KR101518022B1 - Method for producing cycloamylose using 4-alpha-glucanotransferase - Google Patents

Abstract

The present invention relates to a method of manufacturing cycloamylose and a use thereof, wherein the method enables to inhibit coupling reaction destroying cycloamylose and facilitate production reaction of cycloamylose for glucanotransferase derived from Deinococcus geothermalis. According to the present invention, a method comprises the steps of: manufacturing recombinant vectors including genes to encode 4-α-glucanotransferase consisting of the amino acid sequence derived from Deinococcus geothermalis (SEQ ID NO: 2); and processing additionally the same.

Description

[0001] The present invention relates to a method for producing cycloamylose using 4-alpha-glucanotransferase,

The present invention relates to a process for preparing cycloamylase using 4-alpha-glucanotransferase, and more particularly to a process for producing cycloamylase from 4-alpha-glucanotransferase derived from Deinococcus geothermalis , The present invention relates to a method for producing cycloamylase by using a reaction between a contained substrate.

4-alpha-glucanotransferase (EC 2.4.1.25) is an enzyme having sugar-exchange activity. It is an enzyme that transfers sugar chains from one linear? -Glucan molecule to another linear? -Glucan molecule A disproportionation reaction, a cyclization reaction in which a cyclic glucan is generated in a single linear α-glucan molecule by reaction, and a coupling reaction in which cyclic glucan is converted into a linear glucan by opening a cyclic glucan, do. Amylose is used as a substrate to generate cycloamylase by the action of 4-alpha-glucanotransferase.

Cycloamylose has hydrophilic outer and annular inner cavities, and captures hydrophobic compounds to improve the solubility, reactivity, and stability of the material. Due to these specific properties, cycloamylose is useful in the food, pharmaceutical, chemical and cosmetic industries. For example, cycloamylose having a degree of polymerization (DP) of 6 to 8 is widely used as a dietary fiber, an emulsifying agent for mayonnaise, a stabilizer for flavoring agents, and a deodorant. On the other hand, the presence of cycloamylose with a degree of polymerization of 20 or more was known in the 1990s and industrial applications began. As the artificial chaperone function involved in the protein refolding of these cyclamyloses has become known, the industrial application of cycloamylose has been widening. Until now, chemical synthesis methods of some cycloamyloses have been known, but the method has not been useful in industrial methods because it has undergone many processing steps. Accordingly, in the art, a method of producing various cycloamyloses using a biosynthetic enzyme is performed.

A method of producing cycloamylose, which is mainly used at present, is a method using the sugar transfer activity of 4-alpha-glucanotransferase. The 4-alpha-glucanotransferase was first cloned in E. coli and is present in plants and microorganisms. Alpha-glucanotransferase derived from a microorganism is industrially used. For example, there is a glucanotransferase (TS alpha GT) (Korean Patent No. 10-0645993) derived from Thermus scotoductus . Further, there is a method of simultaneously reacting a glucanotransferase derived from a conventionally known microorganism with an additional secondary enzyme to improve the yield of cycloamylose (Korean Patent Nos. 10-1214572 and 10-1045357). However, a method for improving the yield of cycloamylose of various compositions by the novel glucanotransferase of the present invention has not been described.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a method for increasing the cyclization / coupling ratio using glucanotransferase (Dg? GT) derived from Deinococcus geothermalis And amylose having a polymerization degree as high as 26 in a short reaction time with amylose as a substrate can be produced. The present invention has been completed based on this finding.

In order to solve the above problems, the present invention provides a method for producing cycloamylase, which comprises reacting 4-alpha-glucanotransferase with amylose.

The Deinococcus of the present invention It was obtained in a cycle amylose glue Kano improved yield by using a transferase enzyme, in particular alpha-4 of the present invention - - geothermalis) derived from 4-alpha glue Kano transferase enzyme is derived from gluconic sseomeoseu seukoto virtue Tuscan (Thermus scotoductus) It is possible to increase the cyclization / coupling ratio with respect to the cano transferase (TS alpha GT) and to produce a cycloamylase having a high degree of polymerization such as a degree of polymerization of 26 within a short reaction time with the substrate amylose. Therefore, the 4-alpha-glucanotransferase of the present invention is expected to be useful for the industrial production of cycloamylose.

