KR101938366B1 - Method for production of 1-deoxynojirimycin derivative with a single glucose - Google Patents

Abstract

The present invention relates to a method for producing 1-deoxynojirimycin (DNJ) compound, which is one-deoxynojirimycin (DNJ) compound, A novel DNJ compound (G1-DNJ) in which one glucose was bound was obtained by transferring glucose to a DNJ (1-deoxynojirimycin) compound one time by using alpha-glucanotransferase comprising the amino acid sequence of SEQ ID NO: Can be manufactured. The thus prepared G1-DNJ compound further inhibits the hydrolysis ability of alpha glucosidease compared to the conventional DNJ, while it does not inhibit alpha amylase. This indicates that G1-DNJ of the present invention has a side effect of existing DNJ Inhibition) and can be actively used as a therapeutic agent for diabetes.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing 1-deoxynojirimycin derivatives,

The present invention relates to a novel process for preparing 1-deoxynojirimycin derivatives, and more particularly, to a process for producing 1-deoxynojirimycin derivatives by transferring glucose to a 1-deoxynojirimycin (DNJ) Deoxynojirimycin in which glucose is bound in one molecule.

1-deoxynojirimycin (DNJ) is a molecule in which an oxygen atom is substituted with a nitrogen atom, unlike aza-sugar or a common monosaccharide having a structure similar to a monosaccharide.

DNJ has been reported to inhibit the activity of α-amylase and α-glucosidase in the intestinal tract, thereby reducing blood sugar in the body (Narita, Y., & Inouye, K. (2009) , J. Agric . Food Chem ., 57 (19), 9218-9225). ≪ / RTI >

Among the two enzymes, alpha glucosidase is the final step enzyme that hydrolyzes the alpha-1,4 bond of bovine sugars to produce glucose from the non-reducing end. Therefore, the inhibition of alpha glucosideases plays an important role in the rapid increase of blood glucose and (. Yao, Y., Cheng, X., & Ren, G. (2014). α-Glucosidase inhibitory activity of protein-rich extracts from extruded adzuki bean in diabetic KK-Ay mice. Food Funct, 5 (5) , 966-971). Thus, DNJ can be used with significant benefit to diabetic patients in which an abrupt increase in blood glucose should be avoided. However, DNJ's inferiority to alpha amylase can interfere with the digestion of the absorbed starch in the body, which can lead to rather negative effects.

Thus, DNJ has both positive effects to prevent the rapid increase of blood sugar level in diabetic patients and negative effects that may interfere with starch digestion. Thus, there is a need to develop a DNJ analogue that can inhibit alpha glutathione without inhibiting alpha amylase.

Korean Patent Publication No. 10-2014-0047992 (Apr. 23, 2014) discloses a strain of Bacillus amyloliquefaciens 335N (KCCM11285P) producing 1-deoxynojirimycin (DNJ) A functional food having an antidiabetic, anticancer and antiviral effect including oxinojirimycin is described. Korean Patent Publication No. 10-2004-0071953 (published on Aug. 16, 2004) discloses a novel microorganism producing 1-deoxynojirimycin having blood glucose lowering and antiviral activity and And a composition containing the same, which is capable of effectively inhibiting alpha-glycosidase and thus being useful for the prevention and treatment of diabetes mellitus. Korean Patent Publication No. 10-2002-0071334 (published on September 12, 2002) discloses a method in which a silkworm powder is extracted with ethanol, filtered, and then subjected to a series of ion exchange column chromatography to obtain an excellent blood- Discloses a technique for separating 1-deoxynojirimycin and its hypoglycemic activity.

Narita, Y., & Inouye, K. (2009). Kinetic analysis and mechanism of the inhibition of chlorogenic acid and its components against porcine pancreas alpha-amylase isozymes I and II. J. Agric. Food Chem., 57 (19), 9218-9225.

In the present invention, a method for producing DNJ analogs capable of inhibiting alpha glutathione without inhibiting alpha amylase is developed and provided.

The present invention relates to a transglucose 1-deoxy-1-deoxyglucosyltransferase, which is characterized in that 1 glucose is transferred to 1-deoxynojirimycin using an alpha glucanotransferase enzyme comprising the amino acid sequence of SEQ ID NO: A method for producing nojirimycin is provided.

In the method for producing glucose-1-deoxynojirimycin of the present invention, the glucose may be present in a polymer formed by polymerizing glucose, for example. At this time, the polymer may be, for example, starch.

In the method for producing glucose-1-deoxynojirimycin of the present invention, the 1-deoxynojirimycin is, for example, at a concentration of 1 to 3.0% (w / v) in the reaction solution And the starch may be prepared, for example, at a concentration of 0.5 to 1.5% (w / v) in the reaction solution.

In the method for preparing glucose trans-1-deoxynojirimycin of the present invention, the reaction solution is preferably a 40 to 60 mM sodium acetate buffer solution having a pH of 5.5 to 6.5 as a solvent.

In the method for producing glucose-1-deoxynojirimycin of the present invention, it is preferable that the above-mentioned α-glucanotransferase enzyme is used in an amount of 0.5 to 1.5 U of an α-glucanotransferase enzyme per 1 mL of the reaction solution .

In the method for producing glucose-1-deoxynojirimycin of the present invention, it is preferable that the method for producing glucose-1-deoxynojirimycin is carried out at 50 to 60 ° C for 5 to 9 hours.

In the present invention, a novel DNJ compound (G1-DNJ) in which one glucose is bound is obtained by transferring glucose to a DNJ (1-deoxynojirimycin) compound by using alpha-glucanotransferase comprising the amino acid sequence of SEQ ID NO: . ≪ / RTI >

It was confirmed that the G1-DNJ compound thus prepared further inhibited the hydrolysis ability of alpha glucosidease compared to the conventional DNJ, but did not inhibit alpha amylase. This property means that G1-DNJ of the present invention can reduce the adverse effect (alpha amylase inhibition) of existing DNJ and can be actively used as a therapeutic agent for diabetes.

Figure 1 shows the bt_2146 gene (A) amplified by PCR and the recombinant expression plasmid pTKNdbt_2146 (B) containing the gene.
FIG. 2 is a graph showing SDS-PAGE analysis (Lane S, lane 1, lane 2, soluble fraction, lane 3, insoluble fraction, lane 1) of the purified alpha-glucanotransferase of the present invention (Bt? lane 4, purified Bt [alpha] GTase).
Figure 3 shows the results of the optimal temperature and pH analysis of recombinant Bt [alpha] gTase.
Fig. 4 shows the results of the comparison of the effects of the recombinant Bt [alpha] GTase with the cations.
Figure 5 shows the results of the reaction analysis of Bt [alpha] gTase enzyme for various donors.
FIG. 6 is a photograph showing a comparison of the results of the reaction between the common alpha-glucanotransferase and the Bt alpha GTase used in the present invention.
FIG. 7 shows the results of analysis of the sugar compound using HPAEC.
Fig. 8 shows the results of analysis of the sugar compound using MALDI-TOF.
FIG. 9 shows the results of the comparison of the inhibition of DNJ and G1-DNJ against the alpha Glucosidease using the Lineweaver-Burk plot.
FIG. 10 shows a result of reconstructing a Linewiever Burk plot with a Dixon plot and confirming the extent of inhibition of the substrate.
FIG. 11 shows the results of the comparison of the inhibition of DNJ and G1-DNJ for the alpha amylase using the Lineweaver-Burk plot.
12 shows the results of the comparison of the inhibition against alpha amylase using the Dixon plot.

In the present invention, a method for producing 1-deoxynojirimycin in which 1 glucose is bound to 1-deoxynojirimycin by transferring 1 glucose to it, comprising the steps of: preparing an alpha glue comprising the amino acid sequence of SEQ ID NO: 2 A method using cano transferase enzyme is provided.

Alpha glucanotransferase (Bt [alpha] GTase) comprising the amino acid sequence of SEQ ID NO: 2 of the present invention is derived from Bacteroides thetaiotaomicron . Unlike the conventional alpha glucanotransferase, It was found that the ability to chain is significantly low.

The common alpha-glucanotransferase transforms other glucose to a 'chain' in glucose (G1) to form a variety of long malto-oligosaccharides. However, BtαGTase does not use products of various lengths during the sugar chain reaction, (See Example 6 of the present invention below).

This means that only a specific product can be produced intensively, thereby enhancing the productivity of the product. The present invention can easily produce a novel compound in which one glucose is bound to 1-deoxynojirimycin You can. In addition, when the alpha glucano transferase of the present invention is used, a single kind of substance is produced mainly rather than producing various kinds of substances, so that it is advantageous that the purification can be facilitated.

On the basis thereof, the present invention provides a method for producing 1-deoxynojirimycin having the following structural formula 1 by using an alpha-glucanotransferase enzyme comprising the amino acid sequence of SEQ ID NO: 2, (For example, a compound represented by the following formula (2)). The compound represented by the following formula (2) is formed by ether bonding of the carbon at the 1-position of glucose and the 4-carbon of 1-deoxynojirimycin. Usually, It is also said to be combined.

[Chemical Formula 1]

(2)

In the method for producing glucose-1-deoxynojirimycin of the present invention, the glucose may be present in a polymer formed by polymerizing glucose, for example.

The present invention is characterized in that 1 glucose is transferred to 1-deoxynojirimycin. The donor glucose used herein may be used as a single molecule of glucose, but various polymers formed by combining glucose ) May be used after separation of glucose.

The alpha glucanotransferase having the amino acid sequence of SEQ ID NO: 2 of the present invention has the ability to remove glucose from a glucose polymer (e.g., starch), so that a glucose polymer can be used as a substrate for the reaction. However, the polymer containing glucose is not necessarily made of only glucose, and a polymer formed by bonding other molecules to glucose may be used.

On the other hand, in the method for producing glucose-1-deoxynojirimycin of the present invention, 1-deoxynojirimycin is dissolved in a concentration of 1 to 3.0% (w / v) in the reaction solution And the starch serving as a glucose donor can be prepared, for example, at a concentration of 0.5 to 1.5% (w / v) in the reaction solution. At this time, the reaction solution, that is, the solvent, can preferably use a 40 to 60 mM sodium acetate buffer solution having a pH of 5.5 to 6.5.

On the other hand, in the method for producing glucose-1-deoxynojirimycin of the present invention, the α-glucanotransferase enzyme preferably uses 0.5 to 1.5 U of an α-glucanotransferase enzyme per 1 mL of the reaction solution good.

On the other hand, the process for producing glucose-1-deoxynojirimycin of the present invention is preferably performed at 50 to 60 ° C for 5 to 9 hours.

The glucose trans-1-deoxynojirimycin prepared in the present invention is more effective than the 1-deoxynojirimycin compound, which is known to have a hypoglycemic effect, as compared to the α-glucosidase (1-deoxynojirimycin) The activity against α-amylase is removed while maintaining the activity for the α-amylase.

1-deoxynojirimycin has an inhibitory effect on alpha-glucosidase and can inhibit the rapid increase of blood sugar, but also has an inhibitory activity against alpha amylase and thus has a negative side that can interfere with the digestive action of starch have. However, the 1-deoxynojirimycin (G1-DNJ) in which one glucose is linked to? -1,4 bond developed by the present invention can inhibit only alpha glucosidease without inhibiting alpha amylase Which can overcome the negative problem of 1-deoxynojirimycin as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[Example 1: Cloning of novel? -1,4-alpha glucanotransferase]

The genomic DNA of Bacteroides thetaiotaomicron was amplified with two primers ( bt_2146 ) encoding alpha-1,4-alpha glucanotransferase. The sequence of the primers was as follows.

Primer name order bt-Forward 5'-AAAACCATGGCCACTGTATCATTTAAC-3 ' bt-Reverse 5'-AAAACTCGAGTTTCTTGGGAGCTCTGCC-3 '

The amplification conditions of the PCR were 98 ° C for 10 seconds, 53 ° C for 30 seconds, and 72 ° C for 1 minute and 30 seconds, followed by 30 cycles of denaturation at 98 ° C for 1 minute. The amplified gene was ligated to pTKNd6xHis vector by treatment with Nco I and Xho I restriction enzyme.

Figure 1 shows the bt_2146 gene (A) amplified by PCR and the recombinant expression plasmid pTKNdbt_2146 (B) containing the gene. As a result of the nucleotide sequence analysis, the bt_2146 gene was confirmed to have the nucleic acid sequence of SEQ ID NO: 1 and was confirmed to encode the alpha glucano transferase having the amino acid sequence of SEQ ID NO: 2.

[Example 2: Expression of recombinant protein]

The recombinant expression plasmid pTKNdbt_2146 was transformed into E. coli using calcium ion treatment and then transformed into LB medium (10 g / L bacto-tryptone, 5 g / L yeast extract, 5 g / L) supplemented with kanamycin g / L NaCl), and the mixture was stirred at 30 rpm for 30 hours at 150 rpm.

The cultured strains were harvested by centrifugation (7,000 × g, 20 min, 4 ° C) and lysed in a lysis buffer (300 mM NaCl, 50 mM Tris-HCl buffer pH 7.5), 10 mM imidazole). Then, the cells were disrupted using an ultrasonic disintegrator (output 12, 5 min, 4 times) and used as crude enzyme solution. The crude enzyme solution was purified using Ni-NTA affinity adsorption chromatography and purified and its purity was confirmed by SDS-PAGE.

Figure 2 shows the results of SDS-PAGE analysis (Lane S, lane 1, lane 2, soluble fraction, lane 3, insoluble fraction, lane 4) of purified alpha-glucanotransferase , purified BtαGTase).

[Example 3: Measurement of potency of recombinant BtαGTase]

BtαGTase was prepared by dissolving amylose (0.02%, (w / v)) maltose (0.05%, (w / v)) as a substrate in citric-NaOH buffer solution (pH 3.0) The reaction was carried out at 60 ° C for 10 minutes. Then, 100 μL of the enzyme reaction mixture was mixed with 1 mL of Lugol solution (0.02% (w / v) iodine and 0.2% (w / v) potassium iodide) and the absorbance at 620 nm was measured. 1 unit was defined as the amount by which 1 μg of amylose was degraded and converted to maltose by enzyme for 1 minute.

Table 2 summarizes the various factors of recombinant BtαGTase.

Step Volume (mL) Enzyme activity (U) Protein concentration (mg / mL) Protein amount (mg) Specific activity (U / μg) Yield (%) Purification fold Crude enzyme solution 104 3.57 169.44 17621.76 0.20 ^-3 100 1.00 Ni-NTA purification 1.5 2.03 2.26 3.39 0.59 56.8 2953.20

[ Example  4: Recombinant enzyme Of BtαGTase  Comparison of Optimum Reaction Conditions]

To determine the optimal reaction conditions for the recombinant enzyme BtαGTase, the activity was measured at various temperatures (40-70 ° C) and pH (2.5-6.0). The highest value was 100% and the relative titer was shown. BtαGTase enzyme showed the highest activity at 60 ℃ and pH 3.0.

Figure 3 shows the results of the optimal temperature and pH analysis of recombinant Bt [alpha] gTase. The optimum temperature was 60 ℃ and the optimum pH was 3.

[ Example  5: Recombinant enzyme Of BtαGTase  Ion effect comparison]

To determine whether the recombinant enzyme BtαGTase is affected by the ions, the enzyme activities were compared using various divalent cations and EDTA. (100%), and the final concentration of each divalent cation was set to 5 mM.

Fig. 4 shows the results of the comparison of the effects of the recombinant Bt [alpha] GTase with the cations. As shown in FIG. 4, the addition of Ca ²⁺ , Mg ²⁺ , and Cu ²⁺ showed an increase in potency.

[Example 6: Reaction characteristics of recombinant BtαGTase enzyme]

Tissue transfer reactions were performed on various acceptor materials, and their pattern was analyzed by thin layer chromatography (Thin layer chromatorgraphy). The reaction was carried out on a receptor (0.3% (w / v)) from glucose (G1) to maltoheptaose (G7) with 1% (w / v) starch as a donor molecule.

As shown in FIG. 5, BtαGTase used in the present invention has a significantly lower ability to chain the glycosylation reaction unlike the conventional alpha glutaryl transferase. This may seem like a disadvantage at first glance, but if you look at the details below, you can see that this is a great advantage. Figure 5 shows the results of the reaction analysis of Bt [alpha] gTase enzyme for various donors.

On the other hand, for more clear comparison, the reaction was carried out under the same conditions as those of ordinary alpha glucano transferase (MalQ). FIG. 6 is a photograph showing a comparison of the results of the reaction between the common alpha-glucanotransferase and the Bt alpha GTase used in the present invention. As shown in FIG. 6, the common alpha-glucan transferase transforms other glucose to 'chain' in glucose (G1) to form various long maltooligosaccharides. However, the BtαGTase of the present invention uses various lengths of products It can be seen that only one party of the party can produce mainly the product.

This means that it is possible to increase the productivity of the product by producing only specific products in an 'intensive' manner. In addition, it means that it is advantageous to make the purification easier because it mainly produces a single kind of substance as compared with producing various kinds of substances.

[ Example  7: New DNJ The party  Production and purification of the compound]

In order to carry out the reaction, 1.5% (w / v) DNJ and 1% (w / v) starch were added to a pH 6.0, 50 mM sodium acetate buffer solution, and 1 U of αglucanotransferase enzyme And the reaction was carried out at 55 ° C for 7 hours. Then, the reaction solution was boiled for 10 minutes and the enzyme reaction was allowed to take place.

The DNJ conjugate was purified by preparative high performance liquid chromatography (YMC Korea, Sungnam, Korea) combined with a UV detector (200 and 210 nm) on a Triart-C18 column (250 × 20 mm, YMC Korea) . The amount of sample injected per experiment was 1 mL, deionized water containing 0.1% ammonium was used as the mobile phase, and the flow rate was 12.0 mL / min and separated at room temperature.

[ Example  8: New DNJ The party  Analysis of compound]

A high performance anion exchange chromatography (HPAEC) assay was used to determine the purification and purity of the precursor compound produced in Example 7 above.

HPAEC was instrumented with a pulsed aerometric detector (ED40; Dionex) detector on a CarboPac ™ PA1 column (4 × 250 mm; Dionex, Sunnyvale, CA, USA). A 20 μ sample was used for the analysis and the reaction products were analyzed by sodium acetate concentration gradient on a 150 mM sodium hydroxide solution mobile phase.

Sodium acetate concentration gradient was as follows. 0-2% for 0-20 min, 2-40% for 20-58 min, 40-100% for 58-68 min, 100% for 68-70 min, 100-0% for 70-78 min. The flow rate of the mobile phase was set at 1.0 mL / min.

In addition, the molecular weight of the purified precursor product was confirmed by using Matrix Assisted Laser Desorption / Ionization Time-of-Flight (MALDI-TOF). The compound dissolved in methanol was mixed with the matrix at a ratio of 1: 1, and then the maltitol was analyzed at room temperature. The matrix used was? -Cyano-4-hydroxycinnamic acid (CHCA) and the accelerating voltage was set at 20,000 volts.

FIG. 7 shows the results of analysis of the glycidyl compound using HPAEC, and FIG. 8 shows the results of analysis of the glycidyl compound using MALDI-TOF. The resulting precursor compound was confirmed to be a compound having one more glucose added to the conventional DNJ compound, and it was confirmed to have the structural formula shown in the above formula (2).

The newly generated prodrug compound was named G1-DNJ and the following enzyme inhibition experiment was carried out.

[ Example  9: Alpha Glucose Days and Alpha Amylase's  Check inhibitory effect]

Inhibition kinetics was performed to confirm the inhibition of enzyme activity of DNJ and G1-DNJ. In the experiment of alpha glucosides, pNPG ( p- nitrophenyl-α-D-glucopyranoside) was used as a substrate and pNPG2 ( p- nitrophenyl-α-D-maltoside ) Was used. The amount of pNP produced by hydrolysis of each substrate was measured at 405 nm using an ELISA plate reader.

To determine the Ki value as an indicator of substrate inhibition, enzymatic reactions were performed using various concentrations (0.1-1 μmol) of inhibitor and the values were calculated using Dixon-plot.

FIG. 9 shows the results of the comparison of the inhibition of DNJ and G1-DNJ against the alpha Glucosidease using the Lineweaver-Burk plot. As shown in Fig. 9, it was confirmed that both DNJ and G1-DNJ had an inhibitory effect on alpha-glucosidase. In addition, it was confirmed that these materials are competitive inhibition. For reference, both graphs were collected at one point on the left, which means that even with varying concentrations of inhibitor ultimately having the same K _m value, inhibitors that are similar to the substrate are "'Competitiveinhibitors' that compete with substrates to inhibit enzyme activity. In general, many pharmaceutical materials act as competitive inhibitors of certain proteins.

On the other hand, a Dixon plot was constructed to determine whether any of the two substances (DNJ and G1-DNJ) were more effective inhibitors, and the inhibition constant (K _i value) was calculated. FIG. 10 is a result of modifying the Linewiever Burk plot and restructuring it with Dixon plot, thereby confirming the degree of substrate inhibition. The K _i values of DNJ and G1-DNJ were 1.1 and 0.48, respectively. This means that G1-DNJ is an effective inhibitor than DNJ. For reference, K _i The lower the value, the higher the inhibition efficiency.

DNJ and G1-DNJ both act as inhibitors against alpha-glucosidase through the kinetic study described above, indicating that G1-DNJ is a more potent inhibitor of alpha glucosidase.

On the other hand, a kinetic study on alpha amylase was performed by the same method as described above, and the results were as shown in FIG. FIG. 11 shows the results of the comparison of the inhibition of DNJ and G1-DNJ for the alpha amylase using the Lineweaver-Burk plot. As shown in FIG. 11, it was confirmed that the slope of the graph was increased (the reaction rate was inhibited) as the concentration increased. However, although the concentration of the inhibitor was increased, There was little change. This means that G1-DNJ has no specific inhibitory effect on alpha amylase.

Dixon-plot was modified for more definite proof, and the result was as shown in Fig. 12 shows the results of the comparison of the inhibition against alpha amylase using the Dixon plot. As shown in FIG. 12, the DNJ has an inferiority to the alpha amylase and can calculate the K _i value. However, since G 1 -DNJ does not have an inferiority, K _i Value can not be obtained. This means that G1-DNJ lacks the ability to inhibit alpha amylase.

The results of the comparison between DNJ and G1-DNJ against Alpha Glucosides and Alpha Amylase are summarized in Table 3 below.

inhibitor Target enzyme K _i inhibition type DNJ
α-glucosidase 1.1 competitive α-amylase 4.4 competitive G1-DNJ
α-glucosidae 0.48 competitive α-amylase - No inhibition

<110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of 1-deoxynojirimycin derivative with a          단 glucose <130> YP-17-076 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 2682 <212> DNA <213> Bacteroides thetaiotaomicron <400> 1 atgactgtat catttaacat cgaatatcgc accagttggg gagaagaagt caggattgcc 60 gggctgctgc cggagtcgat ccccatgcat accacagacg gcatatactg gacggcagac 120 gtggaactgg aagttcccaa agaaggaatg actatcaatt acagctatca gatagaacaa 180 aaccaaatta tcatccgcaa agaatgggac agctttccga gacgtctctt tttatccggt 240 aactctaaaa agaagtatca gattaaagac tgctggaaaa atatccccga acaattgtat 300 tactacagtt cggccttcac ggaagccttg ttagcccatc ccgacagagc tgaaattccg 360 ccttgtcaca gaaaagggct ggtcatcaag gcatacgccc cacgcatcaa caaagactat 420 tgcctggcaa tttgcggaaa tcagaaagca ttgggaaact gggacccgga caaagcgatt 480 cccatgagcg atgccaactt tccggaatgg cagatcgagc tggatgcaag taagctgaaa 540 ttcccgctgg aatacaaatt catcctttac cataaagaag aaaagaaagc ggactgctgg 600 gaaaataacc ccaaccgcta tcttgccgac ccggaactga aaacaaatga gacgctggtc 660 atctccgacc gatatgccta tttcgatatt ccggtatgga aaggggccgg tatagccatc 720 cctgtctttt cactgaaatc agagaacagt ttcggagtag gagatttcgg agatttgaaa 780 cgcatgattg actgggctgt aagtacccaa caaaaggtta tacagatatt accgatcaat 840 gatacgacca tgactcatgc gtggaccgat tcttatccat ataatagtat ctccatttat 900 gctttccacc cgatgtatgc agatataaag cagatgggaa cactgaaaga caagtcagcc 960 gcagcgaagt tcaacaaaaa acagaaagaa ctcaacggcc tgccggcgat ggattatgaa 1020 gctgtcaacc aaacgaaatg ggaatacttc cgactgatct tcaagcagga aggagaaaaa 1080 gtattagcct ccggagagtt cggcgaattc tttaatgcca acaaggaatg gctgcaacct 1140 tacgctgttt tcagctatct gcgcgatgcc tttcagacac ctaatttccg tgaatggccc 1200 agacattccg tttataatgc acaagatata gagaagatgt gccggccaga atctgtagat 1260 tacccgcata tcgcacttta ttattatatc cagtttcacc tgcacctgca attggtagcc 1320 gctacgaaat acgcccgcga acacggtgtc gtactcaaag gagatatccc tatcggcatc 1380 agccgcaaca gtgtggaagc atggaccgaa ccttattact tcaatctcaa cgggcaggcg 1440 ggcgcccctc ccgatgattt ctcggtgaac ggacaaaact ggggattccc cacgtacaac 1500 tgggacgtca tggaaaagga cggctaccgc tggtggatga agcgtttcca aaaaatgtca 1560 gagtacttcg acgcttaccg gattgaccac atcctcggtt tcttccgtat ctgggaaatc 1620 ccgatgcatg ccgtacacgg attactcgga cagttcattc cctccatccc tatgagtcgg 1680 gaggaaatcg aaagttacgg actccccttt cgggaagaat atttgattcc atatattcat 1740 gaatccttcc tcgggcaagt attcggtccg cataccgatt atgtgaaaca gacttttctg 1800 cttcctgccg aaacacccgg tgtttatcac atgaaaccgg agttcaccac ccaacgggaa 1860 gtggagagtt tctttgcagg aaagaatgat gaaaacagtc tttggattcg ggatggacta 1920 tacactttga tcagtgatgt tctttttgta ccggacacga aagaaaaaga taaatatcat 1980 ccgcgcatcg gaatacaacg cgatttcatc ttccgttcac tcaatgaaca ggagcagaat 2040 gcgtttaaca gactgtatga tcagtattat tatcaccgcc acaacgagtt ctggcgccaa 2100 caggctatga agaaactacc gcaactgaca caatcgacac gtatgctggt ttgcggagaa 2160 gacctgggta tgattccgga ttgtgtttcg tccgtcatga atgatcttcg tatcctcagt 2220 ctggagattc agcggatgcc aaagaatccg atgcacgagt tcggatattt aaatgaatac 2280 ccataccggt cggtttgtac catctccact catgatatgt ctactttgcg aggctggtgg 2340 gaagaagatt acctgcagac acagcgttac tacaacacga tgctggggca ttacggaacc 2400 gcaccgacag tcgccactcc cgaattgtgc gaagaagtgg tgcgcaatca tctgaaaagc 2460 aattccatac tttgtattct gtccctgcaa gactggctga gcattgatgg caaatggaga 2520 aatccgaatg tgcaggaaga acggattaat gtaccatcca atccgcgcaa ttattggcgt 2580 taccggatgc acctcactct ggaacagttg atgaaagcag aagaactcaa tgacaagata 2640 cgtgagctaa taaaatacac cggcagagct cccaagaaat aa 2682 <210> 2 <211> 893 <212> PRT <213> Bacteroides thetaiotaomicron <400> 2 Met Thr Val Ser Phe Asn Ile Glu Tyr Arg Thr Ser Trp Gly Glu Glu   1 5 10 15 Val Arg Ile Ala Gly Leu Leu Pro Glu Ser Ile Pro Met His Thr Thr              20 25 30 Asp Gly Ile Tyr Trp Thr Ala Asp Val Glu Leu Glu Val Pro Lys Glu          35 40 45 Gly Met Thr Ile Asn Tyr Ser Tyr Gln Ile Glu Gln Asn Gln Ile Ile      50 55 60 Ile Arg Lys Glu Trp Asp Ser Phe Pro Arg Arg Leu Phe Leu Ser Gly  65 70 75 80 Asn Ser Lys Lys Lys Tyr Gln Ile Lys Asp Cys Trp Lys Asn Ile Pro                  85 90 95 Glu Gln Leu Tyr Tyr Tyr Ser Ser Ala Phe Thr Glu Ala Leu Leu Ala             100 105 110 His Pro Asp Arg Ala Glu Ile Pro Pro Cys His Arg Lys Gly Leu Val         115 120 125 Ile Lys Ala Tyr Ala Pro Arg Ile Asn Lys Asp Tyr Cys Leu Ala Ile     130 135 140 Cys Gly Asn Gln Lys Ala Leu Gly Asn Trp Asp Pro Asp Lys Ala Ile 145 150 155 160 Pro Met Ser Asp Ala Asn Phe Pro Glu Trp Gln Ile Glu Leu Asp Ala                 165 170 175 Ser Lys Leu Lys Phe Pro Leu Glu Tyr Lys Phe Ile Leu Tyr His Lys             180 185 190 Glu Glu Lys Lys Ala Asp Cys Trp Glu Asn Asn Pro Asn Arg Tyr Leu         195 200 205 Ala Asp Pro Glu Leu Lys Thr Asn Glu Thr Leu Val Ile Ser Asp Arg     210 215 220 Tyr Ala Tyr Phe Asp Ile Pro Val Trp Lys Gly Ala Gly Ile Ala Ile 225 230 235 240 Pro Val Phe Ser Leu Lys Ser Glu Asn Ser Phe Gly Val Gly Asp Phe                 245 250 255 Gly Asp Leu Lys Arg Met Ile Asp Trp Ala Val Ser Thr Gln Gln Lys             260 265 270 Val Ile Gln Ile Leu Pro Ile Asn Asp Thr Thr Met Thr His Ala Trp         275 280 285 Thr Asp Ser Tyr Pro Tyr Asn Ser Ile Ser Ile Tyr Ala Phe His Pro     290 295 300 Met Tyr Ala Asp Ile Lys Gln Met Gly Thr Leu Lys Asp Lys Ser Ala 305 310 315 320 Ala Ala Lys Phe Asn Lys Lys Gln Lys Glu Leu Asn Gly Leu Pro Ala                 325 330 335 Met Asp Tyr Glu Ala Val Asn Gln Thr Lys Trp Glu Tyr Phe Arg Leu             340 345 350 Ile Phe Lys Gln Glu Gly Glu Lys Val Leu Ala Ser Gly Glu Phe Gly         355 360 365 Glu Phe Phe Asn Ala Asn Lys Glu Trp Leu Gln Pro Tyr Ala Val Phe     370 375 380 Ser Tyr Leu Arg Asp Ala Phe Gln Thr Pro Asn Phe Arg Glu Trp Pro 385 390 395 400 Arg His Ser Val Tyr Asn Ala Gln Asp Ile Glu Lys Met Cys Arg Pro                 405 410 415 Glu Ser Val Asp Tyr Pro His Ile Ala Leu Tyr Tyr Tyr Ile Gln Phe             420 425 430 His Leu His Leu Gln Leu Val Ala Ala Thr Lys Tyr Ala Arg Glu His         435 440 445 Gly Val Val Leu Lys Gly Asp Ile Pro Ile Gly Ile Ser Arg Asn Ser     450 455 460 Val Glu Ala Trp Thr Glu Pro Tyr Tyr Phe Asn Leu Asn Gly Gln Ala 465 470 475 480 Gly Ala Pro Pro Asp Phe Ser Val Asn Gly Gln Asn Trp Gly Phe                 485 490 495 Pro Thr Tyr Asn Trp Asp Val Met Glu Lys Asp Gly Tyr Arg Trp Trp             500 505 510 Met Lys Arg Phe Gln Lys Met Ser Glu Tyr Phe Asp Ala Tyr Arg Ile         515 520 525 Asp His Ile Leu Gly Phe Phe Arg Ile Trp Glu Ile Pro Met Met His Ala     530 535 540 Val His Gly Leu Leu Gly Gln Phe Ile Pro Ser Ile Pro Met Ser Arg 545 550 555 560 Glu Glu Ile Glu Ser Tyr Gly Leu Pro Phe Arg Glu Glu Tyr Leu Ile                 565 570 575 Pro Tyr Ile His Glu Ser Phe Leu Gly Gln Val Phe Gly Pro His Thr             580 585 590 Asp Tyr Val Lys Gln Thr Phe Leu Leu Pro Ala Glu Thr Pro Gly Val         595 600 605 Tyr His Met Lys Pro Glu Phe Thr Thr Gln Arg Glu Val Glu Ser Phe     610 615 620 Phe Ala Gly Lys Asn Asp Glu Asn Ser Leu Trp Ile Arg Asp Gly Leu 625 630 635 640 Tyr Thr Leu Ile Ser Asp Val Leu Phe Val Pro Asp Thr Lys Glu Lys                 645 650 655 Asp Lys Tyr His Pro Arg Ile Gly Ile Gln Arg Asp Phe Ile Phe Arg             660 665 670 Ser Leu Asn Glu Gln Glu Gln Asn Ala Phe Asn Arg Leu Tyr Asp Gln         675 680 685 Tyr Tyr Tyr His Arg His Asn Glu Phe Trp Arg Gln Gln Ala Met Lys     690 695 700 Lys Leu Pro Gln Leu Thr Gln Ser Thr Arg Met Leu Val Cys Gly Glu 705 710 715 720 Asp Leu Gly Met Ile Pro Asp Cys Val Ser Ser Val Met Asn Asp Leu                 725 730 735 Arg Ile Leu Ser Leu Glu Ile Gln Arg Met Pro Lys Asn Pro Met His             740 745 750 Glu Phe Gly Tyr Leu Asn Glu Tyr Pro Tyr Arg Ser Val Cys Thr Ile         755 760 765 Ser Thr His Asp Met Ser Thr Leu Arg Gly Trp Trp Glu Glu Asp Tyr     770 775 780 Leu Gln Thr Gln Arg Tyr Tyr Asn Thr Met Leu Gly His Tyr Gly Thr 785 790 795 800 Ala Pro Thr Val Ala Thr Pro Glu Leu Cys Glu Glu Val Val Arg Asn                 805 810 815 His Leu Lys Ser Asn Ser Ile Leu Cys Ile Leu Ser Leu Gln Asp Trp             820 825 830 Leu Ser Ile Asp Gly Lys Trp Arg Asn Pro Asn Val Gln Glu Glu Arg         835 840 845 Ile Asn Val Pro Ser Asn Pro Arg Asn Tyr Trp Arg Tyr Arg Met His     850 855 860 Leu Thr Leu Glu Gln Leu Met Lys Ala Glu Glu Leu Asn Asp Lys Ile 865 870 875 880 Arg Glu Leu Ile Lys Tyr Thr Gly Arg Ala Pro Lys Lys                 885 890

