KR101261004B1 - Method for production of lactulose from lactose using by cellobiose 2-epimerase - Google Patents

Abstract

The present invention relates to a method for producing lactulose from lactose using cellobiose 2-epimerase, and more particularly, to the cellobiose 2-epimerase gene. It relates to a recombinant expression vector comprising, microorganisms transformed therewith and a production method for obtaining lactulose using them.
According to the present invention, it is possible to produce lactulose directly from inexpensive and easily secured lactose by using cellobiose 2-epimerase, and can be usefully used in manufacturing functional foods and pharmaceuticals.

Description

Method for production of lactulose from lactose using by cellobiose 2-epimerase}

The present invention relates to a method for producing lactulose from lactose using cellobiose 2-epimerase, and more particularly, to the cellobiose 2-epimerase gene. It relates to a production method for obtaining lactulose using a recombinant expression vector comprising, microorganisms and enzymes transformed therewith.

Lactulose is an isomeric sugar of lactose in which the glucose portion of lactose is replaced by fructose and is a sugar rarely present in nature. Lactulose is not degraded by beta galactosidase in the small intestine and reaches the large intestine and is used by various lactic acid bacteria including Bifidobacterium, which lowers the pH of the large intestine, thus inhibiting the growth of harmful bacteria and promoting the colonization of the large intestine. The effect is to improve. In addition, lactulose is a non-digestible sugar that acts as a soluble fiber and is used for the treatment of brain diseases caused by constipation and chronic liver disease. Recently, lactulose promotes calcium absorption in postmenopausal women (Eurpean Journal of Clinical Nutrition Vol 58 no 3 pp 462-466 (2004); Biochimica Biophysica Acta Vol 110 pp 635-636 (1965); Journal of Clinical Investigation Vol 75 no 2 pp 608-613 (1985) ;; Journal of Bone and Mineral Research Vol 14 no 7 pp 1211-1216 (1999)); and industrially, lactulose has a higher sweetness and solubility than lactose, leading to baking and It can be usefully used in the confectionery industry (Bulletin of the International Dairy Federation Vol 212 pp 69-76 (1987)).

Such lactulose does not exist in crude oil but is contained in small amounts in heat-treated milk. Lactulose can be produced by chemical methods of isomerizing lactose under strong alkaline conditions and enzymatic methods using the galactose transfer activity of β-galactosidase (Trend in Food Science and Technology Vol 18 no 7 pp 356 -364 (2007); Enzyme and Microbial Technology Vol 38 no 4 pp 903-908 (2006)). The chemical method reacts by adding strong alkali compounds such as sodium hydroxide, calcium hydroxide and potassium hydroxide, and in the process of neutralization, not only causes the environmental pollution by using strong acid, but also decomposes lactulose produced under strong alkali conditions and produces by-products. The process and the lactulose purification process have complex disadvantages. On the contrary, the enzymatic method using beta galactosidase has a simple advantage because it is environmentally friendly and there are no by-products, but low yield has been a problem. In addition, lactose as well as fructose as a substrate to be used in the reaction, and the reaction is economically inefficient because the reaction is performed only at a high substrate concentration. Therefore, research is needed to find and apply enzymes that are eco-friendly and can produce lactulose in high yield using only one substrate of lactose.

The present invention solves the above problems, and the object of the present invention is to provide an enzyme capable of producing lactulose (lacactose) from lactose.

Another object of the present invention is to provide a method capable of producing lactulose from lactose.

Another object of the present invention is to provide a composition capable of producing lactulose from lactose.

In order to achieve the above object, the present invention provides a cellobiose 2-epimerase used in the production of lactulose.

In one embodiment of the present invention, the cellobiose 2-epimerase preferably has an amino acid sequence as set out in SEQ ID NO: 1, one or more substitutions, deletions, inversions, translocations, etc. All mutant polypeptides capable of achieving the object of the present invention through mutations are also included in the scope of the present invention.

In another embodiment of the present invention, the cellobiose 2-epimerase is preferably encoded by a gene having the nucleotide sequence set forth in SEQ ID NO: 2, but considering the genetic code degeneracy, All gene sequences having at least 80% homology, preferably at least 90% homology with the nucleotide sequences set forth in SEQ ID NO: 2, taking into account the mutants, are included in the scope of the present invention.

