CN113699138A - Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application - Google Patents
Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application Download PDFInfo
- Publication number
- CN113699138A CN113699138A CN202110952632.8A CN202110952632A CN113699138A CN 113699138 A CN113699138 A CN 113699138A CN 202110952632 A CN202110952632 A CN 202110952632A CN 113699138 A CN113699138 A CN 113699138A
- Authority
- CN
- China
- Prior art keywords
- bcp
- alkaline protease
- seq
- artificial sequence
- recombinant expression
- 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
- 108091005658 Basic proteases Proteins 0.000 title claims abstract description 101
- 239000013604 expression vector Substances 0.000 title claims abstract description 59
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 42
- 241000894006 Bacteria Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 24
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 68
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 66
- 239000002773 nucleotide Substances 0.000 claims abstract description 56
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 56
- 230000014509 gene expression Effects 0.000 claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 38
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 230000008569 process Effects 0.000 claims description 10
- 108010065511 Amylases Proteins 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000007065 protein hydrolysis Effects 0.000 claims 1
- 108700026220 vif Genes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 50
- 108090000790 Enzymes Proteins 0.000 description 50
- 230000000694 effects Effects 0.000 description 49
- 238000000855 fermentation Methods 0.000 description 28
- 230000004151 fermentation Effects 0.000 description 28
- 239000001963 growth medium Substances 0.000 description 18
- 101150058790 bcp gene Proteins 0.000 description 17
- 239000007788 liquid Substances 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 241000193752 Bacillus circulans Species 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 108091005804 Peptidases Proteins 0.000 description 9
- 239000004365 Protease Substances 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 239000004382 Amylase Substances 0.000 description 7
- 102000013142 Amylases Human genes 0.000 description 7
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 7
- 235000019418 amylase Nutrition 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 108010050848 glycylleucine Proteins 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 4
- 102000012286 Chitinases Human genes 0.000 description 4
- 108010022172 Chitinases Proteins 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 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 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 102000004400 Aminopeptidases Human genes 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 108700040634 Bacillus subtilis Vpr Proteins 0.000 description 2
- 241000193388 Bacillus thuringiensis Species 0.000 description 2
- 108010019119 Bombyx acid cysteine proteinase Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 2
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- JPIDMRXXNMIVKY-VZFHVOOUSA-N Ser-Ala-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPIDMRXXNMIVKY-VZFHVOOUSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 108010070944 alanylhistidine Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940097012 bacillus thuringiensis Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- BSQTUVXJJUHMPJ-USJZOSNVSA-N (2s)-2-[[2-[[(2s)-2-[[2-[[(2s)-1-[(2s)-2-aminopropanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-3-methylbutanoyl]amino]acetyl]amino]-3-methylbutanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N BSQTUVXJJUHMPJ-USJZOSNVSA-N 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- NXSFUECZFORGOG-CIUDSAMLSA-N Ala-Asn-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXSFUECZFORGOG-CIUDSAMLSA-N 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 1
- 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 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- XUCHENWTTBFODJ-FXQIFTODSA-N Ala-Met-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O XUCHENWTTBFODJ-FXQIFTODSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- PEEYDECOOVQKRZ-DLOVCJGASA-N Ala-Ser-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PEEYDECOOVQKRZ-DLOVCJGASA-N 0.000 description 1
- VRTOMXFZHGWHIJ-KZVJFYERSA-N Ala-Thr-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VRTOMXFZHGWHIJ-KZVJFYERSA-N 0.000 description 1
- KTXKIYXZQFWJKB-VZFHVOOUSA-N Ala-Thr-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O KTXKIYXZQFWJKB-VZFHVOOUSA-N 0.000 description 1
- XSLGWYYNOSUMRM-ZKWXMUAHSA-N Ala-Val-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XSLGWYYNOSUMRM-ZKWXMUAHSA-N 0.000 description 1
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 1
- GXMSVVBIAMWMKO-BQBZGAKWSA-N Asn-Arg-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N GXMSVVBIAMWMKO-BQBZGAKWSA-N 0.000 description 1
- HAJWYALLJIATCX-FXQIFTODSA-N Asn-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N HAJWYALLJIATCX-FXQIFTODSA-N 0.000 description 1
- ACRYGQFHAQHDSF-ZLUOBGJFSA-N Asn-Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ACRYGQFHAQHDSF-ZLUOBGJFSA-N 0.000 description 1
- BHQQRVARKXWXPP-ACZMJKKPSA-N Asn-Asp-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N BHQQRVARKXWXPP-ACZMJKKPSA-N 0.000 description 1
- IKLAUGBIDCDFOY-SRVKXCTJSA-N Asn-His-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O IKLAUGBIDCDFOY-SRVKXCTJSA-N 0.000 description 1
- NPZJLGMWMDNQDD-GHCJXIJMSA-N Asn-Ser-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NPZJLGMWMDNQDD-GHCJXIJMSA-N 0.000 description 1
- CLUMZOKVGUWUFD-CIUDSAMLSA-N Asp-Leu-Asn Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O CLUMZOKVGUWUFD-CIUDSAMLSA-N 0.000 description 1
- 241001328122 Bacillus clausii Species 0.000 description 1
- 241001661918 Bartonia Species 0.000 description 1
- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 description 1
- TWTWUBHEWQPMQW-ZPFDUUQYSA-N Gln-Ile-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWTWUBHEWQPMQW-ZPFDUUQYSA-N 0.000 description 1
- JILRMFFFCHUUTJ-ACZMJKKPSA-N Gln-Ser-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O JILRMFFFCHUUTJ-ACZMJKKPSA-N 0.000 description 1
- OTQSTOXRUBVWAP-NRPADANISA-N Gln-Ser-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O OTQSTOXRUBVWAP-NRPADANISA-N 0.000 description 1
- SZXSSXUNOALWCH-ACZMJKKPSA-N Glu-Ala-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O SZXSSXUNOALWCH-ACZMJKKPSA-N 0.000 description 1
- VAZZOGXDUQSVQF-NUMRIWBASA-N Glu-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)O VAZZOGXDUQSVQF-NUMRIWBASA-N 0.000 description 1
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 1
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 1
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 1
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 1
- JDUKCSSHWNIQQZ-IHRRRGAJSA-N Glu-Phe-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JDUKCSSHWNIQQZ-IHRRRGAJSA-N 0.000 description 1
- DMYACXMQUABZIQ-NRPADANISA-N Glu-Ser-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O DMYACXMQUABZIQ-NRPADANISA-N 0.000 description 1
- SFKMXFWWDUGXRT-NWLDYVSISA-N Glu-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N)O SFKMXFWWDUGXRT-NWLDYVSISA-N 0.000 description 1
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 1
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 1
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 1
- XCLCVBYNGXEVDU-WHFBIAKZSA-N Gly-Asn-Ser Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O XCLCVBYNGXEVDU-WHFBIAKZSA-N 0.000 description 1
- HAXARWKYFIIHKD-ZKWXMUAHSA-N Gly-Ile-Ser Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HAXARWKYFIIHKD-ZKWXMUAHSA-N 0.000 description 1
- 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 1
- BAYQNCWLXIDLHX-ONGXEEELSA-N Gly-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN BAYQNCWLXIDLHX-ONGXEEELSA-N 0.000 description 1
- RGPWUJOMKFYFSR-QWRGUYRKSA-N His-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RGPWUJOMKFYFSR-QWRGUYRKSA-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
- FHGVHXCQMJWQPK-SRVKXCTJSA-N His-Lys-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O FHGVHXCQMJWQPK-SRVKXCTJSA-N 0.000 description 1
- QLRMMMQNCWBNPQ-QXEWZRGKSA-N Ile-Arg-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)O)N QLRMMMQNCWBNPQ-QXEWZRGKSA-N 0.000 description 1
- WZDCVAWMBUNDDY-KBIXCLLPSA-N Ile-Glu-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)O)N WZDCVAWMBUNDDY-KBIXCLLPSA-N 0.000 description 1
- WUKLZPHVWAMZQV-UKJIMTQDSA-N Ile-Glu-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N WUKLZPHVWAMZQV-UKJIMTQDSA-N 0.000 description 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- KKXDHFKZWKLYGB-GUBZILKMSA-N Leu-Asn-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKXDHFKZWKLYGB-GUBZILKMSA-N 0.000 description 1
- KTFHTMHHKXUYPW-ZPFDUUQYSA-N Leu-Asp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KTFHTMHHKXUYPW-ZPFDUUQYSA-N 0.000 description 1
- CLVUXCBGKUECIT-HJGDQZAQSA-N Leu-Asp-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CLVUXCBGKUECIT-HJGDQZAQSA-N 0.000 description 1
- KVMULWOHPPMHHE-DCAQKATOSA-N Leu-Glu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KVMULWOHPPMHHE-DCAQKATOSA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- 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 1
- QJXHMYMRGDOHRU-NHCYSSNCSA-N Leu-Ile-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O QJXHMYMRGDOHRU-NHCYSSNCSA-N 0.000 description 1
- KYIIALJHAOIAHF-KKUMJFAQSA-N Leu-Leu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 KYIIALJHAOIAHF-KKUMJFAQSA-N 0.000 description 1
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 1
- 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 1
- VWPJQIHBBOJWDN-DCAQKATOSA-N Lys-Val-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O VWPJQIHBBOJWDN-DCAQKATOSA-N 0.000 description 1
- HUKLXYYPZWPXCC-KZVJFYERSA-N Met-Ala-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HUKLXYYPZWPXCC-KZVJFYERSA-N 0.000 description 1
