CN114958813B - 一种碱性蛋白酶sp4及其突变体、编码基因和应用 - Google Patents
一种碱性蛋白酶sp4及其突变体、编码基因和应用 Download PDFInfo
- Publication number
- CN114958813B CN114958813B CN202210570089.XA CN202210570089A CN114958813B CN 114958813 B CN114958813 B CN 114958813B CN 202210570089 A CN202210570089 A CN 202210570089A CN 114958813 B CN114958813 B CN 114958813B
- Authority
- CN
- China
- Prior art keywords
- protease
- mutant
- ala
- gly
- ser
- 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
- 108091005658 Basic proteases Proteins 0.000 title claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 title abstract description 22
- 108091005804 Peptidases Proteins 0.000 claims abstract description 81
- 239000004365 Protease Substances 0.000 claims abstract description 78
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 73
- 230000000694 effects Effects 0.000 claims abstract description 31
- 239000003599 detergent Substances 0.000 claims abstract description 21
- 230000003197 catalytic effect Effects 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 5
- 150000001413 amino acids Chemical class 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 13
- 238000002741 site-directed mutagenesis Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 abstract description 34
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 51
- 108090000790 Enzymes Proteins 0.000 description 51
- 229940088598 enzyme Drugs 0.000 description 51
- 239000000243 solution Substances 0.000 description 20
- 239000000843 powder Substances 0.000 description 11
- 108010050848 glycylleucine Proteins 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 108010047495 alanylglycine Proteins 0.000 description 7
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000004094 surface-active agent Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 108010037850 glycylvaline Proteins 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 241001451172 Fusarium pseudograminearum Species 0.000 description 4
- MXOODARRORARSU-ACZMJKKPSA-N Glu-Ala-Ser Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)O)N MXOODARRORARSU-ACZMJKKPSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000004744 fabric Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 3
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- KVYVOGYEMPEXBT-GUBZILKMSA-N Gln-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O KVYVOGYEMPEXBT-GUBZILKMSA-N 0.000 description 3
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 3
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 3
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 3
- OJNZVYSGVYLQIN-BQBZGAKWSA-N Gly-Met-Asp Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O OJNZVYSGVYLQIN-BQBZGAKWSA-N 0.000 description 3
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 3
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 3
- 108010065920 Insulin Lispro Proteins 0.000 description 3
- 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 3
- WVJNGSFKBKOKRV-AJNGGQMLSA-N Lys-Leu-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVJNGSFKBKOKRV-AJNGGQMLSA-N 0.000 description 3
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HXOLCSYHGRNXJJ-IHRRRGAJSA-N Pro-Asp-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HXOLCSYHGRNXJJ-IHRRRGAJSA-N 0.000 description 3
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 3
- ZVBCMFDJIMUELU-BZSNNMDCSA-N Ser-Tyr-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CO)N ZVBCMFDJIMUELU-BZSNNMDCSA-N 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 3
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 108010051242 phenylalanylserine Proteins 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 108010026333 seryl-proline Proteins 0.000 description 3
- 108010071207 serylmethionine Proteins 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 2
- UQJUGHFKNKGHFQ-VZFHVOOUSA-N Ala-Cys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UQJUGHFKNKGHFQ-VZFHVOOUSA-N 0.000 description 2
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 2
- 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 2
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 2
- BLTRAARCJYVJKV-QEJZJMRPSA-N Ala-Lys-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(O)=O BLTRAARCJYVJKV-QEJZJMRPSA-N 0.000 description 2
- IETUUAHKCHOQHP-KZVJFYERSA-N Ala-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)[C@@H](C)O)C(O)=O IETUUAHKCHOQHP-KZVJFYERSA-N 0.000 description 2
- LYILPUNCKACNGF-NAKRPEOUSA-N Ala-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N LYILPUNCKACNGF-NAKRPEOUSA-N 0.000 description 2
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 2
- NUHQMYUWLUSRJX-BIIVOSGPSA-N Asn-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N NUHQMYUWLUSRJX-BIIVOSGPSA-N 0.000 description 2
