CN111057686B - 一种醇脱氢酶突变体及应用 - Google Patents
一种醇脱氢酶突变体及应用 Download PDFInfo
- Publication number
- CN111057686B CN111057686B CN201911342186.8A CN201911342186A CN111057686B CN 111057686 B CN111057686 B CN 111057686B CN 201911342186 A CN201911342186 A CN 201911342186A CN 111057686 B CN111057686 B CN 111057686B
- Authority
- CN
- China
- Prior art keywords
- gly
- alcohol dehydrogenase
- escherichia coli
- val
- leu
- 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
- 108010021809 Alcohol dehydrogenase Proteins 0.000 title claims abstract description 42
- 102000007698 Alcohol dehydrogenase Human genes 0.000 title claims abstract description 39
- 241000588724 Escherichia coli Species 0.000 claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 241000894006 Bacteria Species 0.000 claims abstract description 29
- 108010006519 Molecular Chaperones Proteins 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 39
- 238000006243 chemical reaction Methods 0.000 claims description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 18
- 239000005515 coenzyme Substances 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 8
- 150000002576 ketones Chemical class 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 58
- 108090000790 Enzymes Proteins 0.000 abstract description 58
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 27
- 239000000758 substrate Substances 0.000 abstract description 16
- 102000005431 Molecular Chaperones Human genes 0.000 abstract description 14
- 238000010353 genetic engineering Methods 0.000 abstract description 13
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 210000003000 inclusion body Anatomy 0.000 abstract description 9
- 230000035772 mutation Effects 0.000 abstract description 5
- 230000003197 catalytic effect Effects 0.000 abstract description 4
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
- 230000002708 enhancing effect Effects 0.000 abstract description 3
- 101000780495 Clostridium beijerinckii NADP-dependent isopropanol dehydrogenase Proteins 0.000 description 37
- 239000000243 solution Substances 0.000 description 34
- 239000013612 plasmid Substances 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 241000193454 Clostridium beijerinckii Species 0.000 description 9
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical compound CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 description 9
- 238000006555 catalytic reaction Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- QQZOPKMRPOGIEB-UHFFFAOYSA-N n-butyl methyl ketone Natural products CCCCC(C)=O QQZOPKMRPOGIEB-UHFFFAOYSA-N 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 239000002184 metal Substances 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- QNVRIHYSUZMSGM-UHFFFAOYSA-N hexan-2-ol Chemical compound CCCCC(C)O QNVRIHYSUZMSGM-UHFFFAOYSA-N 0.000 description 6
- 229930027917 kanamycin Natural products 0.000 description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 6
- 229960000318 kanamycin Drugs 0.000 description 6
- 229930182823 kanamycin A Natural products 0.000 description 6
- JYVLIDXNZAXMDK-UHFFFAOYSA-N pentan-2-ol Chemical compound CCCC(C)O JYVLIDXNZAXMDK-UHFFFAOYSA-N 0.000 description 6
- XNLICIUVMPYHGG-UHFFFAOYSA-N pentan-2-one Chemical compound CCCC(C)=O XNLICIUVMPYHGG-UHFFFAOYSA-N 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical group NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 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 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical group C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 4
- XJROSHJRQTXWAE-XGEHTFHBSA-N Pro-Cys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XJROSHJRQTXWAE-XGEHTFHBSA-N 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 108010092114 histidylphenylalanine Proteins 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- QNVRIHYSUZMSGM-LURJTMIESA-N 2-Hexanol Natural products CCCC[C@H](C)O QNVRIHYSUZMSGM-LURJTMIESA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 3
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 3
- 239000004471 Glycine Chemical group 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 108010079364 N-glycylalanine Proteins 0.000 description 3
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Chemical group OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical group CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Chemical group OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Chemical group OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 3
- 239000004474 valine Chemical group 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- KDZIGQIDPXKMBA-UHFFFAOYSA-N 2-[[2-[[2-[(2-amino-3-methylbutanoyl)amino]acetyl]amino]-3-hydroxypropanoyl]amino]pentanedioic acid Chemical compound CC(C)C(N)C(=O)NCC(=O)NC(CO)C(=O)NC(C(O)=O)CCC(O)=O KDZIGQIDPXKMBA-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 2
- FOWHQTWRLFTELJ-FXQIFTODSA-N Ala-Asp-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N FOWHQTWRLFTELJ-FXQIFTODSA-N 0.000 description 2