- XMQZLGBUJMMODC-AVGNSLFASA-N Met-His-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O XMQZLGBUJMMODC-AVGNSLFASA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 1
- VXCHGLYSIOOZIS-GUBZILKMSA-N Pro-Ala-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 VXCHGLYSIOOZIS-GUBZILKMSA-N 0.000 description 1
- FZHBZMDRDASUHN-NAKRPEOUSA-N Pro-Ala-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1)C(O)=O FZHBZMDRDASUHN-NAKRPEOUSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- 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 1
- XSXABUHLKPUVLX-JYJNAYRXSA-N Pro-Ser-Trp Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O XSXABUHLKPUVLX-JYJNAYRXSA-N 0.000 description 1
- DMNANGOFEUVBRV-GJZGRUSLSA-N Pro-Trp-Gly Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)O)C(=O)[C@@H]1CCCN1 DMNANGOFEUVBRV-GJZGRUSLSA-N 0.000 description 1
- HQTKVSCNCDLXSX-BQBZGAKWSA-N Ser-Arg-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O HQTKVSCNCDLXSX-BQBZGAKWSA-N 0.000 description 1
- UGJRQLURDVGULT-LKXGYXEUSA-N Ser-Asn-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UGJRQLURDVGULT-LKXGYXEUSA-N 0.000 description 1
- VDVYTKZBMFADQH-AVGNSLFASA-N Ser-Gln-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VDVYTKZBMFADQH-AVGNSLFASA-N 0.000 description 1
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 description 1
- 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 1
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 1
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-CIUDSAMLSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- 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 1
- ZKOKTQPHFMRSJP-YJRXYDGGSA-N Ser-Thr-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZKOKTQPHFMRSJP-YJRXYDGGSA-N 0.000 description 1
- OQSQCUWQOIHECT-YJRXYDGGSA-N Ser-Tyr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OQSQCUWQOIHECT-YJRXYDGGSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- SKHPKKYKDYULDH-HJGDQZAQSA-N Thr-Asn-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SKHPKKYKDYULDH-HJGDQZAQSA-N 0.000 description 1
- DCCGCVLVVSAJFK-NUMRIWBASA-N Thr-Asp-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O DCCGCVLVVSAJFK-NUMRIWBASA-N 0.000 description 1
- WLDUCKSCDRIVLJ-NUMRIWBASA-N Thr-Gln-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O WLDUCKSCDRIVLJ-NUMRIWBASA-N 0.000 description 1
- YUOCMLNTUZAGNF-KLHWPWHYSA-N Thr-His-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N)O YUOCMLNTUZAGNF-KLHWPWHYSA-N 0.000 description 1
- 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 1
- LCCSEJSPBWKBNT-OSUNSFLBSA-N Thr-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N LCCSEJSPBWKBNT-OSUNSFLBSA-N 0.000 description 1
- YJCVECXVYHZOBK-KNZXXDILSA-N Thr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H]([C@@H](C)O)N YJCVECXVYHZOBK-KNZXXDILSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- JONPRIHUYSPIMA-UWJYBYFXSA-N Tyr-Ala-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JONPRIHUYSPIMA-UWJYBYFXSA-N 0.000 description 1
- SZEIFUXUTBBQFQ-STQMWFEESA-N Tyr-Pro-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SZEIFUXUTBBQFQ-STQMWFEESA-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
- LTFLDDDGWOVIHY-NAKRPEOUSA-N Val-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N LTFLDDDGWOVIHY-NAKRPEOUSA-N 0.000 description 1
- CFSSLXZJEMERJY-NRPADANISA-N Val-Gln-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CFSSLXZJEMERJY-NRPADANISA-N 0.000 description 1
- OQWNEUXPKHIEJO-NRPADANISA-N Val-Glu-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N OQWNEUXPKHIEJO-NRPADANISA-N 0.000 description 1
- 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 1
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- VIKZGAUAKQZDOF-NRPADANISA-N Val-Ser-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O VIKZGAUAKQZDOF-NRPADANISA-N 0.000 description 1
- UGFMVXRXULGLNO-XPUUQOCRSA-N Val-Ser-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O UGFMVXRXULGLNO-XPUUQOCRSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010090037 glycyl-alanyl-isoleucine Proteins 0.000 description 1
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010078580 tyrosylleucine Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an alkaline protease gene, a hybrid promoter, a recombinant expression vector, recombinant expression engineering bacteria, alkaline protease, a method and application thereof, wherein the nucleotide sequence of an alkaline protease gene bcp is shown as SEQ ID NO. 1. The total length of the alkaline protease gene BCP is 1143bp, and the alkaline protease gene BCP is efficiently expressed in a bacillus subtilis expression strain, so that the production cost of the alkaline protease BCP is reduced.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to an alkaline protease gene. In addition, the invention also relates to a hybrid promoter, a recombinant expression vector, a recombinant expression engineering bacterium, a preparation method and application thereof.
Background
The protease can decompose protein to form polypeptide and free amino acid, and has wide application prospect in many fields. The protease source is wide, the protease mainly comes from microorganisms in the current industrialized application, and the protease derived from the microorganisms has the characteristics of multiple types, various enzymological characteristics and the like. Proteases derived from microorganisms can be classified into acid proteases, neutral proteases and alkaline proteases according to the pH of action. The three proteases are applied to different industrial fields according to different enzymological properties. The alkaline protease is used as a serine protease, has good activity and stability, can effectively decompose various proteins, and is mainly applied to the fields of food processing, washing, feed and the like at present. Alkaline protease is a commonly used industrial enzyme preparation, and the main alkaline protease products in the market are derived from Bacillus alkalophilus, Bacillus licheniformis and Bacillus subtilis. The production modes of the alkaline protease are mainly divided into two categories of natural bacteria fermentation culture and heterologous high-efficiency expression. The natural bacteria fermentation culture has the advantages of short fermentation period and the disadvantages of low enzyme activity, unstable fermentation process, complex culture medium composition, easy degeneration of strains and the like. Compared with natural bacteria, the heterologous recombinant expression has the advantages of high fermentation enzyme activity, stable and controllable fermentation process and the like, so that the heterologous recombinant expression becomes a mainstream production mode. The alkaline protease heterologous expression hosts currently include Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, of which Bacillus subtilis expression hosts have been the most studied. The bacillus subtilis used as a food-grade expression host has the advantages of simple genetic operation, clear gene background, extracellular secretion of protein and the like.
Disclosure of Invention
The invention provides an alkaline protease gene, a heterozygous promoter, a recombinant expression vector, recombinant expression engineering bacteria, alkaline protease, a method and application, and aims to solve the technical problems of high production cost and low fermentation activity of the alkaline protease from bacillus.
The technical scheme adopted by the invention is as follows:
an alkaline protease gene bcp, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The Bacillus circulans alkaline protease gene bcp was obtained by homologous cloning. Extracting a Bacillus circulans genome sy1 (Bacillus circulans sy1 is obtained by screening bean dregs in the early stage of a laboratory) by referring to a bacterial genome extraction kit, designing a pair of primers according to a genome sequence of a Bacillus circulans strain NCTC2610 (GenBank: UAPZ 01000012.1: 120935-122077), namely the nucleotide sequence of a forward primer is shown as SEQ ID NO:20 (bc1-fw, 5'-TCACGGCCG ATGAAGAAACTGTTGACGAAA-3', underlined is Ec52I cleavage site), and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:21 (bc 1-rev: 5'-ACGTCTAGAGCGTGTTGCCGCTTCTGC-3', XbaI restriction sites underlined). The extracted genome is used as a template, and the alkaline protease gene bcp is obtained by PCR amplification.
The alkaline protease gene BCP is bacillus circulans alkaline protease gene BCP, the total length of the alkaline protease gene BCP is 1143bp, 380 amino acids of the encoding protease BCP are provided, and the nucleotide sequence of the encoding protease BCP is shown as SEQ ID NO. 1.
According to another aspect of the invention, the alkaline protease BCP is provided, and the amino acid sequence of the alkaline protease BCP is shown as SEQ ID NO. 2. By analysis, the alkaline protease BCP encoded by the gene BCP can be divided into three segments, the first 27 amino acids being its signal peptide sequence, amino acids 28 to 111 being its leader peptide, and amino acids 112 to 269 being its mature peptide (please confirm whether modifications are required here).
The NCBI protein comparison analysis shows that the similarity of the alkaline protease BCP and the Bacillus circulans NCTC2610 alkaline protease (protein accession number: No. SPU21234.1) is the highest and is 90 percent; followed by the Bacillus clausii alkaline protease (protein accession No.: WP-094423791) and the Bacillus bartonia alkaline protease (protein accession No.: WP-094190329.1), with similarities of 89.4% and 78.7%, respectively. The three-dimensional structure of the alkaline protease BCP protein is obtained by homologous modeling, and as can be seen from FIG. 1, the BCP catalytic active site consists of a catalytic triad of aspartic acid (D143) at position 143, histidine (H173) at position 173 and serine (S326) at position 326. Two amino acid loops (G) on the surface of proteins209To S215,L233To S239) Is responsible for reacting with the substrate. In addition, two Ca are also present in the mature peptide of BCP2+The binding sites, one on the surface of the protein and one in the middle of the protein.
