GB2169902A - Polypeptide possessing cyclomaltodextrin glucanotransferase activity - Google Patents

Abstract

The sequence of cyclomaltodextrin glucanotransferase (CGTase) gene derived from a microorganism of the genus Bacillus and the amino acid sequence of CGTase are determined. A recombinant DNA carrying the CGTase gene is introduced by an in vitro genetic engineering technique into a host microorganism of species Bacillus subtilis or Escherichia coli. The recombinant microorganism carrying the recombinant DNA automically proliferates to secrete a large amount of CGTase. The enzymes are used to produce glycosides having low calorie low cariogenic properties.

Description

SPECIFICATION Polypeptide possessing cyclomaltodextrin glucanotransferase activity The present invention relates to a polypeptide, and more particularly to a polypeptide possessing cyclomaltodextrin glucanotrarisferase activity.

Throughout the present specification, amino acids, peptides, etc. are designated with abbreviations which are commonly used in the art. Examples of such abbreviations are as follows: DNA is the abbreviation for deoxytibonucleic acid; RNA, ribonuclei acid; A, adenine; T, thymine; G, guanine; C, cytosine; dNTP, deoxynucleotide triphosphate; ddNTP, dideoxynucleotide triphosphate; dCTP, deoxycytidin 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; lIe, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threoinine; Trp, tryptophan; Tyr, tyrosine; Val, valine; and CGTase, cyclomaltodextrin glycanotransferase.

Wherever optical isomers are possible, the abbreviation used to designate an amino acid refers to the L-isomer, unless specified otherwise.

The term "polypeptide" is used throughout this specification to mean "polypeptide possessing CGTase activity".

CGTase, or macerans amylase, has been known for sometime as any enzyme produced by Bacillus macerans.

Recently, it has been found that CGTase is produced by other microorganisms such as those of species Bacillus stearothermophilus and Bacillus circulans. The saccharide transfer activity of CGTase now has many industrial applications.

For example, cyclodextrins are produced by subjecting gelatinized starch to the action of CGTase, while glycosylsucrose production utilizes the saccharide transfer reaction from starch to sucrose which is effected by subjecting a solution of liquefied starch and sucrose to the action of CGTase.

There is an expanding demand for cyclodextins for use as a hose to form a stable inclusion complex with an organic compound which is volatile or susceptive to oxidation. There is also an increasing demand for glycosylsucrose as a mildly-sweet low-cariogenic sweetener. This sweetener is sold by Hayashibara Co., Ltd., Okayama, Japan, under the Registered Trademark of "Coupling Sugar".

In order to meet these demands, there is an urgent need to provide a stationary CGTase supply. This requires determination of the amino acid sequence of the polypeptide that possesses CGTase activity.

The amino acid sequence af CGTase has not been known hitherto.

The Applicants have performed investigations to determine the amino acid sequence of the polypeptide possessing CGTase activity and have found that the polypeptide comprises one or more partial amino acid sequences selected from (a) Asn-Lys-lle-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-lle-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) lle-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly, and more particularly, that these partial amino acids sequences (a), (b), (c), (d) and (e) are located in sequence of nearness to the N-terminal end of polypeptide.

The polypeptide is characterized by the fact that it forms cyclodextrin from soluble starch; that it exhibits a molecular weight of 70,000+10,000 daltons on SDS-polyacrylamide electrophoresis; and that it has a specific activity of 200+30 units/mg protein.

We have also found that polypeptides having CGTase activity derived from Bacillus stearother mophilus and Bacillus macerans have the amino acid sequences as shown in Tables 2-1 and 5-1 respectively. Both amino acid sequences will be detailed more fully below.

In addition we have determined the amino acid sequences of the signal peptides which regulate secretion of the polypeptide from producer microorganisms.

In the present invention, the amino acid sequence of the polypeptide is determined by cloning the polypeptide gene from a CGTase producer microorganism, and sequencing the polypeptide gene.

By amino acid sequence containing Nqerminal end is determined by analyzing a highly-purified polypeptide with a gas phase protein sequencer.

Cloning of polypeptide gene In the invention, a DNA fragment (obtained by separating DNA from a donor microorganism capable of producing the polypeptide, and digesting the DNA, for example, with ultrasonic means or restricion enzymes), and a vector fragment (obtained by cleaving a vector in the same way) are ligated, for example, with DNA ligase to obtain a recombinant DNA carrying polypeptide gene The donor microorganism is chosen from bacteria which produce the polypeptide.Examples of such bacteria are those of genus Bacillus such as Bacillus macerans, Bacillus megaterium, Bacillus circulans, Bacillus polymyxa, and Bacillus stearothermophilus, and those of genus Klebsiella such as Klebsilla pneumoniae, as described, for example, in Japan Patent Kokai No. 20,373/72, Japan Patent Kokai No. 63,189/75, Japan Patent Kokai No. 88,290/75, and Hans Bender, Archives of Microbiology, Vol. 111, pp. 271-282 (1977).

Recombinant microorganisms in which polypeptide producibility has been introduced by genetic engineering techniques can be used as the donor microorganism.

The DNA of the donor microorganism can be prepared by culturing the donor microorganism, for example, with a liquid culture medium for about 1-3 days under aeration-agitation conditions, centrifugally collecting the microorganism from the culture, and lysing the microorganism.

Examples of bacteriolytic procedures are cytohydrolist treatment using lysozyme or ssglucanase, and ultrasonic treatment.

Other enzymes, such as protease, and/or surface active agents, such as sodium lauryl sulfate, can be used in combination, if necessary. Freezing-thawing treatments may also be carried out, if necessary.

In order to isolate DNA from the resultant lysate, two or more conventional procedures such as phenol extraction, protein removal, protease treatment, ribonuclease treatment, alcohol sedimentation, and centrifugation are combined.

Although DNA ligation can be effected by treating DNA- and vector-fragments, for example, with ultrasonic means or restriction enzymes, it is desirable to use restriction enzymes, in particular, those acting specifically on a prescribed nucleotide sequence, for smooth ligation.

Specifically suited are Type II restriction enzymes, for example, EcoRI, Hindlil, BamHI, Sall, Slal, Xmal, Mbol, Xbal, Mbol, Xba!, Sacl, Pstl, etc.

Bacteriophages and plasmids which autonomically proliferate in the host microorganism are suitable as vectors.

When a microorganism of species Escherichia coli is used as the host, bacteriophages such as Agt.AC and Agt.AB may be used, while p11, yll and to105 are suitable when a microorganism of species Bacillus subtitis is used as the host.

As regards plasmids, when a microorganism of species Escherichia coli is used as the host, plasmids such as pBR322 and pBR325 are suitable, while pUB110, pTZ4 (pTP4) and pC194 may be used for a host microorganism of the specis Bacillus subtilis. Plasmids which autonomically proliferate in two or more different host microoganisms, for example, pHV14, TRp7, YEp7 and pBS7, can be used as the vector. These vectors are cleaved with the same types of restriction enxymes as used in DNA digestion to obtain a vector fragment.

DNA- and vector-fragments are ligated by conventional procedures using DNA ligase. For example, DNA- and vector-fragments are first annealed, then subjected in vitro to the action of a suitable DNA ligase to obtain a recombinant DNA. If necessary such recombinant DNA can be prepared by introducing the annealed fragments into the host microorganism to subject them to in vivo DNA ligase.

The host microorganisms usable in the invention are those in which reombinant DNA automatically and consistently proliferates to express its characteristics. Specifically, microorganisms which are not capable of producing a-amylase (EC 3.2.1.1.) can be advantageously used because the use of such microorganisms facilitates isolation and purification of the secreted polypeptide.

The recombinant DNA can be introduced into the host microorganism by conventional procedures. For example, when the host microorganism belongs to the species Escherichia coli, introduction of recombinant DNA is effected in the presecne of calcium ions, while the competent cell- and protoplast-methods are employed when host microorganisms of the genus Bacillus are used.

The recombinant microorganism in which recombinant DNA has been introduced is selected by collecting clones which grow on a plate culture containing starch to convert the starch into cyclodextrin.

We have found that the recombinant DNA carrying the polypeptide gene cloned in this way can be easily introduced, after isolation from the recombinant microorganism, into a different host microorganism. We have also found that a DNA fragment carrying the polypeptide gene, obtained by digesting with restriction enzymes a recombinant DNA carrying the gene, can be easily ligated with a vector fragment which has been obtained in the same manner.

Furthermore, we have found that the polypeptide gene in the recombinant DNA obtained according to the invention is cleaved by the restriction enzyme Pvull, purchased from Toyobo Co., Ltd., Osaka, Japan, to lose its ability of expressing the polypeptide gene because the recombinarn DNA has a Pvull restriction cleavage site.

Sequence of the polypeptide gene The polypeptide gene was sequenced by the chain-terminator method as described in Gene, Vol. 9, pp. 259-268 (1982).

This method contains the step of using restriction enzymes to insert a cloned DNA fragment carrying the polypeptide gene into the Insertion site of a suitable plasmid such as pUC18. The obtained recombinant plasmid is introduced by transformation into a suitable Escherichia coli strain such aS Escherichia coli JM83, followed by selection of the recombinant microorganism that contains the plasmid.

The recombinant plasmid is prepared from the proliferated recombinant microorganism.

The obtained recombinant plasmid is annealed together with a synthetic primer, and the Klenow fragment is then allowed to act on the mixture to extend the primer, as well as to form the complementary DNA.

Thereafter, the mixture is subjected sequentially to polyacrylamide-electrophoresis and autoradiography from which the polypeptide gene sequence can be determined.

The signal peptide which regulates secretion of the polypeptide from the cell can be sequenced in the same manner.

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

The amino acid sequence of the signal peptide is determined in the same manner.

Partial amino acid sequence df polypeptide containing N-terminal end A polypeptide producer microorganism of genus Bacillus is cultured with a nutrient culture medium to produce the polypeptide. The supernatant, centrifugally obtained from the culture, is purified by ammonium sulfate fractionation, ion exchange chromatography and high-performance liquid chromatography to obtain a high-purity polypeptide specimen. The specimen is then degraded using a gas phase protein sequencer in accordance with the method described in Journal of Biological Chemistry, Vol. 256, pp. 7990-7997 (1981), and fixed with high-performance liquid chromatograpy, followed by determination of the partial amino acid sequence containing N-terminal end.

Preparation of the polypeptide with recombination microorganism We have found that a large amount of the polypeptide can be consistently produced by culturing a recombinant microorganism with a nutrient culture medium.

To the nutrient culture medium is incorporated, for example, a carbon source, a nitrogen source, minerals, and, if necessary, small amounts of organic nutrients such as amino acids and vitamins.

Starch, partial starch hydrolsate, and saccharides such as glucose, fructose and sucrose are suitable as the carbon source. Examples of suitable sources of nitrogen are inorganic nitrogen sources such as ammonia gas, ammonia water, ammonium salts and nitrates; and organic nitrogen sources such as peptone, yeast extract, and defatted soy-bean; corn steep liquor and meat extracts.

The recombinatnt microorganism is cultured with a nutrient culture medium for about 1-4 days under aearation-agitation conditions to accumulate the polypeptide while keeping the culture medium, for example, at pH 4-10 and 25-65"C.

Although the polypeptide in the culture may be used intact, generally, the culture is separated into a polypeptide solution and cells by conventional procedures such as filtration and centrifugation, prior to its use. Thus, for example, the cells are first treated with ultrasonic waves, surface active agents and/or cytohydrolyst, filtered and centrifuged to separate a solution containing the polypeptide.

The solution containing the polypeptide thus obtained is purified, for example, by a combination of concentration in vacuo, concentration using a membrane filter, salting-out using ammonium sulfate or sodium sulfate, fractional sedimentation using methanol, ethanol or acetone, to obtain a highly-purified polypeptide specimen which is advantageously usable as industrial polypeptide material.

To further improve the quality of the polypeptide, prior to its use, the amino acid sequence of the polypeptide may be partially substituted, removed, added, or modified in such a manner that the polypeptide does not lose its CGTase activity.

One unit of CGTase activity is defined as the amount of polypeptide that diminishes completely the iodine-coloration of 15 mg soluble starch at 40"C over a period of 10 minutes under the following reaction conditions: To 5 ml of 0.3 w/w % soluble starch solution containing 0.02 M acetate buffer (pH 5.5) and 2X 10-3 M calcium chloride is added 0.2 ml of a diluted enzyme solution, and the mixture is incubated at 40"C for 10 minutes. Thereafter, 0.5 ml of the reaction mixture is sampled and added with 15 ml of 0.02 N aqueous sulfuric acid solution to suspend the enzymatic reaction. The reaction mixture is then added with 0.2 ml of 0.1 N l2-Kl solution to effect coloration, and determined for the absorbance at a wavelength of 660 nm.

Deposition of recombinant microorganisms Becdmbinant microorganisms Escherichia coli TCH201, Escherichia coli MAH2, Bacillus subtilis MAU21O, and Bacillus subtilis TCU21 1 referred to in this specification were deposited as from November 2, 1984 under the accession numbers of FERM P-7924, 7925, 7926 and 7927 respectively as the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Yatabemachi, Tsukuba-gun, Ibaraki-ken, Japan.

In the accompanying drawings:- Figure 1 shows the restriction map of recombinant DNA pTCH201, in particular, that of the DNA fragment which carries the polypeptide gene derived from Bacillus stearothermophilus.

Figure 2 shows the restriction map of recombinant DNA pTCU211, in particular, that of the DNA fragment which carries the polypeptide gene derived from Bacillus stearothermophilus.

Figure 3 shows the restriction map of recombinant DNA pMAH2, in particular, that of the DNA fragment which carries the polypeptide gene derived from Bacillus macerans.

Figure 4 shows the restriction map of recombinant DNA pMAU210, in particular, that of the DNA fragment which carries the polypeptide gene derived from Bacillus macerans.

The present invention is illustrated further by the following Examples.

Example 1 Cloning of Bacillus stearotherntophilus polypeptide gene into Escherichia coli Example 1-(1) Preparation of chromosome DNA carrying heat-resistant-polypeptide gene of Bacillus stearother mophilus The chromosome DNA carrying heat-resistant-polypeptide gene of Bacillus stearothermophilus was prepared in accordance with the method described by Saito and Miura, Biochimica et Biophisica Acta, Vol. 72, pp. 619-629 (1963). A seed culture of Bacillus stearothermophilus FERM-P No. 2225 was cultured with brain heart infusion medium at 50"C overnight under vigorous shaking conditions.The cell, centrifugally collected from the culture, was suspended with TES buffer (pH 8.0) containing Tris-aminomethane, hydrochloric acid, EDTA and sodium chloride, added with 2 mg/ml of lysozyme, and incubated at 37"C for 30 minutes. The incubated mixture was freezed, allowed to stand at -200C overnight, added with TSS buffer (pH 9.0) comaining Trisaminomethane, hydrochloric acid, sodium lauryl sulfate and sodium chloride, heated to 60"C, added with a mixture of TES buffer (pH 7.5) and phenol (1:4 by volume), cooled in ice-chilled water, and centrifuged to obtain a supernatant.To the supernatant was added two volumes of cold ethanol to recover a crude chromosome DNA which was then dissolved in SSC buffer (pH 7.1) containihg sodium chloride and trisodium citrate, thereafter, the mixture was subjected to both "RNase A", a ribonuclease commerialized by Sigma Chemical Co., MO, USA, and "Pronase E", a protease commercialized by Kaken Pharmaceutical Co., Ltd., Tokyo, Japan, added with a fresh preparation of TES buffer and phenol mixture, cooled, centrifuged, and added with two volumes of cold ethanol to recover a purified chromosome DNA. The chromosome DNA was dissoved in a buffer (pH 7.5) containing Tris-aminomethane, hydrochloric acid and ED TA, and stored at -200C.

Example 1-(2) Preparation of plasmid pBR322 Plasmid pBR322 (ATCC 37013) was isolated from Escherichia coli in accordance with the method described by J. Meyer et al. in Journal of Bacteriology, Vol. 127, pp. 1524-1537 (1976).

Example 1-(3) Preparation of recombinant DNA carrying polypeptide gene The purified chromosome DNA carrying heat-resistant-polypeptide gene, prepared in Example J, was partially digested with restriction enzyme Mbol, purchased from Nippon Gene Co., Ltd., Toyarna, Japan, to give a DNA fragment of 1-20 kbp. Separately, the pBR322 specimen, prepared in Example 1-(2), was completely cleaved with restriction enzyme BamHI, purchased from Nippon Gene Co., Ltd., and the cleaved product was subjected to Escherichia coli alkali phosphatase, purchased from Takara Shuzo Co., Ltd., Kyoto, Japan, to prevent self-ligation of the plasmid fragment as well as to dephosphorize the 5'-terminal end of the fragment.

Both fragments were then ligated by subjecting them to T4 DNA ligase, purchased from Nippon Gene Co., Ltd., at 4"C overnight to obtain a recombinant DNA.

Example 1-(4) Introduction of recombinant DNA into Escherichia coil Escherichia coli HB101 (ATCC 33694), a strain incapable of producing amylase, was used as the host.

the microorganism was cultured with L-broth at 37"C for 4 hours, and the cell, centrifugally collected from the culture, was suspended with 10 mM acetate buffer (pH 5.6) containing 50 mM manganese chloride, centrifugally collected again, resuspended with 10 mM acetate buffer (pH 5.6) containing 125 mM manganese chloride, addd with the recombinant DNA prepared in Example 1-(3), and allowed to stand in an ice-chilled water bath for 30 minutes. The mixture was then warmed to 37"C, added with L-broth, spread on L-broth agar plate medium containing 50 g/ml of ampicillin and 2 mg/ml starch, and incubated at 37"C for 24 hours to form colonies, the colony which had degraded the starch into cyclodextrin was selected by the iodinecoloration method.Thus, the microorganism in which the recombinant DNA carrying polypeptide gene had been introduced was selected. The recombinant microorganism was then proliferated, and the recombinant DNA was extracted from the proliferated microorganism by the plasmid preparation method in Example 1-(2), subjected to restriction enzymes to determine the restric- tion cleavage sites, and completely digested with restriction enzyme EcoRI purchased from Nippon Gene Co., Ltd. The digested product was subjected to T4 DNA ligase similarly as in Example 1-(3) to obtain a recombinant DNA, followed by selection of recombinant microorganism in accordance wth the method in Example 1-(4). The recombinant microorganism contained a recombinant DNA of a relatively small-size that carries polypeptide gene.

The recombinant DNA and plasmid pBR322 were then completely digested with restriction enzyme Sall, purchased from Nippon Gene Co., Ltd., and treated similarly as in the case of EcoRI to select recombinant microorganisms containing a recombinant DNA of a much smaller-size that carries polypeptide gene.

One of these microorganisms and its recombinant DNA were named as "Escherichia coli TCH201 (FERM P-1924) and "pTCH201".

The restriction map of recombinant DNA pTCH201, in particular, that of the DNA fragment derived from Bacillus stearothermophilus microorganism was as shown in Fig. 1.

Fig. 1 clearly shows that this recombinant DNA is cleaved by either restriction enzyme Pvull purchased from Toyobo Co., Ltd., Kpnl, Hindlll purchased from Nippon Gene Co., Ltd., or Xbal purchased from Takara Shuzo Co., Ltd, but not by EcoRI, BamHI, Pstl, Xhol, Brill or Accl, all purchased from Nippon Gene Co., Ltd.

Example 2 Cloning of polypeptide gene of Bacillus stearothermophilus into Bacillus subtilis Example 2-(1) Preparation of recombinant DNA pTCH201 Recombinant DNA pTCH201 was isolated from Escherichia coli TCH201 (FERM P-7924) in accordance with the method in Example 1-(2).

Example 2-(2) Preparation of plasmid pUB 110 Plasmid pUB110 (ATCC 37015) was isolated from Bacillus subtilis in accordance with the method described by Gryczan et al. in Journal of Bacteriology, Vol. 134, pp. 318-329 (1978).

Example 2-(3) Preparation of recombinant DNA carrying polypeptide gene The recombinant DNA pTCH201 carrying heat-resistant-polypeptide gene, prepared in Example 2-(1), was completely digested by subjecting it simultaneously to restriction enzymes EcoRI and Xbal.

Separately, the plasmid pUB110 specimen, prepared in Example 2-(2), was completely cleaved by subjecting it to restriction enxymes EcoRI and Xbal in the same manner.

The resultant fragments were subjected to T4 DNA ligase similarly as in Example 1-(3) to obtain a recombinant DNA.

Example 2-(4) Introduction of recombinant DNA into Bacillus subtilis In this Example, Bacillus subtilis 715A, a strain incapable of producing amylase, was used as the host. The microorganism was cultured with brain heart infusion medium at 28"C for 5 hours, and the cell, centrifugally collected from the culture, was then prepared into protoplast suspen sion in accordance with the method described by Schaeffer et al. in Preceeding of the National Academy of Sciences of the USA, Vol. 73, pp. 2151-2155 (1976).