- 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 2
- YNOCMHZSWJMGBB-GCJQMDKQSA-N Ala-Thr-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O YNOCMHZSWJMGBB-GCJQMDKQSA-N 0.000 description 2
- XKHLBBQNPSOGPI-GUBZILKMSA-N Ala-Val-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N XKHLBBQNPSOGPI-GUBZILKMSA-N 0.000 description 2
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 2
- FFEUXEAKYRCACT-PEDHHIEDSA-N Arg-Ile-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(O)=O FFEUXEAKYRCACT-PEDHHIEDSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- PNHQRQTVBRDIEF-CIUDSAMLSA-N Asn-Leu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)N)N PNHQRQTVBRDIEF-CIUDSAMLSA-N 0.000 description 2
- ORJQQZIXTOYGGH-SRVKXCTJSA-N Asn-Lys-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ORJQQZIXTOYGGH-SRVKXCTJSA-N 0.000 description 2
- KTDWFWNZLLFEFU-KKUMJFAQSA-N Asn-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O KTDWFWNZLLFEFU-KKUMJFAQSA-N 0.000 description 2
- XBQSLMACWDXWLJ-GHCJXIJMSA-N Asp-Ala-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XBQSLMACWDXWLJ-GHCJXIJMSA-N 0.000 description 2
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 2
- ZSJFGGSPCCHMNE-LAEOZQHASA-N Asp-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N ZSJFGGSPCCHMNE-LAEOZQHASA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Chemical group OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- NITLUESFANGEIW-BQBZGAKWSA-N Cys-Pro-Gly Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O NITLUESFANGEIW-BQBZGAKWSA-N 0.000 description 2
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 2
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 2
- PMSDOVISAARGAV-FHWLQOOXSA-N Glu-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 PMSDOVISAARGAV-FHWLQOOXSA-N 0.000 description 2
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 2
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 2
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 2
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 2
- ORXZVPZCPMKHNR-IUCAKERBSA-N Gly-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 ORXZVPZCPMKHNR-IUCAKERBSA-N 0.000 description 2
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-N 0.000 description 2
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 2
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 2
- WTUSRDZLLWGYAT-KCTSRDHCSA-N Gly-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN WTUSRDZLLWGYAT-KCTSRDHCSA-N 0.000 description 2
- LKJCZEPXHOIAIW-HOTGVXAUSA-N Gly-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN LKJCZEPXHOIAIW-HOTGVXAUSA-N 0.000 description 2
- YADRBUZBKHHDAO-XPUUQOCRSA-N His-Gly-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](C)C(O)=O YADRBUZBKHHDAO-XPUUQOCRSA-N 0.000 description 2
- VXZZUXWAOMWWJH-QTKMDUPCSA-N His-Thr-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O VXZZUXWAOMWWJH-QTKMDUPCSA-N 0.000 description 2
- GYXDQXPCPASCNR-NHCYSSNCSA-N His-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N GYXDQXPCPASCNR-NHCYSSNCSA-N 0.000 description 2
- JHCVYQKVKOLAIU-NAKRPEOUSA-N Ile-Cys-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)O)N JHCVYQKVKOLAIU-NAKRPEOUSA-N 0.000 description 2
- WNQKUUQIVDDAFA-ZPFDUUQYSA-N Ile-Gln-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N WNQKUUQIVDDAFA-ZPFDUUQYSA-N 0.000 description 2
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 2
- TWYOYAKMLHWMOJ-ZPFDUUQYSA-N Ile-Leu-Asn Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O TWYOYAKMLHWMOJ-ZPFDUUQYSA-N 0.000 description 2
- YCKPUHHMCFSUMD-IUKAMOBKSA-N Ile-Thr-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCKPUHHMCFSUMD-IUKAMOBKSA-N 0.000 description 2
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 2
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- ZRLUISBDKUWAIZ-CIUDSAMLSA-N Leu-Ala-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O ZRLUISBDKUWAIZ-CIUDSAMLSA-N 0.000 description 2
- HVJVUYQWFYMGJS-GVXVVHGQSA-N Leu-Glu-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVJVUYQWFYMGJS-GVXVVHGQSA-N 0.000 description 2
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-N 0.000 description 2
- AAORVPFVUIHEAB-YUMQZZPRSA-N Lys-Asp-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O AAORVPFVUIHEAB-YUMQZZPRSA-N 0.000 description 2
- HAUUXTXKJNVIFY-ONGXEEELSA-N Lys-Gly-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAUUXTXKJNVIFY-ONGXEEELSA-N 0.000 description 2
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 2
- GAELMDJMQDUDLJ-BQBZGAKWSA-N Met-Ala-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O GAELMDJMQDUDLJ-BQBZGAKWSA-N 0.000 description 2
- WPTDJKDGICUFCP-XUXIUFHCSA-N Met-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CCSC)N WPTDJKDGICUFCP-XUXIUFHCSA-N 0.000 description 2
- AFFKUNVPPLQUGA-DCAQKATOSA-N Met-Leu-Ala Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O AFFKUNVPPLQUGA-DCAQKATOSA-N 0.000 description 2
- AXHNAGAYRGCDLG-UWVGGRQHSA-N Met-Lys-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O AXHNAGAYRGCDLG-UWVGGRQHSA-N 0.000 description 2
- HSJIGJRZYUADSS-IHRRRGAJSA-N Met-Lys-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HSJIGJRZYUADSS-IHRRRGAJSA-N 0.000 description 2
- ILKCLLLOGPDNIP-RCWTZXSCSA-N Met-Met-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ILKCLLLOGPDNIP-RCWTZXSCSA-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
- JNRFYJZCMHHGMH-UBHSHLNASA-N Phe-Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JNRFYJZCMHHGMH-UBHSHLNASA-N 0.000 description 2
- VUYCNYVLKACHPA-KKUMJFAQSA-N Phe-Asp-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N VUYCNYVLKACHPA-KKUMJFAQSA-N 0.000 description 2
- BNRFQGLWLQESBG-YESZJQIVSA-N Phe-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O BNRFQGLWLQESBG-YESZJQIVSA-N 0.000 description 2
- GCFNFKNPCMBHNT-IRXDYDNUSA-N Phe-Tyr-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)NCC(=O)O)N GCFNFKNPCMBHNT-IRXDYDNUSA-N 0.000 description 2
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 2
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- NRCJWSGXMAPYQX-LPEHRKFASA-N Ser-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N)C(=O)O NRCJWSGXMAPYQX-LPEHRKFASA-N 0.000 description 2
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 2
- YMEXHZTVKDAKIY-GHCJXIJMSA-N Ser-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO)C(O)=O YMEXHZTVKDAKIY-GHCJXIJMSA-N 0.000 description 2
- KAAPNMOKUUPKOE-SRVKXCTJSA-N Ser-Asn-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KAAPNMOKUUPKOE-SRVKXCTJSA-N 0.000 description 2
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 2
- 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 2
- ZTPXSEUVYNNZRB-CDMKHQONSA-N Thr-Gly-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZTPXSEUVYNNZRB-CDMKHQONSA-N 0.000 description 2
- AHOLTQCAVBSUDP-PPCPHDFISA-N Thr-Ile-Lys Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)[C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O AHOLTQCAVBSUDP-PPCPHDFISA-N 0.000 description 2
- NDXSOKGYKCGYKT-VEVYYDQMSA-N Thr-Pro-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O NDXSOKGYKCGYKT-VEVYYDQMSA-N 0.000 description 2
- TWJDQTTXXZDJKV-BPUTZDHNSA-N Trp-Arg-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O TWJDQTTXXZDJKV-BPUTZDHNSA-N 0.000 description 2
- GITNQBVCEQBDQC-KKUMJFAQSA-N Tyr-Lys-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O GITNQBVCEQBDQC-KKUMJFAQSA-N 0.000 description 2
- CVUDMNSZAIZFAE-TUAOUCFPSA-N Val-Arg-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N CVUDMNSZAIZFAE-TUAOUCFPSA-N 0.000 description 2
- CVUDMNSZAIZFAE-UHFFFAOYSA-N Val-Arg-Pro Natural products NC(N)=NCCCC(NC(=O)C(N)C(C)C)C(=O)N1CCCC1C(O)=O CVUDMNSZAIZFAE-UHFFFAOYSA-N 0.000 description 2
- UKEVLVBHRKWECS-LSJOCFKGSA-N Val-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)N UKEVLVBHRKWECS-LSJOCFKGSA-N 0.000 description 2
- 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 2
- 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 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
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 2
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 2
- -1 alcohol compound Chemical class 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 108010060035 arginylproline Proteins 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010015792 glycyllysine Proteins 0.000 description 2
- 108010037850 glycylvaline Proteins 0.000 description 2
- 108010036413 histidylglycine Proteins 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 108010000761 leucylarginine Proteins 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 108010038320 lysylphenylalanine Proteins 0.000 description 2
- 108010005942 methionylglycine Proteins 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000005839 oxidative dehydrogenation reaction Methods 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical group OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 229960000268 spectinomycin Drugs 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 108010000998 wheylin-2 peptide Proteins 0.000 description 2
- 101150007523 32 gene Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- YHKANGMVQWRMAP-DCAQKATOSA-N Ala-Leu-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YHKANGMVQWRMAP-DCAQKATOSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 1
- QEHMMRSQJMOYNO-DCAQKATOSA-N Arg-His-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N QEHMMRSQJMOYNO-DCAQKATOSA-N 0.000 description 1
- SSZGOKWBHLOCHK-DCAQKATOSA-N Arg-Lys-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N SSZGOKWBHLOCHK-DCAQKATOSA-N 0.000 description 1
- VIINVRPKMUZYOI-DCAQKATOSA-N Arg-Met-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VIINVRPKMUZYOI-DCAQKATOSA-N 0.000 description 1
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 1
- VHWNKSJHQFZJTH-FXQIFTODSA-N Asp-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N VHWNKSJHQFZJTH-FXQIFTODSA-N 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 241000224432 Entamoeba histolytica Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- GPISLLFQNHELLK-DCAQKATOSA-N Gln-Gln-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N GPISLLFQNHELLK-DCAQKATOSA-N 0.000 description 1
- OXEMJGCAJFFREE-FXQIFTODSA-N Glu-Gln-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O OXEMJGCAJFFREE-FXQIFTODSA-N 0.000 description 1
- XBWMTPAIUQIWKA-BYULHYEWSA-N Gly-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN XBWMTPAIUQIWKA-BYULHYEWSA-N 0.000 description 1
- HHSOPSCKAZKQHQ-PEXQALLHSA-N Gly-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN HHSOPSCKAZKQHQ-PEXQALLHSA-N 0.000 description 1
- JNGHLWWFPGIJER-STQMWFEESA-N Gly-Pro-Tyr Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JNGHLWWFPGIJER-STQMWFEESA-N 0.000 description 1
- FKYQEVBRZSFAMJ-QWRGUYRKSA-N Gly-Ser-Tyr Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FKYQEVBRZSFAMJ-QWRGUYRKSA-N 0.000 description 1
- RYAOJUMWLWUGNW-QMMMGPOBSA-N Gly-Val-Gly Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O RYAOJUMWLWUGNW-QMMMGPOBSA-N 0.000 description 1
- SKOKHBGDXGTDDP-MELADBBJSA-N His-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N SKOKHBGDXGTDDP-MELADBBJSA-N 0.000 description 1
- PMMMQRVUMVURGJ-XUXIUFHCSA-N Ile-Leu-Pro Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O PMMMQRVUMVURGJ-XUXIUFHCSA-N 0.000 description 1
- 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 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical group CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical group C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- LAPSXOAUPNOINL-YUMQZZPRSA-N Leu-Gly-Asp Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O LAPSXOAUPNOINL-YUMQZZPRSA-N 0.000 description 1
- KGCLIYGPQXUNLO-IUCAKERBSA-N Leu-Gly-Glu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O KGCLIYGPQXUNLO-IUCAKERBSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- SSJBMGCZZXCGJJ-DCAQKATOSA-N Lys-Asp-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O SSJBMGCZZXCGJJ-DCAQKATOSA-N 0.000 description 1
- DRCILAJNUJKAHC-SRVKXCTJSA-N Lys-Glu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DRCILAJNUJKAHC-SRVKXCTJSA-N 0.000 description 1
- AFLBTVGQCQLOFJ-AVGNSLFASA-N Lys-Pro-Arg Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O AFLBTVGQCQLOFJ-AVGNSLFASA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- FIRWJEJVFFGXSH-RYUDHWBXSA-N Phe-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 FIRWJEJVFFGXSH-RYUDHWBXSA-N 0.000 description 1
- SFKOEHXABNPLRT-KBPBESRZSA-N Phe-His-Gly Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)NCC(O)=O SFKOEHXABNPLRT-KBPBESRZSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- CMOIIANLNNYUTP-SRVKXCTJSA-N Pro-Gln-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O CMOIIANLNNYUTP-SRVKXCTJSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- CRZRTKAVUUGKEQ-ACZMJKKPSA-N Ser-Gln-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CRZRTKAVUUGKEQ-ACZMJKKPSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000186339 Thermoanaerobacter Species 0.000 description 1
- NQVDGKYAUHTCME-QTKMDUPCSA-N Thr-His-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O NQVDGKYAUHTCME-QTKMDUPCSA-N 0.000 description 1
- YUPVPKZBKCLFLT-QTKMDUPCSA-N Thr-His-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N)O YUPVPKZBKCLFLT-QTKMDUPCSA-N 0.000 description 1
- VMBBTANKMSRJSS-JSGCOSHPSA-N Trp-Glu-Gly Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VMBBTANKMSRJSS-JSGCOSHPSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- MVYRJYISVJWKSX-KBPBESRZSA-N Tyr-His-Gly Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)NCC(=O)O)N)O MVYRJYISVJWKSX-KBPBESRZSA-N 0.000 description 1
- IEBGHUMBJXIXHM-AVGNSLFASA-N Val-Lys-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)O)N IEBGHUMBJXIXHM-AVGNSLFASA-N 0.000 description 1
- LJSZPMSUYKKKCP-UBHSHLNASA-N Val-Phe-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 LJSZPMSUYKKKCP-UBHSHLNASA-N 0.000 description 1
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 1
- DLLRRUDLMSJTMB-GUBZILKMSA-N Val-Ser-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)O)N DLLRRUDLMSJTMB-GUBZILKMSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940007078 entamoeba histolytica Drugs 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Chemical group CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
-
- 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
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种醇脱氢酶突变体及应用。所述醇脱氢酶突变体的氨基酸序列如SEQ ID NO.2所示。所述醇脱氢酶突变体由野生型酶经22个位点的点突变获得，显著提高了在大肠杆菌中活性表达能力。以异丙醇为底物，本发明的醇脱氢酶突变体的催化活力和热稳定性显著提高。通过强化大肠杆菌中分子伴侣GroES‑EL蛋白的表达，构建了一种全新的大肠杆菌基因工程菌，用于表达所述醇脱氢酶突变体，基本解决了包涵体的问题，发酵酶活进一步提高至38.20U/mL。
Description
技术领域
本发明涉及酶工程技术领域，特别是涉及一种醇脱氢酶突变体及应用。
背景技术
醇脱氢酶(alcohol dehydrogenase，ADH)是广泛存在于细菌和真核生物中的一种酶。醇脱氢酶在一定条件下，可以催化醇的氧化脱氢，生成相应的酮，与此同时将氧化型辅酶NAD(P)+转化为还原型辅酶NAD(P)H；也可以在消耗等量的还原型辅酶NAD(P)H的同时、将酮还原为相应的醇。因此，醇脱氢酶在辅酶的循环再生、手性醇的不对称合成等领域中有广泛的应用。来自拜氏梭菌Clostridium beijerinckii的醇脱氢酶(下称CbADH)是一种NADPH依赖型醇脱氢酶，以异丙醇为底物再生NADPH时、其单位蛋白的活力是目前文献报道的较高水平。