According to another aspect of the present invention, there is also provided a hybrid promoter for efficient expression of alkaline protease in Bacillus subtilis, the nucleotide sequence of the hybrid promoter being shown in SEQ ID NO. 11. The construction method of the hybrid promoter comprises the following steps: (1) obtaining alkaline protease gene bcp by PCR amplification, carrying out agarose gel purification, carrying out restriction enzyme digestion by restriction enzymes XbaI and Eco521, purifying the product after enzyme digestion, connecting the product to an expression vector pHY, and finally obtaining the expression vector pHY-bcp by screening and sequencing. (2) And respectively constructing different promoter expression vectors by using the constructed pHY-bcp as a template. The promoters selected included: pcryIIIA(Bacillus thuringiensis crystallin promoter), PBsamy(Bacillus subtilis mesophilic amylase promoter), PBaamy(Bacillus amyloliquefaciens mesophilic amylase promoter), PHpaII(Derived from expression vector pMA5q), Pgsib(derived from Bacillus subtilis) PBsapr(Bacillus subtilis alkaline protease promoter),PBsnpr(Bacillus subtilis neutral protease promoter) and PBlapr(the lichen spore alkaline protease promoter) and the nucleotide sequence of the promoter is shown in SEQ ID NO. 3-SEQ ID NO. 10. (3) And transferring all different promoter expression vectors into the bacillus subtilis RIK1285 by adopting an electrical transformation method to obtain a plurality of transformants. (4) And performing enzyme activity screening after culturing a plurality of transformants to determine a single optimal promoter. (5) And (4) constructing a hybrid promoter by using the single optimal promoter as a template, and repeating the steps (3) and (4) to obtain the hybrid promoter. The hybrid promoter is pHYPBsapr-cryIIIAThe enzyme activity of the corresponding recombinant engineering bacteria is 2210U/mL, and the nucleotide sequence of the recombinant engineering bacteria is shown as SEQ ID NO. 11.
According to another aspect of the present invention, there is also provided a recombinant expression vector comprising the above-mentioned alkaline protease gene bcp, the hybrid promoter of claim 3 and a signal peptide, the nucleotide sequence of which is shown in SEQ ID NO. 15. The promoter and the signal peptide are used as core elements for the recombinant expression of the bacillus subtilis and play an important role in the process of the recombinant expression of heterologous proteins. Different enzymes are selective to promoters and signal peptides, so that the protease is heterologously expressed in the bacillus subtilis, and the optimal promoter and signal peptide are obtained by screening. The signal peptide is optimized to further improve the expression level of the alkaline protease BCP in the bacillus subtilis RIK 1285. Selected signal peptides include: SPBsamy(Bacillus subtilis medium temperature amylase signal peptide), SPBsapr(Bacillus subtilis alkaline protease signal peptide), SPBsnpr(Bacillus subtilis neutral protease signal peptide), SPBschi(Bacillus subtilis chitinase Signal peptide), SPDacB(Bacillus subtilis DacB signal peptide), SPVpr(Bacillus subtilis Vpr signal peptide), SPYncM(Bacillus subtilis YncM signal peptide) and SPAp(Bacillus subtilis aminopeptidase signal peptide), the nucleotide sequence of which is shown in SEQ ID NO. 12-SEQ ID NO. 19. Respectively transferring different signal peptide expression vectors into bacillus subtilis RIK1285, screening transformants, determining the best signal peptide, and obtaining the SPBschi(Bacillus subtilis chitinase signal peptide) the best effect, it is onThe fermentation enzyme activity of the corresponding recombinant engineering bacteria is the highest, the enzyme activity is 2650U/mL after 48 hours of culture, and then the recombinant engineering bacteria are corresponding to the SPBsamy and the SPBsnpr, and the enzyme activities are 2452U/mL and 2345U/mL respectively. The recombinant expression vector is pHYPBsapr-cryIIIA-SPBschi-bcp。
According to another aspect of the invention, a recombinant expression engineering bacterium is also provided, which comprises the recombinant expression vector. In the mode of free plasmid expression, the fermentation enzyme activity of the recombinant engineering bacteria is obviously reduced after multiple passages, so that an optimal recombinant expression vector pHYP needs to be obtainedBsapr-cryIIIA-SPBschiIntegration of bcp into the B.subtilis RIK1285 genome. After the recombinant expression engineering strain Bs16-5 is cultured in a 7-liter fermentation tank for 48 hours, the fermentation activity reaches the highest value of 51807U/mL; the fermentation enzyme activity after 48 hours of culture in a 50L fermentor was 53708U/mL.
According to another aspect of the present invention, there is also provided a method for preparing the above alkaline protease BCP, comprising the steps of:
providing an alkaline protease gene bcp, an expression vector and an expression strain;
amplifying alkaline protease gene bcp, and connecting an amplification product with an expression vector to obtain a recombinant expression vector;
integrating the recombinant expression vector gene into an expression strain to obtain recombinant expression engineering bacteria;
culturing the recombinant expression engineering bacteria to obtain alkaline protease BCP;
wherein, the nucleotide sequence of the alkaline protease gene bcp is shown as SEQ ID NO:1 is shown.
The preparation method of the alkaline protease BCP firstly obtains the alkaline protease BCP coding gene BCP through homologous cloning, secondly obtains the high-efficiency recombinant bacillus subtilis engineering bacteria Bs16-5 through promoter and signal peptide optimization and integrated expression, and finally realizes the high-efficiency preparation of the alkaline protease BCP through high-density fermentation, thereby laying a foundation for the industrial application of the alkaline protease BCP.
Further, the expression vector comprises a vector pHY, a hybrid promoter and a signal peptide; and/or, the expression strain is bacillus subtilis RIK 1285.
Further, the recombinant expression vector gene is integrated into the site of the mesophilic amylase gene of bacillus subtilis RIK 1285.
Further, in the amplification process of the alkaline protease gene bcp, primers for amplification include a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is shown as SEQ ID NO:20, the nucleotide sequence of the reverse primer is shown as SEQ ID NO: shown at 21.
According to another aspect of the invention, the application of the above alkaline protease BCP in hydrolyzing protein is also provided, wherein the reaction temperature of the alkaline protease BCP is 20-70 ℃; the reaction pH value of the alkaline protease BCP is 7-11.
The invention has the following beneficial effects:
the total length of the alkaline protease gene BCP is 1143bp, and the alkaline protease gene BCP is efficiently expressed in a bacillus subtilis expression strain, so that the production cost of the alkaline protease BCP is reduced.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a three-dimensional structural view of the alkaline protease BCP according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the optimization of the recombinant expression vector according to the preferred embodiment of the present invention;
FIG. 3 is a graph showing the enzyme production of Bacillus subtilis Bs16-5 in a 7-liter fermentor culture in accordance with a preferred embodiment of the present invention;
FIG. 4 is a graph showing the enzyme production of Bacillus subtilis Bs16-5 in a 50-liter fermentor culture in accordance with a preferred embodiment of the present invention;
FIG. 5 is a graph showing reaction temperature characteristics of BCP, an alkaline protease according to a preferred embodiment of the present invention; and
FIG. 6 is a pH profile of BCP, an alkaline protease according to a preferred embodiment of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
FIG. 1 is a three-dimensional structural view of the alkaline protease BCP according to a preferred embodiment of the present invention; FIG. 2 is a schematic diagram of the optimization of the recombinant expression vector according to the preferred embodiment of the present invention; FIG. 3 is a graph showing the enzyme production of Bacillus subtilis Bs16-5 in a 7-liter fermentor culture in accordance with a preferred embodiment of the present invention; FIG. 4 is a graph showing the enzyme production of Bacillus subtilis Bs16-5 in a 50-liter fermentor culture in accordance with a preferred embodiment of the present invention; FIG. 5 is a graph showing reaction temperature characteristics of BCP, an alkaline protease according to a preferred embodiment of the present invention; and FIG. 6 is a pH profile of BCP, an alkaline protease according to a preferred embodiment of the present invention.
Examples
Coli strains Topl0 and Bacillus subtilis RIK1285 were purchased from Takara Shuzo Co.
Bacillus circulans sy1 was obtained from the screening of bean dregs in the early laboratory.
The expression vector pHY was constructed from the preliminary experiments in this subject group and was routinely stored in a freezer at-80 ℃.
High fidelity Q5 enzyme, available from NEB.
Plasmid extraction, gel purification, restriction enzyme, and kit were purchased from Shanghai Biotech.
The culture medium of the escherichia coli and the bacillus subtilis RIK1285 is LB liquid and solid culture medium, and the components of the LB liquid culture medium are as follows: 1% peptone, 0.5% yeast extract, 1% NaCl, ph 7.0; the solid culture medium is prepared by adding 2.5% agar powder to liquid culture medium.
The screening culture medium of the escherichia coli and the bacillus subtilis RIK1285 is a liquid LBK culture medium and a solid LBK culture medium, and is mainly used for screening the escherichia coli recombinant transformant and the bacillus subtilis recombinant transformant. Liquid LBK configuration: adding 30 mu g/mL kanamycin into the sterilized LB liquid culture medium to obtain a liquid LBK culture medium; solid LBK medium: heating and dissolving the sterilized solid LB culture medium, cooling the culture medium to 50 ℃, adding kanamycin according to 30 mu g/mL, and inverting the culture medium to obtain the solid LBK culture medium.
The bacillus subtilis fermentation medium is a maltose culture medium and comprises the following components: 2.5% of yeast extract, 1.5% of peptone, 4% of maltose, 1% of sodium citrate, 0.3% of calcium chloride and 1% of dipotassium phosphate.
The high-density fermentation medium comprises the following components: 7% of maltose, 2.5% of soybean meal, 1.5% of yeast powder, 2% of bran, 1% of dipotassium phosphate, 0.3% of trisodium citrate and 0.3% of calcium chloride.
Example 1
Cloning of alkaline protease gene bcp gene and construction of expression vector
Extracting a Bacillus circulans genome sy1 by referring to a bacterial genome extraction kit, designing a pair of primers according to a genome sequence (GenBank: UAPZ 01000012.1: 120935-122077) of a Bacillus circulans strain NCTC2610, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO:20 (bc1-fw, 5'-TCACGGCCGATGAAGAAACTGTTGACGAAA-3', underlined is Ec52I cleavage site), and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:21 (bc 1-rev: 5'-ACGTCTAGAGCGTGTTGCCGCTTCTGC-3', underlined XbaI cleavage site) for amplification of the Bacillus circulans alkaline protease gene bcp. The extracted genome is used as a template, and the alkaline protease gene bcp is obtained by PCR amplification.
The amplified alkaline protease gene bcp was purified by agarose gel digestion and digested with restriction enzymes XbaI and Eco521, the digested product was purified, ligated to an expression vector pHY, the ligated product was transformed into E.coli Top10, and the transformed product was spread uniformly on LBK solid medium. The transformant is screened by a bacterial liquid PCR method, which comprises the following steps: (1) the transformant was inoculated in a single colony form into a 2ml sterilized centrifuge tube containing 300. mu.l of LBK liquid medium, and cultured at 37 ℃ and 200rpm for 5 hours; (2) taking the cultured bacterial liquid as a template, carrying out PCR amplification by using primers bc1-fw and bc1-rev, inoculating a transformant with a correct amplification result into an LBK liquid culture medium for culture, and extracting plasmids; (3) through sequencing verification, the expression vector pHY-bcp is finally obtained.
Example 2
Single promoter optimization
And respectively constructing different promoter expression vectors by using the constructed expression vector pHY-bcp as a template. The promoters selected included: pcryIIIA(Bacillus thuringiensis crystallin promoter), PBsamy(Bacillus subtilis mesophilic amylase promoter), PBaamy(Bacillus amyloliquefaciens mesophilic amylase promoter), PHpaII(derived from the expression vector pMA5q), Pgsib(derived from Bacillus subtilis) PBsapr(Bacillus subtilis alkaline protease promoter), PBsnpr(Bacillus subtilis neutral protease promoter) and PBlapr(lichenified spore alkaline protease promoter), the nucleotide sequence of the promoter is shown in SEQ ID NO. 3-SEQ ID NO.10, all the promoter sequences are synthesized by general biology company, and Eco52I and MluI enzyme cutting sites are added at both ends of the promoter sequence in the gene synthesis process.
As shown in FIG. 2, the specific procedure is as follows (in pHY)PcryIIIA-bcp for example, other analogous): the expression vector pH was first digested with restriction enzymes Eco52I and MluI, respectivelyY-bcpAnd synthetic promoter PcryIIIAA plasmid; then, main frame pHY (containing no pHY self-contained promoter P43) and P were recovered by agarose gel aggregationcryIIIAA promoter, and PcryIIIAThe promoter is connected to the main frame pHY, and is transferred into escherichia coli Top 10; finally, the pHYP of the expression vector is determined and obtained through colony PCR and sequencing verificationcryIIIA-bcp. Respectively obtaining pHYP by sequential methodcryIIIA-bcp、pHYPBsamy-bcp、pHYPBaamy-bcp、pHYPHpaII-bcp、pHYPgsib-bcp、pHYPBsapr-bcp、pHYPBsnpr-bcp、pHYPBlaprBcp, 8 expression vectors in total.
8 expression vectors are transferred into bacillus subtilis RIK1285 by adopting an electrical transformation method. The transformation process is as follows: (1) picking a single colony (diameter is 2 mm-3 mm) from a plate cultured for 16 h-20 h at 37 ℃, transferring the single colony into a 50mL EP tube containing 5mL of LB culture medium, and violently shaking at 37 ℃ for overnight; (2) inoculating 1% of the inoculum size in 50ml GM (LB +0.5M sorbitol), measuring OD in a shaking tube, and controlling the inoculum size to ensure that the OD of the culture medium after inoculation is between 0.19 and 0.2, culturing at 37 ℃ and 200rpm until the OD600 is about 0.8 to 1.0 (about 3 to 4 hours); (3) taking all bacteria liquid, carrying out ice-water bath for 10min, then centrifuging at 5000rpm for 8min at 4 ℃ and collecting thalli; (4) washing thallus with 30ml precooled electrotransfer buffer ETM (0.5M sorbitol, 0.5M mannitol, 10% glycerol, and optionally 0.5M trehalose to improve efficiency), centrifuging at 5000rpm for 8min at 4 deg.C to remove supernatant, and rinsing for 3 times; (5) resuspending the washed cells in 500. mu.l ETM, and packaging 100. mu.l each tube; (6) add 1-6. mu.l plasmid into 100. mu.l competent cell, incubate for 5min in ice bath, add into precooled electric rotating cup (1mm), shock once, the setting of electric rotating instrument: 1.5-2.0 kv, 25 muF, 200 omega, 1mm, 1 time of electric shock (duration time is between 4.5ms and 5 ms); (7) after the electric shock, 0.5ml of recovery medium RM (LB +0.5M sorbitol +0.38 mannitol) was added immediately, and after recovery for 3 hours at 37 ℃ and 120rpm, the mixture was plated on an LBK solid plate and cultured overnight at 37 ℃.
And (3) screening transformants by adopting shake flask culture, respectively inoculating bacillus subtilis RIK1285 containing different promoter expression vectors into a 50mL centrifuge tube containing 5mL of bacillus subtilis fermentation medium, culturing at 37 ℃ and 200rpm for 24 hours, and then inoculating the bacillus subtilis RIK into a 500mL shake flask containing 50mL of bacillus subtilis fermentation medium according to the inoculation amount of 5%. The enzyme activity was measured after culturing at 37 ℃ and 200rpm for 48 hours. The enzyme activity determination method is carried out by referring to the national standard GB/T23527-2009 alkaline protease.
The results of the experiments are shown in Table 1, and it can be seen from Table 1 that the promoter P is containedcryIIIAThe highest enzyme activity of the bacillus subtilis is 1852U/mL, and the second is promoter PBsaprAnd promoter PBaamyThe enzyme activity is 1455U/mL and 1415U/mL respectively.
TABLE 1 enzymatic Activity of expression from different promoters
Example 3
Dual promoter optimization
Double promoter optimization was performed based on the results of example 2, using expression vector pHYP, since the promoter PcryIIIA enzyme activity was highestcryIIIAConstruction of double-promoter expression vector with-bcp as template, and other four promoters PBsapr、PBaamy、PBsamyAnd PHpaIIAnd (4) carrying out matching. The construction process of the double-promoter expression vector is as follows (with the double-promoter expression vector pHYP)Bsapr-cryIIIA-bcp as an example): (1) using expression vector pHYPcryIIIABcp is a template, and PCR amplification is carried out by primers of double-1-fw and double-1-rev, wherein the nucleotide sequence is shown in SEQ ID NO. 22-SEQ ID NO.23, so as to obtain a main frame 1; (2) using expression vector pHYPBsapr-bcp as template by primer PBsapr-fw and PBsaprPCR amplification of rev to obtain promoter PBsapr(ii) a (3) Promoter P by the seamless cloning methodBsaprFusing with main frame 1 to obtain dual-promoter expression vector pHYPBsapr-cryIIIA-bcp。
All primer sequences of this part are shown below (5 '-3'):
PBsaprPrimer, PBsapr-the nucleotide sequence of fw (SEQ ID NO. 24): AAAAACTGGTCTGAACGCGTTATTTCTTCCTCCCTCTCAAT, PBsapr-the nucleotide sequence of rev (SEQ ID No. 25): GGTTTCATCTTACGTTTCGATCTTTACCCTCTCCTTTTAAA are provided.
PBaamyPrimer, PBaamy-the nucleotide sequence of fw (SEQ ID NO. 26): AAAAACTGGTCTGAACGCGTGCCCCGCACATACGAAAAGAC, PBaamy-the nucleotide sequence of rev (SEQ ID No. 27): GGTTTCATCTTACGTTTCGAGTTTCCTCTCCCTCTCATTTT are provided.
PBsamyPrimer, PBsamy-the nucleotide sequence of fw (SEQ ID NO. 28): AAAAACTGGTCTGAACGCGTCCGAGAATGGACACCAAAGA, PBsamy-the nucleotide sequence of rev (SEQ ID No. 29): GGTTTCATCTTACGTTTCGATCTTGACACTCCTTATTTGAT are provided.
PHpaIIPrimer, PHpaII-the nucleotide sequence of fw (SEQ ID NO. 30): AAAAACTGGTCTGAACGCGTGGGCGCGATTGCTGAATAAAAG, PHpaII-the nucleotide sequence of rev (SEQ ID No. 31): GGTTTCATCTTACGTTTCGAATGTAAATCGCTCCTTTTTA
Respectively expressing the two promoter expression vectors pHYPBsapr-cryIIIA-bcp、pHYPBaamy-cryIIIA-bcp、pHYPBsamy-cryIIIABcp and pHYPHpaII-cryIIIAThe-bcp was transformed into Bacillus subtilis RIK1285, the transformation process and the selection method of the recombinant transformant were the same as in example 2, and the enzyme activity was measured.
