CA1335183C - Polypeptide possessing cyclomaltodextrin glucanotransferase activity - Google Patents

Abstract

The sequence of cyclomaltodextrin glucanotransferase (CGTase) gene derived from microorganism of genus Bacillus microorganism and the amino acid sequence of CGTase are determined. A recombinant DNA carrying the CGTase gene is introduced by in vitro genetic engineering technique into a host micro-organism of species Bacillus subtilis or Escherichia coli. The recombinant microorganism carrying the recombinant DNA autonomically proliferates to secrete a large amount of CGTase.

Description

133~183 POLYPEPTIDE POSSESSING CYCLOMALTODEXTRIN
GLUCANOTRANSFERASE ACTIVITY

Field of the Invention The present invention relates to a polypeptide, particularly, a polypeptide possessing cyclomaltodextrin glucanotransferase activity.

Abbreviations Throughout the invention, amino acids, peptides, etc. are designated with abbreviations which are commonly used in the art. Examples of such abbreviations are as follows.
When optical isomers are possible, the abbreviations of amino acids mean L-isomers, unless specified otherwise.
DNA is the abbreviation of deoxyribonucleic acid; RNA, ribonucleic acid; A, adenine; T, thymine; G, guanine; C, cytosine; dNTP, deoxynucleo-tide triphosphate; ddNTP, dideoxynucleotide triphosphate; dCTP, deoxycytidin triphosphate; SD S, sodium dodecyl sulfate; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Cys, cysteine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine;

Met, methionine; Phe, phenyl~l~nine; Pro, proline; Ser, serine; Thr, threo-nine; Trp, tryptophan; Tyr, tyrosine; Val, valine; and CGTase, cyclomalto-dextrin glucanotransferase.
The wording of "polypeptide" means "polypeptide possessing CGTase activity " .

13~183 Description of the Prior Art CGTase, or macerans amylase, is known for years as the enzyme produced by Bacillus macerans.
Recently, it was found that CGTase is produced by other micro-organisms such as those of species Bacillus stearothermophilus and Bacillus circulans. The saccharide transfer activity of CGTase is now in many indus-trial uses.
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 sub-jecting a mixture solution of liquefied starch and sucrose to CGTase.
Cyclodextrins is now in an expanding demand as the host to form a stable inclusion complex with an organic compound which is volatile or suscep-tive to oxidation, while glycosylsucrose is in an expanding demand as a mildly-sweet low-cariogenic sweetener which is commercialized by Hayashibara Co., Ltd., Okayama, Japan, under the Registered Trademark of "Coupling Sugar".
In order to meet these demands, development of means to provide a stationary CGTase supply is one of the urgent necessities. This requires determination of the amino acid sequence of polypeptide that possesses CGTase activity .
Such amino acid sequence was, however, so far unknown.

Brief Description of the Accompanying Drawings FIG. 1 shows the restriction map of recombinant DNA pTCH201, in particular, that of the DNA fragment which carries the polypeptide gene deriv-ed from Bacillus stearothermophilus.
FIG. 2 show the restriction map of recombinant DNA pTCU211, in particular, that of the DNA fragment which carries the polypeptide gene deriv-ed from Bacillus stearothermophilus.
FIG. 3 shows the restriction map of recombinant DNA pMAH2, in particular, that of the DNA fragment which carries the polypeptide gene deriv-ed from Bacillus macerans.
FIG . 4 shows the restriction map of recombinant DNA pMAU210, in particular, that of the DNA fragment which carries the polypeptide gene deriv-ed from Bacillus macerans.

Summary of the Invention The present inventors carried out investigations to determine the amino acid sequence of polypeptide; to assure a wide polypeptide source by gene recombinant technique; and also to improve polypeptide productivity.
As the result, the present inventors found that polypeptide com-prises one or more partial amino acid sequences selected from the group con-sisting of (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Thr, (b ) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, ( d ) Ile-Tyr-Tyr- Gly-Thr- Glu-Gln- Tyr-Met- Thr- Gly-Asn - Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly, and that, more particularly, these partial amino acids sequences (a), (b), (c), (d) and (e) are located in sequence of nearness to the N-terminal end of poly-peptide .
Polypeptide is characterized by the facts that it forms cyclodextrin from soluble starch; that it shows 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.
The present inventors also found that polypeptides derived from Bacillus stearothermophilus and Bacillus macerans have the amino acid se-quences as shown in Tables 2-1 and 5-I respectively. Both amino acid se-quences will be explained hereafter.
In addition, the present inventors determined the amino acid se-quences of the signal peptides which regulate polypeptide secretion from producer microorganisms.
The present invention and features thereof will hereinafter be ex-plained .

