WO2015021690A1 - 一种酶法制备瑞鲍迪甙m的方法 - Google Patents
一种酶法制备瑞鲍迪甙m的方法 Download PDFInfo
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- WO2015021690A1 WO2015021690A1 PCT/CN2013/084644 CN2013084644W WO2015021690A1 WO 2015021690 A1 WO2015021690 A1 WO 2015021690A1 CN 2013084644 W CN2013084644 W CN 2013084644W WO 2015021690 A1 WO2015021690 A1 WO 2015021690A1
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- Prior art keywords
- udp
- sequence
- ugt
- rebaudioside
- reaction
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- 238000000034 method Methods 0.000 title claims abstract description 37
- GSGVXNMGMKBGQU-PHESRWQRSA-N rebaudioside M Chemical compound C[C@@]12CCC[C@](C)([C@H]1CC[C@@]13CC(=C)[C@@](C1)(CC[C@@H]23)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O)C(=O)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GSGVXNMGMKBGQU-PHESRWQRSA-N 0.000 title claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 title abstract description 3
- 102000004190 Enzymes Human genes 0.000 title abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 45
- 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 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 101710204244 Processive diacylglycerol beta-glucosyltransferase Proteins 0.000 claims abstract description 12
- RPYRMTHVSUWHSV-CUZJHZIBSA-N rebaudioside D 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]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 RPYRMTHVSUWHSV-CUZJHZIBSA-N 0.000 claims abstract description 12
- 239000001512 FEMA 4601 Substances 0.000 claims abstract description 6
- 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 claims abstract description 6
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 6
- 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 claims abstract description 6
- 235000019203 rebaudioside A Nutrition 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 20
- 241000544066 Stevia Species 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 14
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 11
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical group 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 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 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 description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 235000007164 Oryza sativa Nutrition 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 241000209094 Oryza Species 0.000 claims description 8
- 235000009566 rice Nutrition 0.000 claims description 8
- 238000006911 enzymatic reaction Methods 0.000 claims description 6
- 230000008929 regeneration Effects 0.000 claims description 6
- 238000011069 regeneration method Methods 0.000 claims description 6
- 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 5
- 239000008103 glucose Substances 0.000 claims description 5
- 108010043934 Sucrose synthase Proteins 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 241000235058 Komagataella pastoris Species 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 239000012634 fragment Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000008176 lyophilized powder Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 238000002425 crystallisation Methods 0.000 description 6
- 230000008025 crystallization Effects 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000000741 silica gel Substances 0.000 description 6
- 229910002027 silica gel Inorganic materials 0.000 description 6
- 235000019202 steviosides Nutrition 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 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 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 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 description 4
- 238000007036 catalytic synthesis reaction Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 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 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 229940013618 stevioside Drugs 0.000 description 4
- 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 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 239000000386 donor Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 229930188195 rebaudioside Natural products 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 235000021096 natural sweeteners Nutrition 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- QSRAJVGDWKFOGU-WBXIDTKBSA-N rebaudioside c Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]1(CC[C@H]2[C@@]3(C)[C@@H]([C@](CCC3)(C)C(=O)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)CC3)C(=C)C[C@]23C1 QSRAJVGDWKFOGU-WBXIDTKBSA-N 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- -1 0.145 g Reb A Chemical compound 0.000 description 1
- 101000630755 Arabidopsis thaliana Sucrose synthase 1 Proteins 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 239000001776 FEMA 4720 Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 239000008122 artificial sweetener Substances 0.000 description 1
- 235000021311 artificial sweeteners Nutrition 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000000348 glycosyl donor Substances 0.000 description 1
- 235000019534 high fructose corn syrup Nutrition 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- QRGRAFPOLJOGRV-UHFFFAOYSA-N rebaudioside F Natural products CC12CCCC(C)(C1CCC34CC(=C)C(CCC23)(C4)OC5OC(CO)C(O)C(OC6OCC(O)C(O)C6O)C5OC7OC(CO)C(O)C(O)C7O)C(=O)OC8OC(CO)C(O)C(O)C8O QRGRAFPOLJOGRV-UHFFFAOYSA-N 0.000 description 1
- HYLAUKAHEAUVFE-AVBZULRRSA-N rebaudioside f 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)CO1)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 HYLAUKAHEAUVFE-AVBZULRRSA-N 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000003944 tolyl group Chemical group 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
- A23L27/36—Terpene glycosides
-
- 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
- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- 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)
- C12Y204/01017—Glucuronosyltransferase (2.4.1.17)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the invention relates to a preparation method of rebaudioside M, in particular to a biological preparation method of rebaudioside M.