As a result of the experiment, pHYP was foundBsapr-cryIIIA-bcp,pHYPBaamy-cryIIIA-bcp,pHYPBsamy-cryIIIABcp and pHYPHpaII-cryIIIAThe enzyme activity of the recombinant engineering bacteria corresponding to the bcp is 2210U/mL, 1985U/mL, 1850U/mL and 2042U/mL respectively.
TABLE 2 enzymatic Activity of expression from different promoters
Example 4
Signal peptide optimization
The signal peptide is selected from the group consisting of: SPBsamy(Bacillus subtilis medium temperature amylase signal peptide), SPBsapr(Bacillus subtilis alkaline protease signal peptide), SPBsnpr(Bacillus subtilis neutral protease signal peptide), SPBschi (Bacillus subtilis chitinase signal peptide), SPDacB(Bacillus subtilis DacB signal peptide), SPVpr(Bacillus subtilis Vpr signal peptide), SPYncM(Bacillus subtilis YncM signal peptide) and SPAp(Bacillus subtilis aminopeptidase signal peptide) and the nucleotide sequence is shown in SEQ ID NO. 12-SEQ ID NO. 19.
Optimized vector pHYP with double promotersBsapr-cryIIIAConstruction of different signal peptide expression vectors using-bcp as template (with expression vector pHYP)Bsapr-cryIIIA-SPBschi-bcp as an example): (1) using expression vector pHYPBsapr-cryIIIABcp is a template, and a main frame 2 is obtained by amplification of primers, namely, signal-fw and signal-rev; (2) the primer SP was prepared by PCRBschi-fw and SPBschiRev Synthesis of Signal peptide SPBschi(ii) a (3) SP is cloned by seamless cloningBschiConnecting with main frame 2 to obtain expression vector pHYPBsapr-cryIIIA-SPBschi-bcp。
All primer sequences for this part are shown below:
SPBsamyPrimer, SPBsamy-the nucleotide sequence of fw (SEQ ID NO. 34): TATTATGAAAAGCGGCCGATGTTTGCAAAACGATTCAAAACCTCTTTACTGCCGTTATTCGCTGGATTTTTAT, SPBsamy-the nucleotide sequence of rev (SEQ ID No. 35): TTCATTTGCTTCCTCAGCAGCACTCGCAGCCGCCGGTCCTGCCAGAACCAAATGAAACAGCAATAAAAATCCA are provided.
SPBsaprPrimer, SPBsapr-the nucleotide sequence of fw (SEQ ID NO. 36): TATTATGAAAAGCGGCCGGTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTA, SPBsapr-the nucleotide sequence of rev (SEQ ID No. 37): TTCATTTGCTTCCTCAGCAGCCTGCGCAGACATGTTGCTGAACGCCATCGTAAAGATTAACGTTAA are provided.
SPBsnprPrimer, SPBsnpr-the nucleotide sequence of fw (SEQ ID NO. 38): TATTATGAAAAGCGGCCGGTGGGTTTAGGTAAGAAATTGTCTGTTGCTGTCGCTGCTTCGTTT, SPBsnpr-the nucleotide sequence of rev (SEQ ID No. 39): TTCATTTGCTTCCTCAGCAGCCTGAACACCTGGCAGGCTGATTGATAAACTCATAAACGAAGC are provided.
SPBschiPrimer, SPBschi-the nucleotide sequence of fw (SEQ ID No. 40): TATTATGAAAAGCGGCCGATGAAAAAAGTGTTTTCAAACAAAAAGTTTCTCGTTTTTTCTTTCATTTTTGCGA, SPBschi-the nucleotide sequence of rev (SEQ ID No. 41): TTCATTTGCTTCCTCAGCGGCTTTTGCACTTTCCCCATTAAAAAAAGACAGACTTAAAATCATCGCAAAAATG are provided.
SPDacBPrimer, SPDacB-the nucleotide sequence of fw (SEQ ID No. 42): TATTATGAAAAGCGGCCGATGCGCATTTTCAAAAAAGCAGTATTCGTGATCATGATTTCTTTT,SPDacB-the nucleotide sequence of rev (SEQ ID No. 43): TTCATTTGCTTCCTCAGCAGCATGTGCTGTATTCACATTTACGGTTGCAATAAGAAAAGAAAT are provided.
SPVprPrimer, SPVpr-the nucleotide sequence of fw (SEQ ID NO. 44): TATTATGAAAAGCGGCCGATGAAAAAGGGGATCATTCGCTTTCTGCTTGTAAGTTTCGTCTTATTTTTT, SPVpr-the nucleotide sequence of rev (SEQ ID No. 45): TTCATTTGCTTCCTCAGCAGCCGGAGCTGCCTGAACGCCCGTAATGCCTGTGGATAACGCAAAAAATAAG are provided.
SPYncMPrimer, SPYncM-the nucleotide sequence of fw (SEQ ID NO. 46): TATTATGAAAAGCGGCCGATGGCGAAACCACTATCAAAAGGGGGAATTTTGGTGAAAAAAGTATTGATTGCAGGTGCAGTAGGAACAG, SPYncM-the nucleotide sequence of rev (SEQ ID No. 47): TTCATTTGCTTCCTCAGCAGCGTCTGCCGCGGGTAAACCTGGTATACCTGATGAAAGGGTTCCGAAAAGAACTGCTGTTCCTACTGCA are provided.
SPApPrimer, SPAp-the nucleotide sequence of fw (SEQ ID NO. 48): TATTATGAAAAGCGGCCGATGAAAAAGCTTTTGACTGTCATGACGATGGCTGTTTTAACTGCCGGCAC, SPAp-the nucleotide sequence of rev (SEQ ID No. 49): TTCATTTGCTTCCTCAGCAGCGTGCGCGGCAGGGGTGACACTCTGTGCCGGCAAGAGCAGTGTGCCGGC are provided.
Different signal peptide expression vectors are respectively transferred into bacillus subtilis RIK1285, the conversion process and the screening method of the recombinant transformant are the same as the embodiment 2, and the enzyme activity is measured.
As shown in Table 3, it can be seen from Table 3 that SPBschiThe (Bacillus subtilis chitinase signal peptide) has the best effect, the corresponding recombinant engineering bacteria have the highest fermentation enzyme activity, the enzyme activity is 2650U/mL after 48 hours of culture, and SP is used for the second timeBsamyAnd SPBsnprThe enzyme activities of the corresponding recombinant engineering bacteria are 2452U/mL and 2345U/mL respectively.
TABLE 3 enzymatic Activity of different Signal peptide expressions
Example 5
Expression of integrated genes
Expression ofThe vector pHY can be used as a gene integration vector for integration and expression, the 5 'end and the 3' homology arm of the mesophilic amylase of the bacillus subtilis exist on the pHY vector, and in addition, a replicon of the pHY vector is a temperature-sensitive replicon. Will contain pHYPBsapr-cryIIIA-SPBschi-bcp recombinant bacillus subtilis to perform a gene integration experiment, the process of which is approximately as follows: (1) inducing at 45 deg.C to make expression vector pHYPBsapr-cryIIIA-SPBschiInactivation of the replicon of bcp, thus promoting the expression vector pHYPBsapr-cryIIIA-SPBschiHomologous recombination of bcp and B.subtilis RIK 1285; (2) extracting homologous recombination single colony, inoculating the extracted homologous recombination single colony into a 2ml sterilization centrifugal tube containing 400 mul LB liquid culture medium, culturing for 4 hours at 37 ℃ and 200rpm, and then treating bacterial liquid at 95 ℃ to be used as a PCR amplification template; (3) and taking the heat-treated bacterial liquid as a template, and identifying the nucleotide sequence of the forward primer at the 5 end as shown in SEQ ID NO: 50 (5-fw: 5' -TTCAAAACCTCTTTACTGCCG) and the nucleotide sequence of the reverse primer identified at the 5-terminal is shown in SEQ ID NO: 51 (5-end identification-rev: 5'-GGTCTTTCACATCTTCTGGGC-3'), and the nucleotide sequence of the forward primer identified by the 3-end is shown in SEQ ID NO: 52 (3-terminal identification-fw: 5'-GATGGCTACTCCTCATGTTGC-3') and the nucleotide sequence of the reverse primer identified by the 3-terminal is shown in SEQ ID NO: 53 (primer 3 end identification-rev: 5'-CGCCGTCTCTGGTCCATTATT-3'), 3' end homologous arm amplification is carried out, and the position where the homologous arm single exchange occurs is determined; (3) carrying out nonresistant subculture on the transformants at the position where the homologous arm single exchange occurs, carrying out continuous subculture for 10 times, and carrying out a dot plate experiment, wherein transformants which do not grow on the resistant plate and grow on the nonresistant plate are preliminarily determined as successful gene integration; (4) extracting the genome of the bacillus subtilis with successfully integrated genes, performing PCR amplification by using primer 5 end identification-fw and primer 3 end identification-rev, sequencing the amplified product, and successfully integrating the genes of the bacillus subtilis. Finally, 5 recombinant expression engineering bacteria (respectively named as bacillus subtilis Bs16-1, bacillus subtilis Bs16-2, bacillus subtilis Bs16-3, bacillus subtilis Bs16-4 and bacillus subtilis Bs16-5) with successfully integrated genes are obtained.
