CN109750013B - 一种脂肪酶突变体及其制备方法和应用 - Google Patents
一种脂肪酶突变体及其制备方法和应用 Download PDFInfo
- Publication number
- CN109750013B CN109750013B CN201910235199.9A CN201910235199A CN109750013B CN 109750013 B CN109750013 B CN 109750013B CN 201910235199 A CN201910235199 A CN 201910235199A CN 109750013 B CN109750013 B CN 109750013B
- Authority
- CN
- China
- Prior art keywords
- lipase
- thr
- val
- gly
- ala
- 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 80
- 102000004882 Lipase Human genes 0.000 title claims abstract description 71
- 239000004367 Lipase Substances 0.000 title claims abstract description 70
- 235000019421 lipase Nutrition 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000003674 animal food additive Substances 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 claims 1
- 102000053602 DNA Human genes 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 241000235528 Rhizopus microsporus var. chinensis Species 0.000 abstract description 17
- 108090000623 proteins and genes Proteins 0.000 abstract description 16
- 150000001413 amino acids Chemical group 0.000 abstract description 10
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 abstract description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 abstract description 3
- 229960001230 asparagine Drugs 0.000 abstract description 3
- 235000009582 asparagine Nutrition 0.000 abstract description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 abstract 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000004474 valine Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 27
- 229940088598 enzyme Drugs 0.000 description 27
- 230000000694 effects Effects 0.000 description 16
- 230000035772 mutation Effects 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 235000019626 lipase activity Nutrition 0.000 description 9
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 238000009776 industrial production Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 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
- 239000000872 buffer Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000000758 substrate Substances 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 239000006228 supernatant Substances 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
- 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 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
- 108010011667 Ala-Phe-Ala Proteins 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
- 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
- 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
- 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
- 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 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
- 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 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
- 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 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
- 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
- 239000004472 Lysine Substances 0.000 description 2
- 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 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
- 238000012408 PCR amplification Methods 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
- 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
- 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
- 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
- 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 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
- 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
- 108010013835 arginine glutamate Proteins 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 108010060199 cysteinylproline Proteins 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 108010047926 leucyl-lysyl-tyrosine Proteins 0.000 description 2
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 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
- 108010020432 prolyl-prolylisoleucine Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- 108010073969 valyllysine Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- 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
- 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
- 102100031375 Endothelial lipase Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- FKESCSGWBPUTPN-FOHZUACHSA-N Gly-Thr-Asn Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O FKESCSGWBPUTPN-FOHZUACHSA-N 0.000 description 1
- 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 1
- 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 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 238000005915 ammonolysis reaction Methods 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
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960002303 citric acid monohydrate Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000001962 electrophoresis 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
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001139 pH measurement Methods 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
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 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
发明属于生物工程技术领域,公开了一种脂肪酶突变体及其制备方法和应用,所述的脂肪酶突变体的氨基酸序列如SEQ ID NO.2所示,脂肪酶突变体由编码华根霉脂肪酶氨基酸序列序列2中的131位缬赖氨酸变为天冬酰胺而获得,其编码基因如SEQ ID NO.3所示。该突变体具有较理想耐热特性,因此特别适合工业化大规模生产。
Description
技术领域
本发明属于生物工程技术领域,涉及一种脂肪酶突变体,具体为一种脂肪酶突变体及其制备方法和应用。
背景技术
脂肪酶(triacylglycerol acylhydrolase,EC 3.1.1.3,)属羧基酯水解酶,是一类特殊的酯酶,能逐步地将甘油三酯水解成脂肪酸、二酸甘油酯、单酸甘油酯及甘油;同时它还能催化酯的酸解、醇解、氨解以及转酯化和酯合成等反应。它以其特殊的生理生化特性被广泛地应用到生物能源、油脂加工、食品加工、皮革加工以及饲料添加等过程中。脂肪酶在生物柴油制备产业、再生能源的开发和环境保护中起着至关重要。在工业生产过程中,酶制剂往往会经历高温环境,而天然脂肪酶耐热性普遍较差,生产过程损失率高达70%—80%。天然脂肪酶在高温下失活的这一缺点,致使工业生产的成本大幅度升高,限制了脂肪酶在工业中的应用。
华根霉脂肪酶的热稳定性比较差,工业生产损失量比较大,因此生产成本也会大幅度提高。若能将华根霉脂肪酶的热稳定性提高,则其生产成本也会随之下降。因此,本发明通过对华根霉脂肪酶基因上的改造使其成为一种耐热性比较高的脂肪酶。
发明内容
本发明的目的在于提供一种脂肪酶突变体及其应用,旨在解决华根霉脂肪酶的耐热性较差和在工业生产上不理想的问题。
本发明具体通过以下技术方案实现:
一种脂肪酶突变体,该突变体由华根霉脂肪酶的氨基酸序列经取代得到,所述的华根霉脂肪酶氨基酸序列如SEQ ID NO.1所示。
具体的,所述的脂肪酶突变体由华根霉脂肪酶氨基酸序列SEQ ID NO.1中第131位赖氨酸变为天冬酰胺而获得。
本发明所述的脂肪酶突变体的氨基酸序列如SEQ ID NO.2所示。
所述的脂肪酶突变体的酶促反应最适pH值为9.0;最适温度为35℃;在pH4-pH10、37℃条件下,pH耐受1小时,残活还在50%,在pH7-pH10、37℃条件下,pH耐受1小时,残活还在80%以上,脂肪酶在50℃耐受75min、60℃时耐受30min残活都在50%以上。
在本发明另一方面,所述的氨基酸序列SEQ ID NO.2经修饰、缺失或添加一或几个氨基酸获得氨基酸序列,且保持只有90%的同源性的序列也在本发明的保护范围内。
所述的脂肪酶突变体编码基因的核苷酸序列如SEQ ID NO.3所示。
在本发明另一方面,与所述的编码基因SEQ ID NO.3编码相同的蛋白质,但因遗传密码的简并性而与SEQ ID NO.3所示的核苷酸序列或其互补序列不同的核苷酸序列也在本发明的保护范围内。
在本发明另一方面,本发明还包括携带有编码基因序列为SEQ ID NO.3的脂肪酶突变体的质粒。
本发明一并提供上述脂肪酶突变体的构建方法,包括以下步骤:
1)以华根霉脂肪酶基因连接到载体上的重组质粒为模板,分别设计引物进行突变PCR扩增;
2)添加lμLDMT消化酶于PCR产物中,混匀,孵育70min;
3)加入l0μL消化产物于DMT感受态细胞中,经冰浴冷却、热激、冷却后,加500μL LB培养基,37℃180转培养1h,离心保留部分上清液,悬浮沉淀,取全部菌液涂板,37℃过夜培养;
4)进行阳性克隆子筛选验证,挑取单菌落于相应抗性的LB培养基中,培养2-3h后PCR鉴定,将筛选出的阳性克隆子送出测序,测序结果与原序列比对。
在本发明另一方面,本发明还提供了一种脂肪酶突变体的基因的工程菌,所述的工程菌含有具有SEQ ID NO.3所示基因的载体。
所述的工程菌通过将SEQ ID NO.3所示的基因克隆到表达载体上,然后进行细胞转化,获得重组基因工程工程菌。
本发明所述的编码基因SEQ ID NO.3的表达载体选自pPIC9K、pPIC9、pPICZaA\B\C、pPICZA\B\C或PGAPZaA\B\C。
在本发明另一方面,本发明提供的脂肪酶突变体在饲料添加剂中的应用也在本发明的保护范围之内。
本发明的有益效果为:
本发明提供一种脂肪酶突变体,该突变后的基因除与pPIC9K构建重组质粒外,还可以与pPIC9、pPICZaA\B\C、pPICZA\B\C、PGAPZaA\B\C等表达载体构建重组质粒,转化相应宿主菌中,通过在平板中加入G418、Zeocin等抗生素,筛选获得脂肪酶突变体基因工程菌,然后通过发酵获得新的脂肪酶突变体。使其成为耐热性高的脂肪酶,该脂肪酶的突变体与脂肪酶在高温下耐受经实验证明,无论在任何温度和时间下,突变后的相对酶活都高于突变前的,在50℃耐受120min脂肪酶突变体相对酶活大约还有66.12%,而野生型脂肪酶仅仅剩余28.24%。在60℃时野生型脂肪酶相对酶活剩余一半时的时间大约为60min,而突变体相对酶活剩余一半时的时间大约为25min。该突变体的耐热性有了明显的提高,工业化生产中损失率有一定的降低。
附图说明
图1是本发明实施例提供的脂肪酶突变体的构建方法流程图;
图2是本发明实施例提供的最适pH的测定曲线图;
图3是本发明实施例提供的最适温度曲线图;
图4是本发明实施例提供的pH耐受曲线图;
图5是本发明实施例提供的50℃耐受曲线图;
图6是本发明实施例提供的60℃耐受曲线图。
具体实施方式
下面将结合本发明具体的实施例,对本发明技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
通过对华根霉脂肪酶的蛋白质空间结构进行模拟分析,确定其突变位点为第131位赖氨酸突变成天冬酰胺。具体实施方案为以华根霉脂肪酶基因为模板,通过基因的定点突变方法,将华根霉脂肪酶基因发生突变,获得了新的脂肪酶基因,将该突变基因与pPIC9、pPICZaA\B\C、pPICZA\B\C、PGAPZaA\B\C等载体相连构建重组质粒,转入相应宿主菌(GS115或X33、SMD1168、PICHIAPINK)中进行异源表达,发酵可以获得该脂肪酶突变体。该突变体能在酸性环境中很好发生作用,并且具有较理想耐热特性,适合耐高温制粒,因此适合工业上生产。
本发明实施例的脂肪酶突变后的突变体的氨基酸序列如SEQ ID NO.2所示。
1、实验材料和试剂
基因来源菌株:由本实验室筛选并保存的华根霉Rhizopus chinensis CCTCCM201021;表达宿主菌及载体:GS115和pPIC9K均购于Novagen公司;脂肪酶重组质粒由实验室构建;宿主菌:DMT感受态细胞购于北京全式金公司。
主要试剂:DNAMarker、蛋白Marker(TaKaRa公司);定点突变试剂盒(北京全式金公司),PlasmidMini Kit I(Omega公司)。
实验仪器:离心机(Eppendorf);PCR扩增仪(Bio-Rad);核酸电泳仪(Bio-Rad);蛋白电泳仪(Amersham Bioscience);凝胶成像仪(Bio-Rad)。
主要培养基:YPD、LB、酵母发酵培养基(FA与FB)均按照“Invitrogen公司操作手册”的推荐方法来配制。
2、脂肪酶酶活力的测定
在一定条件下每分钟水解p-NP底物而生成1μmoL的对硝基苯酚所需要的酶量,即一个酶活单位,以U表示。
1)实验仪器:恒温水浴锅;pH仪器;酶标仪(Bio-Rad)等。
2)实验材料:对硝基苯酚棕榈酸酯(p-NPC16)(Sigma公司)。
3)溶液配制:
pH缓冲液:0.1mol/L一水合柠檬酸缓冲液和0.1mol/L磷酸缓冲液(pH2-7);
0.1mol/L Tris-HCl缓冲液(pH7-9);
0.1mol/L甘氨酸-NaOH缓冲液(pH9-12)。
底物溶液:10mmol/L对硝基苯酚棕榈酸酯(p-NPC16)。
4)采用对硝基苯酚法(p-nitrophenol):总体系为500μL,其中含有50mmol/L缓冲液420μL,10mmol/L底物p-NP 30μL和稀释的酶液50μL。底物和缓冲液混合后在反应温度下预热2min,加入稀释酶液混匀,反应5min加入50μL 1.0mol/L的SDS终止反应,并加入500μL1.0mol/L的Na2CO3显色;在波长为405nm下测定其OD值。
实施例1脂肪酶突变体的制备
如图1所示,本发明实施例的脂肪酶突变体的获得方法包括以下步骤:
(1)定点突变:华根霉脂肪酶基因连接到载体上的重组质粒为模板,按照试剂盒说明书配制50μL突变体系进行突变PCR扩增。
(2)突变PCR验证:取10μL突变产物,进行0.8%琼脂糖凝胶电泳检测。条带正确后加1μLDMT消化酶于突变产物中,轻弹混匀,PCR仪中37℃孵育70min。
(3)转化:加入5μL突变后的产物于50μL DMT感受态细胞中,轻弹混匀,冰浴30min;42℃准确热激45s后立即置于冰上冷却l0min;加500μL LB培养基,180转,37℃培养lh;7000rpm离心3min,弃上部分清液,保留100-150μL上清轻弹悬浮菌体,取全部菌液涂板,37℃过夜培养。
(4)阳性克隆子验证:挑取单菌落于500μL相应抗性的LB培养基中,200rpm培养2-3h后PCR鉴定;将筛选出的阳性克隆子送出测序,测序结果与原序列比对。
(5)找出突变正确的重组质粒;将突变质粒转入毕赤酵母GS115或X33、SMD1168、PICHIAPINK中进行表达,发酵并进行对比测酶活,研究酶学及应用特性。
其中,步骤(1)中定点突变引物如下:
F:TGGGACTGTAAGCAACGTTCCAGGTACCAAT;
R:ATTGGTACCTGGAACGTTGCTTACAGTCCCA。
华根霉脂肪酶基因突变,按上实验方法进行突变后送华大基因公司测序,结果如序列SEQ ID NO.2,对应脂肪酶核苷酸序列如序列SEQ ID NO.3,转化的酵母菌株具有脂肪酶活性,选取一株发酵酶活性单位高的菌株进行发酵获得酶液进行酶学性质测定。
实施例2脂肪酶最适pH测定
将缓冲液pH调成2、3、4、5、6、7、8、9、10、11、12,将酶液稀释到适应的倍数,依照脂肪酶活测定方法在37℃测出最适pH之后在最大值两侧补半点继续检测最适值(例如最适pH为9,则再取pH8、8.5、9、9.5和10按照脂肪酶活测定方法进行测定)。
脂肪酶酶促反应最适pH值结果如图2所示。突变体与脂肪酶最适均为9,无明显变化。
实施例3脂肪酶最适温度测定
依照脂肪酶活测定方法测定,在上述最适pH的条件下,将反应物放在不同温度下反应0℃、10℃、20℃、30℃、40℃、50℃、60℃、70℃、80℃,测出最适温度后,在最大值两侧补半点(例如最适温度是40℃,则补充30℃、35℃、40℃、45℃、50℃按照脂肪酶活测定方法测定)。
脂肪酶酶促反应最适温度值如图3所示,突变体与脂肪酶最适温度为40。
实施例4脂肪酶pH耐受测定
将缓冲液调节到不同pH:2、3、4、5、6、7、8、9、10、11、12,用这些不同的缓冲液稀释酶液,从放入酶液之时开始计时,稀释好的酶液放入37℃水浴锅中耐受1小时放后在冰上,立即按照脂肪酶活测定方法在最适pH和最适温度下进行反应。对照组的酶液是未耐受过的酶液。
由图4可知,两种脂肪酶的耐受曲线趋势是相同的,在pH为4-9之间37℃耐受1小时相对酶活有了明显的升高。
实施例5脂肪酶温度耐受测定
将酶液稀释到相应倍数,然后放入不同温度:50℃下耐受5min、10min、15min、20min、30min、40min、50min、65min、80min、100min、120min;和60℃下耐受1min、3min、6min、10min、15min、20min、25min、30min、35min、45min、60min、75min、90min之后按照脂肪酶活测定方法在最适pH和最适温度下反应。对照实验组酶液是未温度耐受过的酶液。
高温下脂肪酶的温度耐受情况如图5图6所示,随着温度的升高,相对酶活不断降低,随着时间的增加,相对酶活也逐渐降低。无论在任何温度和时间下,突变后的相对酶活都高于突变前的,在50℃耐受120min脂肪酶突变体相对酶活大约还有66.12%,而野生型脂肪酶仅仅剩余28.24%。在60℃时野生型脂肪酶相对酶活剩余一半时的时间大约为60min,而突变体相对酶活剩余一半时的时间大约为25min。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
<110> 云南师范大学
<120> 一种脂肪酶突变体及其制备方法和应用
<160> 5
<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> 363
<212> PRT
<213> Lipase mutant
<400> 2
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 Asn 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> 3
<211> 1092
<212> DNA
<213> Coding gene
<400> 3
tgggactgta agcaatgtct caagtatgtt cctgatggta agcttatcaa gaccttcact 60
tctcttctca ctgataccaa tggttttatc ttgagaagtg atgctcaaaa gaccatctat 120
gttactttca gaggtactaa ttccttcaga agcgctatta ctgacatggt cttcaccttt 180
actgattatt ctcctgtcaa gggtgccaaa gttcacgctg gtttcctttc ctcatacaac 240
caagttgtca aagactactt ccctgtcgtt caagaccaat tgaccgctta ccctgactat 300
aaggtcatcg tcaccggtca ctctctcggt ggtgcccaag ccttgctcgc tggtatggat 360
ctctaccaaa gagaaaagag attatctcct aagaacttga gcatctatac tgttggttgt 420
cctagagtcg gtaacaatgc attcgcttac tacgtcgaca gcaccggaat tcctttccac 480
agaaccgttc acaagagaga tatcgtccct catgttcctc ctcaagcctt cggttatctt 540
caccctggtg tcgaatcttg gatcaaggaa gaccctgctg atgttcaaat ctgtacttcc 600
aacattgaaa ccaaacaatg cagtaactct atcgttcctt tcacctctat cgctgatcac 660
ttaacctact ttggtattaa cgaaggaagc tgtttgtaag ttcctgttgc tggtcataaa 720
ggttcagtca aggcaactaa tggtactgac ttccaactcc ctcctctcat ctctagcaga 780
tgtactcctc cttcccatcc tgaaaccaca ggtgatcctg atgccgaagc ttactatatt 840
aacaagagcg ttcaatggta ccaagctcac ggtggtaact acactgctct tatcaagaga 900
gatactgaaa ccgtcggtgg tatgaccttg gatttgcctg agaaccctcc tcctattcct 960
gccacgtcca ctgctcctag ctctgattca ggtgaagttg tcacagccac tgctgctcaa 1020
atcaaagagc tcactaacta cgctggtgtt gctgctactg cttactgtag aagtgtcgtt 1080
ccaggtacca at 1092
<210> 4
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 4
tgggactgta agcaacgttc caggtaccaa t 31
<210> 5
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 5
attggtacct ggaacgttgc ttacagtccc a 31
Claims (3)
1.一种脂肪酶突变体,其特征在于,所述的脂肪酶突变体的氨基酸序列如SEQ ID NO.2所示。
2.权利要求1所述的脂肪酶突变体的DNA分子。
3.权利要求1所述的脂肪酶突变体在饲料添加剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910235199.9A CN109750013B (zh) | 2019-03-27 | 2019-03-27 | 一种脂肪酶突变体及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910235199.9A CN109750013B (zh) | 2019-03-27 | 2019-03-27 | 一种脂肪酶突变体及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109750013A CN109750013A (zh) | 2019-05-14 |
CN109750013B true CN109750013B (zh) | 2023-01-13 |
Family
ID=66409469
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910235199.9A Active CN109750013B (zh) | 2019-03-27 | 2019-03-27 | 一种脂肪酶突变体及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109750013B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109161538B (zh) * | 2018-09-29 | 2021-10-15 | 云南师范大学 | 一种热稳性提高的脂肪酶突变体及其应用 |
CN112574974B (zh) * | 2019-06-17 | 2022-04-19 | 云南师范大学 | 一种催化活性提高的脂肪酶突变体d163f及其应用 |
CN112375751B (zh) | 2021-01-18 | 2021-04-06 | 凯莱英生命科学技术(天津)有限公司 | 脂肪酶突变体及其应用 |
CN113637653B (zh) * | 2021-08-05 | 2023-05-23 | 云南师范大学 | 一种活性提高的酯酶突变体Est8-XL及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102604909A (zh) * | 2009-11-11 | 2012-07-25 | 江南大学 | 定向进化构建的催化活力提高的脂肪酶突变体 |
CN109161538A (zh) * | 2018-09-29 | 2019-01-08 | 云南师范大学 | 一种热稳性提高的脂肪酶突变体及其应用 |
CN109750012A (zh) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | 一种脂肪酶突变体及其应用 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60124616T2 (de) * | 2000-05-08 | 2007-09-13 | Pfizer Products Inc., Groton | Enzymatische Spaltung von selektiven Modulatoren des Östrogenrezeptors |
ES2395582B1 (es) * | 2011-06-29 | 2013-12-26 | Consejo Superior De Investigaciones Científicas (Csic) | Procedimiento de acilación para la obtención de compuestos de interés alimenticio y/o farmacéutico utilizando esterol esterasas fúngicas. |
US10731110B2 (en) * | 2013-12-20 | 2020-08-04 | Novozymes A/S | Compositions and processes for treatment with lipases |
CN107567329B (zh) * | 2015-03-05 | 2022-04-26 | 路博润先进材料公司 | 皮壳正青霉的发酵提取物和其美容用途 |
EP3384019B1 (en) * | 2015-12-01 | 2020-06-24 | Novozymes A/S | Methods for producing lipases |
US20180271805A1 (en) * | 2017-03-27 | 2018-09-27 | NanoBiotech Pharma, Inc. | Composition for the treatment of human and mammalian atherosclerosis and pathological calcification in tissue |
CN112375751B (zh) * | 2021-01-18 | 2021-04-06 | 凯莱英生命科学技术(天津)有限公司 | 脂肪酶突变体及其应用 |
-
2019
- 2019-03-27 CN CN201910235199.9A patent/CN109750013B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102604909A (zh) * | 2009-11-11 | 2012-07-25 | 江南大学 | 定向进化构建的催化活力提高的脂肪酶突变体 |
CN109161538A (zh) * | 2018-09-29 | 2019-01-08 | 云南师范大学 | 一种热稳性提高的脂肪酶突变体及其应用 |
CN109750012A (zh) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | 一种脂肪酶突变体及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN109750013A (zh) | 2019-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109750013B (zh) | 一种脂肪酶突变体及其制备方法和应用 | |
CN109750012B (zh) | 一种脂肪酶突变体及其应用 | |
CN109161538B (zh) | 一种热稳性提高的脂肪酶突变体及其应用 | |
CN110129301B (zh) | 一种催化活性提高的脂肪酶突变体及其应用 | |
CN109776686B (zh) | 一种热稳性提高的融合型脂肪酶及其制备方法和应用 | |
CN109355275B (zh) | 高热稳定性β-葡萄糖苷酶突变体及其应用 | |
CN113862233B (zh) | 提高葡萄糖氧化酶的酸稳定性的方法及突变体q241e/r499e、基因和应用 | |
CN117625581B (zh) | 一种N-乙酰氨基葡萄糖苷酶突变体Ea2F及其应用 | |
CN113846074A (zh) | 一种疏棉状嗜热丝孢菌脂肪酶突变体g91c及其应用 | |
CN108841809A (zh) | 具有高比活及热稳定性的淀粉酶突变体及其基因和应用 | |
CN107916257B (zh) | T1脂肪酶突变体和应用 | |
CN106635941B (zh) | 一种来源于Aquifex aeolicus菌株的嗜热酯酶及其功能验证 | |
CN116640746A (zh) | 具有高耐热性的α-淀粉酶突变体、重组菌株及应用 | |
CN110117586B (zh) | 一种超耐热的木聚糖酶XynGold及基因与应用 | |
Ningsih et al. | Cloning and expression of gene encoding lipase from local isolate Bacillus cereus isolated from compost Jambangan Indonesia | |
CN108315312B (zh) | 一种热稳定性提高的脂肪酶ttl突变体及其编码基因和应用 | |
CN108277212B (zh) | 脂肪酶突变体Gly183Cys/Gly212Cys及其基因和应用 | |
CN108359655B (zh) | 一种热稳定性高的脂肪酶突变体TDL-mut及其编码基因 | |
CN116286748A (zh) | 具有高比活力的耐热α-淀粉酶突变体及其基因和应用 | |
CN109750014B (zh) | 一种热稳性提高的融合型华根霉脂肪酶及其应用 | |
Luo et al. | Effect of propeptide variation on properties of Rhizomucor miehei lipase | |
CN112501148B (zh) | 一种纤维素酶及其应用 | |
CN114807091A (zh) | 一种耐热性提高的疏棉状嗜热丝孢菌脂肪酶及应用 | |
CN112574975A (zh) | 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 | |
CN108165540B (zh) | 一种米黑根毛霉α-淀粉酶及其编码基因与应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
OL01 | Intention to license declared |