Description of the Preferred Embodiments In the present invention, the amino acid sequence of polypeptide is determined by cloning the polypeptide gene from a CGTase producer microor-ganism, and sequencing the polypeptide gene.
The amino acid sequence containing N-terminal end is determined by analyzing a highly-purified polypeptide with gasphase protein sequencer.
Cloning of polypeptide gene In the invention, a DNA fragment, obtained by separating DNA from a donor microorganism capable of producing polypeptide, and digesting the DNA, for example, with ultrasonic or restriction enzymes, and a vector frag-ment, 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 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 Klebsiella 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, Archieves of Microbiology, Vol.lll, pp.271-282 (1977).
Recombinant microorganisms in which polypeptide producibility has been introduced by genetic engineering technique can be used as the donor microorg anism .
The DNA of 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 micro-organism from the culture, and lysing the microorganism. Examples of bacteri-olytic procedures are cytohydrolist treatment using lysozyme or ~-glucanase, and ultrasonic treatment.
Other enzyme, such as protease, and/or surface active agent, such as sodium lauryl sulfate, can be used in combination, if necessary. Of course, freezing-thawing treatment can 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 or restriction enzymes, it is desirable to use restriction enzymes, in particular, those acting specifically on a pre-scribed nucleotide sequence, for smooth ligation. Specifically suited are Type II restriction enzymes, for example, EcoRI, HindIII, BamHI, SalI, SlaI, XmaI, MboI, XbaI, SacI, PstI, etc.
Bacteriophages and plasmids which autonomically proliferate in host microorganism are suitable for vector.
When a microorganism of species Escherichia coli is used as the host, bacteriophages such as Agt-~C and Agt-~B are employable, while pll, ~1 and ~105 are usable when a microorganism of species Bacillus subtilis 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 employable, while pUB110, pTZ4 (pTP4) and pC194 are usable for host microorganism of species Bacillus subtilis. Plasmids which autonomically proliferate in two or more different host microorganisms, for example, pHV14, TRp7, YEp7 and pB S7, can be used as the vector . These vectors are cleaved with the same types of restriction enzymes as used in DNA digestion to obtain a vector fragment .
DNA- and vector-fragments are ligated with conventional procedure using DNA ligase. For example, DNA- and vector-fragments are first anneal-ed, 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 13~S183 toin vivo DNA ligase.
The host microorganisms usable in the invention are those in which recombinant DNA autonomically and consistently proliferates to express its characteristics. Specifically, microorganisms which are not capable of pro-ducing ~-amylase (EC 3.2.1.1) can be advantageously used because the use of such microorganisms facilitates isolation and purification of the secreted poly-peptide .
The recombinant DNA can be introduced into host microorganism with conventional procedure. For example, when host microorganism belongs to species Escherichia coli, introduction of recombinant DNA is effected in the presence of calcium ion, while the competent cell- and protoplast-methods are employed when host microorganism of genus Bacillus is used.
The recombinant microorganism in which recombinant DNA has been introduced is selected by collecting clone ( s ) which grows on plate culture con-taining starch to convert the starch into cyclodextrin.
The present inventors found that the recombinant DNA carrying polypeptide gene cloned in this way can be easily introduced, after isolation from the recombinant microorganism, into different host microorganism. Also was found that a DNA fragment carrying polypeptide gene, obtained by digest-ing a recombinant DNA carrying the gene with restriction enzymes, can be easily ligated with a vector fragment which has been obtained in the same manner .
Furthermore, the present inventors found that the polypeptide gene in the recombinant DNA obtained according to the invention is cleaved by restriction enzyme PvuII, purchased from Toyobo Co., Ltd., Osaka, Japan, to lose the ability of expressing the polypeptide gene because the recombinant 133~183 DNA has a PvuII restriction cleavage site.
Sequence of polypeptide gene Polypeptide gene is sequenced by the chain-terminator method as described in Gene, Vol.9, pp.259-268 (1982).
This method contains the step of inserting a cloned DNA fragment carrying polypeptide gene into the insertion site of a suitable plasmid such as pUC18 using restriction enzymes. The obtained recombinant plasmid is intro-duced by transformation into a suitable Escherichia coli strain such as Esch-erichia coli JM83, followed by selection of the recombinant microorganism that contains the plasmid.
The recombinant plasmid is prepared from the proliferated recombi-nant microorganism.
The obtained recombinant plasmid is annealed together with a syn-thetic 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 subj ected sequentially to polyacrylamide-electrophoresis and radioautography, followed by sequencing polypeptide gene.
Signal peptide which regulates polypeptide secretion from the cell can be sequenced in the same manner.
Amino acid sequence of polypeptide The amino acid sequence of polypeptide is determined from DNA
sequence of polypeptide gene.
The amino acid sequence of signal peptide is determined in the same manner .
Partial amino acid sequence of polypeptide containing N-terminal end A polypeptide producer microorganism of genus Bacillus is cultured with a nutrient culture medium to produce polypeptide. The supernatant, cen-trifugally obtained from the culture, is purified by ammonium sulfate frac-tionation, ion exchange chromatography and high-performance liquid chromato-graphy to obtain a high-purity polypeptide specimen. The specimen is then degraded with gasphase 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 chromatography, followed by determina-tion of the partial amino acid sequence containing N-terminal end.
Preparation of polypeptide with recombinant microorganism The present inventors found that a large amount of polypeptide can be consistently produced by culturing a recombinant microorganism with a nutrient culture medium.
To the nutrient culture medium is incorporated, for example, carbon source, nitrogen source, minerals, and, if necessary, small amounts of organic nutrients such as amino acid and vitamin.
Starch, partial starch hydrolysate, and saccharides such as glucose, fructose and sucrose are suitable for carbon source. Inorganic nitrogen sources such as ammonia gas, ammonia water, ammonium salts and nitrates;