- PCKRJVZAQZWNKM-WHFBIAKZSA-N Asn-Asn-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O PCKRJVZAQZWNKM-WHFBIAKZSA-N 0.000 description 2
- XQQVCUIBGYFKDC-OLHMAJIHSA-N Asn-Asp-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XQQVCUIBGYFKDC-OLHMAJIHSA-N 0.000 description 2
- NKTLGLBAGUJEGA-BIIVOSGPSA-N Asn-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N)C(=O)O NKTLGLBAGUJEGA-BIIVOSGPSA-N 0.000 description 2
- ULRPXVNMIIYDDJ-ACZMJKKPSA-N Asn-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N ULRPXVNMIIYDDJ-ACZMJKKPSA-N 0.000 description 2
- XTMZYFMTYJNABC-ZLUOBGJFSA-N Asn-Ser-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N XTMZYFMTYJNABC-ZLUOBGJFSA-N 0.000 description 2
- HCZQKHSRYHCPSD-IUKAMOBKSA-N Asn-Thr-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HCZQKHSRYHCPSD-IUKAMOBKSA-N 0.000 description 2
- WCFCYFDBMNFSPA-ACZMJKKPSA-N Asp-Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O WCFCYFDBMNFSPA-ACZMJKKPSA-N 0.000 description 2
- BKXPJCBEHWFSTF-ACZMJKKPSA-N Asp-Gln-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O BKXPJCBEHWFSTF-ACZMJKKPSA-N 0.000 description 2
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 2
- ITGFVUYOLWBPQW-KKHAAJSZSA-N Asp-Thr-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ITGFVUYOLWBPQW-KKHAAJSZSA-N 0.000 description 2
- XMKXONRMGJXCJV-LAEOZQHASA-N Asp-Val-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XMKXONRMGJXCJV-LAEOZQHASA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- IXPSSIBVVKSOIE-SRVKXCTJSA-N Cys-Ser-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N)O IXPSSIBVVKSOIE-SRVKXCTJSA-N 0.000 description 2
- HILMIYALTUQTRC-XVKPBYJWSA-N Glu-Gly-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HILMIYALTUQTRC-XVKPBYJWSA-N 0.000 description 2
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 description 2
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 2
- FUTAPPOITCCWTH-WHFBIAKZSA-N Gly-Asp-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FUTAPPOITCCWTH-WHFBIAKZSA-N 0.000 description 2
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 2
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 2
- FQKKPCWTZZEDIC-XPUUQOCRSA-N Gly-His-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 FQKKPCWTZZEDIC-XPUUQOCRSA-N 0.000 description 2
- 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 2
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 2
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- KFQDSSNYWKZFOO-LSJOCFKGSA-N His-Val-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KFQDSSNYWKZFOO-LSJOCFKGSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 2
- KWURTLAFFDOTEQ-GUBZILKMSA-N Leu-Cys-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KWURTLAFFDOTEQ-GUBZILKMSA-N 0.000 description 2
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 2
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 2
- RIHIGSWBLHSGLV-CQDKDKBSSA-N Leu-Tyr-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O RIHIGSWBLHSGLV-CQDKDKBSSA-N 0.000 description 2
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 2
- YFQSSOAGMZGXFT-MEYUZBJRSA-N Lys-Thr-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YFQSSOAGMZGXFT-MEYUZBJRSA-N 0.000 description 2
- WXHHTBVYQOSYSL-FXQIFTODSA-N Met-Ala-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O WXHHTBVYQOSYSL-FXQIFTODSA-N 0.000 description 2
- FPTXMUIBLMGTQH-ONGXEEELSA-N Phe-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 FPTXMUIBLMGTQH-ONGXEEELSA-N 0.000 description 2
- CDHURCQGUDNBMA-UBHSHLNASA-N Phe-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDHURCQGUDNBMA-UBHSHLNASA-N 0.000 description 2
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 2
- LPGSNRSLPHRNBW-AVGNSLFASA-N Pro-His-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 LPGSNRSLPHRNBW-AVGNSLFASA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 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
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 2
- 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 2
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- OJRNZRROAIAHDL-LKXGYXEUSA-N Thr-Asn-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OJRNZRROAIAHDL-LKXGYXEUSA-N 0.000 description 2
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 2
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 2
- 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 2
- QYDKSNXSBXZPFK-ZJDVBMNYSA-N Thr-Thr-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYDKSNXSBXZPFK-ZJDVBMNYSA-N 0.000 description 2
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 2
- PELIQFPESHBTMA-WLTAIBSBSA-N Thr-Tyr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 PELIQFPESHBTMA-WLTAIBSBSA-N 0.000 description 2
- DNUJCLUFRGGSDJ-YLVFBTJISA-N Trp-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CNC2=CC=CC=C21)N DNUJCLUFRGGSDJ-YLVFBTJISA-N 0.000 description 2
- WMIUTJPFHMMUGY-ZFWWWQNUSA-N Trp-Pro-Gly Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)NCC(=O)O WMIUTJPFHMMUGY-ZFWWWQNUSA-N 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- USYGMBIIUDLYHJ-GVARAGBVSA-N Tyr-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 USYGMBIIUDLYHJ-GVARAGBVSA-N 0.000 description 2
- IZFVRRYRMQFVGX-NRPADANISA-N Val-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N IZFVRRYRMQFVGX-NRPADANISA-N 0.000 description 2
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 2
- NWDOPHYLSORNEX-QXEWZRGKSA-N Val-Asn-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N NWDOPHYLSORNEX-QXEWZRGKSA-N 0.000 description 2
- ISERLACIZUGCDX-ZKWXMUAHSA-N Val-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N ISERLACIZUGCDX-ZKWXMUAHSA-N 0.000 description 2
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 2
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-N 0.000 description 2
- LKUDRJSNRWVGMS-QSFUFRPTSA-N Val-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LKUDRJSNRWVGMS-QSFUFRPTSA-N 0.000 description 2
- YTNGABPUXFEOGU-SRVKXCTJSA-N Val-Pro-Arg Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O YTNGABPUXFEOGU-SRVKXCTJSA-N 0.000 description 2
- QTPQHINADBYBNA-DCAQKATOSA-N Val-Ser-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN QTPQHINADBYBNA-DCAQKATOSA-N 0.000 description 2
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000013492 plasmid preparation Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- -1 washing Substances 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 108010011619 6-Phytase Proteins 0.000 description 1
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-N 0.000 description 1
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 1
- VCSABYLVNWQYQE-SRVKXCTJSA-N Ala-Lys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O VCSABYLVNWQYQE-SRVKXCTJSA-N 0.000 description 1
- JNLDTVRGXMSYJC-UVBJJODRSA-N Ala-Pro-Trp Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O JNLDTVRGXMSYJC-UVBJJODRSA-N 0.000 description 1
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 1
- JNJHNBXBGNJESC-KKXDTOCCSA-N Ala-Tyr-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JNJHNBXBGNJESC-KKXDTOCCSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 1
- OOWSBIOUKIUWLO-RCOVLWMOSA-N Asn-Gly-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O OOWSBIOUKIUWLO-RCOVLWMOSA-N 0.000 description 1
- KEUNWIXNKVWCFL-FXQIFTODSA-N Asn-Met-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O KEUNWIXNKVWCFL-FXQIFTODSA-N 0.000 description 1
- NJIKKGUVGUBICV-ZLUOBGJFSA-N Asp-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O NJIKKGUVGUBICV-ZLUOBGJFSA-N 0.000 description 1
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- XJQRWGXKUSDEFI-ACZMJKKPSA-N Asp-Glu-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XJQRWGXKUSDEFI-ACZMJKKPSA-N 0.000 description 1
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 description 1
- JUWZKMBALYLZCK-WHFBIAKZSA-N Asp-Gly-Asn Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O JUWZKMBALYLZCK-WHFBIAKZSA-N 0.000 description 1
- HAFCJCDJGIOYPW-WDSKDSINSA-N Asp-Gly-Gln Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O HAFCJCDJGIOYPW-WDSKDSINSA-N 0.000 description 1
- BWJZSLQJNBSUPM-FXQIFTODSA-N Asp-Pro-Asn Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O BWJZSLQJNBSUPM-FXQIFTODSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- AOZBJZBKFHOYHL-AVGNSLFASA-N Cys-Glu-Tyr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O AOZBJZBKFHOYHL-AVGNSLFASA-N 0.000 description 1
- CAXGCBSRJLADPD-FXQIFTODSA-N Cys-Pro-Asn Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O CAXGCBSRJLADPD-FXQIFTODSA-N 0.000 description 1
- KFYPRIGJTICABD-XGEHTFHBSA-N Cys-Thr-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N)O KFYPRIGJTICABD-XGEHTFHBSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000631857 Fusarium pseudograminearum CS3096 Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- QYTKAVBFRUGYAU-ACZMJKKPSA-N Gln-Asp-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O QYTKAVBFRUGYAU-ACZMJKKPSA-N 0.000 description 1
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 1
- CLROYXHHUZELFX-FXQIFTODSA-N Glu-Gln-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O CLROYXHHUZELFX-FXQIFTODSA-N 0.000 description 1
- LRPXYSGPOBVBEH-IUCAKERBSA-N Glu-Gly-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O LRPXYSGPOBVBEH-IUCAKERBSA-N 0.000 description 1
- QJVZSVUYZFYLFQ-CIUDSAMLSA-N Glu-Pro-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O QJVZSVUYZFYLFQ-CIUDSAMLSA-N 0.000 description 1
- HJTSRYLPAYGEEC-SIUGBPQLSA-N Glu-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCC(=O)O)N HJTSRYLPAYGEEC-SIUGBPQLSA-N 0.000 description 1
- 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 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 1
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 1
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 1
- WMGHDYWNHNLGBV-ONGXEEELSA-N Gly-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 WMGHDYWNHNLGBV-ONGXEEELSA-N 0.000 description 1
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 1
- 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 1
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- KZTLOHBDLMIFSH-XVYDVKMFSA-N His-Ala-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O KZTLOHBDLMIFSH-XVYDVKMFSA-N 0.000 description 1
- BDFCIKANUNMFGB-PMVVWTBXSA-N His-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 BDFCIKANUNMFGB-PMVVWTBXSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- HDOYNXLPTRQLAD-JBDRJPRFSA-N Ile-Ala-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)O)N HDOYNXLPTRQLAD-JBDRJPRFSA-N 0.000 description 1
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 1
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 1
- UIEZQYNXCYHMQS-BJDJZHNGSA-N Ile-Lys-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)O)N UIEZQYNXCYHMQS-BJDJZHNGSA-N 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-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
- 241000880493 Leptailurus serval Species 0.000 description 1
- 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 1
- REPBGZHJKYWFMJ-KKUMJFAQSA-N Leu-Lys-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N REPBGZHJKYWFMJ-KKUMJFAQSA-N 0.000 description 1
- XOQMURBBIXRRCR-SRVKXCTJSA-N Lys-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN XOQMURBBIXRRCR-SRVKXCTJSA-N 0.000 description 1
- CUHGAUZONORRIC-HJGDQZAQSA-N Lys-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N)O CUHGAUZONORRIC-HJGDQZAQSA-N 0.000 description 1
- IMDJSVBFQKDDEQ-MGHWNKPDSA-N Lys-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCCCN)N IMDJSVBFQKDDEQ-MGHWNKPDSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- SSSFPISOZOLQNP-GUBZILKMSA-N Pro-Arg-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSSFPISOZOLQNP-GUBZILKMSA-N 0.000 description 1
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- POQFNPILEQEODH-FXQIFTODSA-N Pro-Ser-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O POQFNPILEQEODH-FXQIFTODSA-N 0.000 description 1
- OQSGBXGNAFQGGS-CYDGBPFRSA-N Pro-Val-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OQSGBXGNAFQGGS-CYDGBPFRSA-N 0.000 description 1
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 1
- KNZQGAUEYZJUSQ-ZLUOBGJFSA-N Ser-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N KNZQGAUEYZJUSQ-ZLUOBGJFSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- OJFFAQFRCVPHNN-JYBASQMISA-N Ser-Thr-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O OJFFAQFRCVPHNN-JYBASQMISA-N 0.000 description 1
- HAYADTTXNZFUDM-IHRRRGAJSA-N Ser-Tyr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HAYADTTXNZFUDM-IHRRRGAJSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- JMZKMSTYXHFYAK-VEVYYDQMSA-N Thr-Arg-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O JMZKMSTYXHFYAK-VEVYYDQMSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 1
- LVRFMARKDGGZMX-IZPVPAKOSA-N Thr-Tyr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=C(O)C=C1 LVRFMARKDGGZMX-IZPVPAKOSA-N 0.000 description 1
- BKIOKSLLAAZYTC-KKHAAJSZSA-N Thr-Val-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O BKIOKSLLAAZYTC-KKHAAJSZSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- DXYWRYQRKPIGGU-BPNCWPANSA-N Tyr-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DXYWRYQRKPIGGU-BPNCWPANSA-N 0.000 description 1
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 1
- CTDPLKMBVALCGN-JSGCOSHPSA-N Tyr-Gly-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O CTDPLKMBVALCGN-JSGCOSHPSA-N 0.000 description 1
- CLEGSEJVGBYZBJ-MEYUZBJRSA-N Tyr-Thr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CLEGSEJVGBYZBJ-MEYUZBJRSA-N 0.000 description 1
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 1
- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 1
- YDPFWRVQHFWBKI-GVXVVHGQSA-N Val-Glu-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N YDPFWRVQHFWBKI-GVXVVHGQSA-N 0.000 description 1
- 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 1
- APQIVBCUIUDSMB-OSUNSFLBSA-N Val-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N APQIVBCUIUDSMB-OSUNSFLBSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-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
- 238000002835 absorbance Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 108010083912 bleomycin N-acetyltransferase Proteins 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 229940085127 phytase Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- KMPHTYSTEHXSTL-UHFFFAOYSA-M sodium;2-hydroxypropanoate;2-hydroxypropanoic acid Chemical compound [Na+].CC(O)C(O)=O.CC(O)C([O-])=O KMPHTYSTEHXSTL-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 108010084932 tryptophyl-proline Proteins 0.000 description 1
- 108010038745 tryptophylglycine 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
- 108010073969 valyllysine Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及基因工程领域,具体涉及一种碱性蛋白酶SP4及其突变体、编码基因和应用。所述碱性蛋白酶的氨基酸序列如SEQ ID NO:1所示。蛋白酶SP4及其突变体SP4‑M1的最适温度、最适pH、pH和热稳定性并未发生明显变化,但SP4‑M1的比活力较野生型提高了50%。此外洗涤环境稳定性检测结果也显示,蛋白酶SP4及其催化活性提高的突变体SP4‑M1洗涤剂稳定性良好,具有洗涤用蛋白酶制剂的应用潜力。
Description
技术领域
本发明涉及基因工程领域,具体涉及一种碱性蛋白酶SP4及其突变体、编码基因和应用。
背景技术
蛋白酶是一类重要的肽键水解酶类,其广泛的存在于动物、植物和微生物中。长久以来,蛋白酶一直占据全球酶制剂市场60%以上的销售份额,被广泛的应用于食品、饲料、皮革和、洗涤和医药等众多工业领域。而洗涤用酶占到全工业领域用酶制剂的2/5左右,是不可忽视的重要应用领域。欧美等发达国家的衣料用洗涤剂中加酶洗涤剂的占比高达80%。1913年,德国化学家Otto最早提出了加酶洗涤剂这一应用策略,并将从动物胰脏中提取到的胰蛋白酶和胰凝乳蛋白酶加入到洗涤剂中用于实际生产。但是由于动物源胰蛋白酶和胰凝乳蛋白酶在碱性洗涤剂中的不稳定性和生产成本过高,洗涤用蛋白酶并未得到有效的发展。直到七八十年代,解决了酶制剂生产成本过高的源头问题,洗涤用酶制剂得以获得了飞速发展的机会。酶制剂作为一种绿色、高效的优良添加剂被广泛应用到洗涤行业中,时至今日,蛋白酶依然是洗涤行业中应用最多的酶种。这主要得益于日常生活中,蛋白质污渍是主要的污渍种类,并且难以完全去除,如奶渍、汗渍、血渍、各种食物污渍以及人体皮肤细胞新陈代谢产生的角质等。蛋白酶能够高效水解蛋白质中的肽键,将难溶大分子降解为易溶小分子而被清除。
目前洗涤剂市场上添加的主要是碱性蛋白酶,大多以细菌来源的碱性蛋白酶为主,这些酶的最适pH范围主要在9.0-10.0之间,最适温度范围主要集中在50-60℃。但是在实际洗涤过程中,家用洗涤环境一般控制温度在30-40℃,而商用洗涤环境甚至更低。此外,随着人们对衣料品质的追求,碱性洗涤剂正在慢慢退出历史舞台,取而代之的是更为柔和的中性洗涤剂。因此洗涤用酶的实际应用效果大打折扣。相较于细菌来源的碱性蛋白酶,真菌来源的蛋白酶有着更为丰富的资源库。因此,挖掘真菌来源的中低温碱性蛋白酶来丰富洗涤用酶制剂市场,为行业发展奠定基础尤为重要。
酶的催化活性一直是衡量酶工业化重要指标之一,是酶制剂领域的研究重点。除了从根源上挖掘得到高酶活的野生型蛋白酶外,还可以通过蛋白质工程的手段对蛋白酶分子进行分子改良,以获得催化活性更为优异的突变体来满足工业生产的需要。植酸酶、葡萄糖氧化酶、淀粉酶等重要工业酶种都通过蛋白质工程化改造得到了大量催化活性优异的酶种用以工业生产中。
发明内容
本发明的目的是提供一种洗涤用碱性蛋白酶SP4。
本发明在于目的是提供上述碱性蛋白酶SP4的突变体SP4-M1。
本发明的再一目的是提供上述蛋白酶SP4及突变体SP4-M1的基因。
本发明的再一目的是提供包含上述蛋白酶SP4及突变体SP4-M1编码的重组载体。
本发明的再一目的是提供包含上述蛋白酶基因sp4及突变体基因sp4-m1的重组菌株。
本发明的再一目的是提供上述蛋白酶及突变体SP4-M1的应用。
本发明提供一种来源于Fusarium pseudograminearum的中碱性蛋白酶SP4,其在温度、pH和稳定性方面表现优异,在洗涤剂行业有着巨大的应用潜力,弥补了市场中洗涤用蛋白酶不适合中低温洗涤和中性洗涤剂的技术不足,其氨基酸序列如SEQ ID NO.1:
其中,该酶全长为389个氨基酸,N端前15个氨基酸为信号肽序列,使用双下划线作为标注;第16位到108位氨基酸为蛋白酶前导肽序列,使用单下环线标注。碱性蛋白酶SP4以成熟酶形式存在于发酵液上清中,因此其成熟酶理论分子量为28.3kDa,其氨基酸序列如SEQ ID NO.2所示:
该蛋白酶的最适pH为8.5,在pH 7.0-pH 10.0范围内,相对酶活在80%以上;25℃条件下在各个pH缓冲液中孵育1小时,蛋白酶SP4在pH 4.0-pH 12.0可以保持90%以上剩余酶活。最适温度40℃,在30-45℃条件下具有70%以上的相对酶活力;其在25-35℃条件下孵育1h可以保持70%以上剩余酶活。
本发明还提供了编码上述蛋白酶的基因。本发明通过提取RNA和反转录的方法分离克隆了这个蛋白酶基因sp4,其cDNA全长为1170bp,编码信号肽的核苷酸序列使用双下划线作为标注,编码前导肽的核苷酸序列使用单下划线作为标注,其序列如SEQ ID NO.3所示:
根据本发明的具体实施方式,将上述中碱性蛋白酶SP4的第70位氨基酸Gln突变为Asn,第142位氨基酸Asp突变为Ser和第143位Ala突变为Ser。结果表明,与野生型蛋白酶SP4相比,突变体SP4-M1的最适pH值、最适温度并未发生变化,但比活力相较于野生型提高了约50%。
根据本发明的催化活性提高的蛋白酶突变体SP4-M1,其氨基酸序列SEQ ID NO:4所示:
本发明提供了编码上述催化活性提高的蛋白酶突变体的基因sp4-m1,所述基因编码通过以下定点突变获得的突变体,(2)通过对氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶进行Q178N/D250S/A251S定点突变而得,或者(2)通过对氨基酸序列如SEQ ID NO:2的碱性蛋白酶进行Q70N/D142S/A143S定点突变而得;或者(3)将氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶去除N端前15个氨基酸后进行Q163N/D235S/A236S定点突变而得。
根据本发明的编码上述催化活性提高的蛋白酶突变体的基因sp4-m1,将氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶去除N端前15个氨基酸后进行Q163N/D235S/A236S定点突变而得的突变体的编码基因的核苷酸序列如SEQ ID NO:5所示。
本发明还提供了包含上述蛋白酶和突变体基因的重组载体,优选为pPICZαA-sp4和pPICZαA-sp4-m1。
本发明还提供了包含上述蛋白酶和突变体基因载体的重组菌株GS115/sp4、GS115/sp4-m1。
本发明还提供了一种制备蛋白酶的方法,包括以下步骤:
(1)以上述重组表达载体转化宿主细胞;
(2)培养并诱导宿主细胞异源表达蛋白酶SP4及其突变体SP4-M1;
(3)分离纯化获得蛋白酶SP4及其催化活性提高的蛋白酶SP4-M1。
本发明还提供了洗涤用蛋白酶洗涤环境稳定性的检测方法,通过表面活性剂、有机溶剂和洗涤剂稳定性来初步评估蛋白酶是否具有洗涤用酶制剂应用前景。
根据本发明的具体实施方式,将克隆得到的真菌来源的碱性蛋白酶基因sp4及含有突变体基因sp4-m1重组载体线性化后电转化进毕赤酵母GS115菌株中在基因组中相对应的整合位点发生同源重组,通过牛奶板对阳性转化子进行筛选。挑选牛***解圈最明显的转化子进行摇瓶水平诱导,对粗酶液进行蛋白浓缩及离子柱纯化。利用SDS-PAGE电泳分析检测相应蛋白酶纯度。采用福林酚法测定野生型与突变体的基本酶学性质,并进行洗涤环境稳定性检测。结果表明,蛋白酶SP4及其突变体SP4-M1的最适温度、最适pH、pH和热稳定性并未发生明显变化,但SP4-M1的比活力较野生型提高了50%。此外洗涤环境稳定性检测结果也显示,蛋白酶SP4及其催化活性提高的突变体SP4-M1洗涤剂稳定性良好,具有洗涤用蛋白酶制剂的应用潜力。
附图说明
图1显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的SDS-PAGE分析结果;
图2显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的最适pH值;
图3显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的pH稳定性;
图4显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的最适反应温度;
图5显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的热稳定性;
图6显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的有机溶剂稳定性;
图7显示本发明重组表达的蛋白酶SP4及突变体SP4-M1的表面活性剂稳定性;
图8显示本发明重组表达的蛋白酶突变体SP4-M1的污布洗涤性能实验。
具体实施方式
试验材料和试剂
1、菌株及载体:毕赤酵母(Pichia pastoris GS115)和表达载体(pPICZαA)为本实验室保存;
2、酶类及其它生化试剂:限制性内切酶购自Thermofisher公司,同源重组和点突变试剂盒购自全式金公司,酪蛋白购自Sigma公司,真菌总RNA提取试剂盒购自OMEGA公司,其它都为国产试剂(均从普通生化试剂公司购买);
3、培养基:
LB培养基:酵母浸提物5g/L,蛋白胨10g/L,NaCl 10g/L,琼脂粉20g/L,
YPD培养基:酵母浸提物10g/L,蛋白胨20g/L,葡萄糖20g/L,琼脂粉20g/L,
BMGY培养基:酵母浸提物10g/L,蛋白胨20g/L,甘油1%(V/V),YNB 1.34g/L,生物素0.0004g/L,
BMMY培养基:酵母浸提物10g/L,蛋白胨20g/L,甲醇0.5%(V/V),YNB 1.34g/L,生物素0.0004g/L,
牛奶双层筛选固体培养基:
上层:酵母浸提物10g/L,蛋白胨20g/L,甲醇0.5%(V/V),YNB 1.34g/L,生物素0.0004g/L,琼脂粉20g/L,
下层:脱脂奶粉80g/L,琼脂粉20g/L。
以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例1蛋白酶编码基因sp4及突变体sp4-m1基因克隆
(1)Fusarium pseudograminearum CS3096基因组RNA提取
真菌总RNA提取试剂盒使用的是OMEGA公司产品,具体操作如下:
取少量菌丝接种于PDB培养基中,以28℃、200rpm条件培养3-4天。收集适量菌丝体放入经过高温灭菌的研钵中,用液氮快速研磨成粉末。取50mg研磨后的菌丝体于2mL EP管中,加入500ul裂解液,上下颠倒混匀。12000rpm,4℃条件下离心5min,将上清转移到预装柱中发,12000rpm,4℃条件下离心2min。吸取500ul上清液转移到新的EP管中,加入250ul无水乙醇,上下颠倒混匀,将液体转移到RNA吸附柱上,12000rpm,4℃条件下离心1min。加入300ul washing bufferⅠ,12000rpm,4℃条件下离心1min。加入75ul加有DNA酶的消化液,室温放置15min。加入400ul RNA washing bufferⅠ,12000rpm,4℃条件下离心1min。加入500ul RNA washing bufferⅡ,12000rpm,4℃条件下离心1min,重复两次。将吸附柱室温放置并晾干。加入50ul ddH2O,静置3min,12000rpm、4℃条件下离心1min收集RNA。使用琼脂糖凝胶电泳检测RNA提取质量。
(2)蛋白酶RNA反转录和cDNA的获取
以提取到的总RNA为模板进行反转录。使用2.5ul Oligo(dT)20(10umol/L)和27.5ul的RNA合成cDNA第一条链。使用蛋白酶基因sp4引物(见表1)扩增cDNA序列,获得的DNA片段连接到T载体上送至测序。
表1基因克隆引物表
实施例2蛋白酶表达载体pPICZαA-sp4及pPICZαA-sp4-m1构建
(1)蛋白酶野生型表达载体构建pPICZαA-sp4
以测序正确的蛋白酶sp4的cDNA为模板,设计合成了带有EcoR I和Not I限制性酶切位点的引物sp4-ZF和sp4-ZR(见表2),对sp4的成熟蛋白的编码区进行扩增。使用EcoR I和Not I对表达载体pPICZαA进行双酶切,通过同源重组试剂盒将扩增片段连接于表达载体信号肽序列下游,形成正确的开放式阅读框,成功构建蛋白酶SP4表达载体pPICZαA-sp4,热激转化进入大肠杆菌克隆宿主XL10(南京诺唯赞生物科技股份有限公司)。使用博来霉素进行阳性克隆子的筛选并送到测序公司测序。挑选测序正确的转化子进行表达质粒的制备。
表2表达载体构建引物表
(2)蛋白酶突变体表达载体构建pPICZαA-sp4-m1
以重载载体pPICZαA-sp4为模板,通过点突变引物(见表2)对载体进行扩增,使用DMT酶消化具有甲基化修饰的模板,获得含有突变的重组载体转化进克隆宿主,筛选并送到测序公司测序。挑选测序正确的转化子进行表达质粒的制备。
实施例3蛋白酶表达菌株的构建
(1)表达载体电转化表达宿主
使用限制性内切酶Dra I线性化表达载体,电击转化进表达宿主GS115感受态细胞中,使用山梨醇孵育30min后涂布于带有博来霉素抗性(100ul/100mL)的YPD平板上,30℃培养2天。
(2)高蛋白酶活性转化子的筛选
用灭过菌的牙签从长有转化子的YPD板上挑取单菌落,接种于牛奶双层筛选固体平板上,30℃培养过夜。牛奶板上出现肉眼可见的牛***解透明圈,挑选水解圈最大的转化子接种于30mLYPD培养基中。
实施例4重组蛋白酶的表达与纯化
(1)蛋白酶SP4及突变体SP4-M1的摇瓶水平表达
将筛选得到的酶活较高的转化子按1%接菌量接种于300mL的BMGY培养基中,在30℃、200rpm摇床振荡培养48h后,以4500rpm离心5min,去掉上清。用200mL BMMY培养基振荡重悬,在30℃、200rpm摇床继续振荡培养48h。培养期间,每24h向培养基中补加0.5%甲醇。培养结束后,以12000rpm离心10min收集上清发酵液,用以进行纯化。
(2)蛋白酶SP4及SP4-M1的纯化
将收集的发酵液使用5kDa膜包进行浓缩,浓缩至15mL。使用3kDa透析袋在20mM柠檬酸-磷酸盐缓冲液(pH 6.0)过夜透析去除盐离子。使用20mM柠檬酸-磷酸盐缓冲液(pH6.0)A液和B液进行阴离子交换层析进行纯化。对1M NaCl线性洗脱液进行酶活检测和SDS-PAGE电泳分析(图1)。
实施例5重组蛋白酶酶学性质分析
采用福林酚法对蛋白酶SP4及突变体SP4-M1进行酶学性质测定,缓冲体系分别为乳酸-乳酸钠缓冲体系(50mM,pH 1.0-5.0)、磷酸盐缓冲体系(200mM,pH 5.0-8.0)和硼砂-氢氧化钠缓冲体系(100mM,pH 8.0-12.0)具体方法如下:
以酪蛋白为底物,500ul底物(1%W/W)和500ul适当稀释倍数的酶液,在不同温度下反应20min,加入1mL 0.4M三氯乙酸溶液终止反应。将该反应体系转入2mL EP管中,使用12000rpm离心3min后,吸取500ul上清加入2.5mL 0.4M碳酸钠溶液中,再加入500ul福林酚试剂,40℃下显色30min,冷却后吸取250ul显色体系读取其在680nm的吸光值。蛋白酶活性单位定义:在一定条件下,每分钟分解底物酪蛋白生成lμmol酪氨酸所需的酶量为1个活性单位(U)。
(1)最适pH及pH稳定性
利用不同pH的缓冲液将酶液稀释至适当倍数,在适当温度下与对应pH的底物反应20min,测定其酶活,以探究其最适pH。结果表明(图2),蛋白酶SP4的最适pH为8.5,突变体SP4-M1的最适pH未发生变化。蛋白酶在pH 7.0-pH 10.0范围内,相对酶活在80%以上。
将纯化后的酶液用不同pH缓冲液稀释至100ng/mL在25℃下孵育1h,对处理后的酶液进行适当稀释,在最适温度及pH下测定其pH稳定性。结果表明(图3),蛋白酶SP4及突变体SP4-M1的pH稳定性基本一致,在25℃条件下具有很宽的pH稳定性范围,适合在中性洗衣液和碱性洗衣粉中的应用。
(2)最适温度及热稳定性
将纯化后的酶液用最适pH条件下的缓冲液稀释适当倍数,测定不同温度下酶活,以探究其最适温度。结果表明(图4),蛋白酶SP4及突变体SP4-M1的最适温度均为40℃,在在30-45℃条件下具有70%以上的相对酶活力,符合家庭洗涤环境温度。
将纯化后的酶液稀释至100ng/mL在不同温度下孵育1h,不同时间取样,对处理后的酶液进行适当稀释,在最适温度及pH下测定其热稳定性。结果表明(图5),其在25-35℃条件下孵育1h可以保持70%以上剩余酶活。
(3)动力学常数测定
分别配制浓度为0.5、0.8、1.0、1.3、1.5、2.0、2.5、5.0、8.0、10.0、12.0、15.0mg/mL的酪蛋白为底物,对纯化后的酶液适当稀释与不同浓度底物在最适温度和pH下反应10min,测定其酶活力。使用GraphPad Prism version 5.01软件拟合米氏方程常数,计算Km和Vmax。
比活测定结果如表3所示,蛋白酶SP的比活力为416.62±5.87U·mg-1,而突变体SP4-M1的比活为615.84±8.46U·mg-1。相较于SP4,突变体SP4-M1的Km值明显变小,Q70N点突变造成的底物亲和力的改变。Vmax值变化明显,由625.2±6.78U·mg-1提高到875.2±8.57U·mg-1。转换数由295.16±3.20s-1提高至413.18±4.05s-1,最终kcat/Km由51.40±0.63mL·s–1·mg–1提高至93.02±1.48mL·s–1·mg–1。
表3蛋白酶SP4和SP4-M1动力学参数
实施例6重组蛋白酶洗涤环境稳定性和洗涤能力检测
(1)有机溶剂、表面活性剂、洗涤剂稳定性测定
将纯化后的酶液稀释至100ng/mL,分别与终浓度10%有机溶剂、0.5%表面活性剂在25℃下孵育30min,以未处理酶液作为对照,在最适温度及pH下测定其有机溶剂和表面活性剂稳定性。结果表明(图6和图7),除了正丁醇和H2O2对蛋白酶的酶活由较大影响外,其他有机试剂和表面活性剂对蛋白酶酶活影响不大。特别是0.5%SDS(约等于17mM SDS)孵育后,剩余酶活仍然在70%以上。
将纯化后的酶液稀释至100ng/mL,分别与终浓度1%洗衣液和7mg/mL洗衣粉在25℃下孵育30min、60min和90min,以未处理酶液作为对照,在最适温度及pH下测定其洗涤剂稳定性。结果表明,蛋白酶SP4及SP4-M1在洗衣液和洗衣粉中孵育90min后,剩余酶活在80%以上,适合作用洗涤用酶加入到洗涤剂中。
(2)蛋白酶污布洗涤性能实验
取2.5cm2的棉布,向其滴加50ul鸡血,室温干燥48h后,以50mL离心管作为处理容器,加入5mL自来水,30℃条件下以200rpm处理10min洗去松散和多余的血液,室温风干12h。取风干后的棉布,分别在各个处理组中添加自来水、洗衣液1%、洗衣粉7mg/mL、SP4-M1(250U)+洗衣液1%和SP4-M1(250U)+洗衣粉7mg/mL,各个处理组总体积均控制在5mL,在30℃和200rpm的条件下处理1h后,用清水冲洗干净残留液体,室温干燥并观察。从实验的结果(图8)中可以看出,水洗棉布被血液染成谈黄褐色,洗衣液和洗衣粉处理组有肉眼可见的圆形血渍,而添加了蛋白酶的处理组棉布的白度明显优于未添加蛋白酶的处理组。
以上实施例仅用于解释本申请的技术方案,不限定本申请的保护范围。
序列表
<110> 山东隆科特酶制剂有限公司
<120> 一种碱性蛋白酶SP4及其突变体、编码基因和应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 389
<212> PRT
<213> 小麦冠腐病菌(Fusarium pseudograminearum)
<400> 1
Met Arg Ser Ala Thr Leu Leu Ala Leu Leu Pro Phe Ala Leu Ala Ala
1 5 10 15
Pro Ser Ala Ser Val Ser Arg Arg Thr Glu Pro Ala Pro Val Ile Arg
20 25 30
Pro Arg Gly Val Lys Leu Val Asp Gly Lys Tyr Ile Val Lys Met Lys
35 40 45
Ala Gly Val Arg Ala Ala Ser Val Asp Ser Ala Val Ser Thr Ile Gln
50 55 60
Ala Asp Ala Asp Tyr Thr Tyr Thr Lys Ser Phe Ser Gly Phe Ala Ala
65 70 75 80
Ser Leu Lys Asp Glu Glu Ile Glu Thr Leu Lys His Asp Pro Asn Val
85 90 95
Glu Tyr Ile Glu Gln Asp Ala Val Ile Thr Ile Lys Ala Thr Val Asp
100 105 110
Gln Asp Asn Ala Pro Trp Gly Ile Ala Arg Leu Ser Ser Ser Lys Pro
115 120 125
Gly Ser Lys Thr Tyr Thr Tyr Asp Glu Ser Ala Gly Glu Gly Thr Cys
130 135 140
Ser Tyr Val Ile Asp Thr Gly Ile Asp Val Glu His Pro Asp Phe Asp
145 150 155 160
Gly Arg Ala Lys Phe Leu Lys Asn Phe Ala Gly Gly Arg Asp Gly Asp
165 170 175
Gly Gln Gly His Gly Thr His Val Ala Gly Thr Ile Gly Ser Thr Thr
180 185 190
Tyr Gly Val Ala Lys Lys Thr Thr Leu Tyr Ala Val Lys Val Leu Gly
195 200 205
Asp Asp Gly Ser Gly Thr Asn Ser Ala Val Ile Ala Gly Met Asp Phe
210 215 220
Val Ala Gly His Ala Asp Asp Glu Asn Cys Pro Asn Gly Ala Val Val
225 230 235 240
Asn Met Ser Leu Gly Gly Glu Ala Ser Asp Ala Val Asn Ser Ala Ala
245 250 255
Lys Ser Ile Val Asp Ala Gly Leu Phe Leu Ala Val Ala Ala Gly Asn
260 265 270
Glu Ala Val Asp Ala Ser Gly Ser Ser Pro Ala Ser Glu Ala Ser Ala
275 280 285
Cys Thr Val Gly Ala Thr Thr Arg Asn Asp Thr Leu Ser Tyr Phe Ser
290 295 300
Asn Phe Gly Asp Leu Val Asp Val Leu Ala Pro Gly Thr Asp Ile Leu
305 310 315 320
Ser Thr Trp Pro Gly Gly Lys Thr Asn Thr Ile Ser Gly Thr Ser Met
325 330 335
Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr Phe Leu Gly Leu Gly
340 345 350
Lys Lys Ala Glu Gly Leu Cys Glu Tyr Ile Ala Ser Gln Ala Leu Glu
355 360 365
Gly Val Val Ala Gln Val Pro Arg Asp Thr Val Asn Lys Leu Ile Asn
370 375 380
Asn Gly Val Ser Lys
385
<210> 2
<211> 281
<212> PRT
<213> 小麦冠腐病菌(Fusarium pseudograminearum)
<400> 2
Ala Thr Val Asp Gln Asp Asn Ala Pro Trp Gly Ile Ala Arg Leu Ser
1 5 10 15
Ser Ser Lys Pro Gly Ser Lys Thr Tyr Thr Tyr Asp Glu Ser Ala Gly
20 25 30
Glu Gly Thr Cys Ser Tyr Val Ile Asp Thr Gly Ile Asp Val Glu His
35 40 45
Pro Asp Phe Asp Gly Arg Ala Lys Phe Leu Lys Asn Phe Ala Gly Gly
50 55 60
Arg Asp Gly Asp Gly Gln Gly His Gly Thr His Val Ala Gly Thr Ile
65 70 75 80
Gly Ser Thr Thr Tyr Gly Val Ala Lys Lys Thr Thr Leu Tyr Ala Val
85 90 95
Lys Val Leu Gly Asp Asp Gly Ser Gly Thr Asn Ser Ala Val Ile Ala
100 105 110
Gly Met Asp Phe Val Ala Gly His Ala Asp Asp Glu Asn Cys Pro Asn
115 120 125
Gly Ala Val Val Asn Met Ser Leu Gly Gly Glu Ala Ser Asp Ala Val
130 135 140
Asn Ser Ala Ala Lys Ser Ile Val Asp Ala Gly Leu Phe Leu Ala Val
145 150 155 160
Ala Ala Gly Asn Glu Ala Val Asp Ala Ser Gly Ser Ser Pro Ala Ser
165 170 175
Glu Ala Ser Ala Cys Thr Val Gly Ala Thr Thr Arg Asn Asp Thr Leu
180 185 190
Ser Tyr Phe Ser Asn Phe Gly Asp Leu Val Asp Val Leu Ala Pro Gly
195 200 205
Thr Asp Ile Leu Ser Thr Trp Pro Gly Gly Lys Thr Asn Thr Ile Ser
210 215 220
Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr Phe
225 230 235 240
Leu Gly Leu Gly Lys Lys Ala Glu Gly Leu Cys Glu Tyr Ile Ala Ser
245 250 255
Gln Ala Leu Glu Gly Val Val Ala Gln Val Pro Arg Asp Thr Val Asn
260 265 270
Lys Leu Ile Asn Asn Gly Val Ser Lys
275 280
<210> 3
<211> 1170
<212> DNA
<213> 小麦冠腐病菌(Fusarium pseudograminearum)
<400> 3
atgcgttccg ctactcttct cgctcttttg ccctttgcgc tggccgctcc ttcagcctca 60
gtctctcgac gaactgagcc cgcgcctgtc atccgccctc gcggtgtcaa gcttgtcgat 120
ggcaagtaca tcgtcaagat gaaggccggt gtccgagctg cttctgttga cagcgctgtt 180
tcgaccatcc aggctgatgc cgattacacc tacaccaaga gcttcagtgg ctttgctgct 240
agtctcaagg atgaggagat tgagaccctc aagcacgacc ctaatgtcga gtacattgag 300
caggatgctg tcatcaccat caaggctact gtcgaccagg acaatgctcc ttggggtatc 360
gcccgtcttt ctagctccaa gcccggtagc aagacttaca cctatgacga gagcgctggc 420
gagggtactt gctcctacgt catcgacacc ggtattgatg ttgagcatcc cgactttgat 480
ggacgtgcca agttcctcaa gaactttgct ggtggtcgtg atggtgatgg tcaaggtcac 540
ggcacccacg tcgctggaac tatcggctcc accacctacg gtgtcgccaa gaagaccacc 600
ctctacgccg ttaaggtcct tggtgacgac ggctccggta ccaactctgc tgtcattgct 660
ggtatggact ttgtcgctgg tcatgccgat gacgagaatt gccctaacgg tgccgttgtc 720
aacatgtccc ttggtggcga agcctccgat gctgtcaaca gcgctgccaa gagcatcgtc 780
gatgctggtc tcttccttgc cgtagcagct ggcaacgaag ccgtcgacgc ttccggctca 840
tcccctgctt ccgaggccag cgcctgcacc gtcggcgcaa ccacccgcaa cgatactctt 900
tcctacttct ccaactttgg cgacctcgtc gatgttcttg ctcctggtac cgacatcctg 960
agcacctggc ccggcggcaa gaccaacacc atctccggaa cttccatggc ttcgccccat 1020
gttgctggtc ttggtgctta cttccttggt ctgggcaaga aggccgaggg tctctgcgag 1080
tacattgctt cccaggctct tgagggagtt gtggctcagg tgccccgcga cactgttaac 1140
aagctcatca acaacggtgt tagcaagtaa 1170
<210> 4
<211> 281
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Thr Val Asp Gln Asp Asn Ala Pro Trp Gly Ile Ala Arg Leu Ser
1 5 10 15
Ser Ser Lys Pro Gly Ser Lys Thr Tyr Thr Tyr Asp Glu Ser Ala Gly
20 25 30
Glu Gly Thr Cys Ser Tyr Val Ile Asp Thr Gly Ile Asp Val Glu His
35 40 45
Pro Asp Phe Asp Gly Arg Ala Lys Phe Leu Lys Asn Phe Ala Gly Gly
50 55 60
Arg Asp Gly Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Ile
65 70 75 80
Gly Ser Thr Thr Tyr Gly Val Ala Lys Lys Thr Thr Leu Tyr Ala Val
85 90 95
Lys Val Leu Gly Asp Asp Gly Ser Gly Thr Asn Ser Ala Val Ile Ala
100 105 110
Gly Met Asp Phe Val Ala Gly His Ala Asp Asp Glu Asn Cys Pro Asn
115 120 125
Gly Ala Val Val Asn Met Ser Leu Gly Gly Glu Ala Ser Ser Ser Val
130 135 140
Asn Ser Ala Ala Lys Ser Ile Val Asp Ala Gly Leu Phe Leu Ala Val
145 150 155 160
Ala Ala Gly Asn Glu Ala Val Asp Ala Ser Gly Ser Ser Pro Ala Ser
165 170 175
Glu Ala Ser Ala Cys Thr Val Gly Ala Thr Thr Arg Asn Asp Thr Leu
180 185 190
Ser Tyr Phe Ser Asn Phe Gly Asp Leu Val Asp Val Leu Ala Pro Gly
195 200 205
Thr Asp Ile Leu Ser Thr Trp Pro Gly Gly Lys Thr Asn Thr Ile Ser
210 215 220
Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr Phe
225 230 235 240
Leu Gly Leu Gly Lys Lys Ala Glu Gly Leu Cys Glu Tyr Ile Ala Ser
245 250 255
Gln Ala Leu Glu Gly Val Val Ala Gln Val Pro Arg Asp Thr Val Asn
260 265 270
Lys Leu Ile Asn Asn Gly Val Ser Lys
275 280
<210> 5
<211> 1125
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gctccttcag cctcagtctc tcgacgaact gagcccgcgc ctgtcatccg ccctcgcggt 60
gtcaagcttg tcgatggcaa gtacatcgtc aagatgaagg ccggtgtccg agctgcttct 120
gttgacagcg ctgtttcgac catccaggct gatgccgatt acacctacac caagagcttc 180
agtggtttcg ctgctagtct caaggatgag gagattgaga ccctcaagca cgaccccaat 240
gtcgagtaca ttgagcagga tgctgtcatc accatcaagg ctactgttga ccaggacaat 300
gctccttggg gtatcgcccg tctttccagc tccaagcccg gtagcaagac ttacacctac 360
gacgagagcg ctggtgaggg tacttgctct tacgtcatcg acaccggcat tgatgttgag 420
catcccgact ttgatggacg tgccaagttc ctcaagaact ttgctggtgg ccgtgatggt 480
gacggcaacg gtcacggcac ccacgttgct ggaactatcg gctccaccac ctacggtgtc 540
gccaagaaga ccaccctcta cgccgtcaag gtcctgggtg atgacggttc cggtaccaac 600
tctgctgtca ttgctggcat ggactttgtc gctggccacg ccgatgacga gaactgccct 660
aacggtgccg ttgtcaacat gtctcttggt ggcgaagcct cgtcttctgt caacagcgct 720
gccaagagca ttgttgatgc tggtctcttc cttgccgtcg cagctggcaa cgaagccgtc 780
gacgcttccg gctcatcccc tgcttccgag gccagcgcct gcaccgttgg cgcaaccact 840
cgaaacgaca ccctttccta cttctccaac tttggcgacc tcgtcgatgt tcttgctcct 900
ggtaccgaca tcctgagcac ctggcccggc ggcaagacca acaccatctc cggaacttcc 960
atggcttcgc cccatgtcgc tggtctgggc gcttacttcc ttggacttgg aaagaaggcc 1020
gagggtctct gcgagtacat tgcttctcag gctcttgagg gagttgtggc ccaggtgcct 1080
cgcgacactg ttaacaagct catcaacaac ggtgttagca agtaa 1125
Claims (8)
1.一种比活和催化效率提高的蛋白酶突变体SP4-M1,其特征在于,所述突变体通过对氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶进行Q178N、D250S和 A251S定点突变而得;
所述突变体通过将氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶去除N端前15个氨基酸后进行Q163N、 D235S和A236S定点突变而得;或者
所述突变体通过对氨基酸序列如SEQ ID NO:2的碱性蛋白酶进行Q70N、D142S和A143S定点突变而得。
2.一种碱性蛋白酶基因,其特征在于,编码权利要求1所述的比活和催化效率提高的蛋白酶突变体SP4-M1。
3.根据权利要求2所述的碱性蛋白酶基因,其特征在于,其核苷酸序列如SEQ ID NO:5所示。
4.包含权利要求2所述碱性蛋白酶基因的重组表达载体。
5.包含权利要求2所述碱性蛋白酶基因的重组菌株。
6.一种制备碱性蛋白酶的方法,其特征在于,所述包括以下步骤:
(1)以权利要求4所述的重组表达载体转化宿主细胞;
(2)培养并诱导宿主细胞异源表达碱性蛋白酶;
(3)分离纯化获得碱性蛋白酶。
7.权利要求1所述的比活和催化效率提高的蛋白酶突变体SP4-M1的在制备洗涤剂中的应用。
8.一种提高蛋白酶的比活和催化效率的方法,其特征在于,所述方法包括以下步骤:对氨基酸序列如SEQ ID NO:1所示的碱性蛋白酶进行Q178N、 D250S和 A251S定点突变,或者去除N端前15个氨基酸后进行Q163N、D235S和A236S定点突变;
或者,
对氨基酸序列如SEQ ID NO:2的碱性蛋白酶进行Q70N、D142S和A143S定点突变。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210029528 | 2022-01-12 | ||
CN2022100295286 | 2022-01-12 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114958813A CN114958813A (zh) | 2022-08-30 |
CN114958813B true CN114958813B (zh) | 2023-08-04 |
Family
ID=82956559
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210570089.XA Active CN114958813B (zh) | 2022-01-12 | 2022-05-24 | 一种碱性蛋白酶sp4及其突变体、编码基因和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114958813B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116904428B (zh) * | 2023-09-11 | 2024-01-02 | 中国农业科学院北京畜牧兽医研究所 | 一种改良高温碱性蛋白酶AprThc的稳定性的方法及突变体 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109722428B (zh) * | 2018-12-11 | 2022-05-31 | 中国农业科学院北京畜牧兽医研究所 | 比活和热稳定性提高的碱性蛋白酶突变体prok-m及其编码基因和应用 |
-
2022
- 2022-05-24 CN CN202210570089.XA patent/CN114958813B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN114958813A (zh) | 2022-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5475101A (en) | DNA sequence encoding endoglucanase III cellulase | |
US9512409B2 (en) | Thermostable catalase | |
CN102839165B (zh) | 基因突变型重组蛋白酶k及其工业化生产方法 | |
CN107012130B (zh) | 一种葡萄糖氧化酶突变体及其编码基因和应用 | |
CN111676210B (zh) | 一种提高纤维素酶活性的方法及纤维素酶突变体5i77-m和应用 | |
CN110527677B (zh) | 玉米赤霉烯酮水解酶突变体zhdm2及其编码基因和应用 | |
CN114958813B (zh) | 一种碱性蛋白酶sp4及其突变体、编码基因和应用 | |
CN109280656A (zh) | 重组布氏白僵菌蛋白酶k突变体pk-m1及制备方法 | |
CN113684198B (zh) | 一种提高纤维素酶催化效率的方法及突变体5i77-m2 | |
CN110218709B (zh) | 一种耐热漆酶及其基因与应用 | |
CN109371004B (zh) | 热稳定性提高的酸性蛋白酶Bs2688突变体K203E及其基因和应用 | |
CN107988190B (zh) | 一种酸性蛋白酶及其编码基因和应用 | |
CN111676209B (zh) | 一种提高双功能纤维素酶的甘露聚糖酶活的方法及纤维素酶突变体rmx-m和应用 | |
CN111733149B (zh) | 一种转换纤维素与甘露聚糖活力的纤维素酶突变体及其基因和应用 | |
CN109207460A (zh) | 重组布氏白僵菌蛋白酶k突变体pk-m2及制备方法 | |
CN114317495B (zh) | 一种热稳定性提高的葡聚糖酶突变体及其应用 | |
CN113980940B (zh) | 一种提高双功能纤维素酶的催化效率的方法及突变体、基因和应用 | |
CN115247165A (zh) | 一种比活和热稳定性提高的纤维素酶突变体 | |
CN114836408A (zh) | 含有前肽突变体的碱性蛋白酶及应用 | |
CN113755473A (zh) | 分泌表达量提高的葡萄糖淀粉酶突变体m5及其基因和应用 | |
CN113073057A (zh) | 耐高温毕赤酵母菌株 | |
CN114350643B (zh) | 一种产氨肽酶的重组菌株及其在高效蛋白水解中的应用 | |
CN114958785B (zh) | 漆酶重组蛋白及其应用 | |
CN116218822A (zh) | 一种提高碱性蛋白酶热稳定性的方法及突变体ThAPT3-M5和应用 | |
Kiribayeva et al. | Cloning, purification and study of the biochemical properties of α-amylase from Bacillus licheniformis T5 strain |
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 |