In another embodiment of the present invention, the cellobiose 2-epimerase is preferably derived from a Caldicellulosiruptor saccharolyticus strain, but is not limited thereto.

In another aspect, the present invention provides a recombinant expression vector comprising a cellobiose 2-epimerase gene having a nucleotide sequence of SEQ ID NO: 2 derived from a Caldicellulosiruptor saccharolyticus strain.

In one embodiment of the invention, the vector preferably has a cleavage map described in Figure 5, but is not limited thereto.

If the recombinant expression vector of the present invention can be used for the production of lactulose, any vector used in the genetic recombination method, including the expression vector pET24a (+), can be used, and as the microorganism transformed with the recombinant expression vector E. coli It is preferable to use ER 2566, but any strain can be used as long as it can be transformed with a recombinant expression vector to overexpress the desired gene and produce an active enzyme protein.

The present invention also provides a recombinant expression vector of the present invention;

b) culturing the microorganism transformed with the recombinant vector;

c) induce expression of said cellobiose 2-epimerase gene; And

d) isolating and purifying the expressed recombinant protein; It provides a method for obtaining the cellobiose 2-epimerase of claim 1 comprising in the process.

The present invention also provides a method for producing lactulose using the cellobiose 2-epimerase of the present invention.

In one embodiment of the present invention, the lactulose production method is preferably to use lactose as a substrate, but is not limited thereto.

In another aspect, the present invention provides a composition for producing lactulose comprising the cellobiose 2-epimerase of the present invention.

Hereinafter, the present invention will be described.

The present inventors have made many efforts, kaldi cellulose with low-saccharide syrup Sat ET carcass (Caldicellulosiruptor to more effectively stable in an environmentally friendly way to develop a method for producing a lactulose (lactulose) It was first confirmed that cellobiose 2-epimerase found in saccharolyticus can produce lactulose from lactose, and the present invention was completed.

The invention kaldi cellulose with low-saccharide syrup Sat ET carcass (Caldicellulosiruptor saccharolyticus) a cellobiose 2-epi Murray's fabric is joined to a recombinant expression vector and the resulting transformed microorganism and, by using this epi-2-cellobiose Murray's derived from It provides a method for producing, and a method for producing lactulose using the cellobiose 2-epimerase.

The invention as it is capable of producing a cellulose kaldi lactulose (lactulose) with lactose (lactose) as a substrate saccharide syrup soil roller ET carcass (Caldicellulosiruptor saccharolyticus ) cellopose 2-epimerase from the strain is provided.

In one preferred embodiment of the invention, a) a cellulose kaldi saccharide syrup sat Laura ET carcass (Caldicellulosiruptor a genomic DNA of the saccharolyticus strain was used as a template, and PCR was carried out using primers of SEQ ID NOs: 3 and 4 to amplify a DNA fragment containing the cellobiose 2-epimerase gene (SEQ ID NO: 2). ; B) DNA fragments containing the amplified cellobiose 2-epimerase gene were treated with restriction enzymes Nhe I and Eco RI, and then cloned into the expression vector pET24a (+) to clone the recombinant expression vector pET24a / cellobiose 2-epimerlay. Fabricate; C) transform it into E. coli ER 2566 strain by a conventional transformation method; D) culturing the transformed Escherichia coli and inducing overexpression to produce cellobiose 2-epimerase; And e) separate and purify the expressed cellobiose 2-epimerase.

In addition, the cellobiose 2-epimerase protein expressed in the step e), (a) disrupting the microorganism; (b) the cell debris is centrifuged to obtain a supernatant; (c) separating the supernatant by fast protein liquid chromatography; Enzyme liquid can be separated and purified by the process.

In addition, the present invention provides a production method for obtaining lactulose in high yield using a cellobiose 2-epimerase prepared by the above method.

Specifically, the present invention provides a method for producing lactulose using the cellobiose 2-epimerase capable of characteristically synthesizing lactulose without adding coenzyme.

Cellobiose 2-epimerase of the present invention is cultured E. coli transformed with a recombinant expression vector comprising a cellobiose 2-epimerase gene as described above to induce the expression of the recombinant enzyme gene and then expressed the recombinant protein expressed Preference is given to using those prepared in the process of separation and purification.