Figure 1 shows the results of SDS-PAGE of Dg < alpha > GT enzyme expressed in E. coli BL21 (DE3). M, size marker; 1, cell extract; 2, insoluble fraction; 3, fraction after Ni-NTA affinity chromatography.
FIG. 2 is a graph showing the relative activity of Dg? GT enzyme according to temperature change.
FIG. 3 is a graph showing the relative activity of Dg? GT enzyme according to pH change.
FIG. 4 shows the results of analysis of cycloamylase produced using the DgαGT (A) and TSαGT (B) enzymes by the high performance anion exchange chromatography (HPAEC).
FIG. 5 shows the results obtained by comparing quantitative changes of cycloamylase having various degrees of polymerization produced using Dg? GT (A) and TS? GT (B) enzymes at reaction time intervals.
FIG. 6 shows the results obtained by comparing quantitative changes of cycloamylase having polymerization degrees of 6 and 26 produced using Dg? GT (A) and TS? GT (B) enzymes at reaction time intervals.

In order to accomplish the object of the present invention, the present invention provides a method for producing cycloamylase, which comprises reacting 4-alpha-glucanotransferase with amylose.

The method according to an embodiment of the present invention is more particularly

Preparing a recombinant vector comprising a gene encoding 4-a-glucanotransferase;

Transforming the prepared recombinant vector into E. coli;

Isolating and purifying the 4-alpha-glucanotransferase enzyme from the transformed E. coli; And

And then reacting the purified 4-alpha-glucanotransferase enzyme with amylose.

In a method according to an embodiment of the present invention, the 4-a-glucanotransferase is preferably a Deinococcus strain comprising the amino acid sequence of SEQ ID NO: 2 geothermalis derived 4-alpha-glucanotransferase, but are not limited thereto. The 4-alpha-glucanotransferase The range of the protein includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous bioactivity" means an activity that produces cycloamylose.

Preferably, the gene encoding the 4-alpha-glucanotransferase of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the method according to one embodiment of the present invention, the 4-α-glucanotransferase is characterized by increasing the cyclization / coupling ratio, but is not limited thereto. For example, the 4-alpha-glucanotransferase enzyme of the present invention has a higher cyclization / coupling ratio than the glucanotransferase (TS alpha GT ) derived from Thermus scotoductus .

The method according to an embodiment of the present invention comprises reacting the purified 4-a-glucanotransferase enzyme with amylose, wherein the reaction temperature is preferably 50-55 ° C, and most preferably Lt; RTI ID = 0.0 > 50 C, < / RTI > Further, the reaction time may be preferably 3 to 12 hours, more preferably 3 to 6 hours, but most preferably 6 hours, but is not limited thereto. In addition, the pH of the reaction may be 6.0 to 9.0, and more preferably 6.0, but is not limited thereto.

In the method according to one embodiment of the present invention, the substrate used to prepare the cycloamylose may be selected without limitation including amylose or amylose, preferably amylose can be used as the substrate.

Chromatography can be used to purify the cycloamylase after the step of reacting the glucanotransferase of the present invention (hereinafter, used in combination with 4-alpha-glucanotransferase) and the substrate containing amylose, A high performance anion exchange chromatography method may be used, but the present invention is not limited thereto.

In the method according to one embodiment of the present invention, the obtained cycloamylose may be amylose with a degree of polymerization of 5 to 37, but is not limited thereto. In addition, the produced cycloamylose may be contained in an amount of 10% or less, preferably 5 to 10%, based on the weight of the total produced cycloamylase, but not limited thereto.

The method of the present invention is preferably characterized in that the reaction time is 3 to 4 hours, and the amylose of cycloamyl is a main component of cycloamylase having a degree of polymerization of 24 to 28. However, the present invention is not limited thereto. For example, Thermus < RTI ID = 0.0 > glucanotransferase (TSαGT) derived from the scotoductus can hardly produce amylose having a high degree of polymerization, such as a polymerization degree of 26, in a short reaction time of about 3 hours, while the 4-alpha-glucanotransferase enzyme Can produce a large amount of cycloamylose having a high degree of polymerization such as a polymerization degree of 26 within a short reaction time of about 3 hours (see FIG. 6). Therefore, the 4-alpha-glucanotransferase enzyme of the present invention is very useful for producing a large amount of cycloamylose having a high degree of polymerization within a short reaction time.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: 4- Of glucanotransferase (? GT)  Produce

Deinococcus A recombinant vector containing a glucanotransferase (Dg? GT) gene derived from geothermal was prepared. Genomic DNA of DYNOCOCCUS geothermally was used as a PCR template for cloning the glucanotransferase gene and p6xH119 was used as a cloning vector. The DgαGT gene cloned into the recombinant vector p6xHis119_DgαGT produced a 57.2 kDa glucanotransferase consisting of 511 amino acids.

The recombinant vector p6xHis119_Dg? GT was transformed into E. coli After transforming BL21 (DE3), the transformed E. coli was inoculated and cultured in 1 L of LB medium containing ampicillin. The culture was centrifuged at 4 ° C under 3699xg for 20 minutes to collect the cells. The recovered cells were suspended in 100 ml of lysis buffer (300 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM imidazole), followed by ultrasonic pulverization. After obtaining a cell extract, Ni-NTA affinity chromatography was performed to purify the enzyme. The purified enzyme was concentrated to 70% ammonium sulfate and diluted with 50 mM Tris-HCl (pH 7.5) buffer. Enzyme concentration was determined by the Bradford assay. Purification was confirmed by SDS-PAGE and the results are shown in Fig.

Glucanotransferase ( TSαGT ) derived from Thermus scotoductus , which is well known in the art, was obtained and used in the following experiment for comparison of the enzymatic activity of the glucanotransferase and the yield of cycloamylase.

Example  2: Glucanotransferase  Enzyme activity measurement

2-1. Measurement of temperature and pH stability

0.05 U / mg of glucanotransferase was added to 50 mM sodium acetate buffer (pH 6.0) containing 1 mg / ml amylose substrate and reacted at 30 to 60 ° C for 30 minutes at 5 ° C intervals, Were measured. As shown in FIG. 2, the test results showed the maximum activity at 55 ° C and the activity was almost lost at 60 ° C.

Glucanotransferase was added to the buffers of different pH including amylose substrate and reacted for 30 minutes, and the effect of pH on enzyme activity was measured. 50 mM sodium acetic acid at pH 3.5 to 6.5, MOPs buffer at pH 6.5 to 8.5, and CHEs buffer at pH 8.5 to 10 were used. The results of the experiment are shown in FIG. and showed optimum activity at pH 6.0 to 9.0.

2-2. Measurement of cyclization activity

The cyclization activity was determined by measuring the reduction of linear sugars using amylose as a substrate. The purified glucanotransferase was added to the reaction solution containing the substrate, treated for 30 minutes, and then the reaction was terminated for 10 minutes and β-amylase was added to remove the linear saccharide. After the reaction was terminated for 5 minutes, the absorbance at 575 nm was measured to determine the weight of cycloamylose. One unit of enzyme activity is defined as the amount of enzyme that catalyzes a 1 μmole substrate per minute.

2-3. Measurement of coupling activity

Coupling activity was achieved using cycloamylose and glucose. The activity was determined by measuring the decrease in glucose weight with the GOD-POD method (Werner et al., 1970, Analytical Chemistry 252: 224-228). After the reaction was terminated, the absorbance at 500 nm was measured by treatment with GOD-POD solution for 30 minutes.

Table 1 below shows the cyclization and coupling reaction rates of glucotransferase. DgαGT showed a significantly slower coupling reaction rate than TSαGT.

U / nmole protein Coupling (U / nmole protein) Cyclization / Coupling DgαGT 599x10 ² 564x10 ² 1.06 TSαGT 125x10 ³ 452x10 ³ 0.276

room Sime  3: Cycloamylic  Produce

3-1. Cycloamylase yield determination

The purified 0.05 U / mg glucanotransferase was added to a 50 mM sodium acetate buffer (pH 6.0) containing 1 mg / ml amylose and incubated at 50 ° C for 0.5, 1, 2, 3, 6, 12, 30 hours. After the reaction, the resulting cycloamylose was precipitated with 80% ethanol, dried and dissolved in distilled water, treated with 0.25 U / mg of β-amylase at pH 5.5 and 35 ° C. for 2 days. To remove small maltodextrins such as maltose, cycloamylose was precipitated with 80% ethanol and the precipitate was washed with 80% ethanol. Cycloamylase precipitate was dried and dissolved in distilled water.

The yield was calculated as the percentage (w / w) of cycloamylase produced after reaction to the amount of amylose added before the reaction. Table 2 below shows the change in the yield of amylose production in cycles by reaction time. DgαGT accumulated cycloamylase more rapidly than TSαGT and showed a maximum yield of 53% after 6 hours of reaction and then decreased.

Reaction time (hr) DgαGT TSαGT 0.5 14% Less than 1% One 14% Less than 1% 2 16% Less than 1% 3 39% One% 6 53% 43% 12 45% 46% 24 23% 46% 30 37% 44%

3-2. Analysis using high performance anion exchange chromatography

Cycloamylase produced after 3-1 reaction was analyzed by high performance anion exchange chromatography (HPAEC) method. HPAEC analysis was performed using a Dionex (USA) DX-500 system with ED40 electrochemical detector, CarboPac column (Carbopac PA-1) and guard column. The column was equilibrated with 150 mM of caustic soda solution and then eluted with 600 mM sodium acetate at a flow rate of 1 ml / min.

FIG. 4 shows the results obtained by performing the above method on the cycloamylase produced from the Dg? GT (A) and TS? GT (B) enzymes by reaction time scale. The cycloamylase produced in DgαGT was produced faster than the cycloamylose produced in TSαGT and showed a cycloamylose peak after 30 minutes of reaction.

3-3. Quantitative analysis of cycloamylase using MALDI-TOF / MS

The molecular weight composition of the cycloamylose produced after the 3-1 reaction was measured using MALDI-TOF / MS. The system was a Bruker ultraflexXtreme ^TM system (USA).

The results compared and analyzed the total cycloamylase produced from the Dg? GT (A) and TS? GT (B) enzymes in FIG. As shown in Fig. 5, the amylose exhibited two peaks in the degree of polymerization of 5 to 36, and the amount of amylose in the medium size was small. After 3 hours of reaction, TSαGT produced almost no cycloamylase whereas DgαGT enzyme produced a significant amount of cycloamylose (FIG. 5). In particular, as shown in FIG. 6, amylose production was remarkably high in a cycle with a degree of polymerization of 26 at 3 hours of reaction. 5 and 6, it can be seen that the Dg &ggr; GT enzyme efficiently produces amylose in a cycle of 24 to 28 in the reaction time of 3 to 4 hours.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Method for producing cycloamylose using          4-alpha-glucanotransferase <130> PN14017 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1536 <212> DNA <213> Deinococcus geothermalis <400> 1 atggaacacc atcaccatca ccatatgctc accaagcgtt ccagcggcgt gctgctgcat 60 cccaccagcc ttcccggtcc ctacggcatt ggggaactcg gcgcgcaggc gcggcatttc 120 gtggactggc tggcgcgggc cggtcagacg tactggcagg tgatgccgct tggcccaacc 180 gggtacggcg acagcccgta tcaggctttt agcgcctttg ccggcaatcc ctatctgatc 240 gacctcacca cgctgcgcga ggaaggcctg ctgcacgcga gcgacttcga gggcacgccc 300 gacttcgacg caggccgagt ggactttggg ttgcagtacg tgtggcggat gcagatgctg 360 ggccgtgcct acgcacattt cgctttcggg caccaccccg agctgaaagc cgcgtttgaa 420 gcctttaagg ctgaggaggc ggcctggctg gacgactacg cgctctttat ggcgctcaag 480 gcgcgcacg gtggcttgcc ctggaacgcc tgggaacccg gcacacgcga ccgcgagccg 540 caggcgctgg cggcagcaca ggaacagctc gcgcccaaca tcgagcgcgt gaagttcatc 600 cagttcctgt tctttcggca gtggcgagcg ctgcgcacct acgctcgcga gcgcggcgtg 660 ggggtgatcg gggacatccc gattttcgtg gccatggact ccagtgatgc ctgggccaac 720 cgcgagcagt tctatttcga cgaacagggc cagcctaccg tcgtggcggg cgtcccgcca 780 gctacttca gcgagacggg gcagctgtgg ggcaatcccc tttaccgctg ggacgtgatg 840 gagcaagacg gcttccactg gtggatcgag cgcttccgcg gcagcctgaa gctctacgac 900 ctcatccgga tcgaccactt ccgcgggttt gccgcctact gggagattcc ctaccccgcc 960 ggaacgcga ttcatggccg ctgggtgccc gcgccgggcc acgcgctgct ggaggcggtg 1020 cgccgggcgc tggggcagat gcccatcatc gcagaagact tgggggtcat cacgcccgac 1080 gtcgaacagc tgcgtgatga ttttgggttg cccggtatgg ccgtcttgca cttcgctttt 1140 ggcggcggcg actttagcgt gaatgccttc ctgccgcaca acctcaaggc caatcaggtc 1200 gtgtacaccg gcacccacga caacgacacc tcgcgcggct ggtggcagca tgccgatgag 1260 caagagcggc agaacttccg cctctacacc cacagtgacc ccagcgagga gaccttcgcc 1320 tggcagctga ccgagatcgc cctggaaagc cgccccaatc tggcgattgt tcccttgcag 1380 gacctcctga acctgggcag cgaggcgcgc atgaacttcc ccggcaccac tggcccccac 1440 aactggacct ggcgttacct cgccgcggac ctgcgccccg atctggcgac aaagctgcgg 1500 gcgctgacgg agagaacagg ccgagtcgaa cggtaa 1536 <210> 2 <211> 511 <212> PRT <213> Deinococcus geothermalis <400> 2 Met Glu His His His His His Met Leu Thr Lys Arg Ser Ser Gly   1 5 10 15 Val Leu Leu His Pro Thr Ser Leu Pro Gly Pro Tyr Gly Ile Gly Glu              20 25 30 Leu Gly Ala Gln Ala Arg His Phe Val Asp Trp Leu Ala Arg Ala Gly          35 40 45 Gln Thr Tyr Trp Gln Val Met Pro Leu Gly Pro Thr Gly Tyr Gly Asp      50 55 60 Ser Pro Tyr Gln Ala Phe Ser Ala Phe Ala Gly Asn Pro Tyr Leu Ile  65 70 75 80 Asp Leu Thr Thr Leu Arg Glu Glu Gly Leu Leu His Ala Ser Asp Phe                  85 90 95 Glu Gly Thr Pro Asp Phe Asp Ala Gly Arg Val Asp Phe Gly Leu Gln             100 105 110 Tyr Val Trp Arg Met Gln Met Leu Gly Arg Ala Tyr Ala His Phe Ala         115 120 125 Phe Gly His His Pro Glu Leu Lys Ala Ala Phe Glu Ala Phe Lys Ala     130 135 140 Glu Glu Ala Ala Trp Leu Asp Asp Tyr Ala Leu Phe Met Ala Leu Lys 145 150 155 160 Asp Ala His Gly Gly Leu Pro Trp Asn Ala Trp Glu Pro Gly Thr Arg                 165 170 175 Asp Arg Glu Pro Gln Ala Leu Ala Ala Ala Gln Glu Gln Leu Ala Pro             180 185 190 Asn Ile Glu Arg Val Lys Phe Ile Gln Phe Leu Phe Phe Arg Gln Trp         195 200 205 Arg Ala Leu Arg Thr Tyr Ala Arg Glu Arg Gly Val Gly Val Ile Gly     210 215 220 Asp Ile Pro Ile Phe Val Ala Met Asp Ser Ser Asp Ala Trp Ala Asn 225 230 235 240 Arg Glu Gln Phe Tyr Phe Asp Glu Gln Gly Gln Pro Thr Val Val Ala                 245 250 255 Gly Val Pro Pro Asp Tyr Phe Ser Glu Thr Gly Gln Leu Trp Gly Asn             260 265 270 Pro Leu Tyr Arg Trp Asp Val Met Glu Gln Asp Gly Phe His Trp Trp         275 280 285 Ile Glu Arg Phe Arg Gly Ser Leu Lys Leu Tyr Asp Leu Ile Arg Ile     290 295 300 Asp His Phe Arg Gly Phe Ala Ala Tyr Trp Glu Ile Pro Tyr Pro Ala 305 310 315 320 Glu Asn Ala Ile His Gly Arg Trp Val Pro Ala Pro Gly His Ala Leu                 325 330 335 Leu Glu Ala Val Arg Ala Leu Gly Gln Met Pro Ile Ile Ala Glu             340 345 350 Asp Leu Gly Val Ile Thr Pro Asp Val Glu Gln Leu Arg Asp Asp Phe         355 360 365 Gly Leu Pro Gly Met Ala Val Leu His Phe Ala Phe Gly Gly Gly Asp     370 375 380 Phe Ser Val Asn Ala Phe Leu Pro His Asn Leu Lys Ala Asn Gln Val 385 390 395 400 Val Tyr Thr Gly Thr His Asp Asn Asp Thr Ser Arg Gly Trp Trp Gln                 405 410 415 His Ala Asp Glu Gln Glu Arg Gln Asn Phe Arg Leu Tyr Thr His Ser             420 425 430 Asp Pro Ser Glu Glu Thr Phe Ala Trp Gln Leu Thr Glu Ile Ala Leu         435 440 445 Glu Ser Arg Pro Asn Leu Ala Ile Val Pro Leu Gln Asp Leu Leu Asn     450 455 460 Leu Gly Ser Glu Ala Arg Met Asn Phe Pro Gly Thr Thr Gly Pro His 465 470 475 480 Asn Trp Thr Trp Arg Tyr Leu Ala Ala Asp Leu Arg Pro Asp Leu Ala                 485 490 495 Thr Lys Leu Arg Ala Leu Thr Glu Arg Thr Gly Arg Val Glu Arg             500 505 510

Claims (6)

Preparing a recombinant vector comprising a gene encoding 4-alpha-glucanotransferase consisting of the amino acid sequence of SEQ ID NO: 2 derived from Deinococcus geothermalis ;
Transforming the prepared recombinant vector into E. coli;
Isolating and purifying the 4-alpha-glucanotransferase enzyme from the transformed E. coli; And
Reacting the purified 4-α-glucanotransferase enzyme with amylose at a pH of 6.0 and a temperature of 50 ° C. for 3 to 6 hours, wherein the amylose having a degree of polymerization of 5 is completely produced Wherein the amylose is a main component comprising 5 to 10% by weight of cycloamylose and a cycloamylose having a degree of polymerization of 24 to 28. [ delete 2. The method according to claim 1, wherein the 4-alpha-glucanotransferase increases the rate of cyclization reaction rate to the coupling reaction rate. delete delete delete

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