CN112175924A - 一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 - Google Patents
一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 Download PDFInfo
- Publication number
- CN112175924A CN112175924A CN202011089477.3A CN202011089477A CN112175924A CN 112175924 A CN112175924 A CN 112175924A CN 202011089477 A CN202011089477 A CN 202011089477A CN 112175924 A CN112175924 A CN 112175924A
- Authority
- CN
- China
- Prior art keywords
- carboxypeptidase
- phenyl
- benzidine
- stirring
- filtering
- 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.)
- Pending
Links
- 102000000496 Carboxypeptidases A Human genes 0.000 title claims abstract description 24
- 108010080937 Carboxypeptidases A Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 230000000694 effects Effects 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 15
- FZERHIULMFGESH-UHFFFAOYSA-N N-phenylacetamide Chemical compound CC(=O)NC1=CC=CC=C1 FZERHIULMFGESH-UHFFFAOYSA-N 0.000 claims description 14
- 239000000047 product Substances 0.000 claims description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 229960001413 acetanilide Drugs 0.000 claims description 7
- PKJBWOWQJHHAHG-UHFFFAOYSA-N 1-bromo-4-phenylbenzene Chemical group C1=CC(Br)=CC=C1C1=CC=CC=C1 PKJBWOWQJHHAHG-UHFFFAOYSA-N 0.000 claims description 6
- -1 N-phenyl-4-acetylbenzidine Chemical compound 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 3
- 239000004927 clay Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000004973 liquid crystal related substance Substances 0.000 description 14
- 239000000463 material Substances 0.000 description 10
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 6
- 238000006069 Suzuki reaction reaction Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 3
- 239000004305 biphenyl Substances 0.000 description 3
- 235000010290 biphenyl Nutrition 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 229910052763 palladium Inorganic materials 0.000 description 3
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- SPSSULHKWOKEEL-UHFFFAOYSA-N 2,4,6-trinitrotoluene Chemical compound CC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O SPSSULHKWOKEEL-UHFFFAOYSA-N 0.000 description 1
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 1
- YNOCMHZSWJMGBB-GCJQMDKQSA-N Ala-Thr-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O YNOCMHZSWJMGBB-GCJQMDKQSA-N 0.000 description 1
- VEAIMHJZTIDCIH-KKUMJFAQSA-N Arg-Phe-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VEAIMHJZTIDCIH-KKUMJFAQSA-N 0.000 description 1
- OVQJAKFLFTZDNC-GUBZILKMSA-N Arg-Pro-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O OVQJAKFLFTZDNC-GUBZILKMSA-N 0.000 description 1
- UGXVKHRDGLYFKR-CIUDSAMLSA-N Asn-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(N)=O UGXVKHRDGLYFKR-CIUDSAMLSA-N 0.000 description 1
- OPEPUCYIGFEGSW-WDSKDSINSA-N Asn-Gly-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OPEPUCYIGFEGSW-WDSKDSINSA-N 0.000 description 1
- QCLHLXDWRKOHRR-GUBZILKMSA-N Asp-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N QCLHLXDWRKOHRR-GUBZILKMSA-N 0.000 description 1