The 5 recombinant transformants were cultured and screened by the culture method of example 2, and after 48 hours of culture, the enzyme activities of the 5 recombinant expression engineering bacteria were 2510U/mL, 2515U/mL, 2351U/mL, 2462U/mL and 2615U/mL, respectively. As the Bs16-5 has the highest fermentation enzyme activity, the recombinant expression engineering bacteria Bs16-5 are selected for high-density fermentation culture.
Example 6
High density fermentation
Carrying out high-density fermentation experiments on recombinant expression engineering strains of bacillus subtilis Bs16-5 in 7-liter and 50-liter fermentation tanks, wherein the culture medium for high-density fermentation is as follows: 7% of maltose, 2.5% of soybean meal, 1.5% of yeast powder, 2% of bran, 1% of dipotassium phosphate, 0.3% of trisodium citrate and 0.3% of calcium chloride, wherein the culture temperature is 37 ℃, and the culture pH is 6.0. Taking fermentation liquor at intervals during the culture process for enzyme activity determination.
As shown in FIG. 3, the fermentation enzyme activity of the recombinant expression engineering strain Bacillus subtilis Bs16-5 reaches the highest value of 51807U/mL after culturing for 48 hours in a 7-liter fermentor. As shown in FIG. 4, the maximum level of the fermentative enzyme activity was 53708U/mL after 48 hours of culture in a 50-liter fermentor.
Example 7
Measurement of temperature characteristics
Centrifuging the high-density fermentation liquor to obtain liquid alkaline protease BCP enzyme liquid, wherein the temperature characteristic of the alkaline protease BCP is determined as follows: measuring the enzyme activity of the alkaline protease BCP at different temperatures of 20-70 ℃ under the condition of pH10.0, and calculating the relative enzyme activity at other temperatures by taking the enzyme activity at the highest temperature of the enzyme activity as 100%; and (3) carrying out water bath heat treatment at different temperatures of 50-70 ℃ for 10min, then determining residual enzyme activity, taking the enzyme activity without heat treatment as 100%, and calculating the relative residual enzyme activity at other temperatures.
The experimental result shows that the temperature characteristic of the alkaline protease BCP is shown in figure 5, the relative enzyme activities of the alkaline protease BCP at 20 ℃ and 70 ℃ are respectively 9.7% and 31.6%, the optimal reaction temperature is 60 ℃, the alkaline protease has good stability in terms of thermal stability, the BCP has good stability at different temperatures of 20 ℃ to 50 ℃, after 10min of heat treatment, the residual enzyme activities are both more than 90%, when the temperature is increased to be higher than 50 ℃, the thermal stability of the BCP is sharply reduced, and after 10min of heat treatment at 60 ℃ and 70 ℃, the residual enzyme activities are only 21% and 3%.
Example 8
Determination of pH characteristics
Centrifuging the high-density fermentation liquor to obtain liquid alkaline protease BCP enzyme liquid, wherein the pH characteristic of the alkaline protease BCP is determined as follows: measuring the enzyme activity of alkaline protease BCP at the pH value of 6.0-11.0 at the temperature of 60 ℃, and calculating the relative enzyme activity at other pH values by taking the enzyme activity under the highest enzyme activity pH value as 100%; and (3) under the condition of pH6.0-11.0, storing at room temperature for 5 hours, determining residual enzyme activity, taking the enzyme activity which is not subjected to any treatment as 100%, and calculating the relative residual enzyme activity at other temperatures.
According to the experimental result, the pH characteristic of the alkaline protease BCP is shown in figure 6, the relative enzyme activity of the alkaline protease BCP is more than 70% within the range of pH 7.0-11.0, and the optimal reaction pH is 10.0; in the aspect of pH stability, the residual enzyme activity is more than 80% after the mixture is stored for 5 hours at room temperature under the condition of pH6.0-11.0, and the alkaline protease BCP has good stability in the pH range.
The methods used in the present invention are conventional methods unless otherwise specified, and are carried out as described in molecular cloning, a laboratory Manual (J. SammBruk, D.W. Lassel, Huang Petang, Wang Jia seal, Zhu Hou, et al, 3 rd edition, Beijing: scientific Press, 2002). Meanwhile, the amino acids in the invention are marked by their abbreviations or codes unless otherwise specified (see Table 4 for the names of the amino acids in English and their abbreviations and codes).
TABLE 4 Chinese and English names of amino acids, their abbreviations and codes
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shaoyang college
<120> alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application
<160> 53
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1143
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaagaaac tgttgacgaa aattgtcgca agcgccgcac tactcctttc tgttgctttt 60
acttcatcga tcgtatcggc tgctgaggaa gcaaatgaaa aatatttaat tggccttaat 120
gagcaggaag ctgtcagtga gtttgtagaa tccatagagg caaatgacga ggtcgccatt 180
ctctctgagg atgagtcagt cgaaattgaa ttgcttcatg agtttgaaac gattcctgtt 240
gtatccgttg agttaagccc agaagatgtg aaagaccttg aaaaagatcc agcgatttct 300
tatactgaag aggatattga agtaacgata atggcgcaat cagtgccatg gggaattagc 360
cgtgtgcaag ccccagctgc ccataaccgt ggattgacag gttctggtga aaaagttgct 420
gtcctcgata caggtatttc cactcatcca gacttaaata ttcgtggtgg cgctagcttt 480
gtaccagggg caccatccac tcaagatggg aatgggcatg gcacgcatgt ggccgggacg 540
attgctgctt taaacaattc gattggcgtt cttggcgtag cgccgagtgc ggaactatac 600
gctgttaatg ttttaggagc cgacgggaga ggtgcaatca gctcgattgc ccatgggttg 660
gaatggacag ggaacaataa catgcacgtt gctaatttaa gtttaggaag cccttcgcca 720
agtgccacac ttgagcaagc tgttaatagc gcgacttcta gaggcgttct tgttgtagcg 780
gcatctggga attcaggtgc aagctcaatc agctatccgg cccgttatgc gaacgcaatg 840
gcagtcggag ctactgacca aaacaacaac cgcgccagct tttcacagta tggcgcaggg 900
cttgacattg tcgcaccagg ggtaggcgtg cagagctcat acccaggttc aacgtatgcc 960
agcttaaacg gtacatcgat ggctactcct catgttgcag gtgtcgcagc ccttgttaaa 1020
cacaagaacc catcttggtc caatacacaa atccgcaacc atctagagaa tacggcaacg 1080
agcttaggaa gcacgaactt gtatgtaagc ggacttgtca aagcagaagc ggcaacacgc 1140
taa 1143
<210> 2
<211> 380
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Lys Lys Leu Leu Thr Lys Ile Val Ala Ser Ala Ala Leu Leu Leu
1 5 10 15
Ser Val Ala Phe Thr Ser Ser Ile Val Ser Ala Ala Glu Glu Ala Asn
20 25 30
Glu Lys Tyr Leu Ile Gly Leu Asn Glu Gln Glu Ala Val Ser Glu Phe
35 40 45
Val Glu Ser Ile Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Asp
50 55 60
Glu Ser Val Glu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val
65 70 75 80
Val Ser Val Glu Leu Ser Pro Glu Asp Val Lys Asp Leu Glu Lys Asp
85 90 95
Pro Ala Ile Ser Tyr Thr Glu Glu Asp Ile Glu Val Thr Ile Met Ala
100 105 110
Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His
115 120 125
Asn Arg Gly Leu Thr Gly Ser Gly Glu Lys Val Ala Val Leu Asp Thr
130 135 140
Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe
145 150 155 160
Val Pro Gly Ala Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His
165 170 175
Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly
180 185 190
Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Asn Val Leu Gly Ala Asp
195 200 205
Gly Arg Gly Ala Ile Ser Ser Ile Ala His Gly Leu Glu Trp Thr Gly
210 215 220
Asn Asn Asn Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro
225 230 235 240
Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val
245 250 255
Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser Tyr
260 265 270
Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn
275 280 285
Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val
290 295 300
Ala Pro Gly Val Gly Val Gln Ser Ser Tyr Pro Gly Ser Thr Tyr Ala
305 310 315 320
Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val Ala
325 330 335
Ala Leu Val Lys His Lys Asn Pro Ser Trp Ser Asn Thr Gln Ile Arg
340 345 350
Asn His Leu Glu Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr
355 360 365
Val Ser Gly Leu Val Lys Ala Glu Ala Ala Thr Arg
370 375 380
<210> 3
<211> 426
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgtcgacgtg catgcaggcc ggggcatatg ggaaacagcg cggacggagc ggaatttcca 60
atttcatgcc gcagccgcct gcgctgttct catttgcggc ttccttgtag agctcagcat 120
tattgagtgg atgattatat tccttttgat aggtggtatg ttttcgcttg aacttttaaa 180
tacagccatt gaacatacgg ttgatttaat aactgacaaa catcaccctc ttgctaaagc 240
ggccaaggac gctgccgccg gggctgtttg cgtttttacc gtgatttcgt gtatcattgg 300
tttacttatt tttttgccaa agctgtaatg gctgaaaatt cttacattta ttttacattt 360
ttagaaatgg gcgtgaaaaa aagcgcgcga ttatgtaaaa tataaagtga tagcggtacc 420
attata 426
<210> 4
<211> 510
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgagaatgg acaccaaaga agaactgcaa aaacgggtga agcagcagcg aatagaatca 60
attgcggtcg cctttgcggt agtggtgctt acgatgtacg acagggggat tccccataca 120
ttcttcgctt ggctgaaaat gattcttctt tttatcgtct gcggcggcgt tctgtttctg 180
cttcggtatg taattgtgaa gctggcttac agaagagcgg taaaagaaga aataaaaaag 240
aaatcatctt gaaaaataga tggtttcttt ttttgtttgg aaagcgaggg aagcgttcac 300
agtttcgggc agcttttttt ataggaacat tgatttgtat tcactctgcc aagttgtttt 360
gatagagtga ttgtgataat ttaaaatgta agcgttaaca aaattctcca gtcttcacat 420
cagtttgaaa ggaggaagcg gaagaatgaa gtaagaggga tttttgactc cgaagtaagt 480
cttcaaaaaa tcaaataagg agtgtcaaga 510
<210> 5
<211> 249
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60
ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc 120
atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180
ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240
agaggaaac 249
<210> 6
<211> 479
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gggcgcgatt gctgaataaa agatacgaga gacctctctt gtatcttttt tattttgagt 60
ggttttgtcc gttacactag aaaaccgaaa gacaataaaa attttattct tgctgagtct 120
ggctttcggt aagctagaca aaacggacaa aataaaaatt ggcaagggtt taaaggtgga 180
gattttttga gtgatcttct caaaaaatac tacctgtccc ttgctgattt ttaaacgagc 240
acgagagcaa aacccccctt tgctgaggtg gcagagggca ggtttttttg tttctttttt 300
ctcgtaaaaa aaagaaaggt cttaaaggtt ttatggtttt ggtcggcact gccgacagcc 360
tcgcagagca cacactttat gaatataaag tatagtgtgt tatactttac ttggaagtgg 420
ttgccggaaa gagcgaaaat gcctcacatt tgtgccacct aaaaaggagc gatttacat 479
<210> 7
<211> 371
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gatcaagacc gtacatataa gaatgtcgct tctcaaatcc aaggctggcg agaagtcgtt 60
ttgggctatc gagacacgtt tggctggaaa aaacttttcc agatagtgcc ggttgccgga 120
atggtttttg gcgccgctgc caatcgctca acattaaacg acattaccga gacaggcatg 180
atgctgtaca aaaagaggcg cattcttgaa cgactgaaag aaacagaacg agagatggaa 240
tagcagaaag cagacggaca ccgcgatccg cctgcttttt ttagtggaaa catacccaat 300
gtgttttgtt tgtttaaaag aattgtgagc gggaatacaa caaccaacac caattaaagg 360
aggaattcaa a 371
<210> 8
<211> 432
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tatttcttcc tccctctcaa taattttttc attctatccc ttttctgtaa agtttatttt 60
tcagaatact tttatcatca tgctttgaaa aaatatcacg ataatatcca ttgttctcac 120
ggaagcacac gcaggtcatt tgaacgaatt ttttcgacag gaatttgccg ggactcagga 180
gcatttaacc taaaaaagca tgacatttca gcataatgaa catttactca tgtctatttt 240
cgttcttttc tgtatgaaaa tagttatttc gagtctctac ggaaatagcg agagatgata 300
tacctaaata gagataaaat catctcaaaa aaatgggtct actaaaatat tattccatct 360
attacaataa attcacagaa tagtctttta agtaagtcta ctctgaattt ttttaaaagg 420
agagggtaaa ga 432
<210> 9
<211> 520
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gattgcacct ggtgaataag ttcaacagac actcccgcca gcagcacaat ccgcaatata 60
acacccgcca agaacattgt gcgctgccgg tttattttgg gatgatgcac caaaagatat 120
aagcccgcca gaacaacaat tgaccattga atcagcaggg tgctttgtct gcttaatata 180
aaataacgtt cgaaatgcaa tacataatga ctgaataact ccaacacgaa caacaatcct 240
ttacttctta ttaaggcctc attcggttag acagcggact tttcaaaaag tttcaagatg 300
aaacaaaaat atctcatctt ccccttgata tgtaaaaaac ataactcttg aatgaaccac 360
cacatgacac ttgactcatc ttgatattat tcaacaaaaa caaacacagg acaatactat 420
caattttgtc tagttatgtt agtttttgtt gagtattcca gaatgctagt ttaatataac 480
aatataaagt tttcagtatt ttcaaaaagg gggatttatt 520
<210> 10
<211> 406
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgcctttca catgagctga tttcatatct tacacccgtt tctgtatgcg atatattgca 60
tattttaata gatgatcgac taggccgcaa cctccttcgg caaaaaatga tctcataaaa 120
taaatgaata gtattttcat aaaatgaatc agacgaagca atctcctgtc attcacggac 180
cccgggacct ctttccctgc caggttgaag cggtctattc atactttcga accgaatatt 240
tttctaaaac agttattaat aaccaataaa tttaaattgg ccgttcaaaa aaatgggtct 300
accatataat tcattttttt tctataataa attaacagaa taattggaat agagtatatt 360
attcttctat ttcaattatt ctgaataaaa cggaggagag tgagta 406
<210> 11
<211> 858
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tatttcttcc tccctctcaa taattttttc attctatccc ttttctgtaa agtttatttt 60
tcagaatact tttatcatca tgctttgaaa aaatatcacg ataatatcca ttgttctcac 120
ggaagcacac gcaggtcatt tgaacgaatt ttttcgacag gaatttgccg ggactcagga 180
gcatttaacc taaaaaagca tgacatttca gcataatgaa catttactca tgtctatttt 240
cgttcttttc tgtatgaaaa tagttatttc gagtctctac ggaaatagcg agagatgata 300
tacctaaata gagataaaat catctcaaaa aaatgggtct actaaaatat tattccatct 360
attacaataa attcacagaa tagtctttta agtaagtcta ctctgaattt ttttaaaagg 420
agagggtaaa gatgtcgacg tgcatgcagg ccggggcata tgggaaacag cgcggacgga 480
gcggaatttc caatttcatg ccgcagccgc ctgcgctgtt ctcatttgcg gcttccttgt 540
agagctcagc attattgagt ggatgattat attccttttg ataggtggta tgttttcgct 600
tgaactttta aatacagcca ttgaacatac ggttgattta ataactgaca aacatcaccc 660
tcttgctaaa gcggccaagg acgctgccgc cggggctgtt tgcgttttta ccgtgatttc 720
gtgtatcatt ggtttactta tttttttgcc aaagctgtaa tggctgaaaa ttcttacatt 780
tattttacat ttttagaaat gggcgtgaaa aaaagcgcgc gattatgtaa aatataaagt 840
gatagcggta ccattata 858
<210> 12
<211> 99
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgct 99
<210> 13
<211> 87
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60
gcgttcagca acatgtctgc gcaggct 87
<210> 14
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgggtttag gtaagaaatt gtctgttgct gtcgctgctt cgtttatgag tttatcaatc 60
agcctgccag gtgttcaggc t 81
<210> 15
<211> 99
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgaaaaaag tgttttcaaa caaaaagttt ctcgtttttt ctttcatttt tgcgatgatt 60
ttaagtctgt ctttttttaa tggggaaagt gcaaaagcc 99
<210> 16
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgcgcattt tcaaaaaagc agtattcgtg atcatgattt cttttcttat tgcaaccgta 60
aatgtgaata cagcacatgc t 81
<210> 17
<211> 93
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgaaaaagg ggatcattcg ctttctgctt gtaagtttcg tcttattttt tgcgttatcc 60
acaggcatta cgggcgttca ggcagctccg gct 93
<210> 18
<211> 126
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atggcgaaac cactatcaaa agggggaatt ttggtgaaaa aagtattgat tgcaggtgca 60
gtaggaacag cagttctttt cggaaccctt tcatcaggta taccaggttt acccgcggca 120
gacgct 126
<210> 19
<211> 93
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atgaaaaagc ttttgactgt catgacgatg gctgttttaa ctgccggcac actgctcttg 60
ccggcacaga gtgtcacccc tgccgcgcac gct 93
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tcacggccga tgaagaaact gttgacgaaa 30
<210> 21
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
acgtctagag cgtgttgccg cttctgc 27
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tcgaaacgta agatgaaacc t 21
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acgcgttcag accagttttt a 21
<210> 24
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aaaaactggt ctgaacgcgt tatttcttcc tccctctcaa t 41
<210> 25
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ggtttcatct tacgtttcga tctttaccct ctccttttaa a 41
<210> 26
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaaaactggt ctgaacgcgt gccccgcaca tacgaaaaga c 41
<210> 27
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggtttcatct tacgtttcga gtttcctctc cctctcattt t 41
<210> 28
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aaaaactggt ctgaacgcgt ccgagaatgg acaccaaaga 40
<210> 29
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ggtttcatct tacgtttcga tcttgacact ccttatttga t 41
<210> 30
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aaaaactggt ctgaacgcgt gggcgcgatt gctgaataaa ag 42
<210> 31
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggtttcatct tacgtttcga atgtaaatcg ctccttttta 40
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gctgaggaag caaatgaaaa at 22
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cggccgcttt tcataataca taa 23
<210> 34
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tattatgaaa agcggccgat gtttgcaaaa cgattcaaaa cctctttact gccgttattc 60
gctggatttt tat 73
<210> 35
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ttcatttgct tcctcagcag cactcgcagc cgccggtcct gccagaacca aatgaaacag 60
caataaaaat cca 73
<210> 36
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tattatgaaa agcggccggt gagaagcaaa aaattgtgga tcagcttgtt gtttgcgtta 60
acgtta 66
<210> 37
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ttcatttgct tcctcagcag cctgcgcaga catgttgctg aacgccatcg taaagattaa 60
cgttaa 66
<210> 38
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tattatgaaa agcggccggt gggtttaggt aagaaattgt ctgttgctgt cgctgcttcg 60
ttt 63
<210> 39
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ttcatttgct tcctcagcag cctgaacacc tggcaggctg attgataaac tcataaacga 60
agc 63
<210> 40
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tattatgaaa agcggccgat gaaaaaagtg ttttcaaaca aaaagtttct cgttttttct 60
ttcatttttg cga 73
<210> 41
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttcatttgct tcctcagcgg cttttgcact ttccccatta aaaaaagaca gacttaaaat 60
catcgcaaaa atg 73
<210> 42
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tattatgaaa agcggccgat gcgcattttc aaaaaagcag tattcgtgat catgatttct 60
ttt 63
<210> 43
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ttcatttgct tcctcagcag catgtgctgt attcacattt acggttgcaa taagaaaaga 60
aat 63
<210> 44
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tattatgaaa agcggccgat gaaaaagggg atcattcgct ttctgcttgt aagtttcgtc 60
ttatttttt 69
<210> 45
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ttcatttgct tcctcagcag ccggagctgc ctgaacgccc gtaatgcctg tggataacgc 60
<210> 46
<211> 88
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tattatgaaa agcggccgat ggcgaaacca ctatcaaaag ggggaatttt ggtgaaaaaa 60
gtattgattg caggtgcagt aggaacag 88
<210> 47
<211> 88
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ttcatttgct tcctcagcag cgtctgccgc gggtaaacct ggtatacctg atgaaagggt 60
tccgaaaaga actgctgttc ctactgca 88
<210> 48
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tattatgaaa agcggccgat gaaaaagctt ttgactgtca tgacgatggc tgttttaact 60
gccggcac 68
<210> 49
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ttcatttgct tcctcagcag cgtgcgcggc aggggtgaca ctctgtgccg gcaagagcag 60
tgtgccggc 69
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ttcaaaacct ctttactgcc g 21
<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggtctttcac atcttctggg c 21
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gatggctact cctcatgttg c 21
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cgccgtctct ggtccattat t 21
Claims (10)
