KR20080113624A - Yeast expressing cyclodextrin glucanotransferase and method for using the same - Google Patents

Abstract

A yeast expressing cyclodextrin glucanotransferase and a method using the same are provided to improve the quality of starch food by restraining the aging of the starch material processed foodstuff. A yeast cell expression vector comprises cyclodextrin glucanotransferase gene, promoter, and delta sequence. The promoter is linked on the top of the cyclodextrin glucanotransferase gene. The delta sequence is an integration site to integrate the cyclodextrin glucanotransferase gene with the genome of the yeast. The cyclodextrin glucanotransferase is expressed on the cell surface of the yeast.

Description

YEAST EXPRESSING CYCLODEXTRIN GLUCANOTRANSFERASE AND METHOD FOR USING THE SAME}

1 is a diagram illustrating a process for preparing a recombinant plasmid pδCGT [3-18] for transforming a cyclodextrin glucanotransferase gene into yeast according to an embodiment of the present invention.

2 is a diagram showing a recombinant plasmid pδCGT [3-18] -2 prepared for transforming a cyclodextrin glucanotransferase gene into yeast according to an embodiment of the present invention.

Figure 3 is a diagram showing the freeze stability of the yeast and wild-type control yeast transformed cyclodextrin glucanotransferase gene according to an embodiment of the present invention.

4 is a recombinant recombinant S. cerevisiae / pδCGT and a recombinant vector pδYD1 which does not contain cyclodextrin glucanotransferase expressed on the surface of the cyclodextrin glucanotransferase according to an embodiment of the present invention This figure shows the relative volume of bread prepared using yeast S. cerevisiae / pδYD1.

5 is a recombinant yeast transformed with a recombinant vector pδYD1 that does not contain S. cerevisiae / pδCGT, which is a recombinant yeast expressed on the surface of a cyclodextrin glucanotransferase, and a cyclodextrin glucanotransferase according to an embodiment of the present invention. The relative volume of Jeungpyun prepared using S. cerevisiae / pδYD1 was measured.

6 is a recombinant yeast transformed from a recombinant vector containing S. cerevisiae / pδCGT, which is a recombinant yeast expressed on the surface of the cyclodextrin glucanotransferase according to one embodiment of the present invention, and which does not include the cyclodextrin glucanotransferase The aging degree of bread prepared using S. cerevisiae / pδYD1 was measured using a differential scanning calorimeter.

7 is a recombinant yeast transformed with a recombinant vector containing S. cerevisiae / pδCGT, which is a recombinant yeast expressed on the surface of a cyclodextrin glucanotransferase, and a cyclodextrin glucanotransferase according to an embodiment of the present invention. The aging degree of Jeungpyun prepared using S. cerevisiae / pδYD1 was measured by differential scanning calorimetry.

8 is a high-performance liquid chromatography (HPLC) of small sugars present in bread prepared using S. cerevisiae / pδCGT, which is a recombinant yeast expressed on a surface of cyclodextrin glucanotransferase according to an embodiment of the present invention. It is a picture analyzed using.

9 is pR ₂ CGT [3-18] ( Protein according to an embodiment of the present invention) Eng . Des . Sel . , 17, pp205-211 (2004)) is a figure showing the polynucleotide sequence encoding the cyclodextrin glucanotransferase.

10 is a diagram showing the sequence of the δ sequence (δ sequence) which is an integration site according to an embodiment of the present invention.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a yeast expressing cyclodextrin glucanotransferase having an anti-aging effect of starch and a method for preparing bread or enlargement using the yeast, and aging of a product without additional enzyme treatment when using the yeast. This can be very useful for the food industry.

[Private Technology]

Starch widely used in the food field is composed of amylase and amylopectin, and when heated above a certain temperature in aqueous solution, the starch particles swell and become disorganized, resulting in a loss of crystallinity. . Typically, food containing starch is prepared and distributed in a digestible state by heating with moisture. However, foods containing starch tend to age the gelatinized starch during storage.

The aging of the starch refers to a phenomenon in which amylopectin crystals are formed in a process of decreasing moisture in the food when the food is stored for a long time. More specifically, when gelatinized starch is left at room temperature for a long time, the starch molecules are associated again by hydrogen bonding, that is, a phenomenon in which fine crystals are formed, that is, alpha starch (gelatinized starch) becomes beta starch. It means a return phenomenon. Factors affecting aging of starch are influenced by starch type, starch concentration, pH, moisture, temperature, presence of salts and various ions. For example, starch with high amylose content is aging well, and aging is promoted under acidic conditions or below 60 ° C., whereas aging is suppressed when salts and various ions are present.

Many problems arise when products containing starch, especially food, age. For example, when food containing starch ages, deterioration of product quality due to deterioration of taste and flavor, increase in toughness or increase in water retention capacity is a problem. .

In particular, aging of starch in the food containing the starch has a great influence on the shelf life of the product as well as quality. In particular, the aging rate of starch is very fast in Jeungpyeon or bread, which is a traditional food, and aging of starch is a great difficulty in mass production and commercialization of Jeungpyeon and bread.

 In order to solve the above problems, a variety of starch measures (preventive measures of retorogradation) has been used for starch-containing products. As a method of inhibiting aging of starch, there are control of water content, freezing, addition of sugar and emulsifier, etc. Recently, a method using an enzyme has been widely used.

In relation to the enzyme treatment method, the starch aging inhibitor of commonly used starch is a commercially available enzyme such as Novamyl, most of which are imported from foreign manufacturers. As the anti-aging enzymes as described above, various alpha amylase, cyclodextrin glucanotransferase, and the like have been studied. However, the enzyme treatment method described above has a problem in that its use is limited to some products because of the increased cost due to the additional process such as the enzyme purchase and the enzyme addition process, since the enzyme must be added during the manufacture of the product.

Thus, there is an increasing need for research into a method that can process the enzyme treatment in a simpler process.

It is an object of the present invention to provide a transformed yeast expressing cyclodextrin glucanotransferase that is effective in inhibiting aging of starch.

Another object of the invention relates to a method of using the transformed yeast.

In order to achieve the above object, the present invention provides a transformed yeast expressing cyclodextrin glucanotransferase that is effective in inhibiting aging of starch.

In addition, the present invention relates to a method of using the transformed yeast, and more particularly to a bread or a Jeungpyun using the transformed yeast in order to achieve the above object.

The present inventors have continuously studied to overcome the above problems, and as a result of using the yeast expressing the starch aging inhibitor enzyme on the surface of the yeast in the production of food containing starch, aging of the product without the addition of additional enzyme The present invention was completed by discovering that it can be suppressed.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a vector for producing a transformed yeast comprising a cyclodextrin glucanotransferase, a promoter and an integration site, which are effective in inhibiting aging of starch.

More specifically, the cyclodextrin glucanotransferase gene, the promoter operably linked to the upper part of the cyclodextrin glucanotransferase gene, and the cyclodextrin glucanotransferase gene, which are effective in inhibiting the aging of starch, are genomes of yeast. Yeast cell expression vector or transformed yeast comprising an integration site, i.e., an integration polynucleotide sequence, that is integrated so as to be integrated into the yeast cell, wherein the cyclodextrin glucanotransferase is expressed on the cell surface of the yeast. It relates to a production vector.

The yeast cell expression vector may further include a selective marker, and the selective marker may be a conventional selective marker capable of confirming transformation, and preferably an ampicillin resistance gene (Ampicillin resistance). gene, Amp (r)) and / or Neomycin resistance gene (Neo (r)).

The cyclodextrin glucanotransferase may be a conventional cyclodextrin glucanotransferase, preferably may be a cyclodextrin glucanotransferase (Accesion No. AY478421), more preferably pR ₂ CGT [3-18] ] (Cyclodextrin glucanotransferase obtained from Protein Eng . Des . Sel . , 17, pp 205-211 (2004)), most preferably the cyclodextrin glucanotransase of SEQ ID NO: 6. .

PR ₂ CGT [3-18] ( Protein Eng . Des . Sel . , 17, pp 205-211 (2004)), the cyclodextrin glucanotransferase preferably comprises the threonine (Methionine) of the 234th amino acid sequence of the cyclodextrin glucanotransferase (Accesion No. AY478421). Threonine), the 259th amino acid sequence from phenylalanine (Phenylalanine) to isoleucine (Isoleucine), and the 591th amino acid sequence from cyclone (Methionine) to alanine (Cylodextrin glucanotransferase) Specifically, it may be a cyclodextrin glucanotransferase of SEQ ID NO: 6, and preferably a cyclodextrin glucanotransferase encoded by the polyneutrophilide described in FIG. 9 and SEQ ID NO: 5.

The integration site may be a δ sequence, preferably a polynucleotide having a sequence of SEQ ID NO.

The transformed yeast production vector may be prepared by inserting the cyclodextrin glucanotransferase gene into a vector including a promoter and a surface expression sequence.

More specifically,

a) obtaining a cyclodextrin glucanotransferase;

b) preparing a first vector by inserting the cyclodextrin glucanotransferase obtained in step a) into a surface expression vector;

c) inserting a nucleotide sequence for inserting a foreign gene into the chromosome of the host organism into the first vector prepared in step b);

It can be prepared by a vector production method for producing a transformed yeast comprising.

Specifically, step a) is pR ₂ CGT [3-18] ( Protein Eng . Des . Sel . , 17, pp 205-211 (2004)) as a template can be carried out by a method for amplifying a polynucleotide encoding a cyclodextrin glucanotransferase. Amplification of the polynucleotide encoding the cyclodextrin glucanotransferase of pR ₂ CGT [3-18] can be performed using PCR amplification, and the cyclodextrin amplified by the polymerase chain reaction. The polynucleotide encoding the glucanotransferase may include a restriction enzyme cutting site at both ends. The restriction enzyme may be Kpn I or Xho I. The polynucleotide encoding the cyclodextrin glucanotransferase may preferably be a polynucleotide encoding the protein of SEQ ID NO: 6, more preferably the polynucleotide of SEQ ID NO: 5.

Primers for performing the polymerase chain reaction may be SEQ ID NO: 1 and SEQ ID NO: 2, for example.

The surface expression vector of step b) may preferably be pYD1 (Invitrogen, Carlsbad, CA, USA), and step b) may be performed by treating the obtained cyclodextrin glucanotransferase with Kpn I and Xho I. This can be done by inserting into one pYD1.

The first vector may also be used as a vector for preparing a transformed yeast for expressing cyclodextrin glucanotransferase on the surface of the yeast, but in order to express more stable and sustained enzyme, the first vector is foreign to the chromosome of the host organism. The base sequence for inserting the gene can be further inserted.

Step c) may be performed by amplifying the first vector, treating with a restriction enzyme, and inserting a nucleotide sequence for inserting a foreign gene into the chromosome of the host organism. Amplification of the first vector may be performed using a polymerase chain reaction, and the restriction enzyme may be treated with Sac I. A base sequence for inserting a foreign gene into the chromosome of the host organism, specifically, a base sequence for integrating the cyclodextrin glucanotransferase gene into the genome of the yeast seed, is integrated into the pδ Neo (integration) vector. Biotechol. Bioeng . 49, pp 45-51 (1996)) can be obtained by restriction enzyme treatment, which restriction enzyme may preferably be Sac I. In addition, primers for performing the polymerase chain reaction may be, for example, SEQ ID NO: 3 and SEQ ID NO: 4. The base sequence for inserting a foreign gene into the chromosome of the host organism may be an integration site, preferably a δ sequence, and more preferably pδNeo ( Biotechol . Bioeng . 49, pp45). -51 (1996)), and most preferably, may be a polynucleotide having a sequence of SEQ ID NO: 7.

The prepared vector for producing a transformed yeast may be, for example, pδCGT [3-18] -2 or pδCGT [3-18].

The present invention relates to a transformed yeast capable of inhibiting aging of starch by expressing cyclodextrin glucanotransferase on the surface.

The transformed yeast of the present invention can stably and continuously express cyclodextrin glucanotransferase on the surface, thereby inhibiting the aging of gelatinized starch, and therefore, when using the transformed yeast, the quality of food containing starch It has an excellent effect that can improve the, and extend the shelf life of the product.

The transformed yeast may be transformed by introducing a recombinant expression vector into a host organism. The recombinant expression vector may be a vector for preparing a transformed yeast comprising a cyclodextrin glucanotransferase, a promoter and an integration site, preferably in addition to pYD1 into which the cyclodextrin glucanotransferase is inserted. The base sequence for inserting the foreign gene into the chromosome of the host organism may be inserted, and the base sequence for inserting the foreign gene into the chromosome of the host organism may be pδNeo.

The recombinant expression vector is more preferably a cyclodextrin glucanotransferase gene that is effective in inhibiting aging of starch, a promoter operably linked to an upper portion of the cyclodextrin glucanotransferase gene, and the cyclodextrin glucanotransferase gene. Vector comprising an integration site, ie, an integration polynucleotide sequence, which integrates so as to be able to integrate into the genome of the yeast, and wherein the cyclodextrin glutatotransferase is expressed on the cell surface of the yeast. Or, for example, pδCGT [3-18] -2 or pδCGT [3-18].

The host organism may be a yeast, preferably Saccharomyces cerevisiae , Saccharomyces rose rosei ), Saccharomyces uvarum ), Saccharomyces chevalieri , Saccharomyces Torulaspora delbrueckii or Kluyveromyces thermotolerans ), and more preferably Saccharomyces cerbizi . For example, the host organism is Saccharomyces servizier EBY100 ( Saccharomyces cerevisiae EBY100).

Specifically, the transgenic yeast is Saccharomyces cerevisiae E. Saccharomyces cerevisiae PδCGT may be introduced into the chromosome of EBY100). The production of the transformed yeast can be carried out by treating pδCGT with Sal I and introducing the gene of EBY100 into Saccharomyces cerbiz (J. Agric. Food Chem. 55, pp 4735-4740 (2007). )). All content set forth in this document is incorporated herein by reference. The yeast transformed by introducing pδCGT to EBY100 in Saccharomyces cervage was YNB medium lacking tryptophan [0.67% yeast nitrogen base without amino acid (YNB; Sigma Aldrich, St. Louis, MO, USA), 0.19 % yeast synthetic drop-out medium supplement without tryptophan (YNB; Sigma Aldrich, St. Louis, MO, USA), 2% galactose, 1% starch, and 1.5% agar]. The selected transformed yeast was named S. cerevisiae / pδCGT. The S. cerevisiae / pδCGT was deposited at the Korea Research Institute of Bioscience and Biotechnology, located at 52, Eui-dong, Yuseong-gu, Daejeon, Korea on January 5, 2007, and received the accession number KCTC 11060BP on January 11, 2007.

The present invention relates to a starch-containing food dough prepared using yeast that stably and continuously expresses the cyclodextrin glucanotransferase effective on the aging of the starch. The starch-containing dough for food production preferably relates to bread dough or thickened dough.

The present invention also relates to a starch-containing food prepared using the starch-containing food preparation dough. The food containing starch may preferably be bread or thickening.

The present invention relates to a method for preparing starch-containing foods using yeast that stably and continuously expresses cyclodextrin glucanotransferase, which is effective in inhibiting aging of the starch. The food containing starch may preferably be bread or thickening.

The method for preparing a food containing starch comprises the steps of preparing a dough for preparing food containing starch and preparing a food containing starch. can do.

For example, the method of manufacturing the bread

a) preparing a bread dough; And

b) preparing bread by applying heat to the bread dough of step a)

It may be a bread manufacturing method comprising a.

Step a) may be prepared by mixing the flour, water containing starch powder, yeast expressing the cyclodextrin glucanotransferase stably and continuously on the surface, additional dough ingredients and additives. The grain flour may be preferably wheat flour.

The additional dough component may be one or more selected from the group consisting of sugar, salt, fat or oil, diet dietary ingredients, milk or milk powder, gluten and the like.

The additive may be at least one selected from the group consisting of emulsifiers, flavors, oxidizing agents, minerals, vitamins, hydrocolloids or enzymes.

The emulsifier is added to strengthen the dough and soften the bread, lecithin, polyethylene stearate, edible fatty acid monoglyceride or diglyceride, acetic acid ester of edible fatty acid, diacetyl tartaric acid ester of edible fatty acid, edible fatty acid Sucrose ester, sodium stearoyl-2-lactylate or calcium stearoyl-2-lactylate.

The enzyme may be an oxidoreductase such as glucose oxidase or ascorbate oxidase, a hydrolase such as lipase, a glucosidase such as alpha-amylase or an esterase.

The amount of yeast that stably and continuously expresses the cyclodextrin glucanotransferase according to the present invention on the surface is 0.5 g to 50 g, preferably 1 g to 30 g, more preferably 1 kg to 1 kg of the grain flour based on dry weight. Preferably from 5 g to 20 g, most preferably from 7.5 g to 12.5 g, specifically the amount of grain flour, water and yeast added is 30 ml to 80 ml, preferably based on 100 g of the grain flour. 50 g to 75 ml and yeast 0.5 g to 2 g, preferably 0.75 g to 1.25 g, more preferably 0.9 g to 1.1 g, which stably and continuously express the cyclodextrin glucanotransferase on the surface. .

The step b) may be performed by applying heat to the bread dough prepared in step a), and may be performed by applying heat to the bread dough using a conventional oven to prepare bread.

For example, a method of manufacturing bread by applying the heat, and after dividing the obtained bread dough, 10 ℃ to 40 ℃, preferably 18 ℃ to 30 ℃ and relative humidity 60% to 95%, preferably Primary fermentation under conditions of 70% to 90% for 1 hour to 10 hours, preferably 2 hours to 4 hours, and after molding, 15 ° C to 35 ° C, preferably 20 ° C to 30 ° C and 65% relative humidity. To secondary fermentation for 30 minutes to 5 hours, preferably 1 hour to 3 hours at a condition of from 95%, preferably 70% to 90%, and then 150 ° C to 250 ° C, preferably 170 ° C to 230 ° C, More preferably, it can be prepared by baking in an oven at 170 ° C. to 230 ° C. for 10 minutes to 180 minutes, preferably 30 minutes to 90 minutes.

For example, the preparation of the thickener of the present invention is to produce a thickening dough by adding grain flour, dough components and yeast or additional additives that stably and continuously express the cyclodextrin glucanotransferase on the surface, and the thickening dough After fermentation, it may be molded and then streamed.

The grain flour may preferably be rice flour, and the additional dough component may be sugar, salt, diet dietary ingredients, and the like. In addition, the additive may be at least one selected from the group consisting of emulsifiers, flavors, oxidizing agents, mummies, vitamins, hydrocolloids or enzymes.

Fermentation of the thickening dough may be carried out by a method of fermentation for 1 hour to 10 hours, preferably 2 hours to 5 hours at 20 ℃ to 50 ℃, preferably 30 ℃ to 40 ℃ conditions.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

< Example >

< Example  1> Preparation of Recombinant Yeast

In order to prepare a recombinant yeast that stably and continuously expresses the cyclodextrin glucanotransferase on the surface, a site having a nucleotide sequence including a cyclodextrin glucanotransferase, a promoter and an integration site may be inserted, and the recombinant Yeast manufacturing method is the same as FIG.

The manufacturing method of FIG. 1 is as follows.

Example  1-1 Cyclodextrin Glucanotransferase  purchase

Cyclo dextrin glue Kano in order to obtain a transfer-raised, pR ₂ CGT [3-18] which includes the gene encoding the CGTase [3-18] (Protein Eng . Des . Sel . , 17, pp 205-211 (2004)) was used as a template to conduct a polymerase chain reaction. The polymerase chain reaction was performed using the primers of Table 1, PCR was carried out in the composition ratio of the PCR composition of Table 2 and the PCR conditions of Table 3 below. The primers of SEQ ID NO: 1 and SEQ ID NO: 2 of Table 1 have restriction enzyme cleavage sites of restriction enzymes Kpn I or Xho I. The polymerase (ExTaq polymerase) described in Table 2 below was purchased from Takara (Japan) and used.

order SEQ ID NO: Forward primer KpnI-N 5'-CACCATCACCAGGTACCTATGAGGAAAGAAGCC-3 ' One Reverse primer XhoI-C 5'-GTAACAAGCGGC CTCGAGCCTCTACCCGGAGCC-3 2

Example  1-2 Preparation of Recombinant Vectors and Recombinant Yeast

To prepare the recombinant vector, the cyclodextrin glucanotransferase obtained in Example 1-1 was recombined with a commercially available surface expression vector pYD1 (Invitrogen, Carlsbad, CA, USA) to obtain pYCGT [3-18]. Prepared. More specifically, the restriction enzyme the vector pYD1 Kpn I (Takara, Japan) and Xho I (Takara, Japan) to the process, and the restriction enzyme treated vector pYD1 a cycle dextrin glue obtained in the above Examples 1-1 Kano The transferase was inserted to prepare recombinant vector pYCGT [3-18].

For more stable and sustained expression of enzymes, the integration between the GAL1 promoter and the TRP1 gene region, which is a key site related to the surface expression of pYCGT [3-18], is not used directly, and the recombinant vector pYCGT [3-18] is used. ) PδNeo ( Biotechol. Bioeng . 49, pp 45-51 (1996)) was inserted to complete pδCGT.

Specifically, in order to obtain the cyclodextrin glucanotransferase and the GAL1 promoter region of pYCGT [3-18], pYCGT [3-18] was used as a template, and the primers of SEQ ID NO: 3 and SEQ ID NO: 4 of Table 4 below were used. The polymerase chain reaction was carried out using the composition ratio of the PCR composition of Table 2 and the PCR conditions of Table 3. However, unlike Table 2, primers of SEQ ID NO: 3 and SEQ ID NO: 4 were used instead of the primers of SEQ ID NO: 1 and SEQ ID NO: 2. The primers of SEQ ID NO: 3 and the primers of SEQ ID NO: 4 of the following Table 4 have restriction enzyme cleavage sites of restriction enzyme Sac I.

The integration vector of 4.2 kb of pYCGT [3-18] cyclodextrin glucanotransferase and GAL1 promoter site obtained by the polymerase chain reaction was treated with restriction enzyme Sac I (Takara, Japan). The recombinant vector was prepared by inserting into pδNeo, and the recombinant vector prepared by the above method was named pδCGT.

order SEQ ID NO: Forward Primer Int-F 5'- ACCCTCACTAAAGAGCTCAAAAGCTGGCTAG-3 ' 3 Reverse Primer Int-R 5'- GTGATTAAGCACACAGAGCTCGCTTGGAGTATG-3 4

On the other hand, in order to confirm the usefulness of the yeast transformed with the recombinant vector of the present invention, a recombinant vector was prepared that does not contain cyclodextrin glucanotransferase as a control example, unlike the present invention. Specifically, unlike pδCGT, except that a 2.1 kb DNA fragment was obtained by using pYD1 as a template DNA and performing a polymerase chain reaction, the recombinant vector prepared by the above method was prepared. Was designated as pδYD1.

The recombinant vectors pδCGT and pδYD1 prepared above were transformed into E. coli DH5α, and LB medium (1% bacto-tryptone, 0.5% yeast extract, added with ampicillin (100 μg / ml) at 37 ° C. After incubation in 0.5% NaCl, yeast were transformed using an alkali-cation kit (Bio101, USA) according to the manufacturer's instructions (manufacurer's protocol). S. cerevisiae EBY100 (Invitrogen, USA) was used as a host organism to be transformed to produce the recombinant yeast. In order to screen the yeast stably expressing the vector among the transformed yeast, YNB medium lacking tryptophan [0.67% yeast nitrogen base without amino acid (YNB; Sigma Aldrich, St. Louis, MO, USA), 0.19 % yeast synthetic drop-out medium supplement without tryptophan (YNB; Sigma Aldrich, St. Louis, MO, USA), 2% galactose, 1% starch, and 1.5% agar]. Among the yeast confirmed that the cyclodextrin glucanotransferase was stably cultured, was selected as S. cerevisiae / pδCGT, and as a control, the yeast identified as transformed with the recombinant vector was selected as S. cerevisiae / pδYD1. Named

In order to confirm the effect of the recombinant yeast transformed with the recombinant vector, the recombinant yeast cultured in YNB medium was shaken at 200 rpm and 200 rpm in YPG medium (1% yeast extract, 2% peptone and 2% galactose) Incubated.

< Example  2> of recombinant yeast Cold thaw  stability

In order to confirm the freeze thaw stability of the recombinant yeast, by measuring the number of yeast cells at a wavelength of 600 nm using an absorbometer, the same amount of S. cerevisiae EBY100 and the transformant S. cerevisiae / pδCGT prepared in Example 1 Were each stored frozen at -20 ° C for 12 hours and then thawed at 27 ° C for 1 hour. After diluting the thawed cells by 10 ^-7 times, incubated in YPD solid medium (2% peptone, 1% yeast extract, 2% glucose, and 1% agar) for 60 hours at 30 ℃ to determine the percentage of viable cells It confirmed, and the result is shown in FIG.

As shown in FIG. 3, S. cerevisiae / pδCGT showed 98% viability during cold thaw four times, but wild type S. cerevisiae EBY100 showed 78% viability. Yeast was found to be very good in freeze thaw stability.

< Example  3> Preparation of Bread and Jeungpyun Using Recombinant Yeast

3-1 Manufacturing of Bread

In order to confirm the anti-aging effect of the recombinant yeast, bread was prepared using the recombinant yeast. More specifically, 247 g of white pan bread Mix II (CJ, South Korea) and 173 ml of water are mixed, and 2.5 g of the recombinant yeast is added based on dry weight, and then to the baking machine (SHB-600). , Samsung Co., South Korea) was prepared.

If the experiment example, in which the recombinant yeast cyclo dextrin glue transfer Kano were raised the use of the S. cerevisiae / pδCGT expressed on the surface, for example the control transformed pδYD1 which are not containing the cyclo dextrin glue transfer Kano raised to the recombinant yeast S. cerevisiae / pδYD1 was used, and the ratio of each volume increase was compared, and the results are shown in FIG. 4.

Wherein as shown in FIG. 4, cyclo dextrin glue Kano transfer case is raised by the S. cerevisiae / pδCGT expressed on the surface of S. was transformed pδYD1 which are not containing the cyclo dextrin glue transfer Kano raised to cerevisiae recombinant yeast / Compared with the case of using pδYD1, the volume ratio was confirmed to increase by about 25%.

From the above results, when using S. cerevisiae / pδCGT expressing cyclodextrin glucanotransferase on the surface, it was confirmed that starch inhibits the aging of starch as in the case of adding an anti-aging enzyme through an additional process. It became.

3-2 Preparation of enlargement

In order to confirm the anti-aging effect of the recombinant yeast, Jeungpyun was prepared using the recombinant yeast. More specifically, 100 g of rice flour, 15 g of sugar, 1 g of salt, and recombinant yeast were mixed with 1.5 g based on dry weight, and then fermented at 35 ° C. for 4 hours. 25 mm, height 30 mm) was streamed for 30 minutes.

In the experimental example, S. cerevisiae / pδCGT was used as the recombinant yeast, and S. cerevisiae / pδYD1 was used as the recombinant yeast, and the ratio of the volume increase was compared, and the results are shown in FIG. 5.

Wherein as shown in FIG. 5, cyclo dextrin glue Kano transfer case is raised by the S. cerevisiae / pδCGT expressed on the surface of S. was transformed pδYD1 which are not containing the cyclo dextrin glue transfer Kano raised to cerevisiae recombinant yeast / Compared to the case of using pδYD1, the volume ratio was found to increase by 45%.

< Example  4> Anti-aging Effect of Bread and Jeungpyun Prepared Using Recombinant Yeast

Aging degree was measured using a differential scanning calorimeter to measure the aging inhibitory effect of bread and Jeungpyun prepared using recombinant yeast.

Specifically, the bread prepared in Example 3-1 and the thickener prepared in Example 3-2 were each packed in a plastic pack and refrigerated at 4 ° C., while 10 mg of each sample was stored at 20 ° C. to 130 ° C. The aging degree of starch was measured using a differential scanning caloriemeter (DSC) at a rate of 5 ° C. per minute in the range, the aging degree of bread is shown in FIG. 6, and the aging degree of thickening is shown in FIG. 7.

As shown in FIG. 6, the aging degree of bread was measured at 3 and 7 days after cold storage, and when S. cerevisiae / pδCGT expressing cyclodextrin glucanotransferase was expressed on the surface. Showed a significant decrease in aging compared to the case of using S. cerevisiae / pδYD1 transformed with pδYD1 without the cyclodextrin glucanotransferase with recombinant yeast, and 50% compared to the control at 7 days. When the bread is produced with the recombinant yeast of the present invention showing the following degree of aging, it was confirmed that there is a significant aging reduction effect.

In addition, as shown in FIG. 7, the degree of aging of the enlargement was carried out after 12 hours and 24 hours after refrigerated storage, and compared to the case of using S. cerevisiae / pδYD1 as a control example, cyclodextrin glucanotransfer. In the present invention using the S. cerevisiae / pδCGT expressed on the surface, there was a marked decrease in aging, and after 24 hours, the aging degree was less than 30% compared to the control, especially, 12 of the control. It was confirmed that the aging degree at the time of 24 hours of the present invention is lower than the aging degree at the time elapsed time, and when producing the enlargement with the recombinant yeast of the present invention, it was confirmed that there is a significant aging reduction effect.

< Example  5> of bread made using recombinant yeast Small sugars  analysis

10 g of the bread prepared using S. cerevisiae / pδCGT of Example 3 was added to 100 ml of distilled water and stirred at room temperature for 1 hour, followed by centrifugation for 10 minutes under conditions of 7,000 xg, followed by centrifugation. The obtained supernatant was filtered with 0.45 μm filtration membrane, and then the small sugars were analyzed by high performance liquid chromatography (HPLC, SLC-100, Samsung Electronics Inc., Korea), and the results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that glucose, maltose, and maltotriose were mainly present in the bread of Example 3.

From the above results, it was confirmed that the recombinant yeast of the present invention can stably and continuously express cyclodextrin glucanotransferase on its surface, thereby producing glucose and the like, and further, in the process of manufacturing bread using the recombinant yeast. Since cyclodextrins, which are not allowed to be used as additives in Europe and the like, were not generated, it was confirmed that the recombinant yeast could be widely used in the industry, particularly in the food industry.

Recombinant yeast of the present invention is excellent in freezing thaw stability compared to wild-type yeast, the yeast survivability of the frozen or refrigerated dough containing the starch added yeast, the volume increase ratio of the product produced compared to wild-type yeast In addition, in the case of bread or Jeungpyun manufactured using recombinant yeast, cyclodextrin glucanotransferase is stably and continuously expressed on the surface of the recombinant yeast of the present invention, so processing starch material such as bread or Jeungpung containing starch By suppressing the aging of food to improve the quality of the food containing starch, and the shelf life of the product can be extended, its usefulness in the industrial, in particular with respect to the food industry.

