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WO2012115390A2 - Procédé de préparation de lactulose à partir de lactose en utilisant de la cellobiose 2-épimerase ou de la n-acétyl glucosamine 2-épimerase - Google Patents

Procédé de préparation de lactulose à partir de lactose en utilisant de la cellobiose 2-épimerase ou de la n-acétyl glucosamine 2-épimerase Download PDF

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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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WO2012115390A9 (fr
WO2012115390A3 (fr
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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/ko
Priority claimed from KR1020110081385A external-priority patent/KR101361688B1/ko
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Publication of WO2012115390A2 publication Critical patent/WO2012115390A2/fr
Publication of WO2012115390A9 publication Critical patent/WO2012115390A9/fr
Publication of WO2012115390A3 publication Critical patent/WO2012115390A3/fr
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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

La présente invention concerne un procédé de préparation de lactulose à partir de lactose en utilisant de la cellobiose 2-épimerase ou de la N-acétyl glucosamine 2-épimerase, et concerne plus spécifiquement un procédé de production permettant d'obtenir du lactulose en utilisant des vecteurs d'expression recombinants contenant ces gènes, des microorganismes transformés de ce fait, et des enzymes.
PCT/KR2012/001177 2011-02-23 2012-02-16 Procédé de préparation de lactulose à partir de lactose en utilisant de la cellobiose 2-épimerase ou de la n-acétyl glucosamine 2-épimerase Ceased WO2012115390A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2011-0016067 2011-02-23
KR1020110016067A KR101261004B1 (ko) 2011-02-23 2011-02-23 셀로비오스 2-에피머레이즈를 이용한 유당으로부터 락툴로스의 제조방법
KR1020110081385A KR101361688B1 (ko) 2011-08-16 2011-08-16 엔아세틸 글루코사민 2-에피머레이즈를 이용한 유당으로부터 락툴로스의 제조방법
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JPS56102774A (en) * 1980-01-22 1981-08-17 Shikamitsu Honsha:Kk Production of food product controlling intestinal function and giving trace amount nutrients
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