organic nitrogen sources such as peptone, yeast extract, and defatted soy-bean, corn steep liquor and meat extract are suitable for nitrogen source.
The recombinant microorganism is cultured with a nutrient culture medium for about 1-4 days under aeration-agitation conditions to accumulate polypeptide while keeping the culture medium, for example, at pH 4-10 and 25-65C .
Although the polypeptide in the culture may be used intact, general-ly, the culture is separated into polypeptide solution and cell with conventional procedure such as filtration and centrifugation, prior to its use.
When polypeptide is present in the cell, the cell is first treated with ultrasonic, surface active agent and/or cytohydrolyst, then with filtration and centrifugation to separate a solution containing polypeptide.
The solution containing polypeptide thus obtained is purified, for example, by combination of concentration in vacuo, concentration using mem-brane 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 polypeptide, the amino acid se-quence of polypeptide may be partially substituted, removed, added, or modifi-ed in such a manner that the polypeptide does not lose its CGTase activity, prior to its use.
The one unit of CGTase activity is defined as the amount of poly-peptide that ~limini~hes completely the iodine-coloration of 15 mg soluble starch at 40C 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 2X10 3 M calcium chloride is added 0.2 ml of a diluted enzyme solution, and the mixture is incubated at 40C 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 . Z ml of 0 . l N I2-KI solution to effect coloration, and determined for the absorbance at a wavelength of 660 nm.
Deposition of recombinant microorganisms Recombinant microorganisms Escherichia coli TCH201, Escherichia coli MAH2, Bacillus subtilis MAU210, and Bacillus subtilisTCU211 have been de-posited from November 2, 1984 under the accession numbers of FERM P-7924, 7925, 7926 and 7927 respectively at the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Yatabe-machi, Tsukuba-gun, Ibaraki-ken, Japan.
Several embodiments according to the invention will be disclosed.
Example 1 Cloning of Bacillus stearothermophilus polypeptide gene into Escherichia coli Example 1-(1) Preparation of chromosome DNA carrying heat-resistant-polypeptide gene of Bacillus stearothermophilus The chromosome DNA carrying heat-resistant-polypeptide gene of Bacillus stearothermophilus was prepared in accordance with the method de-scribed 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 50C overnight under vigorous shaking conditions. The cell, centrifugally collected from the culture, was suspended with TES buffer (pH 8.0) containing Tris-aminomethane, hydro-chloric acid, EDTA and sodium chloride, added with 2 mg/ml of lysozyme, and incubated at 37C for 30 minutes. The incubated mixture was freezed, allowed to stand at -20C overnight, added with TSS buffer (pH 9.0) containing Tris-aminomethane, hydrochloric acid, sodium lauryl sulfate and sodium chloride, heated to 60C, 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 super-natant. 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) containing sodium chloride and trisodium citrate, thereafter, the mixture was subjected to both "RNase A", a ribonuclease commercialized by Sigma Chemical Co., MO, USA, and "Pronase E", a protease commercialized by Kaken Pharma-ceutical 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 dissolved in a buffer (pH 7.5) containing Tris-aminomethane, hydrochloric acid and EDTA, and stored at -20C.
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 B acteri-ology, 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 1- (1), was partially digested with restriction en-zyme MboI, purchased from Nippon Gene Co., Ltd., Toyama, 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, pur-chased 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 4C overnight to obtain a * trade mark - 133~183 recombinant DNA.
Example 1 - (4) Introduction of recombinant DNA into Escherichia coli Escherichia coli HB101 (ATCC 33694), a strain incapable of produc-ing amylase, was used as the host.
The microorganism was cultured with L-broth at 37C 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, added 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 37C, added with L-broth, spread on L-broth agar plate medium containing 50 llg/ml of ampicillin and 2 mg/ml starch, and incubated at 37C for 24 hours to form colonies.
The colony which had degraded the starch into cyclodextrin was selected by the iodine-coloration 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 re-combinant DNA was extracted from the proliferated microorganism by the plasmid preparation method in Example 1-(2), subjected to restriction enzymes to determine the restriction cleavage sites, and completely digested with re-striction 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 with the method in Example 1-(4). The recombinant microorganism contained a recombinant DNA of a relatively small-size that carries polypeptide - 133~183 gene .
The recombinant DNA and plasmid pBR322 were then completely digested with restriction enzyme SalI , purchased from Nippon Gene Co ., Ltd ., and treated similarly as in the case of EcoRI to select recombinant microorga-nisms containing a recombinant DNA of a much smaller-size that carries poly-peptide gene.
One of these microorganisms and its recombinant DNA were named as "Escherichia coli TCH201 (FERM P-7924) and "pTCH201".
The restriction map of recomblnant DNA pTCH201, in particular, that of the DNA fragment derived from Bacillus stearothermophilus microorganism was as shown in FIG. l .
FIG. l clearly shows that this recombinant DNA is cleaved by either restriction enzyme PvuII purchased from Toyobo Co., Ltd., ~, HindIII
purchased from Nippon Gene Co., Ltd., or XbaI purchased from Takara Shuzo Co., Ltd, but not by EcoRI, BamHI, PstI, XhoI, BglII or AccI, 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 pUB110 Plasmid pUB110 (ATCC 37015) was isolated from Bacillus subtilis in accordance with the method described by Gryczan et al. in Journal of Bacteri-ology, 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 XbaI.
Separately, the plasmid pUB110 specimen, prepared in Example 2-(2), was completely cleaved by subjecting it to restriction enzymes EcoRI
and XbaI 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 pro-ducing amylase, was used as the host. The microorganism was cultured with brain heart infusion medium at 28C for 5 hours, and the cell, centrifugally collected from the culture, was then prepared into protoplast suspension in accordance with the method described by Schaeffer et al. in Proceeding 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 llg/ml of kanamycin and 10 mg/ml of starch, and incubated at 28C for 72 hours to form colonies.
From these colonies, recombinant microorganisms in which the re--16- 13~18~

