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WO2012115390A2 - Method for preparing lactulose from lactose using cellobiose 2-epimerase or n-acetyl glucosamin 2-epimerase - Google Patents

Method for preparing lactulose from lactose using cellobiose 2-epimerase or n-acetyl glucosamin 2-epimerase Download PDF

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Publication number
WO2012115390A2
WO2012115390A2 PCT/KR2012/001177 KR2012001177W WO2012115390A2 WO 2012115390 A2 WO2012115390 A2 WO 2012115390A2 KR 2012001177 W KR2012001177 W KR 2012001177W WO 2012115390 A2 WO2012115390 A2 WO 2012115390A2
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lactulose
epimerase
enzyme
caldicellulosiruptor
cellobiose
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Korean (ko)
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WO2012115390A3 (en
WO2012115390A9 (en
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오덕근
김영수
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University Industry Cooperation Corporation of Konkuk University
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University Industry Cooperation Corporation of Konkuk University
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Priority claimed from KR1020110016067A external-priority patent/KR101261004B1/en
Priority claimed from KR1020110081385A external-priority patent/KR101361688B1/en
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Publication of WO2012115390A9 publication Critical patent/WO2012115390A9/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C12Y501/03011—Cellobiose epimerase (5.1.3.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C12Y501/03014—UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing) (5.1.3.14)

Definitions

  • the present invention is to prepare lactulose (lacactose) from lactose using cellobiose 2-epimerase or N-acetyl glucosamin 2-epimerase
  • the present invention relates to a method for producing lactulose using a recombinant expression vector, a microorganism and an enzyme transformed thereto, and more particularly, a method of producing lactulose.
  • 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 colon 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.
  • 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)).
  • 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 ⁇ -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.
  • 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.
  • 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.
  • the present invention provides a cellobisoe 2-epimerase used in the production of lactulose.
  • the cellobiose 2-epimerase preferably has an amino acid sequence as set out in SEQ ID NO: 41, but at least one substitution, deletion, inversion, translocation, 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.
  • the cellobiose 2-epimerase is preferably encoded by a gene having the nucleotide sequence set forth in SEQ ID NO: 42, but considering a genetic code degeneracy, All gene sequences having at least 80% homology, preferably at least 90% homology with the nucleotide sequence set forth in SEQ ID NO: 42, in consideration of mutants, are included within the scope of the present invention.
  • the cellobiose 2-epimerase is preferably derived from a Caldicellulosiruptor saccharolyticus strain, but is not limited thereto.
  • the present invention also provides a recombinant expression vector comprising a cellobiose 2-epimerase gene having a nucleotide sequence of SEQ ID NO: 42 derived from a Caldicellulosiruptor saccharolyticus strain.
  • the vector preferably has a cleavage map described in Figure 5, but is not limited thereto.
  • 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 method for producing lactulose using the cellobiose 2-epimerase of the present invention.
  • the lactulose production method is preferably to use lactose as a substrate, but is not limited thereto.
  • the present invention provides a composition for producing lactulose comprising the cellobiose 2-epimerase of the present invention.
  • the inventors have found in the cell cases efforts result in kaldi cellulosic soil saccharide syrup Laura ET carcass (Caldicellulosiruptor saccharolyticus) to develop a method for producing a lactulose (lactulose) more effectively stable in an environmentally friendly way agarobiose 2-epi It was confirmed for the first time that cellobiose 2-epimerase 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 present invention stems in capable kaldi cellulose for producing a lactulose (lactulose) with lactose (lactose) as a substrate from a saccharide syrup soil roller ET carcass (Caldicellulosiruptor saccharolyticus) strain cellobiose 2-epi Murray's (cellobiose 2 epimerase).
  • 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.
  • the present invention provides a production method for obtaining lactulose in high yield using a cellobiose 2-epimerase prepared by the above method.
  • 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.
  • 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.
  • the enzyme reaction is preferably made 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 °C to 80 °C, the temperature of 75 °C Most preferably done at temperature.
  • the enzyme reaction time may be appropriately adjusted according to conventional methods.
  • the present invention comprises the steps of a) preparing a recombinant vector comprising one gene selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 10; b) transforming the preparation vector into a microorganism; And c) obtaining an enzyme solution used for producing lactulose from the transformed microorganism and treating the substrate.
  • the gene is an aeroline thermophila ( Anaerolinea thermophila ), Caldicellulose Syrup To Besi ( Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal ( Caldicellulosiruptor hydrothermalis ), Caldicellulose Syrup To Cristzanson ( Caldicellulosiruptor kristjanssonii ), Caldicellulose Syrup To Obsidiansis ( Caldicellulosiruptor obsidiansis ), Dictioglumos Tergium ( Dictyoglomus turgidum ), Penivacillus ( Paenibacillus sp.), Rhodon Somers Mariners ( Rhodothermus marinus DSM 4252), spiroketa thermophila Spirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium ( Thermo
  • the substrate is preferably lactose, but is not limited thereto.
  • the enzyme and substrate reaction is preferably made in the range of pH 6.5 to pH 8.5, preferably in the range of temperature 65 to 90 °C, substrate concentration is preferably in the range of 50 to 700 g / L.
  • 'enacetylglucosamine 2-epimerase' may be used interchangeably as 'cellobiose 2-epimerase'.
  • the present invention provides a composition for the production of lactulose (lactulose) comprising an enzyme selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 31 to 40 as an active ingredient.
  • the present invention comprises the steps of a) preparing a recombinant vector comprising one gene selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 10; b) transforming the preparation vector into a microorganism; And c) obtaining an enzyme solution used for epilactose production from the transformed microorganism and treating the substrate.
  • the present invention provides a composition for producing epilactose comprising an enzyme selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 31 to 40 as an active ingredient.
  • the present invention is an air aero thermophila ( Anaerolinea thermophila ), Caldicellulose Syrup To Besi ( Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal ( Caldicellulosiruptor hydrothermalis ), Caldicellulose Syrup To Cristzanson ( Caldicellulosiruptor kristjanssonii ), Caldicellulose Syrup To Obsidiansis ( Caldicellulosiruptor obsidiansis ), Dictioglumos Tergium ( Dictyoglomus turgidum ), Penivacillus ( Paenibacillus sp.), Rhodon Somers Mariners ( Rhodothermus marinus DSM 4252), spiroketa thermophila Spirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium ( Therm
  • the present invention also provides a microorganism transformed with a recombinant expression vector comprising an N-acetyl glucosamin 2-epimerase gene.
  • the present invention is an unairline thermopil ( Anaerolinea thermophila ), Caldicellulose Syrup To Besi ( Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal ( Caldicellulosiruptor hydrothermalis ), Caldicellulose Syrup To Cristzanson ( Caldicellulosiruptor kristjanssonii ), Caldicellulose Syrup To Obsidiansis ( Caldicellulosiruptor obsidiansis ), Dictioglumos Tergium ( Dictyoglomus turgidum ), Penivacillus ( Paenibacillus sp.), Rhodon Somers Mariners ( Rhodothermus marinus DSM 4252),
  • any vector used in gene recombination methods including the expression vector pET28a (+), 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 (1) to produce a recombinant expression vector comprising an N-acetyl glucosamin 2-epimerase gene; (2) culturing the microorganism transformed with the recombinant expression vector; (3) induce expression of the N-acetyl glucosamin 2-epimerase gene; And (4) isolating and purifying the expressed recombinant protein; It provides a process for obtaining N-acetyl glucosamin 2-epimerase comprising a process.
  • a) Unairrone thermophila Anaerolinea thermophila
  • Caldicellulose Syrup To Besi Caldicellulosiruptor bescii DSM 6725
  • Caldicellulose Syrup To Hydrothermal Caldicellulosiruptor hydrothermalis
  • Caldicellulose Syrup To Cristzanson Caldicellulosiruptor kristjanssonii
  • Caldicellulose Syrup To Obsidiansis Caldicellulosiruptor obsidiansis
  • Dictioglumos Tergium Dictyoglomus turgidum
  • Penivacillus Paenibacillus sp.
  • Rhodon Somers Mariners Rhodothermus marinus DSM 4252
  • spiroketa thermophila Spirochaeta thermophila DSM 6192 or Thermoaerobacterium Thermosacc
  • the englucosamine 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.
  • the present invention provides a production method for obtaining lactulose in high yield using the enacetylglucosamine 2-epimerase prepared by the above method.
  • the present invention provides a method for producing lactulose using the enacetylglucosamine 2-epimerase capable of characteristically synthesizing lactulose without adding coenzyme.
  • Enacetylglucosamine 2-epimerase of the present invention is cultured E. coli transformed with a recombinant expression vector comprising the enacetylglucosamine 2-epimerase gene as described above to induce the expression of the recombinant enzyme gene and then expressed recombinant It is preferable to use those prepared by the process of separating and purifying proteins.
  • the lactulose is preferably synthesized using lactose as a substrate, 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.
  • the enzyme reaction time may be appropriately adjusted according to conventional methods.
  • the present invention relates to recombinant expression vectors comprising cellobiose 2-epimerase gene or enacetylglucosamine 2-epimerase, microorganisms transformed therefrom and cellobiose 2-epimerase or enacetylglucosamine 2-epi.
  • 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 °C; ⁇ : 70 °C; ⁇ : 75 °C and ⁇ : 80 °C).
  • 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).
  • FIG. 5 is a diagram showing a cleavage map of the expression vector of the present invention.
  • FIG. 6 to 15 show the production of lactulose with reaction time at substrate concentration of 700 g / L by the enacetylglucosamine 2-epimerase of the present invention ( ⁇ : Lactose; ⁇ : Lactulose; ⁇ : Epilactose ).
  • FIG. 6 is Anaerolinea thermophila
  • FIG. 7 is Caldicellulosiruptor bescii DSM 6725
  • FIG. 8 is Caldicellulosiruptor hydrothermalis
  • FIG. 9 is caldicellulose .
  • FIG. 10 shows Caldicellulosiruptor obsidiansis
  • FIG. 11 shows Dictyoglomus turgidum
  • FIG. 12 shows Paenibacillus sp
  • FIG. 13 shows a Rhodothermus marinus DSM 4252
  • FIG. 14 shows a Spirochaeta thermophila DSM 6192
  • FIG. 15 shows a Thermoaerobacterium thermosaccharolyticum DSM 571. .
  • 2-epi-cellobiose Murray's of the present invention the cellobiose was isolated 2-epi Murray's first gene derived from a cellulose kaldi saccharide syrup sat Laura ET carcass (Caldicellulosiruptor saccharolyticus) strain (DSMZ four strains).
  • the gene sequence and the amino acid sequence kaldi cell rules syrup that has already been specified Sat saccharide Laura ET carcass (Caldicellulosiruptor saccharolyticus) selecting a strain, and a kaldi cellulose comprising the nucleotide sequence of SEQ ID NO: 42 derived therefrom syrup Sat LAURA ET coarse saccharide (Caldicellulosiruptor saccharolyticus) sequence of the gene [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: 43 and SEQ ID NO: 44 were designed and manufactured.
  • SEQ ID NO: 43 (Forward primer): 5'-AA GCTAGC ATGGATATTACAAGGTTTAAG-3 '
  • SEQ ID NO: 44 (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 gene gene obtained in large quantities was inserted into the restriction enzymes Nhe I and Eco RI of the vector pET24a (+) (manufactured by Novagen) using the 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.
  • 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.
  • 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 ⁇ g / ml kanamycin antibiotic to carry out the main culture.
  • IPTG IPtage
  • 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.
  • the optimum temperature was confirmed to be 75 °C (see Figure 1b).
  • Anaerolinea thermophila Caldicellulose Syrup To Besi ( Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal ( Caldicellulosiruptor hydrothermalis ), Caldicellulose Syrup To Cristzanson ( Caldicellulosiruptor kristjanssonii ), Caldicellulose Syrup To Obsidiansis ( Caldicellulosiruptor obsidiansis ), Dictioglumos Tergium ( Dictyoglomus turgidum ), Penivacillus ( Paenibacillus sp.), Rhodon Somers Mariners ( Rhodothermus marinus DSM 4252), spiroketa thermophila Spirochaeta thermophila DSM 6192), or Thermoaerobacterium
  • unairrene thermophila in which the gene sequence and the amino acid sequence are already specified ( Anaerolinea thermophila ), Caldicellulose Syrup To Besi ( Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal ( Caldicellulosiruptor hydrothermalis ), Caldicellulose Syrup To Cristzanson ( Caldicellulosiruptor kristjanssonii ), Caldicellulose Syrup To Obsidiansis ( Caldicellulosiruptor obsidiansis ), Dictioglumos Tergium ( Dictyoglomus turgidum ), Penivacillus ( Paenibacillus sp.), Rhodon Somers Mariners ( Rhodothermus marinus DSM 4252), spiroketa thermophila Spirochaeta thermophila DSM 6192), or Thermoaerobacterium
  • Caldicellulose Syrupto Cristianzoni Caldicellulosiruptor kristjanssonii
  • Dictioglumos Tergium Dictyoglomus turgidum
  • Rhodon Somers Mariners Rhodothermus marinus DSM 4252
  • thermopile Caro lithium Thermoanaerobacterium thermosaccharolyticum DSM 571
  • the primers are each endiyi source (Nde I, underlined), yen eyichiyi source (Nhe I, underlined), X H. oh source (Xho I, underlined), and Ico alwon (Eco RI, underlined) restriction enzyme digestion It was designed as a part, and the polymerase chain reaction (PCR) using the primers was performed to amplify the base sequence of the gene.
  • PCR polymerase chain reaction
  • the enacetyl glucosamine 2-epimerase gene obtained in large quantities was inserted into the same restriction enzyme site of the vector pET28a (+) (manufactured by Novagen) using the respective restriction enzymes to generate the recombinant expression vector pET28a (+) / enacetylglucosamine 2 -Epimerase was produced.
  • the recombinant expression vector thus obtained was transformed into E. coli ER 2566 strain by a conventional transformation method.
  • 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 as E. Coli ER2566 pET28a (+) / enacetylglucosamine 2-epimerase strain.
  • the recombinant E. coli ER 2566 strains of Example 6 which were stored in frozen form, were 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 ⁇ g / ml kanamycin antibiotic to carry out the main culture.
  • the enacetylglucosamine 2-epimerase produced by overexpression as described above was centrifuged at 6,000 g for 30 minutes at 6,000 g of the culture medium of the transformed strain, and washed twice with 0.85% sodium chloride (NaCl). The cell solution was then 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 minutes at 4 ° C., cell pellets were removed, and only cell supernatant was obtained for fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA).
  • a HisTrap HP adsorption column using a His-tag was attached to and separated as an enzyme solution used for lactulose production.
  • the optimum temperature was found to be 65-80 °C (see Table 2).
  • lactose was also produced in the production of lactose, in addition to lactose, epilactose, in which glucose of lactose was converted into mannose, was produced as a production sugar, and about 60-100 g / L was produced for 2 hours.
  • This is a method for producing lactulose using cellobiose 2-epimerase or enacetylglucosamine 2-epimerase according to the present invention is an environmentally friendly method of producing lactulose from lactose in a high yield without additional cost due to the mixing of fructose. Because of its economic feasibility, it can be said to have more competitiveness than the existing method of producing lactulose.

