CN114574454A - 一种短链脱氢酶及其突变体和应用 - Google Patents
一种短链脱氢酶及其突变体和应用 Download PDFInfo
- Publication number
- CN114574454A CN114574454A CN202011398423.5A CN202011398423A CN114574454A CN 114574454 A CN114574454 A CN 114574454A CN 202011398423 A CN202011398423 A CN 202011398423A CN 114574454 A CN114574454 A CN 114574454A
- Authority
- CN
- China
- Prior art keywords
- gly
- ala
- leu
- thr
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000009105 Short Chain Dehydrogenase-Reductases Human genes 0.000 title claims abstract description 54
- 108010048287 Short Chain Dehydrogenase-Reductases Proteins 0.000 title claims abstract description 54
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- MJZYTEBKXLVLMY-RXMQYKEDSA-N (2r)-1-(6-aminopurin-9-yl)propan-2-ol Chemical compound N1=CN=C2N(C[C@H](O)C)C=NC2=C1N MJZYTEBKXLVLMY-RXMQYKEDSA-N 0.000 claims abstract description 28
- 239000000758 substrate Substances 0.000 claims abstract description 24
- 239000005515 coenzyme Substances 0.000 claims abstract description 16
- 230000008929 regeneration Effects 0.000 claims abstract description 16
- 238000011069 regeneration method Methods 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 4
- 239000011942 biocatalyst Substances 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 102220198654 rs199566527 Human genes 0.000 claims description 12
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims description 4
- 239000004280 Sodium formate Substances 0.000 claims description 4
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims description 4
- 235000019254 sodium formate Nutrition 0.000 claims description 4
- 102000004316 Oxidoreductases Human genes 0.000 claims description 2
- 108090000854 Oxidoreductases Proteins 0.000 claims description 2
- 230000035772 mutation Effects 0.000 claims description 2
- 238000010531 catalytic reduction reaction Methods 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 106
- 108010050848 glycylleucine Proteins 0.000 description 106
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 76
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 68
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 68
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 68
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 68
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 68
- 108010057821 leucylproline Proteins 0.000 description 68
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 34
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 34
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 34
- OKEWAFFWMHBGPT-XPUUQOCRSA-N Ala-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CN=CN1 OKEWAFFWMHBGPT-XPUUQOCRSA-N 0.000 description 34
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 34
- OPZJWMJPCNNZNT-DCAQKATOSA-N Ala-Leu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)O)N OPZJWMJPCNNZNT-DCAQKATOSA-N 0.000 description 34
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 34
- 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 34
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 34
- 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 34
- NLYYHIKRBRMAJV-AEJSXWLSSA-N Ala-Val-Pro Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N NLYYHIKRBRMAJV-AEJSXWLSSA-N 0.000 description 34
- 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 34
- AEJSNWMRPXAKCW-WHFBIAKZSA-N Cys-Ala-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O AEJSNWMRPXAKCW-WHFBIAKZSA-N 0.000 description 34
- YMUFWNJHVPQNQD-ZKWXMUAHSA-N Gly-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN YMUFWNJHVPQNQD-ZKWXMUAHSA-N 0.000 description 34
- IXKRSKPKSLXIHN-YUMQZZPRSA-N Gly-Cys-Leu Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IXKRSKPKSLXIHN-YUMQZZPRSA-N 0.000 description 34
- ADZGCWWDPFDHCY-ZETCQYMHSA-N Gly-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 ADZGCWWDPFDHCY-ZETCQYMHSA-N 0.000 description 34
- HMHRTKOWRUPPNU-RCOVLWMOSA-N Gly-Ile-Gly Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O HMHRTKOWRUPPNU-RCOVLWMOSA-N 0.000 description 34
- LPHQAFLNEHWKFF-QXEWZRGKSA-N Gly-Met-Ile Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LPHQAFLNEHWKFF-QXEWZRGKSA-N 0.000 description 34
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 34
- MKWSZEHGHSLNPF-NAKRPEOUSA-N Ile-Ala-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O)N MKWSZEHGHSLNPF-NAKRPEOUSA-N 0.000 description 34
- YBKKLDBBPFIXBQ-MBLNEYKQSA-N Ile-Thr-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)O)N YBKKLDBBPFIXBQ-MBLNEYKQSA-N 0.000 description 34
- 241000186840 Lactobacillus fermentum Species 0.000 description 34
- QPRQGENIBFLVEB-BJDJZHNGSA-N Leu-Ala-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O QPRQGENIBFLVEB-BJDJZHNGSA-N 0.000 description 34
- DQPQTXMIRBUWKO-DCAQKATOSA-N Leu-Ala-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(C)C)N DQPQTXMIRBUWKO-DCAQKATOSA-N 0.000 description 34
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 34
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 34
- 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 34
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 34
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 34
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 34
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 34
- VDIARPPNADFEAV-WEDXCCLWSA-N Leu-Thr-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O VDIARPPNADFEAV-WEDXCCLWSA-N 0.000 description 34
- IUYCGMNKIZDRQI-BQBZGAKWSA-N Met-Gly-Ala Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O IUYCGMNKIZDRQI-BQBZGAKWSA-N 0.000 description 34
- 108010079364 N-glycylalanine Proteins 0.000 description 34
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 34
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 34
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 34
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 34
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 34
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 34
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 34
- XOWKUMFHEZLKLT-CIQUZCHMSA-N Thr-Ile-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O XOWKUMFHEZLKLT-CIQUZCHMSA-N 0.000 description 34
- XNTVWRJTUIOGQO-RHYQMDGZSA-N Thr-Met-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O XNTVWRJTUIOGQO-RHYQMDGZSA-N 0.000 description 34
- MXDOAJQRJBMGMO-FJXKBIBVSA-N Thr-Pro-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O MXDOAJQRJBMGMO-FJXKBIBVSA-N 0.000 description 34
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 34
- QNXZCKMXHPULME-ZNSHCXBVSA-N Thr-Val-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O QNXZCKMXHPULME-ZNSHCXBVSA-N 0.000 description 34
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 34
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 34
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 34
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 34
- 108010047495 alanylglycine Proteins 0.000 description 34
- 108010090037 glycyl-alanyl-isoleucine Proteins 0.000 description 34
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 34
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 34
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 34
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 34
- 229940012969 lactobacillus fermentum Drugs 0.000 description 34
- KMGOBAQSCKTBGD-DLOVCJGASA-N Ala-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CN=CN1 KMGOBAQSCKTBGD-DLOVCJGASA-N 0.000 description 33
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 28
- CPGJELLYDQEDRK-NAKRPEOUSA-N Val-Ile-Ala Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](C)C(O)=O CPGJELLYDQEDRK-NAKRPEOUSA-N 0.000 description 26
- DEGCBBCMYWNJNA-RHYQMDGZSA-N Thr-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O DEGCBBCMYWNJNA-RHYQMDGZSA-N 0.000 description 25
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 23
- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 description 21
- 239000000047 product Substances 0.000 description 16
- 229940125904 compound 1 Drugs 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- 230000009467 reduction Effects 0.000 description 10
- 238000006722 reduction reaction Methods 0.000 description 10
- 241001052560 Thallis Species 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion 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](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 229950006238 nadide Drugs 0.000 description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 6
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) 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-NNYOXOHSSA-O 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- YGCDFAJJCRVQKU-RCWTZXSCSA-N Thr-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O YGCDFAJJCRVQKU-RCWTZXSCSA-N 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 description 4
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 4
- SFTZTYBXIXLRGQ-JBDRJPRFSA-N Ser-Ile-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SFTZTYBXIXLRGQ-JBDRJPRFSA-N 0.000 description 4
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 4
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 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 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- WTMPKZWHRCMMMT-KZVJFYERSA-N Thr-Pro-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WTMPKZWHRCMMMT-KZVJFYERSA-N 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 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 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- RUOJZAUFBMNUDX-GSVOUGTGSA-N (4r)-4-methyl-1,3-dioxolan-2-one Chemical compound C[C@@H]1COC(=O)O1 RUOJZAUFBMNUDX-GSVOUGTGSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 2
- HBTCFCHYALPXME-HTFCKZLJSA-N Ser-Ile-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HBTCFCHYALPXME-HTFCKZLJSA-N 0.000 description 2
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229960004556 tenofovir Drugs 0.000 description 2
- VCMJCVGFSROFHV-WZGZYPNHSA-N tenofovir disoproxil fumarate Chemical compound OC(=O)\C=C\C(O)=O.N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N VCMJCVGFSROFHV-WZGZYPNHSA-N 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229940126656 GS-4224 Drugs 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound 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
- QJUWBDPGGYVRHY-YUMQZZPRSA-N Leu-Gly-Cys Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N QJUWBDPGGYVRHY-YUMQZZPRSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- GOOHAUXETOMSMM-GSVOUGTGSA-N R-propylene oxide Chemical compound C[C@@H]1CO1 GOOHAUXETOMSMM-GSVOUGTGSA-N 0.000 description 1
- JCLAFVNDBJMLBC-JBDRJPRFSA-N Ser-Ser-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JCLAFVNDBJMLBC-JBDRJPRFSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VTMGKRABARCZAX-OSUNSFLBSA-N Thr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O VTMGKRABARCZAX-OSUNSFLBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 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
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- GKIRPKYJQBWNGO-OCEACIFDSA-N clomifene Chemical compound C1=CC(OCCN(CC)CC)=CC=C1C(\C=1C=CC=CC=1)=C(\Cl)C1=CC=CC=C1 GKIRPKYJQBWNGO-OCEACIFDSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- BRHPBVXVOVMTIQ-ZLELNMGESA-N l-leucine l-leucine Chemical compound CC(C)C[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O BRHPBVXVOVMTIQ-ZLELNMGESA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000010587 phase diagram Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- LDEKQSIMHVQZJK-CAQYMETFSA-N tenofovir alafenamide Chemical compound O([P@@](=O)(CO[C@H](C)CN1C2=NC=NC(N)=C2N=C1)N[C@@H](C)C(=O)OC(C)C)C1=CC=CC=C1 LDEKQSIMHVQZJK-CAQYMETFSA-N 0.000 description 1
- 229960004946 tenofovir alafenamide Drugs 0.000 description 1
- 229960001355 tenofovir disoproxil Drugs 0.000 description 1
- JFVZFKDSXNQEJW-CQSZACIVSA-N tenofovir disoproxil Chemical compound N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N JFVZFKDSXNQEJW-CQSZACIVSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012137 tryptone Substances 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
- 239000004474 valine Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
-
- 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)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明属于医药技术领域，涉及一种短链脱氢酶及其突变体和应用，具体涉及短链脱氢酶及其突变体在(R)‑(+)‑9‑(2‑羟丙基)腺嘌呤合成中的应用，具体涉及利用一种短链脱氢酶及其突变体在催化还原底物6‑氨基‑9‑(2‑丙酮基)嘌呤，获得手性纯度极高的(R)‑(+)‑9‑(2‑羟丙基)腺嘌呤中的应用。本发明所述的短链脱氢酶，其氨基酸序列如SEQ ID NO:1所示，其突变体如SEQ ID NO:2‑34所示。所述的短链脱氢酶及其突变体作为生物催化剂，以6‑氨基‑9‑(2‑丙酮基)嘌呤(1)为底物，搭载辅酶再生系统，以NAD(P)+作为辅酶，实现(R)‑(+)‑9‑(2‑羟丙基)腺嘌呤(2)的酶法合成。与现有(R)‑(+)‑9‑(2‑羟丙基)腺嘌呤化学合成工艺相比，该法的产物得率高、立体选择性好且绿色环保。化合物的转化率和立体选择性均较高，最高可达到99％。
Description
技术领域
本发明属于医药技术领域，涉及一种短链脱氢酶及其突变体和应用，具体涉及短链脱氢酶及其突变体在(R)-(+)-9-(2-羟丙基)腺嘌呤合成中的应用，具体涉及利用一种短链脱氢酶及其突变体在催化还原底物6-氨基-9-(2-丙酮基)嘌呤，获得手性纯度极高的(R)-(+)-9-(2-羟丙基)腺嘌呤中的应用。
背景技术
(R)-(+)-9-(2-羟丙基)腺嘌呤是抗病毒药物替诺福韦二吡呋酯和替诺福韦艾拉酚胺的关键手性中间体，替诺福韦二吡呋酯和替诺福韦艾拉酚胺是吉利德(Gilead)公司开发的新型核苷酸类逆转录酶抑制剂，可有效对抗多种病毒，主要用于治疗艾滋病(HIV)和慢性乙型肝炎(HBV)。在多条替诺福韦的合成路线中，(R)-(+)-9-(2-羟丙基)腺嘌呤都是关键中间体。
专利(CN103848868A)报道了以R-碳酸丙烯酯为起始原料，在碱性条件下与腺嘌呤发生缩合反应，得到(R)-(+)-9-(2-羟丙基)腺嘌呤。目前R-碳酸丙烯酯几乎处于停产状态，没有需求，没有大批量现货供应，R-碳酸丙烯酯一般以环氧丙烷作为原料，通过水解拆分法获得，整步合成路线反应条件苛刻，成本高昂，此外，其合成原料环氧丙烷低沸点、易燃易爆，且有致癌性，极大地制约了此路线的工业化应用。
文献[Die Pharmazie,2015,70(5):283-8]报道了利用腺嘌呤与R-环氧丙烷进行开环缩合反应，直接得到(R)-(+)-9-(2-羟丙基)腺嘌呤，但是环氧丙烷化学性质活泼，沸点低、毒性大的问题依然存在。
短链脱氢酶家族是脱氢酶超家族中的重要一员，是作为涵盖从原核生物到真核生物的几乎所有生物体的最大的蛋白质家族之一，受到最广泛的关注。
现有技术中没有采用本发明的短链脱氢酶制备(R)-(+)-9-(2-羟丙基)腺嘌呤的相关报道。
发明内容
为了克服现有技术的缺陷，本发明提供了一种短链脱氢酶。
本发明的第二个目的是提供了所述短链脱氢酶的突变体。
本发明的第三个目的是提供所述的短链脱氢酶及其突变体在(R)-(+)-9-(2-羟丙基)腺嘌呤合成中的应用。
本发明通过如下技术方案实现：
本发明提供一种短链脱氢酶，其氨基酸序列如SEQ ID NO:1所示。
进一步地，本发明提供了所述的短链脱氢酶的突变体，所述的突变体是通过短链脱氢酶(SEQ ID NO:1)的92位、140位、141位、146位、186位、191位、202位中的一个或几个位置突变得到。
更进一步地，本发明优选如下短链脱氢酶的突变体中的一种或几种：G92E(V)，I140L(S)，E141I(L)，D146S(V)，V186S(A)，L191V(A)，F202Q(S)。
具体地，本发明优选SEQ ID NO:2-34所示的短链脱氢酶的突变体。
本发明提供了所述的短链脱氢酶及其突变体在(R)-(+)-9-(2-羟丙基)腺嘌呤合成中的应用。
本发明以所述的短链脱氢酶及其突变体作为生物催化剂，以6-氨基-9-(2-丙酮基)嘌呤(1)为底物，搭载辅酶再生系统，以NAD(P)+作为辅酶，实现(R)-(+)-9-(2-羟丙基)腺嘌呤(2)的酶法合成。
反应的合成路线为：
本发明所述短链脱氢酶及其突变体可以多种可行的方式如细胞、粗酶粉、酶溶液、固定化酶等多种形式存在。
本发明所述的辅酶再生系统包括底物耦联再生系统和酶耦联再生系统。底物耦联再生系统以异丙醇作为辅底物再生辅酶，异丙醇体积占系统的1-30％；酶耦联再生系统以葡萄糖脱氢酶或甲酸脱氢酶作为额外添加的氧化还原酶，葡萄糖或甲酸钠作为辅底物再生辅酶，葡萄糖脱氢酶或甲酸脱氢酶10-100g/L，葡萄糖或甲酸钠为1-10摩尔当量。
如上反应的合成方法如下：在磷酸盐缓冲液中，加入底物1，搭载辅酶再生系统，以NAD(P)+作为辅酶，之后加入短链脱氢酶或其突变体催化还原反应，得到(R)-(+)-9-(2-羟丙基)腺嘌呤(2)；
或底物1搭载辅酶再生系统，以NAD(P)+作为辅酶，之后加入短链脱氢酶或其突变体催化还原反应，反应过程中调节pH 6.0-7.0，得到(R)-(+)-9-(2-羟丙基)腺嘌呤(2)。
本发明所述反应底物浓度为0.5-200g/L，底物浓度优选为0.5-100g/L，更优选为0.5-30g/L。
本发明所述反应烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷磷酸NAD(P)+终浓度为0.05-1.0mM，优选0.05-0.3mM。
本发明所述磷酸盐缓冲液的pH值为6.0-7.0。
本发明所述的温度为能够保持在短链脱氢酶可以表达其活性的温度范围，优选温度为30-40℃。
当反应底物浓度为0.5-100g/L，NAD(P)+终浓度为0.05-0.3mM，pH值为6.0-7.0，温度为30-40℃时，转化率和立体选择性高达95％以上。
本发明的有益技术效果：
本发明通过对一种短链脱氢酶进行改造，获得了立体选择性优良且能高效催化目标反应的短链脱氢酶突变体，实现替诺福韦药物关键手性中间体(R)-(+)-9-(2-羟丙基)腺嘌呤(2)的高浓度底物的酶法合成，极富应用价值。与现有(R)-(+)-9-(2-羟丙基)腺嘌呤化学合成工艺相比，该法的产物得率高、立体选择性好且绿色环保。化合物的转化率和立体选择性均较高，最高可达到99％。
附图说明
图1为短链脱氢酶突变体V186S/G92E催化还原底物6-氨基-9-(2-丙酮基)嘌呤(1)的液相图。
具体实施方式
下面通过具体的实施方案，描述本发明的短链脱氢酶突变体，以及利用该酶还原底物6-氨基-9-(2-丙酮基)嘌呤(1)获取(R)-(+)-9-(2-羟丙基)腺嘌呤(2)。除特别指明，本发明中所用的实验方案均为本领域技术人员所公知的方法。此外，实施例应理解为说明性的，而不是限制本发明。
某些术语的定义。
不对称还原是还原前手性化合物得到光学纯的相应的还原产物的一种方法，在本发明中指短链脱氢酶催化还原化合物1选择性地得到S或R构型的相应的手性醇。
对映体过量定义为对映体混合物中一个异构体A比另一个异构体B多出来的量占总量的百分数，缩写为ee，公式为(A-B)/(A+B)*100％，对映体过量值用于表示一种手性化合物的光学纯度。ee值越高，光学纯度也越高。
20种氨基酸缩写如下
Asp D 天冬氨酸 Ile I 异亮氨酸
Thr T 苏氨酸 Leu L 亮氨酸
Ser S 丝氨酸 Tyr Y 酪氨酸
Glu E 谷氨酸 Phe F 苯丙氨酸
Pro P 脯氨酸 His H 组氨酸
Gly G 甘氨酸 Lys K 赖氨酸
Ala A 丙氨酸 Arg R 精氨酸
Cys C 半胱氨酸 Trp W 色氨酸
Val V 缬氨酸 Gln Q 谷氨酰胺
Met M 甲硫氨酸 Asn N 天冬酰胺
本发明中涉及的本发明中涉及的氨基酸为Gly,Phe,Glu,Val,Leu,Ile,Asp,Ser,Gln和Ala中的一种或几种。
实施例涉及的配方。
种子培养基：采用LB培养基作为种子培养基，其配方包含0.5％酵母提取物(W/V)、1％胰蛋白胨(W/V)与1％氯化钠(W/V)(如配置固体培养基，在灭菌前加入1.5％琼脂)，115℃高压灭菌30min。
抗生素：卡那霉素或氨苄青霉素，无菌水溶解，配制浓度50-100g/L，接种抗生素用量依据所接种培养基体系终浓度为50-100μg/mL而定(0.20μm微孔滤膜过滤除菌备用)。
诱导剂：异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)，无菌水溶解，配制浓度0.1mM，诱导剂用量依据所接种培养基体系终浓度为0.1μM而定(0.20μm微孔滤膜过滤除菌备用)。
实施例1突变体的构建
以含有短链脱氢酶野生型基因的质粒为模板，通过重叠延伸PCR方法获取突变体基因。
设计的引物如下：
PCR反应体系如下：
扩增程序：94℃：10min，(94℃：30s，48℃：30s，72℃：90s)35个循环，72℃，10min。
实施例2构建突变体基因重组质粒
将重叠延伸PCR获得的突变体基因与载体质粒pET22b进行双酶切，反应体系如下：
37℃培养箱温育4-8h后，利用普通DNA产物凝胶回收试剂盒，回收经酶切后的核酸片段；以T4 DNA连接酶将经过双酶切，具有粘性末端的突变基因片段及载体质粒pET22b片段连接(16℃静置2-6h)获取重组质粒。
实施例3感受态细胞E.coli Rosetta(DE3)的制备及转化
a)从种子培养基中取1mL接种于100mL LB液体培养基中，37℃，200r/min振荡培养3h；
b)3000r/min，10min富集菌体，弃上清；
c)加入400μL预冷0.1M CaCl2溶液，重新悬浮菌体，等分分装到4个EP管中，冰浴30min获得感受态细胞悬浮液；
d)向感受态细胞悬浮液中加入20μL重组质粒连接液轻轻混匀，冰浴30min；
e)42℃热休克90s，冰水浴3min，加入800μL LB液体培养基，37℃培养箱温育1-2h，200r/min振荡培养1-2h；
f)分别取150μL培养液涂布于带有卡那霉素或氨苄青霉素抗性的LB固体培养基，37℃培养10-20h后挑取阳性转化子，获得突变体重组转化子。
实施例4短链脱氢酶与其突变体的表达
a)取突变体重组转化子接种于4mL卡那霉素或氨苄青霉素抗性LB液体培养基中，37℃，200rpm培养6-8h后得到种子液；
b)取1mL种子液接种于100mL卡那霉素或氨苄青霉素抗性LB液体培养基中，37℃，200rpm培养至培养液OD600达到0.8-1.0时加入终浓度0.1mM IPTG，并将温度降至15-20℃诱导表达15-20h；
c)3000rpm×10min离心培养液收集菌体，生理盐水洗涤两次后用0.1M pH 6.0磷酸钠缓冲液重新悬浮菌体(菌体湿重浓度为0.1g/mL)，冰水浴环境下超声破碎菌体，4℃环境下，12000rpm离心收集上清获得短链脱氢酶与其突变体(SEQ ID NO:1-34)粗酶液。
实施例5葡萄糖脱氢酶表达
将含有pET22b-GDH质粒的E.coli Rosetta(DE3)按照实施例4所述表达获得葡萄糖脱氢酶。
实施例6粗酶液催化还原
反应体系：如实施例4中所述的突变体粗酶上清液500μL，葡萄糖脱氢酶粗酶上清液100μL，终浓度0.3mM NADP+，2.4mg葡萄糖，DMSO溶解的浓度为10g/L的化合物1用量为50μL，35℃、220rpm摇床反应12h。
实施例7高效液相色谱法分析底物和产物
1)立体选择性检测
a)样品预处理
取1mL反应液，12000rpm离心5min；精密吸取10μL上清液转移至安瓿瓶中；加入100μL无水乙醇，30℃旋干；取1mL流动相溶解样品，微孔滤膜过滤后进样10μL。
b)样品检测
HPLC检测底物6-氨基-9-(2-丙酮基)嘌呤及其还原产物(R)-(+)-9-(2-羟丙基)腺嘌呤ee％的检测条件如下：
其中，流动相依据上述配方配制，100mL流动相配制过程如下：量取60mL色谱级正己烷、40mL色谱级乙醇和100μL二乙胺，混匀后超声20min待用。
2)转化率检测
a)样品预处理
取1ml反应液，12000rpm离心5min；精密吸取10μL上清液转移至进样瓶中；加入990μL纯净水混匀，微孔滤膜过滤后进样10μL。
b)样品检测
HPLC检测底物6-氨基-9-(2-丙酮基)嘌呤及其还原产物(R)-(+)-9-(2-羟丙基)腺嘌呤conv％的检测条件如下：
其中，流动相依据上述配方配制，配制1L流动相过程如下：称取4.68g NaH2PO4·2H2O(30mM)，采用970mL纯净水溶解，加入30mL色谱甲醇和480μL CF3COOH，混匀后超声20min待用。
野生型短链脱氢酶及其突变体催化化合物1的检测结果如下(底物浓度为0.5g/L)：
注：a)还原产物对映体过量值[ee％]；b)还原产物的绝对构型；c)转化率[conv％]；
实施例8短链脱氢酶突变体V186S/G92E与葡萄糖脱氢酶催化化合物1(反应体系10mL)
反应体系：短链脱氢酶突变体V186S/G92E全细胞湿菌体2g，葡萄糖脱氢酶全细胞湿菌体400mg，终浓度0.3mM NADP+，0.86-2.35g葡萄糖，0.3-1g化合物1，35℃、220rpm摇床反应12h，反应过程中用2M Na2CO3溶液调节pH维持于6.0-7.0。经HPLC检测，短链脱氢酶突变体V186S/G92E催化化合物1的检测结果如下：
注：a)还原产物对映体过量值[ee％]b)还原产物的绝对构型c)转化率[conv％]
实施例9短链脱氢酶突变体V186S/G92E与葡萄糖脱氢酶催化化合物1(反应体系100mL)
反应体系：短链脱氢酶突变体V186S/G92E全细胞湿菌体20g，葡萄糖脱氢酶全细胞湿菌体4g，终浓度0.3mM NADP+，23.54g葡萄糖，10g化合物1，35℃、220rpm摇床反应12h，反应过程中用2M Na2CO3溶液调节pH维持于6.0-7.0。经HPLC检测，短链脱氢酶突变体V186S/G92E催化化合物1的检测结果如下：
注：a)还原产物对映体过量值[ee％]b)还原产物的绝对构型c)转化率[conv％]
实施例10短链脱氢酶突变体V186S/G92E-异丙醇体系催化化合物1(反应体系10mL)
反应体系：短链脱氢酶突变体V186S/G92E全细胞湿菌体2g，终浓度0.3mM NADP+，异丙醇5％(V/V)，0.3-0.5g化合物1，35℃、220rpm摇床反应72h。经HPLC检测，短链脱氢酶突变体V186S/G92E催化化合物1的检测结果如下：
注：a)还原产物对映体过量值[ee％]b)还原产物的绝对构型c)转化率[conv％]
实施例11短链脱氢酶突变体V186S/G92E-异丙醇体系催化化合物1(反应体系100mL)
反应体系：短链脱氢酶突变体V186S/G92E全细胞湿菌体20g，终浓度0.3mM NADP+，异丙醇5％(V/V)，5g化合物1，35℃、220rpm摇床反应72h。经HPLC检测，短链脱氢酶突变体催化化合物1的检测结果如下：
注：a)还原产物对映体过量值[ee％]b)还原产物的绝对构型c)转化率[conv％]。
序列表
<110> 沈阳药科大学
<120> 一种短链脱氢酶及其突变体的应用
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 1
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 2
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 2
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Ala His
180 185 190
Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro Leu Ile
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 3
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 3
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Val Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 4
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 4
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Leu Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 5
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 5
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ser Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 6
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 6
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Ile Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 7
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 7
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Leu Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 8
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 8
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ser Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 9
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 9
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Val Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 10
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 10
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ser Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 11
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 11
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ala Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 12
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 12
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Val Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 13
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 13
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Ala Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 14
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 14
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 15
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 15
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Gly Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 16
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 16
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Val Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 17
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 17
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Val Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ser Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 18
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 18
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Leu Gly Gly Met Ile
130 135 140
Gly Ser Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 19
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 19
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ser Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ala Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 20
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 20
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Ile Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 21
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 21
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Leu Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Gly Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 22
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 22
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Val Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 23
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 23
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Ala Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Gly Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 24
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 24
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ala Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Cys Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 25
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 25
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ser Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 26
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 26
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Leu Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Leu Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 27
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 27
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Leu Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 28
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 28
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ser Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Val Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 29
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 29
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Val Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Gly Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 30
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 30
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ser Ile Ala Thr Pro Ile Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 31
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 31
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Val Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Leu Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Ala Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 32
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 32
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ile Gly Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Val Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 33
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 33
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Leu Gly Gly Met Ile
130 135 140
Gly Ser Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Pro Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
<210> 34
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 34
Met Gly Gly Pro Ala Ala Leu Val Ala Leu Val Thr Gly Gly Thr Leu
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Gly Leu Pro Leu Leu Gly Gly Ala Leu
20 25 30
Gly Val Ala Pro Thr Gly Ala His Gly Ala Gly Gly Leu Ala Val Gly
35 40 45
Gly Ala Leu Gly Gly Ala Ser Leu Pro Ile Thr Gly Ala Val Ser Leu
50 55 60
Gly Gly Ala Thr Gly Ala Ala Thr Leu Ala Val Val Gly Leu Pro Gly
65 70 75 80
Gly Leu Ala Ala Ile Val Ala Ala Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Gly Gly Met Thr Leu Ala His Thr Ala Ala Gly Ile Ala Ile Ala
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Leu Thr Gly Val Leu Ala Met Leu
115 120 125
Gly His Gly Gly Ala Ile Val Ala Ile Ser Ser Ser Ile Gly Met Ile
130 135 140
Gly Ala Pro Thr Val Pro Ala Thr Ala Ala Ala Leu Gly Gly Val Ala
145 150 155 160
Leu Leu Thr Leu Ser Val Ala Leu Gly Cys Ala Gly Leu Gly Thr Ala
165 170 175
Ile Ala Val Ala Ser Ile Thr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Ala His Leu Ala Ala Ala Thr Leu Gly Ser Thr Ile Ala Leu His Pro
195 200 205
Met Gly Ala Leu Gly Leu Pro Gly Gly Val Ala Leu Met Ala Val Pro
210 215 220
Val Ala Ser Ala Gly Ala Ser Pro Ser Thr Gly Ser Gly Pro Val Val
225 230 235 240
Ala Gly Gly Thr Thr Ala Gly
245
Claims (10)
1.一种短链脱氢酶，其特征在于，其氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述的短链脱氢酶的突变体，其特征在于，其氨基酸序列通过SEQ ID NO:1的92位、140位、141位、146位、186位、191位、202位中的一个或几个位置突变得到。
3.如权利要求2所述的短链脱氢酶的突变体，其特征在于，所述突变体为G92E(V)，I140L(S)，E141I(L)，D146S(V)，V186S(A)，L191V(A)，F202Q(S)中的一种或几种。
4.如权利要求3所述的短链脱氢酶的突变体，其特征在于，其氨基酸序列如SEQ ID NO:2-34所示。
5.权利要求1所述的短链脱氢酶或权利要求2-4任何一项所述的短链脱氢酶的突变体在作为生物催化剂中的应用。
6.如权利要求5所述的应用，其特征在于，以6-氨基-9-(2-丙酮基)嘌呤(1)为底物，搭载辅酶再生系统，以NAD(P)+作为辅酶，实现(R)-(+)-9-(2-羟丙基)腺嘌呤(2)的酶法合成：
7.如权利要求6所述的应用，其特征在于，所述的辅酶再生系统包括底物耦联再生系统和酶耦联再生系统；底物耦联再生系统以异丙醇作为辅底物再生辅酶，异丙醇体积占系统的1-30％；酶耦联再生系统以葡萄糖脱氢酶或甲酸脱氢酶作为额外添加的氧化还原酶，葡萄糖或甲酸钠作为辅底物再生辅酶；葡萄糖脱氢酶或甲酸脱氢酶10-100g/L，葡萄糖或甲酸钠1-10摩尔当量。
8.如权利要求6所述的应用，其特征在于，底物浓度为0.5-200g/L，底物浓度优选为0.5-100g/L，更优选为0.5-30g/L。
9.如权利要求6所述的应用，其特征在于，所述反应NAD(P)+终浓度为0.05-1.0mM，优选0.05-0.3mM。
10.如权利要求6所述的应用，其特征在于，反应体系的pH值为6.0-7.0，温度为30-40℃。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011398423.5A CN114574454B (zh) | 2020-12-02 | 2020-12-02 | 一种短链脱氢酶及其突变体和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011398423.5A CN114574454B (zh) | 2020-12-02 | 2020-12-02 | 一种短链脱氢酶及其突变体和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114574454A true CN114574454A (zh) | 2022-06-03 |
| CN114574454B CN114574454B (zh) | 2023-11-17 |
Family
ID=81770121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011398423.5A Active CN114574454B (zh) | 2020-12-02 | 2020-12-02 | 一种短链脱氢酶及其突变体和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114574454B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546690A (zh) * | 2018-04-24 | 2018-09-18 | 沈阳药科大学 | 一种短链脱氢酶及其突变体与基因的制备及应用 |
| CN111041008A (zh) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | 短链脱氢酶突变体及其应用 |
| CN111041009A (zh) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | 一种短链脱氢酶及其突变体与基因的制备及应用 |
-
2020
- 2020-12-02 CN CN202011398423.5A patent/CN114574454B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546690A (zh) * | 2018-04-24 | 2018-09-18 | 沈阳药科大学 | 一种短链脱氢酶及其突变体与基因的制备及应用 |
| CN111041008A (zh) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | 短链脱氢酶突变体及其应用 |
| CN111041009A (zh) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | 一种短链脱氢酶及其突变体与基因的制备及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114574454B (zh) | 2023-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110564788B (zh) | 一种利用亚胺还原酶生产麻黄碱的方法 | |
| CN112143764B (zh) | 一种生物酶催化制备布瓦西坦中间体化合物的方法 | |
| CN111996176B (zh) | 羰基还原酶突变体及其应用 | |
| CN112831488B (zh) | 一种谷氨酸脱羧酶及γ-氨基丁酸高产菌株 | |
| CN111235123B (zh) | 一种醇溶液高浓度耐受的羰基还原酶及其应用 | |
| CN112175971A (zh) | 密码子优化的krd基因和gdh基因及其应用 | |
| CN109679978B (zh) | 一种用于制备l-2-氨基丁酸的重组共表达体系及其应用 | |
| CN111500600B (zh) | 3-甾酮-1,2-脱氢酶及其基因序列和应用 | |
| CN113583988B (zh) | 氨基酸脱氢酶突变体及其应用 | |
| CN112143688B (zh) | 一种重组大肠杆菌的构建及其应用 | |
| CN114921392B (zh) | 一种高效联产葡萄糖酸和蒜糖醇的方法 | |
| CN114574454B (zh) | 一种短链脱氢酶及其突变体和应用 | |
| CN114686451B (zh) | 胺脱氢酶突变体及其在制备(s)-5-甲基-2-吡咯烷酮中的应用 | |
| CN105950595B (zh) | (-)-γ-内酰胺酶、基因、突变体、载体及其制备与应用 | |
| CN115838697A (zh) | 亚胺还原酶突变体及其在拉罗替尼手性中间体合成中的应用 | |
| CN112626144B (zh) | 一种替诺福韦中间体(r)-9-(2-羟丙基)腺嘌呤的生物合成方法 | |
| CN109971730A (zh) | 一种来源于黑曲霉的单胺氧化酶用于手性胺中间体的制备 | |
| CN111254181B (zh) | 一种化学酶法制备(s)-1,2,3,4-四氢异喹啉-3-甲酸的方法 | |
| CN111254170B (zh) | 一种多酶耦合制备(s)-1,2,3,4-四氢异喹啉-3-甲酸的方法 | |
| CN111254180B (zh) | 一种酶法拆分制备(s)-1,2,3,4-四氢异喹啉-3-甲酸的方法 | |
| CN109897872B (zh) | 酶法制备(2s,3s)-n-叔丁氧羰基-3-氨基-1-氯-2-羟基-4-苯基丁烷 | |
| CN113174377A (zh) | 羰基还原酶、突变体及其在制备地尔硫卓中间体中的应用 | |
| CN112813040B (zh) | 老黄酶oye1突变体蛋白及在酶催化不对称还原2-芳基丙烯酸衍生物中的应用 | |
| CN110358804B (zh) | R-3-氨基正丁醇的酶法生产工艺 | |
| CN111500549B (zh) | 制备c1,2-位脱氢甾体化合物的酶及其应用 |
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 |