1. An alkaline protease gene bcp, characterized in that its nucleotide sequence is shown in SEQ ID NO. 1.
2. An alkaline protease BCP, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
3. A hybrid promoter for efficient expression of alkaline protease in Bacillus subtilis, wherein the nucleotide sequence of the hybrid promoter is as shown in SEQ ID No. 11.
4. A recombinant expression vector comprising the alkaline protease gene bcp of claim 1, the hybrid promoter of claim 3 and a signal peptide, the nucleotide sequence of which is shown in SEQ ID No. 15.
5. A recombinant expression engineered bacterium comprising the recombinant expression vector of claim 4.
6. The method for preparing BCP, an alkaline protease, according to claim 2, comprising the steps of:
providing an alkaline protease gene bcp, an expression vector and an expression strain;
amplifying alkaline protease gene bcp, and connecting an amplification product with an expression vector to obtain a recombinant expression vector;
integrating the recombinant expression vector gene into an expression strain to obtain recombinant expression engineering bacteria;
culturing the recombinant expression engineering bacteria to obtain alkaline protease BCP;
wherein, the nucleotide sequence of the alkaline protease gene bcp is shown as SEQ ID NO. 1.
7. The method for producing BCP, an alkaline protease, according to claim 6,
the expression vector comprises a vector pHY, a hybrid promoter and a signal peptide; and/or
The expression strain is bacillus subtilis RIK 1285.
8. The method for producing BCP, an alkaline protease, according to claim 6,
the recombinant expression vector gene is integrated to the mesophilic amylase gene site of bacillus subtilis RIK 1285.
9. The method for producing BCP, an alkaline protease, according to claim 6,
in the process of amplifying the alkaline protease gene bcp, primers for amplification comprise a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 20, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 21.
10. The use of the alkaline protease BCP according to claim 2 for the hydrolysis of proteins,
the reaction temperature of the alkaline protease BCP is 20-70 ℃;
the reaction pH value of the alkaline protease BCP is 7-11.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110952632.8A CN113699138B (en) | 2021-08-19 | 2021-08-19 | Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110952632.8A CN113699138B (en) | 2021-08-19 | 2021-08-19 | Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113699138A true CN113699138A (en) | 2021-11-26 |
CN113699138B CN113699138B (en) | 2023-10-24 |
Family
ID=78653456
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110952632.8A Active CN113699138B (en) | 2021-08-19 | 2021-08-19 | Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113699138B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114540330A (en) * | 2022-04-21 | 2022-05-27 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
CN114574514A (en) * | 2022-03-30 | 2022-06-03 | 威海迪普森生物科技有限公司 | Nattokinase recombinant expression vector, recombinant gene engineering bacterium and application |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101511998A (en) * | 2006-07-18 | 2009-08-19 | 丹尼斯科美国公司 | Protease variants active over a broad temperature range |
KR20180003252A (en) * | 2016-06-30 | 2018-01-09 | 명지대학교 산학협력단 | Recombinant bacillus subtilis strain producing beta agarase and uses thereof |
CN111893126A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Alkaline protease gene, alkaline protease, preparation method and application thereof |
CN112226451A (en) * | 2020-10-23 | 2021-01-15 | 中国科学院上海高等研究院 | Bacillus subtilis expression system and method for producing alpha-L-AFs by using same |
-
2021
- 2021-08-19 CN CN202110952632.8A patent/CN113699138B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101511998A (en) * | 2006-07-18 | 2009-08-19 | 丹尼斯科美国公司 | Protease variants active over a broad temperature range |
KR20180003252A (en) * | 2016-06-30 | 2018-01-09 | 명지대학교 산학협력단 | Recombinant bacillus subtilis strain producing beta agarase and uses thereof |
CN111893126A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Alkaline protease gene, alkaline protease, preparation method and application thereof |
CN112226451A (en) * | 2020-10-23 | 2021-01-15 | 中国科学院上海高等研究院 | Bacillus subtilis expression system and method for producing alpha-L-AFs by using same |
Non-Patent Citations (3)
Title |
---|
MEERA VENUGOPAL 等: "An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations", 《INDIAN J MICROBIOL》, vol. 47, no. 4, pages 298 - 303 * |
NDERJEET KAUR 等: "Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906", 《INDIAN JOURNAL OF MICROBIOLOGY》, vol. 52, pages 630 - 637 * |
李怡欣 等: "碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达", 《微生物学通报》, pages 1 - 16 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114574514A (en) * | 2022-03-30 | 2022-06-03 | 威海迪普森生物科技有限公司 | Nattokinase recombinant expression vector, recombinant gene engineering bacterium and application |
CN114540330A (en) * | 2022-04-21 | 2022-05-27 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
CN114540330B (en) * | 2022-04-21 | 2022-07-12 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN113699138B (en) | 2023-10-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1128221C (en) | Gene encoding alkaline liquefying alpha-amylase | |
CN114591935B (en) | Protease mutant BLAPR3 with improved thermal stability, and encoding gene and application thereof | |
CN112522173B (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
WO2022134236A1 (en) | Alkaline protease mutant, gene thereof, engineering bacteria thereof, preparation method therefor and use thereof | |
CN113699138A (en) | Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application | |
CN114540330B (en) | Alkaline protease mutant AprBpM and application thereof | |
CN112410322B (en) | Bacillus licheniformis beta-mannase mutant and application thereof | |
CN111893126A (en) | Alkaline protease gene, alkaline protease, preparation method and application thereof | |
CN111153968B (en) | Signal peptide mutant for improving expression quantity of exogenous alkaline protease and construction method and application thereof | |
CN114107266B (en) | Protease mutant with improved heat resistance, encoding gene and application thereof | |
CN110904174B (en) | Application of bacillus licheniformis with deletion of leucine dehydrogenase gene in production of heterologous protein | |
WO2005045013A2 (en) | Recombinant microorganism | |
CN111944790B (en) | Neutral protease gene, neutral protease, preparation method and application thereof | |
CN112094781B (en) | Bacillus amyloliquefaciens and application thereof | |
CN116790564A (en) | Heat-resistant protease mutant and encoding gene and application thereof | |
CN110106128B (en) | Genetically engineered bacterium for producing recombinant alkaline protease and construction method thereof | |
CN109182309B (en) | Heat-resistant aminopeptidase and high-yield pichia pastoris engineering bacteria thereof | |
JPWO2018021324A1 (en) | Polypeptide having stable lipase activity | |
CN108410786B (en) | Bacillus subtilis engineering bacteria for efficiently expressing plasmin and preparation method thereof | |
CN112725315B (en) | Application of chitosanase and mutant thereof in preparation of chitosan oligosaccharide | |
EP1680502B1 (en) | Recombinant microorganism | |
CN110878293B (en) | Application of bacillus licheniformis with deletion of yceD gene in production of heterologous protein | |
CN114807100B (en) | Alkaline protease gene sequence suitable for bacillus licheniformis expression and application | |
CN114350643B (en) | Recombinant strain for producing aminopeptidase and application of recombinant strain in efficient proteolysis | |
CN115125248B (en) | Combined promoter pctsR-alpha 2 and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |