JP2016508378A - ステビオシドからレバウジオシドaを製造する方法 - Google Patents
ステビオシドからレバウジオシドaを製造する方法 Download PDFInfo
- Publication number
- JP2016508378A JP2016508378A JP2015560087A JP2015560087A JP2016508378A JP 2016508378 A JP2016508378 A JP 2016508378A JP 2015560087 A JP2015560087 A JP 2015560087A JP 2015560087 A JP2015560087 A JP 2015560087A JP 2016508378 A JP2016508378 A JP 2016508378A
- Authority
- JP
- Japan
- Prior art keywords
- glycosyltransferase
- reaction
- rebaudioside
- stevioside
- sucrose synthase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 title claims abstract description 75
- 235000019202 steviosides Nutrition 0.000 title claims abstract description 67
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 title claims abstract description 62
- 229940013618 stevioside Drugs 0.000 title claims abstract description 62
- OHHNJQXIOPOJSC-UHFFFAOYSA-N stevioside Natural products CC1(CCCC2(C)C3(C)CCC4(CC3(CCC12C)CC4=C)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O)C(=O)OC7OC(CO)C(O)C(O)C7O OHHNJQXIOPOJSC-UHFFFAOYSA-N 0.000 title claims abstract description 62
- 239000001512 FEMA 4601 Substances 0.000 title claims abstract description 61
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 title claims abstract description 61
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 235000019203 rebaudioside A Nutrition 0.000 title claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 35
- 238000006243 chemical reaction Methods 0.000 claims abstract description 88
- 108010043934 Sucrose synthase Proteins 0.000 claims abstract description 58
- 108700023372 Glycosyltransferases Proteins 0.000 claims abstract description 56
- 102000051366 Glycosyltransferases Human genes 0.000 claims abstract description 47
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 19
- 229930006000 Sucrose Natural products 0.000 claims abstract description 19
- 239000005720 sucrose Substances 0.000 claims abstract description 19
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 23
- 240000007594 Oryza sativa Species 0.000 claims description 22
- 235000007164 Oryza sativa Nutrition 0.000 claims description 22
- 239000008103 glucose Substances 0.000 claims description 22
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 21
- 239000001177 diphosphate Substances 0.000 claims description 21
- 235000011180 diphosphates Nutrition 0.000 claims description 21
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 20
- 244000228451 Stevia rebaudiana Species 0.000 claims description 16
- 235000009566 rice Nutrition 0.000 claims description 11
- 241000588724 Escherichia coli Species 0.000 claims description 10
- GIPHUOWOTCAJSR-UHFFFAOYSA-N Rebaudioside A. Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC1OC(CO)C(O)C(O)C1OC(C1O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O GIPHUOWOTCAJSR-UHFFFAOYSA-N 0.000 claims description 9
- 235000006092 Stevia rebaudiana Nutrition 0.000 claims description 9
- 241000209140 Triticum Species 0.000 claims description 8
- 235000021307 Triticum Nutrition 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 6
- 241000589158 Agrobacterium Species 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 241000186216 Corynebacterium Species 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 241000209128 Bambusa Species 0.000 claims description 3
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims description 3
- 235000017491 Bambusa tulda Nutrition 0.000 claims description 3
- 241001330002 Bambuseae Species 0.000 claims description 3
- 240000008620 Fagopyrum esculentum Species 0.000 claims description 3
- 235000009419 Fagopyrum esculentum Nutrition 0.000 claims description 3
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 240000006394 Sorghum bicolor Species 0.000 claims description 3
- 235000007230 Sorghum bicolor Nutrition 0.000 claims description 3
- 244000062793 Sorghum vulgare Species 0.000 claims description 3
- 235000007244 Zea mays Nutrition 0.000 claims description 3
- 239000011425 bamboo Substances 0.000 claims description 3
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 claims description 3
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 claims description 3
- 235000019713 millet Nutrition 0.000 claims description 3
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 36
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 22
- 238000004128 high performance liquid chromatography Methods 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 238000006911 enzymatic reaction Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 239000000758 substrate Substances 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000035484 reaction time Effects 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 6
- 239000004383 Steviol glycoside Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 235000019411 steviol glycoside Nutrition 0.000 description 5
- 229930182488 steviol glycoside Natural products 0.000 description 5
- 150000008144 steviol glycosides Chemical class 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 229920002558 Curdlan Polymers 0.000 description 3
- 239000001879 Curdlan Substances 0.000 description 3
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940078035 curdlan Drugs 0.000 description 3
- 235000019316 curdlan Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002213 purine nucleotide Substances 0.000 description 2
- 125000000561 purinyl group Chemical class N1=C(N=C2N=CNC2=C1)* 0.000 description 2
- 239000002719 pyrimidine nucleotide Substances 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 2
- 229940045145 uridine Drugs 0.000 description 2
- RGOFBPTWSOFRNJ-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphono dihydrogen phosphate Chemical compound OP(O)(=O)OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O RGOFBPTWSOFRNJ-BTVCFUMJSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 244000289276 Bambusa oldhamii Species 0.000 description 1
- 235000004270 Bambusa oldhamii Nutrition 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 241000743774 Brachypodium Species 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 101000763602 Manilkara zapota Thaumatin-like protein 1 Proteins 0.000 description 1
- 101000763586 Manilkara zapota Thaumatin-like protein 1a Proteins 0.000 description 1
- 101000966653 Musa acuminata Glucan endo-1,3-beta-glucosidase Proteins 0.000 description 1
- 101710204244 Processive diacylglycerol beta-glucosyltransferase Proteins 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- -1 and among these Chemical compound 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-L diphosphate(2-) Chemical compound OP([O-])(=O)OP(O)([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-L 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 229930188195 rebaudioside Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- DZLFLBLQUQXARW-UHFFFAOYSA-N tetrabutylammonium Chemical compound CCCC[N+](CCCC)(CCCC)CCCC DZLFLBLQUQXARW-UHFFFAOYSA-N 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- DRSKVOAJKLUMCL-MMUIXFKXSA-N u2n4xkx7hp Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(O)=O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O DRSKVOAJKLUMCL-MMUIXFKXSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- 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
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
-
- 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
-
- 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
- C12N9/10—Transferases (2.)
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
- C12N9/1062—Sucrose synthase (2.4.1.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01013—Sucrose synthase (2.4.1.13)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
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Abstract
Description
(1)スクロースとヌクレオチドジホスフェートとをスクロース合成酵素の存在下で反応させ、ブドウ糖が結合されたヌクレオチドジホスフェートを製造するステップ;
(2)前記ブドウ糖が結合されたヌクレオチドジホスフェートを糖転移酵素の存在下でステビオシドと反応させ、レバウジオシドAを製造するステップ;を含む、レバウジオシドAの製造方法が提供される。
(1)スクロースとヌクレオチドジホスフェートとをスクロース合成酵素の存在下で反応させ、ブドウ糖が結合されたヌクレオチドジホスフェートを製造するステップ;及び
(2)前記ブドウ糖が結合されたヌクレオチドジホスフェートを糖転移酵素の存在下でステビオシドと反応させ、レバウジオシドAを製造するステップ;を含む、レバウジオシドAの製造方法に関する。前記(1)ステップ及び前記(2)ステップは、順次行われてもよく、同一の反応系で連続的に行われてもよいが、同一の反応系で連続的に行われることが好ましい。
・
米(Oryza sativa)由来のスクロース合成酵素(sucrose synthase)によって生産されたブドウ糖が結合されたウリジンジホスフェート(Uridine diphosphate)は、糖転移酵素によってステビオシドと反応してレバウジオシドAに転換され、ウリジンジホスフェート(Uridine diphosphate)を解離することができる。前記2種の酵素が一つの反応器に存在し、レバウジオシドAが生産されるときの最適なpHを確認した。最適なpHを確認するためのHPLC分析条件は、次の通りである。
Claims (14)
- (1)スクロースとヌクレオチドジホスフェートとをスクロース合成酵素の存在下で反応させ、ブドウ糖が結合されたヌクレオチドジホスフェートを製造するステップ;
(2)前記ブドウ糖が結合されたヌクレオチドジホスフェートを糖転移酵素の存在下でステビオシドと反応させ、レバウジオシドAを製造するステップ;を含む、レバウジオシドAの製造方法。 - 前記スクロース合成酵素は、米、トウモロコシ、小麦、タケ、シロイヌナズナ、シバ、麦、キビまたはジャガイモに由来したスクロース合成酵素である、請求項1に記載の製造方法。
- 前記スクロース合成酵素は、スクロース合成酵素遺伝子を含有するベクターに形質転換された組換え大腸菌、バシラス、酵母、コリネバクテリウムまたはアグロバクテリウムから生産されたものである、請求項1または2に記載の製造方法。
- 前記スクロース合成酵素が配列番号3の配列を有する、請求項3に記載の製造方法。
- 前記糖転移酵素は、オリザ・サティバ(Oryza sativa)、ステビア・レバウディアナ・ベルトニー(Stevia rebaudiana Bertoni)、バンブサ・オルダミイ(Bambusa oldhamii)、ブラキポディウム(Brachypodium distachyon)、ホルデウム・ウルガレ(Hordeum vulgare)、ソルガム・バイカラー(Sorghum bicolor)、ジー・メイズ(Zea mays)、またはアラビドプシス・タリアナ(Arabidopsis thaliana) 由来の糖転移酵素である、請求項1から4のいずれか1項に記載の製造方法。
- 前記糖転移酵素は、糖転移酵素遺伝子を含有するベクターに形質転換された組換え大腸菌、バシラス、酵母、コリネバクテリウムまたはアグロバクテリウムから生産されたものである、請求項1から5のいずれか1項に記載の製造方法。
- 前記糖転移酵素が配列番号4の配列を有する、請求項1から6のいずれか1項に記載の製造方法。
- 前記(1)ステップ及び前記(2)ステップが同一の反応系で連続的に行われる、請求項1から7のいずれか1項に記載の製造方法。
- 前記同一の反応系では、反応温度が20℃〜60℃で、反応pHが5〜10の範囲である、請求項8に記載の製造方法。
- スクロース、ヌクレオチドジホスフェート、ステビオシド、スクロース合成酵素及び糖転移酵素を同一の反応系で反応させ、レバウジオシドAを製造するステップを含む、ステビオシドからレバウジオシドAを製造する方法。
- 前記反応温度が20℃〜60℃で、反応pHが5〜10の範囲である、請求項10に記載の方法。
- 前記スクロース合成酵素は、米、トウモロコシ、小麦、タケ、シロイヌナズナ、シバ、麦、キビまたはジャガイモに由来したスクロース合成酵素である、請求項10または11に記載の方法。
- 前記糖転移酵素は、オリザ・サティバ(Oryza sativa)、ステビア・レバウディアナ・ベルトニー(Stevia rebaudiana Bertoni)、バンブサ・オルダミイ(Bambusa oldhamii)、ブラキポディウム(Brachypodium distachyon)、ホルデウム・ウルガレ(Hordeum vulgare)、ソルガム・バイカラー(Sorghum bicolor)、ジー・メイズ(Zea mays)、またはアラビドプシス・タリアナ(Arabidopsis thaliana)由来の糖転移酵素である、請求項10から12のいずれか1項に記載の製造方法。
- 請求項1から13のいずれか1項による製造方法によって製造されたレバウジオシドA。
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