<110> SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION <120> YEAST EXPRESSING CYCLODEXTRIN GLUCANOTRANSFERASE AND METHOD FOR          USING THE SAME <130> dpp20064534kr <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> KpnI-N for <400> 1 caccatcacc aggtacctat gaggaaagaa gcc 33 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> XhoI-C re <400> 2 gtaacaagcg gcctcgagcc tctacccgga gcc 33 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Int-F <400> 3 accctcacta aagagctcaa aagctggcta g 31 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Int-R <400> 4 gtgattaagc acacagagct cgcttggagt atg 33 <210> 5 <211> 2055 <212> DNA <213> Artificial Sequence <220> <223> CGT [3-18] -2 <220> <221> CDS (222) (1) .. (2025) <400> 5 gcc ccg gat acc tcg gta tcc aac aag cag aat ttc agc acg gat gtc 48 Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val   1 5 10 15 ata tat cag atc ttc acc gac cgg ttc tcg gac ggc aat ccg gcc aac 96 Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn              20 25 30 aat ccg acc ggc gcg gca ttt gac gga tca tgt acg aat ctt cgc tta 144 Asn Pro Thr Gly Ala Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu          35 40 45 tac tgc ggc ggc gac tgg caa ggc atc atc aac aaa atc aac gac ggt 192 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly      50 55 60 tat ttg acc ggc atg ggc att acg gcc atc tgg att tca cag cct gtc 240 Tyr Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val  65 70 75 80 gag aat atc tac agc gtg atc aac tac tcc ggc gtc cat aat acg gct 288 Glu Asn Ile Tyr Ser Val Ile Asn Tyr Ser Gly Val His Asn Thr Ala                  85 90 95 tat cac ggc tac tgg gcg cgg gac ttc aag aag acc aat ccg gcc tac 336 Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr             100 105 110 gga acg atg cag gac ttc aaa aac ctg atc gac acc gcg cat gcg cat 384 Gly Thr Met Gln Asp Phe Lys Asn Leu Ile Asp Thr Ala His Ala His         115 120 125 aac ata aaa gtc atc atc gac ttt gca ccg aac cat aca tct ccg gct 432 Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala     130 135 140 tct tcg gat gat cct tcc ttt gca gag aac ggc cgc ttg tac gat aac 480 Ser Ser Asp Asp Pro Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn 145 150 155 160 ggc aac ctg ctc ggc gga tac acc aac gat acc caa aat ctg ttc cac 528 Gly Asn Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His                 165 170 175 cat tat ggc ggc acg gat ttc tcc acc att gag aac ggc att tat aaa 576 His Tyr Gly Gly Thr Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys             180 185 190 aac ctg tac gat ctg gct gac ctg aat cat aac aac ag agc agc gtc gat 624 Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp         195 200 205 gtg tat ctg aag gat gcc atc aaa atg tgg ctc gac ctc ggg gtt gac 672 Val Tyr Leu Lys Asp Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp     210 215 220 ggc att cgc gtg gac gcg gtc aag cat acg cca ttc ggc tgg cag aag 720 Gly Ile Arg Val Asp Ala Val Lys His Thr Pro Phe Gly Trp Gln Lys 225 230 235 240 agc ttt atg tcc act att aac aac tac aag ccg gtc ttc acc att ggc 768 Ser Phe Met Ser Thr Ile Asn Asn Tyr Lys Pro Val Phe Thr Ile Gly                 245 250 255 gaa tgg atc ctt ggc gtc aat gag att agt ccg gaa tac cat caa ttc 816 Glu Trp Ile Leu Gly Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe             260 265 270 gct aac gag tcc ggg atg agc ctg ctc gat ttc cgc ttt gcc cag aag 864 Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys         275 280 285 gcc cgg caa gtg ttc agg gac aac acc gac aat atg tac ggc ctg aaa 912 Ala Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys     290 295 300 gcg atg ctg gag ggc tct gaa gta gac tat gcc cag gtg aat gac cag 960 Ala Met Leu Glu Gly Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln 305 310 315 320 gtg acc ttc atc gac aat cat gac atg gag cgt ttc cac acc agc aat 1008 Val Thr Phe Ile Asp Asn His Asp Met Glu Arg Phe His Thr Ser Asn                 325 330 335 ggc gac aga cgg aag ctg gag cag gcg ctg gcc ttt acc ctg act tca 1056 Gly Asp Arg Arg Lys Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser             340 345 350 cgc ggt gtg cct gcc atc tat tac ggc agc gag cag tat atg tct ggc 1104 Arg Gly Val Pro Ala Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly         355 360 365 ggg aat gat ccg gac aac cgt gct cgg att cct tcc ttc tcc acg acg 1152 Gly Asn Asp Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Thr     370 375 380 acg acc gca tat caa gtc atc caa aag ctc gct ccg ctc cgc aaa tcc 1200 Thr Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser 385 390 395 400 aac ccg gcc atc gct tac ggt tcc aca cag gag cgc tgg atc aac aac 1248 Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn                 405 410 415 gat gtg atc atc tat gaa cgc aaa ttc ggc aat aac gtg gcc gtt gtt 1296 Asp Val Ile Ile Tyr Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val             420 425 430 gcc att aac cgc aat atg aac aca ccg gct tcg att acc ggc ctt gtc 1344 Ala Ile Asn Arg Asn Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val         435 440 445 act tcc ctc ccg cag ggc agc tat aac gat gtg ctc ggc gga att ctg 1392 Thr Ser Leu Pro Gln Gly Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu     450 455 460 aac ggc aat acg cta acc gtg ggt gct ggc ggt gca gct tcc aac ttt 1440 Asn Gly Asn Thr Leu Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe 465 470 475 480 act ttg gct cct ggc ggc act gct gta tgg cag tac aca acc gat gcc 1488 Thr Leu Ala Pro Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala                 485 490 495 aca gct ccg atc atc ggc aat gtc ggc ccg atg atg gcc aag cca ggg 1536 Thr Ala Pro Ile Ile Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly             500 505 510 gtc acg att acg att gac ggc cgc gct tcg gct cgg caa gga acg gtt 1584 Val Thr Ile Thr Ile Asp Gly Arg Ala Ser Ala Arg Gln Gly Thr Val         515 520 525 tac ttc ggt aca acg gca gtc act ggc gcg gac atc gta gct tgg gaa 1632 Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu     530 535 540 gat aca caa atc cag gtg aaa atc ctg cgg gtc cct ggc ggc atc tat 1680 Asp Thr Gln Ile Gln Val Lys Ile Leu Arg Val Pro Gly Gly Ile Tyr 545 550 555 560 gat atc aga gtt gcc aac gca gcc gga gca gcc agc aac atc tac gac 1728 Asp Ile Arg Val Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp                 565 570 575 aat ttc gag gtg ctg acc gga gac cag gtc acc gtt cgg ttc gca atc 1776 Asn Phe Glu Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe Ala Ile             580 585 590 aac aat gcc aca acg gcg ctg gga cag aat gtg ttc ctc acg ggc aat 1824 Asn Asn Ala Thr Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn         595 600 605 gtc agc gag ctg ggc aac tgg gat ccg aac aac gcg atc ggc ccg atg 1872 Val Ser Glu Leu Gly Asn Trp Asp Pro Asn Asn Ala Ile Gly Pro Met     610 615 620 tat aat cag gtc gtc tac caa tac ccg act tgg tat tat gat gtc agc 1920 Tyr Asn Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser 625 630 635 640 gtt ccg gca ggc caa acg att gaa ttt aaa ttc ctg aaa aag caa ggc 1968 Val Pro Ala Gly Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly                 645 650 655 tcc acc gtc aca tgg gaa ggc ggc gcg aat cgc acc ttc acc acc cca 2016 Ser Thr Val Thr Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro             660 665 670 acc agc ggc acggc aacggtgaat gtgaactggc agcct 2055 Thr Ser Gly         675 <210> 6 <211> 675 <212> PRT <213> Artificial Sequence <400> 6 Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val   1 5 10 15 Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn              20 25 30 Asn Pro Thr Gly Ala Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu          35 40 45 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly      50 55 60 Tyr Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val  65 70 75 80 Glu Asn Ile Tyr Ser Val Ile Asn Tyr Ser Gly Val His Asn Thr Ala                  85 90 95 Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr             100 105 110 Gly Thr Met Gln Asp Phe Lys Asn Leu Ile Asp Thr Ala His Ala His         115 120 125 Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala     130 135 140 Ser Ser Asp Asp Pro Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn 145 150 155 160 Gly Asn Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His                 165 170 175 His Tyr Gly Gly Thr Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys             180 185 190 Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp         195 200 205 Val Tyr Leu Lys Asp Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp     210 215 220 Gly Ile Arg Val Asp Ala Val Lys His Thr Pro Phe Gly Trp Gln Lys 225 230 235 240 Ser Phe Met Ser Thr Ile Asn Asn Tyr Lys Pro Val Phe Thr Ile Gly                 245 250 255 Glu Trp Ile Leu Gly Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe             260 265 270 Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys         275 280 285 Ala Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys     290 295 300 Ala Met Leu Glu Gly Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln 305 310 315 320 Val Thr Phe Ile Asp Asn His Asp Met Glu Arg Phe His Thr Ser Asn                 325 330 335 Gly Asp Arg Arg Lys Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser             340 345 350 Arg Gly Val Pro Ala Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly         355 360 365 Gly Asn Asp Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Thr     370 375 380 Thr Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser 385 390 395 400 Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn                 405 410 415 Asp Val Ile Ile Tyr Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val             420 425 430 Ala Ile Asn Arg Asn Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val         435 440 445 Thr Ser Leu Pro Gln Gly Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu     450 455 460 Asn Gly Asn Thr Leu Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe 465 470 475 480 Thr Leu Ala Pro Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala                 485 490 495 Thr Ala Pro Ile Ile Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly             500 505 510 Val Thr Ile Thr Ile Asp Gly Arg Ala Ser Ala Arg Gln Gly Thr Val         515 520 525 Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu     530 535 540 Asp Thr Gln Ile Gln Val Lys Ile Leu Arg Val Pro Gly Gly Ile Tyr 545 550 555 560 Asp Ile Arg Val Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp                 565 570 575 Asn Phe Glu Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe Ala Ile             580 585 590 Asn Asn Ala Thr Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn         595 600 605 Val Ser Glu Leu Gly Asn Trp Asp Pro Asn Asn Ala Ile Gly Pro Met     610 615 620 Tyr Asn Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser 625 630 635 640 Val Pro Ala Gly Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly                 645 650 655 Ser Thr Val Thr Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro             660 665 670 Thr Ser Gly         675 <210> 7 <211> 543 <212> DNA <213> Artificial Sequence <220> <223> delta sequence <400> 7 ggatccgcct acctttcacg agttgcgcag tttgtctgca agactctatg agaagcagat 60 aagcgataag tttgctcaac atcttctcgg gcataagtcg gacaccatgg catcacagta 120 tcgtgatgac agaggcaggg agtgggacaa aattgaaatc aaataatgat tttattttga 180 ctgatagtga cctgttcgtt gcaacaaatt gataagcaat gctttttttg agaaatgggt 240 gaatgttgag ataattgttg ggattccatt gttgataaag gctataatat taggtatcgg 300 tcgaccgata cagaatatac tagaagttct cctcaaggat ttaggaatcc ataaaaggga 360 atctgcaatt ctacacaatt ctataaatat tattatcatc attttatatg tttatattca 420 ttgatcctat tacattatca atccttgcgt ttcagcttcc actaatttag atgactattt 480 ctcatcattt gcgtcatctt ctaacaccgt atatgataat atactagtaa tgtaaatact 540 agt 543  

Claims (8)

Cyclodextrin glucanotransferase gene; A promoter operably linked to the top of the cyclodextrin glucanotransferase gene; And The delta sequence sequence, which is an integration site for integrating the cyclodextrin glucanotransferase gene into the yeast genome, The yeast cell expression vector, wherein the cyclodextrin glucanotransferase is expressed on the cell surface of the yeast. The method of claim 1, The cyclodextrin glucanotransferase gene is a polynucleotide encoding the protein of SEQ ID NO: 6, wherein the integration site (integration site) having a δ sequence sequence of SEQ ID NO: 7.  The method of claim 1, The yeast cell expression vector adds one or more selective markers selected from the group consisting of ampicillin resistance gene (Amp (r)) and neomycin resistance gene (Neo (r)). Yeast cell expression vector comprising a. A transformed yeast characterized by transforming with the yeast cell expression vector according to claim 1 to express the cyclodextrin glucanotransferase on the cell surface of the yeast. The method of claim 4, wherein The yeast cell expression vector adds one or more selective markers selected from the group consisting of Ampicillin resistance gene (Amp (r)) and Neomycin resistance gene (Neo (r)). It is to include a transgenic yeast. The method according to claim 4 or 5, The yeast is transformed yeast Saccharomyces cerevisiae ( Saccharomyces cerevisiae ). The method of claim 6, The yeast is transformed yeast Saccharomyces cerevisiae KCTC 11060BP to Saccharomyces cerbiz. Food containing starch prepared using the yeast according to any one of claims 4 to 6.

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