KR940003652B1 - Method of preparing of polypeptide possessing cyclomaltodextrin glucano transferase activity - Google Patents

Abstract

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Description

Method for preparing a polypeptide having cyclomaltodextrin glucanotransferase activity

1 is a restriction plot of DNA fragments having polypeptide genes derived from recombinant DNA pTCH201, particularly Bacillus stearotermophilus.

2 is a restriction plot of DNA fragments having polypeptide genes derived from recombinant DNA pTCU211, particularly Bacillus stearotermophilus.

3 is a restriction plot of DNA fragments having polypeptide genes derived from recombinant DNA pMAH2, in particular Bacillus maserans.

4 is a restriction plot of DNA fragments having polypeptide genes derived from recombinant DNA pMAU210, in particular Bacillus maserans.

FIG. 5 is a graph showing isoelectric point fractionation results at a pH range of 4 to 6 in a polyacrylamide gel column of an E. coli TCH201-derived polypeptide sample.

The present invention relates to a polypeptide and a method for producing the same. In particular, the present invention relates to a polypeptide having cyclomaltodextrin glucanotransferase activity and a method for producing the same.

Throughout the present invention, amino acids, peptides, and the like are indicated as abbreviations commonly used in the art, and examples of such abbreviations are as follows.

Where optical isomers are present, unless otherwise indicated, the abbreviation for amino acid means L-isomer.

DNA is deoxyribonucleic acid; RNA is ribonucleic acid; A is adenine; T is thymine; G is guanine; C is cytosine; dNTP is deoxynucleotide triphosphate; ddNTP is didioxynucleotide triphosphate; dCTP is dioxycytidine triphosphate; SDS is sodium dodecyl sulfate; Ala is alanine; Arg is arginine; Asn is asparagine; Asp is aspartic acid; Cys is cysteine; Gln is glutamine; Glu is glutamic acid; Gly is glycine; His is histidine and Ile isoleucine; Leu leucine; Lys is Lysine; Met is methionine; Phe is phenylalanine; Pro is proline; Ser is serine; Thr is threonine; Trp is tryptophan; Tyr is tyrosine; Val is valine; CGTase is an abbreviation of cyclomaltodextrin glucanotransferase.

The term “polypeptide” means “polypeptide with CGTase activity”.

CGTase, or macerans amirase, has been known for many years as an enzyme produced by Bacillus macerans.

It has recently been discovered that CGTase can be produced by other microorganisms such as Bacillus stearothermophilus and Bacillus circulans species.

The sugar transfer activity of CGTase is currently used in many industries.

For example, cyclodextrins can be prepared by applying CGTase to gelatinized starch, while glycosyl sucrose can be prepared by the action of CGTase on a mixture of liquefied starch and sucrose to change sugars from starch to sucrose. Will be used.

Cyclodextrins are currently increasing in demand as a main agent for forming stable inclusion complexes with organic compounds which are volatile or susceptible to oxidation, while glycosyl sucrose is manufactured by Hayashibara Co., Ltd., Okamayama, Japan. Coupling Sugar ”is a registered trademark that has a light sweet taste on the market and is low in corrosion resistance.

In order to meet this demand, the urgent need for the development of a means to enable a stable supply of CGTase, which requires the determination of the amino acid sequence of a polypeptide having CGTase activity.

However, such an amino acid sequence is not well known.

We determine the amino acid sequence of the polypeptides; Securing a wide range of polypeptide sources by gene recombination; In addition, studies were conducted to increase polypeptide productivity.

As a result, the inventors have shown that the polypeptide consists of one or more partial amino acid sequence sequences selected from the following groups, and in particular, these partial amino acid sequence sequences (a), (b), (c), (d) and (e) was found to be arranged in the N-terminal proximal region of the polypeptide.

(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

The polypeptide forms a cyclodextrin from soluble starch; SDS-polyacrylamide electrophoresis shows a molecular weight of 70,000 ± 10,000 Daltons; It can also be characterized as having a specific activity of 200 ± 30 units / mg protein.

We also found that Bacillus stearotermophilus and polypeptides derived from Bacillus maserans have the amino acid sequence listed in Tables 2a and 5a, respectively. These two amino acid sequences will be described later.

In addition, the inventors determined the amino acid sequence of the coding peptides that regulate the secretion of polypeptides from producer microorganisms.

The invention and its features will be described next.

In the present invention, the amino acid sequence of the polypeptide was determined by cloning the polypeptide gene from the CGTase-generating microorganism and examining the sequence of the polypeptide gene.

The amino acid sequence comprising the N-terminus can be determined by analyzing the highly purified polypeptide as a gas phase protein sequence sequencer.

Cloning of Polypeptide Genes

In the present invention, DNA fragments obtained by separating DNA from donor microorganisms having the ability to produce a polypeptide, and digesting the DNA with, for example, ultrasound or restriction enzymes, and the same method. The vector fragment obtained by dividing the vector into the above was combined with, for example, a DNA ligase to obtain a recombinant DNA containing a polypeptide gene.

Donor microorganisms are selected from bacteria that produce polypeptides. Examples of such bacteria include bacteria of the genus Bacillus, such as Bacillus maserans, Bacillus megaterium, Bacillus circulans, Bacillus polymyxa, and Bacillus stearotermophilus, for example Japanese Patent Application 47-20373, 50-63, 189, 50-88, 290, and Hans Bender, Archives of Microbiology, Vol. Bacteria of the genus Klebsiella, such as Klebsiella pneumoniae, described on pages 111, 271 to 282 (1977).

Recombinant microorganisms whose polypeptide generation capacity has been introduced by genetic engineering methods can be used as donor microorganisms.

Donor microorganisms can be prepared by incubating the donor microorganisms, for example, in a liquid medium under aeration-stirring conditions for about 1 to 3 days, centrifuging the microorganisms from the medium, and then dissolving these microorganisms. . Examples of the Ryongyong bacterium method include cell membrane hydrolase treatment and ultrasonic treatment using lysozyme or β-glucanase.

If necessary, other enzymes such as proteases and / or surfactants such as sodium lauryl sulfate may be used in combination. Of course, freeze-thaw treatment can be performed if necessary.

In order to separate DNA from the obtained lysate, two or more conventional methods such as phenol extraction, protein removal, protease treatment, ribonuclease treatment, alcohol precipitation and centrifugation can be used in combination.

DNA ligation can be carried out by, for example, sonication or restriction enzyme treatment of DNA fragments and vector fragments, but slow insertion using restriction enzymes, in particular restriction enzymes which are characteristic of the above-described nucleotides. Preferred for

In particular, suitable restriction enzymes are Type II restriction enzymes, and examples thereof include EcoRI, HindIII, BamHI, SalI, SlaI, XmaI, MboI, XbaI, SacI, PstI and the like.

Bacteriophages and plasmids that autonomously proliferate in host microorganisms are suitable as vectors.

When microorganisms of Escherichia coli species are used as hosts, bacteriophages such as λgt · λc and λgt · λB may be used, while microorganisms of Bacillus subtilis species are used as hosts. Ρ11, Ψ1, and Ψ105 can be used for this.

Referring to the plasmids, when using microorganisms of Escherichia coli as a host, plasmids such as pBR 322 and pBR 325 can be used, while pUB110, pTZ4 (pTP4) for host microorganisms of Bacillus subtilis species. And pC194 can be used. Plasmids that autonomously proliferate in two or more different host microorganisms, such as pHV14, TRp7, YEp7 and pBS7, can be used as vectors. These vectors can be cleaved as restriction enzymes of the same type as those used in DNA digestion to obtain vector fragments.

DNA fragments and vector fragments are combined according to conventional methods using DNA ligase. For example, DNA fragments and vector fragments are first annealed and then ex vivo to effect the appropriate DNA ligase to obtain recombinant DNA. If necessary, the annealed fragments can be introduced into a host microorganism and reacted with the DNA ligase in vivo to produce such recombinant DNA.

Host microorganisms usable in the present invention are microorganisms capable of expressing their characteristics by autonomously and continuously proliferating recombinant DNA. In particular, microorganisms lacking the ability to produce α-amylases (EC 3.2.1.1) can be conveniently used because such microorganisms can facilitate the isolation and purification of secreted polypeptides.

Recombinant DNA can be introduced into host microorganisms according to conventional methods. For example, when the host microorganism belongs to Escherichia coli species, the introduction of recombinant DNA is carried out in the presence of calcium ions, while the cell- and protoplasm-methods possible can be applied when using a host microorganism of the genus Bacillus.

Recombinant microorganisms into which recombinant DNA has been introduced are collected by screening clones that grow on platelets containing starch and convert the starch to cyclodextrins.

The inventors have found that recombinant DNA with polypeptide genes cloned in this manner can be easily introduced into other host microorganisms after separation from the recombinant microorganism. It has also been found that DNA fragments containing polypeptide genes obtained by aging recombinant DNA containing this gene with restriction enzymes can be easily combined with vector fragments obtained in the same manner.

In addition, the inventors of the present invention, the polypeptide gene in the recombinant DNA obtained according to the present invention is divided by the restriction enzyme PvuII purchased from Toyo Co., Ltd., Osaka, Japan loses the ability to express the polypeptide gene The reason for this is that the recombinant DNA has a PvuII restriction fragment.

Array of polypeptide genes

Polypeptide genes are described in Gene Vol. 9, 259-268 (1982), arranged in a chain-termination method.

This method involves inserting a cloned DNA fragment containing a polypeptide gene into a site of insertion of a suitable plasmid such as pUC18 using restriction enzymes. The recombinant plasmid obtained is transformed into a suitable Escherichia coli strain such as Escherichia coli JM 83, followed by screening for recombinant microorganisms comprising the plasmid.

Recombinant prasmids are made from propagated recombinant microorganisms.

The resulting recombinant plasmid is annealed with synthetic primers, and then the Klenow fragment is acted on this mixture to form supplemental DNA as well as to enlarge the primers.

Thereafter, the mixture is sequentially subjected to polyacrylamide electrophoresis or the like by radiograph, and then the sequence of polypeptide genes is confirmed.

Coding peptides that regulate the secretion of polypeptides from cells can also 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 coding peptide is also determined in the same manner.

Partial amino acid sequence of the polypeptide containing the N-terminus

Polypeptide-generating microorganisms belonging to the genus Bacillus are cultured with nutrient medium to produce polypeptide. The supernatant obtained by centrifugation of the culture was purified by ammonium sulfate fractionation, ion exchange chromatography, and high performance liquid chromatography to obtain a high purity polypeptide sample. This sample was reviewed in Journal of Biological Chemistry, Vol. Partial amino acid sequences containing the N-terminus are determined after cleavage as a gas phase protein sequencer and fixed as high performance liquid chromatography according to the methods described in pages 256, 7990 to 7997 (1981).

Preparation of Polypeptides by Recombinant Microorganisms

The inventors have discovered that large amounts of polypeptides can be continuously produced by culturing recombinant microorganisms in a nutrient medium.

The nutrient medium contains, for example, carbon sources, nitrogen sources, minerals and, if necessary, organic nutrients such as small amounts of amino acids and vitamins.

Suitable carbon sources are starch, starch hydrolyzate and sugars such as glucose, fructose and sucrose. Examples of nitrogen sources include inorganic nitrogen sources such as ammonia gas, ammonia water, ammonium salts and nitrates; Organic nitrogen sources such as peptone and yeast extract; And skim soybeans, corn steep liquor and gravy are suitable.

Recombinant microorganisms accumulate polypeptide by incubating the medium in a nutrient medium for about 1 to 4 days under aeration-stirring tidal phase while maintaining the medium at pH 4-10, 25-65 ° C.

The polypeptide in the culture may generally be used as it is, but the culture is separated into a polypeptide solution and a cell according to conventional methods such as filtration and centrifugation before use.

If polypeptides are present in the cells, these cells are first treated with ultrasound, surfactants and / or Cytohydrolysts, followed by filtration and centrifugation to separate the polypeptide-containing solution.

The polypeptide-containing solution thus obtained is used as an industrial polypeptide by, for example, purifying a combination of vacuum concentration, concentration using a membrane filter, salt precipitation using ammonium sulfate or sodium sulfate, and fractional precipitation using methanol, ethanol or acetone. Obtain a high purity polypeptide sample as much as possible.

In order to further improve the quality of this polypeptide, the amino acid sequence of the polypeptide can be partially substituted, eliminated, added or modified prior to its use without losing its CGTase activity.

One unit of CGTase activity is defined as the amount of polypeptide that completely decolorizes iodine coloration of 15 mg soluble starch at 40 ° C. over 10 minutes under the following reaction conditions. That is, 0.2 ml of diluted enzyme solution is added to 5 ml of 0.3 w / w% soluble starch solution containing 0.02 M acetate buffer (pH 5.5) and 2 × 10 ⁻³ M calcium chloride, and the mixture is stirred at 40 ° C. for 10 minutes. After incubation, 0.5 ml of the reaction mixture is taken as a sample, and 15 ml of 0.02 M aqueous sulfuric acid solution is added to stop the enzymatic reaction. 0.2 ml of 0.1NI ₂ -KI solution was added to the reaction mixture for color development, and then the absorbance was measured at a wavelength of 660 nm.

Deposit of Recombinant Microorganisms

Recombinant microorganisms Escherichia coli TCH 201, Escherichia coli MAH 2, Bacillus subtilis MAU 210 and Bacillus subtilis TCU 211 are the fermentation of 1-3 Chuo, Higashi, Ibarakigen, Japan. Deposited on November 2, 1984 to the Research Institute, Agency of Industrial Science and Technology with access numbers FERM BP-2109, 2110, 2111 and 2112, respectively.

Some embodiments according to the present invention are described below.

Example 1

Cloning of Bacillus stearotermophilus polypeptide gene against Escherichia coli

(1) Preparation of chromosomal DNA having heat resistant polypeptide gene of Bacillus stearotermophilus

The heat resistant polypeptide gene of Bacillus stearotermophilus has a chromosomal DNA having Saito and Miura by Biochimica et Biophisica Acta, Vol. Prepared according to the method described in pages 72, 619-629 (1963).

Bacillus stearotermophilus FERM-P No. Seed culture of 2225 was performed overnight in brain-heart leaching medium at 50 ° C. under vigorous stirring conditions. Cells collected by centrifugation from this culture were suspended in TES buffer (pH 8.0) containing tris-aminomethane, hydrochloric acid, EDTA and sodium chloride, and incubated at 37 ° C for 30 minutes with the addition of 2 mg / ml lysozyme. The culture mixture was frozen, left overnight at -20 ° C and then heated to 60 ° C by addition of TSS buffer (pH 9.0) containing tris-aminomethane, hydrochloric acid, sodium lauryl sulfate and sodium chloride, TES buffer (pH 7.5) and a mixture of phenol (1: 4 volume ratio) were added, cooled with ice cooling water, and then centrifuged to obtain a supernatant. Two volumes of cold ethanol was added to the supernatant to recover crude chromosomal DNA, which was then dissolved in SSC buffer containing pH 7.1 and sodium chloride and trisodium citrate. RNase A, a ribonuclease marketed by Sigma Chemical Company, and Pronase E, a protease marketed by Kaken Pharmaceutical Co., Ltd. of Tokyo, Japan. After cooling and centrifugation, two volumes of cold ethanol were added to recover purified chromosomal DNA. This chromosomal DNA was dissolved in buffer containing pH-aminomethane, hydrochloric acid and EDTA (pH 7.5) and stored at -20 ° C.

(2) Preparation of Plasmid pBR 322

Journal of Bacteriology, Vol. Plasmid pBR 322 (ATCC 37013) was isolated from Escherichia coli according to the method described by J. Meyer et al. On pages 127, 1524-1537 (1976).

(3) Preparation of Recombinant DNA Having Polypeptide Genes

The purified chromosomal DNA having the heat resistant polypeptide gene prepared in Example 1- (1) was partially digested with restriction enzyme Mbo I purchased from Nippon Gene Co., Ltd., Toyama, Japan, DNA fragments were obtained. Separately, the pBR 322 specimen prepared in Example 1- (2) was completely divided into restriction enzyme BamH I purchased from Nippon Gene Co., Ltd., and the split product was obtained from Takara Shuzo Co., Kyoto, Japan. E. coli alkaline phosphatase purchased from., Ltd. is added to dephosphorize the 5′-end of the plasmid fragment as well as to prevent self-bonding of the plasmid fragment.

T ₄ , DNA ligase purchased from Nippon Gene Co., Ltd. was added to these two fragments overnight at 4 ° C. to bind these two fragments to obtain recombinant DNA.

(4) Introduction of Recombinant DNA into E. coli

E. coli HB 101 (ATCC 33694), a strain having amylase production capacity, was used as a host.

The microorganisms were incubated for 4 hours in L-juice at 37 ° C., and the cells collected by centrifugation from this culture were suspended in 10 mM acetate buffer (pH 5.6) containing 50 mM manganese chloride, and then again centrifuged. The mixture was collected and resuspended in 10 mM acetate buffer (pH 5.6) containing 125 mM manganese chloride, and the recombinant DNA prepared in Example 1- (3) was added and left in ice-cooled water for 30 minutes. The mixture was then warmed to 37 ° C. to add L-juice, developed on L-juice agar plate medium containing 50 μg / ml ampicillin and 2 mg / ml starch, and then colonized in a 24-hour incubation at 37 ° C. colony).

Colonies whose starches were changed to cyclodextrins were selected by the iodine-coloring method. In this way, microorganisms into which recombinant DNA having a polypeptide gene were introduced were selected. Subsequently, the recombinant microorganism was grown, and the recombinant DNA was extracted from the proliferating microorganism according to the plasmid preparation method in Example 1- (2) and added to the restriction enzyme to determine the restriction division site. Complete digestion with restriction enzyme EcoR I purchased from. Digestion products were added to T ₄ DNA ligase similarly as in Example 1- (3) to obtain recombinant DNA, followed by selection of recombinant microorganisms according to the method in Example 1- (4). These recombinant microorganisms contained a relatively small size of recombinant DNA carrying a polypeptide gene.

This recombinant DNA and plasmid pBR 322 were then completely digested with the restriction enzyme Sal I purchased from Nippon Gene Co., Ltd., and treated much like the case of EcoR I to deliver a polypeptide gene. Recombinant microorganisms containing recombinant DNA were selected.

These microorganisms and their recombinant DNA were named "Esheria coli TCH 201 (FERM BP-2109)" and "pTCH 291", respectively.

Restriction diagrams of DNA fragments derived from this recombinant DNA pTCH 201, in particular Bacillus stearotermophilus microorganisms, are shown in FIG.

Figure 1 shows that this recombinant DNA is purchased from Kpn I, Hind III, or Takara Shuzo Co., Ltd., a restriction enzyme Pvu II purchased from Toyobo Co., Ltd., from Nippon Gene Co., Ltd. It can be divided by any one of Xba I, but not by Ecor I, BamH I, Pst I, Xho I, Bgl II or Acc I available from Nippon Gene Co., Ltd. .

Example 2

Cloning of Bacillus Stearotermophilus Polypeptide Gene Against Bacillus Subtilis

(1) Preparation of Recombinant DNA pTCH 201

Recombinant DNA pTCH 201 was isolated from Escherichia coli TCH 201 (FERM BP-2109) according to the method of Example 1- (2).

(2) Preparation of the plasmid pUB 110

Journal of Bacteriology, Vol. Plasmid pUB 110 (ATCC 37015) was isolated from Bacillus subtilis according to the method described by Gryczan et al. On pages 134, 318-329 (1978).

(3) Preparation of Recombinant DNA Having Polypeptide Genes

Restriction enzymes EcoR I and Xba I were simultaneously added to the recombinant DNA pTCH 201 having the heat-resistant polypeptide gene prepared in Example 2- (1) to completely digest it.

Separately, the restriction enzymes EcoR I and Xba I were added to the plasmid pUB 110 specimen prepared in Example 2- (2) in the same manner to divide them completely.

T ₄ DNA ligase was added to the obtained fragments similarly as in Example 1- (3) to obtain recombinant DNA.

(4) Introduction of recombinant DNA into Bacillus subrelease

In this example, Bacillus subtilis 715A, a strain having the ability to generate amiralase, was used as a host. These microorganisms were incubated at 28 ° C. in brain-heart leaching medium for 5 hours, and the cells collected by centrifugation from the culture were collected from the Proceedings of the National Academy of Sciences of the USA, Vol, 73, 2151-2155. 1976) to produce a plasma suspension according to the method described by Schaeffer et al.

To this suspension was added the recombinant DNA prepared in Example 2- (3), and then the mixture was added to Agricultural and Biological Chemistry, Vol. Treated and transformed according to the method described by Sekiguchi et al. On pages 46, 1617 to 1621 (1982), developed in HCP medium containing 250 μg / ml kanamycin and 10 mg / ml starch and incubated at 28 ° C. for 72 hours to colonize Was formed.

Recombinant microorganisms into which recombinant DNA having a heat resistant polypeptide gene were introduced from these colonies were selected by the method of Example 1- (4).

These microorganisms and their recombinant DNA were named "Bacillus subtilis TCU 211 (FERM BP-2112)" and "pTCU 211", respectively.

Restriction diagrams of DNA fragments derived from recombinant DNA pTCU 211, particularly Bacillus stearotermophilus microorganisms, are as shown in FIG. Figure 2 shows that this recombinant DNA can be cleaved by any of the restriction enzymes Pvu II, Kpn I or Hind III, but by EcoR I, BamH I, Pst I, Xho I, Bgl II, Acc I or Xba I. It is not shown.

Example 3

Partial Amino Acid Sequences of Bacillus Stearotermophilus Polypeptide Containing N-terminus

(1) Preparation of Polypeptides

Bacillus stearotermophilus FERM-P No. 2225 was cultured in a liquid medium by the method of Example 5 to prepare a polypeptide. The supernatant obtained by centrifugation from this culture was salted with ammonium sulfate to obtain a polypeptide fraction, which was obtained from Toyo Soda Manufacturing Co., Ltd., Japan. Purified by column chromatography using the commercially available anion exchanger, DEAE Toyopearl 650, and chromatofocusing using “Mono P” from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Obtained.

Journal of Biological Chemistry, Vol. Polypeptide samples in SDS-polyacrylamide electrophoresis according to the method described by K. Weber and M. Osborn on pages 244, 4406 (1969) showed a molecular weight of 70,000 ± 10,000 Daltons.

The native activity of this polypeptide is 200 ± 30 units / mg Table 1b.

(2) partial amino acid sequence of polypeptide comprising N-terminus

Polypeptide samples prepared by the method of Example 3- (1) were prepared using Applied Biosystems Inc., California, USA. The product was supplied to a gaseous protein sequence analyzer "Model 470 A", followed by high performance liquid chromatography analysis to determine the partial amino acid sequence containing the N-terminus. This partial amino acid sequence was Ala-Gly-Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr.

Example 4

Sequence of Polypeptide Gene Derived from Bacillus Stearotermophilus and Sequence of Amino Acids of Polypeptide

(1) Preparation of Plasmid pUC 18

Plasmid pUC 18 was prepared following the method of Example 1- (2) from Escherichia coli JM 83 (ATCC 35607) introduced with plasmid.

(2) Preparation of Recombinant DNA Having Polypeptide Genes

A fragment obtained by aging a fragment having a polypeptide gene prepared by the method of Example 2- (3) with restriction enzymes and a pUC 18 sample prepared by the method of Example 4- (1) were divided in the same manner. The obtained plasmid fragment was added to T ₄ DNA ligase to obtain recombinant DNA.

(3) introduction of recombinant DNA into Escherichia coli

In this example Escherichia coli JM 83 was used as the host. Recombinant DNA was introduced into this microorganism according to the method of Example 1- (4) to transform the microorganism.

Recombinant microorganisms were inoculated in a medium containing 5-bromo-4-chloro-3-indoyl-β-galactoside (Xgal) and microorganisms forming colorless spots were selected.

(4) Preparation of recombinant DNA from recombinant microorganisms

Cells obtained by culturing the recombinant microorganisms in L-juice medium containing 50 μg / ml of ampicillin were treated by an alkali reduction method to obtain recombinant DNA.

(5) Sequence of recombinant DNA

Recombinant DNA was arranged by the Dideoxy chain terminator method.

After mixing the recombinant DNA prepared in Example 4- (4) and the synthetic primer consisting of 17 bases and annealing at 60 ° C. for 20 minutes, dNTP, ddNTP, (α-32P) dCTP and Klenow fragment were added. The primer was extended from 5′-site to 3′-site by reaction at 37 ° C. for 30 minutes. In this way, supplementary DNA was obtained. After excess dNTP was added to the supplemental DNA, the mixture was allowed to react at 37 DEG C for 30 minutes, followed by addition of formamide solution of the dye mixture to stop the reaction. The reaction mixture was boiled for 3 minutes and electrophoresed at about 25 mA (about 2000 volts) on a 6% polyacrylamide gel to separate expanded supplemental DNA. After the end of electrophoresis, the gel was fixed and dehydrated.

The dehydrated gel was subjected to autograph analysis, and the polypeptide gene was measured by analyzing the base band on the radio autogram.

The results obtained were as listed in Table 1a.

The sequence of coding peptide genes located on the 5'-terminal top of the polypeptide gene was measured in the same manner.

The results obtained are shown in Table 1b.

(6) amino acid sequence of polypeptide

The amino acid sequence of the polypeptide was determined with reference to the sequence listed in Table 1a, and the results are shown in Table 2a.

The amino acid sequence of the coding peptide was determined in the same manner, and the results are shown in Table 2b.

These data confirm that the polypeptide derived from Bacillus stearotermophilus has the amino acid sequence shown in Table 2a.

TABLE 1a

TABLE 1b

TABLE 2a

TABLE 2b

Example 5

Preparation of Polypeptides by Recombinant Microorganisms

Polypeptides as recombinant microorganisms Escherichia coli TCH 201 (FERM BP-2109) and Bacillus subtilis TCU 211 (FERM BP-2112) introduced with recombinant DNA carrying a heat resistant polypeptide gene derived from Bacillus stearotermophilus. Prepared.

The polypeptide-generating ability of these recombinant microorganisms was compared with that of host microorganisms and that of donor Bacillus stearotermophilus microorganisms for their CGTase activity.

PH of liquid medium consisting of 1.0w / v% corn steep liquor, 0.1w / v% ammonium sulfate, 1.0w / v% calcium carbonate, 1w / v% starch and water was adjusted to 7.2 and heated at 120 ° C. for 20 minutes. After sterilization and cooling. In the case of Escherichia coli TCH 201, 50 μg / ml of ampicillin was added to the liquid medium and then microorganisms were inoculated into the liquid medium. Escherichia coli HB 101 was inoculated in liquid medium without the addition of antibiotics. In each case the microorganisms were incubated for 48 hours under vigorous stirring conditions at 37 ° C.

Separately, Bacillus subtilis TCU 211 was incidentally inoculated into liquid busys containing 5 μg / ml kanamycin while Bacillus subtilis 715A was inoculated into liquid medium without the addition of antibiotics. In each case the microorganisms were incubated at 28 ° C. for 72 hours.

Bacillus stearotermophilus RFRM-P No. 2225 was incubated for 48 hours in a 50 ℃ liquid medium without the addition of antibiotics. Each culture was centrifuged to separate the supernatant from the cells, and the supernatant was subjected to the CGTase activity test as it was, while the cells were sonicated to measure CGTase activity per culture. The results obtained are shown in Table 3a.

These data indicate that recombinant microorganisms may be useful for industrial scale production of polypeptides because of their ability to produce polypeptides.

The supernatant was salted out with ammonium sulfate with a saturation of 0.6 to obtain a crude polypeptide sample. Sugar-transition from starch to sucrose, from starch to cyclodextrin, ratio of α-, β- and γ-cyclodextrin, optimal temperature, optimal pH, stable temperature range and stable pH for polypeptide samples prepared with recombinant microorganisms As a result of examining enzyme properties such as range, etc., the properties of these samples were superior to those of polypeptides prepared with a donor Bacillus stearotermophilus microorganism.

TABLE 3a

[Experiment 1]

Polypeptide and CGTase Preparation

Polypeptide having CGTase activity was prepared in the same manner as in Example 5. A liquid medium consisting of 1.0w / v% corn steep liquor, 0.1w / v% ammonium sulfate, 1.0w / v% calcium carbonate, 1w / v% starch and water was adjusted to pH 7.2 and sterilized by heating at 120 ° C. for 20 minutes. After cooling, 50 μg / ml of ampicillin was added. This liquid medium was inoculated with a seed culture of Escherichia coli TCH201 (FERM BP-2109) and incubated with vigorous stirring at 37 ° C. for 48 hours. The obtained culture was sonicated and centrifuged. The obtained supernatant was mixed with ammonium sulfate, and the fractions which precipitated within the saturation range of 0.3-0.6 were recovered and column chromatography was performed using "DEAE Toyopearl 650" (anion exchange resin manufactured by Toyo Soda Manufacturing, Japan). Thereafter, chromatofocusing treatment was performed using “Mono P” (manufactured by Pharmacia Fine Chemicals AB of Sweden) to prepare a high purity polypeptide. Another polypeptide sample was prepared by incubating Bacillus subtilis TCU211 (FERM BP-2112) in the same manner as above using kanamycin 5 μg / ml instead of ampicillin.

CGTase samples were prepared by purifying the culture solution obtained by culturing Bacillus stearotermophilus TC-60 (FERM-P No. 2222) according to the method disclosed in US Pat. No. 3,988,206.

[Experiment 2]

Enzyme Properties

[Experiment 2-1]

Effect of pH on the Activity of Polypeptides and CGTase

The polypeptide prepared in Experiment 1 from Escherichia coli TCH201 was compared with CGTase derived from Bacillus stearotermophilus TC-60 for enzyme properties such as optimum pH, pH stability, optimal temperature and thermal stability. These enzyme properties are described in the Journal of the Japanese Society of Starch Science, Vol. 29, No. 1, pp. 7-12 (1982)].

0.5 ml of the polypeptide solution or CGTase solution was added to a mixture of 3 ml of 0.5% soluble starch aqueous solution and 2 ml of 50 mM acetate buffer (pH 3.5-6.0) or 2 ml of 50 mM Tris-maleate buffer (pH 6.5-8.0) and 12.5 mM CaCl ₂ and Incubated at 40 ° C. for 10 minutes at pH. The reaction mixture was then iodine stained in the usual manner and then absorbance at 660 nm was measured. The results are shown in Table 3b.

TABLE 3b

The results of Table 3b shows that the optimum pH of the polypeptide sample is 5.5, while the optimal pH of the CGTase sample is 6.0.

[Experiment 2-2]

Effect of pH on Stability of Polypeptide and CGTase

Polypeptide and CGTase were incubated for 2 hours at different pH and 40 ° C in 50mM McIvavaine buffer (pH 4.0-8.0) or 50mM borate-Na ₂ CO ₃ buffer (pH 9.0-10.0) and the residual activity was measured according to standard methods. The results are shown in Table 3c.

TABLE 3c

From the results of Table 3c, it can be seen that the polypeptide samples are stable in the pH range of 6-9 and the CGTase samples are stable in the range of pH 7-9.

[Experiment 2-3]

Effect of temperature on the activity of polypeptides and CGTase

Polypeptide and CGTase samples were incubated for 10 min at different temperature and pH 5.5 in the presence of 5 mM CaCl ₂ and then the remaining activity was measured. The results are shown in Table 3d.

Table 3d

From the results of Table 3d, it can be seen that the optimum temperature of the polypeptide sample is 75 ° C and the optimal temperature of the CGTase sample is 70 ° C.

[Experiment 2-4]

Effect of temperature on the stability of polypeptide and CGTase

Polypeptide and CGTase samples were incubated for 30 min at different temperatures in 50 mM Tris-maleate buffer (pH 7.0) containing 5 mM CaCl ₂ and the remaining activity was measured. The results are shown in Table 3e.

Table 3e

The results of Table 3e show that the polypeptide sample is stable up to 70 ° C. and the CGTase sample is stable up to 50 ° C.

From these results, it can be seen that the polypeptide samples maintained nearly 100% of their activity when incubated at pH 7.0 for 30 minutes in the presence of 5 mM CaC1 ₂ , while the CGTase samples maintained about 30% of their activity. .

Polypeptide samples derived from Bacillus subtilis TCU211 were studied in the same manner as above, and the polypeptide samples showed almost the same enzyme properties as polypeptide samples derived from Escherichia coli TCH201.

It is clear from the above data that polypeptide samples derived from Escherichia coli TCH201 and Bacillus subtilis TCU211 have been shown to be more effective in conventional CGTase, especially Bacillus stea, in terms of enzyme properties such as optimum pH, pH stability, optimal temperature and thermal stability. It is remarkably different from Rotermophilus TC-60.

[Experiment 3]

Enzyme Properties

[Experiment 3-1]

Polypeptide and CGTase Preparation

Polypeptide having CGTase activity was prepared in the same manner as in Example 5. A liquid medium consisting of 1.0w / v% corn steep liquor, 0.1w / v% ammonium sulfate, 1.0w / v% calcium carbonate, 1w / v% starch and water was adjusted to pH 7.2 and sterilized by heating at 120 ° C. for 20 minutes. It was then cooled and 50 μg / ml of ampicillin was added. Species of Escherichia coli TCH201 (FERM BP-2109) were inoculated into the liquid medium and incubated at 37 ° C. with vigorous stirring for 48 hours. The obtained culture was sonicated and centrifuged. The obtained supernatant was mixed with ammonium sulfate, and the fractions precipitated within a saturation range of 0.3 to 0.6 were recovered and column chromatographed using “DEAE Toyopearl 650” (anion exchange resin manufactured by Toyo Soda Manufacturing, Japan). High purity polypeptides were prepared by chromatofocusing treatment using “Mono P” (manufactured by Pharmacia Fine Chemicals AB, Sweden). Another chromatofocusing treatment produced a high purity polypeptide. Another polypeptide sample was prepared by incubating Bacillus subtilis TCU211 (FERM BP-2112) as above using 5 μg / ml kanamycin instead of ampicillin.

CGTase samples were incubated with Bacillus stearotermophilus TC-60 (FERM-P No. 2222), Bacillus megaterium T-5 (ATCC 21737), Bacillus circle lance (ATCC 9995) and Bacillus maserans (IFO 3490). The cultures obtained were prepared by purifying according to the methods disclosed in US Pat. Nos. 3,703,440, 3,812,011 and 3,988,206.

[Experiment 3-2]

Isoelectric point measurement

The isoelectric point was measured by isoelectric point fractionation of the polypeptide and CGTase sample prepared in Experiment 3-1. Isoelectric point fractions are described in N. Catsimpoolas, Analytical Biochemistry, Vol. 26, pp. 480 to 482 (1968).

Gel medium contains 15 g of acrylamide, 0.15 ml of N, N, N ', N'-tetramethylethylenediamine, 0.6 g of N, N'-methylenebisacrylamide, 0.003 g of riboflavin, 40% amphoteric electrolyte "Ampholine" (Sweden) LKB-Producter AB Co., Ltd.) 15 ml of the mother liquor and distillation were mixed to make 300 ml in total. A sample solution containing 30 μg of E. coli TCH201-derived polypeptide per gel column and also containing 2% Ampholine and 30% glycerol was added to the gel medium and then added to sixteen cylindrical glass columns, each 65 mm in length, and then to light. Polymerized by exposure. Isoelectric point fractionation was performed in a bath (btah) used for gel disc electrophoresis (using “Modedl TOYO CD8” manufactured by Advancd Toyo Kaisha, Japan). The upper bath was charged with 0.02 M NaOH and the lower bath with 0.01 MH ₃ PO ₄ . Subsequently, the upper electrode was connected to the positive electrode of the constant voltage supply and the lower electrode was also connected to the negative electrode. First, electrofocusing was performed by passing a 200 V direct current through each electrode for 16 hours and then a 400 V direct current for 1 hour.

After the electrofraction, the column was removed from the electrophoresis apparatus, the gel was removed from the column, and 8 gels were cut into 5 mm, each placed in a small glass container, mixed with 1 ml of water, extracted with Ampholine, and the pH was measured with a micro electrode. pH gradient was determined.

The remaining 8 gels were immersed in a 12% trichloroacetic acid solution for 2 hours to fix protein components in the gel, and then washed with a mixture containing methanol: acetic acid: water = 25: 8: 65 for 24 hours to extract Ampholine. . The gel was washed several times with 12% trichloroacetic acid solution and then stained each of the electric fraction bands with 0.1% Coomassie Blue. Decolorization was carried out by repeatedly washing the gel with 10% methanol and 7% acetic acid aqueous solution. The relative intensity of each band dyed was scanned at a wavelength of 600 nm with a chromatoscanner "Model CS-930" (manufactured by Shimdzu Seisakusho, Japan), and the representative gel scattering pattern is shown in FIG. The dashed line in FIG. 5 shows the pH slope along the polyacrylamide gel column after the electrical fraction.

±SD, n=8)이었다.As is apparent from FIG. 5, the isoelectric point of the polypeptide derived from Escherichia coli TCU211 is 5.0 ± 0.1 (

± SD, n = 8).

±SD, n=8)이었다.As a result of the electro-fractionation of the polypeptide derived from Bacillus subtilis TCU211, the isoelectric point of this polypeptide was 5.0 ± 0.1 (

± SD, n = 8).

SD, n=8), 바실러스 메가테르움 CGTase의 등전점은 6.1±0.1 및 6.8±0.1(

SD, n=8), 바실러스 서클란스 CGTase의 등전점은 5.8±0.1 및 6.6±0.1(

SD, n=8), 그리고 바실러스 마세란스 CGTase의 등전점은 4.6±0.1(

SD, n=8)이었다.CGTase samples derived from strains such as Bacillus stearotermophilus TC-60, Bacillus megaterium T-5, Bacillus circle lance and Bacillus maserans were subjected to electrofraction as above. As a result, the isoelectric point of Bacillus stearotermophilus CGTase was 4.5 ± 0.1 (

SD, n = 8), and the isoelectric points of Bacillus megaterium CGTase were 6.1 ± 0.1 and 6.8 ± 0.1 (

SD, n = 8), and the isoelectric points of Bacillus circle lance CGTase were 5.8 ± 0.1 and 6.6 ± 0.1 (

SD, n = 8), and the isoelectric point of Bacillus maseran CGTase is 4.6 ± 0.1 (

SD, n = 8).

It is clear from these data that polypeptides derived from Escherichia coli TCH201 and Bacillus subtilis TCU211 differed significantly in isoelectric point from conventional CGTase. This means that these polypeptides differ from conventional CGTases.

Example 6

Cloning of Bacillus maseran polypeptide gene against Escherichia coli

(1) Preparation of Chromosome DNA Having Bacillus Maseran Polypeptide Gene

Polypeptide genes were prepared according to the method of Example 1- (1) except that Bacillus maserans 17A was incubated at 28 ° C.

(2) Preparation of Recombinant DNA Having Polypeptide Genes

Chromosomal DNA having a polypeptide gene derived from Bacillus maserans prepared in Example 6- (1) was obtained from Nippon Gene Co. The partial digestion was carried out similarly as in Example 1- (3) with restriction enzyme Hind III purchased from Ltd.

Separately, the plasmid pBR 322 sample prepared by the method of Example 1- (2) was completely divided by the restriction enzyme Hind III, and the 5′-end of the split product was dephosphorized by the method of Example 1- (3). I) A treatment was performed. The fragments thus obtained were combined in accordance with the method of Example 1- (3) to obtain recombinant DNA.

(3) introduction of recombinant DNA into Escherichia coli

Recombinant microorganism into which recombinant DNA was introduced was cloned according to the method of Example 1- (4), using Escherichia coli HB101 (ATCC 33694), a strain capable of producing amylase, as a host, and then recombinant DNA was isolated from the microorganism. The restriction sites were determined by isolation and addition to restriction enzymes, followed by partial digestion with restriction enzyme Sau 3 AI, which is commercially available from Nippon Gene Co., Ltd.

Separately, the plasmid pBR 322 sample obtained by the method of Example 1- (2) was completely divided as restriction enzyme BamHI and the 5′-end of the obtained product was subjected to dephosphorization in a similar manner as in Example 1- (3). . The obtained fragments were combined with T ₄ DNA ligase to obtain recombinant DNA, and then recombinant microorganisms were selected according to the method of Example 1- (4). These recombinant microorganisms contained a relatively small size of recombinant DNA containing polypeptide genes.

These recombinant microorganisms and their recombinant DNA were named "Esheria coli MAH2 (FERM BP-2110)" and "pMAH2", respectively.

Restriction diagrams of DNA fragments having polypeptide genes derived from recombinant DNA pMAH2, in particular Bacillus maserans, are listed in FIG.

Figure 3 shows that the recombinant DNA can be cleaved by any of the restriction enzymes Pvu II, Sal I, Ava I or Pst I sold by Nippon Gene Co., Ltd., but EcoR I, Hind III, Kpn I, It is not divided into BamH I, Xba I, Xho I or Sma I.

Example 7

Cloning of Bacillus maseran polypeptide gene against Bacillus subtilis

(1) Preparation of Recombinant DNA pMAH2

Recombinant DNA pMAH2 was isolated from Escherichia coli MAH2 (FERM BP-2110) according to the method of Example 1- (2).

(2) Preparation of Recombinant DNA Having Polypeptide Genes

Restriction enzymes EcoR I and BamH I were simultaneously added to the recombinant DNA pMAH2 having the polypeptide gene prepared in Example 7- (1) for complete digestion.

Separately, restriction enzymes EcoR I and BamH I were simultaneously added to the plasmid pUB110 specimen prepared by the method of Example 2- (2) to perform complete cleavage.

T ₄ DNA ligase was added to the fragment thus obtained similarly to Example 1- (3) to obtain a recombinant DNA.

(3) Introduction of recombinant DNA into Bacillus subtilis

Recombinant microorganisms into which recombinant DNA having a polypeptide gene derived from Bacillus maserans were introduced were cloned according to the method of Example 2- (A) using Bacillus subtilis 715A, a strain capable of amylase production.

The recombinant microorganism and its recombinant DNA were named "Bacillus subtilis MAU210 (FERM BP-2111)" and "pMAU210", respectively. Recombinant DNA Restriction diagrams of DNA fragments with polypeptide genes derived from pMAU210, in particular Bacillus maserans, are listed in FIG. 4 shows that recombinant DNA can be cleaved by any of the restriction enzymes Pva II, Sal I, Ava I or Pst I, but with EcoR I, Hind III, Kpn I, BamH I, Xba I, Xho I or Sma I. It is not divided.

Example 8

Amino Acid Sequence of Polypeptides Derived from Bacillus maserans Containing N-terminus

(1) Preparation of Polypeptides

Similarly to Example 10, the polypeptide was prepared by culturing Bacillus subtilis MAU210 (FERM BP-2111) in a liquid medium, and then purified according to the method of Example 4- (1) to obtain a high purity polypeptide sample.

Polypeptide samples by SDS-polyacrylamide electrophoresis showed a molecular weight of 70,000 ± 10,000 Daltons and a high activity of 200 ± 30 units / mg protein.

(2) partial amino acid sequence including N-terminus

With the polypeptide sample prepared in Example 8- (1), the partial amino acid sequence including the N-terminus was determined according to the method of Example 3- (2).

This partial amino acid sequence was Ser-Pro-Asp-Thr-Ser-Val-Asn-Asn-Lys-Len.

Example 9

Sequence of Polypeptide Gene Derived from Bacillus maserans and Sequence of Amino Acids of Polypeptide

(1) Preparation of Recombinant DNA Having Polypeptide Genes

Recombinant DNA was prepared according to the method of Example 4- (3). In particular, the DNA fragment obtained by digesting the DNA fragment containing the polypeptide gene prepared by the method of Example 7- (2) with the restriction enzymes and the plasmid pUC18 sample prepared by the method of Example 4- (2) are identical. The plasmid fragment obtained by cleavage by the method was combined with T4 DNA ligase to obtain recombinant DNA.

(2) introduction of recombinant DNA into Escherichia coli

Recombinant DNA was introduced into the host microorganism Escherichia coli JM83 according to the method of Example 4- (3) to obtain a recombinant microorganism.

(3) Preparation of DNA prepared from recombinant microorganisms

Recombinant DNA was prepared according to the method of Example 4- (4).

(4) Sequence of recombinant DNA

The sequence of polypeptide genes was measured according to the method of Example 4- (5). The measurement results are shown in Table 4a.

The coding peptides located on the 5′-site of the polypeptide genes were measured in the same order. The measurement results are shown in Table 4b.

(5) amino acid sequence of polypeptide

The amino acid sequence of the polypeptide was determined with reference to the polypeptide gene sequence. The results are shown in Table 5a.

The amino acid sequence of the coding peptide was determined in the same manner. The results are shown in Table 5b.

These data confirmed that the polypeptide derived from Bacillus maserans has the amino acid sequence as shown in Table 5a.

As data contained in Tables 2a and 5b, each polypeptide usually has the amino acid sequence of the following (a), (b), (c), (d) and (e), and these partial amino acid sequences (a), ( It can be seen that b), (c), (d) and (e) are arranged adjacent to the N-terminus of the polypeptide.

(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

TABLE 4a

TABLE 4b

TABLE 5a

TABLE 5b

Example 10

Preparation of Polypeptides by Recombinant Microorganisms

Polypeptides were prepared from Escherichia coli MAH2 (FERM BP-2110) and Bacillus subkillis MAU210 (FERM BP-2111) into which recombinant DNA containing polypeptide genes derived from Bacillus maserans was introduced.

The polypeptide-producing ability of these recombinant microorganisms, host microorganisms and donor Bacillus maserans was compared as their CGTase activity. The used liquid medium was prepared by the method of Example 5.

Escherichia coli MAH2 was inoculated in liquid medium additionally containing 50 μg / ml of ampicillin, while Escherichia coli HB101 was inoculated in liquid medium without the addition of antibiotics. In each case the microorganisms were incubated for 24 hours at 35 ° C. under vigorous stirring conditions.

Bacillus subtilis MAU210 was additionally inoculated into liquid medium containing 5 μg / ml of kanamycin, while Bacillus subtilis 715A was inoculated into liquid medium without the addition of antibiotics. In each case the microorganisms were incubated at 28 ° C. for 72 hours.

Bacillus maserans 17A was incubated in 28 ° C. liquid medium for 72 hours without the addition of antibiotics.

Each culture was treated similarly to Example 5 and then its CGTase activity was determined. The results obtained are shown in Table 6.

These data indicate that recombinant microorganisms can be flowed to industrial scale production of polypeptides because of their ability to produce polypeptides.

The supernatant was salted out with ammonium sulfate with a saturation of 0.6 to obtain a crude polypeptide sample.

Enzymatic properties of these crude polypeptide samples were examined similarly to Example 5, and the enzyme properties of the polypeptides prepared as these recombinant microorganisms were superior to those of the polypeptides produced by the microorganisms on donor Bacillus maserans.

TABLE 6

Important uses of the polypeptides of the invention are described below.

The polypeptide undergoes an intramolecular or intermolecular saccharide transfer reaction between the appropriate saccharide donor and the saccharide receptor.

One feature of the present invention is that various sugar transfer products can be prepared by utilizing the strength of such a sugar transfer reaction.

For example, starch-containing substances such as starch, dextrose equivalent (DE) of less than 10, or starch-containing substances such as amylase are used as amylase substrates to act on polypeptides by using intramolecular saccharide transfer reactions. -And a starch hydrolyzate containing γ-cyclodextrin can be prepared. If necessary, each cyclodextrin can be isolated from this starch partial hydrolyzate.

α-glycosylated saccharide sweeteners such as α-glucosyl-, α-maltosyl and α-maltotriosyl saccharides, for example, starch, liquefied starch, dextrin, cyclodextrin or starch substances such as amylose Intermolecular saccharide transfer reaction by reacting a polypeptide to a mixture of a sugar donor and a saccharide receptor such as monosaccharides such as xylose, sorbose, or fructose, or disaccharides such as sucrose, maltulose or isomaltulose It can be prepared by advancing. α-glucosylated saccharide sweeteners may be advantageous for use in foods and beverages because they are very mild in taste and better soluble in water compared to the saccharide sweeteners themselves, but are difficult to crystallize. The use of sugar sweeteners as these materials will be greatly expanded.

In the intermolecular rectification transfer reaction, by using a glycoside such as steviol glycoside, such as stevioside or rebaudioside, glycyrrizine, soyasaponin, tinaponin, rutin or esculin, as a sugar receptor, α-glucosyl α-glycosylated glycosides can be prepared, such as α-maltosyl and α-maltotriosyl-glycosides. This α-glycosylated glycoside removes unpleasant tastes such as bitterness and astringent taste derived from the glycoside itself and is more easily dissolved in water than glycoside itself. These materials will greatly expand the use of glycosides.

