JPH03218400A - Sweetness-inducing substance - Google Patents
Sweetness-inducing substanceInfo
- Publication number
- JPH03218400A JPH03218400A JP63318437A JP31843788A JPH03218400A JP H03218400 A JPH03218400 A JP H03218400A JP 63318437 A JP63318437 A JP 63318437A JP 31843788 A JP31843788 A JP 31843788A JP H03218400 A JPH03218400 A JP H03218400A
- Authority
- JP
- Japan
- Prior art keywords
- added
- miraculin
- amino acid
- resultant
- sweetness
- 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
- 239000000126 substance Substances 0.000 title claims abstract description 10
- 230000001939 inductive effect Effects 0.000 title abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 101710084933 Miraculin Proteins 0.000 claims description 30
- 150000001413 amino acids Chemical class 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 20
- 239000000243 solution Substances 0.000 abstract description 17
- 235000011341 Sideroxylon dulcificum Nutrition 0.000 abstract description 13
- 240000007326 Thaumatococcus daniellii Species 0.000 abstract description 13
- 235000005266 Thaumatococcus daniellii Nutrition 0.000 abstract description 13
- 239000011780 sodium chloride Substances 0.000 abstract description 10
- 239000006228 supernatant Substances 0.000 abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 7
- 239000002244 precipitate Substances 0.000 abstract description 6
- 239000013543 active substance Substances 0.000 abstract description 5
- 235000003599 food sweetener Nutrition 0.000 abstract description 4
- 238000004255 ion exchange chromatography Methods 0.000 abstract description 4
- 239000003765 sweetening agent Substances 0.000 abstract description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 abstract description 3
- 238000001042 affinity chromatography Methods 0.000 abstract description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 abstract description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 abstract description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 abstract description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 2
- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 abstract 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 abstract 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 abstract 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 abstract 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 abstract 1
- 229930182830 galactose Natural products 0.000 abstract 1
- 229960002442 glucosamine Drugs 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 239000011369 resultant mixture Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 230000029087 digestion Effects 0.000 description 12
- 238000010828 elution Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 229920002684 Sepharose Polymers 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 235000019605 sweet taste sensations Nutrition 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 108010053725 prolylvaline Proteins 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000005185 salting out Methods 0.000 description 4
- 108090000317 Chymotrypsin Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 229960002376 chymotrypsin Drugs 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- 108010059378 Endopeptidases Proteins 0.000 description 2
- 102000005593 Endopeptidases Human genes 0.000 description 2
- YLJHCWNDBKKOEB-IHRRRGAJSA-N Glu-Glu-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YLJHCWNDBKKOEB-IHRRRGAJSA-N 0.000 description 2
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- NBWATNYAUVSAEQ-ZEILLAHLSA-N His-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O NBWATNYAUVSAEQ-ZEILLAHLSA-N 0.000 description 2
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 2
- AWJGUZSYVIVZGP-YUMQZZPRSA-N Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 AWJGUZSYVIVZGP-YUMQZZPRSA-N 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- SSJMZMUVNKEENT-IMJSIDKUSA-N Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CO SSJMZMUVNKEENT-IMJSIDKUSA-N 0.000 description 2
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 description 2
- YLXAMFZYJTZXFH-OLHMAJIHSA-N Thr-Asn-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O YLXAMFZYJTZXFH-OLHMAJIHSA-N 0.000 description 2
- MSIYNSBKKVMGFO-BHNWBGBOSA-N Thr-Gly-Pro Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N)O MSIYNSBKKVMGFO-BHNWBGBOSA-N 0.000 description 2
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 2
- FJBCEFPCVPHPPM-STECZYCISA-N Tyr-Ile-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O FJBCEFPCVPHPPM-STECZYCISA-N 0.000 description 2
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 108010041407 alanylaspartic acid Proteins 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 108010016616 cysteinylglycine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000019640 taste Nutrition 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- BRPMXFSTKXXNHF-IUCAKERBSA-N (2s)-1-[2-[[(2s)-pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H]1NCCC1 BRPMXFSTKXXNHF-IUCAKERBSA-N 0.000 description 1
- PQMRRAQXKWFYQN-UHFFFAOYSA-N 1-phenyl-2-sulfanylideneimidazolidin-4-one Chemical class S=C1NC(=O)CN1C1=CC=CC=C1 PQMRRAQXKWFYQN-UHFFFAOYSA-N 0.000 description 1
- QMOQBVOBWVNSNO-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(O)=O QMOQBVOBWVNSNO-UHFFFAOYSA-N 0.000 description 1
- HPGXYQZKIMWRAM-UHFFFAOYSA-N 4-ethylmorpholin-4-ium;acetate Chemical compound CC(O)=O.CCN1CCOCC1 HPGXYQZKIMWRAM-UHFFFAOYSA-N 0.000 description 1
- JAQNUEWEJWBVAY-WBAXXEDZSA-N Ala-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 JAQNUEWEJWBVAY-WBAXXEDZSA-N 0.000 description 1
- NGYHSXDNNOFHNE-AVGNSLFASA-N Arg-Pro-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O NGYHSXDNNOFHNE-AVGNSLFASA-N 0.000 description 1
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
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- PQHYZJPCYRDYNE-QWRGUYRKSA-N Cys-Gly-Phe Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PQHYZJPCYRDYNE-QWRGUYRKSA-N 0.000 description 1
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- VOUSELYGTNGEPB-NUMRIWBASA-N Gln-Thr-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O VOUSELYGTNGEPB-NUMRIWBASA-N 0.000 description 1
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- BDYBHQWMHYDRKJ-UNQGMJICSA-N Thr-Phe-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(=O)O)N)O BDYBHQWMHYDRKJ-UNQGMJICSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- UBTBGUDNDFZLGP-SRVKXCTJSA-N Val-Arg-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](C(C)C)C(=O)O)N UBTBGUDNDFZLGP-SRVKXCTJSA-N 0.000 description 1
- DIOSYUIWOQCXNR-ONGXEEELSA-N Val-Lys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O DIOSYUIWOQCXNR-ONGXEEELSA-N 0.000 description 1
- IOUPEELXVYPCPG-UHFFFAOYSA-N Valylglycine Chemical compound CC(C)C(N)C(=O)NCC(O)=O IOUPEELXVYPCPG-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 210000003323 beak Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- AWADHHRPTLLUKK-UHFFFAOYSA-N diazanium sulfuric acid sulfate Chemical compound [NH4+].[NH4+].OS(O)(=O)=O.[O-]S([O-])(=O)=O AWADHHRPTLLUKK-UHFFFAOYSA-N 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 1
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 108091005708 gustatory receptors Proteins 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 108010076756 leucyl-alanyl-phenylalanine Proteins 0.000 description 1
- 108010091798 leucylleucine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- HOVAGTYPODGVJG-ZFYZTMLRSA-N methyl alpha-D-glucopyranoside Chemical compound CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HOVAGTYPODGVJG-ZFYZTMLRSA-N 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- -1 phenylthiocarbamyl Chemical group 0.000 description 1
- 229940012982 picot Drugs 0.000 description 1
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019614 sour taste Nutrition 0.000 description 1
- 238000003325 tomography Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Jellies, Jams, And Syrups (AREA)
- Seasonings (AREA)
- Peptides Or Proteins (AREA)
- General Preparation And Processing Of Foods (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は天然のミラクルフルーツから分離した新規な甘
味誘導物質ミラクリンに関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to miraculin, a novel sweetness-inducing substance isolated from natural miracle fruit.
西アフリカ原産のミラクルフルーツ(Richarde
lla Dulcifica)には、これを口に含んで
から酸味のものを味わうと甘く感じるという性質がある
。Miracle fruit (Richard) native to West Africa
lla Dulcifica) has the property that if you put it in your mouth and then taste something sour, it will taste sweet.
ミラクルフルーツのこの味覚を変える活性成分(味覚変
性物質)は(分子量45,000前後の)糖タンパク質
であることがすでに知られている。また、この糖タンパ
ク質に含まれる糖の割合はおよそ10〜15%前後であ
り、糖の種類は、L−アラビノース、L−ラムノース、
D−キシロース、D−マンノース、D−ガラクトース、
D−グルコースであることが確認されている。It is already known that this taste-altering active ingredient (taste modifier) of the miracle fruit is a glycoprotein (with a molecular weight of around 45,000). In addition, the proportion of sugar contained in this glycoprotein is approximately 10 to 15%, and the types of sugar are L-arabinose, L-rhamnose,
D-xylose, D-mannose, D-galactose,
It has been confirmed that it is D-glucose.
このミラクルフルーツの甘味発現の機構はいまだ解明さ
れていないが、ミラクルフルーツの果肉をあらかじめ酸
味物質と混ぜて一定時間放置したのち口に含んでも甘味
を示さないことから、ミラクルフルーツの活性成分であ
る甘味誘導物質は、従来の甘味剤とは異なって、酸味そ
のものを甘味に変えるのではなく、活性成分が舌上皮の
甘味受容体と何らかの相互作用をすることにより甘味が
発現すると考えられている。The mechanism behind the sweetness of this miracle fruit has not yet been elucidated, but the pulp of the miracle fruit is mixed with sour substances in advance and does not exhibit any sweetness even if it is left in the mouth for a certain period of time, so it is believed to be the active ingredient in the miracle fruit. Unlike conventional sweeteners, sweet taste inducers are thought to produce sweet taste through some kind of interaction between their active ingredients and sweet taste receptors in the epithelium of the tongue, rather than by converting sour taste itself into sweet taste.
このようなことからミラクルフルーツの甘味誘導物質は
新しいタイプの甘味剤として、特に、糖分の摂取を制限
されている人にも有効な甘味剤として期待されているも
のである。For these reasons, miracle fruit sweetness inducers are expected to be a new type of sweetener, especially as a sweetener that is effective for people who have limited sugar intake.
本発明者から先に、ミラクルフルーツから甘味誘導物質
を活性を失うことなく高純度且つ高収率に製造する方法
を提供したが、本発明では、更にこの甘味誘導物質につ
いてその一次構造を解明した、甘味度の優れた新規な高
純度ミラクリンを提供することにある。The present inventors have previously provided a method for producing sweetness-inducing substances from miracle fruits with high purity and high yield without losing activity, but in the present invention, the primary structure of this sweetness-inducing substance has been further elucidated. The purpose of the present invention is to provide a novel high-purity miraculin with excellent sweetness.
本発明の新規な高純度ミラクリンは次の理化学的性質を
有するもの、
(1)アミノ酸組成は第1表に記載したとおりである。The novel high-purity miraculin of the present invention has the following physical and chemical properties: (1) The amino acid composition is as shown in Table 1.
(本頁以下余白)
第1表
アミノ酸組成
アミノ酸
%
*数値は全残基100としたときのアミノ酸残基の和対
数を示す。(Margins below this page) Table 1 Amino acid composition Amino acid % *Numbers indicate the sum of logarithms of amino acid residues when the total residues are 100.
(2) I! 組 成 :第2表
(3)分子量:約25,000
(4)等電点:約pH 9.0
第2表
ミラクリンの糖組成
八sp Ser Ala ProAsp Gly
Glu Lys
Tyr Tyr Ile Val
Gly Gly Gly Leu
八sn Pro Val LeuLeu Arg
Thr Gly
Pro Val Leu Arg
Thr Vat Ser AlaAsp He
Thr Asn
Asp His
Thr Thr
Val Vat Gln ThrAsp Arg
Pro Leu
Pro Lys Glu AspAsp Le
u Asn TieArg Lys Glu V
al
Ala Phe Phe ProVal Va
l Arg VatAsn Phe Ser
AlaAsp旧S
Glu Asn
Ser Thr
Phe Met
Arg Leu Asp Lys Tyr Asp
Glu Ser Thr GlyGly Asn
Pro Gly Pro Glu Thr11
e Ser Ser
Trp Phe Lys
Tie Glu Glu Phe CysGl.
y Ser
Val Cys Gly Ser Cys Lys
Val Lys Cys GlyPhe
本発明の新規な高純度ミラクリンはミラクルフルーツの
果肉を洗液が着色しなくなるまで水洗し、次いで塩化ナ
トリウム溶液で抽出し、次いでこの抽出物を精製するこ
とにより得られる。(2) I! Composition: Table 2 (3) Molecular weight: Approximately 25,000 (4) Isoelectric point: Approximately pH 9.0 Table 2 Sugar composition of Miraculin 8sp Ser Ala ProAsp Gly
Glu Lys Tyr Tyr Ile Val Gly Gly Gly Leu Eight sn Pro Val LeuLeu Arg
Thr Gly Pro Val Leu Arg Thr Vat Ser AlaAsp He Thr Asn Asp His Thr Thr Thr Val Vat Gln ThrAsp Arg
Pro Leu Pro Lys Glu AspAsp Le
u Asn TieArg Lys Glu V
al Ala Phe Phe ProVal Va
l Arg VatAsn Phe Ser
AlaAsp Old S Glu Asn Ser Thr Phe Met Arg Leu Asp Lys Tyr Asp
Glu Ser Thr GlyGly Asn
Pro Gly Pro Glu Thr11
e Ser Ser Trp Phe Lys Tie Glu Glu Phe CysGl.
y Ser Val Cys Gly Ser Cys Lys
Val Lys Cys GlyPhe The novel high-purity miraculin of the present invention can be obtained by washing the pulp of miracle fruit with water until the washings are no longer colored, then extracting with a sodium chloride solution, and then purifying this extract.
具体的には、ミラクルフルーツの凍結乾燥果肉に水を加
えてホモジナイズし、遠心分離する。この時上清は濃い
ピンク色を示す。この上清には酸味を甘味に変える活性
はない。このときの沈渣に当初と等量の水を加えてホモ
ジナイスし、遠心分離する。この操作を上清が無色にな
るまで繰り返す。無色となったときの上清にも活性はな
い。Specifically, water is added to the freeze-dried pulp of the miracle fruit, homogenized, and centrifuged. At this time, the supernatant shows a deep pink color. This supernatant has no activity to change sourness into sweetness. Add the same amount of water as the initial amount to the resulting sediment, homogenize it, and centrifuge. Repeat this operation until the supernatant becomes colorless. There is no activity in the supernatant when it becomes colorless.
次に塩化ナトリウム溶液を用いて抽出する。すなわち、
水洗操作でのあとの沈渣に塩化ナトリウム溶液を加えて
ホモジナイズし、遠心分離する。Then extract using sodium chloride solution. That is,
A sodium chloride solution is added to the precipitate after the water washing operation, homogenized, and centrifuged.
中性付近での抽出のための、得られる抽出液は活性を失
うことなく高活性を示す。 抽出液の精製は、抽出液を
硫酸アンモニウムで塩析したのち、通常のクロマトグラ
フ法によって行うことができる。たとえば、塩析で得ら
れた沈澱をCM−セファロースイオン交換クロマトグラ
フィーにかけ、さらにアフィニティーク口マトグラフィ
ーにより精製する。得られる高純度ミラクリンのアミノ
酸配列の決定はこの高純度ミラクリンを酵素(トリプシ
ン、キモトリプシン、リジルエンドベプチダーゼ)で加
水分解した後、ODS 120Tのカラムを用いHPL
Cで各ベプチドフラグメントを分画することにより行っ
た。For extraction near neutrality, the resulting extract exhibits high activity without loss of activity. The extract can be purified by salting out the extract with ammonium sulfate and then using a conventional chromatography method. For example, a precipitate obtained by salting out is subjected to CM-Sepharose ion exchange chromatography and further purified by affinity chromatography. The amino acid sequence of the obtained high-purity miraculin was determined by hydrolyzing the high-purity miraculin with enzymes (trypsin, chymotrypsin, lysyl endobeptidase), and then performing HPL using an ODS 120T column.
This was done by fractionating each peptide fragment with C.
ミラクリンは、全く新しいタイプの甘味物質として、食
品、医薬品など応用されるもので、この発明によって、
その実用化は一層加速される。Miraculin is a completely new type of sweet substance that can be applied to foods, medicines, etc. With this invention,
Its practical application will be further accelerated.
参考例
(1)水洗および塩化ナトリウム溶液による抽出ミラク
ルフルーツの凍結乾燥果肉10gをとり、40ccの水
を加えてホモジナイズし、遠心分離(12.50O r
pm 、20分間)した。このとき上清は濃いピンク色
を呈し、これには酸味を甘味に変える活性はなかった。Reference Example (1) Water washing and extraction with sodium chloride solution Take 10 g of freeze-dried pulp of miracle fruit, add 40 cc of water, homogenize, and centrifuge (12.50 O r
pm, for 20 minutes). At this time, the supernatant had a deep pink color and had no activity to change sourness into sweetness.
この沈渣に40ccの水を加えてホモジナイズし、遠心
分離(12.50O rpm、20分間)する。この上
清には味覚を変更する活性はなかった。Add 40 cc of water to this sediment, homogenize it, and centrifuge (12.50 rpm, 20 minutes). This supernatant had no taste-altering activity.
つぎに、0.5M塩化ナトリウム溶液を加えてホモジナ
イズし、遠心分離(12.50O rpm、20分間)
した。これによりえられた上滑は無色で、甘味を誘導す
る活性を示した。40ccの0.5M塩化ナトリウム溶
液による抽出操作を3回繰返し、3回分の上清を合わせ
た。Next, 0.5M sodium chloride solution was added, homogenized, and centrifuged (12.50 rpm, 20 minutes).
did. The resulting curd was colorless and exhibited sweetness-inducing activity. The extraction operation using 40 cc of 0.5M sodium chloride solution was repeated three times, and the supernatants from the three times were combined.
(2)硫酸アンモニウムによる塩析
上記の操作で得られた抽出液に40%飽和になるように
硫酸アンモニウムを加え、活性物質を折出させた。遠心
分離(13.00O rpm、20分間)して得た沈澱
を0.OIMリン酸緩衝液に溶かす。(2) Salting out with ammonium sulfate Ammonium sulfate was added to the extract obtained in the above procedure to achieve 40% saturation to precipitate the active substance. The precipitate obtained by centrifugation (13.00 rpm, 20 minutes) was Dissolve in OIM phosphate buffer.
(3)CM−セファロースイオン交換クロマトグラフイ
ー
(2)で調整した溶液をあらかじめ十分に0.01Mリ
ン酸緩衝液により平衡化したCM−セファロース力ラム
にのせた。次いで、最初にリン酸緩衝液(pH 6.8
)で十分溶出した後、塩化ナトリウム溶液O〜1.0M
の直線濃度勾配溶出法で溶出した(流速5cc/15m
in、1分画5cc、全溶出液量400cc)。蛋白質
は280nmの吸収によりモニターした。その結果を示
したものが第1図である。この第1図のピーク0が甘味
誘導活性物質である。(3) CM-Sepharose ion exchange chromatography The solution prepared in (2) was placed on a CM-Sepharose column that had been sufficiently equilibrated with 0.01M phosphate buffer in advance. Then, first phosphate buffer (pH 6.8
) After sufficient elution with sodium chloride solution O~1.0M
Elution was performed using a linear concentration gradient elution method (flow rate 5cc/15m).
in, 1 fraction 5 cc, total eluate volume 400 cc). Protein was monitored by absorption at 280 nm. Figure 1 shows the results. Peak 0 in FIG. 1 is the sweet taste-inducing active substance.
(4) アフィニティーク口マトグラフィー上記のピ
ーク0の部分を、限外濾過で10ccに濃縮し、さらに
溶媒を0.5M塩化ナトリウムを含む0.OIMリン酸
緩衝液(pH 6.8)に溶媒置換した。この濃縮液を
ConA−セファロース4Bカラムにより処理した。は
じめに0.5M塩化ナトリウムを含む0.OIMリン酸
緩衝液(pH6.8)で洗い、ついでα−メチルーD−
グルコシド溶液0〜0.15Mの直線濃度勾配溶出法で
溶出した(流速3 cc/14min、全溶出液量20
0 cc)。蛋白質は10
280nmの吸光度でモニターした。その結果を示した
ものが第2図である。ピークIが活性を示し、その収量
は35mgであった。(4) Affinity oral tomography The above peak 0 portion was concentrated to 10 cc by ultrafiltration, and the solvent was added to 0.0 cc containing 0.5 M sodium chloride. The solvent was replaced with OIM phosphate buffer (pH 6.8). This concentrate was processed through a ConA-Sepharose 4B column. First, 0.5M sodium chloride was added. Wash with OIM phosphate buffer (pH 6.8), then α-methyl-D-
Glucoside solution was eluted using a linear concentration gradient elution method of 0 to 0.15M (flow rate 3 cc/14 min, total eluate volume 20
0 cc). Protein was monitored by absorbance at 10280 nm. Figure 2 shows the results. Peak I showed activity and the yield was 35 mg.
(5)高速液体クロマトグラフイー
活性物質(第2図のピーク■)の純度を逆相カラムを用
いた高速液体クロマトグラフイー(H P L C )
により確認した。(5) High performance liquid chromatography The purity of the active substance (peak ■ in Figure 2) was determined by high performance liquid chromatography (HPLC) using a reversed phase column.
Confirmed by.
カラムはTSK gel TMS−250を用い、0.
05%トリフルオロ酢酸を含むアセトニトリル(20〜
70%)の直線濃度勾配溶出法で溶出し、210nmの
吸光度を測定することによりモニターした。The column used was TSK gel TMS-250.
Acetonitrile containing 0.5% trifluoroacetic acid (20~
70%) using a linear concentration gradient elution method and monitored by measuring absorbance at 210 nm.
その結果は、第3図に示すように、鋭い単一ピークを示
し、きわめて純度が高いことが確認できた。As shown in FIG. 3, the results showed a sharp single peak, and it was confirmed that the purity was extremely high.
この高純度ミラクリンの分子量は、SOS−PAGEに
より28,000を示した。一方、糖の含有率は14%
(フェノールー硫酸法とLowry法から計算した。)
であるから、(P2)のアミノ酸一次構造から計算する
と、約25,000をとなる。糖の組成は第2表に示し
た通りである。等電点11
pHは9.0前後である。アミノ酸組成は第1表の通り
であり、アミノ酸配列は前述の通りである。The molecular weight of this highly purified miraculin was 28,000 by SOS-PAGE. On the other hand, the sugar content is 14%
(Calculated from the phenol-sulfuric acid method and Lowry method.)
Therefore, when calculated from the amino acid primary structure of (P2), it is approximately 25,000. The composition of the sugar is shown in Table 2. Isoelectric point 11 pH is around 9.0. The amino acid composition is as shown in Table 1, and the amino acid sequence is as described above.
この高純度ミラクリンの活性テストの結果を第8図に示
す。この第8図からこのアミノ酸配列を確定した高純度
ミラクリンの活性は0.5M庶誠に相当し、一方従来の
ちは最高0.4M庶糖に相当することから本発明のミラ
クリンが従来のものに比して優れた活性を示すことが判
る。The results of the activity test of this highly purified miraculin are shown in FIG. From FIG. 8, the activity of high-purity miraculin whose amino acid sequence has been determined corresponds to 0.5M sucrose, while the conventional one corresponds to a maximum of 0.4M sucrose, so the miraculin of the present invention has a higher activity than the conventional one. It can be seen that it shows excellent activity.
この発明によりアミノ酸配列の決定した活性の高い高純
度ミラクリンを提供することができた。This invention has made it possible to provide highly active and highly purified miraculin whose amino acid sequence has been determined.
また、この高純度ミラクリンは遺伝子工学的手法により
大量にミラクリンを製造するときの構造遺伝子の供給源
として有用である。Furthermore, this highly purified miraculin is useful as a source of structural genes when miraculin is produced in large quantities by genetic engineering techniques.
実施例
(1)S一カルポキシアミドメチル化ミラクリンの調製
参考例により調製した高純度ミラクリン7mgを6Mグ
アニジン塩酸塩、2mM EDTA及び60mMジチオ
スレイトールを含む0.4M I−リス緩衝l2
液5 mflに溶解する。この溶液を窒素ガス中で37
゜C、24時間インキユベートした。この溶液にヨード
アセトアミド0.2gを加え、室温で10分間静置し、
次いで氷水浴中で60分間静置した。得られるS一カル
ボキシアミドメチル化ミラクリンをセファデックスG−
25を用い2M尿素及び2mM EDTAを含む50m
M重炭酸ナトリウム緩衝液(pH 8.0)を溶媒とし
て脱塩した。Example (1) Preparation of S-carpoxyamidomethylated miraculin 7 mg of high purity miraculin prepared according to Reference Example was added to 5 mfl of 0.4M I-Lys buffer 12 containing 6M guanidine hydrochloride, 2mM EDTA and 60mM dithiothreitol. dissolve in This solution was dissolved in nitrogen gas for 37
℃, incubated for 24 hours. Add 0.2 g of iodoacetamide to this solution and let stand at room temperature for 10 minutes.
Then, it was left standing in an ice water bath for 60 minutes. The resulting S-carboxyamidomethylated miraculin was treated with Sephadex G-
25 with 2M urea and 2mM EDTA.
Desalting was carried out using M sodium bicarbonate buffer (pH 8.0) as a solvent.
(2)S一カルボキシアミドメチル化ミラクリンの酵素
開裂
S一カルボキシアミドメチル化ミラクリンのりジルエン
ドペプチダーゼ消化を2M尿素及び2 mM EDTA
を含む50mM重炭酸アンモニウム緩衝液中で37゜C
120時間行った。(2) Enzymatic cleavage of S-carboxyamidomethylated miraculin Glycine endopeptidase digestion of S-carboxyamidomethylated miraculin with 2 M urea and 2 mM EDTA
at 37°C in 50mM ammonium bicarbonate buffer containing
I went for 120 hours.
蛋白質濃度はlIIlg/rdで酵素:基質比ば1:1
00 (w/w)である。消化反応はHCLを加えpt
+ 2.0とすることにより停止した。消化物には不溶
性物質は存在しなかった。また、S一カルポキシアミド
メチル化ミラクリンのキモトリプシン消化を上記消化と
同じ緩衝液、蛋白質濃度及び酵13
素:基質化の下、37゜C、90分間行った。消化反応
は上記と同じ方法で停止した。消化物には不溶性物質は
形成されなかった。The protein concentration is lIIlg/rd and the enzyme:substrate ratio is 1:1.
00 (w/w). For the digestion reaction, add HCL and pt
It was stopped by setting the value to +2.0. No insoluble material was present in the digestate. In addition, chymotrypsin digestion of S-carpoxyamidomethylated miraculin was carried out at 37°C for 90 minutes using the same buffer, protein concentration, and enzyme:substrate as in the above digestion. Digestion reactions were stopped in the same manner as above. No insoluble material was formed in the digestate.
S一カルポキシアミドメチル化ミラクリンを2M尿素及
び2mMEDT八を含む50mM重炭酸アンモニウム(
pH 7.8)中、スタフィ口コツカス・アウレウスの
V B (Staphylococcus aureu
s V8)プロテアーゼで37゜C、3時間消化した。S-carpoxyamidomethylated miraculin was added to 50mM ammonium bicarbonate (containing 2M urea and 2mM EDT8).
V B (Staphylococcus aureus) in pH 7.8).
s V8) Digested with protease at 37°C for 3 hours.
蛋白質濃度はl mg/mfl,酵素:基質比は1 :
30 w7wである。消化後沈澱が形成された。反応
混合物に溶液が透明になるまで固形の尿素を加えた。The protein concentration was 1 mg/mfl, and the enzyme:substrate ratio was 1:
30w7w. A precipitate formed after digestion. Solid urea was added to the reaction mixture until the solution became clear.
(3)ペプチドの分離
上記三種類の酵素により消化して得られる各ベプチドは
TSK−003−120T (東洋曹達■製)カラムを
用いHPLC (東洋曹達■PC 8000)により分
離した。各ベプチドは0.05%トリフルオロ酢酸を含
むアセトニトリルの直線濃度匂配溶出法で溶出した。ベ
プチドは210nmの吸収により検知され、各ピークが
集められた。(3) Separation of peptides Each peptide obtained by digestion with the three types of enzymes mentioned above was separated by HPLC (Toyo Soda PC 8000) using a TSK-003-120T (manufactured by Toyo Soda) column. Each peptide was eluted using a linear gradient elution method using acetonitrile containing 0.05% trifluoroacetic acid. Veptides were detected by absorption at 210 nm and each peak was collected.
(4)アミノ酸分析およびアミノ酸配列の決定14
各ベプチドのアミノ酸組成はWaters Picot
agsystemにより決定した。即ち、ペブチドはH
CL蒸気で110゜C、22時間加水分解した。得られ
るアミノ酸をフェニルチオカルバミル(PTC) 誘導
体とし、TSK − OI]S−80TMカラム0.4
6 X 15cm(東洋曹達■製)を用いたllPLf
l:により分析した。(4) Amino acid analysis and determination of amino acid sequence 14 The amino acid composition of each peptide is determined by Waters Picot.
Determined by agsystem. That is, the peptide is H
Hydrolysis was carried out using CL steam at 110°C for 22 hours. The obtained amino acid was used as a phenylthiocarbamyl (PTC) derivative, and the TSK-OI]S-80TM column 0.4
llPLf using 6 x 15cm (manufactured by Toyo Soda)
Analyzed by l:.
アミノ酸のPTC誘導体は50lIIMリン酸緩衝液(
p117.0)の3.0%アセトニトリルで20分間、
次いで40%アセト二トリルにより5分間それぞれ流速
1d/minでカラムから流出された。流出されたPT
C−アミノ酸は254nmの吸光度により検知した。ア
ミノ酸配列の決定は470A Applied Bio
sytem Protein Sequencerで行
われた。すなわちフェニルチオヒダントイン誘導体(P
TH−アミノ酸)としてTSK 一〇DS −120
Tカラム(東洋曹達)を用いた+1 P L Cにより
分析された。PTC derivatives of amino acids were prepared in 50 lIIM phosphate buffer (
p117.0) in 3.0% acetonitrile for 20 minutes;
The column was then flushed with 40% acetonitrile for 5 minutes each at a flow rate of 1 d/min. Leaked PT
C-amino acids were detected by absorbance at 254 nm. Amino acid sequence was determined using 470A Applied Bio
System Protein Sequencer was used. That is, phenylthiohydantoin derivative (P
TH-amino acid) as TSK 10DS-120
Analysis was performed by +1 PLC using a T column (Toyo Soda).
C一末端アミノ酸配列は八mblerにより記載されて
いるカルボキシペプチターゼを用いて決定された。ミラ
クリン200 tt gを0.1MN−エチルモルホリ
ン酢酸緩衝液(pH 8.0) 0.9 mlに溶15
解する。この溶液にカルボキシペプチダーゼA,1μg
を加え、反応混合物を室温でインキユベートする。反応
液の一部を、15, 30, 60, 120分毎に採
取する。これらの反応液にトリクロル酢酸を加え蛋白質
を沈澱させ、これを遠心分離により除き、上清にある遊
離したアミノ酸をHaters Picotag sy
stemにより、分析した。The C-terminal amino acid sequence was determined using carboxypeptidase as described by Yambler. Dissolve 200 tt g of miraculin in 0.9 ml of 0.1M N-ethylmorpholine acetate buffer (pH 8.0). Add 1 μg of carboxypeptidase A to this solution.
is added and the reaction mixture is incubated at room temperature. Aliquots of the reaction solution are collected every 15, 30, 60, and 120 minutes. Trichloroacetic acid was added to these reaction solutions to precipitate proteins, which were removed by centrifugation, and the free amino acids in the supernatant were purified using Haters Picotag Sy.
It was analyzed by stem.
以上の方法により決定されたアミノ酸配列は次の通りで
ある。The amino acid sequence determined by the above method is as follows.
八sp Ser Ala ProAsp Gly
Glu Lys
Tyr Tyr Ile Val
Gly Gly Gly Leu
^sn Pro Val LeuLeu Ar
g Thr Gly
Pro Val Leu Arg
Thr Val Ser AlaAsp Il
e
Thr Asn
Asp His
Thr Thr
Asp Arg Pro Leu Ala Phe
Phe Pro Glu AsnPro Lys
Glu Asp Val Val Arg Val
Ser ThrArg Leu Asp Lys
Tyr Asp Glu Ser Thr Gly16
Gln Tyr Phe Val Thr I
le Gly GIy Val LysGly A
sn Pro Gly Pro Glu Thr
Ile Ser SerTrp Phe Lys
Ile Glu Glu Phe Cys Gly
SerAsp Val
Gly
11e Tyr
11e Asp Gin Lys Gly8sp Ser Ala ProAsp Gly
Glu Lys Tyr Tyr Ile Val Gly Gly Gly Gly Leu ^sn Pro Val LeuLeu Ar
g Thr Gly Pro Val Leu Arg Thr Val Ser AlaAsp Il
e Thr Asn Asp His Thr Thr Asp Arg Pro Leu Ala Phe
Phe Pro Glu AsnPro Lys
Glu Asp Val Val Arg Val
Ser ThrArg Leu Asp Lys
Tyr Asp Glu Ser Thr Gly16 Gln Tyr Phe Val Thr I
le Gly GIy Val LysGly A
sn Pro Gly Pro Glu Thr
Ile Ser SerTrp Phe Lys
Ile Glu Glu Phe Cys Gly
SerAsp Val Gly 11e Tyr 11e Asp Gin Lys Gly
第1図は、ミラクルフルーツから水洗、抽出、塩析操作
によって得た甘味誘導物質のCM−セファロースイオン
交換クロマトグラフィーの溶出パターンである。
第2図は、第1図のビーク■部分をConA−セファロ
ース4Bカラムにより処理したアフィニティーク口マト
グラフィーの溶出パターンである。
第3図は、第2図のビークIの高速液体クロマ17
トグラフフィーの溶出パターンである。
第4図はS一カルボキシアミドメチル化ミラクリンのり
ジルエンドペプチターゼ消化により得られるペプチドの
}IPLc分離を示す。第5図はS−カルボキシアミド
メチル化ミラクリンのキモトリプシン消化により得られ
るペプチドのHPLCを示す。
第6図はS一カルボキシアミドメチル化ミラクリンのス
タフィロコッカスアウレスV B (Staphy1o
coccus aureus V8)プロテアーゼ消化
により得られるHPLC分離を示す。第7図はミラクリ
ンのアミノ酸配列を示す。第7図において、LEP及び
chはリジルエンドペブチターゼ及びキモトリプシン消
化からのベプチド、■は■8−プロテアーゼ消化からの
べブチドを、NはN末端から決定したアミノ酸配列をそ
れぞれ示す。←←はC一末端からの配列を示す。
また、アルファベットとアミノ酸との関係は、次の通り
である。
G :G1y,A :A1a,S :Ser,T :T
hr,C :Cys,N :Asn,Q :GIn,L
:Leu, I : Ile,V :Val18
M :Met,F :Phe.Y :Tyr,W :T
rp,P :ProD :Asp.E :G1u,
H :His,K :Lys,R : 八rg
,第8図は本発明の高純度ミラクリンの活性を示す。FIG. 1 shows the elution pattern of a sweet taste-inducing substance obtained from miracle fruit by washing with water, extraction, and salting out by CM-Sepharose ion exchange chromatography. FIG. 2 is an elution pattern of affinity chromatography in which the beak (■) portion of FIG. 1 was treated with a ConA-Sepharose 4B column. FIG. 3 is an elution pattern of high performance liquid chromatography of peak I in FIG. 2. FIG. 4 shows the IPLc separation of peptides obtained by S-carboxyamidomethylated miraculin and dylyl endopeptidase digestion. FIG. 5 shows HPLC of peptides obtained by chymotryptic digestion of S-carboxyamidomethylated miraculin. Figure 6 shows S-carboxyamidomethylated miraculin of Staphylococcus aureus V B (Staphy1o
Figure 3 shows HPLC separation obtained by protease digestion of C. coccus aureus V8). Figure 7 shows the amino acid sequence of miraculin. In FIG. 7, LEP and ch indicate the peptides from lysyl endopeptidase and chymotrypsin digestion, ■ indicates the peptides from ■8-protease digestion, and N indicates the amino acid sequence determined from the N-terminus, respectively. ←← indicates the sequence from the C-terminus. Furthermore, the relationship between alphabets and amino acids is as follows. G: G1y, A: A1a, S: Ser, T: T
hr, C: Cys, N: Asn, Q: GIn, L
:Leu, I:Ile, V:Val18 M:Met, F:Phe. Y: Tyr, W: T
rp,P:ProD:Asp. E:G1u,
H: His, K: Lys, R: 8rg
, FIG. 8 shows the activity of highly purified miraculin of the present invention.
Claims (1)
リン 【遺伝子配列があります】 【遺伝子配列があります】[Claims] 1. High purity miraculin having the following physical and chemical properties (1) Amino acid composition as shown in Table 1 (2) Sugar composition as shown in Table 2 (3) Molecular weight: approximately 25,000 (4) ) Isoelectric point: approximately pH 9.0 2. High purity miraculin of claim 1 having the following amino acid sequence [There is a gene sequence] [There is a gene sequence]
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63318437A JP2620615B2 (en) | 1988-03-12 | 1988-12-19 | Sweetness inducer |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63-57373 | 1988-03-12 | ||
JP5737388 | 1988-03-12 | ||
JP63318437A JP2620615B2 (en) | 1988-03-12 | 1988-12-19 | Sweetness inducer |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH03218400A true JPH03218400A (en) | 1991-09-25 |
JP2620615B2 JP2620615B2 (en) | 1997-06-18 |
Family
ID=26398412
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP63318437A Expired - Lifetime JP2620615B2 (en) | 1988-03-12 | 1988-12-19 | Sweetness inducer |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP2620615B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011072291A1 (en) * | 2009-12-11 | 2011-06-16 | David Posner | Taste-modified consumable products and methods for preparation |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103082260A (en) * | 2013-02-21 | 2013-05-08 | 万福群 | Method for extracting miraculin from miracle fruit |
-
1988
- 1988-12-19 JP JP63318437A patent/JP2620615B2/en not_active Expired - Lifetime
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011072291A1 (en) * | 2009-12-11 | 2011-06-16 | David Posner | Taste-modified consumable products and methods for preparation |
Also Published As
Publication number | Publication date |
---|---|
JP2620615B2 (en) | 1997-06-18 |
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