CN104328092B - 一种谷胱甘肽合成酶突变体、编码基因及应用 - Google Patents
一种谷胱甘肽合成酶突变体、编码基因及应用 Download PDFInfo
- Publication number
- CN104328092B CN104328092B CN201410506648.6A CN201410506648A CN104328092B CN 104328092 B CN104328092 B CN 104328092B CN 201410506648 A CN201410506648 A CN 201410506648A CN 104328092 B CN104328092 B CN 104328092B
- Authority
- CN
- China
- Prior art keywords
- leu
- glu
- ile
- lys
- ser
- 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.)
- Active
Links
- 108010036164 Glutathione synthase Proteins 0.000 title abstract description 79
- 102100034294 Glutathione synthetase Human genes 0.000 title abstract description 79
- 108090000623 proteins and genes Proteins 0.000 title abstract description 24
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 abstract description 99
- 229960003180 glutathione Drugs 0.000 abstract description 47
- 108010024636 Glutathione Proteins 0.000 abstract description 36
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 abstract description 28
- 230000035772 mutation Effects 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 17
- 239000000758 substrate Substances 0.000 abstract description 17
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 abstract description 16
- 239000004471 Glycine Substances 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 13
- 238000006555 catalytic reaction Methods 0.000 abstract description 11
- 108090000364 Ligases Proteins 0.000 abstract description 9
- 239000002126 C01EB10 - Adenosine Substances 0.000 abstract description 8
- 229960005305 adenosine Drugs 0.000 abstract description 8
- YVBGRQLITPHVOP-UHFFFAOYSA-L disodium;[hydroxy-[hydroxy(oxido)phosphoryl]oxyphosphoryl] hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])(=O)OP(O)(=O)OP(O)([O-])=O YVBGRQLITPHVOP-UHFFFAOYSA-L 0.000 abstract description 8
- 230000003197 catalytic effect Effects 0.000 abstract description 7
- 235000018417 cysteine Nutrition 0.000 abstract description 3
- 150000001945 cysteines Chemical class 0.000 abstract 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical class [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 abstract 1
- 235000013923 monosodium glutamate Nutrition 0.000 abstract 1
- 235000003969 glutathione Nutrition 0.000 description 33
- 238000006243 chemical reaction Methods 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 18
- 238000000034 method Methods 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 17
- 239000012634 fragment Substances 0.000 description 17
- 150000001413 amino acids Chemical group 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 15
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 14
- 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 13
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 12
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 12
- 108010038633 aspartylglutamate Proteins 0.000 description 12
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 12
- 108010009298 lysylglutamic acid Proteins 0.000 description 12
- 108010061238 threonyl-glycine Proteins 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 238000012408 PCR amplification Methods 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- OXEMJGCAJFFREE-FXQIFTODSA-N Glu-Gln-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O OXEMJGCAJFFREE-FXQIFTODSA-N 0.000 description 8
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 8
- YYXJFBMCOUSYSF-RYUDHWBXSA-N Gly-Phe-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYXJFBMCOUSYSF-RYUDHWBXSA-N 0.000 description 8
- SJLVSMMIFYTSGY-GRLWGSQLSA-N Ile-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SJLVSMMIFYTSGY-GRLWGSQLSA-N 0.000 description 8
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 8
- HVAUKHLDSDDROB-KKUMJFAQSA-N Lys-Lys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HVAUKHLDSDDROB-KKUMJFAQSA-N 0.000 description 8
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 8
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 8
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 8
- 108010047495 alanylglycine Proteins 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 108010049041 glutamylalanine Proteins 0.000 description 8
- 108010050848 glycylleucine Proteins 0.000 description 8
- 108010085325 histidylproline Proteins 0.000 description 8
- 108010034529 leucyl-lysine Proteins 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 7
- 239000006137 Luria-Bertani broth Substances 0.000 description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 7
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 7
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 7
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 7
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 229910052708 sodium Inorganic materials 0.000 description 5
- CWFMWBHMIMNZLN-NAKRPEOUSA-N (2s)-1-[(2s)-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]propanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CWFMWBHMIMNZLN-NAKRPEOUSA-N 0.000 description 4
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 4
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-N 0.000 description 4
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 4
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 4
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 4
- FVNAUOZKIPAYNA-BPNCWPANSA-N Ala-Met-Tyr Chemical compound CSCC[C@H](NC(=O)[C@H](C)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FVNAUOZKIPAYNA-BPNCWPANSA-N 0.000 description 4
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 4
- MTDDMSUUXNQMKK-BPNCWPANSA-N Ala-Tyr-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N MTDDMSUUXNQMKK-BPNCWPANSA-N 0.000 description 4
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 4
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 4
- RFXXUWGNVRJTNQ-QXEWZRGKSA-N Arg-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N RFXXUWGNVRJTNQ-QXEWZRGKSA-N 0.000 description 4
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 4
- GSUFZRURORXYTM-STQMWFEESA-N Arg-Phe-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 GSUFZRURORXYTM-STQMWFEESA-N 0.000 description 4
- UGZUVYDKAYNCII-ULQDDVLXSA-N Arg-Phe-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UGZUVYDKAYNCII-ULQDDVLXSA-N 0.000 description 4
- 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 4
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 4
- QJWLLRZTJFPCHA-STECZYCISA-N Arg-Tyr-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O QJWLLRZTJFPCHA-STECZYCISA-N 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 4
- SWLOHUMCUDRTCL-ZLUOBGJFSA-N Asn-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N SWLOHUMCUDRTCL-ZLUOBGJFSA-N 0.000 description 4
- BZMWJLLUAKSIMH-FXQIFTODSA-N Asn-Glu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BZMWJLLUAKSIMH-FXQIFTODSA-N 0.000 description 4
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 4
- SZNGQSBRHFMZLT-IHRRRGAJSA-N Asn-Pro-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SZNGQSBRHFMZLT-IHRRRGAJSA-N 0.000 description 4
- REQUGIWGOGSOEZ-ZLUOBGJFSA-N Asn-Ser-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N REQUGIWGOGSOEZ-ZLUOBGJFSA-N 0.000 description 4
- SNYCNNPOFYBCEK-ZLUOBGJFSA-N Asn-Ser-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O SNYCNNPOFYBCEK-ZLUOBGJFSA-N 0.000 description 4
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 4
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 4
- DATSKXOXPUAOLK-KKUMJFAQSA-N Asn-Tyr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DATSKXOXPUAOLK-KKUMJFAQSA-N 0.000 description 4
- JNCRAQVYJZGIOW-QSFUFRPTSA-N Asn-Val-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNCRAQVYJZGIOW-QSFUFRPTSA-N 0.000 description 4
- HMQDRBKQMLRCCG-GMOBBJLQSA-N Asp-Arg-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HMQDRBKQMLRCCG-GMOBBJLQSA-N 0.000 description 4
- SVFOIXMRMLROHO-SRVKXCTJSA-N Asp-Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SVFOIXMRMLROHO-SRVKXCTJSA-N 0.000 description 4
- OEUQMKNNOWJREN-AVGNSLFASA-N Asp-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N OEUQMKNNOWJREN-AVGNSLFASA-N 0.000 description 4
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 4
- KHBLRHKVXICFMY-GUBZILKMSA-N Asp-Glu-Lys Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O KHBLRHKVXICFMY-GUBZILKMSA-N 0.000 description 4
- GISFCCXBVJKGEO-QEJZJMRPSA-N Asp-Glu-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O GISFCCXBVJKGEO-QEJZJMRPSA-N 0.000 description 4
- YDJVIBMKAMQPPP-LAEOZQHASA-N Asp-Glu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O YDJVIBMKAMQPPP-LAEOZQHASA-N 0.000 description 4
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 4
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 4
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 4
- DPNWSMBUYCLEDG-CIUDSAMLSA-N Asp-Lys-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O DPNWSMBUYCLEDG-CIUDSAMLSA-N 0.000 description 4
- YFGUZQQCSDZRBN-DCAQKATOSA-N Asp-Pro-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O YFGUZQQCSDZRBN-DCAQKATOSA-N 0.000 description 4
- DRCOAZZDQRCGGP-GHCJXIJMSA-N Asp-Ser-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DRCOAZZDQRCGGP-GHCJXIJMSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- ODBLJLZVLAWVMS-GUBZILKMSA-N Gln-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)N)N ODBLJLZVLAWVMS-GUBZILKMSA-N 0.000 description 4
- LPYPANUXJGFMGV-FXQIFTODSA-N Gln-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N LPYPANUXJGFMGV-FXQIFTODSA-N 0.000 description 4
- XJKAKYXMFHUIHT-AUTRQRHGSA-N Gln-Glu-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N XJKAKYXMFHUIHT-AUTRQRHGSA-N 0.000 description 4
- HWEINOMSWQSJDC-SRVKXCTJSA-N Gln-Leu-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O HWEINOMSWQSJDC-SRVKXCTJSA-N 0.000 description 4
- MLSKFHLRFVGNLL-WDCWCFNPSA-N Gln-Leu-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MLSKFHLRFVGNLL-WDCWCFNPSA-N 0.000 description 4
- QMVCEWKHIUHTSD-GUBZILKMSA-N Gln-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N QMVCEWKHIUHTSD-GUBZILKMSA-N 0.000 description 4
- 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 4
- SOEXCCGNHQBFPV-DLOVCJGASA-N Gln-Val-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SOEXCCGNHQBFPV-DLOVCJGASA-N 0.000 description 4
- SZXSSXUNOALWCH-ACZMJKKPSA-N Glu-Ala-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O SZXSSXUNOALWCH-ACZMJKKPSA-N 0.000 description 4
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 4
- SBCYJMOOHUDWDA-NUMRIWBASA-N Glu-Asp-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SBCYJMOOHUDWDA-NUMRIWBASA-N 0.000 description 4
- RFDHKPSHTXZKLL-IHRRRGAJSA-N Glu-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N RFDHKPSHTXZKLL-IHRRRGAJSA-N 0.000 description 4
- WPLGNDORMXTMQS-FXQIFTODSA-N Glu-Gln-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O WPLGNDORMXTMQS-FXQIFTODSA-N 0.000 description 4
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 description 4
- PHONAZGUEGIOEM-GLLZPBPUSA-N Glu-Glu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PHONAZGUEGIOEM-GLLZPBPUSA-N 0.000 description 4
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 4
- QXDXIXFSFHUYAX-MNXVOIDGSA-N Glu-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O QXDXIXFSFHUYAX-MNXVOIDGSA-N 0.000 description 4
- VSRCAOIHMGCIJK-SRVKXCTJSA-N Glu-Leu-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VSRCAOIHMGCIJK-SRVKXCTJSA-N 0.000 description 4
- VMKCPNBBPGGQBJ-GUBZILKMSA-N Glu-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N VMKCPNBBPGGQBJ-GUBZILKMSA-N 0.000 description 4
- IRXNJYPKBVERCW-DCAQKATOSA-N Glu-Leu-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IRXNJYPKBVERCW-DCAQKATOSA-N 0.000 description 4
- ATVYZJGOZLVXDK-IUCAKERBSA-N Glu-Leu-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O ATVYZJGOZLVXDK-IUCAKERBSA-N 0.000 description 4
- UERORLSAFUHDGU-AVGNSLFASA-N Glu-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UERORLSAFUHDGU-AVGNSLFASA-N 0.000 description 4
- JDUKCSSHWNIQQZ-IHRRRGAJSA-N Glu-Phe-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JDUKCSSHWNIQQZ-IHRRRGAJSA-N 0.000 description 4
- QNJNPKSWAHPYGI-JYJNAYRXSA-N Glu-Phe-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 QNJNPKSWAHPYGI-JYJNAYRXSA-N 0.000 description 4
- ZIYGTCDTJJCDDP-JYJNAYRXSA-N Glu-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZIYGTCDTJJCDDP-JYJNAYRXSA-N 0.000 description 4
- MIIGESVJEBDJMP-FHWLQOOXSA-N Glu-Phe-Tyr Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 MIIGESVJEBDJMP-FHWLQOOXSA-N 0.000 description 4
- ARIORLIIMJACKZ-KKUMJFAQSA-N Glu-Pro-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ARIORLIIMJACKZ-KKUMJFAQSA-N 0.000 description 4
- DAHLWSFUXOHMIA-FXQIFTODSA-N Glu-Ser-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O DAHLWSFUXOHMIA-FXQIFTODSA-N 0.000 description 4
- WXONSNSSBYQGNN-AVGNSLFASA-N Glu-Ser-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WXONSNSSBYQGNN-AVGNSLFASA-N 0.000 description 4
- UMZHHILWZBFPGL-LOKLDPHHSA-N Glu-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O UMZHHILWZBFPGL-LOKLDPHHSA-N 0.000 description 4
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 4
- JVWPPCWUDRJGAE-YUMQZZPRSA-N Gly-Asn-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JVWPPCWUDRJGAE-YUMQZZPRSA-N 0.000 description 4
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 4
- UFPXDFOYHVEIPI-BYPYZUCNSA-N Gly-Gly-Asp Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O UFPXDFOYHVEIPI-BYPYZUCNSA-N 0.000 description 4
- QITBQGJOXQYMOA-ZETCQYMHSA-N Gly-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QITBQGJOXQYMOA-ZETCQYMHSA-N 0.000 description 4
- TVDHVLGFJSHPAX-UWVGGRQHSA-N Gly-His-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 TVDHVLGFJSHPAX-UWVGGRQHSA-N 0.000 description 4
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 4
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 4
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 4
- CLNSYANKYVMZNM-UWVGGRQHSA-N Gly-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N CLNSYANKYVMZNM-UWVGGRQHSA-N 0.000 description 4
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 4
- PCPOYRCAHPJXII-UWVGGRQHSA-N Gly-Lys-Met Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(O)=O PCPOYRCAHPJXII-UWVGGRQHSA-N 0.000 description 4
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 4
- GBYYQVBXFVDJPJ-WLTAIBSBSA-N Gly-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)CN)O GBYYQVBXFVDJPJ-WLTAIBSBSA-N 0.000 description 4
- SYMSVYVUSPSAAO-IHRRRGAJSA-N His-Arg-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O SYMSVYVUSPSAAO-IHRRRGAJSA-N 0.000 description 4
- MFQVZYSPCIZFMR-MGHWNKPDSA-N His-Ile-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N MFQVZYSPCIZFMR-MGHWNKPDSA-N 0.000 description 4
- LQGCNWWLGGMTJO-ULQDDVLXSA-N His-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N LQGCNWWLGGMTJO-ULQDDVLXSA-N 0.000 description 4
- KYFGGRHWLFZXPU-KKUMJFAQSA-N His-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N KYFGGRHWLFZXPU-KKUMJFAQSA-N 0.000 description 4
- VXZZUXWAOMWWJH-QTKMDUPCSA-N His-Thr-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O VXZZUXWAOMWWJH-QTKMDUPCSA-N 0.000 description 4
- SYPULFZAGBBIOM-GVXVVHGQSA-N His-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N SYPULFZAGBBIOM-GVXVVHGQSA-N 0.000 description 4
- VAXBXNPRXPHGHG-BJDJZHNGSA-N Ile-Ala-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)O)N VAXBXNPRXPHGHG-BJDJZHNGSA-N 0.000 description 4
- XENGULNPUDGALZ-ZPFDUUQYSA-N Ile-Asn-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N XENGULNPUDGALZ-ZPFDUUQYSA-N 0.000 description 4
- HOLOYAZCIHDQNS-YVNDNENWSA-N Ile-Gln-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HOLOYAZCIHDQNS-YVNDNENWSA-N 0.000 description 4
- LKACSKJPTFSBHR-MNXVOIDGSA-N Ile-Gln-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N LKACSKJPTFSBHR-MNXVOIDGSA-N 0.000 description 4
- MQFGXJNSUJTXDT-QSFUFRPTSA-N Ile-Gly-Ile Chemical compound N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)O MQFGXJNSUJTXDT-QSFUFRPTSA-N 0.000 description 4
- DMSVBUWGDLYNLC-IAVJCBSLSA-N Ile-Ile-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 DMSVBUWGDLYNLC-IAVJCBSLSA-N 0.000 description 4
- TWYOYAKMLHWMOJ-ZPFDUUQYSA-N Ile-Leu-Asn Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O TWYOYAKMLHWMOJ-ZPFDUUQYSA-N 0.000 description 4
- HPCFRQWLTRDGHT-AJNGGQMLSA-N Ile-Leu-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O HPCFRQWLTRDGHT-AJNGGQMLSA-N 0.000 description 4
- FFAUOCITXBMRBT-YTFOTSKYSA-N Ile-Lys-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FFAUOCITXBMRBT-YTFOTSKYSA-N 0.000 description 4
- GLYJPWIRLBAIJH-FQUUOJAGSA-N Ile-Lys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N GLYJPWIRLBAIJH-FQUUOJAGSA-N 0.000 description 4
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 4
- BKPPWVSPSIUXHZ-OSUNSFLBSA-N Ile-Met-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N BKPPWVSPSIUXHZ-OSUNSFLBSA-N 0.000 description 4
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 4
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 4
- JZNVOBUNTWNZPW-GHCJXIJMSA-N Ile-Ser-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N JZNVOBUNTWNZPW-GHCJXIJMSA-N 0.000 description 4
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 4
- RQJUKVXWAKJDBW-SVSWQMSJSA-N Ile-Ser-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N RQJUKVXWAKJDBW-SVSWQMSJSA-N 0.000 description 4
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 4
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 4
- 241000880493 Leptailurus serval Species 0.000 description 4
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 4
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 4
- KKXDHFKZWKLYGB-GUBZILKMSA-N Leu-Asn-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKXDHFKZWKLYGB-GUBZILKMSA-N 0.000 description 4
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 4
- DLCOFDAHNMMQPP-SRVKXCTJSA-N Leu-Asp-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DLCOFDAHNMMQPP-SRVKXCTJSA-N 0.000 description 4
- LJKJVTCIRDCITR-SRVKXCTJSA-N Leu-Cys-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LJKJVTCIRDCITR-SRVKXCTJSA-N 0.000 description 4
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 4
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 4
- KVMULWOHPPMHHE-DCAQKATOSA-N Leu-Glu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KVMULWOHPPMHHE-DCAQKATOSA-N 0.000 description 4
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 4
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 4
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 4
- APFJUBGRZGMQFF-QWRGUYRKSA-N Leu-Gly-Lys Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN APFJUBGRZGMQFF-QWRGUYRKSA-N 0.000 description 4
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 description 4
- ZALAVHVPPOHAOL-XUXIUFHCSA-N Leu-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(C)C)N ZALAVHVPPOHAOL-XUXIUFHCSA-N 0.000 description 4
- HRTRLSRYZZKPCO-BJDJZHNGSA-N Leu-Ile-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HRTRLSRYZZKPCO-BJDJZHNGSA-N 0.000 description 4
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 4
- UCNNZELZXFXXJQ-BZSNNMDCSA-N Leu-Leu-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 UCNNZELZXFXXJQ-BZSNNMDCSA-N 0.000 description 4
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 4
- DDVHDMSBLRAKNV-IHRRRGAJSA-N Leu-Met-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O DDVHDMSBLRAKNV-IHRRRGAJSA-N 0.000 description 4
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 4
- VULJUQZPSOASBZ-SRVKXCTJSA-N Leu-Pro-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O VULJUQZPSOASBZ-SRVKXCTJSA-N 0.000 description 4
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 4
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 4
- GOFJOGXGMPHOGL-DCAQKATOSA-N Leu-Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C GOFJOGXGMPHOGL-DCAQKATOSA-N 0.000 description 4
- ONHCDMBHPQIPAI-YTQUADARSA-N Leu-Trp-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N ONHCDMBHPQIPAI-YTQUADARSA-N 0.000 description 4
- UCRJTSIIAYHOHE-ULQDDVLXSA-N Leu-Tyr-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UCRJTSIIAYHOHE-ULQDDVLXSA-N 0.000 description 4
- YIBOAHAOAWACDK-QEJZJMRPSA-N Lys-Ala-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YIBOAHAOAWACDK-QEJZJMRPSA-N 0.000 description 4
- FACUGMGEFUEBTI-SRVKXCTJSA-N Lys-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCCCN FACUGMGEFUEBTI-SRVKXCTJSA-N 0.000 description 4
- OVIVOCSURJYCTM-GUBZILKMSA-N Lys-Asp-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O OVIVOCSURJYCTM-GUBZILKMSA-N 0.000 description 4
- NRQRKMYZONPCTM-CIUDSAMLSA-N Lys-Asp-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NRQRKMYZONPCTM-CIUDSAMLSA-N 0.000 description 4
- CKSBRMUOQDNPKZ-SRVKXCTJSA-N Lys-Gln-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O CKSBRMUOQDNPKZ-SRVKXCTJSA-N 0.000 description 4
- LLSUNJYOSCOOEB-GUBZILKMSA-N Lys-Glu-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O LLSUNJYOSCOOEB-GUBZILKMSA-N 0.000 description 4
- PBIPLDMFHAICIP-DCAQKATOSA-N Lys-Glu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PBIPLDMFHAICIP-DCAQKATOSA-N 0.000 description 4
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 4
- NNKLKUUGESXCBS-KBPBESRZSA-N Lys-Gly-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NNKLKUUGESXCBS-KBPBESRZSA-N 0.000 description 4
- XDPLZVNMYQOFQZ-BJDJZHNGSA-N Lys-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N XDPLZVNMYQOFQZ-BJDJZHNGSA-N 0.000 description 4
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 4
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 4
- BXPHMHQHYHILBB-BZSNNMDCSA-N Lys-Lys-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BXPHMHQHYHILBB-BZSNNMDCSA-N 0.000 description 4
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 4
- QBHGXFQJFPWJIH-XUXIUFHCSA-N Lys-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN QBHGXFQJFPWJIH-XUXIUFHCSA-N 0.000 description 4
- MIROMRNASYKZNL-ULQDDVLXSA-N Lys-Pro-Tyr Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MIROMRNASYKZNL-ULQDDVLXSA-N 0.000 description 4
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 4
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 4
- UAPZLLPGGOOCRO-IHRRRGAJSA-N Met-Asn-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N UAPZLLPGGOOCRO-IHRRRGAJSA-N 0.000 description 4
- NHDMNXBBSGVYGP-PYJNHQTQSA-N Met-His-Ile Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)CC1=CN=CN1 NHDMNXBBSGVYGP-PYJNHQTQSA-N 0.000 description 4
- ULLIQRYQNMAAHC-RWMBFGLXSA-N Met-His-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N ULLIQRYQNMAAHC-RWMBFGLXSA-N 0.000 description 4
- UROWNMBTQGGTHB-DCAQKATOSA-N Met-Leu-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UROWNMBTQGGTHB-DCAQKATOSA-N 0.000 description 4
- OCRSGGIJBDUXHU-WDSOQIARSA-N Met-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 OCRSGGIJBDUXHU-WDSOQIARSA-N 0.000 description 4
- FBLBCGLSRXBANI-KKUMJFAQSA-N Met-Phe-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FBLBCGLSRXBANI-KKUMJFAQSA-N 0.000 description 4
- GWADARYJIJDYRC-XGEHTFHBSA-N Met-Thr-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GWADARYJIJDYRC-XGEHTFHBSA-N 0.000 description 4
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 4
- 108010079364 N-glycylalanine Proteins 0.000 description 4
- MPGJIHFJCXTVEX-KKUMJFAQSA-N Phe-Arg-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O MPGJIHFJCXTVEX-KKUMJFAQSA-N 0.000 description 4
- WGXOKDLDIWSOCV-MELADBBJSA-N Phe-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O WGXOKDLDIWSOCV-MELADBBJSA-N 0.000 description 4
- SWZKMTDPQXLQRD-XVSYOHENSA-N Phe-Asp-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWZKMTDPQXLQRD-XVSYOHENSA-N 0.000 description 4
- OPEVYHFJXLCCRT-AVGNSLFASA-N Phe-Gln-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O OPEVYHFJXLCCRT-AVGNSLFASA-N 0.000 description 4
- UAMFZRNCIFFMLE-FHWLQOOXSA-N Phe-Glu-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N UAMFZRNCIFFMLE-FHWLQOOXSA-N 0.000 description 4
- DVOCGBNHAUHKHJ-DKIMLUQUSA-N Phe-Ile-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O DVOCGBNHAUHKHJ-DKIMLUQUSA-N 0.000 description 4
- CWFGECHCRMGPPT-MXAVVETBSA-N Phe-Ile-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O CWFGECHCRMGPPT-MXAVVETBSA-N 0.000 description 4
- KNYPNEYICHHLQL-ACRUOGEOSA-N Phe-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 KNYPNEYICHHLQL-ACRUOGEOSA-N 0.000 description 4
- BONHGTUEEPIMPM-AVGNSLFASA-N Phe-Ser-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O BONHGTUEEPIMPM-AVGNSLFASA-N 0.000 description 4
- XNQMZHLAYFWSGJ-HTUGSXCWSA-N Phe-Thr-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XNQMZHLAYFWSGJ-HTUGSXCWSA-N 0.000 description 4
- JTKGCYOOJLUETJ-ULQDDVLXSA-N Phe-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JTKGCYOOJLUETJ-ULQDDVLXSA-N 0.000 description 4
- UEHYFUCOGHWASA-HJGDQZAQSA-N Pro-Glu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 UEHYFUCOGHWASA-HJGDQZAQSA-N 0.000 description 4
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 4
- DWGFLKQSGRUQTI-IHRRRGAJSA-N Pro-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 DWGFLKQSGRUQTI-IHRRRGAJSA-N 0.000 description 4
- WOIFYRZPIORBRY-AVGNSLFASA-N Pro-Lys-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O WOIFYRZPIORBRY-AVGNSLFASA-N 0.000 description 4
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 4
- BXHRXLMCYSZSIY-STECZYCISA-N Pro-Tyr-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H]1CCCN1)C(O)=O BXHRXLMCYSZSIY-STECZYCISA-N 0.000 description 4
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 4
- WWXNZNWZNZPDIF-SRVKXCTJSA-N Pro-Val-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 WWXNZNWZNZPDIF-SRVKXCTJSA-N 0.000 description 4
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 4
- WXUBSIDKNMFAGS-IHRRRGAJSA-N Ser-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WXUBSIDKNMFAGS-IHRRRGAJSA-N 0.000 description 4
- VGNYHOBZJKWRGI-CIUDSAMLSA-N Ser-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO VGNYHOBZJKWRGI-CIUDSAMLSA-N 0.000 description 4
- YIUWWXVTYLANCJ-NAKRPEOUSA-N Ser-Ile-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YIUWWXVTYLANCJ-NAKRPEOUSA-N 0.000 description 4
- ZIFYDQAFEMIZII-GUBZILKMSA-N Ser-Leu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZIFYDQAFEMIZII-GUBZILKMSA-N 0.000 description 4
- FPCGZYMRFFIYIH-CIUDSAMLSA-N Ser-Lys-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O FPCGZYMRFFIYIH-CIUDSAMLSA-N 0.000 description 4
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 4
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 4
- SZRNDHWMVSFPSP-XKBZYTNZSA-N Ser-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N)O SZRNDHWMVSFPSP-XKBZYTNZSA-N 0.000 description 4
- HXPNJVLVHKABMJ-KKUMJFAQSA-N Ser-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CO)N)O HXPNJVLVHKABMJ-KKUMJFAQSA-N 0.000 description 4
- GARULAKWZGFIKC-RWRJDSDZSA-N Thr-Gln-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GARULAKWZGFIKC-RWRJDSDZSA-N 0.000 description 4
- UDQBCBUXAQIZAK-GLLZPBPUSA-N Thr-Glu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UDQBCBUXAQIZAK-GLLZPBPUSA-N 0.000 description 4
- WDFPMSHYMRBLKM-NKIYYHGXSA-N Thr-Glu-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O WDFPMSHYMRBLKM-NKIYYHGXSA-N 0.000 description 4
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 4
- FKIGTIXHSRNKJU-IXOXFDKPSA-N Thr-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@H](O)C)CC1=CN=CN1 FKIGTIXHSRNKJU-IXOXFDKPSA-N 0.000 description 4
- XOWKUMFHEZLKLT-CIQUZCHMSA-N Thr-Ile-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O XOWKUMFHEZLKLT-CIQUZCHMSA-N 0.000 description 4
- UYTYTDMCDBPDSC-URLPEUOOSA-N Thr-Ile-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N UYTYTDMCDBPDSC-URLPEUOOSA-N 0.000 description 4
- MCDVZTRGHNXTGK-HJGDQZAQSA-N Thr-Met-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O MCDVZTRGHNXTGK-HJGDQZAQSA-N 0.000 description 4
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 4
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 4
- WBZOZLNLXVBCNW-LTHWPDAASA-N Trp-Thr-Ile Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)[C@@H](C)O)=CNC2=C1 WBZOZLNLXVBCNW-LTHWPDAASA-N 0.000 description 4
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 4
- XHALUUQSNXSPLP-UFYCRDLUSA-N Tyr-Arg-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 XHALUUQSNXSPLP-UFYCRDLUSA-N 0.000 description 4
- QHEGAOPHISYNDF-XDTLVQLUSA-N Tyr-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QHEGAOPHISYNDF-XDTLVQLUSA-N 0.000 description 4
- LOOCQRRBKZTPKO-AVGNSLFASA-N Tyr-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LOOCQRRBKZTPKO-AVGNSLFASA-N 0.000 description 4
- FNWGDMZVYBVAGJ-XEGUGMAKSA-N Tyr-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CC=C(C=C1)O)N FNWGDMZVYBVAGJ-XEGUGMAKSA-N 0.000 description 4
- GULIUBBXCYPDJU-CQDKDKBSSA-N Tyr-Leu-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 GULIUBBXCYPDJU-CQDKDKBSSA-N 0.000 description 4
- BSCBBPKDVOZICB-KKUMJFAQSA-N Tyr-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BSCBBPKDVOZICB-KKUMJFAQSA-N 0.000 description 4
- VPEFOFYNHBWFNQ-UFYCRDLUSA-N Tyr-Pro-Tyr Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 VPEFOFYNHBWFNQ-UFYCRDLUSA-N 0.000 description 4
- DJSYPCWZPNHQQE-FHWLQOOXSA-N Tyr-Tyr-Gln Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(N)=O)C(O)=O)C1=CC=C(O)C=C1 DJSYPCWZPNHQQE-FHWLQOOXSA-N 0.000 description 4
- GOPQNCQSXBJAII-ULQDDVLXSA-N Tyr-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N GOPQNCQSXBJAII-ULQDDVLXSA-N 0.000 description 4
- BYOHPUZJVXWHAE-BYULHYEWSA-N Val-Asn-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N BYOHPUZJVXWHAE-BYULHYEWSA-N 0.000 description 4
- ZSZFTYVFQLUWBF-QXEWZRGKSA-N Val-Asp-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N ZSZFTYVFQLUWBF-QXEWZRGKSA-N 0.000 description 4
- KOPBYUSPXBQIHD-NRPADANISA-N Val-Cys-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KOPBYUSPXBQIHD-NRPADANISA-N 0.000 description 4
- CFSSLXZJEMERJY-NRPADANISA-N Val-Gln-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CFSSLXZJEMERJY-NRPADANISA-N 0.000 description 4
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 0.000 description 4
- SZTTYWIUCGSURQ-AUTRQRHGSA-N Val-Glu-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SZTTYWIUCGSURQ-AUTRQRHGSA-N 0.000 description 4
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 4
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 4
- QSPOLEBZTMESFY-SRVKXCTJSA-N Val-Pro-Val Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O QSPOLEBZTMESFY-SRVKXCTJSA-N 0.000 description 4
- PGQUDQYHWICSAB-NAKRPEOUSA-N Val-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N PGQUDQYHWICSAB-NAKRPEOUSA-N 0.000 description 4
- DLRZGNXCXUGIDG-KKHAAJSZSA-N Val-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O DLRZGNXCXUGIDG-KKHAAJSZSA-N 0.000 description 4
- 108010044940 alanylglutamine Proteins 0.000 description 4
- 108010070783 alanyltyrosine Proteins 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 108010062796 arginyllysine Proteins 0.000 description 4
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 4
- 108010068265 aspartyltyrosine Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 108010079547 glutamylmethionine Proteins 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 108010072405 glycyl-aspartyl-glycine Proteins 0.000 description 4
- 108010023364 glycyl-histidyl-arginine Proteins 0.000 description 4
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 4
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 4
- 108010000761 leucylarginine Proteins 0.000 description 4
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 4
- 108010064235 lysylglycine Proteins 0.000 description 4
- 108010054155 lysyllysine Proteins 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 108010034507 methionyltryptophan Proteins 0.000 description 4
- 108010012581 phenylalanylglutamate Proteins 0.000 description 4
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 4
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 4
- 108010051110 tyrosyl-lysine Proteins 0.000 description 4
- 108010020532 tyrosyl-proline Proteins 0.000 description 4
- 108010073969 valyllysine Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 238000001712 DNA sequencing Methods 0.000 description 3
- 241000672609 Escherichia coli BL21 Species 0.000 description 3
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical class [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 3
- 241000585212 Melissococcus plutonius ATCC 35311 Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 235000011147 magnesium chloride Nutrition 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 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 2
- 241001131785 Escherichia coli HB101 Species 0.000 description 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- 108010063907 Glutathione Reductase Proteins 0.000 description 2
- 102100036442 Glutathione reductase, mitochondrial Human genes 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- CRUSHRATSSNWSB-UHFFFAOYSA-N phenyl-sulfanylidene-sulfidoazanium Chemical compound [S-][N+](=S)C1=CC=CC=C1 CRUSHRATSSNWSB-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- GZQVASFPFGYUAQ-GEMLJDPKSA-N 2-aminoacetic acid;(2s)-2-amino-5-[[(2r)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopentanoic acid Chemical compound NCC(O)=O.OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O GZQVASFPFGYUAQ-GEMLJDPKSA-N 0.000 description 1
- 241000606750 Actinobacillus Species 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 241001468188 Melissococcus plutonius Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001123227 Saccharomyces pastorianus Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- -1 amino acid L-glutamates Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 125000002642 gamma-glutamyl group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
- C12Y603/02003—Glutathione synthase (6.3.2.3)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开一种谷胱甘肽合成酶突变体、编码基因及应用，该谷胱甘肽合成酶突变体由序列表中的序列2经点突变所得，所述点突变为在该序列的第128位、第256位及第320位的至少一个突变。本发明通过对谷胱甘肽合成酶基因序列进行突变，最终获得具有高催化活性的谷胱甘肽合成酶突变体。并且该突变体以三磷酸腺苷二钠盐、L‑谷氨酸钠、L‑半胱氨酸和甘氨酸为底物具有比亲本高出至少50%的谷胱甘肽合成酶催化活性。用固定化谷胱甘肽合成酶突变体制备谷胱甘肽,产生的谷胱甘肽的浓度超过50mM,从而使该谷胱甘肽合成酶突变体可用于工业化生产谷胱甘肽，降低了生产成本，提高了相应产品的市场竞争力。
Description
技术领域
本发明涉及分子生物学与生物技术领域，尤其涉及一种谷胱甘肽合成酶突变体、编码基因及应用。
背景技术
谷胱甘肽，即γ-L-谷氨酰-L-半胱氨酰甘氨酸（glutathione，GSH），是由L-谷氨酸、L-半胱氨酸和甘氨酸经肽键缩合而成的一种同时具有γ-谷氨酰基和巯基的生物活性三肽化合物。
谷胱甘肽有两种形式：还原型GSH和氧化型GSSG，只有GSH才具有活性，而生物体内的GSSG需还原后才能发挥其重要的生理功能。GSH在自然界中广泛分布于动物、植物和微生物细胞内。GSH在生物体内有着多种重要的生理功能，特别是对于维持生物体内适宜的氧化还原环境起着至关重要的作用。GSH还具有独特的生理功能，被称为长寿因子和抗衰老因子。由于GSH在强化食品风味的同时对人体有保健作用，它的应用前景显然要优于其它类型的防腐剂或抗氧化剂。随着GSH的生理生化功能和性质被不断研究发现，GSH作为一种多功能的生物活性添加剂在食品加工业中的应用将会愈来愈广，GSH的需求量正日益增大。
谷胱甘肽的生产方法主要有提取法、发酵法、酶法及化学合成法。提取法是以谷胱甘肽含量丰富的动、植物和酵母为原料，通过加入适当的溶剂或结合淀粉酶、蛋白酶的处理，再经过多步分离获得。由于天然组织中谷胱甘肽的含量低，仅为干重的0.5~1.0%，以及处理步骤多，使得该方法的总体得率较低。化学合成法生产工艺成熟，但化学合成的谷胱甘肽是D-型和L-型的消旋体，其中，具有生理活性的是L-谷胱甘肽，所以，要想得到有活性的谷胱甘肽需要将化学合成的产物进行光学拆分。此外，化学合成法的步骤多，时间较长，污染大。目前工业规模生产谷胱甘肽的主要方法是发酵法，其中以诱变处理获得高谷胱甘肽含量的酵母变异菌种来生产谷胱甘肽最为常见，但利用酵母菌培养来单独生产谷胱甘肽，存在原料利用率低、成本和能耗高等问题。酶法是指用离体酶催化三种氨基酸L-谷氨酸、L-半胱氨酸和甘氨酸形成谷胱甘肽，通常是采用二步酶法，即先用谷氨酰半胱氨酸合成酶将L-谷氨酸和L-半胱氨酸催化合成L-谷氨酰基-L---半胱氨酸, 之后再通过谷胱甘肽合成酶将L-谷氨酰基-L---半胱氨酸与甘氨酸催化合成谷胱甘肽。目前，叶勤等人发现，猪胸膜肺炎放线杆菌等含双功能谷胱甘肽合成酶，与前述二步酶法相比，这些双功能谷胱甘肽合成酶令酶法生产谷胱甘肽工艺变成一步酶法了，效率更高些。但这些双功能谷胱甘肽合成酶效率仍不高，酶法产生的谷胱甘肽浓度较低，致使生产成本高。
因此，现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种谷胱甘肽合成酶突变体、编码基因及应用，旨在解决现有生产谷胱甘肽工艺中谷胱甘肽合成酶催化活性低、产物谷胱甘肽浓度低及生产成本高的问题。
本发明的技术方案如下：
一种谷胱甘肽合成酶突变体，其中，由序列表中的序列2经点突变所得，所述点突变具有选自第128位、第256位及第320位的至少一个突变，并且以三磷酸腺苷二钠盐(ATP)、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物其具有比亲本高出至少50%的谷胱甘肽合成酶催化活性。
所述的谷胱甘肽合成酶突变体，其中，所述谷胱甘肽合成酶突变体还包括其变体，所述变体包括所述序列2所示氨基酸序列中除第128位、第256位及第320位外的其它位点的保守取代形式、增加或缺失一个或几个氨基酸形式、氨基端截断形式、羧基端截断形式，及所述序列2的部分或全部串联重复形式。
所述的谷胱甘肽合成酶突变体，其中，所述点突变具体为：序列2的第128位的谷氨酰胺(Q)突变为精氨酸(R)。
所述的谷胱甘肽合成酶突变体，其中，所述点突变具体为：序列2的第256位的丙氨酸(A)突变为丝氨酸(S)。
所述的谷胱甘肽合成酶突变体，其中，所述点突变具体为：序列2的第320位的亮氨酸(L)突变为赖氨酸(K)。
所述的谷胱甘肽合成酶突变体，其中，所述谷胱甘肽合成酶突变体具有序列表中序列3或序列4或序列5所示的氨基酸序列。
一种编码谷胱甘肽合成酶突变体的基因，其含有编码所述谷胱甘肽合成酶突变体的核苷酸序列。
所述的谷胱甘肽合成酶突变体的应用，其中，所述谷胱甘肽合成酶突变体应用于以三磷酸腺苷二钠盐、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物制备谷胱甘肽。
有益效果：本发明提供一种谷胱甘肽合成酶突变体、编码基因及应用，通过对谷胱甘肽合成酶基因序列进行定点突变，最终获得具有高催化活性的谷胱甘肽合成酶突变体。并且该突变体以L-谷氨酸、L-半胱氨酸、甘氨酸和三磷酸腺苷二钠(ATP)为底物具有比亲本高出至少50%的谷胱甘肽合成酶催化活性, 产物谷胱甘肽浓度大于50mM，从而使该谷胱甘肽合成酶突变体可用于工业化生产谷胱甘肽，降低了生产成本，提高了相应产品的市场竞争力。
具体实施方式
本发明提供一种谷胱甘肽合成酶突变体、编码基因及应用，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明目的在于提供一种谷胱甘肽合成酶突变体。本发明的另一目的还在于提供一种编码本发明所述的谷胱甘肽合成酶突变体的基因。本发明的再一目的在于将本发明的谷胱甘肽合成酶突变体应用于以L-谷氨酸、L-半胱氨酸、甘氨酸和三磷酸腺苷二钠(ATP)为底物催化生成谷胱甘肽。
为实现本发明的上述目的，本发明人进行了大量深入的实验，通过对谷胱甘肽合成酶基因进行定点突变，PCR扩增后插入适当的载体，随后在培养基上筛选，从而获得了一系列具有高催化活性的谷胱甘肽合成酶突变体，该谷胱甘肽合成酶突变体能以L-谷氨酸、L-半胱氨酸、甘氨酸和三磷酸腺苷二钠(ATP)为底物高效催化生成谷胱甘肽。
具体地，为了获得本发明的谷胱甘肽合成酶突变体，可以利用本领域已知的技术，先构建含有亲本谷胱甘肽合成酶基因的载体质粒，然后设定定点突变的位点以及突变后的氨基酸种类，再合成适当的引物，以所述的含亲本谷胱甘肽合成酶基因的载体质粒为模板，PCR扩增DNA片段、装配所扩增的DNA片段以及PCR扩增全长突变基因。然后将该全长突变基因克隆到适当的载体上并转化适当的宿主细胞，经培养筛选出具有谷胱甘肽合成酶活性的阳性克隆。最后从阳性克隆中提取质粒DNA，进行DNA序列测定分析，以确定引入的突变。在确定目的片段插入到载体上后，可通过LB培养基筛选，从而获得具高催化活性的谷胱甘肽合成酶突变体。
本发明提供了一种谷胱甘肽合成酶突变体，其中，由序列表中的序列2经点突变得到，所述点突变具有选自第128位、第256位及第320位的至少一个突变，并且以三磷酸腺苷二钠盐(ATP)、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物其具有比亲本高出至少50%的谷胱甘肽合成酶催化活性。
进一步地，所述谷胱甘肽合成酶突变体还包括其变体，所述变体包括所述序列2所示氨基酸序列中除第128位、第256位及第320位外的其它位点的保守取代形式、增加或缺失一个或几个氨基酸形式、氨基端截断形式、羧基端截断形式，及所述序列2的部分或全部串联重复形式。
优选地，所述点突变具体为：所述亲本序列2第128位的谷氨酰胺(Q)突变为精氨酸(R)，和/或所述亲本序列2第256位的丙氨酸(A)突变为丝氨酸(S)，和/或所述亲本序列2第320位的亮氨酸(L)突变为赖氨酸(K)。亲本序列2第128位的谷氨酰胺(Q)突变为精氨酸(R)形成序列表中序列3所示的氨基酸序列。亲本序列2第256位的丙氨酸(A)突变为丝氨酸(S)形成如序列表中序列4所示的氨基酸序列。亲本序列2第320位的亮氨酸(L)突变为赖氨酸(K)形成如序列表中序列5所示的氨基酸序列。
本发明所述的谷胱甘肽合成酶突变体，其中，所述谷胱甘肽合成酶突变体具有序列表中序列3或序列4或序列5所示的氨基酸序列。
另外，本发明还提供一种基因，其含有编码本发明的谷胱甘肽合成酶突变体的核苷酸序列。
另外，本发明还涉及谷胱甘肽合成酶突变体的应用，其应用以三磷酸腺苷二钠盐(ATP)、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物催化制备谷胱甘肽。所述谷胱甘肽合成酶突变体以L-谷氨酸、L-半胱氨酸、甘氨酸和三磷酸腺苷二钠(ATP)为底物具有比亲本高出至少50%的谷胱甘肽合成酶催化活性, 催化制得的谷胱甘肽浓度大于50mM。
进一步地，在上述制备方法中，所适用的载体可以为原核表达载体, 如pRSET和pES21等; 也可以为克隆载体, 如pUC18/19 和pBluscript-SK。
进一步的，在本发明制备的谷胱甘肽合成酶突变体的方法中，所制得的谷胱甘肽合成酶突变体基因可以在原核细胞或真核细胞胞内表达，当然也可在原核细胞或真核细胞胞外表达。
进一步地，在本发明制备谷胱甘肽合成酶突变体的方法中，所述载体的宿主细胞为原核细胞或真核细胞。所述原核细胞可以为大肠杆菌、凝结芽孢杆菌、枯草芽孢杆菌和链霉菌。所述真核细胞可以为酿酒酵母或毕赤巴斯德酵母。
进一步地，该突变体可以通过Histag纯化方案进行纯化，经酶活测定后发现本发明的谷胱甘肽合成酶突变体具有比亲本高出至少50%的谷胱甘肽合成酶催化活性。
此外，本发明提供的谷胱甘肽合成酶突变体的较高催化活性使其可以未经纯化以粗酶形式使用，也可以是经部分纯化的或完全纯化的酶。当然，还可利用固化技术将本发明谷胱甘肽合成酶突变体制成固相酶或固相细胞形式的固化酶。
本发明的谷胱甘肽合成酶突变体的催化活性较高，即使使用该谷胱甘肽合成酶突变体的粗提物，反应也会以较高的速率进行。另外，本发明的突变体可以在相对较高的温度下用于工业化生产谷胱甘肽，例如40-60℃下生产谷胱甘肽，这样可以保证谷胱甘肽合成酶突变体保持较高的活力，使反应速率保持较高的水平，缩短反应的时间，从而使反应底物因反应时间过长导致的降解得以抑制。
在本申请文本中所用的氨基酸三字母或单字母表达方式，采用IUPAC规定的氨基酸代码(Eur. J. Biochem., 138:9-37, 1984)。
下面结合实施例对本发明进行进一步的说明。
实施例1
亲本谷胱甘肽合成酶基因载体质粒的构建：
根据基因库(GenBank NC_015516)基因序列设计引物MP-F和MP-R。用引物对MP-F和MP-R从Melissococcus plutonius ATCC 35311中扩增谷胱甘肽合成酶编码基因。
扩增条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2 mMMgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP，400 nM引物MP-F，400 nM引物MP-R，1.0 U Pfu DNA聚合酶(Promega，USA)，用接种环挑取少许Melissococcus plutonius ATCC 35311菌体，再用无菌水调反应体积至50 ml。
PCR扩增反应程序为：95℃ 3分钟，40圈循环： 95℃ 50秒、50℃ 30秒和72℃ 1分钟，最后72℃ 10分钟。扩增的产物经限制性内切酶NdeI和AscI酶切后与经同样限制性内切酶NdeI和AscI酶切的载体pRSET-A(源自Invitrogen, USA)连接，得质粒pRSET-MP。经DNA测序，确定该被克隆的谷胱甘肽合成酶的核苷酸序列，具体示于序列表中序列1，相应的氨基酸序列为序列表中的序列2。
表1
实施例2
谷胱甘肽合成酶位点128的定点突变
为了将亲本氨基酸序列中第128位的谷氨酰胺(Q)突变为精氨酸(R)获得突变体Q128R，以质粒pRSET-MP（见实施例1）为模板，设计引物对128RF和128RR (见表1所示)。
用引物对MP-F和128RR，扩增F-RR片段，引物对128RF和MP-R，扩增RF-R片段。引物MP-F和MP-R的具体序列，见表1所示。
上述扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2mM MgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP，400nM引物MP-F和 400 nM引物128RR, 或400 nM引物128RF和 400 nM引物 MP-R，1.5 U PfuDNA聚合酶 (Promega, USA)，20 ng pRSET-MP，用无菌水调反应体积至50微升。
PCR扩增反应程序为：95℃ 3分钟，35圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒 (Promega, USA)回收，分别得到F-RR片段和RF-R片段。然后扩增全长基因。
扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2 mMMgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400nM引物MP-F和400 nM MP-R，1.5 U Pfu DNA聚合酶，20 ng F-RR片段与20 ng RF-R片段，用无菌水调反应体积至50微升。
PCR扩增反应程序为：95℃ 3分钟，40圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒(Promega, USA)回收，得到全长突变基因Q128R。将 Q128R与载体pRSET-A连接，得质粒pRSET-Q128R。将质粒pRSET-Q128R转入感受态细菌细胞E. coli BL21。经DNA测序确定引入的点突变无误。所得突变体的氨基酸序列见序列表1中的序列3。
实施例3
谷胱甘肽合成酶位点256的定点突变
为了将亲本氨基酸序列中第256位点的Ala(A)突变为Ser(S)获得突变体A256S，以实施例1中的质粒pRSET-MP为模板，设计引物对256SF和256SR (见表1所示)。
用引物对MP-F和256SR，扩增F-SR片段，引物对256SF和MP-R，扩增SF-R片段。引物MP-F和MP-R的具体序列见表1所示。
上述扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2mM MgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP，400nM引物MP-F和 400 nM引物256SR,或400 nM引物256SF和 400 nM引物 MP-R，1.5 U PfuDNA聚合酶 (Promega, USA)，20 ng pRSET-MP，用无菌水调反应体积至50微升。
上述PCR扩增反应程序为：95℃ 3分钟，35圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒(Promega, USA)回收，分别得到 F-SR片段和SF-R片段。然后扩增全长基因。
扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2 mMMgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400nM引物MP-F和400 nM MP-R，1.5 U Pfu DNA聚合酶，20 ng F-SR片段与20 ngSF-R 片段，用无菌水调反应体积至50微升。
PCR扩增反应程序为：95℃ 3分钟，35圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒(Promega, USA)回收，得到全长突变基因A256S。将A256S与载体pRSET-A连接(见实施例1)，得质粒pRSET-A256S。将质粒pRSET-A256S转入感受态细菌细胞E. coli BL21。经DNA测序确定引入的点突变无误。A256S的氨基酸序列见序列表中的序列4。
实施例4
谷胱甘肽合成酶位点320的定点突变
为了将亲本氨基酸序列中第320位点的Leu(L)突变为Lys(K)获得突变体L320K，以实施例1中的质粒pRSET-MP为模板，设计引物对320KF和320KR (见表1所示)。
用引物对MP-F和320KR，扩增F-KR片段，引物对320KF和MP-R，扩增KF-R片段。引物MP-F和MP-R的具体序列见表1所示。
上述扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2mM MgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP，400nM引物MP-F和 400 nM引物320KR,或400 nM引物320KF和 400 nM引物 MP-R，1.5 U PfuDNA聚合酶 (Promega, USA)，20 ng pRSET-MP，用无菌水调反应体积至50微升。
上述PCR扩增反应程序为：95℃ 3分钟，35圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒(Promega, USA)回收，分别得到 F-KR片段和KF-R片段。然后扩增全长基因。
扩增反应条件为：20 mM Tris-HCl (pH 8.8)，10 mM KCl，10 mM (NH4)2SO4，2 mMMgSO4，0.1% Triton X-100，50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400nM引物MP-F和400 nM MP-R，1.5 U Pfu DNA聚合酶，20 ng F-KR片段与20 ng KF-R 片段，用无菌水调反应体积至50微升。
PCR扩增反应程序为：95℃ 3分钟，35圈循环：95℃ 50秒、52℃ 30秒和72℃ 3分钟，最后72℃ 5分钟。
经1% 琼脂糖胶电泳分离并用商业试剂盒(Promega, USA)回收，得到全长突变基因L320K。将L320K与载体pRSET-A连接(见实施例1)，得质粒pRSET-L320K。将质粒pRSET-L320K转入感受态细菌细胞E. coli BL21。经DNA测序确定引入的点突变无误。L320K的氨基酸序列见序列表中的序列5。
实施例5
谷胱甘肽合成酶亲本的提取
谷胱甘肽合成酶的提取与纯化具体过程如下：
将含谷胱甘肽合成酶基因的质粒pRSET-MP转化感受态细菌细胞E. coli HB101，在Luria broth(LB)平板(含100 mg/L卡那霉素)上37℃培养24 小时。接种单个克隆于5 毫升LB液体培养基(含100 mg/L卡那霉素)中于30℃培养20-24小时。离心收集菌体，并悬浮于1毫升100mM Tris盐酸缓冲液(pH 7.5)中。然后用超声波裂解细菌细胞。离心(10℃，17,800g，10分钟)并收集上清液, 即为粗提蛋白(或称粗提物)。
实施例6
谷胱甘肽合成酶突变体的提取
谷胱甘肽合成酶的提取与纯化具体过程如下：
将含谷胱甘肽合成酶突变体基因的质粒pRSET-Q128R、pRSET-A256S或pRSET-L320K分别转化感受态细菌细胞E. coli HB101，在Luria broth(LB)平板(含100 mg/L卡那霉素)上37℃培养24 小时。接种单个克隆于5 毫升LB液体培养基(含100 mg/L卡那霉素)中于30℃培养20-24小时。离心收集菌体，并悬浮于1毫升100mM Tris盐酸缓冲液(pH 7.5)中。然后用超声波裂解细菌细胞。离心(10℃，17,800 g，10分钟)并收集上清液, 即为粗提蛋白(或称粗提物)。
实施例7
谷胱甘肽合成酶活性的测定：
谷胱甘肽合成酶活性的测定参考Bjorn Vergauwen, Dirk De Vos, and JozefJ. Van Beeumen. (2006). J.of Biol. Chem. 281(7):4380-4394.
配制底物溶液: 含5mM的三磷酸腺苷二钠盐(ATP)、50mM L-谷氨酸钠、2mM L-半胱氨酸、20mM甘氨酸、20mM 氯化镁、100mM 氯化钠和200mM Tris盐酸缓冲液, 调pH至7.5。取底物溶液900微升，然后加入100微升谷胱甘肽合成酶粗提蛋白,于30℃进行反应。反应产物每隔1分钟取样50微升进行二次反应, 旨在测定反应产物谷胱甘肽的浓度。 50微升反应产物加入至950微升200mM的磷酸缓冲液(含1mM 二硫硝基苯甲酸、500μM NADPH及1个单位的谷胱甘肽还原酶) 中, 用分光光度计测定412nm吸收峰的增加值。
实施例8
谷胱甘肽合成酶突变体活性的测定：
配制底物溶液: 含5mM的三磷酸腺苷二钠盐(ATP)、50mM L-谷氨酸钠、2mM L-半胱氨酸、20mM甘氨酸、20mM 氯化镁、100mM 氯化钠和200mM Tris盐酸缓冲液, 调pH至7.5。取底物溶液900微升，然后加入100微升谷胱甘肽合成酶突变体Q128R、A256S或L320K之粗提蛋白,于30℃进行反应。单个突变体Q128R、A256S或L320K的反应产物每隔1分钟取样50微升进行二次反应, 旨在测定反应产物谷胱甘肽的浓度。 50微升反应产物加入至950微升200mM的磷酸缓冲液(含1mM 二硫硝基苯甲酸、500μM NADPH及1个单位的谷胱甘肽还原酶) 中,用分光光度计测定412nm吸收峰的增加值。结果, 谷胱甘肽合成酶突变体Q128R、A256S和L320K的活力分别比亲本高220%、50%和80%。
实施例9
谷胱甘肽合成酶突变体固定化
取谷胱甘肽合成酶亲本粗提蛋白，用洗酶缓冲液（0.02M Tris-HCl/0.001M EDTA,pH7.0溶液）稀释至蛋白含量5-10mg/ml。将酶稀释液与PB溶液(2.0mol/L磷酸二氢钾,pH7.5)等体积混合, 加入环氧型固定化酶载体LX-3000 (10毫克酶/每克载体)，于摇床(转速100rpm)中25℃反应20小时。反应完成后用滤袋过滤，用洗酶缓冲液清洗5-6次, 得到固定化谷胱甘肽合成酶。
实施例10
用固定化谷胱甘肽合成酶突变体制备谷胱甘肽
配制底物溶液: 含60mM的三磷酸腺苷二钠盐(ATP)、100mM L-谷氨酸钠、55mM L-半胱氨酸、100mM甘氨酸、60mM 氯化镁、50mM 氯化钠和100mM Tris盐酸缓冲液, 调pH至7.5。每升底物溶液中加入400克固定化谷胱甘肽合成酶突变体Q128R,于37℃进行反应。反应2-6小时后, 按实施例8测定谷胱甘肽的浓度。
结果, 突变体Q128R产生了52mM的谷胱甘肽。
本发明提供一种谷胱甘肽合成酶突变体、编码基因及应用，通过对谷胱甘肽合成酶基因序列进行定点突变，最终获得具有高催化活性的谷胱甘肽合成酶突变体。并且该突变体以三磷酸腺苷二钠盐(ATP)、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物具有比亲本高出至少50%的谷胱甘肽合成酶催化活性。用固定化谷胱甘肽合成酶突变体制备谷胱甘肽, 产生的谷胱甘肽的浓度超过50mM, 从而使该谷胱甘肽合成酶突变体可用于工业化生产谷胱甘肽，降低了生产成本，提高了相应产品的市场竞争力。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。
SEQUENCE LISTING
<110> 邦泰生物工程(深圳)有限公司
<120> 谷胱甘肽合成酶突变体及其编码基因和应用
<130> BT-140920-06
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2274
<212> DNA
<213> Melissococcus plutonius ATCC 35311
<400> 1
atgaatttta aaagtttatt acaacaaaag aaggtaaaac cttatattct atccgcacgt 60
tttggtttgg aaaaagaaag ccaacgtagt aaaatagatg gtagacttgc aacgacagaa 120
catcctaaag tgctaggaaa ccgttcgtac catccttata tccaaacaga ttttagtgaa 180
acacaaattg agttaattac acctgtagca gattctatcg atgaagtatc tcgttattta 240
tcagcgattc aggaagttgc ctatcgttca ttggaaaagg aagaaatact ttggccactt 300
agtatgccac caaaactgcc agaaaaagat gaagagatta aaattgcaaa attagatgat 360
tttgaggaaa ctttgtatcg acgctattta gctaaaaagt atggcaaacg aaaacagatg 420
attagtggta ttcactttaa ttttgaatat tcaattgatc ttatccagca tatgtttaat 480
gaacaatcag aatttgaaaa aattgaggaa tttaaaaata cattgtacat gaaaatagct 540
cgtaactatt tacgttatcg ttggttaatt acctatttat ttggtgcttc tccggtttgt 600
gaaaaaggtt actttactga acaggataaa tcattaaatg aaccggtaag aagtattaga 660
aatagttcat ttggttatac aaatgaagaa gttgcagctg tttcttatgc ttcacttaaa 720
aattatttag aagatattca tcgtttggtt gaaaatggta tcttgagcaa agaaaaagaa 780
ttttattcac ctgttcgttt gcgtggtggc aaacaaatat ccgatttatg tcatacgggg 840
attcgatata ttgagctacg aaatttagat ttaaatccgt ttacgtcttt gggaattgat 900
gaagatactt taagattttt gcatattttt cttttgtata tgctatggac agaagaaaaa 960
gaaacacctg atgaatggat aatgactggc aatcttgtta acaaccagat tgctttaatg 1020
catccttttc aatcaattaa cttactttct gaaggtgatc gaatatttag agagatgttt 1080
gaaatgcttg atgaattgga attggttgag gagaaaaaat tggttgatat gtactatcaa 1140
caattaagag cgccagagac gactattgct ggtaaaatgt ggacaattat tcaggaaaat 1200
tctaataaag agttaggaat catttttggc agtgaatatc aagcagctat tctcaatgaa 1260
ccttaccaac taactggttt ccaacaaatg gaattatcta cccaaagctt tttgtttgat 1320
accattcaaa agggaattgc ctttgaaatt ttggatgaac aagaacaatt tttgaagtta 1380
acgcataaaa atcatgttga atacgtgaaa aatgccaata tgaccagtaa agatagttat 1440
attgcaccgt taataatgca gaataagact gttacaaaaa aaatcttggc agatgcagga 1500
tttcaagtac cagttggtga agaatttatc tctctagaac aagcacaaca agcctattta 1560
gattatgaaa ataaagcttt tgtaattaaa cctaaaacaa caaattatgg aattggaatt 1620
acaattttta aacatggcgc ttcgttagca gattttacat tagcattgga attagctttt 1680
aaagaagatc aagtggtaat cattgaagaa tttttagaag gaactgaata tcgcttcttt 1740
gtattagatg gagaagtaaa agctatttta ttacggatac ctgccaatgt tatcggagat 1800
ggcttgcata cagtagaaga attgattatt gaaaaaaatc ttgacccatt aagaggaata 1860
ggacatcgaa aacctttaga agcgattcaa ttagggaaat tagaacaatt aatgttaaaa 1920
gaacaagcat taatctctgt ctctattcct aaaaaagatc agtttgttta tttaagaaaa 1980
aattcaaata ttagtactgg tggagattcc atcgatgtta cagatgaatt taatgaaagc 2040
tataaaaaat tggcagttga agccgttcag gctttggggg ctaaaatttg tggcattgat 2100
tttattcttt cagatgagaa aaaacctata aataaaaata gcaaaagcta tggtattatt 2160
gaggcaaatt tcaatccagc aatgtatatg catatctatc catataaagg aaaaggtagg 2220
ccgcttacaa tggaagtatt aaaatttctc tatccggagt taaatgaaca ataa 2274
<210> 2
<211> 757
<212> PRT
<213> Melissococcus plutonius ATCC 35311
<400> 2
Met Asn Phe Lys Ser Leu Leu Gln Gln Lys Lys Val Lys Pro Tyr Ile
1 5 10 15
Leu Ser Ala Arg Phe Gly Leu Glu Lys Glu Ser Gln Arg Ser Lys Ile
20 25 30
Asp Gly Arg Leu Ala Thr Thr Glu His Pro Lys Val Leu Gly Asn Arg
35 40 45
Ser Tyr His Pro Tyr Ile Gln Thr Asp Phe Ser Glu Thr Gln Ile Glu
50 55 60
Leu Ile Thr Pro Val Ala Asp Ser Ile Asp Glu Val Ser Arg Tyr Leu
65 70 75 80
Ser Ala Ile Gln Glu Val Ala Tyr Arg Ser Leu Glu Lys Glu Glu Ile
85 90 95
Leu Trp Pro Leu Ser Met Pro Pro Lys Leu Pro Glu Lys Asp Glu Glu
100 105 110
Ile Lys Ile Ala Lys Leu Asp Asp Phe Glu Glu Thr Leu Tyr Arg Gln
115 120 125
Tyr Leu Ala Lys Lys Tyr Gly Lys Arg Lys Gln Met Ile Ser Gly Ile
130 135 140
His Phe Asn Phe Glu Tyr Ser Ile Asp Leu Ile Gln His Met Phe Asn
145 150 155 160
Glu Gln Ser Glu Phe Glu Lys Ile Glu Glu Phe Lys Asn Thr Leu Tyr
165 170 175
Met Lys Ile Ala Arg Asn Tyr Leu Arg Tyr Arg Trp Leu Ile Thr Tyr
180 185 190
Leu Phe Gly Ala Ser Pro Val Cys Glu Lys Gly Tyr Phe Thr Glu Gln
195 200 205
Asp Lys Ser Leu Asn Glu Pro Val Arg Ser Ile Arg Asn Ser Ser Phe
210 215 220
Gly Tyr Thr Asn Glu Glu Val Ala Ala Val Ser Tyr Ala Ser Leu Lys
225 230 235 240
Asn Tyr Leu Glu Asp Ile His Arg Leu Val Glu Asn Gly Ile Leu Ala
245 250 255
Lys Glu Lys Glu Phe Tyr Ser Pro Val Arg Leu Arg Gly Gly Lys Gln
260 265 270
Ile Ser Asp Leu Cys His Thr Gly Ile Arg Tyr Ile Glu Leu Arg Asn
275 280 285
Leu Asp Leu Asn Pro Phe Thr Ser Leu Gly Ile Asp Glu Asp Thr Leu
290 295 300
Arg Phe Leu His Ile Phe Leu Leu Tyr Met Leu Trp Thr Glu Glu Leu
305 310 315 320
Glu Thr Pro Asp Glu Trp Ile Met Thr Gly Asn Leu Val Asn Asn Gln
325 330 335
Ile Ala Leu Met His Pro Phe Gln Ser Ile Asn Leu Leu Ser Glu Gly
340 345 350
Asp Arg Ile Phe Arg Glu Met Phe Glu Met Leu Asp Glu Leu Glu Leu
355 360 365
Val Glu Glu Lys Lys Leu Val Asp Met Tyr Tyr Gln Gln Leu Arg Ala
370 375 380
Pro Glu Thr Thr Ile Ala Gly Lys Met Trp Thr Ile Ile Gln Glu Asn
385 390 395 400
Ser Asn Lys Glu Leu Gly Ile Ile Phe Gly Ser Glu Tyr Gln Ala Ala
405 410 415
Ile Leu Asn Glu Pro Tyr Gln Leu Thr Gly Phe Gln Gln Met Glu Leu
420 425 430
Ser Thr Gln Ser Phe Leu Phe Asp Thr Ile Gln Lys Gly Ile Ala Phe
435 440 445
Glu Ile Leu Asp Glu Gln Glu Gln Phe Leu Lys Leu Thr His Lys Asn
450 455 460
His Val Glu Tyr Val Lys Asn Ala Asn Met Thr Ser Lys Asp Ser Tyr
465 470 475 480
Ile Ala Pro Leu Ile Met Gln Asn Lys Thr Val Thr Lys Lys Ile Leu
485 490 495
Ala Asp Ala Gly Phe Gln Val Pro Val Gly Glu Glu Phe Ile Ser Leu
500 505 510
Glu Gln Ala Gln Gln Ala Tyr Leu Asp Tyr Glu Asn Lys Ala Phe Val
515 520 525
Ile Lys Pro Lys Thr Thr Asn Tyr Gly Ile Gly Ile Thr Ile Phe Lys
530 535 540
His Gly Ala Ser Leu Ala Asp Phe Thr Leu Ala Leu Glu Leu Ala Phe
545 550 555 560
Lys Glu Asp Gln Val Val Ile Ile Glu Glu Phe Leu Glu Gly Thr Glu
565 570 575
Tyr Arg Phe Phe Val Leu Asp Gly Glu Val Lys Ala Ile Leu Leu Arg
580 585 590
Ile Pro Ala Asn Val Ile Gly Asp Gly Leu His Thr Val Glu Glu Leu
595 600 605
Ile Ile Glu Lys Asn Leu Asp Pro Leu Arg Gly Ile Gly His Arg Lys
610 615 620
Pro Leu Glu Ala Ile Gln Leu Gly Lys Leu Glu Gln Leu Met Leu Lys
625 630 635 640
Glu Gln Ala Leu Ile Ser Val Ser Ile Pro Lys Lys Asp Gln Phe Val
645 650 655
Tyr Leu Arg Lys Asn Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp
660 665 670
Val Thr Asp Glu Phe Asn Glu Ser Tyr Lys Lys Leu Ala Val Glu Ala
675 680 685
Val Gln Ala Leu Gly Ala Lys Ile Cys Gly Ile Asp Phe Ile Leu Ser
690 695 700
Asp Glu Lys Lys Pro Ile Asn Lys Asn Ser Lys Ser Tyr Gly Ile Ile
705 710 715 720
Glu Ala Asn Phe Asn Pro Ala Met Tyr Met His Ile Tyr Pro Tyr Lys
725 730 735
Gly Lys Gly Arg Pro Leu Thr Met Glu Val Leu Lys Phe Leu Tyr Pro
740 745 750
Glu Leu Asn Glu Gln
755
<210> 3
<211> 757
<212> PRT
<213> Artificial
<220>
<223> The Gln residue at position 128 of the parent has been changed to
Arg.
<400> 3
Met Asn Phe Lys Ser Leu Leu Gln Gln Lys Lys Val Lys Pro Tyr Ile
1 5 10 15
Leu Ser Ala Arg Phe Gly Leu Glu Lys Glu Ser Gln Arg Ser Lys Ile
20 25 30
Asp Gly Arg Leu Ala Thr Thr Glu His Pro Lys Val Leu Gly Asn Arg
35 40 45
Ser Tyr His Pro Tyr Ile Gln Thr Asp Phe Ser Glu Thr Gln Ile Glu
50 55 60
Leu Ile Thr Pro Val Ala Asp Ser Ile Asp Glu Val Ser Arg Tyr Leu
65 70 75 80
Ser Ala Ile Gln Glu Val Ala Tyr Arg Ser Leu Glu Lys Glu Glu Ile
85 90 95
Leu Trp Pro Leu Ser Met Pro Pro Lys Leu Pro Glu Lys Asp Glu Glu
100 105 110
Ile Lys Ile Ala Lys Leu Asp Asp Phe Glu Glu Thr Leu Tyr Arg Arg
115 120 125
Tyr Leu Ala Lys Lys Tyr Gly Lys Arg Lys Gln Met Ile Ser Gly Ile
130 135 140
His Phe Asn Phe Glu Tyr Ser Ile Asp Leu Ile Gln His Met Phe Asn
145 150 155 160
Glu Gln Ser Glu Phe Glu Lys Ile Glu Glu Phe Lys Asn Thr Leu Tyr
165 170 175
Met Lys Ile Ala Arg Asn Tyr Leu Arg Tyr Arg Trp Leu Ile Thr Tyr
180 185 190
Leu Phe Gly Ala Ser Pro Val Cys Glu Lys Gly Tyr Phe Thr Glu Gln
195 200 205
Asp Lys Ser Leu Asn Glu Pro Val Arg Ser Ile Arg Asn Ser Ser Phe
210 215 220
Gly Tyr Thr Asn Glu Glu Val Ala Ala Val Ser Tyr Ala Ser Leu Lys
225 230 235 240
Asn Tyr Leu Glu Asp Ile His Arg Leu Val Glu Asn Gly Ile Leu Ala
245 250 255
Lys Glu Lys Glu Phe Tyr Ser Pro Val Arg Leu Arg Gly Gly Lys Gln
260 265 270
Ile Ser Asp Leu Cys His Thr Gly Ile Arg Tyr Ile Glu Leu Arg Asn
275 280 285
Leu Asp Leu Asn Pro Phe Thr Ser Leu Gly Ile Asp Glu Asp Thr Leu
290 295 300
Arg Phe Leu His Ile Phe Leu Leu Tyr Met Leu Trp Thr Glu Glu Leu
305 310 315 320
Glu Thr Pro Asp Glu Trp Ile Met Thr Gly Asn Leu Val Asn Asn Gln
325 330 335
Ile Ala Leu Met His Pro Phe Gln Ser Ile Asn Leu Leu Ser Glu Gly
340 345 350
Asp Arg Ile Phe Arg Glu Met Phe Glu Met Leu Asp Glu Leu Glu Leu
355 360 365
Val Glu Glu Lys Lys Leu Val Asp Met Tyr Tyr Gln Gln Leu Arg Ala
370 375 380
Pro Glu Thr Thr Ile Ala Gly Lys Met Trp Thr Ile Ile Gln Glu Asn
385 390 395 400
Ser Asn Lys Glu Leu Gly Ile Ile Phe Gly Ser Glu Tyr Gln Ala Ala
405 410 415
Ile Leu Asn Glu Pro Tyr Gln Leu Thr Gly Phe Gln Gln Met Glu Leu
420 425 430
Ser Thr Gln Ser Phe Leu Phe Asp Thr Ile Gln Lys Gly Ile Ala Phe
435 440 445
Glu Ile Leu Asp Glu Gln Glu Gln Phe Leu Lys Leu Thr His Lys Asn
450 455 460
His Val Glu Tyr Val Lys Asn Ala Asn Met Thr Ser Lys Asp Ser Tyr
465 470 475 480
Ile Ala Pro Leu Ile Met Gln Asn Lys Thr Val Thr Lys Lys Ile Leu
485 490 495
Ala Asp Ala Gly Phe Gln Val Pro Val Gly Glu Glu Phe Ile Ser Leu
500 505 510
Glu Gln Ala Gln Gln Ala Tyr Leu Asp Tyr Glu Asn Lys Ala Phe Val
515 520 525
Ile Lys Pro Lys Thr Thr Asn Tyr Gly Ile Gly Ile Thr Ile Phe Lys
530 535 540
His Gly Ala Ser Leu Ala Asp Phe Thr Leu Ala Leu Glu Leu Ala Phe
545 550 555 560
Lys Glu Asp Gln Val Val Ile Ile Glu Glu Phe Leu Glu Gly Thr Glu
565 570 575
Tyr Arg Phe Phe Val Leu Asp Gly Glu Val Lys Ala Ile Leu Leu Arg
580 585 590
Ile Pro Ala Asn Val Ile Gly Asp Gly Leu His Thr Val Glu Glu Leu
595 600 605
Ile Ile Glu Lys Asn Leu Asp Pro Leu Arg Gly Ile Gly His Arg Lys
610 615 620
Pro Leu Glu Ala Ile Gln Leu Gly Lys Leu Glu Gln Leu Met Leu Lys
625 630 635 640
Glu Gln Ala Leu Ile Ser Val Ser Ile Pro Lys Lys Asp Gln Phe Val
645 650 655
Tyr Leu Arg Lys Asn Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp
660 665 670
Val Thr Asp Glu Phe Asn Glu Ser Tyr Lys Lys Leu Ala Val Glu Ala
675 680 685
Val Gln Ala Leu Gly Ala Lys Ile Cys Gly Ile Asp Phe Ile Leu Ser
690 695 700
Asp Glu Lys Lys Pro Ile Asn Lys Asn Ser Lys Ser Tyr Gly Ile Ile
705 710 715 720
Glu Ala Asn Phe Asn Pro Ala Met Tyr Met His Ile Tyr Pro Tyr Lys
725 730 735
Gly Lys Gly Arg Pro Leu Thr Met Glu Val Leu Lys Phe Leu Tyr Pro
740 745 750
Glu Leu Asn Glu Gln
755
<210> 4
<211> 757
<212> PRT
<213> Artificial
<220>
<223> The Ala residue at position 256 of the parent has been changed to
Ser.
<400> 4
Met Asn Phe Lys Ser Leu Leu Gln Gln Lys Lys Val Lys Pro Tyr Ile
1 5 10 15
Leu Ser Ala Arg Phe Gly Leu Glu Lys Glu Ser Gln Arg Ser Lys Ile
20 25 30
Asp Gly Arg Leu Ala Thr Thr Glu His Pro Lys Val Leu Gly Asn Arg
35 40 45
Ser Tyr His Pro Tyr Ile Gln Thr Asp Phe Ser Glu Thr Gln Ile Glu
50 55 60
Leu Ile Thr Pro Val Ala Asp Ser Ile Asp Glu Val Ser Arg Tyr Leu
65 70 75 80
Ser Ala Ile Gln Glu Val Ala Tyr Arg Ser Leu Glu Lys Glu Glu Ile
85 90 95
Leu Trp Pro Leu Ser Met Pro Pro Lys Leu Pro Glu Lys Asp Glu Glu
100 105 110
Ile Lys Ile Ala Lys Leu Asp Asp Phe Glu Glu Thr Leu Tyr Arg Gln
115 120 125
Tyr Leu Ala Lys Lys Tyr Gly Lys Arg Lys Gln Met Ile Ser Gly Ile
130 135 140
His Phe Asn Phe Glu Tyr Ser Ile Asp Leu Ile Gln His Met Phe Asn
145 150 155 160
Glu Gln Ser Glu Phe Glu Lys Ile Glu Glu Phe Lys Asn Thr Leu Tyr
165 170 175
Met Lys Ile Ala Arg Asn Tyr Leu Arg Tyr Arg Trp Leu Ile Thr Tyr
180 185 190
Leu Phe Gly Ala Ser Pro Val Cys Glu Lys Gly Tyr Phe Thr Glu Gln
195 200 205
Asp Lys Ser Leu Asn Glu Pro Val Arg Ser Ile Arg Asn Ser Ser Phe
210 215 220
Gly Tyr Thr Asn Glu Glu Val Ala Ala Val Ser Tyr Ala Ser Leu Lys
225 230 235 240
Asn Tyr Leu Glu Asp Ile His Arg Leu Val Glu Asn Gly Ile Leu Ser
245 250 255
Lys Glu Lys Glu Phe Tyr Ser Pro Val Arg Leu Arg Gly Gly Lys Gln
260 265 270
Ile Ser Asp Leu Cys His Thr Gly Ile Arg Tyr Ile Glu Leu Arg Asn
275 280 285
Leu Asp Leu Asn Pro Phe Thr Ser Leu Gly Ile Asp Glu Asp Thr Leu
290 295 300
Arg Phe Leu His Ile Phe Leu Leu Tyr Met Leu Trp Thr Glu Glu Leu
305 310 315 320
Glu Thr Pro Asp Glu Trp Ile Met Thr Gly Asn Leu Val Asn Asn Gln
325 330 335
Ile Ala Leu Met His Pro Phe Gln Ser Ile Asn Leu Leu Ser Glu Gly
340 345 350
Asp Arg Ile Phe Arg Glu Met Phe Glu Met Leu Asp Glu Leu Glu Leu
355 360 365
Val Glu Glu Lys Lys Leu Val Asp Met Tyr Tyr Gln Gln Leu Arg Ala
370 375 380
Pro Glu Thr Thr Ile Ala Gly Lys Met Trp Thr Ile Ile Gln Glu Asn
385 390 395 400
Ser Asn Lys Glu Leu Gly Ile Ile Phe Gly Ser Glu Tyr Gln Ala Ala
405 410 415
Ile Leu Asn Glu Pro Tyr Gln Leu Thr Gly Phe Gln Gln Met Glu Leu
420 425 430
Ser Thr Gln Ser Phe Leu Phe Asp Thr Ile Gln Lys Gly Ile Ala Phe
435 440 445
Glu Ile Leu Asp Glu Gln Glu Gln Phe Leu Lys Leu Thr His Lys Asn
450 455 460
His Val Glu Tyr Val Lys Asn Ala Asn Met Thr Ser Lys Asp Ser Tyr
465 470 475 480
Ile Ala Pro Leu Ile Met Gln Asn Lys Thr Val Thr Lys Lys Ile Leu
485 490 495
Ala Asp Ala Gly Phe Gln Val Pro Val Gly Glu Glu Phe Ile Ser Leu
500 505 510
Glu Gln Ala Gln Gln Ala Tyr Leu Asp Tyr Glu Asn Lys Ala Phe Val
515 520 525
Ile Lys Pro Lys Thr Thr Asn Tyr Gly Ile Gly Ile Thr Ile Phe Lys
530 535 540
His Gly Ala Ser Leu Ala Asp Phe Thr Leu Ala Leu Glu Leu Ala Phe
545 550 555 560
Lys Glu Asp Gln Val Val Ile Ile Glu Glu Phe Leu Glu Gly Thr Glu
565 570 575
Tyr Arg Phe Phe Val Leu Asp Gly Glu Val Lys Ala Ile Leu Leu Arg
580 585 590
Ile Pro Ala Asn Val Ile Gly Asp Gly Leu His Thr Val Glu Glu Leu
595 600 605
Ile Ile Glu Lys Asn Leu Asp Pro Leu Arg Gly Ile Gly His Arg Lys
610 615 620
Pro Leu Glu Ala Ile Gln Leu Gly Lys Leu Glu Gln Leu Met Leu Lys
625 630 635 640
Glu Gln Ala Leu Ile Ser Val Ser Ile Pro Lys Lys Asp Gln Phe Val
645 650 655
Tyr Leu Arg Lys Asn Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp
660 665 670
Val Thr Asp Glu Phe Asn Glu Ser Tyr Lys Lys Leu Ala Val Glu Ala
675 680 685
Val Gln Ala Leu Gly Ala Lys Ile Cys Gly Ile Asp Phe Ile Leu Ser
690 695 700
Asp Glu Lys Lys Pro Ile Asn Lys Asn Ser Lys Ser Tyr Gly Ile Ile
705 710 715 720
Glu Ala Asn Phe Asn Pro Ala Met Tyr Met His Ile Tyr Pro Tyr Lys
725 730 735
Gly Lys Gly Arg Pro Leu Thr Met Glu Val Leu Lys Phe Leu Tyr Pro
740 745 750
Glu Leu Asn Glu Gln
755
<210> 5
<211> 757
<212> PRT
<213> Artificial
<220>
<223> The Leu residue at position 320 of the parent has been changed to
Lys.
<400> 5
Met Asn Phe Lys Ser Leu Leu Gln Gln Lys Lys Val Lys Pro Tyr Ile
1 5 10 15
Leu Ser Ala Arg Phe Gly Leu Glu Lys Glu Ser Gln Arg Ser Lys Ile
20 25 30
Asp Gly Arg Leu Ala Thr Thr Glu His Pro Lys Val Leu Gly Asn Arg
35 40 45
Ser Tyr His Pro Tyr Ile Gln Thr Asp Phe Ser Glu Thr Gln Ile Glu
50 55 60
Leu Ile Thr Pro Val Ala Asp Ser Ile Asp Glu Val Ser Arg Tyr Leu
65 70 75 80
Ser Ala Ile Gln Glu Val Ala Tyr Arg Ser Leu Glu Lys Glu Glu Ile
85 90 95
Leu Trp Pro Leu Ser Met Pro Pro Lys Leu Pro Glu Lys Asp Glu Glu
100 105 110
Ile Lys Ile Ala Lys Leu Asp Asp Phe Glu Glu Thr Leu Tyr Arg Gln
115 120 125
Tyr Leu Ala Lys Lys Tyr Gly Lys Arg Lys Gln Met Ile Ser Gly Ile
130 135 140
His Phe Asn Phe Glu Tyr Ser Ile Asp Leu Ile Gln His Met Phe Asn
145 150 155 160
Glu Gln Ser Glu Phe Glu Lys Ile Glu Glu Phe Lys Asn Thr Leu Tyr
165 170 175
Met Lys Ile Ala Arg Asn Tyr Leu Arg Tyr Arg Trp Leu Ile Thr Tyr
180 185 190
Leu Phe Gly Ala Ser Pro Val Cys Glu Lys Gly Tyr Phe Thr Glu Gln
195 200 205
Asp Lys Ser Leu Asn Glu Pro Val Arg Ser Ile Arg Asn Ser Ser Phe
210 215 220
Gly Tyr Thr Asn Glu Glu Val Ala Ala Val Ser Tyr Ala Ser Leu Lys
225 230 235 240
Asn Tyr Leu Glu Asp Ile His Arg Leu Val Glu Asn Gly Ile Leu Ala
245 250 255
Lys Glu Lys Glu Phe Tyr Ser Pro Val Arg Leu Arg Gly Gly Lys Gln
260 265 270
Ile Ser Asp Leu Cys His Thr Gly Ile Arg Tyr Ile Glu Leu Arg Asn
275 280 285
Leu Asp Leu Asn Pro Phe Thr Ser Leu Gly Ile Asp Glu Asp Thr Leu
290 295 300
Arg Phe Leu His Ile Phe Leu Leu Tyr Met Leu Trp Thr Glu Glu Lys
305 310 315 320
Glu Thr Pro Asp Glu Trp Ile Met Thr Gly Asn Leu Val Asn Asn Gln
325 330 335
Ile Ala Leu Met His Pro Phe Gln Ser Ile Asn Leu Leu Ser Glu Gly
340 345 350
Asp Arg Ile Phe Arg Glu Met Phe Glu Met Leu Asp Glu Leu Glu Leu
355 360 365
Val Glu Glu Lys Lys Leu Val Asp Met Tyr Tyr Gln Gln Leu Arg Ala
370 375 380
Pro Glu Thr Thr Ile Ala Gly Lys Met Trp Thr Ile Ile Gln Glu Asn
385 390 395 400
Ser Asn Lys Glu Leu Gly Ile Ile Phe Gly Ser Glu Tyr Gln Ala Ala
405 410 415
Ile Leu Asn Glu Pro Tyr Gln Leu Thr Gly Phe Gln Gln Met Glu Leu
420 425 430
Ser Thr Gln Ser Phe Leu Phe Asp Thr Ile Gln Lys Gly Ile Ala Phe
435 440 445
Glu Ile Leu Asp Glu Gln Glu Gln Phe Leu Lys Leu Thr His Lys Asn
450 455 460
His Val Glu Tyr Val Lys Asn Ala Asn Met Thr Ser Lys Asp Ser Tyr
465 470 475 480
Ile Ala Pro Leu Ile Met Gln Asn Lys Thr Val Thr Lys Lys Ile Leu
485 490 495
Ala Asp Ala Gly Phe Gln Val Pro Val Gly Glu Glu Phe Ile Ser Leu
500 505 510
Glu Gln Ala Gln Gln Ala Tyr Leu Asp Tyr Glu Asn Lys Ala Phe Val
515 520 525
Ile Lys Pro Lys Thr Thr Asn Tyr Gly Ile Gly Ile Thr Ile Phe Lys
530 535 540
His Gly Ala Ser Leu Ala Asp Phe Thr Leu Ala Leu Glu Leu Ala Phe
545 550 555 560
Lys Glu Asp Gln Val Val Ile Ile Glu Glu Phe Leu Glu Gly Thr Glu
565 570 575
Tyr Arg Phe Phe Val Leu Asp Gly Glu Val Lys Ala Ile Leu Leu Arg
580 585 590
Ile Pro Ala Asn Val Ile Gly Asp Gly Leu His Thr Val Glu Glu Leu
595 600 605
Ile Ile Glu Lys Asn Leu Asp Pro Leu Arg Gly Ile Gly His Arg Lys
610 615 620
Pro Leu Glu Ala Ile Gln Leu Gly Lys Leu Glu Gln Leu Met Leu Lys
625 630 635 640
Glu Gln Ala Leu Ile Ser Val Ser Ile Pro Lys Lys Asp Gln Phe Val
645 650 655
Tyr Leu Arg Lys Asn Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp
660 665 670
Val Thr Asp Glu Phe Asn Glu Ser Tyr Lys Lys Leu Ala Val Glu Ala
675 680 685
Val Gln Ala Leu Gly Ala Lys Ile Cys Gly Ile Asp Phe Ile Leu Ser
690 695 700
Asp Glu Lys Lys Pro Ile Asn Lys Asn Ser Lys Ser Tyr Gly Ile Ile
705 710 715 720
Glu Ala Asn Phe Asn Pro Ala Met Tyr Met His Ile Tyr Pro Tyr Lys
725 730 735
Gly Lys Gly Arg Pro Leu Thr Met Glu Val Leu Lys Phe Leu Tyr Pro
740 745 750
Glu Leu Asn Glu Gln
755
Claims (4)
1.一种谷胱甘肽合成酶突变体，其特征在于，由序列表中的序列2经点突变所得，所述点突变为选自第128位、第256位及第320位的至少一个突变，并且以三磷酸腺苷二钠盐(ATP)、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物其具有比亲本高出至少50%的谷胱甘肽合成酶催化活性；
所述点突变具体为：序列2的第128位的谷氨酰胺(Q)突变为精氨酸(R)；
序列2的第256位的丙氨酸(A)突变为丝氨酸(S)；序列2的第320位的亮氨酸(L)突变为赖氨酸(K)。
2.根据权利要求1所述的谷胱甘肽合成酶突变体，其特征在于，所述谷胱甘肽合成酶突变体为序列表中序列3或序列4或序列5所示的氨基酸序列。
3.一种编码谷胱甘肽合成酶突变体的基因，其特征在于，其含有编码权利要求1-2任一项所述的谷胱甘肽合成酶突变体的核苷酸序列。
4.根据权利要求1-2任一项所述的谷胱甘肽合成酶突变体的应用，其特征在于，所述谷胱甘肽合成酶突变体应用于以三磷酸腺苷二钠盐、L-谷氨酸钠、L-半胱氨酸和甘氨酸为底物制备谷胱甘肽。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410506648.6A CN104328092B (zh) | 2014-09-28 | 2014-09-28 | 一种谷胱甘肽合成酶突变体、编码基因及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410506648.6A CN104328092B (zh) | 2014-09-28 | 2014-09-28 | 一种谷胱甘肽合成酶突变体、编码基因及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104328092A CN104328092A (zh) | 2015-02-04 |
| CN104328092B true CN104328092B (zh) | 2017-03-15 |
Family
ID=52402899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410506648.6A Active CN104328092B (zh) | 2014-09-28 | 2014-09-28 | 一种谷胱甘肽合成酶突变体、编码基因及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104328092B (zh) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238797B (zh) * | 2015-10-23 | 2018-10-02 | 山东金城生物药业有限公司 | 一种无乳链球菌的gshF基因的突变基因及其应用 |
| US20190330600A1 (en) * | 2016-12-22 | 2019-10-31 | Dsm Ip Assets B.V. | Glutathione reductase |
| CN107267471B (zh) * | 2017-07-05 | 2020-07-14 | 湖南福来格生物技术有限公司 | 双功能谷胱甘肽合成酶突变体、核苷酸序列及其制备方法和应用 |
| CN112280755B (zh) * | 2020-11-06 | 2023-03-14 | 深圳瑞德林生物技术有限公司 | 一种突变酶及其应用和酶催化法制备三胜肽的工艺 |
| CN114317474B (zh) * | 2021-11-12 | 2022-10-11 | 广东省农业科学院农业质量标准与监测技术研究所 | 一种酶活提高的谷胱甘肽s-转移酶突变体及其应用 |
| CN115093470B (zh) * | 2022-06-30 | 2023-03-24 | 广州市乾相生物科技有限公司 | 内含肽Mtu RecA突变体及其在生产谷胱甘肽GSH中的应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1592780A (zh) * | 2001-11-26 | 2005-03-09 | 味之素株式会社 | 产生γ－谷氨酰半胱氨酸的酵母 |
| CN101407768A (zh) * | 2008-11-18 | 2009-04-15 | 常熟理工学院 | 一株酿酒酵母突变菌株及其在发酵生产谷胱甘肽中的应用 |
| WO2013054447A1 (ja) * | 2011-10-14 | 2013-04-18 | 味の素株式会社 | γ‐Ｇｌｕ‐Ｘ‐Ｇｌｙまたはその塩の製造方法、および変異型グルタチオン合成酵素 |
-
2014
- 2014-09-28 CN CN201410506648.6A patent/CN104328092B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1592780A (zh) * | 2001-11-26 | 2005-03-09 | 味之素株式会社 | 产生γ－谷氨酰半胱氨酸的酵母 |
| CN101407768A (zh) * | 2008-11-18 | 2009-04-15 | 常熟理工学院 | 一株酿酒酵母突变菌株及其在发酵生产谷胱甘肽中的应用 |
| WO2013054447A1 (ja) * | 2011-10-14 | 2013-04-18 | 味の素株式会社 | γ‐Ｇｌｕ‐Ｘ‐Ｇｌｙまたはその塩の製造方法、および変異型グルタチオン合成酵素 |
Non-Patent Citations (5)
| Title |
|---|
| A Medicago truncatula Homoglutathione Synthetase Is Derived from Glutathione Synthetase by Gene Duplication;Pierre Frendo等;《Plant Physiol》;20011231;第126卷;1706-1715 * |
| BAK20791.1;Okumura, K.等;《GenBank》;20120822;全文 * |
| Identification of a putative flexible loop in Arabidopsis glutathione synthetase;Chang-Lin WANG等;《Biochemical Journal》;19971231;241-244 * |
| Role of Cysteine Residues in Glutathione Synthetase from Escherichia coli B;Hiroaki Kato等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;19881231;第263卷;11646-11651 * |
| site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the r-L-glutamyl-L-cysteine-binding site;takane hara等;《protein Eng》;19951231;第8卷(第7期);711-716 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104328092A (zh) | 2015-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104328092B (zh) | 一种谷胱甘肽合成酶突变体、编码基因及应用 | |
| KR102132381B1 (ko) | 아스로박터 글로비포미스에 의해 생산되는 케토오스 3-에피머라제 | |
| CN105543201B (zh) | 一种头孢菌素c酰化酶突变体 | |
| CN113151198B (zh) | 一种γ-谷酰胺甲胺合成酶的突变体，其编码基因、氨基酸序列及其应用 | |
| CN112852788A (zh) | 一种碱性底物选择性提高的枯草蛋白酶e突变体及其应用 | |
| CN101134948B (zh) | S-腺苷甲硫氨酸合成酶突变体、编码该突变体的dna以及突变体的应用 | |
| CN112437811B (zh) | 一种重组nad合成酶及其基因和应用 | |
| CN114395541B (zh) | 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 | |
| CN104212757A (zh) | 利用大肠杆菌产γ-谷氨酰甲胺合成酶高效生产L-茶氨酸的方法 | |
| CN103103234A (zh) | 利用固定化酶合成烟酰胺腺嘌呤二核苷酸(nad)的方法 | |
| CN113604445A (zh) | 一种酪氨酸酶及其制备与应用 | |
| CN109022405A (zh) | 一种冷适应性褐藻胶裂解酶AlgA5及其应用 | |
| EP1760148A1 (en) | AMP DEAMINASE ORIGINATING IN STREPTOMYCES&lt;i&gt; &lt;/i&gt;AND UTILIZATION THEREOF | |
| CN102766611B (zh) | 硫化叶菌病毒脱氧尿苷焦磷酸酶和编码这种酶的多核苷酸 | |
| CN103881997B (zh) | 燕窝酸醛缩酶突变体及其编码基因和应用 | |
| EP1960510B1 (en) | Ammonia-specific 5'-xmp aminase mutant | |
| CN109563504A (zh) | 核苷酶 | |
| Lv et al. | A novel approach to cloning and expression of human thymidylate synthase | |
| CN110951717A (zh) | 一种l-阿拉伯糖异构酶异构体及其应用 | |
| Chang et al. | Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli | |
| CN111057698B (zh) | L-阿拉伯糖异构酶、突变体及其应用 | |
| KR0184755B1 (ko) | 재조합 내열성 티로신 페놀리아제 및 이를 이용한 엘-도파의 제조방법 | |
| CN107557348A (zh) | 头孢菌素羟化酶突变体、编码该突变体的dna、使用该突变体的方法及其应用 | |
| CN119162140A (zh) | 一种糖基转移酶突变体及其在红景天苷合成中的应用 | |
| CN102433313B (zh) | 一种l-atc水解酶和编码基因及其重组表达蛋白质的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |