Kn iri←←C-←((←←;ζ←ku--J knee 1 &+>↓-ΔmfJ+飄的--: c g-2 8m Δφ-λ-machibata V- Partial frame 1λ & 1-d-kJ: υ-
-a+V Aimu Archer\(> Vi milli-←←-Lilo=
←←” One kiss←<<
Mu υv-: Rho; Gokou-Vmu ==-ω+l: @J
-m-m-Tame A-v-1--Sho 11110-1-Ii-breath vJ Sugari (←L-ri<L-((Yuku Hibiki=(Shi←Rikuku ■target) Go υ muumu mu wa ki; gourd = fuu to - Vmu
-Kong 飄mutamame L A A + U J: -+ al IQ
-w I/l al temporary al V L' f 11
j &+ Meng @/ L, -AJ (jL l+zu←Piku<<<-Rokushi:EVr (1Q-Riku mouth←ship
AE total!餘：njE＆日=托こYO 二目☆＜ ((
(U −:: S:JIL I+L Vr (k−:
Ll'll L S )-h < Vr ←<@
ff1s+L11+*&-+Lfcmtq>A&al@
JmLa+et+HI:I:LeLU+IJ: -1
: ＞Le 1111 j--1-11-i11-1-
2 Ku←L←>←<<2 ku←-visit<Vr8<-<<Hibiki←B←Ma J+=mouth &+mouth I:J: Yumitomi==muΔmuΔmuhe':5 : e 1: -+ : -j -J+Mite-I+Rii-ii ViiΦiitonka---Doing one-one shouting-V←
t)AI+riL < ((((Z (+4 < Vr
←KU C-kui〉0Σ+-mu part; vv Tamefu 4a i-ra;
Vmu Vru Q-=Rayumi Gong VJ Knee w V++++++++++
＞ILIA& ＞+I:F 6J −Uj−j U
＋qll! ->J-ri←<-m-ζJJIILIL
J←(J -1/J jL -Lyu<<<←ku;-11 ko@J&+Ire=≧:' l:: Cnee L-I?
＜-L'□J: Practice--Practice L n -1 and V-←>←
−Distribution←"←Bekpi" J <": <>CI'i←
I; Ri I knee--0zu 11-Yumi 1 1 Bow 1 4 + Town g
-m2------Bow-Av<><J-<I+kuto<O
-<<-OS><1-ri 0-1 point ≧ 1-1+- Tame Ichiyukiichi-koo-0I+a1e6--〇20-Ichiyukiichipu11
L) -1-〇-1<>(kuku←Xaa><←←-C:!
Sniff 1: &,; t-1+t+- t; this;;-
Tame - nt + υ - - 1e Tame = 1 Pu Tame - 〇 - - Shunyumi - V (ri' mouth C) ←← et al ← - <-Tokuku> Roku! -I:ml-
: &+l: Rossi-cap bow--C-=υ-4)a
J:mL++-bow jll! --Kuriichi-
aotori＞＞S＞＜＜←Takouku←Kui110－tamera＞φ−−
-==Gang---V-> fG 111 m tg -a
b-1+-+ > 16 n F Q 4+ +11
5G A -u))->15←ζ<<-><<>X>>
LC::-Self = mouth-->-〇-weeQ etara-rari, Lll'j1. -IQ-Cap-j-JJS's 9--For≦-<To〈Oku mouth〉〉←-←(L ((L (-←To1;-1-)IuλυaL+ko;1 to C- υtame−
UA>Knee-++ -5-J:Z-111 Tame Tako ==-Yumuta -←-←a-a--sa<<<h<I-o-c>
ll11+φ-1: I+-Ro-Rashima-11 width oC≧-
Ren -υI-υ (1-m j+1 width-111 1111=< Vr ((/i <Vr (Lk-:) I
+lj (>>< L Cal (j hφh kool: +'Itwa fu-hat--g hat-; Koyumi--Q-
Lord-Ul11λ~-V2轡-Me4l-1-6-1aa
IA et al.
λ;-λc-ra--hcb-yum->λ-υ-w
U 4) + Sho - - 2 ← Bow's 1 - - ← ← Kuichi Rokri <
〉(←)L11+Octopus〉-111-1e Ill I
:bko0.吟υ--c＞7ha+-一λzu-zu目+th
target knee λ−−4−−−−=bu−) (><,<<
1+ri Otsu-1) I-CC! J←kuto←C Example 10
Preparation of polypeptide from transformed microorganism KL Recombinant DNA containing polypeptide gene derived from Bacillus macerans? Introduce and then transform microorganisms.
coli MAt(2 (FEBMP-7925) and Bacillus subtilis MAU2) 0 (FEBM P-7
926) was used to produce a polypeptide gene. Furthermore, the amounts of polypeptides produced by these transformed microorganisms, the host microorganism, and the donor microorganism Bacillus macerans were compared in terms of their activity. A liquid medium was prepared according to the method of Example 5.

In the case of Essierhia coli MAH2, the antibiotic ampicillin t''-IJ HBIO was added to this medium at a rate of 950 μV, te, E. coli IJ HBIO
In the case of I, the bacteria were inoculated without adding antibiotics, and each
Culture with aeration at ℃ for 24 hours.

In the case of Bacillus subtilis MAU2), 5μ2 per antibiotic kanamycin is added to the liquid medium.
Bacillus subtilis 7 was added at a ratio of 0 and inoculated.
Antibiotics in case of 15A? They were button-inoculated without addition and cultured at 28°C for 72 hours.

f7t, Bacillus macerans 17A, for 72 hours at 28°C without adding antibiotics to the liquid medium.
Time incubation ratio.

Each culture solution was treated in the same manner as in Example 5, and the activity was measured. The results are shown in Table 6.

As is clear from the results in Table 6, the amount of polypeptide produced from the transformed microorganism is improved, and this method is suitable as an industrial production method.

Then, these supernatants were salted out with 0.6 saturated ammonium sulfate to prepare and collect a crude polypeptide agent.

Using this polypeptide agent, various enzymatic properties were compared in the same manner as in Example 5, and the polypeptide of the transformed microorganism was found to be exactly the same as that of the donor microorganism Bacillus macerans.

Effects of the Invention As is clear from the above, the present invention clarifies the amino acid sequence of a polypeptide and the amino acid sequence of its signal peptide, and also provides in vitro gene recombination from a donor microorganism capable of producing a polypeptide. We have developed a replacement technology,
A recombinant DNA containing a polypeptide gene having a cleavage site with the restriction enzyme Pvu If K is prepared, and the recombinant DNA is introduced into a host microorganism to produce a transformed microorganism. The key to success is that the recombinant DNA can grow stably and autonomously in this material-converting microorganism.

Furthermore, from the standpoint of stably supplying polypeptides, the present invention can secure a wider range of supply sources and easily improve the amount of polypeptides produced, which is of great industrial significance.

[Brief explanation of drawings]

Figure 1 shows the recombinant DNA pTcH201.
A restriction enzyme cleavage map of DNA from Bacillus stearothermophilus is shown. FIG. 2 shows a restriction enzyme cleavage map of the DNA portion derived from Bacillus stearothermophilus for recombinant DNA I)TCU2)1. FIG. 3 shows a restriction enzyme cleavage map of the Bacillus macerans-derived DNA portion of recombinant DNA PMAH2. Figure 4 shows the recombinant DNA pMAU2)0.
A restriction enzyme truncation map of the DNA portion derived from Bacillus machinans is shown.

Claims (13)

[Claims] (1) A polypeptide has a partial amino acid sequence (a
)Asn-Lys-Ile-Asn-Asp-Gly-
Tyr-Leu-Thr, (b) Pro-Val-Ph
e-Thr-Phe-Gly-Glu-Trp-Phe
-Leu, (c) Val-Thr-Phe-Ile-A
sp-Asn-His-Asp-Met-Asp-Ar
g-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Gl
u-Gln-Tyr-Met-Thr-Gly-Asn
-Gly-Asp-Pro-Asn-Asn-Arg,
and (e) Asn-Pro-Ala-Leu-Ala.
A polypeptide having cyclomaltodextrin glucanotransferase activity, characterized by having one or more sequences selected from -Tyr-Gly. (2) The polypeptide has the following partial amino acid sequences in order of proximity from the N-terminus: (a) Asn-Lys-Ile-Asn-Asp-Gl
y-Tyr-Leu-Thr, (b) Pro-Val-
Phe-Thr-Phe-Gly-Glu-Trp-P
he-Leu, (c) Val-Thr-Phe-Ile
-Asp-Asn-His-Asp-Met-Asp-
Arg-Phe, (d) He-Tyr-Tyr-Gly-Thr-Gl
u-Gln-Tyr-Met-Thr-Gly-Asn
-Gly-Asp-Pro-Asn-Asn-Arg,
and (e) Asn-Pro-Ala-Leu-Ala.
2. The polypeptide having cyclomaltodextrin glucanotransferase activity according to claim 1, which has the sequence -Tyr-Gly. (3) Claim 1 or 2, wherein the polypeptide exhibits a molecular weight of 70,000±10,000 by SDS-polyacrylamide gel electrophoresis.
A polypeptide having cyclomaltodextrin glucanotransferase activity as described in 2. (4) The polypeptide has a partial amino acid sequence containing its N-terminus, Ala-Gly-Asn-Leu-A.
Claim 1, characterized in that it has the sequence sn-Lys-Val-Asn-Phe-Thr;
Cyclomaltodextrin according to item 2 or 3
A polypeptide having glucanotransferase activity. (5) Claim 1, characterized in that the polypeptide has the following amino acid sequence:
4. A polypeptide having cyclomaltodextrin glucanotransferase activity according to item 2, 3, or 4. (6) The polypeptide has an amino acid sequence of -P [there is an amino acid sequence] upstream of the N-terminus as a signal peptide for secretion of the polypeptide. A polypeptide having cyclomaltodextrin glucanotransferase activity according to item 4 or 5. (7) The polypeptide has Ser-Pro-Asp-Thr-S as a partial amino acid sequence containing its N-terminus.
Claim 1, characterized in that it has the sequence er-Val-Asn-Asn-Lys-Leu,
Cyclomaltodextrin according to item 2 or 3
A polypeptide having glucanotransferase activity. (8) Claim 1, characterized in that the polypeptide has the following amino acid sequence:
A polypeptide having cyclomaltodextrin glucanotransferase activity according to item 2, 3 or 7. (9) Claim 7, characterized in that the polypeptide has an amino acid sequence of [amino acid sequence] upstream of the N-terminus as a signal peptide for secretion of the polypeptide. or the polypeptide having cyclomaltodextrin glucanotransferase activity according to item 8. (10) The polypeptide is cyclomaltodextrin
Claims 1, 2, 3, 4, 5, 6, and 7, characterized in that the polypeptide is derived from a microorganism that has the ability to produce glucanotransferase. , cyclomaltodextrin glucanotransferase activity according to item 8 or 9. (11) Claims 1, 2, 3, 4, 5, or 6, characterized in that the polypeptide is a polypeptide derived from Bacillus stebrosothermophilus. A polypeptide having cyclomaltodextrin glucanotransferase activity as described in 2. (12) The cyclonucleotide according to claim 1, 2, 3, 7, 8, or 9, wherein the polypeptide is a polypeptide derived from Bacillus macerans. Maltodextrin A polypeptide with glucanotransferase activity. (13) The polypeptide is cyclomaltodextrin
Claims 1, 2, 3, and 4 are polypeptides derived from transformed microorganisms into which DNA containing a glucanotransferase gene has been introduced.
A polypeptide having cyclomaltodextrin glucanotransferase activity according to item 1, item 5, item 6, item 7, item 8 or item 9.