CN109750014B - Fusion type rhizopus chinensis lipase with improved heat stability and application thereof - Google Patents
Fusion type rhizopus chinensis lipase with improved heat stability and application thereof Download PDFInfo
- Publication number
- CN109750014B CN109750014B CN201910235208.4A CN201910235208A CN109750014B CN 109750014 B CN109750014 B CN 109750014B CN 201910235208 A CN201910235208 A CN 201910235208A CN 109750014 B CN109750014 B CN 109750014B
- Authority
- CN
- China
- Prior art keywords
- thr
- lipase
- ala
- pro
- val
- 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
- 108090001060 Lipase Proteins 0.000 title claims abstract description 91
- 102000004882 Lipase Human genes 0.000 title claims abstract description 76
- 239000004367 Lipase Substances 0.000 title claims abstract description 76
- 235000019421 lipase Nutrition 0.000 title claims abstract description 76
- 230000004927 fusion Effects 0.000 title claims abstract description 43
- 241000235528 Rhizopus microsporus var. chinensis Species 0.000 title claims abstract description 29
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 239000003674 animal food additive Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 23
- 238000000034 method Methods 0.000 abstract description 14
- 150000001413 amino acids Chemical class 0.000 abstract description 13
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 31
- 108090000790 Enzymes Proteins 0.000 description 31
- 230000000694 effects Effects 0.000 description 22
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 15
- 101710176384 Peptide 1 Proteins 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 11
- 108010005233 alanylglutamic acid Proteins 0.000 description 10
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 8
- 235000019626 lipase activity Nutrition 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000009776 industrial production Methods 0.000 description 7
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 108010077112 prolyl-proline Proteins 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 4
- JJKKWYQVHRUSDG-GUBZILKMSA-N Glu-Ala-Lys Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O JJKKWYQVHRUSDG-GUBZILKMSA-N 0.000 description 4
- 101710084373 Lipase 1 Proteins 0.000 description 4
- 101710084378 Lipase 2 Proteins 0.000 description 4
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 4
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 108010061238 threonyl-glycine Proteins 0.000 description 4
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 3
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 3
- OSOLWRWQADPDIQ-DCAQKATOSA-N Met-Asp-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OSOLWRWQADPDIQ-DCAQKATOSA-N 0.000 description 3
- 241000235527 Rhizopus Species 0.000 description 3
- ARPONUQDNWLXOZ-KKUMJFAQSA-N Tyr-Gln-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ARPONUQDNWLXOZ-KKUMJFAQSA-N 0.000 description 3
- 108010013835 arginine glutamate Proteins 0.000 description 3
- 108010068265 aspartyltyrosine Proteins 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 108010047926 leucyl-lysyl-tyrosine Proteins 0.000 description 3
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 3
- 108010017391 lysylvaline Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- LZRNYBIJOSKKRJ-XVYDVKMFSA-N Ala-Asp-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LZRNYBIJOSKKRJ-XVYDVKMFSA-N 0.000 description 2
- IKKVASZHTMKJIR-ZKWXMUAHSA-N Ala-Asp-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IKKVASZHTMKJIR-ZKWXMUAHSA-N 0.000 description 2
- FVSOUJZKYWEFOB-KBIXCLLPSA-N Ala-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)N FVSOUJZKYWEFOB-KBIXCLLPSA-N 0.000 description 2
- BTBUEVAGZCKULD-XPUUQOCRSA-N Ala-Gly-His Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BTBUEVAGZCKULD-XPUUQOCRSA-N 0.000 description 2
- LXAARTARZJJCMB-CIQUZCHMSA-N Ala-Ile-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LXAARTARZJJCMB-CIQUZCHMSA-N 0.000 description 2
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 2
- XCIGOVDXZULBBV-DCAQKATOSA-N Ala-Val-Lys Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CCCCN)C(O)=O XCIGOVDXZULBBV-DCAQKATOSA-N 0.000 description 2
- XRNXPIGJPQHCPC-RCWTZXSCSA-N Arg-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)O)C(O)=O XRNXPIGJPQHCPC-RCWTZXSCSA-N 0.000 description 2
- VYZBPPBKFCHCIS-WPRPVWTQSA-N Arg-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N VYZBPPBKFCHCIS-WPRPVWTQSA-N 0.000 description 2
- ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N Asn-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N 0.000 description 2
- DJIMLSXHXKWADV-CIUDSAMLSA-N Asn-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(N)=O DJIMLSXHXKWADV-CIUDSAMLSA-N 0.000 description 2
- NPZJLGMWMDNQDD-GHCJXIJMSA-N Asn-Ser-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NPZJLGMWMDNQDD-GHCJXIJMSA-N 0.000 description 2
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 2
- DJCAHYVLMSRBFR-QXEWZRGKSA-N Asp-Met-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC(O)=O DJCAHYVLMSRBFR-QXEWZRGKSA-N 0.000 description 2
- YRZIYQGXTSBRLT-AVGNSLFASA-N Asp-Phe-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O YRZIYQGXTSBRLT-AVGNSLFASA-N 0.000 description 2
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 2
- GWWSUMLEWKQHLR-NUMRIWBASA-N Asp-Thr-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O GWWSUMLEWKQHLR-NUMRIWBASA-N 0.000 description 2
- CZIVKMOEXPILDK-SRVKXCTJSA-N Asp-Tyr-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O CZIVKMOEXPILDK-SRVKXCTJSA-N 0.000 description 2
- UWZLBXOBVKRUFE-HGNGGELXSA-N Gln-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N UWZLBXOBVKRUFE-HGNGGELXSA-N 0.000 description 2
- KVYVOGYEMPEXBT-GUBZILKMSA-N Gln-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O KVYVOGYEMPEXBT-GUBZILKMSA-N 0.000 description 2
- MFLMFRZBAJSGHK-ACZMJKKPSA-N Gln-Cys-Ser Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N MFLMFRZBAJSGHK-ACZMJKKPSA-N 0.000 description 2
- DAAUVRPSZRDMBV-KBIXCLLPSA-N Gln-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)N)N DAAUVRPSZRDMBV-KBIXCLLPSA-N 0.000 description 2
- VXAIXLOYBPMZPT-JBACZVJFSA-N Gln-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N VXAIXLOYBPMZPT-JBACZVJFSA-N 0.000 description 2
- 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 2
- LXAUHIRMWXQRKI-XHNCKOQMSA-N Glu-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O LXAUHIRMWXQRKI-XHNCKOQMSA-N 0.000 description 2
- CKOFNWCLWRYUHK-XHNCKOQMSA-N Glu-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CKOFNWCLWRYUHK-XHNCKOQMSA-N 0.000 description 2
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 2
- JBRBACJPBZNFMF-YUMQZZPRSA-N Gly-Ala-Lys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN JBRBACJPBZNFMF-YUMQZZPRSA-N 0.000 description 2
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 2
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 2
- LPCKHUXOGVNZRS-YUMQZZPRSA-N Gly-His-Ser Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O LPCKHUXOGVNZRS-YUMQZZPRSA-N 0.000 description 2
- DGKBSGNCMCLDSL-BYULHYEWSA-N Gly-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN DGKBSGNCMCLDSL-BYULHYEWSA-N 0.000 description 2
- UWQDKRIZSROAKS-FJXKBIBVSA-N Gly-Met-Thr Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWQDKRIZSROAKS-FJXKBIBVSA-N 0.000 description 2
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 2
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 2
- SYOJVRNQCXYEOV-XVKPBYJWSA-N Gly-Val-Glu Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SYOJVRNQCXYEOV-XVKPBYJWSA-N 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 2
- GJMHMDKCJPQJOI-IHRRRGAJSA-N His-Lys-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CN=CN1 GJMHMDKCJPQJOI-IHRRRGAJSA-N 0.000 description 2
- PYNPBMCLAKTHJL-SRVKXCTJSA-N His-Pro-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O PYNPBMCLAKTHJL-SRVKXCTJSA-N 0.000 description 2
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 2
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 2
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 2
- APQYGMBHIVXFML-OSUNSFLBSA-N Ile-Val-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N APQYGMBHIVXFML-OSUNSFLBSA-N 0.000 description 2
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- 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 2
- WRLPVDVHNWSSCL-MELADBBJSA-N Leu-His-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N WRLPVDVHNWSSCL-MELADBBJSA-N 0.000 description 2
- 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 2
- ONPJGOIVICHWBW-BZSNNMDCSA-N Leu-Lys-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 ONPJGOIVICHWBW-BZSNNMDCSA-N 0.000 description 2
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 2
- ZJZNLRVCZWUONM-JXUBOQSCSA-N Leu-Thr-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O ZJZNLRVCZWUONM-JXUBOQSCSA-N 0.000 description 2
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 2
- YNNPKXBBRZVIRX-IHRRRGAJSA-N Lys-Arg-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O YNNPKXBBRZVIRX-IHRRRGAJSA-N 0.000 description 2
- NTBFKPBULZGXQL-KKUMJFAQSA-N Lys-Asp-Tyr Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NTBFKPBULZGXQL-KKUMJFAQSA-N 0.000 description 2
- GUYHHBZCBQZLFW-GUBZILKMSA-N Lys-Gln-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N GUYHHBZCBQZLFW-GUBZILKMSA-N 0.000 description 2
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 2
- DCRWPTBMWMGADO-AVGNSLFASA-N Lys-Glu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DCRWPTBMWMGADO-AVGNSLFASA-N 0.000 description 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 2
- WVJNGSFKBKOKRV-AJNGGQMLSA-N Lys-Leu-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVJNGSFKBKOKRV-AJNGGQMLSA-N 0.000 description 2
- MEQLGHAMAUPOSJ-DCAQKATOSA-N Lys-Ser-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O MEQLGHAMAUPOSJ-DCAQKATOSA-N 0.000 description 2
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 description 2
- 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 2
- 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 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- NOFBJKKOPKJDCO-KKXDTOCCSA-N Phe-Ala-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NOFBJKKOPKJDCO-KKXDTOCCSA-N 0.000 description 2
- JEGFCFLCRSJCMA-IHRRRGAJSA-N Phe-Arg-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N JEGFCFLCRSJCMA-IHRRRGAJSA-N 0.000 description 2
- WFHRXJOZEXUKLV-IRXDYDNUSA-N Phe-Gly-Tyr Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 WFHRXJOZEXUKLV-IRXDYDNUSA-N 0.000 description 2
- ZLAKUZDMKVKFAI-JYJNAYRXSA-N Phe-Pro-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O ZLAKUZDMKVKFAI-JYJNAYRXSA-N 0.000 description 2
- BPIMVBKDLSBKIJ-FCLVOEFKSA-N Phe-Thr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 BPIMVBKDLSBKIJ-FCLVOEFKSA-N 0.000 description 2
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 2
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 2
- JFNPBBOGGNMSRX-CIUDSAMLSA-N Pro-Gln-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O JFNPBBOGGNMSRX-CIUDSAMLSA-N 0.000 description 2
- LPGSNRSLPHRNBW-AVGNSLFASA-N Pro-His-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 LPGSNRSLPHRNBW-AVGNSLFASA-N 0.000 description 2
- 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 2
- GNADVDLLGVSXLS-ULQDDVLXSA-N Pro-Phe-His Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O GNADVDLLGVSXLS-ULQDDVLXSA-N 0.000 description 2
- DWPXHLIBFQLKLK-CYDGBPFRSA-N Pro-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 DWPXHLIBFQLKLK-CYDGBPFRSA-N 0.000 description 2
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 2
- IIRBTQHFVNGPMQ-AVGNSLFASA-N Pro-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 IIRBTQHFVNGPMQ-AVGNSLFASA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- JJKSSJVYOVRJMZ-FXQIFTODSA-N Ser-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N)CN=C(N)N JJKSSJVYOVRJMZ-FXQIFTODSA-N 0.000 description 2
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 description 2
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 2
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 2
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 2
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 2
- ATEQEHCGZKBEMU-GQGQLFGLSA-N Ser-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CO)N ATEQEHCGZKBEMU-GQGQLFGLSA-N 0.000 description 2
- PQEQXWRVHQAAKS-SRVKXCTJSA-N Ser-Tyr-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=C(O)C=C1 PQEQXWRVHQAAKS-SRVKXCTJSA-N 0.000 description 2
- OJRNZRROAIAHDL-LKXGYXEUSA-N Thr-Asn-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OJRNZRROAIAHDL-LKXGYXEUSA-N 0.000 description 2
- MROIJTGJGIDEEJ-RCWTZXSCSA-N Thr-Pro-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 MROIJTGJGIDEEJ-RCWTZXSCSA-N 0.000 description 2
- STUAPCLEDMKXKL-LKXGYXEUSA-N Thr-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O STUAPCLEDMKXKL-LKXGYXEUSA-N 0.000 description 2
- NQQMWWVVGIXUOX-SVSWQMSJSA-N Thr-Ser-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NQQMWWVVGIXUOX-SVSWQMSJSA-N 0.000 description 2
- 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 2
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 2
- XVHAUVJXBFGUPC-RPTUDFQQSA-N Thr-Tyr-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XVHAUVJXBFGUPC-RPTUDFQQSA-N 0.000 description 2
- AKHDFZHUPGVFEJ-YEPSODPASA-N Thr-Val-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AKHDFZHUPGVFEJ-YEPSODPASA-N 0.000 description 2
- NBHGNEJMBNQQKZ-UBHSHLNASA-N Trp-Asp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N NBHGNEJMBNQQKZ-UBHSHLNASA-N 0.000 description 2
- PMHLLBKTDHQMCY-ULQDDVLXSA-N Tyr-Lys-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMHLLBKTDHQMCY-ULQDDVLXSA-N 0.000 description 2
- XJPXTYLVMUZGNW-IHRRRGAJSA-N Tyr-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O XJPXTYLVMUZGNW-IHRRRGAJSA-N 0.000 description 2
- XUIOBCQESNDTDE-FQPOAREZSA-N Tyr-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O XUIOBCQESNDTDE-FQPOAREZSA-N 0.000 description 2
- KRXFXDCNKLANCP-CXTHYWKRSA-N Tyr-Tyr-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 KRXFXDCNKLANCP-CXTHYWKRSA-N 0.000 description 2
- SQUMHUZLJDUROQ-YDHLFZDLSA-N Tyr-Val-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O SQUMHUZLJDUROQ-YDHLFZDLSA-N 0.000 description 2
- YCMXFKWYJFZFKS-LAEOZQHASA-N Val-Gln-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCMXFKWYJFZFKS-LAEOZQHASA-N 0.000 description 2
- OXGVAUFVTOPFFA-XPUUQOCRSA-N Val-Gly-Cys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N OXGVAUFVTOPFFA-XPUUQOCRSA-N 0.000 description 2
- FEFZWCSXEMVSPO-LSJOCFKGSA-N Val-His-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](C)C(O)=O FEFZWCSXEMVSPO-LSJOCFKGSA-N 0.000 description 2
- 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 2
- ZXYPHBKIZLAQTL-QXEWZRGKSA-N Val-Pro-Asp Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N ZXYPHBKIZLAQTL-QXEWZRGKSA-N 0.000 description 2
- QIVPZSWBBHRNBA-JYJNAYRXSA-N Val-Pro-Phe Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O QIVPZSWBBHRNBA-JYJNAYRXSA-N 0.000 description 2
- 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 2
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 2
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 108010070783 alanyltyrosine Proteins 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 108010060199 cysteinylproline Proteins 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- LVZSQWIWCANHPF-UHFFFAOYSA-N p-nitrophenyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 LVZSQWIWCANHPF-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108010020432 prolyl-prolylisoleucine Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 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 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- BUANFPRKJKJSRR-ACZMJKKPSA-N Ala-Ala-Gln Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CCC(N)=O BUANFPRKJKJSRR-ACZMJKKPSA-N 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- LGFCAXJBAZESCF-ACZMJKKPSA-N Ala-Gln-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O LGFCAXJBAZESCF-ACZMJKKPSA-N 0.000 description 1
- AWAXZRDKUHOPBO-GUBZILKMSA-N Ala-Gln-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O AWAXZRDKUHOPBO-GUBZILKMSA-N 0.000 description 1
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 1
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 1
- XRUJOVRWNMBAAA-NHCYSSNCSA-N Ala-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 XRUJOVRWNMBAAA-NHCYSSNCSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- XQNRANMFRPCFFW-GCJQMDKQSA-N Ala-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C)N)O XQNRANMFRPCFFW-GCJQMDKQSA-N 0.000 description 1
- KLKARCOHVHLAJP-UWJYBYFXSA-N Ala-Tyr-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CS)C(O)=O KLKARCOHVHLAJP-UWJYBYFXSA-N 0.000 description 1
- ZXKNLCPUNZPFGY-LEWSCRJBSA-N Ala-Tyr-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N ZXKNLCPUNZPFGY-LEWSCRJBSA-N 0.000 description 1
- MUGAESARFRGOTQ-IGNZVWTISA-N Ala-Tyr-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N MUGAESARFRGOTQ-IGNZVWTISA-N 0.000 description 1
- JSHVMZANPXCDTL-GMOBBJLQSA-N Arg-Asp-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JSHVMZANPXCDTL-GMOBBJLQSA-N 0.000 description 1
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 1
- KRQSPVKUISQQFS-FJXKBIBVSA-N Arg-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N KRQSPVKUISQQFS-FJXKBIBVSA-N 0.000 description 1
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 1
- OQPAZKMGCWPERI-GUBZILKMSA-N Arg-Ser-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O OQPAZKMGCWPERI-GUBZILKMSA-N 0.000 description 1
- SRUUBQBAVNQZGJ-LAEOZQHASA-N Asn-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N SRUUBQBAVNQZGJ-LAEOZQHASA-N 0.000 description 1
- GNKVBRYFXYWXAB-WDSKDSINSA-N Asn-Glu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O GNKVBRYFXYWXAB-WDSKDSINSA-N 0.000 description 1
- XVBDDUPJVQXDSI-PEFMBERDSA-N Asn-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N XVBDDUPJVQXDSI-PEFMBERDSA-N 0.000 description 1
- XEGZSHSPQNDNRH-JRQIVUDYSA-N Asn-Tyr-Thr 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]([C@@H](C)O)C(O)=O XEGZSHSPQNDNRH-JRQIVUDYSA-N 0.000 description 1
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 1
- HRGGPWBIMIQANI-GUBZILKMSA-N Asp-Gln-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HRGGPWBIMIQANI-GUBZILKMSA-N 0.000 description 1
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 1
- YIDFBWRHIYOYAA-LKXGYXEUSA-N Asp-Ser-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YIDFBWRHIYOYAA-LKXGYXEUSA-N 0.000 description 1
- UTLCRGFJFSZWAW-OLHMAJIHSA-N Asp-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UTLCRGFJFSZWAW-OLHMAJIHSA-N 0.000 description 1
- BJDHEININLSZOT-KKUMJFAQSA-N Asp-Tyr-Lys Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(O)=O BJDHEININLSZOT-KKUMJFAQSA-N 0.000 description 1
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- UCSXXFRXHGUXCQ-SRVKXCTJSA-N Cys-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N UCSXXFRXHGUXCQ-SRVKXCTJSA-N 0.000 description 1
- GDNWBSFSHJVXKL-GUBZILKMSA-N Cys-Lys-Gln Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O GDNWBSFSHJVXKL-GUBZILKMSA-N 0.000 description 1
- ZOKPRHVIFAUJPV-GUBZILKMSA-N Cys-Pro-Arg Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O ZOKPRHVIFAUJPV-GUBZILKMSA-N 0.000 description 1
- IQXSTXKVEMRMMB-XAVMHZPKSA-N Cys-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N)O IQXSTXKVEMRMMB-XAVMHZPKSA-N 0.000 description 1
- WTXCNOPZMQRTNN-BWBBJGPYSA-N Cys-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CS)N)O WTXCNOPZMQRTNN-BWBBJGPYSA-N 0.000 description 1
- AZDQAZRURQMSQD-XPUUQOCRSA-N Cys-Val-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AZDQAZRURQMSQD-XPUUQOCRSA-N 0.000 description 1
- ZBKUIQNCRIYVGH-SDDRHHMPSA-N Gln-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZBKUIQNCRIYVGH-SDDRHHMPSA-N 0.000 description 1
- GJBUAAAIZSRCDC-GVXVVHGQSA-N Glu-Leu-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O GJBUAAAIZSRCDC-GVXVVHGQSA-N 0.000 description 1
- SJJHXJDSNQJMMW-SRVKXCTJSA-N Glu-Lys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SJJHXJDSNQJMMW-SRVKXCTJSA-N 0.000 description 1
- TZXOPHFCAATANZ-QEJZJMRPSA-N Glu-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N TZXOPHFCAATANZ-QEJZJMRPSA-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
- DLISPGXMKZTWQG-IFFSRLJSSA-N Glu-Thr-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O DLISPGXMKZTWQG-IFFSRLJSSA-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
- 108010033128 Glucan Endo-1,3-beta-D-Glucosidase Proteins 0.000 description 1
- CIMULJZTTOBOPN-WHFBIAKZSA-N Gly-Asn-Asn Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CIMULJZTTOBOPN-WHFBIAKZSA-N 0.000 description 1
- LXXLEUBUOMCAMR-NKWVEPMBSA-N Gly-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)CN)C(=O)O LXXLEUBUOMCAMR-NKWVEPMBSA-N 0.000 description 1
- QPCVIQJVRGXUSA-LURJTMIESA-N Gly-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QPCVIQJVRGXUSA-LURJTMIESA-N 0.000 description 1
- BHPQOIPBLYJNAW-NGZCFLSTSA-N Gly-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN BHPQOIPBLYJNAW-NGZCFLSTSA-N 0.000 description 1
- IBYOLNARKHMLBG-WHOFXGATSA-N Gly-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IBYOLNARKHMLBG-WHOFXGATSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- SOFSRBYHDINIRG-QTKMDUPCSA-N His-Arg-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CN=CN1)N)O SOFSRBYHDINIRG-QTKMDUPCSA-N 0.000 description 1
- FDQYIRHBVVUTJF-ZETCQYMHSA-N His-Gly-Gly Chemical compound [O-]C(=O)CNC(=O)CNC(=O)[C@@H]([NH3+])CC1=CN=CN1 FDQYIRHBVVUTJF-ZETCQYMHSA-N 0.000 description 1
- LVWIJITYHRZHBO-IXOXFDKPSA-N His-Leu-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LVWIJITYHRZHBO-IXOXFDKPSA-N 0.000 description 1
- XKIYNCLILDLGRS-QWRGUYRKSA-N His-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 XKIYNCLILDLGRS-QWRGUYRKSA-N 0.000 description 1
- VSZALHITQINTGC-GHCJXIJMSA-N Ile-Ala-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VSZALHITQINTGC-GHCJXIJMSA-N 0.000 description 1
- FJWYJQRCVNGEAQ-ZPFDUUQYSA-N Ile-Asn-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N FJWYJQRCVNGEAQ-ZPFDUUQYSA-N 0.000 description 1
- CKRFDMPBSWYOBT-PPCPHDFISA-N Ile-Lys-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CKRFDMPBSWYOBT-PPCPHDFISA-N 0.000 description 1
- 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 1
- PMAOIIWHZHAPBT-HJPIBITLSA-N Ile-Tyr-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CS)C(=O)O)N PMAOIIWHZHAPBT-HJPIBITLSA-N 0.000 description 1
- NGKPIPCGMLWHBX-WZLNRYEVSA-N Ile-Tyr-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N NGKPIPCGMLWHBX-WZLNRYEVSA-N 0.000 description 1
- RQZFWBLDTBDEOF-RNJOBUHISA-N Ile-Val-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N RQZFWBLDTBDEOF-RNJOBUHISA-N 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- 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 1
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 1
- SIGZKCWZEBFNAK-QAETUUGQSA-N Leu-Ser-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SIGZKCWZEBFNAK-QAETUUGQSA-N 0.000 description 1
- AEDWWMMHUGYIFD-HJGDQZAQSA-N Leu-Thr-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O AEDWWMMHUGYIFD-HJGDQZAQSA-N 0.000 description 1
- 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 1
- ITWQLSZTLBKWJM-YUMQZZPRSA-N Lys-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCCN ITWQLSZTLBKWJM-YUMQZZPRSA-N 0.000 description 1
- KTINOHQFVVCEGQ-XIRDDKMYSA-N Lys-Trp-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CC(O)=O)C(O)=O KTINOHQFVVCEGQ-XIRDDKMYSA-N 0.000 description 1
- BWECSLVQIWEMSC-IHRRRGAJSA-N Lys-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCCN)N BWECSLVQIWEMSC-IHRRRGAJSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- HGNGAMWHGGANAU-WHOFXGATSA-N Phe-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HGNGAMWHGGANAU-WHOFXGATSA-N 0.000 description 1
- RAGOJJCBGXARPO-XVSYOHENSA-N Phe-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RAGOJJCBGXARPO-XVSYOHENSA-N 0.000 description 1
- APKRGYLBSCWJJP-FXQIFTODSA-N Pro-Ala-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O APKRGYLBSCWJJP-FXQIFTODSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- BRJGUPWVFXKBQI-XUXIUFHCSA-N Pro-Leu-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BRJGUPWVFXKBQI-XUXIUFHCSA-N 0.000 description 1
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 description 1
- RNEFESSBTOQSAC-DCAQKATOSA-N Pro-Ser-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O RNEFESSBTOQSAC-DCAQKATOSA-N 0.000 description 1
- 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 1
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 1
- MMAPOBOTRUVNKJ-ZLUOBGJFSA-N Ser-Asp-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CO)N)C(=O)O MMAPOBOTRUVNKJ-ZLUOBGJFSA-N 0.000 description 1
- DSSOYPJWSWFOLK-CIUDSAMLSA-N Ser-Cys-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O DSSOYPJWSWFOLK-CIUDSAMLSA-N 0.000 description 1
- MQQBBLVOUUJKLH-HJPIBITLSA-N Ser-Ile-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MQQBBLVOUUJKLH-HJPIBITLSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 1
- 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 1
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- CAJFZCICSVBOJK-SHGPDSBTSA-N Thr-Ala-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAJFZCICSVBOJK-SHGPDSBTSA-N 0.000 description 1
- APIQKJYZDWVOCE-VEVYYDQMSA-N Thr-Asp-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O APIQKJYZDWVOCE-VEVYYDQMSA-N 0.000 description 1
- YZUWGFXVVZQJEI-PMVVWTBXSA-N Thr-Gly-His Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O YZUWGFXVVZQJEI-PMVVWTBXSA-N 0.000 description 1
- IHAPJUHCZXBPHR-WZLNRYEVSA-N Thr-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N IHAPJUHCZXBPHR-WZLNRYEVSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- CJXURNZYNHCYFD-WDCWCFNPSA-N Thr-Lys-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O CJXURNZYNHCYFD-WDCWCFNPSA-N 0.000 description 1
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 1
- 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 1
- YKCXQOBTISTQJD-BZSNNMDCSA-N Tyr-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N YKCXQOBTISTQJD-BZSNNMDCSA-N 0.000 description 1
- WURLIFOWSMBUAR-SLFFLAALSA-N Tyr-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O WURLIFOWSMBUAR-SLFFLAALSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- OBKOPLHSRDATFO-XHSDSOJGSA-N Tyr-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N OBKOPLHSRDATFO-XHSDSOJGSA-N 0.000 description 1
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 1
- CPTQYHDSVGVGDZ-UKJIMTQDSA-N Val-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N CPTQYHDSVGVGDZ-UKJIMTQDSA-N 0.000 description 1
- OXVPMZVGCAPFIG-BQFCYCMXSA-N Val-Gln-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N OXVPMZVGCAPFIG-BQFCYCMXSA-N 0.000 description 1
- CHWRZUGUMAMTFC-IHRRRGAJSA-N Val-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CNC=N1 CHWRZUGUMAMTFC-IHRRRGAJSA-N 0.000 description 1
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 description 1
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 1
- YKNOJPJWNVHORX-UNQGMJICSA-N Val-Phe-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YKNOJPJWNVHORX-UNQGMJICSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- VSCIANXXVZOYOC-AVGNSLFASA-N Val-Pro-His Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N VSCIANXXVZOYOC-AVGNSLFASA-N 0.000 description 1
- DOFAQXCYFQKSHT-SRVKXCTJSA-N Val-Pro-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DOFAQXCYFQKSHT-SRVKXCTJSA-N 0.000 description 1
- PDDJTOSAVNRJRH-UNQGMJICSA-N Val-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](C(C)C)N)O PDDJTOSAVNRJRH-UNQGMJICSA-N 0.000 description 1
- VVIZITNVZUAEMI-DLOVCJGASA-N Val-Val-Gln Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(N)=O VVIZITNVZUAEMI-DLOVCJGASA-N 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960002303 citric acid monohydrate Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- -1 diglycerides Chemical class 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of biological engineering, and discloses a fusion type rhizopus chinensis lipase with improved heat stability and application thereof, wherein the amino acid sequence of the fusion type lipase is shown as SEQ ID No.4 or SEQ ID No.5, the fusion type lipase is formed by fusing an amphiphilic short peptide consisting of 30-32 amino acids at the N end in an amino acid sequence of coding rhizopus chinensis lipase, and the amino acid sequence of the amphiphilic short peptide is shown as SEQ ID No.2 or SEQ ID No. 3; the two fusion lipases have ideal heat-resistant property, so the method is particularly suitable for industrial large-scale production.
Description
Technical Field
The invention belongs to the technical field of biological engineering, relates to a fusion type lipase, and particularly relates to a fusion type rhizopus chinensis lipase with improved heat stability and application thereof.
Background
Lipases are capable of gradually hydrolyzing triglycerides into fatty acids, diglycerides, monoglycerides and glycerol; meanwhile, the lipase can also catalyze reactions such as acidolysis, alcoholysis, ammonolysis, transesterification, ester synthesis and the like of ester, is widely applied to processes such as food processing, grease processing, leather processing, biological energy sources, feed addition and the like due to unique catalytic activity, and plays a vital role in development of renewable energy sources and environmental protection. Although a plurality of lipases from rhizopus can be selected in industrial production, most of natural rhizopus lipases are medium-temperature lipases which have low thermal stability and are easy to inactivate at high temperature, and grease processing reaction generally needs to be carried out at higher temperature, so that the industrial production cost is greatly increased due to the defect that the natural rhizopus lipases cannot resist high temperature, and the application of the lipases in industrial production is also limited.
The rhizopus chinensis lipase has poor thermal stability and large loss in industrial production, so the production cost is greatly improved. If the thermal stability of the rhizopus chinensis lipase is improved, the production cost is reduced. Therefore, the rhizopus chinensis lipase gene is transformed into the lipase with relatively high heat resistance by the modification of the rhizopus chinensis lipase gene.
Disclosure of Invention
The invention aims to provide a fusion type lipase and application thereof, and aims to solve the problems of poor heat resistance and undesirable industrial production of rhizopus chinensis lipase.
The invention is realized by the following technical scheme:
a fusion type rhizopus chinensis lipase with improved heat stability is characterized in that a section of amphiphilic short peptide consisting of 30-32 amino acids is fused at the N end of an amino acid sequence of the rhizopus chinensis lipase respectively, wherein the amino acid sequence of the rhizopus chinensis lipase is shown as SEQ ID No. 1.
Specifically, the amphiphilic short peptide is amphiphilic short peptide 1 or amphiphilic short peptide 2, and the fusion type lipase is obtained by fusing a section of amphiphilic short peptide 1 consisting of 30-32 amino acids and amphiphilic short peptide 2 at the N end in the amino acid sequence SEQ ID NO.1 of rhizopus chinensis lipase.
The amino acid sequences of the amphiphilic short peptide 1 and the amphiphilic short peptide 2 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
The amino acid sequence of the fusion type lipase is shown as SEQ ID NO.4 or SEQ ID NO. 5.