- Sweeteners are a class of food additives widely used in the production of food, beverages and confectionery. It can be added either during the production of food or as a substitute for sucrose by proper dilution during home baking.
- Sweeteners include natural sweeteners and artificial sweeteners, such as sucrose, high fructose corn syrup, honey, and the like, such as aspartame, saccharin, and the like.
- Stevioside is a kind of natural sweetener extracted from plant stevia and has been widely used in foods and beverages.
- the extract of Stevia contains a variety of steviosides including rebaudioside, and the naturally-derived stevioside has a large difference in batch composition and requires subsequent purification.
- Rebaudio A contains some other steviosides such as Rebaudioside C, D and F.
- the stevioside prepared by the extraction method is usually mixed with some impurities, which may have a certain influence on its use.
- Rebaudioside M has an advantage over Rebaudioside A, but its content in stevia leaves is extremely low and is only detected in the stevia Morita plant brown (20) 0, J, Appl. Glycosci., 57, 199-209). There is currently no commercial production of Rebaudio M.
- the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for preparing rebaudioside M by enzymatic method, which can produce high purity rebaudioside at a low cost and in a short cycle. M product.
- the present invention adopts the following technical scheme: A method for preparing rebaudioside M by enzymatic method, which uses Rebaudioside A or Rebaudioside D as a substrate to make a substrate in glucose In the presence of a basal donor, it is catalyzed by UDP-glucanosyl alcohol and/or recombinant cells containing UDP-glucosyltransferase to produce rebaudioside M.
- the glucose-based donor may be UDP-glycoside or a UDP-glucose regeneration system consisting of sucrose, sucrose synthase and UDP (2007, FEBS Letters' 581, 2562-2566), and preferably by sucrose, sucrose
- the UDP-glucose regeneration system consisting of synthetase and UDP has a higher UDP glucose price, and the UDP-glycan regeneration system can greatly reduce the cost.
- UDP-glucosyltransferase i.e., ubiquitinuclease-transferase, abbreviated as UGT
- UGT ubiquitinuclease-transferase
- the UDP-glucosyltransferase used in the present invention is UGTA and Z from Stevia rebaudkma or UG B from rice (Oryza sativa).
- the amino acid sequence of UG A may be at least 60% identical to Sequence 2.
- the amino acid sequence of UGT-A has at least 70% identity to sequence 2.
- the amino acid sequence of UG ⁇ C-A has at least 80% identity with Sequence 2.
- the amino acid sequence of UG A has at least 90% identity to sequence 2.
- the amino acid sequence of UG A is identical to that of sequence 2.
- the amino acid sequence of UGT-B can be at least 60% identical to sequence 4. More preferably, the amino acid sequence of UGT-B has at least 70% identity to sequence 4. Further preferably, the amino acid sequence of UGI-B is at least 80% identical to SEQ ID NO:4. Most preferably, the amino acid sequence of UGT-B has at least 90% of sequence 4. Consistency. According to a specific aspect, the amino acid sequence of UGX-B is identical to that of sequence 4.
- the reaction can be carried out in an aqueous phase system having a temperature of from 4 ° C to 50 ° C and a pH of 5.0 9.0.
- the reaction is carried out in an aqueous phase system having a temperature of 25 Torr to 35 Torr and a pH of 6.5 to 7.5.
- reaction is allowed to proceed at a temperature of 30 TTF.
- reaction is carried out at a pH of 7,0.
- the reaction is carried out in a phosphate buffer of pH 7.0.
- the reaction when catalysis is carried out using a recombinant cell containing UDP-glucosyltransferase, the reaction can be carried out in the presence of a cell penetrating agent.
- the cell penetrating agent is toluene, and the volume ratio of the whole reaction system may be from 3% to 3%. More preferably, the volume ratio of toluene is 2%.
- the recombinant cell may preferably be a microbial cell, wherein the microorganism may and preferably is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.
- the preparation method is carried out as follows; all the raw materials used in the reaction are added to the reaction vessel, mixed uniformly, and placed at a set temperature, and the reaction is stirred. After the reaction is completed, the Rebaudio ⁇ product that meets the requirements for use can be obtained by purification treatment.
- a specific purification method is post-treatment including resin separation, according to which a rebaudioside product having a purity of up to 95% can be obtained.
- the substrate is rebaudioside A
- the UDP-glucosyltransferase is a mixture of UGT ⁇ A from Stevia and UGB from rice, and the amino acid sequence and sequence of UGT ⁇ A from Stevia 2 has at least 80% identity
- the amino acid sequence of UGT-B from rice has at least 80% identity to sequence 4.
- the weight ratio of UG' A from stevia to UG' B from rice is 1: 0.8 ⁇ 1, 2, for example, the weight ratio of the two may be i:i.
- the substrate is Rebaudioside D
- the UDP-glucosyltransferase is UG' A from Stevia
- the amino acid sequence of UG' A from Stevia has at least 80% consistency.
- the present invention has the following advantages compared with the prior art:
- the method for preparing rebaudioside M by enzymatic method provided by the invention has important application value. Since the growth rate of microorganisms is much faster than that of plants, the preparation method according to the present invention can greatly reduce the production cost, shorten the production cycle, and greatly improve the competitiveness of the product. In addition, the stevia content in plants is low, and there are many different structures of stevioside, it is difficult to extract a relatively pure product, and the enzymatic synthesis method of the present invention can provide a product with higher purity, which will be further expanded. Application Fan I. Compared with the technology that has extracted Rebaudio M from Stevia, this product has higher productivity, lower cost, and can provide higher purity products.
- Rebaudi ⁇ A, ⁇ L ⁇ M are referred to as Reb A, Reb D il il leb M: respectively, the structural formula of the person is referred to the formula ⁇ , ⁇ and ffl.
- the invention mainly provides four routes for synthesizing Reb M:
- the UGT-A or UG B of ffi may be present in the form of an enzyme jelly or present in recombinant cells.
- the method of obtaining UGT-A or ⁇ is as follows:
- Recombinant Escherichia coli (or other microbial) expression strains of UG A or UGTH3 were obtained by molecular cloning technology and genetic engineering technology, and then recombinant E. coli was fermented to prepare recombinant cells containing UG' A or UG B, or UG was prepared. A or UGT ⁇ B lyophilized powder.
- the recombinant plasmid was transformed into E. coli BL21 (DE3), and the expression of the target protein was induced by IPTG to obtain recombinant E. coli expression UG A or UGTHB.
- sequence 1 and sequence 2 the gene was synthesized into a gene fragment, and Ndelhe il BamHl restriction sites were added to both ends, and ligated into pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.).
- the UGT gene fragment was digested with restriction endo alcohol I and fe HI, and the purified fragment was recovered.
- T4 ligase was added to join the pET30a corresponding restriction site, which was difficult to transform into BL21 (DE3).
- UGT strains were inoculated into 4 ml liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 °C, and the overnight culture was transferred to 50 mi liquid LB medium at a 1% inoculum, 37 ' ⁇ Shake culture (200 rpm) to an OD 6iW value of 0.6-0.8 , and incubate at a final concentration of 0.4 mM IPTG at 20 ° C overnight.
- the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended with 5 mi of 2 mmol/L of phosphate buffer (pH 7.0 7.0) to obtain recombinant cells containing UG' A for catalysis.
- the recombinant cells of UG'I ⁇ -A prepared in Example i were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, lOmin), and the supernatant was collected for 24 h to obtain UGT ⁇ A. Frozen powder.
- the UGT ⁇ B gene fragment was synthesized, and Ndel and ⁇ HI restriction sites were added to both ends, and ligated into pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.).
- the UGT gene fragment was digested with restriction endonucleases Nde and BcrmHl, and the purified fragment was recovered.
- the fragment was ligated into the corresponding cleavage site of P ET30a by T4 ligase, and transformed into BL21 (DE3) strain.