combinant DNA carrying heat-resistant-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 TCU211 (FERM
P-7927) " and "pTCU211" respectively.
The restriction map of recombinant DNA pTCU211, in particular, that of the DNA fragment derived from Bacillus stearothermophilus microorganism, was as shown in FIG .2. FIG .2 clearly shows that this recombinant DNA is cleaved by either restriction enzyme PvuII, KpnI or HindIII, but not by EcoRl, BamHI, PstI, XhoI, BglII, AccI or XbaI.
Example 3 Partial amino acid sequence of Bacillus stearothermophilus polypeptide containing N-terminal end Example 3- (1) Preparation of polypeptide Bacillus stearothermophilus FERM-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 sulfate 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 highly-purified 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+10,000 daltons .

* trade mark >~

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- ( 1 ) Preparation of plasmid pUC18 Plasmid pUC18 was prepared in accordance with the method in Ex-ample 1-(2) from Escherichia coli JM83 (ATCC 35607) in which the plasmid had 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-(3) Introduction of recombinant DNA into Escherichia coli In this example, Escherichia coli JM83 was used as the host.
The recombinant DNA was introduced into this 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-~-galactoside (Xgal), and the microorgan-ism 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 llg/ml of ampicillin, and the obtained cell was then treated with the alkaline mini-preparation method to obtain a recombinant 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 60C for 20 minutes, added with dNTP, ddNTP, (c~-32P) dCTP and Klenow fragment, and reacted at 37C 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 37C 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 electro-phoresis, 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 re-ference 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 Esche-richia coli TCH201 (FERM P-7924) and Bacillus subtilis TCU211 (FERM P-7927) both in which recombinant DNA carrying heat-resistant-polypeptide gene derived from B acillus stearothermophilus had been introduced .
The polypeptide productivities of these recombinant microorganisms were compared with those of host microorganism and donor Bacillus stearo-133~183 ~o ~ 2 ~ ~ 8 ~ ~D " ~ - ~' $ ~ N ~3 ~ ~ ~ 3 _ ~ --O ~-- O ~3 0 ~r O -- O ~ O -- O ~) O C~
~ a N --3 ~ 3 $

D a ,, ~ u ~ y ~ ~

o ~ o ~ o ~ o ~ o ~ o c~ o ~ o '~ o ~
~ ~ 3 ~ ~ 3 ~ ~ ~
C ~ 3 V _ o~- oO o ~ o~ oO o~ o o:~ o~
N O a~ ~ d N ~ N ~ ~ ~' ~ U

- ~ ~ 3 ~ ~ $ ~ ", '31 ~3) ~ O " o ~'S

3 E t ~'3 ~

- ~ ~3 '3 ~3 o ~3 1 , ~ ~ ~ ~ N ~ ~OD

O ~ O ~ O ~ O ,) O-- O ~ O ~ o g a~ ~ ~ o ~ ~OD ~ ~ ~ o ~ O ~

- C~ (3 C~ 3 DO ~ O ~ O C5 O ~J O I-- O C)O 1--0 1-- 0 ~

~, ~ t ~
o ~ o o -- o C~ o ~o ~o V o ~ o ~ ~ O

'6 ~ C~ (') '6 O~J OC~ 0~: 0~ 0~ ~ ~ C~ ~
--_ ~ _ ~ 1_ O~ ~3U~ ~ ~
U~~.D r ~ n _ a) ~ _ ;) ~,.

133~183 O ~ N t 0 ~ ~ o ~o ' N ~

r~ t æ ~ u~ ' - ~ ~ e Y o, "o, O o e 0~ OC~ 0~ 0~ 0~ O~C OC0~ 0~
8 ~ ' Y . ~ t~D ~

,) N ~ N ~

~, 2 3 ~ U

, V _ UN ~N "~ .N ~U~ t C~ 3 ~ ~ '3 ~ ~3 O C O ,. O ~ ~

U

U j~ g ~ 0 ~ _ -- N

~o ~ ~ ~ '~ 8 ~

~1 o ~NO ~ CD S ~ ~ ~ N

C - --CJ ~ ~ j~

L) o C~ o ~ o ~ o ~ o ~; o O

U

~ 2 ' ~,~ ~ ~~ 2t --U) ~ ~

~L ~
t ~ , _ ~W

~o ~ S

. ~

o ~, o W ~

n ~w ~, o U) t Q, A A
-N Q~ C .~ ~ Q~ S ~ ~U ~ U l~

o ~ QU--~o~ J ~ 3 ~ ~r ~ ~ ~ ~ 7 1~

~ S U _ ~ s ~ ~ Q Q ~ C ~

n U~ U~ U~ ~ J ~ ~ a~
a ~ ~ ~ aQ~O ~ C ~ u ~

~ a--~ ~u cn ~ 2 ~ ~ 'S U ~_ ~ I U I ~ ~ S ~ I a ~ I ~ ~ ~ ~ U J

~ (~ U ~ C~ n J U ~ J ~S U U ~ ~ ~ ~ u~ ~ ~lS
N ~ ~ ~ ~ ~ ~ ~r 5~ ~n tn u ~ ~ U 1~

A ~ A ~ A /~ A A A A A A A A A A A A ~ A A A A
, N ~ O _ N ~ In i` ~ O _ ~ ~
_------ _ _ _ N N N ~ N N ~ ~ ~ ~ ~ ~q ~ O ~ C