The optimal pH value of the enzymatic reaction of the fusion type lipase is 9.0; the optimal temperature is 40 ℃; the lipase fusion amphiphilic short peptide 1 has the advantages that the pH is tolerant for 1 hour and the residual activity is 50% under the conditions of pH4-pH10 and 37 ℃, the pH is tolerant for 1 hour and the residual activity is over 80% under the conditions of pH7-pH10 and 37 ℃, and the residual activity of the lipase fusion amphiphilic short peptide 1 is over 50% after the lipase fusion amphiphilic short peptide 1 is tolerant for 45min at 60 ℃; the lipase fused amphiphilic short peptide 2 has the residual activity of over 50 percent after being endured for 30min at 60 ℃.
In another aspect of the present invention, the amino acid sequence SEQ ID NO.4 or SEQ ID NO.5 is modified, deleted or added with one or more amino acids to obtain an amino acid sequence, and a sequence with only 90% homology is also within the protection scope of the present invention.
The invention also provides a construction method of the fusion type lipase, which comprises the following steps:
1) Designing primers F1 and R1, F2 and R2 to perform template-free PCR so as to obtain an amphiphilic short peptide 1 and an amphiphilic short peptide 2; designing primers F3 and R3, and F3 and R4 to perform PCR amplification on the amphiphilic short peptide 1 and the amphiphilic short peptide 2 respectively;
2) Designing primers F4 and R5, and F5 and R5 to respectively carry out PCR amplification on lipase 1 and lipase 2 by taking a recombinant plasmid obtained by connecting rhizopus chinensis lipase genes to a vector as a template;
3) F3 and R5 are used as primers, and the amphiphilic short peptide 1 and the lipase 1, and the amphiphilic short peptide 2 and the lipase 2 are used as templates respectively for carrying out fusion PCR amplification.
4) Recombining the fusion PCR amplification product with a vector;
5) Putting 5 mu L of recombinant product into 50 mu L of DH5 alpha competent cells, cooling by ice bath, thermally exciting, cooling, adding 500 mu L of LB culture medium, culturing for 1h at 37 ℃ for 180 revolutions, centrifuging, reserving partial clear liquid for suspension precipitation, taking all bacterial liquid to coat a plate, and culturing overnight at 37 ℃;
6) And (3) screening and verifying positive clones, selecting single colonies to be cultured in an LB culture medium with corresponding resistance for 2-3h, carrying out PCR identification, sending out sequencing to the screened positive clones, and comparing the sequencing result with the original sequence.
The nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5 and R5 are respectively shown in SEQ ID NO. 8-17.
The expression vector of the coding gene SEQ ID NO.4 and SEQ ID NO.5 is selected from pPIC9K, pPIC, pPICZaA \ B \ C, pPICZA \ B \ C or PGAPZaA \ B \ C.
In another aspect of the present invention, the use of the two fusion lipases in feed additives is also within the scope of the present invention.
The invention has the beneficial effects that:
the fused lipase provided by the invention can construct recombinant plasmids with expression vectors such as pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C, PGAPZaA \ B \ C and the like besides the recombinant plasmids with pPIC9K, and can transform corresponding host bacteria, and through adding antibiotics such as G418, zeocin and the like into a flat plate, lipase gene engineering bacteria are obtained through screening, and then new lipase is obtained through fermentation. The two lipases and wild type lipase are tolerant at high temperature, and experiments prove that the lipase fusion amphiphilic oligopeptide 1 is tolerant at 60 ℃ for 20min and has about 67.98% of enzyme activity, the lipase fusion amphiphilic oligopeptide 2 has about 59.65% of enzyme activity and the wild type lipase only has 50.68% of enzyme activity. The time of half of the relative enzyme activity of the wild-type lipase at 60 ℃ is about 20min, the time of half of the relative enzyme activity of the lipase fusion amphiphilic short peptide 1 is about 45min, and the time of half of the relative enzyme activity of the lipase fusion amphiphilic short peptide 2 is about 30min. The heat resistance of the two lipases is obviously improved, and the loss rate in industrial production is reduced to a certain extent.
Drawings
FIG. 1 is a flow chart of a method for constructing a fused lipase provided in an embodiment of the present invention;
FIG. 2 is a graph showing the determination of the optimum pH according to the example of the present invention;
FIG. 3 is a graph of the optimum temperature profile provided by an embodiment of the present invention;
FIG. 4 is a pH tolerance curve provided by an embodiment of the present invention;
fig. 5 is a 60 ℃ tolerance curve provided by an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Through simulation analysis of the protein space structure of rhizopus chinensis lipase, the amphiphilic short peptide 1 and the amphiphilic short peptide 2 are respectively fused with the rhizopus chinensis lipase. The specific embodiment is that a rhizopus chinensis lipase gene is taken as a template, fusion of the rhizopus chinensis lipase gene and an amphiphilic short peptide is carried out by a gene fusion PCR method to obtain two fusion type lipase genes, the two fusion type lipase genes are connected with vectors such as pPICZaA \ B \ C, pPICZA \ B \ C, PGAPZaA \ B \ C and the like to construct recombinant plasmids, the recombinant plasmids are transferred into corresponding host bacteria (GS 115 or X33, SMD1168 and PICHIAPINK) to carry out heterologous expression, and fermentation can obtain the two lipases. The two lipases can well act in an acid environment, have ideal heat-resistant characteristic and are suitable for high-temperature resistant granulation, so that the lipase is suitable for industrial production.
The amino acid sequence of the lipase fused in the embodiment of the invention is shown in SEQ ID NO.4 or SEQ ID NO. 5.
1. Experimental materials and reagents:
the gene source strain: rhizopus chinensis CCTCC M201021 screened and stored in the laboratory; expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; constructing a lipase recombinant plasmid by a laboratory; host bacteria: DH 5. Alpha. Competent cells were purchased from Tokyo Total gold, inc.
The main reagents are as follows: DNA Marker, protein Marker (TaKaRa corporation); plasmid Mini Kit I (Omega Co.). Agarose was purchased from Tiangen biochemical technologies (beijing) ltd; nucleic acid dyes were purchased from bataxy bio; restriction endonucleases and PCR amplimers were purchased from TaKaRa.
An experimental instrument: centrifuge (Eppendorf); PCR amplificators (Bio-Rad); nucleic acid electrophoresis apparatus (Bio-Rad); protein electrophoresis apparatus (Amersham Bioscience); gel imager (Bio-Rad).
YPD, LB and yeast fermentation media (FA and FB) were prepared according to the method recommended by the Invitrogen operating Manual.
2. Determination of lipase Activity
The amount of enzyme required to hydrolyze the p-NP substrate to form 1. Mu. MoL of p-nitrophenol per minute under certain conditions, i.e., one unit of enzyme activity, is represented by U.
1) An experimental instrument: a constant-temperature water bath kettle; a pH instrument; microplate reader (Bio-Rad), etc.
2) Experimental materials: p-nitrophenol palmitate (p-NPC 16) (Sigma).
3) Solution preparation:
pH buffer solution: 0.1mol/L citric acid monohydrate buffer and 0.1mol/L phosphate buffer (pH 2-7);
0.1mol/L Tris-HCl buffer (pH 7-9);
0.1mol/L glycine-NaOH buffer (pH 9-12).
Substrate solution: 10mmol/L p-nitrophenol palmitate (p-NPC 16).
4) The p-nitrophenol method (p-nitrophenol) was used: the total volume is 500. Mu.L, which contains 420. Mu.L of 50mmol/L buffer, 30. Mu.L of 10mmol/L substrate p-NP, and 50. Mu.L of diluted enzyme solution. Preheating the mixture of substrate and buffer solution at reaction temperature for 2min, adding diluted enzyme solution, mixing, reacting for 5min, adding 50 μ L of 1.0mol/L SDS to terminate the reaction, and adding 500 μ L of 1.0mol/L Na 2 CO 3 Developing color; the OD value was measured at a wavelength of 405 nm.
Example 1 preparation of Lipase fusion amphiphilic short
As shown in fig. 1, the method for obtaining two lipases according to the embodiment of the present invention comprises the following steps:
(1) RCR amplification: designing primers (F1, R1) and (F2, R2) to perform template-free PCR so as to obtain an amphiphilic short peptide 1 and an amphiphilic short peptide 2; designing primers to perform PCR amplification on the amphiphilic short peptide 1 (F3, R3) and the amphiphilic short peptide 2 (F3, R4) respectively; designing primers (F4, R5) (F5, R5) by taking a recombinant plasmid of the rhizopus chinensis lipase gene connected to a vector as a template to perform PCR amplification on a lipase gene 1 and a lipase gene 2;
(2) Respectively carrying out fusion PCR amplification by taking F3 and R5 as primers and using the amphiphilic short peptide 1 and the rhizopus chinensis lipase gene 1, the amphiphilic short peptide 2 and the rhizopus chinensis lipase gene 2 as templates;
(3) Construction of recombinant plasmid: recombining the fusion PCR product with a vector at 37 ℃ for 30min;
(4) And (3) transformation: adding 5 mu L of mutated product into 50 mu L of DH5 alpha competent cell, flicking and mixing evenly, and ice-bathing for 30min; accurately thermally shocking at 42 ℃ for 45s, immediately placing on ice, and cooling for l0min; adding 500 mu L LB culture medium, rotating 180 turns, culturing lh at 37 ℃; centrifuging at 7000rpm for 3min, discarding supernatant, retaining 100-150 μ L supernatant, flicking suspended thallus, collecting all bacteria liquid, plating, and culturing at 37 deg.C overnight;
(5) And (3) verifying positive clones: selecting a single colony in 500 mu L of LB culture medium with corresponding resistance, culturing for 2-3h at 200rpm, and then carrying out PCR identification; sending out sequencing of the screened positive clones, and comparing the sequencing result with the original sequence;
(6) Finding out recombinant plasmids with correct fusion; the mutant plasmid is transferred into pichia pastoris GS115 or X33, SMD1168 and PICHIAPINK for expression, fermentation and enzyme activity comparison are carried out, and the enzymology and application characteristics are researched.
The primers designed in the method are specifically as follows:
the amino acid sequences of the amphiphilic short peptide 1 and the amphiphilic short peptide 2 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
Rhizopus chinensis lipase gene fusion parent short peptides 1 and 2 are fused according to the experimental method and sent to Huada gene company for sequencing, the results are shown as sequences SEQ ID NO.4 and SEQ ID NO.5, the transformed yeast strain has lipase activity, and a strain with high unit of the activity of the zymolase is selected respectively for fermentation to obtain enzyme liquid for enzymological property determination.
Example 2 Lipase optimum pH determination
Adjusting the pH of the buffer solution to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, diluting the enzyme solution to an adaptation multiple, measuring the optimum pH at 37 ℃ according to a lipase activity measuring method, and then continuously detecting the optimum value at half-filling points on both sides of the maximum value (for example, when the optimum pH is 9, measuring the pH at 8, 8.5, 9, 9.5 and 10 according to the lipase activity measuring method).
The optimum pH results of the lipase enzymatic reaction are shown in FIG. 2. The lipase fusion amphiphilic short peptides 1 and 2 and wild lipase are most suitable 9, and no obvious change exists.
Example 3 measurement of optimum temperature of Lipase
According to the determination method of lipase activity, under the condition of the above-mentioned optimum pH, the reactant is placed at different temperatures and reacted at 0 deg.C, 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, after the optimum temperature is determined, half-point is supplemented on both sides of the maximum value (for example, if the optimum temperature is 40 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C are determined according to the determination method of lipase activity).
The optimum temperature values of the lipase enzymatic reactions are shown in FIG. 3, and the optimum temperatures of the lipase gene fusion amphiphilic short peptides 1 and 2 and the wild-type lipase are 40 ℃.
Example 4 Lipase pH tolerance assay
The buffers were adjusted to different pH: 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, diluting the enzyme solution with these different buffers, timing from when the enzyme solution is put in, putting the diluted enzyme solution in a water bath kettle at 37 ℃ for 1 hour, putting on ice, and immediately performing the reaction at the optimum pH and the optimum temperature according to the lipase activity measuring method. The enzyme solution of the control group was an enzyme solution that was not tolerated.
As can be seen from FIG. 4, the tendency of the tolerance curve of the three lipases is the same, and the relative enzyme activity is obviously increased after the lipases are tolerated for 1 hour at 37 ℃ between pH4 and pH 9.
Example 5 Lipase temperature tolerance assay
Diluting the enzyme solution to corresponding times, and then putting the enzyme solution into a reaction kettle at different temperatures: tolerance is carried out at 60 ℃ for 1min, 3min, 5min, 7min, 10min, 15min, 20min, 25min, 30min, 35min, 45min and 60min, and then reaction is carried out at the optimum PH and the optimum temperature according to a lipase activity determination method. The control group enzyme solution was an enzyme solution that was not temperature-tolerant.
The temperature tolerance of the lipase at high temperature is shown in fig. 5, and the relative enzyme activity is continuously reduced along with the increase of temperature, and is gradually reduced along with the increase of time. The relative enzyme activity after fusion is higher than that before mutation at any temperature and time, the relative enzyme activity of the fusion type lipase 1 tolerating 20min at 60 ℃ is about 67.98%, the relative enzyme activity of the fusion type lipase 2 is about 59.65%, and the relative enzyme activity of the wild type lipase is only 50.68%. The time of half of the wild-type lipase remaining relative to the enzyme activity at 60 ℃ is about 20min, the time of half of the fused lipase 1 remaining relative to the enzyme activity is about 45min, and the time of half of the fused lipase 2 remaining relative to the enzyme activity is about 30min.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> university of Yunnan Master
<120> fusion type rhizopus chinensis lipase with improved heat stability and application thereof
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> PRT
<213> Lipase
<400> 1
Val Pro Val Ala Gly His Lys Gly Ser Val Lys Ala Thr Asn Gly Thr
1 5 10 15
Asp Phe Gln Leu Pro Pro Leu Ile Ser Ser Arg Cys Thr Pro Pro Ser
20 25 30
His Pro Glu Thr Thr Gly Asp Pro Asp Ala Glu Ala Tyr Tyr Ile Asn
35 40 45
Lys Ser Val Gln Trp Tyr Gln Ala His Gly Gly Asn Tyr Thr Ala Leu
50 55 60
Ile Lys Arg Asp Thr Glu Thr Val Gly Gly Met Thr Leu Asp Leu Pro
65 70 75 80
Glu Asn Pro Pro Pro Ile Pro Ala Thr Ser Thr Ala Pro Ser Ser Asp
85 90 95
Ser Gly Glu Val Val Thr Ala Thr Ala Ala Gln Ile Lys Glu Leu Thr
100 105 110
Asn Tyr Ala Gly Val Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro
115 120 125
Gly Thr Lys Trp Asp Cys Lys Gln Cys Leu Lys Tyr Val Pro Asp Gly
130 135 140
Lys Leu Ile Lys Thr Phe Thr Ser Leu Leu Thr Asp Thr Asn Gly Phe
145 150 155 160
Ile Leu Arg Ser Asp Ala Gln Lys Thr Ile Tyr Val Thr Phe Arg Gly
165 170 175
Thr Asn Ser Phe Arg Ser Ala Ile Thr Asp Met Val Phe Thr Phe Thr
180 185 190
Asp Tyr Ser Pro Val Lys Gly Ala Lys Val His Ala Gly Phe Leu Ser
195 200 205
Ser Tyr Asn Gln Val Val Lys Asp Tyr Phe Pro Val Val Gln Asp Gln
210 215 220
Leu Thr Ala Tyr Pro Asp Tyr Lys Val Ile Val Thr Gly His Ser Leu
225 230 235 240
Gly Gly Ala Gln Ala Leu Leu Ala Gly Met Asp Leu Tyr Gln Arg Glu
245 250 255
Lys Arg Leu Ser Pro Lys Asn Leu Ser Ile Tyr Cys Val Gly Cys Pro
260 265 270
Arg Val Gly Asn Asn Ala Phe Ala Tyr Tyr Val Asp Ser Thr Gly Ile
275 280 285
Pro Phe His Arg Thr Val His Lys Arg Asp Ile Val Pro His Val Pro
290 295 300
Pro Gln Ala Phe Gly Tyr Leu His Pro Gly Val Glu Ser Trp Ile Lys
305 310 315 320
Glu Asp Pro Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys
325 330 335
Gln Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Ile Ala Asp His Leu
340 345 350
Thr Tyr Phe Gly Ile Asn Glu Gly Ser Cys Leu
355 360
<210> 2
<211> 32
<212> PRT
<213> Amphiphilic short peptide
<400> 2
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
20 25 30
<210> 3
<211> 30
<212> PRT
<213> Amphiphilic short peptide
<400> 3
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Lys Val Ser Pro Glu Ala Val Lys Lys Glu Ala Glu Leu
20 25 30
<210> 4
<211> 395
<212> PRT
<213> Fusion lipase
<400> 4
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
20 25 30
Val Pro Val Ala Gly His Lys Gly Ser Val Lys Ala Thr Asn Gly Thr
35 40 45
Asp Phe Gln Leu Pro Pro Leu Ile Ser Ser Arg Cys Thr Pro Pro Ser
50 55 60
His Pro Glu Thr Thr Gly Asp Pro Asp Ala Glu Ala Tyr Tyr Ile Asn
65 70 75 80
Lys Ser Val Gln Trp Tyr Gln Ala His Gly Gly Asn Tyr Thr Ala Leu
85 90 95
Ile Lys Arg Asp Thr Glu Thr Val Gly Gly Met Thr Leu Asp Leu Pro
100 105 110
Glu Asn Pro Pro Pro Ile Pro Ala Thr Ser Thr Ala Pro Ser Ser Asp
115 120 125
Ser Gly Glu Val Val Thr Ala Thr Ala Ala Gln Ile Lys Glu Leu Thr
130 135 140
Asn Tyr Ala Gly Val Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro
145 150 155 160
Gly Thr Lys Trp Asp Cys Lys Gln Cys Leu Lys Tyr Val Pro Asp Gly
165 170 175
Lys Leu Ile Lys Thr Phe Thr Ser Leu Leu Thr Asp Thr Asn Gly Phe
180 185 190
Ile Leu Arg Ser Asp Ala Gln Lys Thr Ile Tyr Val Thr Phe Arg Gly
195 200 205
Thr Asn Ser Phe Arg Ser Ala Ile Thr Asp Met Val Phe Thr Phe Thr
210 215 220
Asp Tyr Ser Pro Val Lys Gly Ala Lys Val His Ala Gly Phe Leu Ser
225 230 235 240
Ser Tyr Asn Gln Val Val Lys Asp Tyr Phe Pro Val Val Gln Asp Gln
245 250 255
Leu Thr Ala Tyr Pro Asp Tyr Lys Val Ile Val Thr Gly His Ser Leu
260 265 270
Gly Gly Ala Gln Ala Leu Leu Ala Gly Met Asp Leu Tyr Gln Arg Glu
275 280 285
Lys Arg Leu Ser Pro Lys Asn Leu Ser Ile Tyr Thr Val Gly Cys Pro
290 295 300
Arg Val Gly Asn Asn Ala Phe Ala Tyr Tyr Val Asp Ser Thr Gly Ile
305 310 315 320
Pro Phe His Arg Thr Val His Lys Arg Asp Ile Val Pro His Val Pro
325 330 335
Pro Gln Ala Phe Gly Tyr Leu His Pro Gly Val Glu Ser Trp Ile Lys
340 345 350
Glu Asp Pro Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys
355 360 365
Gln Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Ile Ala Asp His Leu
370 375 380
Thr Tyr Phe Gly Ile Asn Glu Gly Ser Cys Leu
385 390 395
<210> 5
<211> 393
<212> PRT
<213> Fusion lipase
<400> 5
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Lys Val Ser Pro Glu Ala Val Lys Lys Glu Ala Glu Leu Val Pro
20 25 30
Val Ala Gly His Lys Gly Ser Val Lys Ala Thr Asn Gly Thr Asp Phe
35 40 45
Gln Leu Pro Pro Leu Ile Ser Ser Arg Cys Thr Pro Pro Ser His Pro
50 55 60
Glu Thr Thr Gly Asp Pro Asp Ala Glu Ala Tyr Tyr Ile Asn Lys Ser
65 70 75 80
Val Gln Trp Tyr Gln Ala His Gly Gly Asn Tyr Thr Ala Leu Ile Lys
85 90 95
Arg Asp Thr Glu Thr Val Gly Gly Met Thr Leu Asp Leu Pro Glu Asn
100 105 110
Pro Pro Pro Ile Pro Ala Thr Ser Thr Ala Pro Ser Ser Asp Ser Gly
115 120 125
Glu Val Val Thr Ala Thr Ala Ala Gln Ile Lys Glu Leu Thr Asn Tyr
130 135 140
Ala Gly Val Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro Gly Thr
145 150 155 160
Lys Trp Asp Cys Lys Gln Cys Leu Lys Tyr Val Pro Asp Gly Lys Leu
165 170 175
Ile Lys Thr Phe Thr Ser Leu Leu Thr Asp Thr Asn Gly Phe Ile Leu
180 185 190
Arg Ser Asp Ala Gln Lys Thr Ile Tyr Val Thr Phe Arg Gly Thr Asn
195 200 205
Ser Phe Arg Ser Ala Ile Thr Asp Met Val Phe Thr Phe Thr Asp Tyr
210 215 220
Ser Pro Val Lys Gly Ala Lys Val His Ala Gly Phe Leu Ser Ser Tyr
225 230 235 240
Asn Gln Val Val Lys Asp Tyr Phe Pro Val Val Gln Asp Gln Leu Thr
245 250 255
Ala Tyr Pro Asp Tyr Lys Val Ile Val Thr Gly His Ser Leu Gly Gly
260 265 270
Ala Gln Ala Leu Leu Ala Gly Met Asp Leu Tyr Gln Arg Glu Lys Arg
275 280 285
Leu Ser Pro Lys Asn Leu Ser Ile Tyr Cys Val Gly Cys Pro Arg Val
290 295 300
Gly Asn Asn Ala Phe Ala Tyr Tyr Val Asp Ser Thr Gly Ile Pro Phe
305 310 315 320
His Arg Thr Val His Lys Arg Asp Ile Val Pro His Val Pro Pro Gln
325 330 335
Ala Phe Gly Tyr Leu His Pro Gly Val Glu Ser Trp Ile Lys Glu Asp
340 345 350
Pro Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys Gln Cys
355 360 365
Ser Asn Ser Ile Val Pro Phe Thr Ser Ile Ala Asp His Leu Thr Tyr
370 375 380
Phe Gly Ile Asn Glu Gly Ser Cys Leu
385 390
<210> 6
<211> 1185
<212> DNA
<213> Coding gene
<400> 6
gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacca 60
actactccaa ctccaccaac tactccaact ccaactgttc ctgttgctgg tcataaaggt 120
tcagtcaagg caactaatgg tactgacttc caactccctc ctctcatctc tagcagatgt 180
actcctcctt cccatcctga aaccacaggt gatcctgatg ccgaagctta ctatattaac 240
aagagcgttc aatggtacca agctcacggt ggtaactaca ctgctcttat caagagagat 300
actgaaaccg tcggtggtat gaccttggat ttgcctgaga accctcctcc tattcctgcc 360
acgtccactg ctcctagctc tgattcaggt gaagttgtca cagccactgc tgctcaaatc 420
aaagagctca ctaactacgc tggtgttgct gctactgctt actgtagaag tgtcgttcca 480
ggtaccaagt gggactgtaa gcaatgtctc aagtatgttc ctgatggtaa gcttatcaag 540
accttcactt ctcttctcac tgataccaat ggttttatct tgagaagtga tgctcaaaag 600
accatctatg ttactttcag aggtactaat tccttcagaa gcgctattac tgacatggtc 660
ttcaccttta ctgattattc tcctgtcaag ggtgccaaag ttcacgctgg tttcctttcc 720
tcatacaacc aagttgtcaa agactacttc cctgtcgttc aagaccaatt gaccgcttac 780
cctgactata aggtcatcgt caccggtcac tctctcggtg gtgcccaagc cttgctcgct 840
ggtatggatc tctaccaaag agaaaagaga ttatctccta agaacttgag catctatact 900
gttggttgtc ctagagtcgg taacaatgca ttcgcttact acgtcgacag caccggaatt 960
cctttccaca gaaccgttca caagagagat atcgtccctc atgttcctcc tcaagccttc 1020
ggttatcttc accctggtgt cgaatcttgg atcaaggaag accctgctga tgttcaaatc 1080
tgtacttcca acattgaaac caaacaatgc agtaactcta tcgttccttt cacctctatc 1140
gctgatcact taacctactt tggtattaac gaaggaagct gtttg 1185
<210> 7
<211> 1167
<212> DNA
<213> Coding gene
<400> 7
gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgtt 60
aagaaggagg ctgagctcgt tcctgttgct ggtcataaag gttcagtcaa ggcaactaat 120
ggtactgact tccaactccc tcctctcatc tctagcagat gtactcctcc ttcccatcct 180
gaaaccacag gtgatcctga tgccgaagct tactatatta acaagagcgt tcaatggtac 240
caagctcacg gtggtaacta cactgctctt atcaagagag atactgaaac cgtcggtggt 300
atgaccttgg atttgcctga gaaccctcct cctattcctg ccacgtccac tgctcctagc 360
tctgattcag gtgaagttgt cacagccact gctgctcaaa tcaaagagct cactaactac 420
gctggtgttg ctgctactgc ttactgtaga agtgtcgttc caggtaccaa gtgggactgt 480
aagcaatgtc tcaagtatgt tcctgatggt aagcttatca agaccttcac ttctcttctc 540
actgatacca atggttttat cttgagaagt gatgctcaaa agaccatcta tgttactttc 600
agaggtacta attccttcag aagcgctatt actgacatgg tcttcacctt tactgattat 660
tctcctgtca agggtgccaa agttcacgct ggtttccttt cctcatacaa ccaagttgtc 720
aaagactact tccctgtcgt tcaagaccaa ttgaccgctt accctgacta taaggtcatc 780
gtcaccggtc actctctcgg tggtgcccaa gccttgctcg ctggtatgga tctctaccaa 840
agagaaaaga gattatctcc taagaacttg agcatctata ctgttggttg tcctagagtc 900
ggtaacaatg cattcgctta ctacgtcgac agcaccggaa ttcctttcca cagaaccgtt 960
cacaagagag atatcgtccc tcatgttcct cctcaagcct tcggttatct tcaccctggt 1020
gtcgaatctt ggatcaagga agaccctgct gatgttcaaa tctgtacttc caacattgaa 1080
accaaacaat gcagtaactc tatcgttcct ttcacctcta tcgctgatca cttaacctac 1140
tttggtatta acgaaggaag ctgtttg 1167
<210> 8
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 8
gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacc 59
<210> 9
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 9
agttggagtt ggagtagttg gtggagttgg agtagttggt ggagttggtt tagctttag 59
<210> 10
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 10
gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgt 59
<210> 11
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 11
gagctcagcc tccttcttaa cagcctccgg gctgactttc ggttcagctt cagctttag 59
<210> 12
<211> 44
<212> DNA
<213> Artificial Sequence
<400> 12
gaggctgaag cttacgtaga attcgctgaa gctgaagcta aagc 44
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 13
accagcaaca ggaacagttg gagttggagt 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 14
accagcaaca ggaacgagct cagcctcctt 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 15
actccaactc caactgttcc tcttgctggt 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 16
gttcctgttg ctggtcataa aggttcagtc 30
<210> 17
<211> 44
<212> DNA
<213> Artificial Sequence
<400> 17
tctaaggcga attaattcgc ggccgcctac aaacagcttc cttc 44
Claims (2)
1. Fusion type rhizopus chinensis (with improved heat stability)Rhizopus chinensis ) The lipase is characterized in that the amino acid sequence of the fusion type rhizopus chinensis lipase is shown as SEQ ID NO. 5.
2. Use of the fused Rhizopus chinensis lipase of claim 1 in feed additives.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910235208.4A CN109750014B (en) | 2019-03-27 | 2019-03-27 | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910235208.4A CN109750014B (en) | 2019-03-27 | 2019-03-27 | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109750014A CN109750014A (en) | 2019-05-14 |
CN109750014B true CN109750014B (en) | 2023-01-13 |
Family
ID=66409261
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910235208.4A Active CN109750014B (en) | 2019-03-27 | 2019-03-27 | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109750014B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109776686A (en) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | A kind of pattern of fusion lipase and its preparation method and application that thermostability improves |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3615512B2 (en) * | 2001-11-12 | 2005-02-02 | 日華化学株式会社 | Lipase activity activator, skin external preparation containing the lipase activity activator, skin external preparation for slimming, and bath preparation for slimming |
CA2822271A1 (en) * | 2010-12-20 | 2012-06-28 | E. I. Du Pont De Nemours And Company | An aqueous stable composition for delivering substrates for a depilatory product using peracetic acid |
CN105316310B (en) * | 2015-11-25 | 2019-03-01 | 江南大学 | A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising |
CN106520733B (en) * | 2016-10-19 | 2020-09-22 | 华南理工大学 | Beta-xylosidase enzyme aggregate and preparation method thereof |
CN106754848B (en) * | 2016-12-27 | 2020-11-03 | 江南大学 | Alkaline pectinase mutant with improved thermal stability |
CN110295159A (en) * | 2018-03-07 | 2019-10-01 | 江南大学 | A kind of enzyme mutant |
CN109161538B (en) * | 2018-09-29 | 2021-10-15 | 云南师范大学 | Lipase mutant with improved heat stability and application thereof |
-
2019
- 2019-03-27 CN CN201910235208.4A patent/CN109750014B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109776686A (en) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | A kind of pattern of fusion lipase and its preparation method and application that thermostability improves |
Also Published As
Publication number | Publication date |
---|---|
CN109750014A (en) | 2019-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109161538B (en) | Lipase mutant with improved heat stability and application thereof | |
CN112574974B (en) | Lipase mutant D163F with improved catalytic activity and application thereof | |
CN109776686B (en) | Fusion type lipase with improved heat stability as well as preparation method and application thereof | |
CN109750012B (en) | Lipase mutant and application thereof | |
CN109750013B (en) | Lipase mutant and preparation method and application thereof | |
CN109468301B (en) | Lipase mutant with improved thermal stability and preparation method and application thereof | |
CN108841809A (en) | With height than amylase mutant and its gene and application living and thermal stability | |
CN113846074A (en) | Thermomyces lanuginosus lipase mutant G91C and application thereof | |
CN111676210A (en) | Method for improving cellulase activity, cellulase mutant 5I77-M and application | |
CN115927250A (en) | Thermomyces lanuginosus lipase mutant with 256-site mutation and application thereof | |
CN107488644B (en) | Lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability and gene and application thereof | |
CN106635941B (en) | A kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strain | |
CN108359655B (en) | Lipase mutant TDL-mut with high thermal stability and coding gene thereof | |
CN109750014B (en) | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof | |
CN108277212B (en) | Lipase mutant Gly183Cys/Gly212Cys and gene and application thereof | |
CN107916257B (en) | T1 lipase mutant and application | |
CN108315312B (en) | Lipase TTL mutant with improved thermal stability and coding gene and application thereof | |
Ningsih et al. | Cloning and expression of gene encoding lipase from local isolate Bacillus cereus isolated from compost Jambangan Indonesia | |
CN107488647B (en) | Lipase TTL mutant TTL-Gly60Glu with improved thermal stability and gene and application thereof | |
CN107488648B (en) | Lipase TTL mutant TTL-Arg59Ser/Gly60Glu/Ser61Asn with improved thermal stability, gene and application | |
CN107488645B (en) | Lipase TTL mutant TTL-Ser61Asn with improved thermal stability and gene and application thereof | |
CN104404012B (en) | A kind of novel phytase | |
CN107151660B (en) | lipase variant Ala36Ser/Asp49His/Ala52Val/Asn55Asp | |
CN106434512B (en) | A kind of thermophilic esterase and its expression from Aquifex aeolicus bacterial strain | |
CN107488646B (en) | Lipase mutant TTL-Arg59Ser/Gly60Glu/Ser61Asn/Ile62Val and gene and application thereof |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
OL01 | Intention to license declared | ||
OL01 | Intention to license declared |