In addition, the lactulose is preferably synthesized using lactose as a substrate, and the concentration of the substrate is preferably in the range of 50 g / L to 700 g / L, more preferably in the range of 700 g / L. Do. In addition, the enzyme reaction is preferably in the range of pH 7.0 to pH 7.5, more preferably in the range of pH 7.5 and more preferably, the enzyme reaction is preferably made in the range of 75 ℃ to 80 ℃, the temperature of 75 ℃ Most preferably done at temperature. In addition, the enzyme reaction time may be appropriately adjusted according to conventional methods.

The present invention provides a recombinant expression vector comprising a cellobiose 2-epimerase gene, a microorganism transformed therefrom, and a method for producing cellobiose 2-epimerase using a large amount thereof, and the cellobiose 2-epimerase. It is effective to provide a production method for obtaining lactulose using a high yield.

In addition, the production method of lactulose according to the present invention can be used in the production of functional foods and medicines, etc., since it can produce lactulose from lactose in high yield without addition of fructose as compared to conventional enzymatic methods.

Figure 1a shows the enzyme activity according to the pH of the cellobiose 2-epimerase of the present invention (●: PIPES buffer; ○: EPPS buffer), Figure 1b shows the enzyme activity with temperature.
Figure 2 shows the results of measuring the temperature stability of the cellobiose 2-epimerase of the present invention (●: 65 ℃; △: 70 ℃; □: 75 ℃ and ○: 80 ℃).
Figure 3a shows the production of lactulose according to the amount of enzyme of cellobiose 2-epimerase of the present invention, Figure 3b shows the production of lactulose according to the substrate concentration (●: Lactulose; ○: Conversion rate).
Figure 4 shows the production of lactulose with reaction time at a substrate concentration of 700 g / L by the cellobiose 2-epimerase of the present invention (○: Lactose; ●: Lactulose; ■: Epilactose).
5 is a diagram showing a cleavage map of the expression vector of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One. Cellobiose  2- Epimeraise  Preparation of Recombinant Expression Vectors and Transgenic Microorganisms Comprising Genes

For the production of 2-epi-cellobiose Murray's of the present invention, a cellulite kaldi saccharide syrup sat Laura ET carcass (Caldicellulosiruptor cellobiose 2-epimerase gene from saccharolyticus ) strain (DSMZ strain) was first isolated.

Specifically, caldicellulose syrup to which a gene sequence and an amino acid sequence are already specified Saccharomyces ( Caldicellulosiruptor saccharolyticus) selecting a strain, and a kaldi cellulose comprising the nucleotide sequence of SEQ ID NO: 2 derived from this low-saccharide syrup Sat ET carcass (Caldicellulosiruptor saccharolyticus ) gene sequence [see sequence list; Based on GenBank Accession No. YP_001179132 (Genebank Accession No. YP_001179132), primers each comprising the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 were designed and manufactured.

SEQ ID NO: 3 (Forward primer): 5'-AA GCTAGC ATGGATATTACAAGGTTTAAG-3 '

SEQ ID NO: 4 (reverse primer): 5'-TT GAATTC TTAGTCAACCCTTTTTATTAT-3 '

The primers were designed with Nhe I (underline) and Eco RI (underline) restriction enzyme cleavage portions, respectively, and were subjected to polymerase chain reaction (PCR) using the primers. The sequence was amplified.

The cellobiose 2-epimerase enzyme gene obtained in a large amount was inserted into the restriction enzymes Nhe I and Eco RI of the vector pET24a (+) (product of Novagen) using restriction enzymes Nhe I and Eco R I and the recombinant expression vector pET24a ( +) / Cellobiose 2-epimerase was prepared.

The recombinant expression vector obtained as described above was transformed into E. coli (ER) 2566 strain (New England Biolabs) by a conventional transformation method. In addition, the transformed microorganism was added to a 20% glycerin (glycerin) solution and stored frozen before performing the culture for the production of lactulose. The recombinant E. coli was named E. Coli ER2566 pET24a (+) / cellobiose 2-epimerase strain.

Example  2. Cellobiose  2- Epimeraise  Produce

In order to mass-produce the cellobiose 2-epimerase of the present invention, the recombinant E. coli ER 2566 strain of Example 1, which was cryopreserved, was inoculated into a test tube containing 3 ml of LB medium and absorbance at 600 nm. The spawn culture was performed with a shake incubator at 37 ° C. until 2.0. The seed cultured culture was then added to a 2,000 ml flask containing 500 ml of LB medium supplemented with 20 µg / ml kanamycin antibiotic to carry out the main culture.

In addition, when the absorbance became 0.5 at 600 nm, 0.1 mM IPtage (IPTG) was added to induce mass expression of cellobiose 2-epimerase enzyme. At this time, the stirring speed was adjusted to 200 rpm, the culture temperature was maintained so that 37 ℃, after the addition of ITPG was stirred at 150 rpm, the culture temperature was adjusted to 16 ℃.

In addition, the cellobiose 2-epimerase produced by overexpression as described above was centrifuged at 6,000 xg for 30 minutes at 6,000 xg, and washed twice with 0.85% sodium chloride (NaCl). The cell solution was disrupted with an ultrasonic sonicator by adding 50 mM sodium monobasic, 300 mM sodium chloride, 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride). The cell lysate was again centrifuged at 13,000 × g for 20 min at 4 ° C., the cell pellet was removed and only the cell supernatant was obtained for a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). The HisTrap HP adsorption column using a His-tag was mounted to separate the enzyme solution used for lactulose production.

Example  3. Cellobiose  2- Epimeraise  Active pH  And temperature effects investigation

In order to investigate the pH and temperature activity change of cellobiose 2-epimerase in the production of lactulose of the present invention, the enzyme and the substrate were reacted under various pH and temperature conditions and the enzyme activity was compared.

3-1. Cellobiose  2- Epimeraise  Active pH  effect

First, in order to investigate the pH effect on cellobiose 2-epimerase activity in lactulose production, 50 mM pipese containing 50 g / L lactose, 1 mg / ml enzyme (piperazine- N , N 50 mM EPI (N- (2-hydroxyethyl piperazine- N- (3-propane) containing '-bis (2-ethane sulfonic acid), PIPES) buffer, 50 g / L lactose and 1 mg / ml enzyme The enzyme reaction was performed using a sulfonic acid (EPPS) buffer in the range of pH 6.5 to 8.5, but the reaction was stopped at 75 ° C. for 20 minutes, and then the reaction was stopped by adding 200 mM hydrogen chloride.

As a result, as shown in Figure 1a, it was confirmed that the optimum pH is 7.5.

3-2. Cellobiose  2- Epimeraise  Temperature effect on activity

To investigate the effect of temperature on cellobiose 2-epimerase activity in lactulose production, a pH containing 50 g / L lactose, 1 mg / ml enzyme was varied with varying enzyme reaction temperatures ranging from 65 ° C to 90 ° C. Each reaction was performed for 20 minutes using 50 mM EPPS buffer, 7.5, and then the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride.

As a result, the optimum temperature was confirmed to be 75 ℃ (see Figure 1b).

3-3. Cellobiose  2- Epimeraise  Temperature stability investigation

To investigate the temperature stability of the cellobiose 2-epimerase of the present invention, 50 mM PIPES buffer solution at pH 7.5 containing 50 g / L lactose and 1 mg / ml enzyme, varying from 60 ° C. to 80 ° C. After the reaction proceeds to the time when the activity of each enzyme is reduced by half, and when the reaction is completed, the reaction is stopped by adding a final concentration of 200 mM hydrogen chloride.

As a result, as shown in Figure 3, it was confirmed that the enzyme activity is reduced by half at the time of 73.9 hours at 70 ℃, 34.4 hours at 70 ℃, 15.8 hours at 75 ℃, and 5.2 hours at 80 ℃ (Fig. 2).

Example  4. Cellobiose  2- Epimeraise  Depending on enzyme amount and substrate concentration Rock Tool Ross Productivity Check

4-1. Cellobiose  2- Epimeraise  According to the amount of enzyme Lactulose  production

50 mM PIPES buffer solution containing 400 g / L lactose and 0.5 to 10 mg / ml enzyme as substrate to confirm lactulose production according to the amount of enzyme of cellobiose 2-epimerase in the production of lactulose of the present invention. After the enzyme was carried out at 75 ° C. for 2 hours at pH 7.5, the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to compare lactulose production.

As a result, the highest lactulose production was confirmed at the enzyme concentration of 5 mg / ml or more as shown in Figure 3a.

4-2. Cellobiose  2- Epimeraise On the substrate concentration  According Lactulose  production

50 mM PIPES containing 10 mg / ml enzyme using 50-400 g / L lactose as substrate to confirm lactulose production according to the amount of enzyme of cellobiose 2-epimerase in the production of lactulose of the present invention. After enzymatically performing the enzyme at a temperature of 75 ° C. for 2 hours in a buffer solution (pH 7.5), the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to compare lactulose production.

As a result, higher lactulose production was confirmed as the substrate concentration was higher as shown in Figure 3b.

Example  5. Cellobiose  2- Epimeraise  Used Lactulose  Productivity Check

In order to confirm the productivity of lactulose using the cellobiose 2-epimerase of the present invention, for 4 hours in 50 mM PIPES buffer solution (pH 7.5) containing 700 g / L lactose and 10 mg / ml enzyme as a substrate The enzyme reaction was carried out at a temperature of 75 ° C. The enzyme reaction solution was collected every hour and the reaction was stopped by addition of a final concentration of 200 mM hydrogen chloride, and then the lactulose production was measured by HPLC.

As a result, as shown in FIG. 4, 414 g / L lactulose was produced in 700 g / L lactose after 2 hours of reaction, which showed 208.5 g / L productivity and 59% conversion yield per hour. In addition to lactulose production, lactose glucose was also produced with lactose epilactose (epilactose) converted to mannose (mannose) (epilactose) was also produced as a production sugar, 100 g / L was produced for 2 hours.

The production of lactulose using enzymes reported to date has been reported on the method for producing lactulose by beta galactosidase by mixing lactose and fructose as substrate (Enzyme and Microbial Technology Vol 38 no 4 pp 903-908 ( 2006); Applied Microbiology and Biotechnology Vol 64 pp 787-793 (2004); Food Resource International Vol 43 pp 716-722 (2010)), and reports of producing lactulose using only enzymes as lactose as substrates This is my first report.

This is a method for producing lactulose using cellobiose 2-epimerase according to the present invention is a method of producing lactulose from lactose in high yield without additional cost due to the mixing of fructose and eco-friendly and economical to produce conventional lactulose It is more competitive than the way it is.

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp. <120> Method for production of lactulose from lactose using by          cellobiose 2-epimerase <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 390 <212> PRT <213> Caldicellulosiruptor saccharolyticus <400> 1 Met Asp Ile Thr Arg Phe Lys Glu Asp Leu Lys Ala His Leu Glu Glu   1 5 10 15 Lys Ile Ile Pro Phe Trp Gln Ser Leu Lys Asp Asp Glu Phe Gly Gly              20 25 30 Tyr Tyr Gly Tyr Met Asp Phe Asn Leu Asn Ile Asp Arg Lys Ala Gln          35 40 45 Lys Gly Cys Ile Leu Asn Ser Arg Ile Leu Trp Phe Phe Ser Ala Cys      50 55 60 Tyr Asn Val Leu Lys Ser Glu Lys Cys Lys Glu Met Ala Phe His Ala  65 70 75 80 Phe Glu Phe Leu Lys Asn Lys Phe Trp Asp Lys Glu Tyr Glu Gly Leu                  85 90 95 Phe Trp Ser Val Ser His Lys Gly Val Pro Val Asp Val Thr Lys His             100 105 110 Val Tyr Val Gln Ala Phe Gly Ile Tyr Gly Leu Ser Glu Tyr Tyr Glu         115 120 125 Ala Ser Gly Asp Glu Glu Ala Leu His Met Ala Lys Arg Leu Phe Glu     130 135 140 Ile Leu Glu Thr Lys Cys Lys Arg Glu Asn Gly Tyr Thr Glu Gln Phe 145 150 155 160 Glu Arg Asn Trp Gln Glu Lys Glu Asn Arg Phe Leu Ser Glu Asn Gly                 165 170 175 Val Ile Ala Ser Lys Thr Met Asn Thr His Leu His Val Leu Glu Ser             180 185 190 Tyr Thr Asn Leu Tyr Arg Leu Leu Lys Leu Asp Asp Val Tyr Glu Ala         195 200 205 Leu Glu Trp Ile Val Arg Leu Phe Val Asp Lys Ile Tyr Lys Lys Gly     210 215 220 Thr Gly His Phe Lys Val Phe Cys Asp Asp Asn Trp Asn Glu Leu Ile 225 230 235 240 Lys Ala Val Ser Tyr Gly His Asp Ile Glu Ala Ser Trp Leu Leu Asp                 245 250 255 Gln Ala Ala Lys Tyr Leu Lys Asp Glu Lys Leu Lys Glu Glu Val Glu             260 265 270 Lys Leu Ala Leu Glu Val Ala Gln Ile Thr Leu Lys Glu Ala Phe Asp         275 280 285 Gly Gln Ser Leu Ile Asn Glu Met Ile Glu Asp Arg Ile Asp Arg Ser     290 295 300 Lys Ile Trp Trp Val Glu Ala Glu Thr Val Val Gly Phe Phe Asn Ala 305 310 315 320 Tyr Gln Lys Thr Lys Glu Glu Lys Tyr Leu Asp Ala Ala Ile Lys Thr                 325 330 335 Trp Glu Phe Ile Lys Glu His Leu Val Asp Arg Arg Lys Asn Ser Glu             340 345 350 Trp Leu Trp Lys Val Asn Glu Asp Leu Glu Ala Val Asn Met Pro Ile         355 360 365 Val Glu Gln Trp Lys Cys Pro Tyr His Asn Gly Arg Met Cys Leu Glu     370 375 380 Ile Ile Lys Arg Val Asp 385 390 <210> 2 <211> 1170 <212> DNA <213> Caldicellulosiruptor saccharolyticus <400> 2 atggatatta caaggtttaa ggaagattta aaagctcatc ttgaagaaaa gataatacca 60 ttttggcaaa gtttaaagga cgatgaattt ggtggctact atggatatat ggactttaat 120 cttaacattg acagaaaagc tcaaaaaggt tgcattttga actcgaggat attgtggttt 180 ttctcagcat gttacaatgt gctgaaaagt gaaaaatgca aagagatggc ttttcatgcg 240 tttgaatttt taaaaaacaa gttttgggac aaagagtatg aaggactttt ctggagtgta 300 tcccacaaag gtgtgcccgt tgatgtgaca aaacatgttt atgttcaggc ttttggcata 360 tacgggcttt ctgagtacta tgaagcatcc ggggacgaag aagctcttca tatggctaag 420 aggcttttcg agattttaga gacaaaatgc aaaagggaaa atggatacac agaacagttt 480 gagagaaact ggcaagaaaa agaaaacagg tttttgagcg aaaatggagt aattgcctca 540 aaaacaatga acacgcatct tcatgtactg gagagctaca caaacctcta caggcttttg 600 aagcttgatg atgtgtatga agcgcttgag tggattgtaa gactctttgt tgacaagatt 660 tacaaaaaag gaacaggtca cttcaaggta ttttgcgatg ataactggaa cgaacttata 720 aaagcagtat catatggaca tgacattgaa gcaagctggc ttttagacca agctgccaag 780 tatctgaagg atgaaaagtt aaaagaggag gttgaaaagc tcgcattaga ggttgcccag 840 ataactttaa aagaagcctt tgatggtcaa agtcttataa atgaaatgat agaagatagg 900 attgacagga gtaaaatctg gtgggttgaa gcagagacgg ttgttggatt tttcaatgca 960 tatcaaaaga caaaagagga aaaatattta gatgcagcca tcaagacatg ggagttcata 1020 aaagagcatc ttgttgacag aagaaagaac tctgaatggc tgtggaaggt aaatgaggat 1080 ttagaagctg taaatatgcc aattgttgag caatggaagt gcccatatca caatggcaga 1140 atgtgtttgg agataataaa aagggttgac 1170 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aagctagcat ggatattaca aggtttaag 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttgaattctt agtcaaccct ttttattat 29

Claims (10)

delete delete delete delete delete delete delete Cellobiose 2-epimerase having the amino acid sequence set forth in SEQ ID NO: 1 is reacted with 700 g / L lactose, and 10 mg / ml in a solution containing the cellobiose 2-epimerase enzyme. A method for producing lactulose using cellobiose 2-epimerase comprising performing an enzymatic reaction at a temperature of 75 ° C. for 2 hours. delete delete

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