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Abstract

The present invention relates to a method for preparing lactulose from lactose using cellobiose 2-epimerase or N-acetyl glucosamin 2-epimerase, and more specifically relates to a production method for obtaining lactulose using recombinant expression vectors containing these genes, microorganisms transformed thereby, and enzymes.

Description

셀로비오스 2-에피머레이즈 또는 엔아세틸 글루코사민 2-에피머레이즈를 이용한 유당으로부터 락툴로스의 제조방법Method for preparing lactulose from lactose using cellobiose 2-epimerase or enacetyl glucosamine 2-epimerase

본 발명은 셀로비오스 2-에피머레이즈 (cellobiose 2-epimerase) 또는 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase)를 이용하여 유당(lactose)으로부터 락툴로스(lactulose)를 제조하는 방법에 관한 것으로, 더욱 상세하게는 이들 유전자를 포함하는 재조합 발현 벡터, 이로 형질전환 된 미생물 및 효소를 이용하여 락툴로스를 얻는 생산 방법에 관한 것이다.The present invention is to prepare lactulose (lacactose) from lactose using cellobiose 2-epimerase or N-acetyl glucosamin 2-epimerase The present invention relates to a method for producing lactulose using a recombinant expression vector, a microorganism and an enzyme transformed thereto, and more particularly, a method of producing lactulose.

락툴로스(lactulose)는 유당(lactose)의 포도당(glucose) 부분이 과당(fructose)로 대체된 유당의 이성질체 당으로서 천연에는 거의 존재하지 않는 당이다. 락툴로스는 소장에서 베타갈락토시다제에 의해 분해되지 않으며 대장에 도달하여 비피도박테리움을 포함한 여러 유산균에 의해 이용되어 대장의 pH를 저하시킴으로서 유해한 균의 성장을 억제하고 대장의 균총을 유익한 방향으로 개선하는 효과를 나타낸다. 또한 락툴로스는 비소화성 당이며 이들 당은 수용성 식이섬유로 작용을 하며 변비와 만성 간질환으로 인한 뇌질병의 치료에 이용되고 있다. 최근에는 lactulose가 폐경기 이후 여성의 칼슘 흡수를 촉진한다는 연구도 있다 (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)).그리고 산업적으로 락툴로스는 유당에 비해 감미도 및 용해성이 우수하여 제빵 및 제과 산업등에 유용하게 사용될 수 있다 (Bulletin of the International Dairy Federation Vol 212 pp 69-76(1987)). 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 colon 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)).

이러한 락툴로스는 원유에는 존재하지 않고 열처리한 우유에 소량 함유되어져 있다. 락툴로스를 유당을 강알칼리 조건에서 이성화시키는 화학적 방법과 베 타 갈락토시다제 (β-galactosidase)의 갈락토스 전이활성을 이용한 효소학적 방법으로 생산할 수 있다 (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)). 화학적 방법은 수산화나트륨, 수산화칼슘, 수산화칼륨등 강알칼리 화합물을 첨가하여 반응을 하고 중화 과정에서 강산을 사용하여 환경 오염을 유발할뿐 아니라 강알칼리 조건에서 생성된 락툴로스를 분해시키고 부산물이 생성되기 때문에 생산후 처리 과정 및 락툴로스 정제 과정이 복잡한 단점을 지니고 있다. 이에 반하여 베타 갈락토시다제를 이용한 효소학적 방법은 친환경적이고 부산물이 나오지 않기 때문에 정제가 간단한 장점을 가지고 있지만 낮은 수율이 문제가 되어져 왔다. 또한 반응에 기질로서 유당뿐 아니라 과당을 함께 사용해야 하며 높은 기질 농도에서만 반응이 이루어 지기 때문에 경제적으로 비효율적이라는 단점이 있다. 그렇기 때문에 친환경적이고 유당 하나의 기질만 사용하여 높은 수율로 락툴로스를 생산할 수 있는 효소를 구하고 적용하는 연구가 필요하다.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.

본 발명은 상기의 문제점을 해결하고, 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 유당(lactose)으로부터 락툴로스(lactulose)를 생산할 수 있는 효소를 제공하는 것이다.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.

본 발명의 다른 목적은 유당(lactose)으로부터 락툴로스(lactulose)를 생산할 수 있는 방법을 제공하는 것이다.Another object of the present invention is to provide a method capable of producing lactulose from lactose.

본 발명의 또 다른 목적은 유당(lactose)으로부터 락툴로스(lactulose)를 생산할 수 있는 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition capable of producing lactulose from lactose.

상기 목적을 달성하기 위하여, 본 발명은 락툴로스(lactulose)의 생산에 사용되는 셀로비오스 2-에피머레이즈(cellobisoe 2-epimerase)를 제공한다.In order to achieve the above object, the present invention provides a cellobisoe 2-epimerase used in the production of lactulose.

본 발명의 일 구현예에 있어서, 상기 셀로비오스 2-에피머레이즈는 서열번호 41에 기재된 아미노산 서열을 가지는 것이 바람직하나, 서열번호 41에 기재된 아미노산 서열에 하나 이상의 치환, 결손, 역위, 전좌 등의 돌연변이를 통하여 본 발명의 목적을 달성할 수 있는 모든 돌연변이체 폴리펩타이드도 본 발명의 범위에 포함된다.In one embodiment of the present invention, the cellobiose 2-epimerase preferably has an amino acid sequence as set out in SEQ ID NO: 41, but at least one substitution, deletion, inversion, translocation, 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.

본 발명의 다른 구현예에 있어서, 상기 셀로비오스 2-에피머레이즈는 서열번호 42에 기재된 염기 서열을 가지는 유전자에 의해서 코딩되는 것이 바람직하나, 유전자 코드 디제너러시를 고려하고, 상기와 같은 효소의 돌연변이체를 고려하여서 서열번호 42에 기재된 염기 서열과 80% 이상의 상동성, 바람직하게는 90% 이상의 상동성을 가지는 모든 유전자 서열이 본 발명의 범위에 포함된다.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: 42, but considering a genetic code degeneracy, All gene sequences having at least 80% homology, preferably at least 90% homology with the nucleotide sequence set forth in SEQ ID NO: 42, in consideration of mutants, are included within the scope of the present invention.

본 발명의 다른 구현예에 있어서,상기 셀로비오스 2-에피머레이즈는 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주로부터 유래한 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the cellobiose 2-epimerase is preferably derived from a Caldicellulosiruptor saccharolyticus strain, but is not limited thereto.

또한 본 발명은 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주로부터 유래한 서열번호 42의 염기서열을 가지는 셀로비오스 2-에피머레이즈 유전자를 포함하는 재조합 발현 벡터를 제공한다.The present invention also provides a recombinant expression vector comprising a cellobiose 2-epimerase gene having a nucleotide sequence of SEQ ID NO: 42 derived from a Caldicellulosiruptor saccharolyticus strain.

본 발명의 일 구현예에 있어서, 상기 벡터는 도 5에 기재된 개열지도를 가지는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the invention, the vector preferably has a cleavage map described in Figure 5, but is not limited thereto.

본 발명의 재조합 발현 벡터는 락툴로스의 생산에 사용될 수 있는 것이라면 발현 벡터 pET24a(+)를 포함하여 유전자 재조합 방법에 이용되는 어느 벡터라도 사용될 수 있고, 상기 재조합 발현 벡터로 형질전환 되는 미생물로는 대장균 ER 2566을 사용하는 것이 바람직하나 재조합 발현 벡터로 형질전환되어 목적하는 유전자를 과발현하고 활성이 있는 효소 단백질을 생산할 수 있는 균주라면 어느 것이라도 사용될 수 있다. 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.

또 본 발명은 상기 본 발명의 셀로비오스 2-에피머레이즈를 이용하여 락툴로스를 생산하는 방법을 제공한다.The present invention also provides a method for producing lactulose using the cellobiose 2-epimerase of the present invention.

본 발명의 일 구현예에 있어서, 상기 락툴로스 생산방법은 기질로서 유당을 사용하는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the lactulose production method is preferably to use lactose as a substrate, but is not limited thereto.

또한 본 발명은 상기 본 발명의 셀로비오스 2-에피머레이즈를 포함하는 락툴로스 생산용 조성물을 제공한다.In another aspect, the present invention provides a composition for producing lactulose comprising the cellobiose 2-epimerase of the present invention.

이하 본 발명을 설명한다.Hereinafter, the present invention will be described.

본 발명자들은 환경 친화적인 방법으로 안정하게 보다 효과적으로 락툴로스(lactulose) 를 생산하는 방법을 개발하기 위하여 예의 노력한 결과, 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus )에서 발견되는 셀로비오스 2-에피머레이즈(cellobiose 2-epimerase)가 유당(lactose)으로부터 락툴로스(lactulose)를 생산할 수 있는 것을 최초로 확인하고, 본 발명을 완성하였다.The inventors have found in the cell cases efforts result in kaldi cellulosic soil saccharide syrup Laura ET carcass (Caldicellulosiruptor saccharolyticus) to develop a method for producing a lactulose (lactulose) more effectively stable in an environmentally friendly way agarobiose 2-epi It was confirmed for the first time that cellobiose 2-epimerase can produce lactulose from lactose, and the present invention was completed.

본 발명은 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus )로부터 유래한 셀로비오스 2-에피머레이즈를 포합하는 재조합 발현 벡터 및 이로 형질전환된 미생물과, 이를 이용하여 셀로비오스 2-에피머레이즈를 제조하는 방법, 그리고 상기 셀로비오스 2-에피머레이즈를 이용한 락툴로스의 제조방법을 제공한다.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.

본 발명은 기질로서 유당(lactose)을 사용하여 락툴로스(lactulose)를 제조하는 것이 가능한 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주로부터 유래한 셀로비오스 2-에피머레이즈(cellobiose 2-epimerase)를 제공한다. The present invention stems in capable kaldi cellulose for producing a lactulose (lactulose) with lactose (lactose) as a substrate from a saccharide syrup soil roller ET carcass (Caldicellulosiruptor saccharolyticus) strain cellobiose 2-epi Murray's (cellobiose 2 epimerase).

본 발명의 바람직한 일 실시예에서는, 가) 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주의 게놈 DNA(genomic DNA)를 주형으로 하고, 서열번호43 및 44의 프라이머를 가지고 PCR을 실시하여 셀로비오스 2-에피머레이즈 유전자(서열번호 42)를 포함하는 DNA 절편을 증폭한 다음; 나) 증폭된 셀로비오스 2-에피머레이즈 유전자를 포함하는 DNA 절편에 제한효소 Nhe I 및 EcoR I를 처리한 후 발현벡터인 pET24a(+)에 클로닝하여 재조합 발현 벡터 pET24a/셀로비오스 2-에피머레이즈를 제작하고; 다) 이를 통상적 형질전환방법으로 대장균 ER 2566 균주에 형질전환하고; 라) 형절전환 된 대장균을 배양한 다음 과발현을 유도하여 셀로비오스 2-에피머레이즈를 생산하고; 및 마) 발현된 셀로비오스 2-에피머레이즈를 분리하여 정제한다. In one preferred embodiment of the invention, a) kaldi the cellulite in syrup Sat saccharide to Lai Atticus (Caldicellulosiruptor saccharolyticus) genomic DNA (genomic DNA) of the strains a template and subjected to PCR with primers of SEQ ID NOS: 43 and 44 Amplifying the DNA fragment containing the cellobiose 2-epimerase gene (SEQ ID NO: 42); B) DNA fragments containing the amplified cellobiose 2-epimerase gene were treated with the 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.

또한, 상기 마) 과정에서 발현된 셀로비오스 2-에피머레이즈 단백질은, (a) 상기 미생물을 파쇄하고; (b) 상기 세포 파쇄물은 원심분리 하여 상등액을 얻고; (c) 상기 상등액을 고속 단백질 액체 크로마토그래피(fast protein liquid chromatography)로 분리하는; 과정으로 효소액을 분리 정제할 수 있다.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.

또한, 본 발명은 상기와 같은 방법으로 제조되는 셀로비오스 2-에피머레이즈를 이용하여 락툴로스를 고수율로 얻는 생산방법을 제공한다.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.

구체적으로, 본 발명은 조효소를 첨가하지 않고 락툴로스를 특징적으로 합성할 수 있는 상기 셀로비오스 2-에피머레이즈를 이용한 락툴로스의 생산 방법을 제공한다.Specifically, the present invention provides a method for producing lactulose using the cellobiose 2-epimerase capable of characteristically synthesizing lactulose without adding coenzyme.

본 발명의 셀로비오스 2-에피머레이즈는 상기와 같이 셀로비오스 2-에피머레이즈 유전자를 포함하는 재조합 발현 벡터로 형질전환된 대장균을 배양하여 재조합 효소 유전자의 발현을 유도한 다음 발현된 재조합 단백질을 분리 및 정제하는 과정으로 제조된 것을 사용하는 것이 바람직하다. 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.

또한, 상기 락툴로스는 기질로서 유당(lactose)를 사용하여 합성되는 것이 바람직하고 상기 기질의 농도는 50 g/ℓ 내지 700 g/ℓ 범위인 것이 바람직하고, 700 g/ℓ 내의 범위인 것은 더욱 바람직하다. 또한, 상기 효소 반응은 pH 7.0 내지 pH 7.5 범위에서 이루어지는 것이 바람직하고, pH 7.5 내외 범위에서 이루어지는 것은 더욱 바람직하며, 상기 효소 반응은 온도 75℃ 내지 80℃ 범위에서 이루어지는 것이 바람직하고, 온도 75℃의 온도에서 이루어지는 것은 가장 바람직하다. 또한, 상기 효소 반응 시간은 통상적인 방법에 따라 적절히 조절될 수 있다. 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 made 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.

또 본 발명은 a)서열번호 1 내지 10의 염기서열로 구성된 군으로부터 선택된 하나의 유전자를 포함한 재조합 벡터를 제조하는 단계;b)상기 제조합 벡터를 미생물에 형질전환하는 단계; 및 c) 상기 형질전환된 미생물로부터 락툴로스 생산에 사용되는 효소액을 얻어서 기질을 처리하는 단계를 포함하는 락툴로스(lactulose)의 제조방법을 제공한다.In another aspect, the present invention comprises the steps of a) preparing a recombinant vector comprising one gene selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 10; b) transforming the preparation vector into a microorganism; And c) obtaining an enzyme solution used for producing lactulose from the transformed microorganism and treating the substrate.

본 발명의 일 구현예에 있어서,In one embodiment of the invention,

상기 유전자는 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주로부터 유래한 것이 바람직하나 이에 한정되지 아니한다.The gene is an aeroline thermophila (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) is preferably derived from, but not limited to.

본 발명의 다른 구현예에 있어서, 상기 기질은 유당 (lactose)인 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the substrate is preferably lactose, but is not limited thereto.

본 발명의 또 다른 구현예에 있어서,In another embodiment of the invention,

상기 효소와 기질 반응은 pH 6.5 내지 pH 8.5 범위에서 이루어지는 것이 바람직하고, 온도 65 내지 90℃의 범위에서 이루어지는 것이 바람직하며, 기질 농도는 50 내지 700 g/L 범위인 것이 바람직하다.The enzyme and substrate reaction is preferably made in the range of pH 6.5 to pH 8.5, preferably in the range of temperature 65 to 90 ℃, substrate concentration is preferably in the range of 50 to 700 g / L.

본 발명에서 '엔아세틸글루코사민 2-에피머레이즈'는 '셀로비오스 2-에피머레이즈'로 혼용될 수도 있다.In the present invention, 'enacetylglucosamine 2-epimerase' may be used interchangeably as 'cellobiose 2-epimerase'.

또 본 발명은 서열번호 31 내지 40에 기재된 아미노산 서열로 구성된 군으로부터 선택된 효소를 유효성분으로 포함하는 락툴로스(lactulose) 제조용 조성물을 제공한다.In another aspect, the present invention provides a composition for the production of lactulose (lactulose) comprising an enzyme selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 31 to 40 as an active ingredient.

또한 본 발명은 a)서열번호 1 내지 10의 염기서열로 구성된 군으로부터 선택된 하나의 유전자를 포함한 재조합 벡터를 제조하는 단계;b)상기 제조합 벡터를 미생물에 형질전환하는 단계; 및 c) 상기 형질전환된 미생물로부터 에피락토스(epilactose) 생산에 사용되는 효소액을 얻어서 기질을 처리하는 단계를 포함하는 에피락토스(epilactose)의 제조방법을 제공한다.In another aspect, the present invention comprises the steps of a) preparing a recombinant vector comprising one gene selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 10; b) transforming the preparation vector into a microorganism; And c) obtaining an enzyme solution used for epilactose production from the transformed microorganism and treating the substrate.

또 본 발명은 서열번호 31 내지 40에 기재된 아미노산 서열로 구성된 군으로부터 선택된 효소를 유효성분으로 포함하는 에피락토스(epilactose) 제조용 조성물을 제공한다.In another aspect, the present invention provides a composition for producing epilactose comprising an enzyme selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 31 to 40 as an active ingredient.

본 발명은 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주로부터 유래하고, 서열번호 1 내지 10 중 어느 하나의 염기서열을 가지는 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase) 유전자를 포함하는 재조합 발현 벡터 pET28a(+)를 제공한다. The present invention is an air aero thermophila (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) a recombinant expression vector pET28a (+) comprising an N-acetyl glucosamin 2-epimerase gene derived from strain and having the nucleotide sequence of any one of SEQ ID NOs: 1-10. to provide.

또한, 본 발명은 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase) 유전자를 포함하는 재조합 발현벡터로 형질전환된 미생물을 제공한다. 구체적으로, 본 발명은 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주로부터 유래한 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase)를 포함하는 재조합 발현 벡터로 형질전환된 대장균 이알(ER) 2566 pET28a(+)/N-acetyl glucosamin 2-epimerase를 제공한다. The present invention also provides a microorganism transformed with a recombinant expression vector comprising an N-acetyl glucosamin 2-epimerase gene. Specifically, the present invention is an unairline thermopil (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) E. coli ER 2566 pET28a (+) / N-acetyl glucosamin 2 transformed with a recombinant expression vector comprising N-acetyl glucosamin 2-epimerase from strain -epimerase is provided.

본 발명의 재조합 발현 벡터는 락툴로스의 생산에 사용될 수 있는 것이라면 발현 벡터 pET28a(+)를 포함하여 유전자 재조합 방법에 이용되는 어느 벡터라도 사용될 수 있고, 상기 재조합 발현 벡터로 형질전환되는 미생물로는 대장균 ER 2566을 사용하는 것이 바람직하나 재조합 발현 벡터로 형질전환되어 목적하는 유전자를 과발현하고 활성이 있는 효소 단백질을 생산할 수 있는 균주라면 어느 것이라도 사용될 수 있다. If the recombinant expression vector of the present invention can be used for the production of lactulose, any vector used in gene recombination methods, including the expression vector pET28a (+), 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.

또한, 본 발명은 (1) 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase) 유전자를 포함하는 재조합 발현 벡터를 제작하고; (2) 상기 재조합 발현 벡터로 형질전환된 미생물을 배양하고; (3) 상기 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase) 유전자의 발현을 유도하고; 및 (4) 발현된 재조합 단백질을 분리 및 정제하는; 과정을 포함하는 엔아세틸 글루코사민 2-에피머레이즈(N-acetyl glucosamin 2-epimerase)를 얻는 제조방법을 제공한다.In addition, the present invention (1) to produce a recombinant expression vector comprising an N-acetyl glucosamin 2-epimerase gene; (2) culturing the microorganism transformed with the recombinant expression vector; (3) induce expression of the N-acetyl glucosamin 2-epimerase gene; And (4) isolating and purifying the expressed recombinant protein; It provides a process for obtaining N-acetyl glucosamin 2-epimerase comprising a process.

본 발명의 바람직한 일 실시예에서는, 가) 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주의 게놈 DNA(genomic DNA)를 주형으로 하고, 서열번호 11 내지 30의 프라이머를 가지고 PCR을 실시하여 엔아세틸 글루코사민 2-에피머레이즈 유전자를 포함하는 DNA 절편을 증폭한 다음; 나) 증폭된 엔아세틸 글루코사민 2-에피머레이즈 유전자를 포함하는 DNA 절편에 제한효소 를 처리한 후 발현벡터인 pET28a(+)에 클로닝하여 재조합 발현 벡터 pET28a/엔아세틸 글루코사민 2-에피머레이즈를 제작하고; 다) 이를 통상적 형질전환방법으로 대장균 ER 2566 균주에 형질전환하고; 라) 형절전환 된 대장균을 배양한 다음 과발현을 유도하여 엔글루코사민 2-에피머레이즈를 생산하고; 및 마) 발현된 엔글루코사민 2-에피머레이즈를 분리하여 정제한다. In a preferred embodiment of the present invention, a) Unairrone thermophila (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) using the genomic DNA of the strain as a template, PCR with primers of SEQ ID NOs: 11 to 30 to amplify a DNA fragment containing the enacetyl glucosamine 2-epimerase gene; B) Recombinant expression vector pET28a (+) was cloned into the expression vector pET28a (+) after the restriction enzyme was treated to the DNA fragment containing the amplified enacetyl glucosamine 2-epimerase gene to prepare a recombinant expression vector pET28a / enacetyl glucosamine 2-epimerase. and; C) transform it into E. coli ER 2566 strain by a conventional transformation method; D) incubating the transformed Escherichia coli and inducing overexpression to produce englucosamine 2-epimerase; And e) isolate and purify the expressed englucosamine 2-epimerase.

또한, 상기 마) 과정에서 발현된 엔글루코사민 2-에피머레이즈 단백질은, (a) 상기 미생물을 파쇄하고; (b) 상기 세포 파쇄물은 원심분리 하여 상등액을 얻고; (c) 상기 상등액을 고속 단백질 액체 크로마토그래피(fast protein liquid chromatography)로 분리하는; 과정으로 효소액을 분리 정제할 수 있다.In addition, the englucosamine 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.

또한, 본 발명은 상기와 같은 방법으로 제조되는 엔아세틸글루코사민 2-에피머레이즈를 이용하여 락툴로스를 고수율로 얻는 생산방법을 제공한다.In addition, the present invention provides a production method for obtaining lactulose in high yield using the enacetylglucosamine 2-epimerase prepared by the above method.

구체적으로, 본 발명은 조효소를 첨가하지 않고 락툴로스를 특징적으로 합성할 수 있는 상기 엔아세틸글루코사민 2-에피머레이즈를 이용한 락툴로스의 생산 방법을 제공한다.Specifically, the present invention provides a method for producing lactulose using the enacetylglucosamine 2-epimerase capable of characteristically synthesizing lactulose without adding coenzyme.

본 발명의 엔아세틸글루코사민 2-에피머레이즈는 상기와 같이 엔아세틸글루코사민 2-에피머레이즈 유전자를 포함하는 재조합 발현 벡터로 형질전환된 대장균을 배양하여 재조합 효소 유전자의 발현을 유도한 다음 발현된 재조합 단백질을 분리 및 정제하는 과정으로 제조된 것을 사용하는 것이 바람직하다. Enacetylglucosamine 2-epimerase of the present invention is cultured E. coli transformed with a recombinant expression vector comprising the enacetylglucosamine 2-epimerase gene as described above to induce the expression of the recombinant enzyme gene and then expressed recombinant It is preferable to use those prepared by the process of separating and purifying proteins.

또한, 상기 락툴로스는 기질로서 유당(lactose)를 사용하여 합성되는 것이 바람직하고 상기 기질의 농도는 50 g/L 내지 700 g/L 범위인 것이 바람직하고, 700 g/L 내의 범위인 것은 더욱 바람직하다. 또한, 상기 효소 반응 시간은 통상적인 방법에 따라 적절히 조절될 수 있다. In addition, the lactulose is preferably synthesized using lactose as a substrate, 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 time may be appropriately adjusted according to conventional methods.

본 발명은 셀로비오스 2-에피머레이즈 유전자 또는 엔아세틸글루코사민 2-에피머레이즈를 포함하는 재조합 발현 벡터, 이로 형질전환된 미생물 및 이들을 이용하여 셀로비오스 2-에피머레이즈 또는 엔아세틸글루코사민 2-에피머레이즈를 대량으로 얻는 제조방법, 그리고 상기 셀로비오스 2-에피머레이즈 또는 엔아세틸글루코사민 2-에피머레이즈를 이용하여 락툴로스를 고수율로 얻는 생산 방법을 제공하는 효과가 있다.The present invention relates to recombinant expression vectors comprising cellobiose 2-epimerase gene or enacetylglucosamine 2-epimerase, microorganisms transformed therefrom and cellobiose 2-epimerase or enacetylglucosamine 2-epi. There is an effect of providing a production method for obtaining a large amount of Murrays, and a production method for obtaining lactulose in high yield using the cellobiose 2-epimerase or enacetylglucosamine 2-epimerase.

또한, 본 발명에 따른 락툴로스의 제조방법은 종래의 효소적 방법과 비교하여 과당의 첨가 없이 유당으로부터 락툴로스를 고수율로 생산할 수 있으므로 기능성 식품 및 의약품 등의 제조시 유용하게 사용될 수 있다.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.

도 1a는 본 발명의 셀로비오스 2-에피머레이즈의 pH에 따른 효소 활성도를 나타낸 것이고(●: PIPES 완충용액; ○: EPPS 완충용액), 도 1b는 온도에 따른 효소 활성도를 나타낸 것이다. 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.

도 2는 본 발명의 셀로비오스 2-에피머레이즈의 온도 안정성 측정 결과를 나타낸 것이다(●: 65℃; △: 70℃; □: 75℃ 및 ○: 80℃).Figure 2 shows the results of measuring the temperature stability of the cellobiose 2-epimerase of the present invention (●: 65 ℃; △: 70 ℃; □: 75 ℃ and ○: 80 ℃).

도 3a는 본 발명의 셀로비오스 2-에피머레이즈의 효소양 에 따른 락툴로스 생산량을 나타낸 것이고, 도 3b는 기질 농도에 따른 락툴로스 생산량을 나타낸 것이다 (●: Lactulose; ○: Conversion rate). 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).

도 4는 본 발명의 셀로비오스 2-에피머레이즈에 의한 기질농도 700 g/ℓ에서 반응 시간에 따른 락툴로스의 생산량을 나타낸 것이다(○: Lactose; ●: Lactulose; ■: Epilactose).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).

도 5는 본 발명의 발현 벡터의 개열지도를 나타낸 그림이다.5 is a diagram showing a cleavage map of the expression vector of the present invention.

도 6부터 15는 본 발명의 엔아세틸글루코사민 2-에피머레이즈들에 의한 기질농도 700 g/L에서 반응 시간에 따른 락툴로스의 생산량을 각각 나타낸 것이다(○: Lactose; ●: Lactulose; ■: Epilactose). 도 6은 에어로리네 써모필라(Anaerolinea thermophila), 도 7은 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725),도 8은 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 도 9는 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 도 10은 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 도 11은 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 도 12는 페니바실러스 (Paenibacillus sp.), 도 13은 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 도 14는 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 도 15는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571)를 나타냄.6 to 15 show the production of lactulose with reaction time at substrate concentration of 700 g / L by the enacetylglucosamine 2-epimerase of the present invention (○: Lactose; ●: Lactulose; ■: Epilactose ). FIG. 6 is Anaerolinea thermophila , FIG. 7 is Caldicellulosiruptor bescii DSM 6725, FIG. 8 is Caldicellulosiruptor hydrothermalis , and FIG. 9 is caldicellulose . Caldicellulosiruptor kristjanssonii , FIG. 10 shows Caldicellulosiruptor obsidiansis , FIG. 11 shows Dictyoglomus turgidum , and FIG. 12 shows Paenibacillus sp. FIG. 13 shows a Rhodothermus marinus DSM 4252, FIG. 14 shows a Spirochaeta thermophila DSM 6192, and FIG. 15 shows a Thermoaerobacterium thermosaccharolyticum DSM 571. .

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예 1. 셀로비오스 2-에피머레이즈 유전자를 포함하는 재조합 발현 벡터 및 형질전환 미생물의 제조 Example 1 Preparation of Recombinant Expression Vector and Transgenic Microorganism Comprising Cellobiose 2-Epimerase Gene

본 발명의 셀로비오스 2-에피머레이즈를 제조하기 위하여, 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주(DSMZ사 균주)로부터 유래한 셀로비오스 2-에피머레이즈 유전자를 먼저 분리하였다.For the production of 2-epi-cellobiose Murray's of the present invention, the cellobiose was isolated 2-epi Murray's first gene derived from a cellulose kaldi saccharide syrup sat Laura ET carcass (Caldicellulosiruptor saccharolyticus) strain (DSMZ four strains).

구체적으로, 유전자 염기서열과 아미노산 서열이 이미 특정되어 있는 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주를 선별하고, 이로부터 유래한 서열번호 42의 염기서열을 포함하는 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 유전자의 서열[서열목록 참조; 진뱅크 수탁번호 YP_001179132(Genebank Accession No. YP_001179132)을 기초로 하여 서열번호 43 및 서열번호 44의 염기서열을 각각 포함하는 프라이머들(primers)을 고안 제작하였다.Specifically, the gene sequence and the amino acid sequence kaldi cell rules syrup that has already been specified Sat saccharide Laura ET carcass (Caldicellulosiruptor saccharolyticus) selecting a strain, and a kaldi cellulose comprising the nucleotide sequence of SEQ ID NO: 42 derived therefrom syrup Sat LAURA ET coarse saccharide (Caldicellulosiruptor saccharolyticus) sequence of the gene [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: 43 and SEQ ID NO: 44 were designed and manufactured.

서열번호 43(정방향 프라이머): 5'-AA GCTAGC ATGGATATTACAAGGTTTAAG-3'SEQ ID NO: 43 (Forward primer): 5'-AA GCTAGC ATGGATATTACAAGGTTTAAG-3 '

서열번호 44(역방향 프라이머): 5'-TT GAATTC TTAGTCAACCCTTTTTATTAT-3'SEQ ID NO: 44 (Reverse primer): 5'-TT GAATTC TTAGTCAACCCTTTTTATTAT-3 '

상기 프라이머는 각각 엔에이치이 원(Nhe I, 언더라인)과 에코알 원(EcoR I, 언더라인) 제한효소 절단부분으로 설계되었으며, 상기 프라이머를 이용한 중합효소 연쇄반응(PCR)을 실시하여 해당 유전자의 염기서열을 증폭하였다.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.

대량으로 얻은 셀로비오스 2-에피머레이즈효소 유전자는 제한효소 Nhe I와 EcoR I을 사용하여 벡터 pET24a(+)(Novagen사 제품)의 제한효소 Nhe I와 EcoR I 부위에 삽입하여 재조합 발현 벡터 pET24a(+)/셀로비오스 2-에피머레이즈를 제작하였다.The cellobiose 2-epimerase gene gene obtained in large quantities was inserted into the restriction enzymes Nhe I and Eco RI of the vector pET24a (+) (manufactured by Novagen) using the restriction enzymes Nhe I and Eco R I and the recombinant expression vector pET24a ( +) / Cellobiose 2-epimerase was prepared.

상기와 같이 얻은 재조합 발현 벡터는 통상적인 형질전환 방법에 의하여 대장균 이알(ER) 2566 균주(New England Biolabs사 제품)에 형질전환하였다. 또한, 상기 형질전환된 미생물은 20% 글리세린(glycerin) 용액에 첨가하여 락툴로스의 생산을 위한 배양을 실시하기 전에 냉동보관 하였다. 상기 재조합 대장균은 E. Coli ER2566 pET24a(+)/셀로비오스 2-에피머레이즈 균주로 명명하였다. 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.

실시예 2. 셀로비오스 2-에피머레이즈의 제조Example 2. Preparation of Cellobiose 2-Epimerase

본 발명의 셀로비오스 2-에피머레이즈를 대량 생산하기 위하여, 냉동 보관된 상기 실시예 1의 재조합 대장균 ER 2566 균주를 LB 배지 3 ml가 들어있는 시험관(test tube)에 접종하고, 600nm에서 흡광도가 2.0이 될 때까지 37℃의 진탕 배양기로 종균 배양을 실시하였다. 그 다음 상기 종균 배양된 배양액을 20㎍/ml의 kanamycin 항생제가 첨가된 LB 배지 500 ml이 들어있는 2,000 ml 플라스크에 첨가하여 본 배양을 실시하였다.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.

또한, 600nm에서 흡광도가 0.5가 될 때, 0.1 mM 아이피티지(IPTG)를 첨가하여 셀로비오스 2-에피머레이즈 효소의 대량 발현을 유도하였다. 이때 교반 속도는 200 rpm, 배양온도는 37℃가 유지될 수 있도록 조절하였으며, ITPG 첨가 후에는 교반 속도 150 rpm, 배양 온도는 16℃로 조정하여 배양하였다.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 ℃.

또한, 상기와 같이 과발현되어 생산된 셀로비오스 2-에피머레이즈는 상기 형질전환된 균주의 배양액을 6,000 xg로 4℃에서 30분 동안 원심분리하고, 0.85% 염화나트륨(NaCl)으로 두 번 세척한 다음 50 mM 제일일산나트륨, 300 mM 염화나트륨, 0.1 mM 단백분해 효소 저해제(phenylmethylsulfonyl fluoride)를 첨가하여 상기 세포 용액을 초음파 파쇄기(sonicator)로 파쇄하였다. 상기 세포 파쇄물을 다시 13,000 xg로 4℃에서 20분 동안 원심분리하고, 세포 펠렛을 제거하고 세포 상등액만 얻어 고속 단백질 액체 크로마토그래피(fast protein liquid chromatography system; Bio-Rad Laboratories, Hercules, CA, USA)에 히스텍(His-tag)을 이용한 히스트랩 에이치피(HisTrap HP) 흡착 컬럼을 장착하여 락툴로스 생산에 사용되는 효소액으로서 분리하였다.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.

실시예 3. 셀로비오스 2-에피머레이즈 활성에 미치는 pH 및 온도 효과 조사Example 3 Investigation of pH and Temperature Effects on Cellobiose 2-Epimerase Activity

본 발명의 락툴로스 생산에 있어 셀로비오스 2-에피머레이즈의 pH 및 온도에 따른 활성 변화를 조사하기 위하여, 다양한 pH 및 온도 조건 하에서 효소와 기질을 반응시키고 효소 활성을 비교하였다.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. 셀로비오스 2-에피머레이즈 활성에 미치는 pH 효과3-1. PH Effect on Cellobiose 2-Epimerase Activity

먼저, 락툴로스 생산에 있어서 셀로비오스 2-에피머레이즈 활성에 대한 pH 효과를 조사하기 위하여, 기질로서 50 g/L 유당, 1 mg/ml 효소가 포함된 50 mM 피페스(piperazine-N,N'-bis(2-ethane sulfonic acid), PIPES) 완충용액과, 50 g/L 유당, 1 mg/ml 효소가 포함된 50 mM 이피피에스(N-(2-hydroxyethyl piperazine-N-(3-propane sulfonic acid, EPPS) 완충용액을 사용하여 pH 6.5에서부터 8.5 범위까지 효소 반응을 실시하되, 75℃에서 20분 동안 효소 반응을 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켰다. 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.

그 결과, 도 1a에 나타난 바와 같이, 최적 pH는 7.5인 것을 확인하였다.As a result, as shown in Figure 1a, it was confirmed that the optimum pH is 7.5.

3-2. 셀로비오스 2-에피머레이즈 활성에 미치는 온도 효과3-2. Temperature Effect on Cellobiose 2-Epimerase Activity

락툴로스 생산에 있어서 셀로비오스 2-에피머레이즈 활성에 대한 온도 효과를 조사하기 위하여, 효소 반응 온도를 65℃에서 90℃ 범위까지 변화시키면서 50 g/L 유당, 1 mg/ml 효소가 포함된 pH 7.5인 50 mM EPPS 완충용액을 사용하여 각각 20분 동안 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켰다. 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.

그 결과, 최적 온도는 75℃ 임을 확인하였다(도 1b 참조).As a result, the optimum temperature was confirmed to be 75 ℃ (see Figure 1b).

3-3. 셀로비오스 2-에피머레이즈의 온도 안정성 조사3-3. Investigation of Temperature Stability of Cellobiose 2-Epimerase

본 발명의 셀로비오스 2-에피머레이즈의 온도 안정성을 조사하기 위하여, 온도 60℃에서 80℃ 범위까지 변화시키면서 50 g/L 유당, 1 mg/ml 효소가 포함된 pH 7.5인 50 mM PIPES 완충용액을 사용하여 각각 효소의 활성이 절반으로 줄어드는 시간까지 반응을 진행시킨 후, 반응이 완료되면 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켰다.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.

그 결과, 도 3에 나타난 바와 같이, 온도 65℃에서는 73.9 시간, 70℃에서는 34.4 시간, 75℃에서는 15.8 시간, 그리고 80℃에서는 5.2 시간이 경과시 효소 활성이 절반으로 줄어드는 것을 확인할 수 있었다 (도 2 참조). 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).

실시예 4. 셀로비오스 2-에피머레이즈의 효소 양 및 기질 농도에 따른 락툴로스 생산성 확인 Example 4 Lactulose Productivity Verification According to Enzyme Amount and Substrate Concentration of Cellobiose 2-Epimerase

4-1. 셀로비오스 2-에피머레이즈 효소 양에 따른 락툴로스 생산4-1. Lactulose Production According to Cellobiose 2-Epimerase Enzyme Amount

본 발명의 락툴로스 생산에 있어 셀로비오스 2-에피머레이즈의 효소양에 따른 락툴로스 생산을 확인하기 위하여 기질로서 400 g/L 유당 및 0.5에서 10 mg/ml 효소가 포함된 50 mM PIPES 완충용액(pH 7.5)에서 2 시간 동안 온도 75℃로 효소 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켜 락툴로스 생산을 비교 하였다. 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.

그 결과, 도 3a에서와 같이 5 mg/ml 이상의 효소 농도에서 최고의 락툴로스 생산을 확인할 수 있었다.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. 셀로비오스 2-에피머레이즈 기질농도에 따른 락툴로스 생산4-2. Lactulose Production According to Cellobiose 2-Epimerase Substrate Concentration

본 발명의 락툴로스 생산에 있어 셀로비오스 2-에피머레이즈의 효소양에 따른 락툴로스 생산을 확인하기 위하여 기질로서 50 - 400 g/L 유당을 사용하여 10 mg/ml 효소가 포함된 50 mM PIPES 완충용액(pH 7.5)에서 2 시간 동안 온도 75℃로 효소 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켜 락툴로스 생산을 비교 하였다. 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.

그 결과, 도 3b에서와 같이 기질 농도가 높을수록 높은 락툴로스 생산을 확인할 수 있었다.As a result, higher lactulose production was confirmed as the substrate concentration was higher as shown in Figure 3b.

실시예 5. 셀로비오스 2-에피머레이즈를 이용한 락툴로스의 생산성 확인Example 5 Productivity Verification of Lactulose Using Cellobiose 2-Epimerase

본 발명의 셀로비오스 2-에피머레이즈를 이용한 락툴로스의 생산성을 확인하기 위하여, 기질로서 700 g/L 유당 및 10 mg/ml 효소가 포함된 50 mM PIPES 완충용액(pH 7.5)에서 4 시간 동안 온도 75℃로 효소 반응을 수행하였다. 매시간 효소 반응액을 수집하고 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시킨 후, HPLC를 통하여 락툴로스 생산량을 측정하였다.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.

그 결과, 도 4에서와 같이 반응 2시간 후에 700 g/ℓ의 유당에서 414 g/ℓ의 락툴로스가 생산되어 시간당 208.5 g/ℓ의 생산성과 59% 전환수율을 나타내었다. 그리고, 락툴로스 생산에 있어서 락툴로스 이외에도 유당의 글루코스가 만노스(mannose)로 전환된 에피락토스(epilactose)도 함께 생산당으로 생산되었으며, 2시간 동안 100 g/L가 생산되었다.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.

실시예 6. 엔아세틸글루코사민 2-에피머레이즈 유전자를 포함하는 재조합 발현 벡터 및 형질전환 미생물의 제조Example 6 Preparation of Recombinant Expression Vector and Transgenic Microorganism Comprising Enacetylglucosamine 2-Epimerase Gene

본 발명의 엔아세틸글루코사민 2-에피머레이즈를 제조하기 위하여, 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주로부터 유래한 엔아세틸글루코사민 2-에피머레이즈 유전자를 먼저 분리하였다.In order to prepare the enacetylglucosamine 2-epimerase of the present invention,Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum The enacetylglucosamine 2-epimerase gene from the DSM 571) strain was first isolated.

구체적으로, 유전자 염기서열과 아미노산 서열이 이미 특정되어 있는 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 균주를 선별하고, 이로부터 유래한 각각 서열번호 1 내지 10의 염기서열을 포함하는 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스 (Paenibacillus sp.), 로도써머스 마리너스 (Rhodothermus marinus DSM 4252), 스피로케타 써모필라 (Spirochaeta thermophila DSM 6192), 또는 써모언에어로박테리운 써모싸카로리튬 (Thermoanaerobacterium thermosaccharolyticum DSM 571) 유전자의 서열을 기초로 하여 하기 서열번호 11 내지 30의 프라이머들(primers)을 고안 제작하였다.Specifically, unairrene thermophila, in which the gene sequence and the amino acid sequence are already specified (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) strains were selected and derived from the unairrone thermophila comprising the nucleotide sequence of SEQ ID NOs: 1 to 10, respectively (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), or Thermoaerobacterium Thermosaccharium Lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571) primers of SEQ ID NOS: 11 to 30 were designed and constructed based on the sequence of the gene.

언에어로리네 써모필라Unaeroline Thermophila (( Anaerolinea thermophilaAnaerolinea thermophila ))

(정방향 프라이머): 5'-TTCATATGATGGAGATCCGTATGCTCAG-3'(서열번호 11)(Forward primer): 5'-TT CATATG ATGGAGATCCGTATGCTCAG-3 '(SEQ ID NO: 11)

(역방향 프라이머): 5'-TTCTCGAGTCACTCTCTTGACGATTGCGAG-3'(서열번호 12)(Reverse primer): 5'-TT CTCGAG TCACTCTCTTGACGATTGCGAG-3 '(SEQ ID NO: 12)

칼디셀룰로시럽토Caldicellulose Syrup 베시 ( Betsy ( Caldicellulosiruptor besciiCaldicellulosiruptor bescii DSM 6725) DSM 6725)

(정방향 프라이머): 5'-TTGCTAGCATGGATATTACAAAGTTTAA-3'(서열번호 13)(Forward primer): 5'-TT GCTAGC ATGGATATTACAAAGTTTAA-3 '(SEQ ID NO: 13)

(역방향 프라이머): 5'-TTGTCGAGTCAACCAACTCTCTTTATTAT-3'(서열번호 14)(Reverse primer): 5'-TT GTCGAG TCAACCAACTCTCTTTATTAT-3 '(SEQ ID NO: 14)

칼디셀룰로시럽토 하이드로써머리스Caldicellulose Syrup To Hydro Summar ( ( Caldicellulosiruptor hydrothermalisCaldicellulosiruptor hydrothermalis ))

(정방향 프라이머): 5'-TTGCTAGCATGGATATTACAAGGTTTAA-3'(서열번호 15)(Forward primer): 5'-TT GCTAGC ATGGATATTACAAGGTTTAA-3 '(SEQ ID NO: 15)

(역방향 프라이머): 5'-TTGAATTCTTAGTCAACCCTTTTTATTATC-3'(서열번호 16)(Reverse primer): 5'-TT GAATTC TTAGTCAACCCTTTTTATTATC-3 '(SEQ ID NO: 16)

칼디셀룰로시럽토 크리스트잔쏘니Caldicellulose Syrupto Cristianzoni ( ( Caldicellulosiruptor kristjanssoniiCaldicellulosiruptor kristjanssonii ))

(정방향 프라이머): 5'-TTGCTAGCATGGATATTACCAGGTTTAA-3'(서열번호 17)(Forward primer): 5'-TT GCTAGC ATGGATATTACCAGGTTTAA-3 '(SEQ ID NO: 17)

(역방향 프라이머): 5'-TTGAATTCTTAACCAACTCTCTTTATTA-3'(서열번호 18)(Reverse primer): 5'-TT GAATTC TTAACCAACTCTCTTTATTA-3 '(SEQ ID NO: 18)

칼디셀룰로시럽토 옵시디안시스 Caldicellulose Syrupto Obsidiansis (( Caldicellulosiruptor obsidiansisCaldicellulosiruptor obsidiansis ))

(정방향 프라이머): 5'-TTGCTAGCATGGATATTACCAGTTTTAA-3'(서열번호 19)(Forward primer): 5'-TT GCTAGC ATGGATATTACCAGTTTTAA-3 '(SEQ ID NO: 19)

(역방향 프라이머): 5'-TTGAATTCTTAACCAACTCTCTTTATTAT-3'(서열번호 20)(Reverse primer): 5'-TT GAATTC TTAACCAACTCTCTTTATTAT-3 '(SEQ ID NO: 20)

딕티오글루모스 터기듐Dictioglumos Tergium ( ( Dictyoglomus turgidumDictyoglomus turgidum ))

(정방향 프라이머): 5'-TTGCTAGCATGGATTTAAAAGTTTTAAA-3'(서열번호 21)(Forward primer): 5'-TT GCTAGC ATGGATTTAAAAGTTTTAAA-3 '(SEQ ID NO: 21)

(역방향 프라이머): 5'-TTGAATTCTTAAATCCTTTTTATTACCT-3'(서열번호 22)(Reverse primer): 5'-TT GAATTC TTAAATCCTTTTTATTACCT-3 '(SEQ ID NO: 22)

페니바실러스Penny Bacillus ( ( PaenibacillusPaenibacillus sp.) sp.)

(정방향 프라이머): 5'-TTGCTAGCATGACAATGACTTTACATAC-3'(서열번호 23)(Forward primer): 5'-TT GCTAGC ATGACAATGACTTTACATAC-3 '(SEQ ID NO: 23)

(역방향 프라이머): 5'-TTGAATTCTTAGAGCTTGCTGCTTACAT-3'(서열번호 24)(Reverse primer): 5'-TT GAATTC TTAGAGCTTGCTGCTTACAT-3 '(SEQ ID NO: 24)

로도써머스 마리너스Rhodon Somers Mariners ( ( Rhodothermus marinusRhodothermus marinus DSM 4252) DSM 4252)

(정방향 프라이머): 5'-TTGCTAGCATGAGCACGGAAACCATCCC-3'(서열번호 25)(Forward primer): 5'-TT GCTAGC ATGAGCACGGAAACCATCCC-3 '(SEQ ID NO: 25)

(역방향 프라이머): 5'-TTGAATTCCTACCGGGATCGAGCGTGTT-3'(서열번호 26)(Reverse primer): 5'-TT GAATTC CTACCGGGATCGAGCGTGTT-3 '(SEQ ID NO: 26)

스피로케타 써모필라Spiroketa thermophila ( ( Spirochaeta thermophilaSpirochaeta thermophila DSM 6192) DSM 6192)

(정방향 프라이머): 5'-TTGCTAGCATGCCTCTTCCCACTACCCT-3'(서열번호 27)(Forward primer): 5'-TT GCTAGC ATGCCTCTTCCCACTACCCT-3 '(SEQ ID NO: 27)

(역방향 프라이머): 5'-TTGAATTCTCATCTTCGTTCCTCCTCGA-3'(서열번호 28)(Reverse primer): 5'-TT GAATTC TCATCTTCGTTCCTCCTCGA-3 '(SEQ ID NO: 28)

써모언에어로박테리운 써모싸카로리튬 ( Thermoanaerobacterium thermosaccharolyticum DSM 571) Thermo frozen aero bacteria cloud inexpensive thermopile Caro lithium (Thermoanaerobacterium thermosaccharolyticum DSM 571)

(정방향 프라이머): 5'-TTGCTAGCATGGAGAAAATAGCACAGGA-3'(서열번호 29)(Forward primer): 5'-TT GCTAGC ATGGAGAAAATAGCACAGGA-3 '(SEQ ID NO: 29)

(역방향 프라이머): 5'-TTCTCGAGTTAAACCTCATTGCCTTTCAC-3'(서열번호 30)(Reverse primer): 5'-TT CTCGAG TTAAACCTCATTGCCTTTCAC-3 '(SEQ ID NO: 30)

상기 프라이머는 각각 엔디이 원(Nde I, 언더라인), 엔에이치이 원 (Nhe I, 언더라인), 엑스에이치오 원(Xho I, 언더라인), 그리고 이코알원 (EcoR I, 언더라인) 제한효소 절단부분으로 설계되었으며, 상기 프라이머를 이용한 중합효소 연쇄반응(PCR)을 실시하여 해당 유전자의 염기서열을 증폭하였다.The primers are each endiyi source (Nde I, underlined), yen eyichiyi source (Nhe I, underlined), X H. oh source (Xho I, underlined), and Ico alwon (Eco RI, underlined) restriction enzyme digestion It was designed as a part, and the polymerase chain reaction (PCR) using the primers was performed to amplify the base sequence of the gene.

대량으로 얻은 엔아세틸 글루코사민 2-에피머레이즈 유전자는 각각의 제한효소를 사용하여 벡터 pET28a(+)(Novagen사 제품)의 동일한 제한효소 부위에 삽입하여 재조합 발현 벡터 pET28a(+)/엔아세틸글루코사민 2-에피머레이즈를 제작하였다.The enacetyl glucosamine 2-epimerase gene obtained in large quantities was inserted into the same restriction enzyme site of the vector pET28a (+) (manufactured by Novagen) using the respective restriction enzymes to generate the recombinant expression vector pET28a (+) / enacetylglucosamine 2 -Epimerase was produced.

상기와 같이 얻은 재조합 발현 벡터는 통상적인 형질전환 방법에 의하여 대장균 이알(ER) 2566 균주에 형질전환하였다. 또한, 상기 형질전환된 미생물은 20% 글리세린(glycerin) 용액에 첨가하여 락툴로스의 생산을 위한 배양을 실시하기 전에 냉동보관 하였다. 상기 재조합 대장균은 E. Coli ER2566 pET28a(+)/엔아세틸글루코사민 2-에피머레이즈 균주로 명명하였다. The recombinant expression vector thus obtained was transformed into E. coli ER 2566 strain 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 as E. Coli ER2566 pET28a (+) / enacetylglucosamine 2-epimerase strain.

실시예 7. 엔아세틸 글루코사민 2-에피머레이즈의 제조Example 7 Preparation of Enacetyl Glucosamine 2-Epimerase

본 발명의 엔아세틸글루코사민 2-에피머레이즈를 대량 생산하기 위하여, 냉동 보관된 상기 실시예 6의 재조합 대장균 ER 2566 균주들을 LB 배지 3ml가 들어있는 시험관(test tube)에 접종하고, 600nm에서 흡광도가 2.0이 될 때까지 37℃의 진탕 배양기로 종균 배양을 실시하였다. 그 다음 상기 종균 배양된 배양액을 20 μg/ml의 kanamycin 항생제가 첨가된 LB 배지 500 ml이 들어있는 2,000 ml 플라스크에 첨가하여 본 배양을 실시하였다.In order to mass-produce the enacetylglucosamine 2-epimerase of the present invention, the recombinant E. coli ER 2566 strains of Example 6, which were stored in frozen form, were 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.

또한, 600nm에서 흡광도가 0.5가 될 때, 0.1 mM 아이피티지(IPTG)를 첨가하여 엔아세틸글루코사민 2-에피머레이즈의 대량 발현을 유도하였다. 이때 교반 속도는 200 rpm, 배양온도는 37℃가 유지될 수 있도록 조절하였으며, ITPG 첨가 후에는 교반 속도 150 rpm, 배양 온도는 16℃로 조정하여 배양하였다.In addition, when the absorbance became 0.5 at 600 nm, 0.1 mM Iptage (IPTG) was added to induce mass expression of enacetylglucosamine 2-epimerase. 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 ℃.

또한, 상기와 같이 과발현 되어 생산된 엔아세틸글루코사민 2-에피머레이즈는 상기 형질전환된 균주의 배양액을 6,000g로 4℃에서 30분 동안 원심분리하고, 0.85% 염화나트륨(NaCl)으로 두 번 세척한 다음 50 mM 제일일산나트륨, 300 mM 염화나트륨, 0.1 mM 단백분해 효소 저해제(phenylmethylsulfonyl fluoride)를 첨가하여 상기 세포 용액을 초음파 파쇄기(sonicator)로 파쇄하였다. 상기 세포 파쇄물을 다시 13,000 g로 4℃에서 20분 동안 원심분리하고, 세포 펠렛을 제거하고 세포 상등액만 얻어 고속 단백질 액체 크로마토그래피(fast protein liquid chromatography system; Bio-Rad Laboratories, Hercules, CA, USA)에 히스텍(His-tag)을 이용한 히스트랩 에이치피(HisTrap HP) 흡착 컬럼을 장착하여 락툴로스 생산에 사용되는 효소액으로서 분리하였다.In addition, the enacetylglucosamine 2-epimerase produced by overexpression as described above was centrifuged at 6,000 g for 30 minutes at 6,000 g of the culture medium of the transformed strain, and washed twice with 0.85% sodium chloride (NaCl). The cell solution was then 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 minutes at 4 ° C., cell pellets were removed, and only cell supernatant was obtained for fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA). A HisTrap HP adsorption column using a His-tag was attached to and separated as an enzyme solution used for lactulose production.

실시예 8. 엔아세틸글루코사민 2-에피머레이즈 활성에 미치는 pH 및 온도 효과 조사 Example 8 Investigation of pH and Temperature Effects on Enacetylglucosamine 2-Epimerase Activity

본 발명의 락툴로스 생산에 있어 엔아세틸글루코사민 2-에피머레이즈의 pH 및 온도에 따른 활성 변화를 조사하기 위하여, 다양한 pH 및 온도 조건 하에서 효소와 기질을 반응시키고 효소 활성을 비교하였다.In order to investigate the change in pH and temperature of the activity of enacetylglucosamine 2-epimerase in the production of lactulose of the present invention, enzymes and substrates were reacted under various pH and temperature conditions and enzyme activities were compared.

8-1. 엔아세틸글루코사민 2-에피머레이즈 활성에 미치는 pH 효과8-1. Effect of pH on Enacetylglucosamine 2-epimerase Activity

먼저, 락툴로스 생산에 있어서 엔아세틸글루코사민 2-에피머레이즈 활성에 대한 pH 효과를 조사하기 위하여, 기질로서 50 g/L 유당, 1 mg/ 효소가 포함된 50 mM 피페스(piperazine-N,N'-bis(2-ethane sulfonic acid), PIPES) 완충용액과, 50 g/L 유당, 1 mg/ 효소가 포함된 50 mM 이피피에스(N-(2-hydroxyethyl piperazine-N-(3-propane sulfonic acid, EPPS) 완충용액을 사용하여 pH 6.5에서부터 8.5 범위까지 효소 반응을 실시하되, 65-80℃에서 10분 동안 효소 반응을 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켰다. First, in order to investigate the pH effect on enacetylglucosamine 2-epimerase activity in lactulose production, 50 mM pipese containing 50 g / L lactose, 1 mg / enzyme (piperazine- N , N '-bis (2-ethane sulfonic acid), PIPES) buffer, 50 g / L lactose, 1 mg / 50 mM IPS containing N- (2-hydroxyethyl piperazine- N- (3-propane sulfonic acid, EPPS) was used to perform the enzyme reaction in the range of pH 6.5 to 8.5, but the enzyme reaction was carried out for 10 minutes at 65-80 ℃ and the reaction was stopped by the addition of 200 mM hydrogen chloride.

그 결과, 표 1 에 나타난 바와 같이, 최적 pH는 7.0-8.0인 것을 확인하였다.As a result, as shown in Table 1, it was confirmed that the optimum pH is 7.0-8.0.

표 1

Figure PCTKR2012001177-appb-T000001
Table 1
Figure PCTKR2012001177-appb-T000001

8-2. 엔아세틸글루코사민 2-에피머레이즈 활성에 미치는 온도 효과8-2. Effect of temperature on enacetylglucosamine 2-epimerase activity

락툴로스 생산에 있어서 엔아세틸글루코사민 2-에피머레이즈 활성에 대한 온도 효과를 조사하기 위하여, 효소 반응 온도를 60에서 90℃ 범위까지 변화시키면서 50 g/L 유당, 1 mg/ml 효소가 포함된 50 mM EPPS 완충용액을 사용하여 각각 20분 동안 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켰다. To investigate the effect of temperature on the enacetylglucosamine 2-epimerase activity in lactulose production, 50 g / L lactose, 50 mg / ml enzyme was added, varying the enzyme reaction temperature in the range from 60 to 90 ° C. Each reaction was performed for 20 minutes using mM EPPS buffer, and then the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride.

그 결과, 최적 온도는 65-80℃임을 확인하였다(표 2 참조).As a result, the optimum temperature was found to be 65-80 ℃ (see Table 2).

표 2

Figure PCTKR2012001177-appb-T000002
TABLE 2
Figure PCTKR2012001177-appb-T000002

실시예 9. 엔아세틸글루코사민 2-에피머레이즈의 효소 양 및 기질 농도에 따른 락툴로스 생산성 확인 Example 9 Lactulose Productivity Verification According to Enzyme Amount and Substrate Concentration of Enacetylglucosamine 2-Epimerase

9-1. 엔아세틸글루코사민 2-에피머레이즈 효소 양에 따른 락툴로스 생산9-1. Lactulose Production According to Enacetylglucosamine 2-epimerase Enzyme Amount

본 발명의 락툴로스 생산에 있어 엔아세틸글루코사민 2-에피머레이즈의 효소양에 따른 락툴로스 생산을 확인하기 위하여 기질로서 400 g/L 유당 및 0.5에서 10 mg/ml 효소가 포함된 50 mM PIPES 완충용액 에서 2 시간 동안 온도 75℃로 효소 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켜 락툴로스 생산을 비교하였다. 50 mM PIPES buffer containing 400 g / L lactose and 0.5 to 10 mg / ml enzyme as substrate to confirm lactulose production according to the amount of enacetylglucosamine 2-epimerase in lactulose production of the present invention. After enzymatic reaction at a temperature of 75 ° C. for 2 hours in solution, the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to compare the production of lactulose.

그 결과, 표 3에서와 같이 3-7 mg/ml 이상의 효소 농도에서 최고의 락툴로스 생산을 확인할 수 있었다.As a result, the highest lactulose production was confirmed at the enzyme concentration of 3-7 mg / ml or more as shown in Table 3.

표 3

Figure PCTKR2012001177-appb-T000003
TABLE 3
Figure PCTKR2012001177-appb-T000003

9-2. 엔아세틸글루코사민 2-에피머레이즈 기질농도에 따른 락툴로스 생산9-2. Lactulose Production According to Enacetylglucosamine 2-Epimerase Substrate Concentration

본 발명의 락툴로스 생산에 있어 엔아세틸글루코사민 2-에피머레이즈의 효소양에 따른 락툴로스 생산을 확인하기 위하여 기질로서 50 - 700 g/L 유당을 사용하여 10 mg/ml 효소가 포함된 50 mM PIPES 완충용액(pH 7.5)에서 2 시간 동안 온도 75℃로 효소 실시한 후 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시켜 락툴로스 생산을 비교하였다. 50 mM containing 10 mg / ml enzyme using 50-700 g / L lactose as a substrate to confirm lactulose production according to the amount of enacetylglucosamine 2-epimerase in lactulose production of the present invention. The enzyme was subjected to enzymatic reaction at 75 ° C. for 2 hours in PIPES buffer (pH 7.5), and then the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to compare lactulose production.

그 결과, 표 4에서와 같이 기질 농도가 높을수록 높은 락툴로스 생산을 확인할 수 있었다.As a result, higher lactulose production was confirmed as the substrate concentration was higher as shown in Table 4.

표 4

Figure PCTKR2012001177-appb-T000004
Table 4
Figure PCTKR2012001177-appb-T000004

실시예 10. 엔아세틸글루코사민 2-에피머레이즈를 이용한 락툴로스의 생산성 확인 Example 10 Confirmation of Productivity of Lactulose Using Enacetylglucosamine 2-Epimerase

본 발명의 엔아세틸글루코사민 2-에피머레이즈를 이용한 락툴로스의 생산성을 확인하기 위하여, 기질로서 700 g/L 유당 및 3-7 mg/ml 효소가 포함된 50 mM PIPES 완충용액(pH 7.5)에서 4 시간 동안 온도 각각 효소의 최적 온도로 효소 반응을 수행하였다. 매시간 효소 반응액을 수집하고 최종 농도 200 mM 염화수소를 첨가하여 반응을 정지시킨 후, HPLC를 통하여 락툴로스 생산량을 측정하였다.In order to confirm the productivity of lactulose using the enacetylglucosamine 2-epimerase of the present invention, in a 50 mM PIPES buffer solution (pH 7.5) containing 700 g / L lactose and 3-7 mg / ml enzyme as a substrate. Enzyme reactions were carried out for 4 hours at the optimum temperature of each enzyme. The enzyme reaction solution was collected every hour and the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride, and then the lactulose production was measured by HPLC.

그 결과, 도 6에서 15에서와 같이 반응 2-3 시간 후에 700 g/L의 유당에서 350-420 g/L의 락툴로스가 생산되어 시간당 150-208.5 g/L의 생산성과 50-59% 전환수율을 나타내었다. 그리고, 락툴로스 생산에 있어서 락툴로스 이외에도 유당의 글루코스가 만노스(mannose)로 전환된 에피락토스(epilactose)도 함께 생산당으로 생산되었으며, 2시간 동안 약 60-100 g/L 가 생산되었다.As a result, 350-420 g / L lactulose was produced in 700 g / L lactose after 2-3 hours of reaction as shown in Fig. 6 to 15, yielding 150-208.5 g / L productivity and 50-59% conversion per hour. Yield is shown. In addition, lactose was also produced in the production of lactose, in addition to lactose, epilactose, in which glucose of lactose was converted into mannose, was produced as a production sugar, and about 60-100 g / L was produced for 2 hours.

현재까지 보고된 효소를 이용한 락툴로스의 생산은 기질로서 유당과 과당을 혼합하여 베타갈락토시다제에 의해서 락툴로스를 생산하는 방법에 대한 보고 (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))가 전부이며 효소를 이용하여 유당만을 기질로 사용하여 락툴로스를 생산하는 보고는 본 보고가 처음이다.The production of lactulose using enzymes reported to date is reported on the method for producing lactulose by beta galactosidase by mixing lactose and fructose as substrates (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.

이는 본 발명에 따른 셀로비오스 2-에피머레이즈 또는 엔아세틸글루코사민 2-에피머레이즈를 이용한 락툴로스 생산 방법은 과당의 혼합으로 인한 추가 비용 없이 유당으로부터 락툴로스를 고수율로 생산하는 방법으로 친환경적이며 경제성이 있어서 기존의 락툴로스를 생산하는 방법보다 많은 경쟁력을 보유하고 있다고 할 수 있다.This is a method for producing lactulose using cellobiose 2-epimerase or enacetylglucosamine 2-epimerase according to the present invention is an environmentally friendly method of producing lactulose from lactose in a high yield without additional cost due to the mixing of fructose. Because of its economic feasibility, it can be said to have more competitiveness than the existing method of producing lactulose.

이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. 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. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

a)서열번호 1 내지 10 및 42의 염기서열로 구성된 군으로부터 선택된 하나의 유전자를 포함한 재조합 벡터를 제조하는 단계;a) preparing a recombinant vector comprising one gene selected from the group consisting of SEQ ID NOs: 1 to 10 and 42; b)상기 제조합 벡터를 미생물에 형질전환하는 단계; 및b) transforming the preparative vector into a microorganism; And c) 상기 형질전환된 미생물로부터 락툴로스 생산에 사용되는 효소액을 얻어서 기질을 처리하는 단계를 포함하는 c) treating the substrate by obtaining an enzyme solution used for producing lactulose from the transformed microorganism. 락툴로스(lactulose)의 제조방법.Method for preparing lactulose. 제 1항에 있어서,The method of claim 1, 상기 유전자는 언에어로리네 써모필라(Anaerolinea thermophila), 칼디셀룰로시럽토 베시 (Caldicellulosiruptor bescii DSM 6725), 칼디셀룰로시럽토 하이드로써머리스 (Caldicellulosiruptor hydrothermalis), 칼디셀룰로시럽토 크리스트잔쏘니 (Caldicellulosiruptor kristjanssonii), 칼디셀룰로시럽토 옵시디안시스 (Caldicellulosiruptor obsidiansis), 딕티오글루모스 터기듐 (Dictyoglomus turgidum), 페니바실러스(Paenibacillus sp.), 로도써머스 마리너스(Rhodothermus marinus DSM 4252), 스피로케타 써모필라(Spirochaeta thermophila DSM 6192), 써모언에어로박테리운 써모싸카로리튬(Thermoanaerobacterium thermosaccharolyticum DSM 571) 또는 칼디셀룰로시럽토 사카로라이티커스(Caldicellulosiruptor saccharolyticus ) 균주로부터 유래한 것을 특징으로 하는 락툴로스(lactulose)의 제조방법.The gene is an aeroline thermophila (Anaerolinea thermophila), Caldicellulose Syrup To Besi (Caldicellulosiruptor bescii DSM 6725), Caldicellulose Syrup To Hydrothermal (Caldicellulosiruptor hydrothermalis), Caldicellulose Syrup To Cristzanson (Caldicellulosiruptor kristjanssonii), Caldicellulose Syrup To Obsidiansis (Caldicellulosiruptor obsidiansis), Dictioglumos Tergium (Dictyoglomus turgidum), Penivacillus (Paenibacillus sp.), Rhodon Somers Mariners (Rhodothermus marinus DSM 4252), spiroketa thermophilaSpirochaeta thermophila DSM 6192), Thermoaerobacterium Thermosacro LithiumThermoanaerobacterium thermosaccharolyticum DSM 571) or Caldicellulose Syrup To Saccharolites (Caldicellulosiruptor saccharolyticus ) Lactulose (Lactulose) method characterized in that derived from the strain. 제 1항에 있어서,The method of claim 1, 상기 기질은 유당 (lactose)인 것을 특징으로 하는 락툴로스(lactulose)의 제조방법.The substrate is lactose (lactose) characterized in that the method for producing lactulose (lactulose). 제 1항에 있어서,The method of claim 1, 상기 효소와 기질 반응은 pH 6.5 내지 pH 8.5 범위에서 이루어지는 것을 특징으로 하는 락툴로스(lactulose)의 제조방법.The enzyme and the substrate reaction is a method of producing lactulose (lactulose), characterized in that made in the range of pH 6.5 to pH 8.5. 제 1항에 있어서,The method of claim 1, 상기 효소와 기질 반응은 온도 65 내지 90℃의 범위에서 이루어지는 것을 특징으로 하는 락툴로스(lactulose)의 제조방법.The enzyme and substrate reaction is a method for producing lactulose (lactulose), characterized in that made in the range of 65 to 90 ℃ temperature. 제 1항에 있어서,The method of claim 1, 상기 효소와 기질 반응에서 기질 농도는 50 내지 700 g/L 범위인 것을 특징으로 하는 락툴로스(lactulose)의 제조방법.The substrate concentration in the enzyme and substrate reaction is a method for producing lactulose (lactulose), characterized in that in the range of 50 to 700 g / L. 서열번호 31 내지 41에 기재된 아미노산 서열로 구성된 군으로부터 선택된 효소를 유효성분으로 포함하는 락툴로스(lactulose) 제조용 조성물.A composition for producing lactulose, comprising an enzyme selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 31 to 41 as an active ingredient. 서열번호 31 내지 40에 기재된 아미노산 서열로 구성된 군으로부터 선택된 효소를 유효성분으로 포함하는 에피락토스(epilactose) 제조용 조성물.Epilactose (epilactose) production composition comprising an enzyme selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 40 as an active ingredient.
PCT/KR2012/001177 2011-02-23 2012-02-16 Method for preparing lactulose from lactose using cellobiose 2-epimerase or n-acetyl glucosamin 2-epimerase Ceased WO2012115390A2 (en)

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KR1020110081385A KR101361688B1 (en) 2011-08-16 2011-08-16 Method for production of lactulose from lactose using N-acetyl glucosamin 2-epimerase
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