- UZFHNLYQWMGUHU-DCAQKATOSA-N Asp-Lys-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UZFHNLYQWMGUHU-DCAQKATOSA-N 0.000 description 1
- JSYULGSPLTZDHM-NRPADANISA-N Gln-Ala-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O JSYULGSPLTZDHM-NRPADANISA-N 0.000 description 1
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 description 1
- VOORMNJKNBGYGK-YUMQZZPRSA-N Glu-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N VOORMNJKNBGYGK-YUMQZZPRSA-N 0.000 description 1
- ITVBKCZZLJUUHI-HTUGSXCWSA-N Glu-Phe-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ITVBKCZZLJUUHI-HTUGSXCWSA-N 0.000 description 1
- CAQXJMUDOLSBPF-SUSMZKCASA-N Glu-Thr-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAQXJMUDOLSBPF-SUSMZKCASA-N 0.000 description 1
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 1
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 1
- XUORRGAFUQIMLC-STQMWFEESA-N Gly-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN)O XUORRGAFUQIMLC-STQMWFEESA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 1
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 1
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 1
- OQDLKDUVMTUPPG-AVGNSLFASA-N His-Leu-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OQDLKDUVMTUPPG-AVGNSLFASA-N 0.000 description 1
- FBCURAVMSXNOLP-JYJNAYRXSA-N His-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N FBCURAVMSXNOLP-JYJNAYRXSA-N 0.000 description 1
- JGFWUKYIQAEYAH-DCAQKATOSA-N His-Ser-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JGFWUKYIQAEYAH-DCAQKATOSA-N 0.000 description 1
- JUCZDDVZBMPKRT-IXOXFDKPSA-N His-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O JUCZDDVZBMPKRT-IXOXFDKPSA-N 0.000 description 1
- WUEIUSDAECDLQO-NAKRPEOUSA-N Ile-Ala-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)O)N WUEIUSDAECDLQO-NAKRPEOUSA-N 0.000 description 1
- BGZIJZJBXRVBGJ-SXTJYALSSA-N Ile-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N BGZIJZJBXRVBGJ-SXTJYALSSA-N 0.000 description 1
- DFFTXLCCDFYRKD-MBLNEYKQSA-N Ile-Gly-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N DFFTXLCCDFYRKD-MBLNEYKQSA-N 0.000 description 1
- TVYWVSJGSHQWMT-AJNGGQMLSA-N Ile-Leu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N TVYWVSJGSHQWMT-AJNGGQMLSA-N 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 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 1
- KSZCCRIGNVSHFH-UWVGGRQHSA-N Leu-Arg-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O KSZCCRIGNVSHFH-UWVGGRQHSA-N 0.000 description 1
- PTRKPHUGYULXPU-KKUMJFAQSA-N Leu-Phe-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O PTRKPHUGYULXPU-KKUMJFAQSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- SUYRAPCRSCCPAK-VFAJRCTISA-N Leu-Trp-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SUYRAPCRSCCPAK-VFAJRCTISA-N 0.000 description 1
- QZONCCHVHCOBSK-YUMQZZPRSA-N Lys-Gly-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O QZONCCHVHCOBSK-YUMQZZPRSA-N 0.000 description 1
- KYXDADPHSNFWQX-VEVYYDQMSA-N Met-Thr-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O KYXDADPHSNFWQX-VEVYYDQMSA-N 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- DDYIRGBOZVKRFR-AVGNSLFASA-N Phe-Asp-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N DDYIRGBOZVKRFR-AVGNSLFASA-N 0.000 description 1
- BAONJAHBAUDJKA-BZSNNMDCSA-N Phe-Tyr-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=CC=C1 BAONJAHBAUDJKA-BZSNNMDCSA-N 0.000 description 1
- GBRUQFBAJOKCTF-DCAQKATOSA-N Pro-His-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O GBRUQFBAJOKCTF-DCAQKATOSA-N 0.000 description 1
- SEZGGSHLMROBFX-CIUDSAMLSA-N Pro-Ser-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O SEZGGSHLMROBFX-CIUDSAMLSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- JZRYFUGREMECBH-XPUUQOCRSA-N Ser-Val-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O JZRYFUGREMECBH-XPUUQOCRSA-N 0.000 description 1
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 1
- SCSVNSNWUTYSFO-WDCWCFNPSA-N Thr-Lys-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O SCSVNSNWUTYSFO-WDCWCFNPSA-N 0.000 description 1
- XJFXZQKJQGYFMM-GUBZILKMSA-N Val-Cys-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)O)N XJFXZQKJQGYFMM-GUBZILKMSA-N 0.000 description 1
- MNSSBIHFEUUXNW-RCWTZXSCSA-N Val-Thr-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N MNSSBIHFEUUXNW-RCWTZXSCSA-N 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 239000012769 display material Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012156 elution solvent Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 239000000015 trinitrotoluene Substances 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17001—Carboxypeptidase A (3.4.17.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及一种羧肽酶A及其在制备N‑苯基‑4‑联苯胺中的应用，属于生物化学合成领域，具有SEQIDNO：1所示的羧肽酶A氨基酸序列，羧肽酶A酶活为90.6U/mL。在N‑苯基‑4‑联苯胺合成的过程中加入羧肽酶A，与传统的单独化学法相比更加绿色环保，三废排放少，合成的N‑苯基‑4‑联苯胺产率和纯度高于化学合成法。
Description
技术领域
本发明属于生物化学合成领域，具体涉及一种羧肽酶A及其在制备N-苯基-4-联苯胺中的应用。
背景技术
苯基-4-联苯胺是一种重要的电子化学品，常用于OLED液晶显示材料。液晶透镜中常用到的高双折射率液晶材料一般为有特定结构的小分子混合物，高双折射率液晶材料的常见分子结构，高双折射率液晶材料中一类常用的联苯类分子结构，其化学结构对温度和紫外光等相对稳定，且其对提升液晶材料的双折射率和清亮点具有显著的效果。因此被广泛应用于液晶材料中，也是研究最多的一类液晶材料。
对于联苯类液晶分子结构的合成，现有技术中一般采用suzuki反应(铃木反应)制备此类单体液晶。suzuki反应在此类结构制备中具有一些不可替代的优势，如反应产率较高，条件相对温和，反应步骤简单等。但是，suzuki反应也有一些不可避免的缺点，如催化剂金属钯不易去除，残留的金属钯将会较大地降低液晶材料的电阻率，并影响其在显示器件中的响应电压；又如反应底物硼酸在suzuki反应条件下会产生自身偶合的副产物，使后处理纯化变得复杂。目前用于suzuki后处理的常用纯化方法是柱层析色谱法，即利用产物与副产物的不同极性来对产物和副产物进行分离，从而达到纯化目的，但由于柱层析色谱法在制备量变大时，所需花费的准备时间，分离时间和洗脱溶剂大大增加，并且相对应的柱层析柱子也要加大，非常不利于工业化的大量生产。
因此需提供一个简便有效又绿色环保的方法来制备联苯类液晶材料，简化产物提纯方法，并且保证产率和纯度较高，便于规模化大生产。
发明内容
基于上述背景技术，本发明拟提供一种N-苯基-4-联苯胺的蛋白酶生物合成新路线。
本发明是通过以下技术方案实现的：
一种羧肽酶A，具有SEQIDNO：1所示的羧肽酶A氨基酸序列。
所述的羧肽酶A氨基酸序列：在N-苯基-4-联苯胺制备过程中的应用。
所述的N-苯基-4-联苯胺制备过程，包括以下步骤：
(1)将乙酰苯胺、4-溴联苯、ZnCl2及pH＝9.5的磷酸二氢钾缓冲溶液和甲醇加入到反应瓶中，搅拌全部溶解后，再加入酶活为65ug/g的羧肽酶A，控制温度28-32℃，并在此温度下搅拌5-6h，经HPLC检验4-溴联苯含量小于0.2％后停止反应，除甲醇后得到N-苯基-4-乙酰基联苯胺粗品，粗品用乙酸乙酯萃取3次，蒸干乙酸乙酯后得到白色的产品；
(2)将以上产品溶解于的醇类溶剂中，再加入氢氧化钾,升温至80-85℃，保温反应5-6h，直至N-苯基-4-乙酰基联苯胺通过HPLC检测含量小于0.10％时停止反应，降温至50-60℃后，趁热倒入冰水混合物中，继续搅拌1-2h，过滤，收集白色固体；
(3)将白色固体加入甲苯中，并加入50g活性物质，升温至60-65℃，搅拌2-3h，然后趁热过滤，收集滤液，浓缩出甲苯，加入目标产品晶种，在25摄氏度下搅拌1-2h，析出晶体，过滤、干燥既得N-苯基-4-联苯胺。
作为优选，步骤(3)中的活性物质为活性白土、活性炭、硅藻土中的一种。
作为优选，步骤(1)中所述羧肽酶A的加入量占乙酰苯胺质量的1/500-1/1000。
反应过程为：
本发明的有益效果
(1)与纯化学合成法相比更加绿色环保，三废排放少。
(2)合成的N-苯基-4-联苯胺产率和纯度高于化学合成法。
具体实施方式
以下实施例用于进一步阐述本发明，但不应理解为对本发明权利要求书请求保护范围的限制。
实施例1
羧肽酶A的制备：
称取10g胰酶，用500ml 0.02M磷酸缓冲液(pH7)搅拌提取1h，10,000r.p.m离心10min，取上清液，上预先用0.02M,pH7磷酸缓冲液平衡好的DEAE-52纤维素柱(柱体积200ml)。然后用上述磷酸缓冲液洗涤不吸咐的部分，再用含有0.4M NaCl的上述磷酸缓冲液梯度洗脱。收集洗脱液，得纯净的羧肽酶A。
实施例2
羧肽酶A活力的测定-三硝基苯磺酸(TNBS)法：
底物为苄氧羰酰-甘氨酰-苯丙氨酸，测定缓冲液是0.05M，pH8Tris-HC1溶液。反应温度为25℃。3ml反应系统中加入20ul酶液后，反应10min，然后加入4％NaHC03 1ml，0.1％TNBS 1ml，37℃保温30min，加入1ml 1N HCl中止显色反应，于340nm测定光密度值，测定浓度为3X10-3，每0.01个光密度增值定为一个酶活力单位。测得羧肽酶A酶活为90.6U/mL。
实施例3
将乙酰苯胺(142.00g，1.05mol)、4-溴联苯(233.10g，1.00mol)、0.5gZnCl2及500ml的5mM磷酸二氢钾缓冲溶液(pH＝9.5)和500ml甲醇加入到2000ml反应瓶中，搅拌全部溶解后，再加入酶活为65ug/g的羧肽酶A，羧肽酶A加入量为乙酰苯胺质量的1/500，控制温度30℃，并在此温度下搅拌5-6h，经HPLC检验4-溴联苯含量小于0.2％后停止反应，整除甲醇后得到N-苯基-4-乙酰基联苯胺粗品，粗品用1000ml乙酸乙酯萃取3次，蒸干乙酸乙酯后得到白色的产品。
将以上产品溶解于600ml的丙醇中，再加入氢氧化钾(60.00g，1.07mol),升温至80-85℃，保温反应5-6h直至N-苯基-4-乙酰基联苯胺通过HPLC检测含量小于0.10％时为止，降温至50-60℃后，趁热倒入2000ml的冰水中，继续搅拌1-2h，过滤，收集白色固体。
将白色固体加入至500ml甲苯中，并加入50g活性白土，升温至60-65℃，搅拌2-3h，然后趁热过滤，收集滤液，并浓缩出甲苯300ml，加入目标产品晶种1.00g，再常温下搅拌1-2h，有大量晶体析出，过滤、干燥得目标产品精品235.25g，收率95.90％，HPLC纯度为99.30％。
对比例1
本实施例与实施例1的不同在于将羧肽酶A替换为金属钯催化剂，金属钯催化剂的加入总质量为乙酰苯胺质量的1/700，其余同实施例1。
得到精品206.23g收率为84％，HPLC纯度为97.23％。
序列表
<110> 江西邦泰绿色生物合成生态产业园发展有限公司
<120> 一种羧肽酶A及其在制备N-苯基-4-联苯胺中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 141
<212> PRT
<213> 人工序列(Synthetic sequence)
<400> 2
Met Thr Asp Ala Pro Lys Glu Glu Val Ile Ala Met Ile Asp Ile Lys
1 5 10 15
Glu Val Val His Thr Lys Val Thr Arg Asp Lys Arg Glu Gly Met Gly
20 25 30
Val Cys Val Glu Phe Thr Asn Asp Leu Phe Asp Glu Lys Gly Asn Lys
35 40 45
Ile Gly Thr Ser Val Gly Thr Gly Val Leu Ser Thr Asn Gly Glu Gly
50 55 60
Asp Glu His Arg Phe Gln Leu Phe Ser Ala Thr Asp His Leu Glu Asp
65 70 75 80
Gly Thr Ile Leu Trp Thr Gly Ala Cys Pro His Asp Pro Ser Gln Ile
85 90 95
Thr Lys Glu His Ser Val Gln Ala Val Gly Thr Ser Gly Arg Tyr Leu
100 105 110
Gly Lys Thr Gly Thr Arg His Phe Gln Phe Tyr Asp Arg Pro Asp Gly
115 120 125
Glu Thr Thr Ile Leu Lys Ser Ser Phe Phe Leu Arg Gly
130 135 140
Claims (5)
1.一种羧肽酶A，其特征在于具有SEQIDNO：1所示的羧肽酶A氨基酸序列。
2.根据权利要求1所述的羧肽酶A氨基酸序列，其特征在于：在N-苯基-4-联苯胺制备过程中的应用。
3.根据权利要求2所述的N-苯基-4-联苯胺制备过程，其特征在于：包括以下步骤：
(1)将乙酰苯胺、4-溴联苯、ZnCl2及pH＝9.5的磷酸二氢钾缓冲溶液和甲醇加入到反应瓶中，搅拌全部溶解后，再加入酶活为65ug/g的羧肽酶A，控制温度28-32℃，并在此温度下搅拌5-6h，经HPLC检验4-溴联苯含量小于0.2％后停止反应，除甲醇后得到N-苯基-4-乙酰基联苯胺粗品，粗品用乙酸乙酯萃取3次，蒸干乙酸乙酯后得到白色的产品；
(2)将以上产品溶解于的醇类溶剂中，再加入氢氧化钾,升温至80-85℃，保温反应5-6h，直至N-苯基-4-乙酰基联苯胺通过HPLC检测含量小于0.10％时停止反应，降温至50-60℃后，趁热倒入冰水混合物中，继续搅拌1-2h，过滤，收集白色固体；
(3)将白色固体加入甲苯中，并加入50g活性物质，升温至60-65℃，搅拌2-3h，然后趁热过滤，收集滤液，浓缩出甲苯，加入目标产品晶种，在25摄氏度下搅拌1-2h，析出晶体，过滤、干燥既得N-苯基-4-联苯胺。
4.根据权利要求3所述的制备方法，其特征在于：步骤(3)中的活性物质为活性白土、活性炭、硅藻土中的一种。
5.根据权利要求3所述的制备方法，其特征在于：步骤(1)中所述羧肽酶A的加入量占乙酰苯胺质量的1/500-1/1000。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011089477.3A CN112175924A (zh) | 2020-10-13 | 2020-10-13 | 一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011089477.3A CN112175924A (zh) | 2020-10-13 | 2020-10-13 | 一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112175924A true CN112175924A (zh) | 2021-01-05 |
Family
ID=73949485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011089477.3A Pending CN112175924A (zh) | 2020-10-13 | 2020-10-13 | 一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112175924A (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160031810A1 (en) * | 2013-04-26 | 2016-02-04 | Postech Academy-Industry Foundation | Benzidine derivative, method for preparing same, and pharmaceutical composition containing benzidine derivative for treating liver disease caused by hepatitis c virus |
-
2020
- 2020-10-13 CN CN202011089477.3A patent/CN112175924A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160031810A1 (en) * | 2013-04-26 | 2016-02-04 | Postech Academy-Industry Foundation | Benzidine derivative, method for preparing same, and pharmaceutical composition containing benzidine derivative for treating liver disease caused by hepatitis c virus |
Non-Patent Citations (6)
| Title |
|---|
| JOSEPH M DENNIS, 等: "Breaking the base barrier: an electron-deficient palladium catalyst enables the use of a common soluble base in C-N coupling", J AM CHEM SOC., vol. 140, no. 13 * |
| 吴静;闵柔;邬敏辰;陈伟;: "羧肽酶研究进展", 食品与生物技术学报, no. 08, pages 1 * |
| 未知: "hypothetical protein [Streptomyces malaysiense]，WP_071387424.1", NCBI GENBANK * |
| 武国军, 郝晓柯, 白玉杰, 张盈华, 药立波, 季少平, 王吉村: "人羧肽酶A的cDNA克隆和原核表达", 细胞与分子免疫学杂志, no. 03 * |
| 王元正: "活性子概念与酶催化反应", 化学世界, no. 06 * |
| 王德解 等: "羧肽酶A/B亚家族", 中国生化药物杂志 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108690854B (zh) | 一种利用化学-酶法生产l-草铵膦的方法 | |
| GB2049703A (en) | Dipeptide preparation utilising an insolubilised-metalloproteinase | |
| CN106632609A (zh) | 一种六胜肽的制备方法及其产品 | |
| CN110002992A (zh) | 一种正长链二元酸的精制方法 | |
| CN110791488A (zh) | 用于拆分手性化合物的脂肪酶及其制备方法和应用 | |
| JP3117157B2 (ja) | 固定化酵素によるアルコールのアシル化方法 | |
| CN112175924A (zh) | 一种羧肽酶a及其在制备n-苯基-4-联苯胺中的应用 | |
| CN102059105B (zh) | 一种类寡肽手性固定相及其制备方法 | |
| Xiao et al. | Two-stage crystallization combining direct succinimide synthesis for the recovery of succinic acid from fermentation broth | |
| CN106905139B (zh) | 一种高纯度己二酸提取工艺 | |
| US4584400A (en) | Refining phenylalanine | |
| CN105153284B (zh) | 一种利那洛肽的纯化方法 | |
| KR890001244B1 (ko) | 성장 호르몬-양 물질의 정제방법 | |
| Shimizu et al. | Synthetic studies on leupeptins and their analogs | |
| Nokihara et al. | Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid | |
| CN114573556A (zh) | 一种吡虫啉的纯化方法 | |
| SU1282816A3 (ru) | Способ получени кристаллической соли @ -6-фтор-4-уреидохроман-4-карбоновой кислоты и @ -(+)-(1-фенетил) амина или @ -(-)-эфедрина | |
| CN1800166A (zh) | 奥硝唑光学对映体的制备及纯化方法 | |
| WO2021062882A1 (zh) | 一种络塞维的合成方法 | |
| Murase et al. | Synthesis of N-Acetyl-O-benzyl-DL-threonine and O-Benzyl-L-threonine | |
| US3716551A (en) | Deuterated l-amino acid mixture | |
| CN112500316B (zh) | 一种制备boc-(r)-3-氨基-4-(2,4,5-三氟苯基)丁酸的方法 | |
| CN1217338A (zh) | 长效芸苔素内酯的合成方法 | |
| Belyaeva et al. | Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel | |
| CN113584096A (zh) | 一种r-2-羟基苯丁腈的制备方法及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210105 |