To the suspension was added the recombinant DNA, prepared in Example 2-(3), and the mixture was then treated in accordance with the method described by Sekiguchi et al. in Agricultural and Biological Chemistry, Vol. 46, pp. 1617-1621 (1982) to effect transformation, spread on HCP medium containing 250,ug/ml of kanamycin and 10 mg/ml of starch, and incubated at 28"C for 72 hours to form colonies.

From these colonies, recombinant microorganism in which the recombinant DNA carrying heatresistant-polypeptide gene had been introduced were selected by the method in Example 1-(4).

One of these microorganisms and its recombinant DNA were named as "Bacillus subtilis TCU 211 (FERM P-79273" and "pTClJ211" respectively.

The restriction map of recombinant DNA pTCU211, in particular, that of the DNA fragment derived frorn Bacillus stearothermophilus microorganism, was as shown in Fig. 2. Fig. 2 clearly shows that this recombinant DNA is cleaved by either restriction enzyme Pvull, Kpnl or Hindlll, but not by EcoRI, BamHI, Pstl, Xhol, Bglll, Accl or Xbal.

Example 3 Partial amino acid sequence of Bacillus stearothermophilus polypeptide containing N-terminal end Exempts 3-(1) Pteparation of polypeptide Bacillus stearothermophilus ERM-P No. 2225 was cultured with a liquid culture medium by the method in Example 5 to produce polypeptide. The supernatant, centrifugally obtained from the culture, was salted out with ammonium sulphate to obtain a polypeptide fraction which was then purified by column chromatography using "DEAE, toyopearl 650", an anion exchanger commercialized by Toyo Soda Manufacturing Co., Ltd., Tokyo, Japan, and chromatofocusing using "Mono P", a product of Pharmacia Fine Chemicals AB, Uppsala, Sweden, to obtain a highlypurified polypeptide specimen.

On SDS-polyacrylamide electrophoresis in accordance with the method described by K. Weber and M. Osborn in Journal of Biological Chemistry, Vol. 244, page 4406 (1969), the polypeptide specimen showed a molecular weight of 70,000+10,00 daltons.

The specific activity of the polypeptide specimen was 200+30 units/mg protein.

Example 3-(2) Partial amino acid sequence of polypeptide containing N-terminal end A polypeptide specimen, prepared by the method in Example 3-(1), was fed to "Model 470A", a gasphase protein sequencer, a product of Applied Biosystems Inc., CA, USA, and then analyzed with high-performance liquid chromatography to determine the partial amino acid sequence containing N-terminal end.

The partial amino acid sequence was Ala-Gly-Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr.

Example 4 Sequence of polypeptide gene derived from Bacillus stearothermophilus and amino acid sequence of polypeptide Example 4-(l) Preparation of plasmid pUC18 Plasmid pUC18 was prepared in accordance with the method of Example 1-(2) from Escherichia coli JM83 (ATCC 35607) in which the plasmid has been introduced.

Example 4-(2) Preparation of recombinant DNA carrying polypeptide gene The recombinant DNA was prepared by the method in Example 1-(3).

A fragment, obtained by digesting a fragment carrying polypeptide gene, prepared by the method in Example 2-(3), with restriction enzymes, and a plasmid fragment, obtained by cleaving a pUC18 specimen, prepared by the method in Example 4-(1), in the same manner, were subjected to T4 DNA ligase to obtain a recombinant DNA.

Example 4-(3J Introduction of recombinant DNA into Escherichia coli In this example, Escherichia coli JM83 was used as the host.

The recombinant DNA was introduced into the microorganism in accordance with the method in Example 1-(4)- to transform the microorganism.

The recombinant microorganisms were inoculated to a culture medium containing 5-bromo-4 chloro-3-indoyl-fl-galactoside (Xgal), and the microorganism forming colorless plaque was selected.

Example 4-(4) Preparation of recombinant DNA from recombinant microorganism The recombinant microorganism was cultured on L-broth containing 50 ,ug/ml of ampicillin, and the obtained cell was then treated with the alkaline mini-preparation method to obtain a recombi nant DNA.

Example 4-(5) Sequence of recombinant DNA The recombinant DNA was sequenced by the dideoxy chain terminator method.

The recombinant DNA, prepared in Example 4-(4), and a synthetic primer composed of 17 bases were mixed, annealed at 60"C for 20 minutes, added with dNTP, ddNTP, (a-32P) dCTP and Klenow fragment, and reacted at 370C for 30 minutes to extend the primer towards the 3' site from the 5' site. Thus, the complementary DNA was obtained. To the complementary DNA was added an excessive amount of dNTP, and the mixture was reacted at 37"C for 30 minutes, followed by addition of a formamide solution of dye mixture to suspend the reaction. The reaction mixture was boiled for 3 minutes, and electrophoresed on 6% polyacrylamide gel at about 25 mA (about 2,000 volts) to separate the extended complementary DNA. After completion of the electrophoresis, the gel was fixed and dehydrated.

The dehydrated gel was then autographed, and the polypeptide gene was determined by analyzing the base bands on the radioautogram.

The results were as shown in Table 1-1.

The signal peptide gene located upstream at the 5'-terminal end of the polypeptide gene was sequenced in the same manner.

The results were as shown in Table 1-2.

Example 4-(6) Amino acid sequence of polypeptide The amino acid sequence of polypeptide was determined with reference to the sequence as shown in Table 1-1, and the results were as shown in Table 2-1.

The amino acid sequence of the signal peptide was determined in the same manner, and the results were as shown in Table 2-2.

These evidences confirmed that the polypeptide derived from Bacillus stearothermophilus has the amino acid sequence as shown in Table 2-1.

Example 5 Preparation of polypeptide with recombinant microorganism Polypeptides were prepared with recombinant microorganisms Escherichia coli TCH201 (FERM P-7924) and Bacillus subtilis TCU211 (FERM P-7927) both in which recombinant DNA carrying heat-resistant-polypeptide gene derived from Bacillus stearothermophilus had been introduced.

The polypeptide productivities of these recombinant microorganisms were compared with those of host microorganism and donor Bacillus stearothermophilus microorganism in relation to their CGTase activity.

Table 1-1 10 20 30 40 50 60 GCTGGAAATC TTAATAAGGT AAACTTTACA TCAGATGTTG TCTATCAAAT TGTAGTGGAT 70 80 90 100 110 120 CGATTTGTGG ATGGAAATAC ATCCAATAAT CCGAGTGGAG CATTATTTAG CTCAGGATGT 130 140 150 160 170 180 ACGAATTTAC GCAAGTATTG CGGTGGAGAT TGGCAAGGCA TCATCAATAA AATTAACGAT 190 200 210 220 230 240 GGGTATTTAA CAGATATGGG TGTGACAGCG ATATGGATTT CTCAGCCTGT AGAAAATGTA 250 260 270 280 290 300 TTTTCTGTGA TGAATGATGC AAGCGGTTCC GCATCCTATC ATGGTTATTG GGCGCGCGAT 310 320 330 340 350 360 TTCAAAAAGC CAAACCCGTT TTTTGGTACC CTCAGTGATT TCCAACGTTT AGTTGATGCC 370 380 390 400 410 420 GCACATGCAA AAGGAATAAA GGTAATTATT GACTTTGCCC CCAACCATAC TTCTCCTGCT 430 440 450 460 470 480 TCAGAAACGA ATCCTTCTTA TATGGAAAAC GGACGACTGT ACGATAATGG GACATTGCTT 490 500 510 520 530 540 GGCGGTTACA CAAATGATGC CAACATGTAT TTTCACCATA ACGGTGGAAC AACGTTTTCC Table 1-1 continued (1) 550 560 570 580 590 600 AGCTTAGAGG ATGGGATTTA TCGAAATCTG TTTGACTTGG CGGACCTTAA CCATCAGAAC 610 620 630 640 650 660 CCTGTTATTG ATAGGTATTT AAAAGATGCA GTAAAAATGT GGATAGATAT GGGGATTGAT 670 680 690 700 710 720 GGTATCCGTA TGGATGCGGT GAAGCACATG CCGTTTGGAT GGCAAAAATC TCTGATGGAT 730 740 750 760 770 780 GAGATTGATA ACTATCGTCC TGTCTTTACG TTTGGGGAGT GGTTTTTGTC AGAAAATGAA 790 800 810 820 830 840 GTGGACGCGA ACAATCATTA CTTTGCCAAT GAAAGTGGAA TGAGTTTGCT CGATTTTCGT 850 860 870 880 890 900 TTCGGACAAA AGCTTCGTCA AGTATTGCGC AATAACAGCG ATAATTGGTA TGGCTTTAAT 910 920 930 940 950 960 CAAATGATTC AAGATACGGC ATCAGCATAT GACGAGGTTC TCGATCAAGT AACATTCATA 970 980 990 1000 1010 1020 GACAACCATG ATATGGATCG GTTTATGATT GACGGAGGAG ATCCGCGCAA GGTGGATATG 1030 1040 1050 1060 1070 1080 GCACTTGCTG TATTATTGAC ATCCCGTGGC GTACCGAATA TTTACTATGG TACAGAGCAA Table 1-1 continued (2) 1090 1100 1110 1120 1130 1140 TACATGACCG GTAACGGCGA TCCAAACAAT CGTAAGATGA TGAGTTCATT CAATAAAAAT 1150 1160 1170 1180 1190 1200 ACTCGCGCGT ATCAAGTGAT TCAAAAACTA TCTTCTCTCC GACGAAACAA TCCGGCGTTA 1210 1220 1230 1240 1250 1260 GCTTATGGTG ATACGGAACA GCGTTGGATC AATGGCGATG TGTATGTGTA TGAGCGACAG 1270 1280 1290 1300 1310 1320 TTTGGCAAAG ATGTTGTGTT AGTTCGGGTT AATCGTAGTT CAAGCAGTAA TTACTCGATT 1330 1340 1350 1360 1370 1380 ACTGGCTTAT TTACAGCTTT ACCAGCAGGA ACATATACGG ATCAGCTTGG CGGTCTTTTA 1390 1400 1410 1420 1430 1440 GACGGAAATA CAATTCAAGT CGGTTCAAAT GGATCAGTTA ATGCATTTGA CTTAGGACCG 1450 1460 1470 1480 1490 1500 GGGGAAGTCG GTGTATGGGC ATACAGTGCA ACAGAAAGCA CGCCAATTAT TGGTCATGTT 1510 1520 1530 1540 1550 1560 GGACCGATGA TGGGGCAAGT CGGTCATCAA GTAACCATTG ATGGCGAAGG ATTCGGAACA 1570 1580 1590 1600 1610 1620 AATACGGGCA CTGTGAAGTT CGGAACGACA GCTGCCAATG TTGTGTCTTG GTCTAACAAT Table 1-1 continued (3) 1630 1640 1650 1660 1670 1680 CAAATCGTTG TGGCTGTACC AAATGTGTCA CCAGGAAAAT ATAATATTAC CGTCCAATCA 1690 1700 1710 1720 1730 1740 TCAAGCGGTC AAACGAGTGC GGCTTATGAT AACTTTGAAG TACTAACAAA TGATCAAGTG 1750 1760 1770 1780 1790 1800 TCAGTGCGGT TTGTTGTTAA TAACGCGACT ACCAATCTAG GGCAAAAT@T ATACATTGTT 1810 1820 1830 1840 1850 1860 GGCAACGTAT ATGAGCTCGG CAACTGGGAC ACTAGTAAGG CAATCGGTCC AATGTTCAAT 1870 1880 1890 1900 1910 1920 CAAGTGGTTT ACTCCTATCC TACATGGTAT ATAGATGTCA GTGTCCCAGA AGGAAAGACA 1930 1940 1950 1960 1970 1980 ATTGAGTTTA AGTTTATTAA AAAAGACAGC CAAGGTAATG TCACTTGGGA AAGTGGTTCA 1990 2000 2010 2020 2030 2040 AATCATGTTT ATACGACACC AACGAATACA ACCGGAAAAA TTATAGTGGA TTGGCAGAAC Table 1-2 10 20 30 40 50 60 ATGAGAAGAT GGCTTTCGCT AGTCTTGAGC ATGTCATTTG TATTTAGTGC AATTTTTATA 70 80 90 100 GTATCTGATA CGCAGAAAGT CACCGTTGAA GCA Table 1-2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Met Arg Arg Trp Leu Ser Leu Val Leu Ser Met Ser Phe Val Phe 10) Ser Ala Ile Phe Ile Val Ser Asp Thr Gln Lys Val Thr Val Glu 31) Ala Table 2-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Ala Gly Asn Leu Asn Lys Val Asn Phe Thr Ser Asp Val Val Tyr 16) Gln Ile Val Val Asp Arg Phe Val Asp Gly Asn Thr Ser Asn Asn 31) Pro Ser Gly Ala Leu Phe Ser Ser Gly Cys Thr Asn Leu Arg Lys 46) Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp 61) Gly Tyr Leu Thr Asp Met Gly Val Thr Ala Ile Trp Ile Ser Gln 76) Pro Val Glu Asn Val Phe Ser Val Met Asn Asp Ala Ser Gly Ser 91) Ala Ser Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Pro Asn 106) Pro Phe Phe Gly Thr Leu Ser Asp Phe Gln Arg Leu Val Asp Ala 121) Ala His Ala Lys Gly Ile Lys Val Ile Ile Asp Phe Ala Pro Asn 136) His Thr Ser Pro Ala Ser Glu Thr Asn Pro Ser Tyr Met Glu Asn 151) Gly Arg Leu Tyr Asp Asn gly Thr Leu Leu Gly Gly Thr Thr Asn 166) Asp Ala Asn Met Tyr Phe His His Asn Gly Gly Thr Thr Phe Ser 1@1) Ser Leu Glu Asp Gly Ile Tyr Arg Asn Leu Phe Asp Leu Ala Asp 196) Leu Asn His Gln Asn Pro Val Ile Asp Arg Tyr Leu Lys Asp Ala 211) Val Lys Met Trp Ile Asp Met Gly Ile Asp Gly Ile Arg Met Asp 226) Ala Val Lys His Met Pro Phe Gly Trp Gln Lys Ser Leu Met Asp 241) Glu Ile Asp Asn Tyr Arg Pro Val Phe Thr Phe Gly Glu Trp Phe 256) Leu Ser Glu Asn Glu Val Asp Ala Asn Asn His Tyr Phe Ala Asn 271) Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Gly Gln Lys Leu 286) Arg Gln Val Leu Arg Asn Asn Ser Asp Asn Trp Tyr Gly Phe Asn 301) Gln Met Ile Gln Asp Thr Ala Ser Ala Tyr Asp Glu Val Leu Asp 316) Gln Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Met Ile 331) Asp Gly Gly Asp Pro Arg Lys Val Asp Met Ala Leu Ala Val Leu 346) Leu Thr Ser Arg Gly Val Pro Asn Ile Tyr Tyr Gly Thr Glu Glu 361) Tyr Met Thr Gly Asn Gly Asp pro Asn Asn Arg Lys Met Met Ser 376) Ser Phe Asn Lys Asn Thr Arg Ala Tyr Gln Val Ile Gln Lys Leu Table 2-1 continued 391) Ser Ser Leu Arg Arg Asn Asn Pro Ala Leu Ala Tyr Gly Asp Thr 406) Glu Gln Arg Trp Ile Asn Gly Asp Val Tyr Val Tyr Glu Arg Gln 421) Phe Gly Lys Asp Val Val Leu Val Arg Val Asn Arg Ser Ser Ser 436) Ser Asn Tyr Ser Ile Thr Gly Leu Phe Thr Ala Leu Pro Ala Gly 451) Thr Tyr Thr Asp Gln Leu Gly Gly Leu Leu Asp Gly Asn Thr Ile 466) Gln Val Gly Ser Asn Gly Ser Val Asn Ala Phe Asp Leu Gly Pro 481) Gly Glu Val Gly Val Trp Ala Tyr Ser Ala Thr Glu Ser Thr Pro 496) Ile Ile Gly His Val Gly Pro Met Met Gly Gln Val Gly His Gln 511) Val Thr Ile Asp Gly Glu Gly Phe Gly Thr Asn Thr Gly Thr Val 526) Lys Phe Gly Thr Thr Ala Ala Asn Val Val Ser Trp Ser Asn Asn 541) Gln Ile Val Val Ala Val pro Asn Val Ser Pro Gly Lys Tyr Asn 566) Ile Thr Val Gln Ser Ser Ser Gly Gln Thr Ser Ala Ala Tyr Asp 571) Asn Phe Glu Val Leu Thr Asn Asp Gln Val Ser Val Arg Phe Val 586) Val Asn Asn Ala Thr Thr Asn Leu Gly Gln Asn Ile Tyr Ile Val 601) Gly Asn Val Tyr Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala Ile 616) Gly Pro Met Phe Asn Gln Val Val Tyr Ser Tyr Pro Thr Trp Tyr 631) Ile Asp Val Ser Val Pro Glu Gly Lys Thr Ile Glu Phe Lys Phe 546) Ile Lys Lys Asp Ser Gln Gly Asn Val Thr Trp Glu Ser Gly Ser 661) Asn His Val Tyr Thr Thr Pro Thr Asn Thr Thr Gly Lys Ile Ile 676) Val Asp Trp Gln Asn A liquid culture medium consisting of 1.0 w/v % corn steep liquor, 0.1 w/v% ammonium sulfate, 1.0 w/v % calcium carbonate, 1 w/v % starch and water was adjusted to pH 7.2, sterilized by heating at 1200C for 20 minutes, and cooled.In the case of Escherichia coli TCH201, the liquid culture medium was added with 50 Hg/ml of ampicillin, and the microorganism was inoculated to the liquid culture medium. Escherichia coli HB101 was inoculated to the liquid culture medium without addition of antibiotic. In the case, microorganism was cultured at 37"C for 48 hours under vigorous shaking conditions.

Separately, Bacillus subtilis TCU211 was inoculated to the liquid culture medium additionally containing 5Jug/ml of kanamycin, while Bacillus subtilis 71 5A was inoculated to the liquid culture medium without addition of antibiotic. In each case, microorganism was cultured at 28"C for 72 hours.

Bacillus stearothermophilus FERM-P No. 2225 was cultured with the liquid culture medium at 50"C for 48 hours without addition of antibiotic. After separation of each culture into supernatant and cell by centrifugation, the supernatant was assayed intact for CGTase activity, while the cell was ultrasonically broken, prior to determination of its CGTase activity per culture. The results were as shown in Table 3.

These evidences clearly show that the recombinant microorganism are advantageously usable in industrial-scale production of polypeptide because these microorganism possess an improved polypeptide productivity.

The supernatants were salted out with ammonium sulfate at a saturation degree of 0.6 to obtain crude polypeptide specimens. After studying these polypeptide specimens on their enzymatic properties such as saccharide transfer from starch to sucrose, cyclodextrin production from starch, ratio of a-, ss and y-cyclodextrins, optimum temperature, optimum pH, stable temperature range and stable pH range, the properties of the polypeptide produced by the recombinant microorganism were in good accordance with those of the polypeptide produced by the donor Bacillus stearothermophilus microorganism.

Table 3 CGTase activity -units/ml) Microorganism Supernatant Cell Total Escherichia coll TCH201 (FERM P-7924) 0.8 13.5 14.3 Present invention Bacillus subtilis TCU211 (FERM P-7927) 46.7 20.5 67.2 Present invention Escherichia coli HB101 0 0 0 Control Bacillus subtilis 715A 0 0 0 Control Bacillus stearothermophilus FERM-P No.2225 8.5 0.3 8.8 Control Example 6 Cloning of Bacillus macerans polypeptide gene into Escherichia coli Example 6-(1) Preparation of chromosome DNA carrying Bacillux macerans polypeptide gene The polypeptide gene was prepared in accordance with the method in Example 1-(1), except that Bacillus macerans 1 7A was cultured at 28"C.

Example 6-(2) Preparation of recombinant DNA carrying polypeptide gene The chromosome DNA carrying polypeptide gene derived from Bacillus macerans, prepared in Example 6-(1), was partially digested similarly as in Example 1-(3) with restriction enzyme Hindlil, purchased from Nippon Gene Co., Ltd.

Separately, a plasmid pBR322 specimen, prepared by the method in Example 1-(2), was completely cleaved with restriction enzyme Hindlll, and the 5'-terminal end of the cleaved product was dephosphrized by the method in Example 1-(3). The fragments thus obtained were ligated in accordance with the method in Example 1-(3) to obtain a recombinant DNA.

Example 6-(3) Introduction of recombinant DNA into Escherichia coli The recombinant microorganism in which recombinant DNA had been introduced was cloned in accordance with cloned in accordance with the method in Example 1-(4) using Escherichia coli HB101 (ATCC 33694), a strain incapable of producing amylase, as the host. Thereafter, the recombinant DNA was isolated from the microorganism, subjected to restriction enzymes to determine the restriction cleavage sites, and partially digested with restriction enzyme Sau3Al commercialized by Nippon Gene Co., Ltd.

Separately, a plasmid pBR322 specimen, obtained by the method in Example 1-(2), was completely cleaved with restriction enzyme BamHI, and the 5'-terminal end of the resultant product was dephosphorized similarly as in Example 1-(3). The obtained fragments were ligated with T4 DNA ligase to obtain a recombinant DNA, followed by selecting recombinant microorganisms in accordance with the method in Example 1-(4). The recombinant microorganisms contained a recombinant DNA of a relatively small-size that carries polypeptide gene.

One of these recombinant microorganisms and its recombinant DNA were named as "Escherichia coli MAH2 (FERM P-7925)" and "pMAH2" respectively.

The restriction map of recombinant DNA pMAH2, in particular, that of the DNA fragment that carries the polypeptide gene derived from Bacillus macerans, was as shown in Fig. 3.

Fig. 3 shows that this recombinant DNA is cleaved by either restriction enzyme Pvull, Sall, Aval commercialized by Nippon Gene Co., Ltd., or Pstl commercialized by Nippon Gene Co., Ltd., but not by EcoRI, Hindlil, Kpnl, BamHI, Xbal, Xhol, or Smal.

Example 7 Cloning of Bacillus macerans polypeptide gene into Bacillus subtilis Example 7-(1) Preparation of recombinant DNA pMAH2 The recombinant DNA pMAH2 was isolated from Escherichia coli MAH2 (FERM P-7925) in accordance with the method in Example 1-(2).

Example 7-(2) Preparation of recombinant DNA carrying polypeptide gene The recombinant DNA pMAH2 specimen carrying polypeptide gene, prepared in Example 7-(1), was completely digested by subjecting it simultaneously to restriction enzymes EcoRI and BamHI.

Separately, a plasmid pUB110 specimen, prepared by the method in Example 2-(2), was completely cleaved by subjecting it simultaneously to restriction enzymes EcoRI and BamHI.

The fragments thus obtained were subjected to T4 DNA ligase similarly as in Example 1-(3) to obtain a recombinant DNA.

Example 7-(3) Introduction of recombinant DNA into Bacillus subtilis Recombinant microorganisms in which recombinant DNA carrying the polypeptide gene derived from Bacillus macerans had been introduced were cloned in accordance with the method in Example 2-(4) using Bacillus subtilis 715A, a strain incapable of producing amylase.

One of the recombinant microorganisms and its recombinant DNA were named as "Bacillus subtilis MAU210 (FERM P-7926)" and "pMAU21O" respectively. The restriction map of recombinant DNA pMAU210, in particular, that of the DNA fragment that carries the polypeptide gene derived from Bacillus macerans, was as shown in Fig. 4. Fig. 4 shows that the recombinant DNA is cleaved by either restriction enzyme Pvull, Sall, Avel or Pstl, but not by EcoRI, Hindlll, Kpnl, BamHl, Xbal, Xhol or Smal.

Example 8 Amino acid sequence of polypeptide derived from Bacillus macerens containing N-terminal end Example 8-(1) Preparation of polypeptide The polypeptide was produced by culturing Bacillus subtilis MAU210 (FERM P-7926) with a liquid culture medium similarly as in Example 10, and then purified in accordance with the method in Example 4-(1) to obtain a high-purity polypeptide specimen.

On SDS-polyacrylamide electrophoresis, the polypeptide specimen showed a molecular weight of 70,000+ 10,000 daltons and a specific activity of 200+30 units/mg protein.

Example 8-(2) Partial amino acid sequence containing N-terminal end The partial amino acid sequence containing N-terminal end was determined with the polypeptide specimen, prepared in Example 8-(1), in accordance with the method in Example 3-(2).

The partial amino acid sequence was Ser-Pro-Asp-Thr-Ser-Val-Asn-Asn-Lys-Leu.

Example 9 Sequence of polypeptide gene derived from Bacillus macerans and amino acid sequence of polypeptide Example 9-(1) Preparation of recombinant DNA carrying polypeptide gene The recombinant DNA was prepared in accordance with the method in Example 4-(3).

More particularly, a DNA fragment, obtained by digesting a DNA fragment carrying polypeptide gene, prepared by the method in Example 7-(2), with restriction enzymes, and a plasmid fragment, obtained by cleaving a plasmid pUC18 specimen, prepared by the method in Example 4-(2), in the same manner, were ligated with T4 DNA ligase to obtain a recombinant DNA.

Example 9-(2) Introduction of recombinant DNA into Escherichia coli The recombinant DNA was introduced in accordance with the method in Example 4-(3) into Escherichia coli JM83 as the host microorganism to obtain a recombinant microorganism.

Example 9-(3) Preparation of recombinant DNA from recombinant microorganism The recombinant DNA was prepared in accordance with the method in Example 4-(4).

Example 9-(4) Sequence of recombinant DNA The polypeptide gene was sequenced in accordance with the method in Example 4-(5).

The results were as shown in Table 4-1.

The signal peptide located upstream at the 5'-site of the polypeptide gene was sequenced in the same manner.

The results were as shown in Table 4-2.

Example 9-(5) Amino acid sequence of polypeptide The amino acid sequence of polypeptide was determined with reference to the sequence of polypeptide gene. The results were as shown in Table 5-1.

The amino acid sequence of the signal peptide was determined in the same manner. The results were as shown in Table 5-2.

These evidences confirmed that the polypeptide derived from Bacillus macerans has the amino acid sequence as shown in Table 5-1.

The evidence as shown in Tables 2-1 and 5-2 show that each polypeptide commonly has the amino acid sequence of (a) Asn-Lys-lle-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-lle-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) lle-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly, as well as that these partial amino acid sequences (a), (b), (c), (d) and (e) are located in sequence of nearness to the N-terminal end of polypeptide.

Table 4-1 10 20 30 40 50 60 TCCCCGGATA CGAGCGTGAA CAACAAGCTC AATTTTAGCA CGGATACGGT TTACCAGATT 70 80 90 100 110 120 AGCGATCATT CCAACCTGAA GCTGTATTTC GGGGGCGACT GGCAGGGGAT CACGAACAAA 130 140 150 160 170 180 AGCGATCATT CCAACCTGAA GCTGTATTTC GGGGGCGACT GGCAGGGGAT CACGAACAAA 190 200 210 220 230 240 ATCAACGACG GCTATCTGAC CGGAATGGGC ATCACCGCCC TCTGGATCTC GCAGCCGGTT 250 260 270 280 290 300 GAGAACATCA CCGCCGTCAT CAATTATTCG GGCGTCAACA ATACAGCTTA CCACCGTTAC 310 320 330 340 350 360 TGGCCTCGCG ACTTCAAGAA GACCAATGCC GCGTTCGGCA GCTTCACCGA CTTCTCCAAT 370 380 390 400 410 420 TTGATCGCCG CAGCGCATTC ACACAATATC AAGGTAGTTA TGGACTTTGC ACCTAATCAC 430 440 450 460 470 480 ACCAACCCGG CTTCGAGTAC GGACCCCTCG TTCGCCGAGA ACGGCGCGCT CTACAACAAC 490 500 510 520 530 540 GGAACGCTGC TCGGCAAGTA TAGCAACGAT ACCGCCGGCC TGTTCCACCA CAATGGCGGC Table 4-1 continued (1) 550 560 570 580 590 600 ACCGATTTCT CGACGACTGA AAGCGGTATC TACAAGAACC TGTACGATCT CGCGGATATC 610 620 630 640 650 660 AATCAGAACA ACAACACCAT CGACTCGTAT CTCAAGGAAT CGATCCAGCT GTGGCTGAAT 670 680 690 700 710 720 CTCGGAGTCG ACGGCATCGG CTTCGACGCC GTGAAGCATA TGCCTCAGGG CTGGCAGAAG 730 740 750 760 770 780 AGCTACGTCT CGTCGATCTA CAGCAGCGCC AATCCGGTGT TCACCTTCGG TGAATGGTTC 790 800 810 820 830 840 CTCGGCCCCG ACGAAATGAC CCAGGACAAC ATCAACTTCG CGAATCAGAG CGGCATGCAC 850 860 870 880 890 900 CTGCTGGACT TTGCGTTTGC GCAGGAAATC CGTGAAGTGT TCCGCGACAA GTCGGAGACG 910 920 930 940 950 960 ATGACCGACC TGAACTCGGT GATCTCCAGC ACCGGCTCCA GCTATAATTA CATCAACAAC 970 980 990 1000 1010 1020 ATGGTTACGT TCATCGACAA CCATGACATG GACCGCTTCC AGCAAGCCGG AGCGAGCACT 1030 1040 1050 1060 1070 1080 CGCCCGACCG AGCAGGCTCT TGCGGTAACG CTGACTTCCC GCGGCGTTCC GGCAATCTAC Table 4-1 continued (2) 1090 1100 1110 1120 1130 1140 TACGGTACAG AGCAATATAT GACCGGCAAC GGCGACCCGA ACAACCGCGG CATGATGACC 1150 1160 1170 1180 1190 1200 GGCTTCGATA CGAACAAGAC AGCGTACAAA GTGATCAAGG CGCTGGCTCC GCTTCGCAAG 1210 1220 1230 1240 1250 1260 TCCAACCCGG CTCTCGCCTA CGGCTCGACG ACCCAGCGTT GGGTGAACAG CGACGTCTAC 1270 1280 1290 1300 1310 1320 GTATATGAAC GCAAGTTCGG AAGCAACGTA GCTCTCGTTG CCGTCAACCG CAGCTCGACG 1330 1340 1350 1360 1370 1380 ACTGCCTATC CGATATCGGG AGCGCTTACT GCTCTGCCAA ACGGAACGTA TACCGACGTT 1390 1400 1410 1420 1430 1440 CTCGGCGGCC TGCTTAATGG CAATTCAATT ACCGTTAACG GCGGCACGGT CAGCAACTTT 1450 1460 1470 1480 1490 1500 ACACTTGCAG CGGGCGGTAC GGCAGTCTGG CAGTACACGA CGACGGAATC CTCGCCGATT 1510 1520 1530 1540 1550 1560 ATCGGCAACG TCGGCCCGAC TATGGGCAAG CCCGGCAACA CCATCACGAT CGACGGACGC 1570 1580 1590 1600 1610 1620 GGCTTCGGTA CTACGAAGAA CAAAGTTACT TTCGGTACGA CAGCCGTTAC CGGCGCGAAC Table 4-1 continued (3) 1630 1640 1650 1660 1670 1680 ATCGTGAGCT GGGAAGATAC CGAAATCAAG GTCAAAGTTC CGAACGTGGC CGCCGGCAAC 1690 1700 1710 1720 1730 1740 ACGGCCGTTA CGGTAACGAA CGCCGCCGGC ACTACCAGCG CAGCGTTCAA CAACTTTAAC 1750 1760 1770 1780 1790 1800 GTACTGACTG CCGATCAGGT CACTGTCCGC TTCAAAGTCA ACAATGCCAC CACGGCCCTG 1810 1820 1830 1840 1850 1860 GGACAAAACG TCTACCTGAC CGGTAACGTC GCCGAGCTTG GCAACTGGAC AGCCGCCAAC 1870 1880 1890 1900 1910 1920 GCAATCGGTC CGATGTACAA CCAGGTAGAA GCCAGCTATC CGACTTGGTA CTTCGACGTC 1930 1940 1950 1960 1970 1980 AGCGTTCCGG CCAACACGGC GCTGCAATTC AAGTTCATCA AAGTGAACGG CTCGACAGTG 1990 2000 2010 2020 2030 2040 ACTTGGGAAG GCGGCAACAA CCACACCTTC ACCTCGCCTT CGAGCGGCGT TGCGACCGTA 2050 2060 ACGGTCGATT GGCAGAAC Table 4-2 10 20 30 40 50 60 ATGAAAAAGC AAGTCAAATG GTTGACGTCG GTGTCGATGT CCGTAGGGAT CGCACTCGGC 70 80 90 GCGGCGCTGC CTGTATGGGC A Table 5-2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Met Lys Lys Gin Val Lys Trp Leu Thr Ser Val Ser Met Ser Val 16) Gly Ile Ala Leu Gly Ala Ala Leu Pro Val Trp Ala Table 5-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Ser Pro Asp Thr Ser Val Asn Asn Lys Leu Asn Phe Ser Thr Asp 16) Thr Val Ty@ Glh Ile Val Thr Asp Arg Phe Val Asp Gly Asn Ser 31) Ala Asn Asn pro Thr Gly Ala Ala Phe Ser Ser Asp His Ser Asn 46) Leu Lys Leu Tyr Phe Gly Gly Asp Trp Gln Gly Ile Thr Asn Lys 61) Ile Asn Asp Gly Tyr Leu Thr Gly Met Gly Ile Thr Ala Leu Trp 76) Ile Ser Gln Pro Val Glu Asn Ile Thr Ala Val Ile Asn Tyr Ser 91) Gly Val Asn Asn Thr Ala Tyr His Gly Tyr Trp pro Arg Asp Phe 106) Lys Lys Thr Asn Ala Ala Phe Gly Ser Phe Thr Asp Phe Ser Asn 121) Leu Ile Ala Ala Ala His Ser His Asn Ile Lys Val Ala Met Asp 136) Phe Ala Pro Asn His Thr Asn Pro Ala Ser Ser Thr Asp Pro Ser 151) Phe Ala Glu Asn Gly Ala Leu Tyr Asn Asn Gly Thr Leu Leu Gly 166) Lys Tyr Ser Asn Asp Thr Ala Gly Leu Phe His His Asn Gly Gly 181) Thr Asp Phe Ser thr Thr Glu Ser Gly Ile Tyr Lys Asn Leu Tyr 196) Asp Leu Ala Asp Ile Asn Gln Asn Asn Asn Thr Ile Asp Ser Tyr 211) Leu Lys Glu Ser Ile Gln Leu Trp Leu Asn Leu Gly Val Asp Gly 226) Ile Arg Phe Asp Ala Val Lys His Met Pro Gln Gly Trp Gln Lys 241) Ser Tyr Val Ser Ser Ile Tyr Ser Ser Ala Asn Pro Val Phe Thr 256) Phe Gly Glu Trp Phe Leu Gly Pro Asp Glu Met Thr Gln Asp Asn 271) Ile Asn Phe Ala Asn Gln Ser Gly Met His Leu Leu Asp Phe Ala 286) Phe Ala Gln Glu Ile Arg Glu Val Phe Arg Asp Lys Ser Glu Thr 301) Met Thr Asp Leu Asn Ser Val Ile Ser Ser Thr Gly Ser Ser Tyr 316) Asn Tyr Ile Asn Asn Met Val Thr Phe Ile Asp Asn His Asp Het 331) Asp Arg Phe Gln Gln Ala Gly Ala Ser Thr Arg Pro Thr Glu Gln 346) Ala Leu Ala Val Thr Leu Thr Ser Arg Gly Val Pro Ala Ile Tyr 361) Tyr Gly Thr Glu Gln Tyr Met Thr Gly Asn Gly Asp Pro Asn Asn 376) Arg Gly Met Met Thr Gly The Asp Thr Asn Lys Thr Ala Tyr Lys Table 5-1 continued 391) Val Ile Lys Ala Leu Ala Pro Leu Arg Lys Ser Asn Pro Ala Leu 406) ala Tyr Gly Ser Thr Thr Gln Arg Trp Val Asn Ser Asp Val Tyr 421) Val Tyr Glu Arg Lys Phe Gly Ser Asn Val Ala Leu Val Ala Val 436) Asn Arg Ser Ser Thr Thr Ala Tyr Pro Ile Ser Gly Ala Leu Thr 451) Ala Leu Pro Asn Gly Thr Tyr Thr Asp Val Leu Gly Gly Leu Leu 466) Asn Gly Asn Ser Ile Thr Val Asn Gly Gly Thr Val Ser Asn Phe 481) Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Thr 496) Glu Ser Ser Pro Ile Ile Gly Asn Val Gly Pro Thr Met Gly Lys 511) Pro Gly Asn Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Thr Thr 526) Lys Asn Lys Val Thr Phe Gly Thr Thr Ala Val Thr Gly Ala Asn 541) Ile Val Ser Trp Glu Asp Thr Glu Ile Lys Val Lys Val Pro Asn 556) Val Ala Ala Gly Asn Thr Ala Val Thr Val Thr Asn Ala Ala Gly 571) Thr Thr Ser Ala Ala Phe Asn Asn Phe Asn Val Leu Thr Ala Asp 586) Gln Val Thr Val Arg Phe Lys Val Asn Asn Ala Thr Thr Ala Leu 601) Gly Gln Asn Val Tyr Leu Thr Gly Asn Val Ala Glu Leu Gly Asn 616) Trp Thr Ala Ala Asn Ala Ile Gly Pro Met Tyr Asn Gln Val Glu 631) Ala Ser Tyr Pro Thr Trp Tyr Phe Asp Val Ser Val Pro Ala Asn 646) Thr Ala Leu Gln Phe Lys Phe Ile Lys Val Asn Gly Ser Thr Val 661) Thr Trp Glu Gly Gly Asn Asn His Thr Phe Thr Ser Pro Ser Ser 678) Gly Val Ala Thr Val Thr Val Asp Trp Gln Asn Example 10 Preparation of polypeptide with recombinant microorganism Polypeptides were prepared with Escherichia coli MAH2 (FERM P-7925) and Bacillus subtilis MAU210 (FERM P-7926) both in which recombinant DNA carrying the polypeptide gene derived from Bacillus macerans had been introduced.The polypeptide productivities of these recombinant microorganisms, host microorganism, and donor Bacillus macerans microorganism were compared in relation to their CGTase activity. The used liquid culture medium was prepared by the method in Example 5.

Escherichia coli MAH2 was inoclated to the liquid culture medium additionally containing 50 ,eeg/ml of amplicillin, while Escherichia coli HB101 was inoculated in the liquid medium culture without addition of antibiotic. In each case, microorganism was cultured at 35"C for 24 hours under vigorous shaking conditions.

Bacillus subtilis MAU210 was inoculated to the liquid culture medium additionally containing 5 ,ag/ml of kanamycin, while Bacillus subtilis 715A was inoculated to the liquid culture medium without addition of antibiotic. In each case, microorganism was cultured at 28"C for 72 hours.

Bacillus macerans 1 7A was cultured with the liquid culture medium at 28"C for 72 hours without addition of antibiotic.

Each culture was treated similarly as in Example 5, and its CGTase activities was then determined. The results were as shown in Table 6.

These evidences clearly show that the recombinant microorganisms are advantageously usable in industriat-scale production of polypeptide because they have improved polypeptide productivity.

Table 6 CGTase activity -units/ml) Microorganism Supernatant Cell Total Escherichia coli MAH2 (FERM P-7925) 0.6 11.8 12.4 Present invention Bacillus subtilis MAU210 (FERM P-7926) 54.6 0.3 54.9 Present invention Escherichia coli HB101 0 0 0 Control Bacillus subtilis 715A 0 0 0 Control Bacillus macerans 17A 7.5 0.4 7.9 Control The supernatants were salted out with ammonium sulfate at a saturation degree of 0.6 to obtain crude polypeptide specimens.

On studying these crude polypeptide specimens on their enzymatic properties similarly as in Example 5, the enzymatic properties of the polypeptide produced by the recombinant microorganisms were in good accordance with those of the polypeptide produced by the donor Bacillus macerans microorganism.

The principal uses af the polypeptide of the invention are more fully discussed below.

The polypeptide effects intra- or intermolecular saccharide transfer between saccharide donors and saccharide acceptors.

According to one aspect of the present invention, various saccharide-transferred products can be produced by taking advantage of these saccharide transfer reactions.

For example, a partial starch hydrolysate containing a-, ss- and y-cyclodextrins is prepared by subjecting an amylaceous substance as the substrate, such as starch, liquefied starch with a Dextrose Equivalent (DE) of below 10, or amylase, to the action of the polypeptide to induce an intramolecular saccharide transfer. Each cyclodextrin can be isolated from the partial starch hydrolysate, if necessary.

a-Glycosyalated saccharide sweeteners, for example, a-glucosyl-, a-maltosyl- and a-maltotriosyl-saccharides, can be prepared by subjecting a mixture of a saccharide donor (for example, an amylaceous substance such as starch, liquefied starch, dextrin, cyclodextrin or amylose) and a saccharide acceptor (for example, a monosaccharide such as xylose, sorbose or fructose, or a disaccharide such as sucrose, maltulose or isomaltulose) to the action of the polypeptide to induce an intermolecular saccharide transfer. The a-glycosylated saccharide sweetener can be advantageously used in foods and beverages because the a-glycosylated saccharide sweetener is much milder in taste, more soluble in water, but less crystallizable in comparison with the unmodified saccharide sweetener. These would expand extremely the use of saccharide sweeteners.

In the intermolecular saccharide transfer redaction, the use of a glycoside, for example, steviol glycoside such as stevioside or rebaudioside, glycyrrhizin, soyasaponin, teasaponin, rutin or esculin, as the saccharide acceptor leads to the formation of a-glycosylated glycosides such as a-glucosyl-, a-maltosyl- and a-maltotriosyl-glycosides. The a-glycosylated glycoside is free of the unpleasant tastes such as bitter- and astringent-tastes which are inherent in the unmodified glycoside, and more readily soluble in water than the unmodified glycoside. These would expand extremely the use of glycosides.Specifically, a-glycosylated steviol glycoside and a-glycosylated glycyrrhizin can be advantageously used in foods, beverages, and phramaceuticals for peroral administration because the taste improvement in these a-glycosylated glycosides is remarkably high, and because their sweetness is comparable to that of sucrose.

The following Examples further illustrate the preparation of such modified saccharide sweeteners.

Example ii Corn syrup containing cyclodextrin A 10 w/w % suspension of potato starch was added with 2 units/g starch of a polypeptide specimen prepared with Bacillus subtilis TCU211 in accordance with the method in Example 5, liquefied by heating to 850C at pH 6.5, cooled to 70"C, further added with the same amount of the polypeptide specimen, and reacted for 40 hours. The reaction mixture was purified by decoloration using activated carbon and deionization using an ion exchange resin, and then concentrated to obtain a corn syrup containing cyclodextrin in a yield of 92% based on the dry solid. The corn syrup can be advantageously incorporated into flavorings and cosmetics wherein fragrance or aroma is one of the important factors because the corn syrup has excellent flavorlocking properties.

The a-, ss- and y-cyclodextrins in the corn syrup can be separated by treating the corn syrup by means of a procedure using an organic precipitant, such as toluene or trichloromethane, or conventional column chromatography.

Example 12 a-Glycosylsucrose A 35 w/w % suspension of cornstarch was added with 0.2 w/w % oxalic acid, autoclaved to 1200C to give a DE of 20, neutralized with calcium carbonate, and filtered to obtain a dextrin solution. The dextrin solution was then added with a half amount of sucrose based on the dry solid, and the resultant mixture was added with 15 units/g starch of a polypeptide specimen prepared with Bacillus subtilis MAU210 in accordance with the method in Example 10, and reacted at pH 6.0 and 55"C for 40 hours. The reaction mixture was purified by decoloration using activated carbon and deionization using ion exchange resin, and then concentrated to obtain a colorless, transparent corn syrup in a yield of 94% based on the dry solid.The corn syrup containing a large amount of a-glycosylsucrose can be advantageously used in confection ery because it is mildly sweet and amorphous.

Example 13 a-Glycosyl stevioside Two-hundred g of stevioside and 600 g of dextrin tDE 8) were dissolved in 3 liters of water by heating, and the resultant solution was cooled to 70"C, added with 5 units/g dextrin of a polypeptide specimen prepared with Bacillus subtilis TCU211 in accordance with the method in Example 5, and reacted at pH 6.0 and 65"C for 35 hours. The reaction mixture was then heated at 95"C for 15 minutes, purified by filtration, concentrated, and pulverized to obtain a pulverulent sweetener containing a-glycosyl stevioside in a yield of about 92% based on the dry solid.

The sweetener free of the unpleasant taste which is inherent in the unmodified parent stevioside was comparable to sucrose in taste quality, and the sweetening power of the sweetener was about 100-times higher than that of scurose. The sweetener can be advantageously used as a diet sweetener or for seasoning foods and beverages because of its low-cariogenic and low-calorific properties.

Example 14 a-Glycosyl ginsenoside Sixty g of a ginseng extract and 180 g of sscyclodextrin were dissolved in 500 ml of water by heating, and the resultant mixture was cooled to 70"C, adjusted to pH 6.0, added with 3 units/g fi-cyclodextrin of a polypeptide specimen prepared with Escherichia coli TCH201 in accordance with the method in Example 5, cooled to 65"C, and reacted at pH 6.0 for 40 hours.

The reaction mixture was heated for 15 minutes to inactivate the polypeptide, followed by filtration. The filtrate was admitted to a column packed with 3 liters of "Amberlite XAD-7", a synthetic adsorbant commercialized by Rohm & Haas Co., Philadelphia. PA, USA. Thereafter, the column was sufficiently washed with water to remove free saccharides. The column was then admitted with 10 liters of 50 v/v % ethanol, and the eluate was concentrated and dehydrated to obtain about 21 g of a pulverulent product that contains a-glycosyl ginsenoside. Since the product is free of the unpleasant tastes such as bitter-, astringent- and harsh-tastes which are manifest in the unmodified parent ginsenoside, the product can be perorally administered intact, or, if necessary, seasoned with any sweetener or sour, prior to its use. In addition, the product can be advantageously used in health foods and medicines for internal administration because the product possesses invigorating, peptic, intestine-regulating, haematic, anti-inflammatory and expectorant effects similar to the parent ginsenoside.

As described above, we have determined the amino acid sequences of the polypeptide gene and its signal peptide, as well as preparing recombinant DNA having a Pvull restriction site from a donor microorganism by in vitro genetic engineering technique. Furthermore, we have prepared recombinant microorganisms in which the recombinant DNA is introduced, as well as confirming that the recombinant microorganisms autonomically and consistently proliferate in a nutrient culture medium.

Claims (39)

1. A polypeptide possessing cyclomaltodextrin glucanotransferase (CGTase) activity, comprising one or more partial amino acid sequences selected from the group consisting of (a) Asn-Lys-lle-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-lle-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) lle-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly.

2. The polypeptide in accordance with claim 1, wherein partial amino acid sequences of (a) Asn-Lys-lle-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-lle-Asp-Asn-His-Asp-Met-Asp.Arg-Phe, (d) lle-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly are located in sequence of nearness to the N-terminal end of said polypeptide.

3. The polypeptide in accordance with claim 1, which shows a molecular weight of 70,000+ 10,000 daltons on SDS-polyacrylamide electrophoresis.

4. The polypeptide in accordance with claim 1, whose partial amino acid sequence containing N-terminal end is Ala-Gly-Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr.

5. The polypeptide in accordance with claim 4, which has the following amino acid sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Ala Gly Asn Leu Asn Lys Val Asn Phe Thr Ser Asp Val Val Tyr 16) Gln Ile Val Val Asp Arg Phe Val Asp Gly Asn Thr Ser Asn Asn 31) Pro Ser gly Ala Leu Phe Ser Ser Gly Cys Thr Asn Leu Arg Lys 46) Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp 61) Gly Tyr Leu Thr Asp Met Gly Val Thr Ala Ile Trp Ile Ser Gla 76) Pro Val Glu Asn Val Phe Ser Val Met Asn Asp Ala Ser Gly Ser 91) Ala Ser Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Pro Asn 106) Pro Phe Phe Gly Th@ Leu Ser Asp Phe Gln Arg Leu val Asp Ala 121) Ala His Ala Lys Gly Ile Lys Val Ile Ile Asp Phe Ala Pro Asn 136) His Thr Ser Pro Ala Ser Glu Thr Asn Pro Ser Tyr Met Glu Asn 151) Gly Arg Leu Tyr Asp Asn Gly Thr Leu Leu Gly Gly Tyr Thr Asn 166) Asp Ala Asn met Tyr Phe His His Asn Gly Gly Thr Thr Phe Ser 181) Ser Leu Glu Asp Gly Ile Tyr Arg Asn Leu Phe Asp Leu Ala Asp 196) Leu Asn His Gln Asn Pro Val Ile Asp Arg Tyr Leu Lys Asp Ala 211) Val Lys Met Trp Ile Asp Met Gly Ile Asp Gly Ile Arg Met Asp 226) Ala Val Lys His Het Pro Phe Gly Trp Gln Lys Ser Leu Met Asp 241) Glu Ile Asp Asn Tyr Arg Pro Val Phe Thr Phe Gly Glu Trp Phe 256) Leu Ser Glu Asn Glu Val Asp Ala Asn Asn His Tyr Phe Ala Asn 271) Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Gly Gln Lys Leu 286) Arg Gln Val Leu Arg Asn Asn Ser Asp Asn Trp Tyr Gly Phe Asn 301) Gln Met Ile Gln Asp Thr Ala Ser Ala Tyr Asp Glu Val Leu Asp 316) Gln Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Met Ile 331) asp Gly Gly Asp Pro Arg Lys Val Asp Met Ala Leu Ala Val Leu 346) Leu Thr Ser Arg Gly Val Pro Asn Ile Tyr Tyr Gly Thr Glu Gln 361) Tyr Met Thr Gly Asn Gly Asp Pro Asn Asn Arg Lys Met Met Ser 376) Ser Phe Asn Lys Asn Thr Arg Ala Tyr Gln Val Ile Gln Lys Leu 391) Ser Ser Leu Arg Arg Asn Asn Pro Ala Leu Ala Tyr Gly Asp Thr 406) Glu Gln Arg Trp Ile Asn Gly Asp Val Tyr Val Tyr Glu Arg Gln 421) Phe Gly Lys Asp Val Val Leu Val Arg Val Asn Arg Ser Ser Ser 436) Ser Asn Tyr Ser Ile Thr Gly Leu Phe Thr Ala Leu Pro Ala Gly 451) Thr Tyr Thr Asp Gln Leu Gly Gly Leu Leu Asp Gly Asn Thr Ile 466) Gln Val Gly Ser Asn Gly Ser Val Asn Ala Phe Asp Leu Gly Pro 481) Gly Glu Val Gly Val Trp Ala Tyr Ser Ala Thr Glu Ser Thr Pro 496) Ile Ile Gly His Val Gly Pro Met Met Gly Gln Val Gly His Gln 511) Val Thr Ile Asp Gly Glu Gly Phe Gly Thr Asn Thr Gly Thr Val 526) Lys Phe Gly Thr Thr Ala Ala Asn Val Val Ser Trp Ser Asn Asn 541) Gln Ile Val val Ala Val Pro Asn Val Ser Pro Gly Lys Tyr Asn 556) Ile Thr Val Gln Ser Ser Ser Gly Gln Thr Ser Ala Ala Tyr Asp 571) Asn Phe Glu Val L@u Thr Asn Asp Gln Val Ser Val Arg Phe Val 586) Val Asn Asn Ala Thr Thr Asn Leu Gly Gln Asn Ile Tyr Ile Val 601) Gly Asn Val Tyr Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala Ile 616) Gly Pro Met Phe Asn Gln Val Val Tyr Ser Tyr Pro Thr Trp Tyr 631) Ile Asp Val Ser Val Pro Glu Gly Lys Thr Ile Glu Phe Lys Phe 646) Ile Lys Lys Asp Ser Gln Gly Asn Val Thr Trp Glu Ser Gly Ser 661) Asn His Val Tyr Thr Thr Pro Thr Asn Thr Thr Gly Lys Ile Ile 676) Val Asp Trp Gln Asn

6. The polypeptide in accordance with claim 4, wherein a signal peptide having an amino acid sequence of Met-Arg-Arg-Trp-Leu-Ser-Leu-Val-Leu-Ser-Met-Ser-Phe-Val-Phe-Ser-Ala-lle-Phe-lle-Val- Ser-Asp-Thr-Gln-Lys-Val-Thr-Val-Glu-Ala is located upstream at the upstream at the N-terminal side of said polypeptide.

7. The polypeptide in accordance with claim 4, whose partial amino acid sequence containing N-terminal end is Ser-Pro-Asp-Thr-Ser-Val-Asn-Asn-Lys-Leu.

8. the polypeptide in accordance with claim 1, which has the following amino acid sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1) Ser Pro Asp Thr Ser Val Asn Asn Lys Leu Asn Phe Ser Thr Asp 16) Thr Val Tyr Gln Ile Val Thr Asp Arg Phe Val Asp Gly Asn Ser 31) Ala Asn Asn Pro Thr Gly Ala Ala Phe Ser Ser Asp His Ser Asn 46) Leu Lys Leu Tyr Phe Gly Gly Asp Trp Gln Gly Ile Thr Asn Lys 61) Ile Asn Asp Gly Tyr Leu Thr Gly Met Gly Ile Thr Ala Leu Trp 76) Ile Ser Gln Pro Val Glu Asn Ile Thr Ala Val Ile Asn Tyr Ser 91) Gly Val Asn Asn Thr Ala Tyr His Gly Tyr Trp Pro Arg Asp Phe 106) Lys Lys Thr Asn Ala Ala Phe Gly Ser Phe Thr Asp Phe Ser Asn 121) Leu Ile Ala Ala Ala His Ser His Asn Ile Lys Val Val Het Asp 136) Phe Ala Pro Asn His Thr Asn Pro Ala Ser Ser Thr Asp Pro Ser 151) Phe Ala Glu Asn Gly Ala Leu Tyr Asn Asn Gly Thr Leu Leu Gly 166) Lys Tyr Ser Asn Asp Thr Ala Gly Leu Phe His His Asn Gly Gly 181) Thr Asp Phe Ser Thr Thr Glu Ser Gly Ile Tyr Lys Asn Leu Tyr 196) Asp Leu Ala Asp Ile Asn Gln Asn Asn Asn Thr Ile Asp Ser Tyr 211) Leu Lys Glu Ser Ile Gln Leu Trp Leu Asn Leu Gly Val Asp Gly 226) Ile Arg Phe Asp Ala Val Lys His Met Pro Gln Gly Trp Gln Lys 241) Ser Tyr Val Ser Ser Ile Tyr Ser Ser Ala Asn Pro Val Phe Thr 256) Phe Gly Glu Trp Phe Leu Gly Pro Asp Glu Met Thr Gln Asp Asn 271) Ile Asn Phe Ala Asn Gln Ser Gly Met His Leu Leu Asp Phe Ala 286) Phe Ala Gln Glu Ile Arg Glu Val Phe Arg Asp Lys Ser Glu Thr 301) Met Thr Asp Leu Asn Ser Val Ile Ser Ser Thr Gly Ser Ser Tyr 316) Asn Tyr Ile Asn Asn Het Val Thr Phe Ile Asp Asn His Asp Met 331) Asp Arg Phe Gln Gln Ala Gly Ala Ser Thr Arg Pro Thr Glu Gln 346) Ala Leu Ala Val Thr Leu Thr Ser Arg Gly Val Pro Ala Ile Tyr 361) Tyr Gly Thr Glu Gln Tyr Met Thr Gly Asn Gly Asp Pro Asn Asn 376) Arg Gly Met Met Thr Gly Phe Asp Thr Asn Lys Thr Ala Tyr Lys 391) Val Ile Lys Ala Leu Ala Pro Leu Arg Lys Ser Asn Pro Ala Leu 406) Ala Tyr Gly Ser Thr Thr Gln Arg Trp Val Asn Ser Asp Val Tyr 421) Val Tyr Glu Arg Lys Phe Gly Ser Asn Val Ala Leu Val Ala Val 436) Asn Arg Ser Ser Thr Thr Ala Tyr Pro Ile Ser Gly Ala Leu Thr 451) Ala Leu Pro Asn Gly Thr Tyr Thr Asp Val Leu Gly Gly Leu Leu 466) Asn Gly Asn Ser Ile Thr Val Asn Gly Gly Thr Val Ser Asn Phe 481) Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Thr 496) Glu Ser Ser Pro Ile Ile Gly Asn Val Gly Pro Thr Met Gly Lys 511) Pro Gly Asn Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Thr Thr 526) Lys Asn Lys Val Thr Phe Gly Thr Thr Ala Val Thr Gly Ala Asn 541) Ile Val Ser Trp Glu Asp Thr Glu Ile Lys Val Lys Val Pro Asn 556) Val Ala Ala Gly Asn Thr Ala Val Thr Val Thr Asn Ala Ala Gly 571) Thr Thr Ser Ala Ala Phe Asn Asn Phe Asn Val Leu Thr Ala Asp 586) Gin Val Thr Val Arg Phe Lys Val Asn Asn Ala Thr Thr Ala Leu 601) Gly Gln Asn Val Tyr Leu Thr Gly Asn Val Ala Glu Leu Gly Asn 616) Trp Thr Ala Ala Asn Ala Ile Gly Pro Met Tyr Asn Gin Val Glu 631) Ala Ser Tyr Pro Thr Trp Tyr Phe Asp Val Ser Val Pro Ala Asn 646) Thr Ala Leu Gln Phe Lys Phe Ile Lys Val Asn Gly Ser Thr Val 661) Thr Trp Glu Gly Gly Asn Asn His Thr Phe Thr Ser Pro Ser Ser 676) Gly Val Ala Thr Val Thr Val Asp Trp Gln Asn

9. The polypeptide in accordance with claim 8, wherein a signal peptide having an amino acid sequence of Met-Lys-Lys-Gln-Val-Lys-Trp-Leu-Thr-Ser-Val-Ser-Met-Ser-Val-Gly-lle-Ala-Leu-Gly-Ala- Ala-Leu-Pro-Val-Trp-Ala is located upstream at the N-terminal side of said polypeptide.

10. The polypeptide in accordance with claim 1, which orginates from a microorganism capable of producing CGTase.

11. The polypeptide in accordance with claim 1, which orginates from a microorganism of species Bacillus stearothermophilus.

12. The polypeptide in accordance with claim 1, which originates from a microorganism of species Bacillus macerans.

13. The polypeptide in accordance with claim 1, which originates from a recombinant microorganism in which a recombinant DNA carrying CGT-ase gene has been introduced.

14. A recombinant DNA having a Pvull restriction cleavage site and carrying a CGTase gene, said recombinant DNA comprising: a DNA fragment, obtained by digesting the DNA of a donor microorganism capable of producing CGTase with a restriction enzyme in vitro; and a vector fragment, obtained by cleaving a vector with the restriction enzyme, these fragments being ligated.

15. The recombinant DNA in accordance with claim 14, wherein said donor microorganism is of genus Bacillus.

16. The recombinant DNA in accordance with claim 14, wherein said donor microorganism is a member selected from the group consisting of Bacillus stearothermophilus and Bacillus macerans.

17. The recombinant DNA in accordance with claim 14, which has restriction cleavage sites as shown in Fig. 1, 2 3 or 4.

18. A biologically-pure culture of a recombinant microorganism in which a recombinant DNA having a Pvull restriction cleavage site and carrying CGTase gene, prepared by ligation of a DNA fragment, obtained by digesting the DNA of a donor microorganism capable of producing CGTase with a restriction enzyme in vitro, and a vector fragment, obtained by cleaving a vector with the restriction enzyme, has been introduced.

19. The culture in accordance with claim 18, wherein said recombinant DNA has restriction cleavage sites as shown in Fig. 1, 2, 3 or 4.

20. The culture in accordance with claim 19, wherein said recombinant microorganism is of Escherichia or Bacillus.

21. The culture in accordance with claim 19, wherein said recombiant microorganism is a member selected from the group consisting of Escherichia coli TCH201 (FERM P-7924) or Escherichia coli MAH2 (FERM P-7925).

22. The culture in accordance with claim 18, wherein said recombinant microorganism is a member selected from the group consisting of Bacillus subtilis MAU210 (FERM P-7926) and Bacillus subtilis TCU211 (FERM P-7927).

23. A process for producing CGTase, comprising: culturing with a nutrient culture medium a recombinant microorganism, in which a recombinant DNA having a Pvull restriction cleavage site and carrying CGTase gene, prepared by ligation of a DNA fragment, obtained by digesting the DNA of a donor microorganism capable of producing a CGTase with a restriction enzyme in vitro, and a vector fragment, obtained by cleaving a vector with the restriction enzyme, has been introduced; and recovering the accumuated CGTase.

24. The process in accordance with claim 23, wherein said recombinant DNA has restriction cleavage sites as shown in Fig. 1, 2, 3 or 4.

25. The process in accordance with claim 23, wherein said recombinant microorganism is of genus Escherichia or Bacillus.

26. The process in accordance with claim 23, wherein said recombinant microorganism is a member selected from the group consisting of Escherichia coli TCH201 (FERM P-7924) or Escherichia coli MAH2 (FERM P-7925).

27. The process in accordance with claim 23, wherein said recombinant microorganism is a member selected from the group consisting of Bacillus subtilis MAU210 (FERM P-7926) and Bacillus subtilis TCU211 (FERM P-7927).

28. The process for producing a saccharide-transferred product, comprising subjecting an amylaceous substance to the action of a polypeptide possessing CGTase activity; said polypeptide being a polypeptide as claimed in any one of claims 1 to 13 and 35.

29. The process in accordance with claim 28, wherein said saccharide-transferred product is cyclodextrin.

30. The process in accordance with claim 28, wherein said amylaceous substance is subjected to the action of said polypeptide in the presence of a saccharide acceptor.

31. The process in accordance with claim 28, wherein said amylaceous substance is selected from the group consisting of starch, amylose, cyclodextrin, dextrin, and mixtures thereof.

32. The process in accordance with claim 30, wherein saidsaccharide acceptor is a member selected from the group consisting of saccharide sweetener, glycoside, and mixtures thereof.

33. The process in accordance with claim 30, wherein the saccharide-transferred product is a member selected from the group consisting of a-glycosylsucrose, a-glycosyl stevioside, and aglycosyl ginsenoside.

34. The process in accordance with claim 30, wherein said saccharide-transferred product is used as a sweetener.

35. A polypeptide according to claim 1, substantially as hereinbefore described with reference to any one of the foregoing Examples 1 to 10.

36. A process according to claim 23, substantially as hereinbefore described with reference to any one of the foregoing Examples 1 to 10.

37. A process according to claim 28, substantially as hereinbefore described with reference to any one of the foregoing Exmples 11 to 14.

38. Cyclomaltodextrin Glucånotransferase whenever obtained by a process as claimed in any one of claims 23 to 27 and 36.

39. A saccharide transferred product whenever obtained by a process as described in any one of claims 28 to 34 and 37.