In particular, since the taste improvement of α-glycosylated steviol glycosides and α-glycosylated glycyrazine is remarkable, and the sweetness thereof is comparable to that of sucrose, these α-glucosylated glycosides are used in food, It can be advantageously used for beverages and oral administration medicaments.

Some embodiments are described below.

Example 11

Corn Syrup Containing Cyclodextrins

Polypeptide samples prepared with Bacillus subtilis TCU211 according to the method of Example 5 were added to a 10 w / w% potato starch suspension at a ratio of 2 units / g starch, liquefied by heating to pH 85 at pH 6.5, 70 After cooling to 占 폚, the same amount of polypeptide sample was further added and reacted for 40 hours. The reaction mixture was purified by decolorization using activated carbon and deionization using ion exchange resin, and then concentrated to give corn syrup containing cyclodextrin in 92% yield on a dry solids basis. Since the corn syrup has excellent flavor retention properties, it is advantageous to use it in perfumes and cosmetics, in which aroma or aroma is one of the important factors.

(Alpha)-, (beta)-, and (gamma) -cyclodextrin in this corn syrup can isolate | separate this corn syrup by the method of using organic precipitants, such as toluene or trichloromethane, or by conventional column chromatography.

Example 12

α-glucosyl sucrose

0.2w / w% oxalic acid was added to 35w / w% cornstarch suspension, and the mixture was autoclaved to 120 DEG C to be DE20, neutralized with calcium carbonate and filtered to obtain a dextrin solution. This dextrin solution was then added to 1/2 the amount of sucrose on the basis of dry solids, and the polypeptide sample prepared with Bacillus subtilis MAU210 according to the method of Example 10 was added to the obtained mixture at a ratio of 15 units / g starch. After the addition, the mixture was reacted at pH 6.0 and 55 ° C. for 40 hours. The reaction mixture was purified by decolorization operation using activated carbon and deionization operation using ion exchange resin, and then concentrated to give a colorless transparent corn syrup in 94% yield based on dry solids. Corn syrups containing large amounts of α-glucosyl sucrose can be advantageously used in confectionery because they have a mild sweetness and are amorphous.

Example 13

α-glycosyl stevioside

After dissolving 200 g of stevioside and 600 g of dextrin (DE8) in 3 L of water, and cooling the obtained solution to 70 DEG C, the sample of polypeptide prepared with Bacillus subtilis TCU211 according to the method of Example 5 was subjected to 5 units / After addition at a ratio of g dextrin, the reaction was carried out at pH 6.0 and 65 ° C. for 35 hours. The reaction mixture was then heated at 95 ° C. for 15 minutes, filtered, purified, concentrated and crushed to yield a powdery sweetener containing α-glycosyl stevioside in about 92% yield on a dry solids basis.

The sweetener from the stevioside itself removed the unpleasant taste similar to sucrose in taste, and the sweetening ability of this sweetener was about 100 times higher than that of sucrose. These sweeteners can be advantageously used in diet sweeteners or seasonings and beverages because of their low calorie and tooth damage properties.

Example 14

α-glycosyl ginsenoside

The mixture obtained by heating and dissolving 60 g of ginseng extract and 180 g of β-cyclodextrin in 500 ml of water was cooled to 70 ° C., and then adjusted to pH 6.0. Polypeptide prepared by Escherichia coli TCH201 according to the method of Example 5. Samples are added at a rate of 3 units / g β-cyclodextrin and then 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 and then filtered. The filtrate was added to a column filled with 3 liters of synthetic adsorbent “Amberlite XAD-7”, commercially available from Rohm & Hass Co., Philadelphia, PA, and then washed with water to remove free saccharides. Then, 10 L of 50v / v% ethanol was added to this column, and the eluate was concentrated and dehydrated to obtain about 21 g of a powder product containing α-glucosyl ginsenoside. Since this product has been removed from unpleasant tastes such as bitterness, astringent taste and coarse taste derived from ginsenoside itself, the product can be used orally as it is or seasoned with sweetener or sour taste before use if necessary. In addition, this product can be advantageously used as a health food and medicine for internal administration because it has the effects of tonic, digestive, intestinal function, blood donor, anti-inflammatory and expectorant such as that of ginsenoside itself.

As described above, the inventors of the present invention have prepared recombinant DNA having a Pvu II restriction site from a donor microorganism as an in vivo genetic engineering technique, as well as determining the amino acid sequence of the polypeptide gene and its coding peptide. In addition, the inventors of the present invention have prepared recombinant microorganisms into which recombinant DNA has been introduced, and have confirmed that the recombinant microorganisms proliferate continuously in nutrient medium.

From the viewpoint of supplying the polypeptide at an appropriate level, the present invention has an industrially significant meaning because the present invention can secure a wide range of polypeptide sources and can easily increase the polypeptide generating ability of the donor microorganism.

While it has been described what has been described as preferred embodiments of the present invention, it will be understood that various modifications are possible in these embodiments, and all such modifications falling within the true spirit and scope of the present invention are included in the following claims. It should be considered to be.

Claims (14)

DNA fragments obtained by digesting the DNA of a donor microorganism with CGTase production in vitro with a restriction enzyme and a vector fragment obtained by cleaving a vector fragment obtained by dividing the vector into a restriction enzyme and a recombinant having a CGTase gene and a Pvu II restriction cleavage site A method for producing CGTase, comprising culturing a recombinant microorganism having DNA introduced therein in an influence medium and recovering accumulated CGTase. The method of producing CGTase according to claim 1, wherein the recombinant DNA has restriction cleavage sites as listed in the first, second, third or fourth degree. The method of claim 1, wherein the recombinant microorganism is Escherichia or Bacillus. The method for producing CGTase according to claim 1, wherein the recombinant microorganism is selected from the group consisting of Escherichia coli TCH201 (FERM BP-2109) or Wesleyia coli MAH2 (FERM BP-2110). The method of claim 1, wherein the recombinant microorganism is selected from the group consisting of Bacillus subtilis MAU210 (FERM BP-2111) and Bacillus subtilis TCU211 (FERM BP-2112). Has one or more partial amino acid sequences selected from the group consisting of the following partial amino acid sequences (a), (b), (c), (d) and (e), and are starchy to polypeptides having CGTase activity Process for the preparation of saccharide transfer products which act on substances. (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. The method of claim 6, wherein the saccharide transfer product is cyclodextrin. The method of claim 6, wherein the starch substance is acted on the polypeptide in the presence of a saccharide receptor. The method of claim 6, wherein the starch material is selected from the group consisting of starch, amylose, cyclodextrin, dextrin, and mixtures thereof. The method of claim 8, wherein the sugar receptor is selected from the group consisting of sugar sweeteners, glycosides, and mixtures thereof. The method of claim 8, wherein the saccharide transfer product is selected from the group consisting of α-glycosyl sucrose, α-glycosyl stevioside and α-glycosyl ginsenoside. The method of claim 8, wherein the saccharide transfer product is used as a sweetener. The method of claim 6, wherein the polypeptide has an isoelectric point of 5.0 ± 0.1. The method of claim 6, wherein the polypeptide is stable for 30 minutes at pH 7.0, 70 ° C. or lower in the presence of CaCl ₂ .

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