Claims (7)

1-deoxynojirimycin was treated with the alpha-glucanotransferase enzyme consisting of the amino acid sequence shown in SEQ ID NO: 2 at 50 to 60 DEG C for 5 to 9 hours to obtain a sugar derivative &Lt; / RTI &gt;
Deoxynojirimycin having a structure represented by the following formula (2): &quot; (1) &quot;
(2)

The method according to claim 1,
The glucose,
Wherein the glucose is present in a polymer formed by polymerization of glucose.
3. The method of claim 2,
The polymer,
Starch. &Lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt;
The method of claim 3,
The 1-deoxynojirimycin may be, for example,
Prepared at a concentration of 1 to 3.0% (w / v) in the reaction solution,
The starch,
Wherein the solution is prepared at a concentration of 0.5 to 1.5% (w / v) in the reaction solution.
5. The method of claim 4,
The reaction solution may contain,
A method for preparing glucose trans- &lt; RTI ID = 0.0 &gt; 1-deoxynojirimycin, &lt; / RTI &gt; characterized by using a buffer solution of 40 to 60 mM sodium acetate at a pH of 5.5 to 6.5 as a solvent.
5. The method of claim 4,
The above-mentioned alpha-glucanotransferase enzyme can be produced,
A method for producing glucose trans-1-deoxynojirimycin, which comprises using 0.5 to 1.5 U of an α-glucanotransferase enzyme per 1 mL of a reaction solution.
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