Q ~ ~ c ~ ,~ '~ C ,~

' Q ~ ~ O, a _ ~" ~ c ~ ~ ~ c ' ' ~" 2 ~, ~ ot s ~ ~ --~

C ~ C

O ~ ~ n S ~ ~ ~! U CJ ~ 1- ~_ J

Q ~ ~ . u ~ a ~ ~ ~ J ~ ~ u ~ U U~ ~r ~2 ~ ~ U ~ U ~

~ ~ U ~ ~ a ~ 00 ~ ,~
C C ~ C ~ C

U I ~ ~ ~ U ~ U

~n ~ U ~ 2L ~Q ~ 0 Q

A A ~ A A A A A A /~ A A A A A A ~\ A A A
_ tD _ ~O _ a~ _ D _ ~D _ ~ _ ~ _ ~D _ ~ _ ~D
o~ o ~ ~ u~ 0 o _ ~

thermophilus microorganism in relation to their CGTase activity.
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 120C for 20 minutes, and cooled. In the case of Escherichia coli TCH201, the liquid culture medium was added with 50 llg/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 each case, microorganism was cultured at 37C for 48 hours under vigorous shaking conditions.
Separately, Bacillus subtilis TCU211 was inoculated to the liquid culture medium additionally containing 5 ~g/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 28C for 72 hours.
Bacillus stearothermophilus FERM-P No.2225 was cultured with the liquid culture medium at 50C 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 cul-ture. The results were as shown in Table 3.
These evidences clearly show that the recombinant microorganisms are advantageously usable in industrial-scale production of polypeptide because these microorganisms possess an improved polypeptide productivity.
The supernatants were salted out with ammonium sulfate at a satura-tion 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 Table 3 CGTase activity (units/ml) Microorganism Supernatant Cell Total Escherichia coli TCH201 (FERM P-79Z4) 0.8 13.5 14.3 ~o Bacillus subtilis TCU211 (FERM P-79Z7) 46.7 20.5 67.2 Escherichia coli liB101 0 0 0 Bacillus subtilis 715A 0 0 0 Bacillus stearothermophilus FERM-P No.ZZZS 8.5 0.3 8.8 C~

- and y-cyclodextrins, optimum temperature, optimum pH, stable tempera-ture 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.
Example 6 Cloning of Bacillus macerans polypeptide gene into Escherichia coli Example 6 - ( 1 ) Preparation of chromosome DNA carrying Bacillus macerans polypeptide gene The polypeptide gene was prepared in accordance with the method in Example 1-(1), except that Bacillus macerans 17A was cultured at 28C.
Example 6- ( 2 ) Preparation of recombinant DNA carrying polypeptide gene The chromosome DNA carrying polypeptide gene derived from Bacil-l_ macerans, prepared in Example 6-(1), was partially digested similarly as in Example 1-(3) with restriction enzyme HindIII, 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 HindIII , and the 5 ' -terminal end of the cleaved product was dephosphorized 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 the method in Example 1-(4) using Escherichia coli HB101 (ATCC 33694), a strain incapable of producing amylase, 133~183 as the host. Thereafter, the recombinant DNA was isolated from the micro-organism, subjected to restriction enzymes to determine the restriction cleavage sites, and partially digested with restriction enzyme Sau3AI 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 w-ere 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" respect-vely .
The restriction map of recombinant DNA pMAH2, in particular, thatof 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 restric-tion enzyme PvuII , SalI , AvaI commercialized by Nippon Gene Co ., Ltd ., or PstI commercialized by Nippon Gene Co ., Ltd ., but not by EcoRI , HindIII , KpnI, BamHI, XbaI, XhoI or SmaI.

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 simulta-neously 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 simi-larly 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 "pMAU210" respective-ly. The restriction map of recombinant DNA pMAU210, in particular, that of the DNA fragment that carries the polypeptide gene derived from Bacillus 133~183 macerans, was as shown in FIG . 4 . FIG . 4 shows that the recombinant DNA is cleaved by either restriction enzyme PvuII, SalI, AvaI or PstI, but not by EcoRI, HindIII, KpnI, BamHI, XbaI, XhoI or SmaI.
Example 8 Amino acid sequence of polypeptide derived from Bacillus macerans 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 enzymès, 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 D NA 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.

133~183 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 re-ference 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 evidences as shown in Tables 2-1 and 5-2 show that each poly-peptide commonly has the amino acid sequence of (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Thr, (b ) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, ( c ) Val-Thr-Phe-Ile-A sp -A sn-His-A sp-M et-Asp -Arg -Phe, ( d ) Ile-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.

r : l Table 4-1 TCCCCGGATA CGAGCGTGAA CAACAAGCTC AAT mAGCA CGGA TACGGT T TACCAGATT

GTAACCGACC GGmGTGGA CGGCAAT TCC GCCAACAACC CGACCGGAGC AGCC l l CAGC

AGCGATCATT CCAACCTGAA GCTGTAI l IC GGGGGCGACT GGCAGGGGAT ~::ACr~CAAA

ATCAACGACG GCTATCTGAC CGGAATGGGC ATCACCGCCC TCTGGA TCTC GCA':rCr':TT

GAGAACATCA CCGCCGTCAT CAATTATTCG GGCGTCAACA ATACAG CTTA CC ACGGTTAC

TGGCCTCGCG ACTTCAAGAA GACCAATGCC GCGI lCGGCA GCTTCACCGA CTTC TCCAAT

TTGATCGCCG CAGCGCATTC ACACAATATC AAGGTAGT TA TGGACTT TGC ACCT AATCAC
UO 440 450 460 470 480 c~
ACCAACCCGG C l l CaAGTAC GGACCCCTCG TTCGCC~ ACGGCGCGCT CTACMCMC
490 500 510 520 530 540 c~
GGAACGCTGC TCGGCAAGTA TAGCAACGAT ACCGCCGGCC TG TTCCACCA ~AATGGCGGC

O~ 0~ 0~3- 0 ~ oC,~ o~3 0 O~- ~
~, æ ~ N o ~
C

~ ~ ~ 1_ ~ ;3 ~ 3 t_ O ~- O ~ o C) o C3 0 U o ~ o ~S O C'' O ') 31` ~3 ~ 3 CO (~ ~ -t3 V ~ - y C _ ~J
~ ~ 8 ~ 8 J ~ t~r 'J

~ 3 ,,~ t~) t~ V t3 o ,~ o o V o C~ o ~ o o .~ o o ~
~') t3 O~ ~ U) t,) c t~ o~

~) 3 ~ C~

9~ t,~ ~ ~ t~ ~3 ~3 'S l-- C~J ~ t~) ~ -- ~ --~D N ~ --o <S a~ ~3 O t~ U a~ 3 O~ t~ O
- u ~3 t3 t V ~J 3 ~ ~ t 8 ~ ~ 3 ol- o~: o~ ol- oC' ol- OO o,~ ot3 t ~ ;3 ~ ~ C) ~3 ~ C
3 ~3 ~ 3 t.) ~ ' ~3 t") ~ U
3 ~ 3 ~3 3 U

N ~ N _ ~ ~ Eg t a~ ~ N
a i~ ~} ~ ~~ j~ O o ~ o O

N ~ 3 -- = ~ :~ '~ ~ ~ ~

N ~-)N ~
--~ L~ t u O ~~D < N < =~ ~ ~ O O

0~0~ 0~ 0~ 0 OC~ 0~ oC~
O N V N-- _ ~ 3 ~ ,~

-- ,~ u ~ V ~ ~ 3 ~ ~ N ;,~

-- ~ v --~ u tD t N ~ ~,) q~ ~ O ~ t~ --J
t u~ ~ o ~ ~o ~ O ~ æ ~ 1n y o '~
E~ tD ~` ~ ~ ~0 ~;~ 0 C~ ~n o--.

~ ~J

~ N ~ ~ o O ~ O ~ O ~ O ~ O ~ O ~ O ~ O

V C~

.''~

o C~ .

~n 0~ ~ _ ~ n ~ O ~ _ '~ o O C~ O CO ~ ~

v ~ ~_ -a O O ~

- ~ C ~"
(~ ~3 o o o C~
3 ~ =

v ~ ~ ~3 =A ~

, `- 1335l83 3 ~ 3 ~ 3 3 ~ 3 3 s s ~ ~ 3 ~ ~ ~ 3 ~
a ~ ~ 3 C

o) ~ 5 s ~ a ~r 3 ~ ~ 3 ~
s ~ s ~L ~ 3 ~ n ~ ~ >--i~
c ~ ~.s 3 ~ ~ 3 . ~ - ~ ~ ~ ~ - ~ c a s ~ ~ S~ ~ 3 ~
a S~ a 3 u ~ ~ ~ ~ ~ ~ ~ ~ q ~ tn ~ 3 ~ " ~ ~ ~ 3 ' 3 ~Q-' ~ ' a ~ ~ 3 ~ ~ ~ S ~
~, ~ ~ 3 ~ a a ~ 3 ~ ~ 3 ~3 a 3 ~ ~ u ~ 3 - ~
~ A ~ A ~ A ~ A A
A ~ D ~ ~ N ~ C~J N N ~

, ,~ ...

~, _ ~ ~ ~ ~ ~ C ~ ~ C~ ~ C ~ C _ --~2J ~

o ~-- ,~,, ~; ~ ~_ . . ~ C o ~ o s-- ~ ~ C ~ C

~ C C
~ ~ Q ~ ~--~, ~S ~ ' 0 C a~ ~ ' Q

~ a ~

--~_ ~ ,~ .' ~L C J ~ ~ ~ ' ~ -- ~ q) ~ ~_-- Q~ W ~ ~ ~ O ~ ~

:'~ -- a~ ` ' ~ s ~ ~ ~ ~ ~
~ C _ _ ~--C ' ~ Q

C _ o ~;~ ~ _ c ~ _ ,Q_ ~ S

A A A A A A A A A A A A A A A A A A A A
~) O ~ ~ 1~ tD ~ O~ _ N ~ In ~ O ~

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 microorgan-isms, 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 MA~12 was inoculated to the liquid culture medium additionally containing 50 llg/ml of ampicillin, while Escherichia coli HB101 was inoculated to the liquid culture medium without addition of antibiotic. In each case, microorganism was cultured at 35C for 24 hours under vigorous shaking conditions .
Bacillus subtilis MAU210 was inoculated to the liquid culture medium additionally containing 5 ~Ig/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 28C for 72 hours.
Bacillus macerans 17A was cultured with the liquid culture medium at 28C 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 industrial-scale production of polypeptide because they have an improved polypeptide productivity.

;~ ? ~`

Table 6 CGTase activity (units/ml) Microorganism Supernatant Cell Total Escherichia coli MAHZ ~FERM P-79ZS) 0.6 11.8 12.4 Bacillus subtilis MAU210 (FERM P-7926 )54 . 6 0 . 3 54 . 9 Escherichia coli HBIOI 0 0 0 Bacillus subtilis 715A 0 0 0 Bacilllls macerans 17A 7.5 0.4 7,9 133~183 The supernatants were salted out with ammonium sulfate at a satura-tion 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 poly-peptide produced by the recombinant microorganisms were in good accordance with those of the polypeptide produced by the donor Bacillus macerans micro-organism .
Principal uses of polypeptide will hereinafter be described.
Polypeptide effects the intra- or intermolecular saccharide transfer reaction between suitable saccharide donor and saccharide acceptor.
According to one aspect of the present invention, various saccha-ride-transferred products can be produced by taking advantages of these saccharide transfer reactions.
For example, a partial starch hydrolysate containing cL-, ~- and r-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 polypeptide utilizing the intramolecular saccharide transfer reaction. Each cyclodextrin can be isolated from the partial starch hydrolysate, if necessary .
o~-Glycosylated saccharide sweetener, for example, -glucosyl-, a-maltosyl- and ~-maltotriosyl-saccharides, is prepared obtained by sub jecting a mixture of a saccharide donor, for example, amylaceous substance such as starch, liquefied starch, dextrin, cyclodextrin or amylose; and a saccharide acceptor, for example, monosaccharide such as xylose, sorbose or fructose, or disaccharide such as sucrose, maltulose or isomaltulose, to the action of poly-peptide utilizing the intermolecular saccharide transfer reaction. The o~-glyco-sylated saccharide sweetener can be advantageously used in foods and bever-ages because the ~-glycosylated saccharide sweetener is much milder in taste, more dissoluble-in water, but less crystallizable in comparison with intact sac-charide sweetener. These would expand extremely the use of saccharide sweeteners .
In the intermolecular saccharide transfer reaction, the use of a gly-coside, for example, steviol glycoside such as stevioside or rebaudioside, glycyrrhizin, soyasaponin, teasaponin, rutin or esculin, as the saccharide acceptor- leads- to--the formation of o~-glycosylated glycosides such as cL-gluco-syl-, ~-maltosyl- and ~-maltotriosyl-glycosides. The c~-glycosylated glycoside is-free of the unpleasant tastes such as bitter- and astringent-tastes which are inherent to intact glycoside, and more readily dissoluble in water than intact glycoside. These would expand extremely the use of glycosides. Specifically, -glycosylated steviol glycoside and o~-glycosylated glycyrrhizin can be advan-tageously used in foods, beverages, and pharmaceutical for peroral administra-tion because the taste improvement in these ~-glycosylated glycosides is re-markably high, as well as because their sweetness is comparable to that of su-crose .
Several embodiments will be disclosed.
Example 1 1 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 85C at pH
6. 5, cooled to 70C, further added with the same amount of the polypeptide specimen, and reacted for 40 hours. The reaction mixture was purified by 1~35183 decoloration using activated carbon and deionization using 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 flavors and cosmetics wherein fragrance or aroma is one of the important factors because the corn syrup is excellent in flavor-locking properties .
The a-, ~- and y-cyclodextrins in the corn syrup can be separated by treating it with a procedure using organic precipitant, such as toluene or trichloromethane, or conventional column chromatography.
Example 12 ~-Glycosylsucrose A 35 w/w % suspension of cornstarch was added with 0 . 2 w/w %
oxalic acid, autoclaved to 120C 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 Ex-ample 10, and reacted at pH 6 . 0 and 55C 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-eries because it is mildly sweet and amorphous.
Example 13 c~-Glycosyl stevioside Two-hundred g of stevioside and 600 g of dextrin (DE 8 ) were dissolved in 3 liters of water by heating, and the resultant solution was cooled to 70C, 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 65C for 35 hours . The reaction mixture was then heated at 95C for 15 minutes, purified by filtration, concentrated, and pul-verized to obtain a pulverulent sweetener containing c~-glycosyl stevioside in a yield of about 92% based on the dry solid.
The sweetener free of the unpleasant taste which is inherent to intact stevioside was comparable to sucrose in taste quality, and the sweeten-ing power of the sweetener was about 100-fold higher than that of sucrose.
The sweetener can be advantageously used as diet sweetener or to season foods and beverages because of its low-cariogenic and low-calorific properties.
Example 14 -Glycosyl ginsenoside Sixty g of a ginseng extract and 180 g of ~-cyclodextrin were dis-solved in 500 ml of water by heating, and the resultant mixture was cooled to 70C, adjusted to pH 6 . 0, added with 3 units/g ~-cyclodextrin of a polypeptide specimen prepared with Escherichla coli TCH201 in accordance with the method in Example 5, cooled to 65C, 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 p acked with 3 liters of "Amberlite XAD-7", a synthetic adsorbent 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 dehy-drated to obtain about 21 g of a pulverulent product that contains o~-glycosyl ginsenoside. Since the product is free of the unpleasant tastes such as bit-ter-, astringent- and harsh-tastes which are inherent to intact 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-infl~mmatory and expectorant effects as intact ginsenoside does.

As described above, the present inventors determined the amino acid sequences of polypeptide gene and its signal peptide, as well as preparing the recombinant DNA having PvuII restriction site from donor microorganism by in vitro genetic engineering technique. Furthermore, the present inventors prepared recombinant microorganisms in which the recombinant DNA is intro-duced, as well as confirming that the recombinant microorganisms autonomically and consistently proliferate in a nutrient culture medium.
In view of adequately supplying polypeptide, the present invention is industrially significant because the present invention assures a wide poly-peptide source and easily improves the polypeptide productivity of donor mlcroorganlsms .

While there has been described what is at present considered to be the preferred embodiments of the invention, it will be understood that various modifications may be made therein, and it is intended to cover in the appended claims all such modifications as fall within the true spirit and scope of the invention .

-48a- 133S183 The microorganisms of the invention have the following international depoæit nos. according to the Budapest Treaty:
New Accession Number Escherichia coli TCH210 FERM P-7924 FERM BP-2109 Escherichia coli MAH2 FERM P-7925 FERM BP-2110 Bacillus subtilis MAU210 FERM P-7926 FERM BP-2111 Bacillus subtilis TCU211 FERM P-7927 FERM BP-2112 The depository is: Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, 305 Japan; and the deposit date was November 2, 1984.

Claims (31)

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-Ile-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly;
said polypeptide having an isoelectric point of 5.0?0.1.