- UGT 3 ⁇ 4 species were inoculated into 4 ml liquid LB medium at a ratio of 1%, 37 (shake culture (200 rpm) overnight, and the overnight culture was transferred to 50 mi liquid LB medium at 1% inoculum, 37 shaking culture ( 200 rpm) to 0D 6 ( 3 ⁇ 4 ) value reached 0.64X8, added to a final concentration of 0.4 mM IPTG and shaken overnight at 2 CTC. After centrifugation, centrifuge the cells (8,000 rpm, 10 niin), H] 5 ml 2 mmol/L phosphate buffer The cells (H value 7.0) were resuspended and recombinant cells containing UGT ⁇ B were obtained for catalysis.
- the recombinant cells of UG ⁇ B prepared in Example 3 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, lOmin), and the supernatant was collected and lyophilized for 24 hours to obtain a frozen powder of UGT B.
- the UGT ⁇ A lyophilized powder prepared according to the method of Example 2 was used to catalyze the synthesis of Reb M.
- 150 mL of 0.05 mol/L phosphate buffer (pH 7.0), 0.255 g UDP glucose, 0.17 g Reb D, and UG A frozen powder 1.5 g were sequentially added, and the mixture was uniformly placed in a 30 ⁇ water bath. ⁇ 60ipm stirred reaction for 2 h. After the reaction, 500 ⁇ of the reaction solution was added to an equal volume of anhydrous methanol, and the mixture was centrifuged at 8,000 rpm for 10 min.
- the supernatant was filtered and detected by high performance liquid chromatography (chromatographic conditions; column; Agilent eclipse sb-C18 4.6X250mm ; detection wavelength: 210 nm; mobile phase: ⁇ 1 ⁇ 2 formic acid aqueous solution: methanol ::: 20%: 80%; flow rate: 1.0m: L / min; column temperature: 25 ': C).
- the conversion rate of Reb D is 40% or more. After separation by silica gel resin, crystallization, etc. After purification by treatment, it was obtained: R_eb M: 0,054 g, purity greater than 95%.
- the lyophilized powder prepared according to the method of Example 2 and the UG' B lyophilized powder prepared according to the method of Example 4 were used for catalytic synthesis of Reb M.
- reaction system 150 niL 0,05 mol/L phosphate buffer (pH 7 ⁇ 0), 0,51 g UDP glucose, 0.145 g Reb A, UGT-A and UGT ⁇ B lyophilized powder were added in sequence. 5 g, mix and mix, place in a 30 C water bath, stir the reaction at 160 rpm for 2 h. After the reaction is completed, take 500 ⁇ ! The reaction solution is mixed with an equal volume of anhydrous methanol, centrifuge at 8,000 rpm for 1O min to remove the supernatant filtration membrane.
- Example 8 Reb M is synthesized by Reb A as a substrate enzymatic method (Route 4)
- sucrose a sucrose synthase from Arabidopsis thaliana (hereinafter referred to as AtSUS i ), and a UDP-glucose regeneration system composed of UDP were used.
- the supernatant was filtered and analyzed by high performance liquid chromatography (chromatographic conditions: column: Agilent eclipse sb-C18 4.6 ⁇ 250 ⁇ ; detecting Jun length: 210 nm; mobile phase: 1% aqueous formic acid: methanol - 20%: 80%; flow rate: 1.0 mL / min: Column temperature: 25 V), the conversion rate 80'1 ⁇ 2 Reb a above. After purification by silica gel resin, crystallization and the like, it was purified to obtain Reb M 0.108 g, and the purity was more than 95%.
- Example 9 Whole cell catalytic synthesis of Reb M using Reb D as substrate
- the UGT ⁇ A-containing recombinant cells prepared according to the method of Example i are used for catalytic synthesis of Reb. M.
- the conversion rate of Reb D is 40% or more.
- Example 0 Whole cell catalytic synthesis of Reb A with Reb A as substrate
- reaction system 150 ml, 0,05 moi/L phosphate buffer (pH 7,0), 0,51 g UDP glucose, 3 mL guanidine, 0.145 g Reb A, and UGH and UG' B were added in sequence.
