KR20050041069A - NOVEL THERMOSTABLE α-GLUCANOTRANSFERASE AND USE THEREOF - Google Patents

Abstract

 The present invention relates to novel heat resistant alpha-glucanotransferases and uses thereof. In particular, the present invention can produce a customized starch, the length of the side chain of the starch is adjusted, and provides a heat-resistant alpha-glucanotransferase that has a remarkable effect on improving the quality of food and extending shelf life.

Description

New heat resistant alpha-glucanotransferases and uses thereof {NOVEL THERMOSTABLE α-GLUCANOTRANSFERASE AND USE THEREOF}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a novel heat-resistant alpha-glucanotransferase and its use, and more particularly to the production of tailored starch, the length of the side chain of the starch can be produced, has a significant effect on improving the quality of food and extending shelf life Heat-resistant alpha-glucanotransferase and its use.

[Private Technology]

Rice starch (70% of brown rice), which accounts for most of the rice, is composed of linear amylose and branched amylopectin. The ratio of amylose and amylopectin, which form rice, is an important factor in determining the stickiness and flavor of heated cooked rice products. For example, non-glutinous rice (10-15%) from low-amylose-derived products is considered to be of superior quality and shelf life compared to non-glutinous rice (17-23%) (N. Inouchi, Food Storage Division Processing industry, 1, 17-25, 2002).

In general, when storing food containing gelatinized starch, recrystallization due to the interaction of amylose and amylopectin, the quality of the product is greatly degraded. As a way to suppress the aging phenomenon, additives such as lipids and fatty acids The method of using and the method of changing the structure of starch itself using amylase are used.

Among the above methods, alpha amylase is used as an antioxidant for starch processed foods such as bread by a method using amylase. However, alpha amylase may weaken the properties of the product even with a slight overuse and produce a large amount of reducing sugars, causing undesired reactions such as Maillard reactions. In addition, rice starch has a higher gelatinization temperature than other starches (68-78 ℃), especially starch particles in rice grains are very dense and firmly attached, which is difficult to process or takes a long time at temperatures lower than gelatinization temperature. There is difficulty. Therefore, for the enzymatic treatment of rice processed foods, development of heat-resistant enzymes with high activity even at temperatures higher than the gelatinization temperature of rice starch is required.

On the other hand, alpha-glucanotransferase is an enzyme belonging to group 13 of alpha amylase and is called D-enzyme in plants such as amylomaltease and potato in E. coli . Alpha glucanotransferase was first cloned in E. coli and is now cloned in potatoes, sweet potatoes, barley and some microorganisms. Alpha-glucanotransferase is an enzyme that transfers glucose units from the non-reducing end of the donor molecule to the non-reducing end of the donor molecule. It is an enzyme that transfers sugars to α-1,4 bonds and reconstitutes amylopectin branches. To date, no application of alpha-glucanotransferase to food has been reported.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the problems of the prior art, and an object of the present invention is to provide a novel heat resistant alpha-glucanotransferase.

It is another object of the present invention to provide a novel heat resistant alpha-glucanotransferase gene.

It is another object of the present invention to provide a heat resistant alpha-glucanotransferase expression system.

It is another object of the present invention to provide a food processing method using heat-resistant alpha-glucanotransferase.

It is another object of the present invention to provide a food processed by heat-resistant alpha-glucanotransferase.

In addition, an object of the present invention is to provide a food having improved physical properties and delayed aging by heat-resistant alpha-glucanotransferase treatment.

In order to achieve the above object, the present invention provides a heat-resistant alpha glucanotransferase consisting of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

The present invention also provides a polynucleotide comprising a nucleic acid sequence encoding the peptide.

The present invention also provides a vector comprising the polynucleotide to be expressed.

The present invention also provides a cell line transformed with the vector comprising a polynucleotide to be expressed.

The present invention also provides a heat resistant alpha-glucanotransferase produced from the transformed cell line.

In another aspect, the present invention provides a method for producing a processed food alpha-glucanotransferase treatment comprising the addition of the heat-resistant alpha-glucanotransferase to starch, starch-containing grains or foods.

In another aspect, the present invention provides a processed food prepared by the processed food manufacturing method.

Hereinafter, the present invention will be described in detail.

We cloned a novel alpha-glucanotransferase with thermostability to analyze its gene nucleic acid sequence and protein amino acid sequence, and established an alpha-glucanotransferase production system. In addition, it was confirmed that the treatment of alpha-glucanotransferase in food has the effect of improving food quality and delaying food aging.

The heat resistant alpha-glucanotransferase of the present invention is a protein derived from Thermus scotoductus ATCC 27978, which consists of 500 amino acids and has a molecular weight of about 57 kDa. Alpha-glucanotransferases consist of a peptide comprising the amino acid sequence of SEQ ID NO: 4.

Heat-resistant alpha-glucanotransferases have a hydrolysis ability to hydrolyze long branches of amylose and amylopectin, and at the same time disproportionate to reconstruct amylopectin branches by transferring the hydrolyzed low molecule glucan to α-1,4 bonds. (disproportionation) has activity. In particular, the enzyme has a good hydrolytic ability to amylose, while the hydrolysis to amylopectin is limited compared to conventional alpha-amylase, so the probability of food or product thinning is low. Alpha-glucanotransferase also removes reducing sugars and reconstitutes amylopectin chains. The enzyme has an activity at room temperature or a high temperature of more than room temperature, particularly exhibits the activity at 45 to 90 ℃, pH range having an enzyme activity may be 5.5 to 8.5. Enzyme reaction time can be appropriately adjusted according to the reaction temperature, the reaction pH or enzyme titer of the enzyme, for example, may be 30 minutes to 12 hours, but the reaction is also made in more than 12 hours is not limited to the numerical limitation. The enzyme preferably shows optimum activity at a reaction temperature of 70 to 85 ° C. and a pH range of 7.0 to 7.5.

The heat resistant alpha-glucanotransferase gene of the present invention consists of a polynucleotide comprising a nucleic acid sequence encoding a peptide of SEQ ID NO: 4. An example of a nucleic acid sequence encoding the peptide of SEQ ID NO: 4 is a nucleic acid sequence set forth in SEQ ID NO: 3.

The heat resistant alpha-glucanotransferase of the present invention can be produced through a microbial expression production system. Accordingly, the present invention provides a recombinant vector in which a heat resistant alpha-glucanotransferase gene is inserted to be expressible in a known vector. The known vector may be any kind of vector proposed for expressing heterologous proteins in microorganisms, in particular yeast, Escherichia coli or fungi, and may further comprise means for facilitating the separation of selection marks, reporter genes or recombinant proteins. Can be. The selection mark may be an antibiotic resistance gene or a gene related to oxotropy. For example, p6xHis119 (Kim Tae-kip, Ph.D. dissertation, Seoul National University, 1998), pUC119 and pET29 (b).

In one embodiment, p6xHisTSαGT was prepared by inserting the nucleic acid sequence of SEQ ID NO: 3 into the Nde I and Hind III restriction sites of the p6xHis119 vector (FIG. 1).

The present invention also provides a cell line transformed with a heat resistant alpha-glucanotransferase gene. The transformed cell line introduces a vector containing a heat resistant alpha-glucanotransferase gene into a host cell. The host cell may be a conventional prokaryote, eukaryote, or a cell derived therefrom, for example, E. coli, yeast or fungus. In one embodiment of the present invention, p6xHisTSαGT was transformed by introducing Escherichia coli MC1061.

Alpha-glucanotransferase recombinant protein expressed from the transformed cell line according to the present invention can be expressed extracellularly or intracellularly, and can be easily isolated and purified through centrifugation or chromatography. For example, Escherichia coli MC1061 transformed with p6xHisTSαGT can be cultured on LB medium, and intracellularly expressed alpha-glucanotransferase recombinant protein can be purified by cell harvest, cell hemolysis, lysis, supernatant separation and chromatography. Do. In particular, p6xHisTSαGT is expressed in a state in which 6 histidines are conjugated to an alpha-glucanotransferase recombinant protein, and thus can be easily purified using a nickel hydrophilic column. Alpha-glucanotransferases expressed from the transformed cell lines disproportionate maltooligosaccharides to produce long linear maltooligosaccharides (FIG. 2).

Alpha-glucanotransferase expressed from the transformed cell line according to the present invention can be used for the purpose of treating food to improve the quality of food or improve food shelf life.

That is, processed foods may be prepared by treating alpha-glucanotransferase with 15 to 200 U based on starch content in starch, grains or foods containing starch. In this case, the reaction temperature may be a temperature range in which alpha-glucanotransferase exhibits activity, and the reaction time may be appropriately adjusted according to the type of food or the degree of modification of the desired starch, and also the hydrolysis and disprophylization of starch by enzymes. If the reaction is to be stopped, the reaction may be stopped by applying heat of 100 ° C. or more.

The grains that can be processed with alpha-glucanotransferases include rice, glutinous rice, potatoes, wheat, corn, and soybeans. Examples of foods include rice cooked rice, gimbap, sushi, rice drinks, rice noodles, rice cakes, tteokbokki, snow white, bread, and biscuits.

In one embodiment of the present invention, the starch is treated with alpha-glucanotransferase to reconstruct the length of the starch side chain, that is, the number of long side chains is reduced and the number of short side chains is increased. It could be prepared (FIG. 4). In addition, rice flour was treated with alpha-glucanotransferase or cooked at the time of preparing rice cake to confirm the change in food quality, shelf life and palatability. As a result, the aging of barley rice cake was extended and the palatability was improved (FIG. 5), the barley rice cake tissue was converted to a dense network structure (FIGS. 6A and 6B), and the cooking taste was also effective in increasing shelf life and palatability.

Thus, alpha-glucanotransferase according to the present invention can improve the quality and flavor of starch-containing food. In addition, alpha-glucanotransferase is stable at high temperatures and can be easily used in food prepared by applying heat.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1 Isolation of Alpha Glucanotransferase Gene from Summers Scothoductus

Thermos scotoductus ATCC 27978 was castenholz TYE medium (0.02% nitrilotriacetic acid, 0.2% Nitschs's trace elements, 0.006% FeCl ₃ solution, 0.012% CaSO ₄ , 0.02% mgSO ₄ , NaCl, KNO ₃ , NaNO ₃ , Na ₂ HPO ₄ , 3% agar, 0.2% trypton and 0.2% yeast extract) were incubated at 70 ℃ and centrifuged to recover the cells. The chromosomal DNA was then isolated from the cells based on known methods (Dubnau, D. and RD Abenson (J. Mol. Biol., 56, 209-221, 1971).

Chromosomal DNA of Summers Scothoductus was cut with Hind III, a restriction enzyme, electrophoresed on agarose gel, and suddenly blown using a probe of SEQ ID NO: 5 containing the homologous region of a known alpha-glucanotransferase. (Molecular cloning, Cold Spring Harbor Laboratory Press, New York, 1989). As a result of sudden blotting, a fragment containing a 3.2 kb gene was obtained and inserted into the p119 vector to prepare a recombinant vector.

In order to clone the open reading frame of alpha-glucanotransferase from the recombinant vector, PCR was performed with primer sets of SEQ ID NOs: 1 and 2, and the PCR product was limited to Nde I and Hind III of the p6xHis119 vector. Inserted into the enzyme site to prepare a recombinant vector p6xHisTSαGT.

The p6xHisTSαGT vector map of the present invention is shown briefly in FIG. The alpha-glucanotransferase gene cloned into p6xHisTSαGT contains ATG as start codon and TAA as stop codon and consists of a total of 1,503 nucleotides as described in SEQ ID NO: 3. The protein expressed therefrom was composed of 500 amino acids (SEQ ID NO: 4).

Example 2: Alpha-Glucanotransferase Production and Its Activity Assay

2-1. Alpha-Glucanotransferase Production

P6xHisTSαGT prepared in Example 1 was transformed into Escherichia coli MC1061, inoculated in LB medium, cultured at 37 ° C for 16 hours, and centrifuged to obtain cells. The recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5, then disrupted by ultrasound and centrifuged to obtain supernatant. The supernatant was passed through Ni-NTA hydrophilic chromatography to obtain purified alpha-glucanotransferase with a yield of 50%, fold 1.5 fold.

2-2. Validation of Disproportionation Activity of Alpha-Glucanotransferase

Alpha-Glucanotransferase 10 U and 1% maltooligosaccharides (maltose, maltotriose, maltotetraose, maltopentase, maltohepsaose or maltoheptaose) were added to 50 mM Tris-HCl buffer (pH 7.0, Sigma). After mixing, the mixture was reacted at 75 ° C. for 3 hours. The reaction was stopped by boiling the reaction solution for 5 minutes after the reaction.

Thin layer chromatography was performed to analyze the reaction product. After completion of the reaction, each sample was loaded into Whatman 1 K5F TLC plate (20 cm x 20 cm) by 1 µl and developed on a developing solution containing 2-propanol: ethyl acetate: distilled water in a 3: 1: 1. After development, the plates were dried, immersed in a developing solution in which 0.3 (w / v)% N- (1-naphthyl) -ethylenediamine and 5 (v / v)% sulfuric acid were dissolved, and then developed for 10 minutes in an oven at 110 ° C. .

Figure 2 shows the result of thin layer chromatography analysis of the reaction product after the alpha-glucanotransferase to maltooligosaccharide. In FIG. 2, alpha-glucanotransferases react with maltose, maltotriose, maltotetraose, maltopentaose, maltohepsaose or maltoheptaose to disproportionate them, respectively, so that long-chain straight maltose It was confirmed that oligosaccharides were produced respectively. Therefore, it was found that the alpha-glucanotransferase of the present invention successfully disproportionates maltooligosaccharides.

2-3. Thermal Stability of Alpha-Glucanotransferases

Thermal inactivation experiments were performed to see the thermal stability of the enzyme.

After adding enzyme solution to 0.25% amylose, 0.05% maltose and 50 mM Tris-HCl (pH 7.5), the activity of alpha glucanotransferase was examined by taking part of temperature and time. 3 is a graph measuring the thermal stability of alpha-glucanotransferases. In FIG. 3, the D-value (the time taken for the activity to decrease to one tenth) was found to be 210 minutes and 90 minutes at 85 ° C. and 90 ° C., respectively, indicating that alpha-glucanotransferase was very stable at high temperatures. I could confirm it.

The thermal stability of the enzyme can be applied even at high temperatures in the process of starch, it is very advantageous for the application of food using the enzyme.

Example 3: Generation of Customized Starch Using Heat Resistant Alpha-Glucanotransferase

Alpha U-Glucanotransferase 10 U and 1% rice starch were mixed in 50 mM Tris-HCl buffer (pH 7.0, Sigma) and reacted at 75 ° C. for 12 hours. The reaction was stopped by boiling the reaction solution for 5 minutes after the reaction.

In order to confirm the length of the side chain of the rice starch modified by alpha-glucanotransferase, a debranching enzyme that cuts α-1,6 bonds into the reaction solution was treated at 60 ° C. for 3 hours. After the reaction, the reaction was stopped by stopping for 5 minutes. The reaction solution was centrifuged to take only the supernatant, and the reaction solution was analyzed using ion exchange chromatography.

Figure 4a is a result of analyzing the side chain of the rice starch by ion exchange chromatography, Figure 4b is a result of analyzing the side chain of the rice starch reacted with alpha-glucanotransparase. In Figures 4a and 4b, it was confirmed that the length of the side chain of the rice starch is significantly reduced by the alpha-glucanotransferase treatment. In particular, the number of long side chains above DP10 was reduced, and the number of short side chains below DP10 was increased.

This experiment confirmed that alpha-glucanotransferases can produce ?? danched starches, in which the length of the side chains of starch is reconstituted.

Example 4 Preparation of Sputum Rice Cake Added with Heat-Resistant Alpha-Glucanotransferase

To investigate the effects of alpha-glucanotransferase treatment on the properties and preferences of rice cakes, the method of Choi, Choi (Chan Ran, Ph.D. Thesis, Chonnam National University, 2002) was added. Application was made.

After washing the rice with water and soaking for 12 hours, water was removed and pulverized twice with a roller mill to prepare rice flour (water content 38.7%). To prepare an enzyme solution, distilled water was added to an enzyme concentrate containing 20 mg of enzyme (20 ml of alpha-glucanotransferase concentrated at 1 mg / ml) to a total of 60 ml, and then 3 g of salt was added thereto. % Salt concentration. After mixing 250 g of rice flour and 60 ml of enzyme solution and left for 1 hour at 37 ℃, lowered in a sieve and steamed for 30 minutes in a steamer containing 3 L of water. Steamed dough was extruded 3 times with the extruder, and the rice cake of diameter 1.5cm was obtained.

Example 5: Sensory Evaluation of Sputumdduk Prepared by Addition of Heat-resistant Alpha-Glucanotransferase

After cooling to room temperature with the control rice cake treated with alpha-glucanotransferase-treated rice cake, the sensory test was performed.

Sensory tests were conducted on 12 graduate students who were trained to recognize the parameters of sputum mochi, and the items were the appearance (color, smoothness), flavor (scent of rice flour, odor) and mouth. They were evaluated in order of feeling inside (hardness, moistness, chewyness, sticking to teeth) and overall preference. The test results were statistically analyzed using ANOVA.

Sensory test results are shown in Table 1 below.

characteristic characteristic Control Enzyme treatment Exterior Color (yellow: 1-15: white) 10.9 ± 1.2 10.4 ± 2.0 Surface smoothness (roughness: 1 to 15: smooth)  8.3 ± 1.5  5.9 ± 1.2 Flavor Unique smell of rice flour (weak: 1 ~ 15: strong)  3.8 ± 1.5 3.4 ± 0.9 Off-flavor (weak: 1 ~ 15: strong)  2.5 ± 0.8 2.4 ± 0.7 Feeling in the mouth Hardness (Fluffy: 1 ~ 15: Hard) 3.9 ± 1.2  6.3 ± 2.1 Chewy (Unpacked: 1 to 15: Chewy)  4.0 ± 0.9  7.5 ± 1.9 Moist (dry: 1 to 15: moist) 11.8 ± 0.6 10.5 ± 0.4 Sticking to teeth (weak: 1 ~ 15: strong)  5.1 ± 1.0  5.7 ± 1.7 Overall preference (Poor: 1 to 15: Good)  7.7 ± 1.7 10.1 ± 1.3

 In Table 1, it can be seen that the rice cake treated with alpha-glucanotransferase significantly improved the chewy level compared to the control. Other significant results were that the rice cakes treated with enzyme were somewhat harder and less smooth than the control. The color of the enzyme treatment did not show a significant difference compared to the control, and in general, it was shown that there was no deepening of browning reaction due to reducing sugar production during amylase enzyme treatment.

Example 6 Determination of Aging Degree of Rice Cake Prepared by Addition of Heat-resistant Alpha-Glucanotransferase

In order to examine the effect of alpha-glucanotransferase treatment on aging of rice cake, the rice cake with alpha-glucanotransferase added in Example 4 and the rice cake control group without enzyme were placed in polyethylene bags. The degree of aging was measured by differential scanning calorimeter on days 5 and 8 while stored at ℃.

About 10 mg of the sample was put in an aluminum container, and the degree of aging was determined by converting the area of the endothermic curve around 40 ° C obtained by heating from 25 ° C to 130 ° C at 5 ° C per minute to the calorific value per unit weight.

Figure 5 confirms the degree of aging of alpha-glucanotransferase treated group (??) and untreated group (??), aging was significantly suppressed in the rice cake treated with alpha-glucanotransferase compared to the control.

Example 7: Observation of Microstructure of Sputumdduk Prepared by Addition of Heat-resistant Alpha-Glucanotransferase

As in Example 4, the microstructure of the rice cake prepared by treatment with alpha-glucanotransferase was observed by scanning electron microscopy (SEM).

FIG. 6A is a scanning electron micrograph of a control sputum rice cake treated with alpha-glucanotransferase and 6b is a sputum scanning electron microscope image of alpha-glucanotransferase treated. As shown in Figure 6, compared to the control barley rice cake, the rice cake treated with alpha-glucanotransferase showed smaller and tighter internal voids and showed a denser network structure.

Example 8 Preparation of Cooked Rice with Heat-Resistant Alpha-Glucanotransferase

To prepare rice, water was added to 100 g of rice at a ratio of 1: 1.3 of the volume of rice. To the rice treated with alpha glucanotransferase, 10 mg (10 mL of alpha-glucanotransferase enzyme solution concentrated to 1 mg / ml, 150 U) of alpha glucanotransferase was added, and the procedure was then identified with the control. After immersing at room temperature for 1 hour, it was cooked with a rice cooker / heat insulating combined electric rice cooker (SJ-M032R, Samsung Electronics Co., Seoul). After steaming for 10 minutes from the moment when the cooking signal of the electric heat cooker turned into heat retention, only the rice in the center of the rice cooker was taken out and left for 1 hour at room temperature to be used for analysis.

Example 9 Analysis of Physical Properties of Cooked Rice Prepared by Addition of Heat-resistant Alpha-Glucanotransferase

In order to investigate the effect of alpha-glucanotransferase treatment on the properties of cooked rice, the hardness of rice grains and the stickiness of the surface using a physical analyzer (TA-XT2i) Was measured.

Five rice grains of the sample prepared in Example 8 were picked up and placed on the physical property analyzer at appropriate intervals, and set at 0.5 mm / min and 50% strain using a 35 mm cylindrical probe, removed at a rate of 10 mm / min, and pressed. The hardness and stickiness of the grains of rice were measured by measuring the force in grams and the force in peeling. The results are shown in Table 2 below.

characteristic Strain Control cooked rice Cooked rice with enzyme Hardness 90% 3,463 g ± 253 4,086 g ± 541 50%  854 g ± 55  793 g ± 68 Stickiness 90% 1,155 g ± 134  985 g ± 79 50%  173 g ± 29  100 g ± 15

 In Table 2, rice grains treated with alpha-glucanotransferase and untreated rice grains exhibited similar hardness, showing no enzymatic retreat and less stickiness on the surface.

Example 10 Determination of Aging Degree of Cooked Rice Prepared by Addition of Heat-resistant Alpha-Glucanotransferase

In order to determine the effect of alpha-glucanotransferase on the inhibition of aging of cooked rice, enzyme treatment was performed as in Example 8, and the degree of aging on the 4th and 8th days of storage at 4 ° C. with the control was measured. It was measured in the same manner as in.

Figure 7 confirms the degree of aging of the alpha-glucanotransferase treated and untreated group, aging was significantly suppressed in the cooked rice treated with alpha-glucanotransferase compared to the control.

As described above, the heat-resistant alpha-glucanotransferase isolated from the high temperature strain provided in the present invention reconstructs the structure of starch in the food, improves the shelf life of the food, and significantly improves the physical properties and palatability of the food. Can be used for purposes.

Figure 1 shows a simplified map of the p6xHisTSαGT vector according to the present invention.

2 is a thin layer chromatography analysis confirming the disproportionation ability of alpha-glucanotransferase.

3 is a graph measuring the thermal stability of alpha-glucanotransferases.

Figure 4a is a result of analyzing the side chain of the rice starch by ion exchange chromatography, Figure 4b is a result of analyzing the side chain of the rice starch reacted with alpha-glucanotransferase.

5 is a measure of the starch aging rate of alpha-glucanotransferase treated (▼) and untreated (●).

FIG. 6A is a scanning electron micrograph of a control sputum rice cake treated with alpha-glucanotransferase and 6b is a scanning electron micrograph of a rice cake treated with alpha-glucanotransferase.

Figure 7 measures the rate of aging prevalence in alpha-glucanotransferase treated and untreated.

<110> PARK, Kwan-Hwa <120> NOVEL THERMOSTABLE alpha-GLUCANOTRANSFERASE AND USE THEREOF <130> dpp20033768kr <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nde-TSaGT primer <400> 1 gggtaaactg ggcatatgga gcttccg 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TSaGT-Hd primer <400> 2 cccttaagct tcccaccctg gccg 24 <210> 3 <211> 1503 <212> DNA <213> Thermus scotoductus <220> <221> CDS <222> (1) .. (1500) <223> alpha-glucanotransferase <400> 3 atg gag ctt ccg cgc gcc tat ggc ctg ctt ctg cac ccc acc agc ctt 48 Met Glu Leu Pro Arg Ala Tyr Gly Leu Leu Leu His Pro Thr Ser Leu 1 5 10 15 ccc gga ccc tat ggg gtt ggc gtt nna aaa nag gag gct ttt gcc ttc 96 Pro Gly Pro Tyr Gly Val Gly Val Xaa Lys Xaa Glu Ala Phe Ala Phe 20 25 30 ctc cga ttc ctg ggg gag tct ggg gcc cgc tac tgg cag gtt ttg ccc 144 Leu Arg Phe Leu Gly Glu Ser Gly Ala Arg Tyr Trp Gln Val Leu Pro 35 40 45 tta ggg ccc aca gga tac gga gac tcc ccc tac cag tcc ttc agc gcc 192 Leu Gly Pro Thr Gly Tyr Gly Asp Ser Pro Tyr Gln Ser Phe Ser Ala 50 55 60 ttt gcc ggg aac ccc tac ctc atc gac ctc cgc cta agg cgg agg cgg 240 Phe Ala Gly Asn Pro Tyr Leu Ile Asp Leu Arg Leu Arg Arg Arg Arg 65 70 75 80 ggg tac ctc cgc ctc gag gac ccc ggt ttt ccc gag ggc cgg gtg gta 288 Gly Tyr Leu Arg Leu Glu Asp Pro Gly Phe Pro Glu Gly Arg Val Val 85 90 95 tac ggc tgg ctc tac gcc tgg aaa tgg ccg gcg ctt agg gaa gcc ttc 336 Tyr Gly Trp Leu Tyr Ala Trp Lys Trp Pro Ala Leu Arg Glu Ala Phe 100 105 110 cgg ggt ttc cag gag cgg gct agc cgg gag gaa aag gag gcc ttt cag 384 Arg Gly Phe Gln Glu Arg Ala Ser Arg Glu Glu Lys Glu Ala Phe Gln 115 120 125 cct ttt ggg acc cgg gaa agg agc tgg ttg gat gac tac acc ctc ttc 432 Pro Phe Gly Thr Arg Glu Arg Ser Trp Leu Asp Asp Tyr Thr Leu Phe 130 135 140 atg gcc ttg aag ccc acc cac gga ggc ctt cct tgg aac cgc tgg ccc 480 Met Ala Leu Lys Pro Thr His Gly Gly Leu Pro Trp Asn Arg Trp Pro 145 150 155 160 atg ccc cta cgc ctg agg gag gaa aag gct tta agg gaa gca tct ttg 528 Met Pro Leu Arg Leu Arg Glu Glu Lys Ala Leu Arg Glu Ala Ser Leu 165 170 175 gcc ctc tcc caa gag gtg gcc ttc cac gcc tgg acc cag tgg ttt ttc 576 Ala Leu Ser Gln Glu Val Ala Phe His Ala Trp Thr Gln Trp Phe Phe 180 185 190 ttc cgg cag tgg cag gcc ttg agg gag gcg gcg gag gcc ttg ggc ctc 624 Phe Arg Gln Trp Gln Ala Leu Arg Glu Ala Ala Glu Ala Leu Gly Leu 195 200 205 tcc ttg atc ggc gac atg ccc atc ttc gtg gcc gag gac tcc gcg gag 672 Ser Leu Ile Gly Asp Met Pro Ile Phe Val Ala Glu Asp Ser Ala Glu 210 215 220 gtc tgg gcc cac ccc gag tgg ttc cac ctg gat gag gag gga agg ccc 720 Val Trp Ala His Pro Glu Trp Phe His Leu Asp Glu Glu Gly Arg Pro 225 230 235 240 acg gtg gtg gcg ggg gtg ccg ccg gat tac ttt tcg gaa acg ggc cag 768 Thr Val Val Ala Gly Val Pro Pro Asp Tyr Phe Ser Glu Thr Gly Gln 245 250 255 cgc tgg ggg aat ccc ctg tac cgc tgg gag gtg ctg gaa agg gag ggg 816 Arg Trp Gly Asn Pro Leu Tyr Arg Trp Glu Val Leu Glu Arg Glu Gly 260 265 270 ttc tcc ttc tgg ata gag cgc ctg agg aag gcc ttg gag ctt ttc cac 864 Phe Ser Phe Trp Ile Glu Arg Leu Arg Lys Ala Leu Glu Leu Phe His 275 280 285 ctg gtg cgc atc gac cac ttc cgg ggc ttt gaa gcc tac tgg gag atc 912 Leu Val Arg Ile Asp His Phe Arg Gly Phe Glu Ala Tyr Trp Glu Ile 290 295 300 cct gcc tcc tgc ccc acg gca gtg gag gga cgc tgg gtg aag gcc ccg 960 Pro Ala Ser Cys Pro Thr Ala Val Glu Gly Arg Trp Val Lys Ala Pro 305 310 315 320 ggg gag aag ctc ttt cag aag att cag gaa acc ttt ggc cgg gtg ccc 1008 Gly Glu Lys Leu Phe Gln Lys Ile Gln Glu Thr Phe Gly Arg Val Pro 325 330 335 atc ctg gcg gag gac ctg ggg gtg atc acc ccc gag gtg gag gcc ctg 1056 Ile Leu Ala Glu Asp Leu Gly Val Ile Thr Pro Glu Val Glu Ala Leu 340 345 350 agg gac cgg ttt ggc ctt ccg ggg atg aag gtc ctg cag ttc gcc ttt 1104 Arg Asp Arg Phe Gly Leu Pro Gly Met Lys Val Leu Gln Phe Ala Phe 355 360 365 gac ggg ggg atg gaa aac ccc ttc ctg ccc cac aac tac ccc tcc cac 1152 Asp Gly Gly Met Glu Asn Pro Phe Leu Pro His Asn Tyr Pro Ser His 370 375 380 ggc cgg gtg gtg gtc tac acc ggc acc cac gac aac gac acc acc ttg 1200 Gly Arg Val Val Val Tyr Thr Gly Thr His Asp Asn Asp Thr Thr Leu 385 390 395 400 ggc tgg tac cgc acc gct acc ccc cac gag cgg gct ttc ctt ggg cgc 1248 Gly Trp Tyr Arg Thr Ala Thr Pro His Glu Arg Ala Phe Leu Gly Arg 405 410 415 tac ctg gcg gag tgg ggg att ggg ttc cag agg gaa gag gag atc ccc 1296 Tyr Leu Ala Glu Trp Gly Ile Gly Phe Gln Arg Glu Glu Glu Ile Pro 420 425 430 tgg gcc ctc atg cac ctg ggg atg aag tcg gtg gcc agg ctg gcc gtc 1344 Trp Ala Leu Met His Leu Gly Met Lys Ser Val Ala Arg Leu Ala Val 435 440 445 tac ccc gtg cag gat gtg ttg gcg ttg gga agc gag gcc cgg atg aac 1392 Tyr Pro Val Gln Asp Val Leu Ala Leu Gly Ser Glu Ala Arg Met Asn 450 455 460 tac cct ggg cgc ccc tcg ggc aac tgg gct tgg cgg ctt agg ccc ggc 1440 Tyr Pro Gly Arg Pro Ser Gly Asn Trp Ala Trp Arg Leu Arg Pro Gly 465 470 475 480 cag ctc ctg ccc gag cac ggg gaa cgg ctt cgc tgg atg gcc gag gcc 1488 Gln Leu Leu Pro Glu His Gly Glu Arg Leu Arg Trp Met Ala Glu Ala 485 490 495 acg ggc agg gtg taa 1503 Thr Gly Arg Val 500 <210> 4 <211> 500 <212> PRT <213> Thermus scotoductus <400> 4 Met Glu Leu Pro Arg Ala Tyr Gly Leu Leu Leu His Pro Thr Ser Leu 1 5 10 15 Pro Gly Pro Tyr Gly Val Gly Val Xaa Lys Xaa Glu Ala Phe Ala Phe 20 25 30 Leu Arg Phe Leu Gly Glu Ser Gly Ala Arg Tyr Trp Gln Val Leu Pro 35 40 45 Leu Gly Pro Thr Gly Tyr Gly Asp Ser Pro Tyr Gln Ser Phe Ser Ala 50 55 60 Phe Ala Gly Asn Pro Tyr Leu Ile Asp Leu Arg Leu Arg Arg Arg Arg 65 70 75 80 Gly Tyr Leu Arg Leu Glu Asp Pro Gly Phe Pro Glu Gly Arg Val Val 85 90 95 Tyr Gly Trp Leu Tyr Ala Trp Lys Trp Pro Ala Leu Arg Glu Ala Phe 100 105 110 Arg Gly Phe Gln Glu Arg Ala Ser Arg Glu Glu Lys Glu Ala Phe Gln 115 120 125 Pro Phe Gly Thr Arg Glu Arg Ser Trp Leu Asp Asp Tyr Thr Leu Phe 130 135 140 Met Ala Leu Lys Pro Thr His Gly Gly Leu Pro Trp Asn Arg Trp Pro 145 150 155 160 Met Pro Leu Arg Leu Arg Glu Glu Lys Ala Leu Arg Glu Ala Ser Leu 165 170 175 Ala Leu Ser Gln Glu Val Ala Phe His Ala Trp Thr Gln Trp Phe Phe 180 185 190 Phe Arg Gln Trp Gln Ala Leu Arg Glu Ala Ala Glu Ala Leu Gly Leu 195 200 205 Ser Leu Ile Gly Asp Met Pro Ile Phe Val Ala Glu Asp Ser Ala Glu 210 215 220 Val Trp Ala His Pro Glu Trp Phe His Leu Asp Glu Glu Gly Arg Pro 225 230 235 240 Thr Val Val Ala Gly Val Pro Pro Asp Tyr Phe Ser Glu Thr Gly Gln 245 250 255 Arg Trp Gly Asn Pro Leu Tyr Arg Trp Glu Val Leu Glu Arg Glu Gly 260 265 270 Phe Ser Phe Trp Ile Glu Arg Leu Arg Lys Ala Leu Glu Leu Phe His 275 280 285 Leu Val Arg Ile Asp His Phe Arg Gly Phe Glu Ala Tyr Trp Glu Ile 290 295 300 Pro Ala Ser Cys Pro Thr Ala Val Glu Gly Arg Trp Val Lys Ala Pro 305 310 315 320 Gly Glu Lys Leu Phe Gln Lys Ile Gln Glu Thr Phe Gly Arg Val Pro 325 330 335 Ile Leu Ala Glu Asp Leu Gly Val Ile Thr Pro Glu Val Glu Ala Leu 340 345 350 Arg Asp Arg Phe Gly Leu Pro Gly Met Lys Val Leu Gln Phe Ala Phe 355 360 365 Asp Gly Gly Met Glu Asn Pro Phe Leu Pro His Asn Tyr Pro Ser His 370 375 380 Gly Arg Val Val Val Tyr Thr Gly Thr His Asp Asn Asp Thr Thr Leu 385 390 395 400 Gly Trp Tyr Arg Thr Ala Thr Pro His Glu Arg Ala Phe Leu Gly Arg 405 410 415 Tyr Leu Ala Glu Trp Gly Ile Gly Phe Gln Arg Glu Glu Glu Ile Pro 420 425 430 Trp Ala Leu Met His Leu Gly Met Lys Ser Val Ala Arg Leu Ala Val 435 440 445 Tyr Pro Val Gln Asp Val Leu Ala Leu Gly Ser Glu Ala Arg Met Asn 450 455 460 Tyr Pro Gly Arg Pro Ser Gly Asn Trp Ala Trp Arg Leu Arg Pro Gly 465 470 475 480 Gln Leu Leu Pro Glu His Gly Glu Arg Leu Arg Trp Met Ala Glu Ala 485 490 495 Thr Gly Arg Val 500 <210> 5 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 5 ggatccagat catcggggac atgcccatct tcgtggccga ggactcggcc gaggtctggg 60 cccaccccga gtggttccac ctggacgagg agggaaggcc cctggtggtg gcgggggtgc 120 cccccgacta cttctcggag accggccagc gctggggcaa ccccctctac cgctgggacg 180 tcctggagag agaggggttc tccttttgga tagcccggct cgccaaggcc ctggagctat 240 tccacctggt gcgcgtggac cacttccggg gctttgaggc ctactgggag atcccggcct 300 cctgccccac ggcggtggag gggcgctggg tgaaggcttc cggggagaag ctctttgacc 360 gcatccagga agttttcggc gaagtgccca tcctggccga agacctgggg gtcatcaccc 420 ccgaggtgga gcccctgcgg gaccgctacg gccttccggg gatgaaagtc ctgcaattcg 480 cctttgacca cggcatggaa aacccctttc ttccccacaa ctaccccgcc catggccggg 540 tggtggttta caccggcacc cacgacaacg acaccaccct gggctggtac c 591

Claims (11)

A heat resistant alpha alpha glucanotransferase consisting of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4. A polynucleotide comprising a nucleic acid sequence encoding the peptide of claim 1. The polynucleotide of claim 2 wherein the nucleic acid sequence is the sequence set forth in SEQ ID NO: 3. A vector comprising the polynucleotide of claim 1 expressably. Cell line transformed with the vector of claim 4. The cell line of claim 5, wherein said cell line is selected from the group consisting of E. coli , yeast and fungi. A heat resistant alpha-glucanotransferase produced from the cell line according to claim 5. A method for preparing a processed food product treated with alpha-glucanotransferase, comprising adding the heat resistant alpha-glucanotransferase of claim 1 to starch, starch-containing grains or foods. The method of claim 8, wherein the starch-containing grain is selected from the group consisting of rice, wheat, potatoes, corn and soybeans. The method of claim 8, wherein the heat resistant alpha-glucanotransferase is added at 150 to 200 U and the reaction is performed at 75 to 80 ° C. 10. Processed food prepared by the method according to any one of claims 8 to 11.