2. The polypeptide in accordance with claim 1, wherein said partial amino acid sequences of (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) 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:

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-Ile-Phe-Ile-Val-Ser-Asp-Thr-Gln-Lys-Val-Thr-Val-Glu-Ala is located 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:

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-Ile-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 originates from a microorganism capable of producing CGTase.

11. The polypeptide in accordance with claim 1, which originates 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 CGTase gene has been introduced.

14. A recombinant DNA having a PvuII restriction cleavage site and carrying CGTase gene, said recombinant DNA
comprising:
a DNA fragment carrying CGTase gene, obtained by digesting the DNA of a microorganism of genus Bacillus 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 a member selected from the group consisting of Bacillus stearothermophilus and Bacillus macerans.

16. The recombinant DNA in accordance with claim 14, which has one of the following restriction cleavage sites:
(a) for enzymes PvuII, KpnI, HindIII and XbaI, but not for enzymes EcoRI, BamHI, PstI, XhoI, BglII and AccI, (b) for enzymes PvuII, KpnI and HindIII, but not for XbaI, EcoRI, BamHI, PstI, XhoI, BglII and AccI, (c) for enzymes PvuII, SalI, AvaI and PstI, but not for enzymes KpnI, HindIII, XbaI, EcoRI, BamHI, XhoI and SmaI, and (d) for enzymes PvuII, PstI, SalI and AvaI, but not for enzymes KpnI, HindIII, XbaI, EcoRI, BamHI, XhoI and SmaI.

17. A biologically-pure culture of a recombinant microorganism of genus Escherichia or Bacillus in which a recombinant DNA having a PvuII restriction cleavage site and carrying CGTase gene, prepared by ligation of a DNA fragment, obtained by digesting the DNA of a microorganism of genus Bacillus 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.

18. The culture in accordance with claim 17, wherein said recombinant DNA has one of the following restriction cleavage sites:
(a) for enzymes PvuII, KpnI, HindIII and XbaI, but not for enzymes EcoRI, BamHI, PstI, XhoI, BglII and AccI, (b) for enzymes PvuII, KpnI and HindIII, but not for XbaI, EcoRI, BamHI, PstI, XhoI, BglII and AccI, (c) for enzymes PvuII, SalI, AvaI and PstI, but not for enzymes KpnI, HindIII, XbaI, EcoRI, BamHI, XhoI and SmaI, and (d) for enzymes PvuII, PstI, SalI and AvaI, but not for enzymes KpnI, HindIII, XbaI, EcoRI, BamHI, XhoI and SmaI.

19. The culture in accordance with claim 17, wherein said recombinant microorganism is a member selected from the group consisting of Escherichia coli TCH201 (FERM BP-2109) and Escherichia coli MAH2 (FERM BP-2110).

20. The culture in accordance with claim 17, wherein said recombinant microorganism is a member selected from the group consisting of Bacillus subtilis MAU210 (FERM BP-2111) and Bacillus subtilis TCU211 (FERM BP-2112).

21. A process for producing CGTase, comprising:
culturing with a nutrient culture medium a recombinant microorganism of genus Escherichia or Bacillus, in which a recombinant DNA having a PvuII restriction cleavage site and carrying CGTase gene, prepared by ligation of a DNA
fragment, obtained by digesting the DNA of a microorganism of genus Bacillus 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; and recovering the accumulated CGTase.

22. The process in accordance with claim 21, wherein said recombinant DNA has restriction cleavage sites as defined in claim 17.

23. The process in accordance with claim 21, wherein said recombinant microorganism is a member selected from the group consisting of Escherichia coli TCH201 (FERM BP-2109) and Escherichia coli MAH2 (FERM BP-2110).

24. The process in accordance with claim 21, wherein said recombinant microorganism is a member selected from the group consisting of Bacillus subtilis MAU210 (FERM BP-2111) and Bacillus subtilis TCU211 (FERM BP-2112).

25. A process for producing a saccharide-transferred product, comprising subjecting an amylaceous substance to the action of a polypeptide possessing CGTase activity; said polypeptide comprising one or more partial amino acid sequences selected from the group consisting of (a) Asn-Lys-Ile-Asn-Asp-Gly-Tyr-Leu-Thr, (b) Pro-Val-Phe-Thr-Phe-Gly-Glu-Trp-Phe-Leu, (c) Val-Thr-Phe-Ile-Asp-Asn-His-Asp-Met-Asp-Arg-Phe, (d) Ile-Tyr-Tyr-Gly-Thr-Glu-Gln-Tyr-Met-Thr-Gly-Asn-Gly-Asp-Pro-Asn-Asn-Arg, and (e) Asn-Pro-Ala-Leu-Ala-Tyr-Gly;
said polypeptide having an isoelectric point of 5.0?0.1.

26. The process in accordance with claim 25, wherein said saccharide-transferred product is cyclodextrin.

27. The process in accordance with claim 25, wherein said amylaceous substance is subjected to the action of said polypeptide in the presence of a saccharide acceptor.

28. The process in accordance with claim 25, wherein said amylaceous substance is selected from the group consisting of starch, amylose, cyclodextrin, dextrin, and mixtures thereof.

29. The process in accordance with claim 27, wherein said saccharide acceptor is a member selected from the group consisting of saccharide sweetener, glycoside, and mixtures thereof.

30. The process in accordance with claim 27, wherein the saccharide-transferred product is a member selected from the group consisting of .alpha.-glycosylsucrose, .alpha.-glycosyl stevioside, and .alpha.-glycosyl ginsenoside.

31. The process in accordance with claim 27, wherein said saccharide-transferred product is used as sweetner.