- the whole cells were 10 g each, mixed uniformly, placed in a 30 ⁇ water bath, and stirred at 160 rpm for 2 h.
- 500 ⁇ l of the reaction solution was added to an equal volume of anhydrous methanol, and the mixture was centrifuged at 8,000 rpm for 10 mill.
- the conversion rate of Reb A is 40% or more.
- the supernatant was purified by silica gel resin separation, crystallization, etc., and then purified to obtain Reb M 0,05 g, and the purity was more than 95%.
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MX2016001986A MX2016001986A (es) | 2013-08-14 | 2013-09-29 | Metodo para preparar rebaudiosido m mediante el uso de un metodo con enzima. |
RU2016108660A RU2658436C2 (ru) | 2013-08-14 | 2013-09-29 | Способ получения ребаудиозида m с использованием ферментативного способа |
JP2016533776A JP6272485B2 (ja) | 2013-08-14 | 2013-09-29 | 酵素法によるレバウジオシドmの製造方法 |
EP13891561.6A EP3034614A4 (en) | 2013-08-14 | 2013-09-29 | Method for preparing rebaudioside m by using enzyme method |
US14/911,876 US10301662B2 (en) | 2013-08-14 | 2013-09-29 | Enzymatic method for preparing rebaudioside M |
CA2921247A CA2921247C (en) | 2013-08-14 | 2013-09-29 | Enzymatic method for preparing rebaudioside m |
AU2013398146A AU2013398146B2 (en) | 2013-08-14 | 2013-09-29 | Method for preparing rebaudioside M by using enzyme method |
BR112016003035-4A BR112016003035A2 (pt) | 2013-08-14 | 2013-09-29 | método para preparação de rebaudiosídeo m através do uso de método enzimático |
US16/380,678 US10428364B2 (en) | 2013-08-14 | 2019-04-10 | Enzymatic method for preparing rebaudioside M |
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US10428364B2 (en) | 2013-08-14 | 2019-10-01 | Pepsico, Inc. | Enzymatic method for preparing rebaudioside M |
US11312985B2 (en) | 2016-10-21 | 2022-04-26 | Pepsico, Inc. | Enzymatic method for preparing Rebaudioside C |
US11352653B2 (en) | 2016-10-21 | 2022-06-07 | Pepsico, Inc. | Enzymatic method for preparing rebaudioside N |
US11359222B2 (en) | 2016-10-21 | 2022-06-14 | Pepsico, Inc. | Enzymatic method for preparing Rebaudioside j |
US11952604B2 (en) | 2016-10-21 | 2024-04-09 | Pepsico, Inc. | Enzymatic method for preparing Rebaudioside J |
US11976312B2 (en) | 2016-10-21 | 2024-05-07 | Pepsico, Inc. | Enzymatic method for preparing Rebaudioside C |
US11976313B2 (en) | 2016-10-21 | 2024-05-07 | Pepsico, Inc. | Enzymatic method for preparing rebaudioside N |
CN110730614A (zh) * | 2017-03-08 | 2020-01-24 | 谱赛科美国股份有限公司 | 高莱鲍迪苷m甜菊植物栽培品种及其产生方法 |
Also Published As
Publication number | Publication date |
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AU2013398146B2 (en) | 2017-07-13 |
CA2921247A1 (en) | 2015-02-19 |
RU2016108660A (ru) | 2017-09-18 |
US20190233866A1 (en) | 2019-08-01 |
US10428364B2 (en) | 2019-10-01 |
AU2013398146A1 (en) | 2016-04-07 |
CN103397064A (zh) | 2013-11-20 |
US10301662B2 (en) | 2019-05-28 |
JP6272485B2 (ja) | 2018-01-31 |
BR112016003035A2 (pt) | 2018-02-06 |
CA2921247C (en) | 2021-04-06 |
RU2658436C2 (ru) | 2018-06-21 |
EP3034614A1 (en) | 2016-06-22 |
CN103397064B (zh) | 2015-04-15 |
US20160298159A1 (en) | 2016-10-13 |
JP2016527892A (ja) | 2016-09-15 |
EP3034614A4 (en) | 2017-04-12 |
HK1226103A1 (zh) | 2017-09-22 |
MX2016001986A (es) | 2016-10-26 |
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