Edi Goihberg等人(A Single Proline Substitution is Critical for theThermostabilization of Clostridium beijerinckii AlcoholDehydrogenase.PROTEINS:Structure,Function,and Bioinformatics 66:196–204(2007))比较了多种NADPH依赖型醇脱氢酶、如来源于布罗基热厌氧菌Thermoanaerobacterbrockii的TbADH，来源于痢疾变形虫Entamoeba histolytica的EhADH1，以及来源于拜氏梭菌Clostridium beijerinckii的CbADH的催化活力，其中，CbADH的Kcat值为9714min-1，远高于TbADH(1724min-1)和EhADH1(3025min-1)，这说明CbADH比活力最高。
由于野生菌Clostridium beijerinckii表达CbADH的总量较低，而且培养过程复杂、产量低，因此，需要对CbADH进行异源重组表达。大肠杆菌基因工程菌是一个重组蛋白生产的首选菌株：它在廉价的培养基质中可以达到较高的细胞生长密度，而且，其遗传学研究有较为透彻，并有大量的改造过的克隆载体来实现包括酶在内的各种蛋白质的生物合成。使用常规的大肠杆菌基因工程菌重组表达CbADH，提高了其总体表达量。但在大肠杆菌基因工程菌中表达CbADH时，重组基因产物的高水平表达导致了酶蛋白的错误折叠，形成了大量的不具有生物活性的聚集体、即包涵体，限制了CbADH的工业化应用。例如，Moshe Peretz等人(Molecular Cloning,Nucleotide Sequencing,and Expression of Genes EncodingAlcohol Dehydrogenases From the Thermophile Thermoanaerobacter brockii andthe Mesophile Clostridium beijerinckii.Anaerobe(1997)3,259–270)在大肠杆菌Escherichia coli strain TG1中异源表达了CbADH，酶活只有3.38U/mL发酵液(底物为异丙醇，测活温度为40℃)。
发明内容
本发明针对来源于拜氏梭菌Clostridium beijerinckii的醇脱氢酶在大肠杆菌中异源表达存在包涵体，活性表达低的问题，提供了22个点突变组合的醇脱氢酶突变体，有效改进了其热稳定性、降低了包涵体的生成量、提高了其活性表达率。此外，通过在大肠杆菌基因工程菌的基因组中引入一种分子伴侣的强启动子基因、构建了一种全新的大肠杆菌基因工程菌，用于表达此醇脱氢酶突变体，基本解决了包涵体的问题，进一步提高了其发酵酶活。
一种醇脱氢酶突变体，氨基酸序列如SEQ ID NO.2所示。来源于拜氏梭菌Clostridium beijerinckii的野生型醇脱氢酶的氨基酸序列如SEQ ID NO.1所示，将该野生型酶进行22个点突变获得氨基酸序列如SEQ ID NO.2所示的醇脱氢酶突变体，具体22个点突变为：第18位谷氨酸突变为脯氨酸；第24位丝氨酸突变为脯氨酸；第45位苯丙氨酸突变为色氨酸；第51位天冬氨酸突变为谷氨酸；第53位赖氨酸突变为组氨酸；第100位谷氨酰胺突变为脯氨酸；第121位甘氨酸突变为丙氨酸；第135位异亮氨酸突变为组氨酸；第138位赖氨酸突变为天冬氨酸；第144位天冬酰胺突变为谷氨酰胺；第182位甘氨酸突变为丙氨酸；第196位甘氨酸突变为丙氨酸；第222位组氨酸突变为天冬氨酸；第250位丝氨酸突变为谷氨酸；第254位丝氨酸突变为赖氨酸；第302位丙氨酸突变为甲硫氨酸；第310位缬氨酸突变为谷氨酰胺；第312位天冬酰胺突变为甘氨酸；第323位缬氨酸突变为精氨酸；第324位酪氨酸突变为苯丙氨酸；第335位亮氨酸突变为精氨酸；第347位丙氨酸突变为缬氨酸。
本发明又提供了编码所述醇脱氢酶突变体的基因。优选的，所述的基因，核苷酸序列如SEQ ID NO.3所示。
本发明又提供了包含所述基因的表达载体。本发明所述的表达载体可以是pET-30a(+)，pET-21a(+)，pET-22b(+)，pET-28a(+)，pETDuet-1，pACYCDuet-1，pCDFDuet-1和RSFDuet-1，但不仅限于所述的这些载体。较佳的是将突变后的醇脱氢酶基因连接在表达载体pET-28a(+)上，形成重组表达载体pET-28a(+)-CbADH-22M。
本发明又提供了包含所述表达载体的基因工程菌。优选的，所述的基因工程菌使用的宿主细胞为大肠杆菌。
更优选的，所述大肠杆菌高表达分子伴侣GroES-EL。现有技术中，来源于拜氏梭菌Clostridium beijerinckii的野生型醇脱氢酶在使用常规的大肠杆菌进行表达时，容易形成包涵体，所以表达产物酶活较低。本发明经研究发现，在大肠杆菌中同时高表达分子伴侣GroES-EL，分子伴侣GroES-EL能够促进表达蛋白的正确折叠，可以提高重组CbADH蛋白的活性表达率。
高表达分子伴侣GroES-EL的方式可以是外源导入携带分子伴侣GroES-EL表达相关基因的质粒，也可以将大肠杆菌原基因组中自带的基因序列进行改造。更优选的，所述大肠杆菌基因组中启动表达分子伴侣GroES-EL的启动子被替换为组成型启动子J23104。将大肠杆菌工程菌基因组中分子伴侣GroES-EL基因前端的原始启动子基因σ26-σ32替换为高强度的新型组成型启动子J23104，进而实现强化表达分子伴侣GroES-EL蛋白。σ26-σ32基因序列为SEQ ID NO.4；所述J23104基因序列为SEQ ID NO.5。
本发明还提供了所述醇脱氢酶突变体或所述基因工程菌在催化醇+氧化型辅酶NADP+与酮+还原型辅酶NADPH的互相转化中的应用。
所述的应用中互相转化的反应如式Ⅰ所示，
式Ⅰ中：
R1为H或具有1-6个C原子的烷基；
R2为H或具有1-6个C原子的烷基；
但R1和R2不能同时为H。
醇脱氢酶在一定条件下，可以催化醇的氧化脱氢，生成相应的酮，与此同时将氧化型辅酶NAD(P)+转化为还原型辅酶NAD(P)H；也可以在消耗等量的还原型辅酶NAD(P)H的同时、将酮还原为相应的醇。
优选的，所述的醇化合物为乙醇、正丙醇、异丙醇、2-丁醇、2-戊醇、2-己醇；对应的酮或醛化合物为乙醛、丙醛、丙酮、2-丁酮、2-戊酮、2-己酮。
与现有技术相比，本发明具有以下有益效果：
(1)本发明通过理性设计的分子改造方法，显著提高了源于拜氏梭菌Clostridiumbeijerinckii的醇脱氢酶在Escherichia coli BL21菌株中活性表达能力，有效解决了包涵体的问题。以异丙醇为底物，本发明的醇脱氢酶突变体的催化活力显著提高，单位LB摇瓶发酵液的酶活最高达16.65U/mL，为野生型的2.01倍；热稳定性显著提高，T5060min(孵育60min失去50％活性的温度)从67.7℃提高到77.1℃。
(2)通过将基因编辑技术改造大肠杆菌基因工程菌的基因组，强化分子伴侣GroES-EL蛋白的表达，从而构建了一种全新的大肠杆菌基因工程菌，用于表达此醇脱氢酶突变体，基本解决了包涵体的问题，将其LB摇瓶发酵酶活进一步提高至38.20U/mL。
(3)本发明改造得到的醇脱氢酶基因，该基因表达后成功获得具有高表达量、高活性以及高稳定性的醇脱氢酶，而且这种醇脱氢酶底物谱广、具手性选择性。
附图说明
图1为CbADH的野生型、突变体和表达分子伴侣的突变体的SDS-PAGE蛋白电泳结果图，其中，箭头所指即为CbADH酶蛋白。
图2为CbADH的野生型(CbADH-WT)和突变体(CbADH-22M)的热稳定性测定结果图。
具体实施方式
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。CRISPR基因组编辑方法参考文献MultigeneEditing in the Escherichia coli Genome via the CRISPR-Cas9 System(Appl.Environ.Microbiol.2015,81(7):2506-14.)。
本发明所涉及带有醇脱氢酶基因的重组大肠杆菌，所用载体为pET-28a(+)，所用宿主为大肠杆菌Escherichia coli BL21(DE3)，使用的基因编辑质粒pCas，pTargetF来源于中国科学院上海生命科学研究院植物生理生态研究所。制备化转感受态细胞的试剂盒购自TAKARA公司
下游催化工艺所用试剂：异丙醇购自自国药集团化学试剂有限公司；NADP+购于邦泰生物工程(深圳)有限公司；其他常用试剂购自国药集团化学试剂有限公司；纯化酶使用的HisPurTM Ni-NTA Resin来自美国Thermo Fisher Scientific公司；NADP+/NADPHQuantitation Kit购自齐一生物科技(上海)有限公司。在本申请文本中所使用的氨基酸三字母或单字母表达方式，采用IUPAC规定的氨基酸代码(Eur.J.Biochem.,138:9-37,1984)。
醇脱氢酶酶活标准检测体系：适量的酶液、50mM底物、1mM NADPH，总体系为1000μL，反应介质为pH 7.5的0.1M磷酸盐缓冲液。35℃反应1min，酶活(即NADPH的生成速度)用1min之内340nm的分光值变化值来计算。
实施例1 微生物培养与酶活测定
1.1微生物的培养
LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。LB固体培养基(平板培养皿)：在LB液体培养基基础上加入20g/L琼脂粉，121℃灭菌、降温后导入培养皿，制作成平板。
将含有相关基因的工程菌E.coli BL21(DE3)接种至含50μg/mL卡那霉素的5mL LB液体培养基中，37℃震荡培养12h。转接至500mL同样含50μg/mL Kan的新鲜LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入IPTG至其浓度为0.3mM，28℃下诱导培养20h。培养结束后，将培养液10000rpm离心10min，弃上清，收集菌体细胞，放到-70℃超低温冰箱中保存，待用。
1.2粗酶液的制备
将培养结束后收集到的菌体细胞，用50mM Tris-HCl(pH 7.0)的缓冲液洗涤菌体两次。之后将菌体重悬于Tris-HCl(50mM，pH 7.5，20mM咪唑，0.3M NaCl，5mM二硫苏糖醇)缓冲液中，超声破碎菌悬液，离心去除沉淀，得到的上清为相应酶的粗酶液。
其他酶类的粗酶液制备方法同上。
1.3酶活力的测定
酶活定义：1961年国际酶学会议规定，1个酶活力单位是指在特定条件下，在1分钟内能转化1微摩尔底物的酶量，或是转化底物中1微摩尔的有关基团的酶量。
醇脱氢酶酶活测定：取底物溶液(100mM异丙醇水溶液，事先用氨水调pH到8.0)950μL，加入10mM NADP+溶液25μL，置于金属浴震荡器中，35℃保温10min；加入25μL粗酶液，迅速取出用手震荡、到入比色皿中，迅速放入分光光度计内，以时间为横坐标(单位min)、340nm吸光值为纵坐标测定吸光值随时间的变化率，根据NADPH摩尔吸光系数，即可计算酶活。
实施例2 构建过表达分子伴侣GroES-EL的大肠杆菌基因工程菌
使用CRISPR基因编辑技术，将大肠杆菌工程菌基因组中表达分子伴侣GroES-EL基因前端的弱启动子σ26-σ32(基因序列为SEQ ID NO.4)替换为强度较高的组成型启动子J23104(来源于http://parts.igem.org/Promoters/Catalog/Anderson，基因序列为SEQID NO.5)。
具体操作步骤如下：
(一)制备携带pCas质粒的大肠杆菌电转感受态细胞
1)制备化转感受态细胞：挑取大肠杆菌E coli BL21(DE3)单菌落于5mL卡那霉素浓度为50μg/mL的LB液体培养基试管中培养6-8h后，按2％接种量接种于50mL三角瓶中，37℃，200rpm培养2h，将三角牌置于冰上冷却30min。取菌液10mL于10mL离心管中，4000rpm，4℃下离心3min，除去上清。加入1mL预冷过的Solution A，用移液器轻轻混匀，4000rpm，4℃下离心3min，除去上清。加入1mL预冷过的Solution B，用移液器轻轻混匀，按每份100μL分装至离心管中，即得到大肠杆菌工程菌感受态细胞。
2)转化操作：将10μL pCas质粒加入100μL的大肠杆菌工程菌感受态细胞中，轻轻混匀，在冰上放置30min。将EP管放在42℃水浴中热击90s，拿出放在冰上冷却2min，在超净工作台中加入890μL LB液体培养基，转移到30℃，200rpm摇床孵育45min。经过6000rpm，2min离心，弃掉900μL上清液，将剩余100μL上清液和沉淀用移液器打匀，转移到卡那霉素浓度为50μg/mL的LB固体平板上，用涂布棒将菌体混匀。将平板倒置放在30℃恒温培养箱中培养12h，挑取单菌落至5mL卡那霉素浓度为50μg/mL LB液体培养基试管中培养6-8h，即完成pCas质粒转化到大肠杆菌工程菌中的操作。
3)制备电转感受态细胞：挑取单菌落于5mL卡那霉素浓度为50μg/mL的LB液体培养基试管中培养6-8h后，按2％接种量接种于50mL三角瓶中，37℃，200rpm培养2h，加入1mL浓度为250mg/mL的阿拉伯糖，在37℃，200rpm诱导1h，等到OD600达到0.6-0.8时取出。将三角瓶置于冰上冷却30min。取菌液10mL于10mL离心管中，4000rpm，4℃下离心3min，除去上清。用1mL 10％甘油重悬，在4℃下4000rpm离心10min，去上清，重复三次。用100μL 10％的甘油重悬，即得到含pCas质粒的大肠杆菌电转感受态细胞。放在-80℃冰箱中保存待用。
(二)更换pTargetF质粒的N20区域
将pTargetF质粒的N20区域更换为大肠杆菌基因组中分子伴侣GroES-EL基因前端的一段带NGG的20bp片段CCATTTCTCTGGTCACCAGC，以实现CRISPR编辑中的定位功能。操作如下：使用引物pTarget-1/pTarget-2对pTargetF模板进行PCR扩增，
引物pTarget-1/pTarget-2如下所示：
pTarget-1：CATTTCTCTGGTCACCAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA
pTarget-2：CTGGTGACCAGAGAAATGGACTAGTATTATACCTAGGACTGAGCTAGCTG
PCR体系如下：
PCR扩增条件：
1)预变性：98℃5min；
2)变性：98℃10s；退火：58℃15s；延伸：72℃30s；共循环35次；
3)延伸：72℃10min；
4)4℃保存2.0h。
PCR扩增结束后，扩增产物用1.0％琼脂糖凝胶电泳来检测，结果显示扩增产物为单一条带，大小为2200bp左右。用DNA回收纯化试剂盒对扩增产物进行纯化回收，具体步骤参照纯化试剂盒说明书。即得到改造后的pTargetF质粒。
(三)制备DonorDNA操作如下：
取含大肠杆菌的菌液20μL，在99℃下加热10min，作为大肠基因组模板待用。分别使用引物UF/DF-104和DR/UR-104对上面得到的大肠杆菌基因组模板进行扩增。
引物UF/DF-104和DR/UR-104如下所示：
UF：CGGCAGCAATTTGTAGCGCGGCAGGC
DR：ATTTATCCAGAACTACGTTACGGCC
DF-104：
TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCATAACAGATACGGACTTTCTC
UR-104：
GCTAGCACAATACCTAGGACTGAGCTAGCTGTCAACCCCTTCAAGGGGGAAAAAG
PCR体系如下：
PCR扩增条件：
1)预变性：98℃5min；
2)变性：98℃10s；退火：58℃15s；延伸：72℃10s；共循环35次；
3)延伸：72℃10min；
4)4℃保存2.0h。
PCR扩增结束后，扩增产物用1.0％琼脂糖凝胶电泳来检测，结果显示扩增产物为单一条带，大小都为500bp左右。用DNA回收纯化试剂盒对扩增产物进行纯化回收，具体步骤参照纯化试剂盒说明书。
将回收的两种扩增产物作为扩增模板，使用引物UF/DR进行扩增。
PCR扩增体系：
PCR扩增条件：
1)预变性：98℃5min；
2)变性：98℃10s；退火：58℃15s；延伸：72℃10s；共循环35次；
3)延伸：72℃10min；
4)4℃保存2.0h。
PCR扩增结束后，扩增产物用1.0％琼脂糖凝胶电泳来检测，结果显示扩增产物为单一条带，大小为1000bp左右。用DNA回收纯化试剂盒对扩增产物进行纯化回收，具体步骤参照纯化试剂盒说明书。即得到DonorDNA，序列如SEQ ID NO.6所示。
(四)电转化
把pTargetF质粒和DonorDNA各10μL加入步骤(一)制备携带pCas质粒的大肠杆菌电转感受态细胞中，用移液器轻轻混匀。转移到电转杯中，在电转仪中以200Ω，25μFd，2.5千伏进行电转操作，结束后加入1mL LB培养基，在30℃，150rpm下培养3h，转移到含卡那霉素浓度为50μg/mL、壮观霉素浓度为100μg/mL的LB固体平板上，用涂布棒将菌体混匀。将平板倒置放在30℃恒温培养箱中培养24h。
(五)菌落PCR验证。
挑步骤(四)中获得的单菌落转移至10μL水中，在99℃下放置10min，作为菌落PCR的模板。使用验证引物U/D对模板进行扩增，并将扩增产物送至测序公司测序，确定基因编辑成功。
验证引物U/D如下所示：
U：CGCCAGCGTACCGAGTTCTGCC
D：TTCCAGTTCGATTTCACGAGCAACGG
PCR体系和条件与步骤(三)相同。
(六)消除质粒
将单菌落挑取至含0.5mM IPTG的5mL LB液体培养基中，在37℃，200rpm下培养12h，取少量菌液在含壮观霉素浓度为100μg/mL的LB固体平板划线，37℃下培养12h，如果没有菌体生长，说明pTarget质粒已经被消除。在含Kan浓度为50μg/mL的LB固体平板划线，37℃下培养12h，如果没有菌体生长，说明pCas质粒已经被消除。即得到强化表达分子伴侣GroES-EL的基因工程菌。
实施例3 构建CbADH野生型及突变体重组菌
委托北京擎科新业生物技术有限公司提供密码子优化和基因合成服务，将CbADH野生型(NCBI登录号WP_077844196.1)及突变体的基因(SEQ ID NO.3)合成在pET-28a(+)质粒上，并带有用于编码6×His标签的序列(位于蛋白的C末端)，便于蛋白纯化，将目的基因置于酶切位点Nco I和Xho I之间。获得pET-28a(+)-CbADH-WT和pET-28a(+)-CbADH-22M重组质粒，并将其分别转化进入实施例2所得到的大肠杆菌基因工程菌(强化表达分子伴侣GroES-EL)中。
野生型基因序列表达的野生型CbADH蛋白的氨基酸序列如SEQ ID NO.1所示，突变体基因序列表达的突变体CbADH蛋白的氨基酸序列如SEQ ID NO.2所示，具体突变位点如表1所示。
表1
实施例4 CbADH野生型及突变体的热稳定性测定
将实施例3得到的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得相应的粗酶液并进行蛋白电泳测定。
酶的温度稳定性用T5060min表征：酶液在不同的温度下保存10min，然后利用酶活检测体系测定剩余酶活，酶活降低至原始酶活的50％所对应的温度即为T5060min。用HisPurTMNi-NTAResin将粗酶液纯化为纯酶液。将野生型和突变体纯酶液分别放置在不同温度下60min，测定剩余酶活的量，计算出T5060min的值。野生型CbADH的T5060min为67.7℃，突变体的T5060min为77.1℃。蛋白电泳结果如图1所示。野生型和突变体纯酶的失活曲线如图2所示。
实施例5 应用CbADH突变体催化乙醇和NADP+生成还原型辅酶NADPH
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。
反应体系为1mL，含有50mM底物乙醇，1mM NADP+，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。通过NADP/NADPH定量测定试剂盒测得该酶在1min之内催化50mM的乙醇和1mM NADP+生成NADPH为0.02mM。
实施例6 催化正丙醇和NADP+生成还原型辅酶NADPH
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方面进行培养，获得粗酶液。
反应体系为1mL，含有50mM底物正丙醇，1mM NADP+，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。通过NADP/NADPH定量测定试剂盒测得该酶在1min之内催化50mM的正丙醇和1mM NADP+生成NADPH为0.01mM。
实施例7 催化异丙醇和NADP+生成还原型辅酶NADPH
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。
反应体系为1mL，含有50mM底物异丙醇，1mM NADP+，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。利用分光光度计检测1min之内340nm下吸光值的变化。通过计算得到CbADH突变体在该反应中粗酶液酶活为38.20U/mL。通过NADP/NADPH定量测定试剂盒测得该酶在1min之内催化50mM的异丙醇和1mM NADP+生成NADPH为0.65mM。
对比例1
将pET-28a(+)-CbADH-WT和pET-28a(+)-CbADH-22M重组质粒分别转化进入常规的、未经改造的大肠杆菌工程菌Escherichia coli BL21(DE3)中。
将得到的表达CbADH野生型和突变体的常规大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。并测定pET-28a(+)-CbADH-WT在常规大肠杆菌工程菌Escherichia coli BL21(DE3)中的蛋白表达(蛋白电泳实验)，蛋白电泳实验结果如图1所示。
反应体系为1mL，含有50mM底物异丙醇，1mM NADP+，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。利用分光光度计检测1min之内340nm下吸光值的变化。通过计算得到CbADH野生型在常规大肠杆菌基因工程菌的表达，粗酶液酶活为8.27U/mL；CbADH突变体在常规大肠杆菌基因工程菌的表达，粗酶液酶活为16.65U/mL。
实施例8 催化2-丁酮为2-丁醇
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。
反应体系为10mL，含有10mM底物2-丁酮、1mM NADP+、15mM异丙醇，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。反应12h气相色谱检测2-丁醇生成量9.8mM，ee值99.1％。
实施例9 催化2-戊酮为2-戊醇
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。
反应体系为10mL，含有10mM底物2-戊酮、1mM NADP+、15mM异丙醇，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。反应12h气相色谱检测2-戊醇生成量9.5mM，ee值99.7％。
实施例10 催化2-己酮为2-己醇
将实施例3得到的表达CbADH突变体的大肠杆菌基因工程菌按照实施例1中的方法进行培养，获得粗酶液。
反应体系为10mL，含有10mM底物2-己酮、1mM NADP+、15mM异丙醇，加入CbADH突变体粗酶液，通过金属浴控制反应温度为35℃，反应过程通过0.1M磷酸盐缓冲液控制pH为7.5。反应12h气相色谱检测2-己醇生成量6.1mM，ee值99.5％。
序列表
<110> 浙江大学
<120> 一种醇脱氢酶突变体及应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 351
<212> PRT
<213> 拜氏梭菌(Clostridium beijerinckii)
<400> 1
Met Lys Gly Phe Ala Met Leu Gly Ile Asn Lys Leu Gly Trp Ile Glu
1 5 10 15
Lys Glu Arg Pro Val Ala Gly Ser Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Leu Gly Asp Arg Lys Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Ile Leu Pro Lys Asp Met Pro Leu Glu Asn
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Gly Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Ile Cys Val Glu Ala Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Leu Asn Tyr Lys Asn Gly His Ile Val
210 215 220
Asp Gln Val Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Ser Glu Thr Leu Ser Gln Ala Val Ser Met Val
245 250 255
Lys Pro Gly Gly Ile Ile Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Ala Leu Leu Ile Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met
290 295 300
Leu Arg Asp Met Val Val Tyr Asn Arg Val Asp Leu Ser Lys Leu Val
305 310 315 320
Thr His Val Tyr His Gly Phe Asp His Ile Glu Glu Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Ala Val Val Ile Leu
340 345 350
<210> 2
<211> 351
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Lys Gly Phe Ala Met Leu Gly Ile Asn Lys Leu Gly Trp Ile Glu
1 5 10 15
Lys Pro Arg Pro Val Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Trp Glu Gly Ala
35 40 45
Leu Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Pro Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Asp Asp Met Pro Leu Glu Gln
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Ala Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Ile Cys Val Glu Ala Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Leu Asn Tyr Lys Asn Gly Asp Ile Val
210 215 220
Asp Gln Val Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Ser Glu Thr Leu Glu Gln Ala Val Lys Met Val
245 250 255
Lys Pro Gly Gly Ile Ile Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Ala Leu Leu Ile Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Met
290 295 300
Leu Arg Asp Met Val Gln Tyr Gly Arg Val Asp Leu Ser Lys Leu Val
305 310 315 320
Thr His Arg Phe His Gly Phe Asp His Ile Glu Glu Ala Leu Arg Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Val Val Val Ile Leu
340 345 350
<210> 3
<211> 1053
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaagggtt ttgccatgct gggcattaat aagctgggtt ggattgaaaa accgcgcccg 60
gttgcaggcc cgtatgatgc aattgttcgc ccgctggccg ttagcccgtg caccagtgat 120
attcataccg tttgggaagg cgcactgggt gaacgtcata atatgattct gggtcatgaa 180
gccgttggtg aagttgtgga agtgggcagt gaagtgaaag attttaaacc gggcgatcgc 240
gtgattgtgc cgtgtaccac cccggattgg cgcagtctgg aagtgcaggc aggctttccg 300
cagcatagca atggcatgct ggccggctgg aaattttcta attttaaaga tggcgtgttc 360
gccgaatatt ttcatgttaa tgatgcagat atgaacctgg cccatctgcc ggatgatatg 420
ccgctggaac aggccgtgat gattaccgat atgatgacca ccggttttca tggtgcagaa 480
ctggccgata ttcagatggg tagcagtgtt gtggttattg gcattggtgc cgtgggcctg 540
atggccattg ccggtgccaa actgcgcggc gccggtagaa ttattgccgt gggcagccgc 600
ccgatttgcg tggaagccgc caaattttat ggcgccaccg atattctgaa ttataaaaat 660
ggcgatatcg tggatcaggt tatgaaactg accaatggta aaggtgttga tcgtgttatt 720
atggcaggtg gcggtagtga aaccctggaa caggccgtga aaatggtgaa accgggtggt 780
attattagca atattaatta tcacggcagc ggcgatgcac tgctgattcc gcgtgttgaa 840
tggggttgtg gtatggccca taaaaccatt aagggtggcc tgtgtccggg cggtcgtctg 900
cgtatggaaa tgctgcgtga tatggtgcag tatggtcgtg tggatctgag taaactggtt 960
acccatcgtt ttcatggctt tgatcatatt gaagaagcac tgcgtctgat gaaagataaa 1020
ccgaaagatc tgattaaggt ggttgttatt ctg 1053
<210> 4
<211> 63
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
cgaagcctca tccccatttc tctggtcacc agccgggaaa ccacgtaagc tccggcgtca 60
ccc 63
<210> 5
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttgacagcta gctcagtcct aggtattgtg ctagc 35
<210> 6
<211> 1036
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 6
cggcagcaat ttgtagcgcg gcaggcaaac ccacgggaat gaccgataaa aacaaccagc 60
cggtgactcg ctcaagccgc gaaccaaaag ccataccgac gaagtgcgcg acaccgcctg 120
cgctgggata gtggcgaccc agaatcgcaa acacaatcgc aatcgggaac actaagataa 180
tcaaaacggg ccacgcccac aggctgttat tgcctgctac cagcgcagct aacgcaggaa 240
cggcaaacac gccagtgcct aataatgacg tcgatagcag gccaatgccc tgggccagcc 300
ccagttcttg tttgagtcca ctcatgggtt gatgtccgat tgcgcccaaa ttttgggcaa 360
ctgcgtagat tttcgatggt agcacaatca gattcgctta tgacggtgat gaagaaattg 420
cgatgaaatg tgaggtgaat cagggttttc acccgatttt gtgctgatca gaattttttt 480
tctttttccc ccttgaaggg gttgacagct agctcagtcc taggtattgt gctagcataa 540
cagatacgga ctttctcaaa ggagagttat caatgaatat tcgtccattg catgatcgcg 600
tgatcgtcaa gcgtaaagaa gttgaaacta aatctgctgg cggcatcgtt ctgaccggct 660
ctgcagcggc taaatccacc cgtggcgaag tgctggctgt cggcaatggc cgtatccttg 720
aaaatggcga agtgaagccg ctggatgtga aagttggcga catcgttatt ttcaacgatg 780
gctacggtgt gaaatctgag aagatcgaca atgaagaagt gttgatcatg tccgaaagcg 840
acattctggc aattgttgaa gcgtaatccg cgcacgacac tgaacatacg aatttaagga 900
ataaagataa tggcagctaa agacgtaaaa ttcggtaacg acgctcgtgt gaaaatgctg 960
cgcggcgtaa acgtactggc agatgcagtg aaagttaccc tcggtccgaa aggccgtaac 1020
gtagttctgg ataaat 1036
Claims (10)
1.一种醇脱氢酶突变体，其特征在于，氨基酸序列如SEQ ID NO.2所示。
2.编码如权利要求1所述醇脱氢酶突变体的基因。
3.如权利要求2所述的基因，其特征在于，核苷酸序列如SEQ ID NO.3所示。
4.包含如权利要求3所述基因的表达载体。
5.包含如权利要求4所述表达载体的基因工程菌，所述基因工程菌为宿主细胞中导入了所述表达载体。
6.如权利要求5所述的基因工程菌，其特征在于，宿主细胞为大肠杆菌。
7.如权利要求6所述的基因工程菌，其特征在于，所述大肠杆菌高表达分子伴侣GroES-EL。
8.如权利要求7所述的基因工程菌，其特征在于，所述大肠杆菌基因组中启动表达分子伴侣GroES-EL的启动子被替换为组成型启动子J23104。
9.如权利要求1所述醇脱氢酶突变体或如权利要求5～8任一所述基因工程菌在催化醇+氧化型辅酶NADP+与酮+还原型辅酶NADPH的互相转化中的应用。
10.如权利要求9所述的应用，其特征在于，互相转化的反应如式Ⅰ所示，
式Ⅰ中：
R1为H或具有1-6个C原子的烷基；
R2为H或具有1-6个C原子的烷基；
但R1和R2不能同时为H。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911342186.8A CN111057686B (zh) | 2019-12-23 | 2019-12-23 | 一种醇脱氢酶突变体及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911342186.8A CN111057686B (zh) | 2019-12-23 | 2019-12-23 | 一种醇脱氢酶突变体及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111057686A CN111057686A (zh) | 2020-04-24 |
| CN111057686B true CN111057686B (zh) | 2021-05-04 |
Family
ID=70302647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911342186.8A Active CN111057686B (zh) | 2019-12-23 | 2019-12-23 | 一种醇脱氢酶突变体及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111057686B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395519A (zh) * | 2021-12-30 | 2022-04-26 | 上海合全药物研发有限公司 | 重组菌和表达酮还原酶的方法 |
| CN116042555B (zh) * | 2022-08-17 | 2023-10-27 | 无锡佰翱得生物科学股份有限公司 | 一种乙醇脱氢酶突变体及其应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009078973A2 (en) * | 2007-12-13 | 2009-06-25 | Glycos Biotechnologies, Incorporated | Microbial conversion of oils and fatty acids to high-value chemicals |
| CN102181368A (zh) * | 2011-02-15 | 2011-09-14 | 中国科学院微生物研究所 | 一种利用蓝藻将co2生物转化为异丙醇的技术 |
| CN104619834A (zh) * | 2012-04-05 | 2015-05-13 | 朗泽科技新西兰有限公司 | 改变代谢活性的酶 |
| CN108486169A (zh) * | 2011-02-25 | 2018-09-04 | 朗泽科技新西兰有限公司 | 重组微生物及其用途 |
| CN110408604A (zh) * | 2019-08-14 | 2019-11-05 | 华东理工大学 | 底物亲和力和辅酶亲和力提高的甲酸脱氢酶突变体 |
| CN110551771A (zh) * | 2018-05-31 | 2019-12-10 | 中国科学院天津工业生物技术研究所 | 一种手性3-氨基-1-丁醇的合成方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359649A (zh) * | 2018-02-12 | 2018-08-03 | 江南大学 | 醇脱氢酶突变体及其在双芳基手性醇合成中的应用 |
-
2019
- 2019-12-23 CN CN201911342186.8A patent/CN111057686B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009078973A2 (en) * | 2007-12-13 | 2009-06-25 | Glycos Biotechnologies, Incorporated | Microbial conversion of oils and fatty acids to high-value chemicals |
| CN102181368A (zh) * | 2011-02-15 | 2011-09-14 | 中国科学院微生物研究所 | 一种利用蓝藻将co2生物转化为异丙醇的技术 |
| CN108486169A (zh) * | 2011-02-25 | 2018-09-04 | 朗泽科技新西兰有限公司 | 重组微生物及其用途 |
| CN104619834A (zh) * | 2012-04-05 | 2015-05-13 | 朗泽科技新西兰有限公司 | 改变代谢活性的酶 |
| CN110551771A (zh) * | 2018-05-31 | 2019-12-10 | 中国科学院天津工业生物技术研究所 | 一种手性3-氨基-1-丁醇的合成方法 |
| CN110408604A (zh) * | 2019-08-14 | 2019-11-05 | 华东理工大学 | 底物亲和力和辅酶亲和力提高的甲酸脱氢酶突变体 |
Non-Patent Citations (3)
| Title |
|---|
| I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones;Nealon, Christopher M.等;《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》;20160915;第606卷;第151-156页，参见全文 * |
| 登录号2B83_A：Chain A, A Single Amino Acid Substitution In The Clostridium Beijerinckii Alcohol Dehydrogenase Is Critical For Thermostabilization;Goihberg,E.等;《GenBank》;20121010;参见序列信息 * |
| 醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响;朱诚等;《化工进展》;20190808;第38卷(第12期);第5504-5511页，参见全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111057686A (zh) | 2020-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109402098B (zh) | 苏氨酸醛缩酶、突变体及其在制备取代苯丝氨酸衍生物中的应用 | |
| CN109609474B (zh) | 一种氨基酸脱氢酶突变体及其在合成l-草铵膦中的应用 | |
| US8124388B2 (en) | Production of 3-hydroxypropionic acid using beta-alanine/pyruvate aminotransferase | |
| JP2007502114A (ja) | 改良されたグルコースデヒドロゲナーゼポリペプチドおよび関連ポリヌクレオチド | |
| CN108728421B (zh) | 一种羰基还原酶突变体及其用途 | |
| CN110791484A (zh) | 一种草铵膦脱氢酶突变体及其在生产l-草铵膦中的应用 | |
| US20220090152A1 (en) | Method for enzymatic preparation of r-3 aminobutyric acid | |
| CN111676203B (zh) | 一种亮氨酸脱氢酶突变体及其应用 | |
| CN109852644B (zh) | 一种制备布瓦西坦中间体的方法 | |
| CN111057686B (zh) | 一种醇脱氢酶突变体及应用 | |
| CN111172142B (zh) | 一种热稳定性高的头孢菌素c酰化酶突变体 | |
| CN113969269A (zh) | D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 | |
| CN109072215B (zh) | 一种头孢菌素c酰化酶突变体及其应用 | |
| US10294461B2 (en) | Modified carbonyl reducing enzyme and gene | |
| CN113969268A (zh) | Glu/Leu/Phe/Val脱氢酶突变体及其在制备L-草铵膦中的应用 | |
| CN111808829A (zh) | 一种γ-谷氨酰甲胺合成酶突变体及其应用 | |
| CN115354034B (zh) | Nadp依赖型甲酸脱氢酶突变体、辅酶再生体系及其在制备l-草铵膦中的应用 | |
| CN113088501A (zh) | 一种用于生产l-草铵膦的谷氨酸脱氢酶突变体及l-草铵膦生产方法 | |
| CN111019982A (zh) | 一种利用羟基酸脱氢酶制备l-草铵膦的方法 | |
| CN111172143B (zh) | D-木糖酸脱水酶及其应用 | |
| KR101564315B1 (ko) | 신규 포름산 탈수소효소 및 이를 이용한 포름산의 생산방법 | |
| CN113913317A (zh) | 一种重组酿酒酵母及提高重组酿酒酵母表面展示葡萄糖脱氢酶的酶活性的方法 | |
| CN114752572B (zh) | 甲酸脱氢酶突变体及其应用 | |
| CN109280651B (zh) | 一种乳酸脱氢酶突变体基因LbLDH1及其在大肠杆菌中高效表达的发酵方法 | |
| CN113913403B (zh) | 一种nadh激酶突变体、编码基因及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |