CN107058265B - 铜绿假单胞菌噬菌体裂解酶及应用 - Google Patents
铜绿假单胞菌噬菌体裂解酶及应用 Download PDFInfo
- Publication number
- CN107058265B CN107058265B CN201710231999.4A CN201710231999A CN107058265B CN 107058265 B CN107058265 B CN 107058265B CN 201710231999 A CN201710231999 A CN 201710231999A CN 107058265 B CN107058265 B CN 107058265B
- Authority
- CN
- China
- Prior art keywords
- lyase
- pseudomonas aeruginosa
- bacteria
- bacillus
- biofilm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 50
- 241001515965 unidentified phage Species 0.000 title claims abstract description 42
- 108090000856 Lyases Proteins 0.000 title abstract description 107
- 102000004317 Lyases Human genes 0.000 title abstract description 98
- 241000894006 Bacteria Species 0.000 claims abstract description 38
- 239000004094 surface-active agent Substances 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000013612 plasmid Substances 0.000 claims description 17
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 claims description 14
- 108010039918 Polylysine Proteins 0.000 claims description 9
- 229920000656 polylysine Polymers 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000006166 lysate Substances 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 14
- 241000194108 Bacillus licheniformis Species 0.000 abstract description 7
- 244000063299 Bacillus subtilis Species 0.000 abstract description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 7
- 150000007523 nucleic acids Chemical group 0.000 abstract description 7
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 4
- 241000589776 Pseudomonas putida Species 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 230000002209 hydrophobic effect Effects 0.000 abstract 1
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 20
- 230000000844 anti-bacterial effect Effects 0.000 description 19
- 230000001580 bacterial effect Effects 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 238000002156 mixing Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000001976 enzyme digestion Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000002504 physiological saline solution Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 239000002054 inoculum Substances 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 230000002195 synergetic effect Effects 0.000 description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 239000004793 Polystyrene Substances 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 6
- 238000005336 cracking Methods 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 229920002223 polystyrene Polymers 0.000 description 6
- 239000012880 LB liquid culture medium Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 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 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 101001014639 Enterobacteria phage T4 Tail fiber assembly helper protein Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 3
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 108010062796 arginyllysine Proteins 0.000 description 3
- 239000003899 bactericide agent Substances 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 239000012459 cleaning agent Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000002101 lytic effect Effects 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 2
- PLTGTJAZQRGMPP-FXQIFTODSA-N Asn-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(N)=O PLTGTJAZQRGMPP-FXQIFTODSA-N 0.000 description 2
- 241001596967 Escherichia coli M15 Species 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- YBGTWSFIGHUWQE-MXAVVETBSA-N Ile-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)CC)CC1=CN=CN1 YBGTWSFIGHUWQE-MXAVVETBSA-N 0.000 description 2
- CKRFDMPBSWYOBT-PPCPHDFISA-N Ile-Lys-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CKRFDMPBSWYOBT-PPCPHDFISA-N 0.000 description 2
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 2
- ZJWIXBZTAAJERF-IHRRRGAJSA-N Lys-Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZJWIXBZTAAJERF-IHRRRGAJSA-N 0.000 description 2
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 108010013639 Peptidoglycan Proteins 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 2
- IVXJODPZRWHCCR-JYJNAYRXSA-N Val-Arg-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IVXJODPZRWHCCR-JYJNAYRXSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 2
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 150000008195 galaktosides Chemical class 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N lysine Chemical compound NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 235000014102 seafood Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108010073969 valyllysine Proteins 0.000 description 2
- VFERDGRYIBXYRG-REVLRCOSSA-N (2s)-1-[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-3-methylbutanoyl]amino]propanoyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 VFERDGRYIBXYRG-REVLRCOSSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000607528 Aeromonas hydrophila Species 0.000 description 1
- YHOPXCAOTRUGLV-XAMCCFCMSA-N Ala-Ala-Asp-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O YHOPXCAOTRUGLV-XAMCCFCMSA-N 0.000 description 1
- ZDYNWWQXFRUOEO-XDTLVQLUSA-N Ala-Gln-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDYNWWQXFRUOEO-XDTLVQLUSA-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
- BVSGPHDECMJBDE-HGNGGELXSA-N Ala-Glu-His Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N BVSGPHDECMJBDE-HGNGGELXSA-N 0.000 description 1
- FBHOPGDGELNWRH-DRZSPHRISA-N Ala-Glu-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FBHOPGDGELNWRH-DRZSPHRISA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- SUMYEVXWCAYLLJ-GUBZILKMSA-N Ala-Leu-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O SUMYEVXWCAYLLJ-GUBZILKMSA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- IHMCQESUJVZTKW-UBHSHLNASA-N Ala-Phe-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 IHMCQESUJVZTKW-UBHSHLNASA-N 0.000 description 1
- MSWSRLGNLKHDEI-ACZMJKKPSA-N Ala-Ser-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O MSWSRLGNLKHDEI-ACZMJKKPSA-N 0.000 description 1
- UCDOXFBTMLKASE-HERUPUMHSA-N Ala-Ser-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N UCDOXFBTMLKASE-HERUPUMHSA-N 0.000 description 1
- XCIGOVDXZULBBV-DCAQKATOSA-N Ala-Val-Lys Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CCCCN)C(O)=O XCIGOVDXZULBBV-DCAQKATOSA-N 0.000 description 1
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 1
- RCAUJZASOAFTAJ-FXQIFTODSA-N Arg-Asp-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N RCAUJZASOAFTAJ-FXQIFTODSA-N 0.000 description 1
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 1
- PDQBXRSOSCTGKY-ACZMJKKPSA-N Asn-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N PDQBXRSOSCTGKY-ACZMJKKPSA-N 0.000 description 1
- IYVSIZAXNLOKFQ-BYULHYEWSA-N Asn-Asp-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IYVSIZAXNLOKFQ-BYULHYEWSA-N 0.000 description 1
- PIABYSIYPGLLDQ-XVSYOHENSA-N Asn-Thr-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PIABYSIYPGLLDQ-XVSYOHENSA-N 0.000 description 1
- SKQTXVZTCGSRJS-SRVKXCTJSA-N Asn-Tyr-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O SKQTXVZTCGSRJS-SRVKXCTJSA-N 0.000 description 1
- SBHUBSDEZQFJHJ-CIUDSAMLSA-N Asp-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O SBHUBSDEZQFJHJ-CIUDSAMLSA-N 0.000 description 1
- AMRANMVXQWXNAH-ZLUOBGJFSA-N Asp-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC(O)=O AMRANMVXQWXNAH-ZLUOBGJFSA-N 0.000 description 1
- YRZIYQGXTSBRLT-AVGNSLFASA-N Asp-Phe-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O YRZIYQGXTSBRLT-AVGNSLFASA-N 0.000 description 1
- AHWRSSLYSGLBGD-CIUDSAMLSA-N Asp-Pro-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AHWRSSLYSGLBGD-CIUDSAMLSA-N 0.000 description 1
- BJDHEININLSZOT-KKUMJFAQSA-N Asp-Tyr-Lys Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(O)=O BJDHEININLSZOT-KKUMJFAQSA-N 0.000 description 1
- XWKBWZXGNXTDKY-ZKWXMUAHSA-N Asp-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O XWKBWZXGNXTDKY-ZKWXMUAHSA-N 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000589875 Campylobacter jejuni Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000193468 Clostridium perfringens Species 0.000 description 1
- SBMGKDLRJLYZCU-BIIVOSGPSA-N Cys-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CS)N)C(=O)O SBMGKDLRJLYZCU-BIIVOSGPSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- FYAULIGIFPPOAA-ZPFDUUQYSA-N Gln-Ile-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(O)=O FYAULIGIFPPOAA-ZPFDUUQYSA-N 0.000 description 1
- ZGHMRONFHDVXEF-AVGNSLFASA-N Gln-Ser-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZGHMRONFHDVXEF-AVGNSLFASA-N 0.000 description 1
- KBKGRMNVKPSQIF-XDTLVQLUSA-N Glu-Ala-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KBKGRMNVKPSQIF-XDTLVQLUSA-N 0.000 description 1
- PBEQPAZRHDVJQI-SRVKXCTJSA-N Glu-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N PBEQPAZRHDVJQI-SRVKXCTJSA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 1
- JGHNIWVNCAOVRO-DCAQKATOSA-N Glu-His-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JGHNIWVNCAOVRO-DCAQKATOSA-N 0.000 description 1
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 1
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 1
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 1
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 1
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 1
- DHNXGWVNLFPOMQ-KBPBESRZSA-N Gly-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN DHNXGWVNLFPOMQ-KBPBESRZSA-N 0.000 description 1
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 1
- RIYIFUFFFBIOEU-KBPBESRZSA-N Gly-Tyr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 RIYIFUFFFBIOEU-KBPBESRZSA-N 0.000 description 1
- DNVDEMWIYLVIQU-RCOVLWMOSA-N Gly-Val-Asp Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O DNVDEMWIYLVIQU-RCOVLWMOSA-N 0.000 description 1
- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 1
- YSMZBYPVVYSGOT-SZMVWBNQSA-N His-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC3=CN=CN3)N YSMZBYPVVYSGOT-SZMVWBNQSA-N 0.000 description 1
- SWTSERYNZQMPBI-WDSOQIARSA-N His-Trp-Met Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(O)=O)C1=CN=CN1 SWTSERYNZQMPBI-WDSOQIARSA-N 0.000 description 1
- HZMLFETXHFHGBB-UGYAYLCHSA-N Ile-Asn-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HZMLFETXHFHGBB-UGYAYLCHSA-N 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- FJWALBCCVIHZBS-QXEWZRGKSA-N Ile-Met-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)O)N FJWALBCCVIHZBS-QXEWZRGKSA-N 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- FIJMQLGQLBLBOL-HJGDQZAQSA-N Leu-Asn-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FIJMQLGQLBLBOL-HJGDQZAQSA-N 0.000 description 1
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 1
- UCDHVOALNXENLC-KBPBESRZSA-N Leu-Gly-Tyr Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UCDHVOALNXENLC-KBPBESRZSA-N 0.000 description 1
- POZULHZYLPGXMR-ONGXEEELSA-N Leu-Gly-Val Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O POZULHZYLPGXMR-ONGXEEELSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 1
- KQFZKDITNUEVFJ-JYJNAYRXSA-N Leu-Phe-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CC=CC=C1 KQFZKDITNUEVFJ-JYJNAYRXSA-N 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- XFIHDSBIPWEYJJ-YUMQZZPRSA-N Lys-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN XFIHDSBIPWEYJJ-YUMQZZPRSA-N 0.000 description 1
- KCXUCYYZNZFGLL-SRVKXCTJSA-N Lys-Ala-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O KCXUCYYZNZFGLL-SRVKXCTJSA-N 0.000 description 1
- SJNZALDHDUYDBU-IHRRRGAJSA-N Lys-Arg-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(O)=O SJNZALDHDUYDBU-IHRRRGAJSA-N 0.000 description 1
- ABHIXYDMILIUKV-CIUDSAMLSA-N Lys-Asn-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ABHIXYDMILIUKV-CIUDSAMLSA-N 0.000 description 1
- PHHYNOUOUWYQRO-XIRDDKMYSA-N Lys-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N PHHYNOUOUWYQRO-XIRDDKMYSA-N 0.000 description 1
- ZXFRGTAIIZHNHG-AJNGGQMLSA-N Lys-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N ZXFRGTAIIZHNHG-AJNGGQMLSA-N 0.000 description 1
- MYZMQWHPDAYKIE-SRVKXCTJSA-N Lys-Leu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O MYZMQWHPDAYKIE-SRVKXCTJSA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- RIJCHEVHFWMDKD-SRVKXCTJSA-N Lys-Lys-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O RIJCHEVHFWMDKD-SRVKXCTJSA-N 0.000 description 1
- GZGWILAQHOVXTD-DCAQKATOSA-N Lys-Met-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O GZGWILAQHOVXTD-DCAQKATOSA-N 0.000 description 1
- UGCIQUYEJIEHKX-GVXVVHGQSA-N Lys-Val-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O UGCIQUYEJIEHKX-GVXVVHGQSA-N 0.000 description 1
- SQUTUWHAAWJYES-GUBZILKMSA-N Met-Asp-Arg Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SQUTUWHAAWJYES-GUBZILKMSA-N 0.000 description 1
- LRALLISKBZNSKN-BQBZGAKWSA-N Met-Gly-Ser Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LRALLISKBZNSKN-BQBZGAKWSA-N 0.000 description 1
- BEZJTLKUMFMITF-AVGNSLFASA-N Met-Lys-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCNC(N)=N BEZJTLKUMFMITF-AVGNSLFASA-N 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 101710097941 N-acetylmuramoyl-L-alanine amidase CwlA Proteins 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
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- JVTMTFMMMHAPCR-UBHSHLNASA-N Phe-Ala-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JVTMTFMMMHAPCR-UBHSHLNASA-N 0.000 description 1
- OPEVYHFJXLCCRT-AVGNSLFASA-N Phe-Gln-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O OPEVYHFJXLCCRT-AVGNSLFASA-N 0.000 description 1
- HOYQLNNGMHXZDW-KKUMJFAQSA-N Phe-Glu-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O HOYQLNNGMHXZDW-KKUMJFAQSA-N 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- DOXQMJCSSYZSNM-BZSNNMDCSA-N Phe-Lys-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O DOXQMJCSSYZSNM-BZSNNMDCSA-N 0.000 description 1
- GOUWCZRDTWTODO-YDHLFZDLSA-N Phe-Val-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O GOUWCZRDTWTODO-YDHLFZDLSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- DEDANIDYQAPTFI-IHRRRGAJSA-N Pro-Asp-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O DEDANIDYQAPTFI-IHRRRGAJSA-N 0.000 description 1
- WVOXLKUUVCCCSU-ZPFDUUQYSA-N Pro-Glu-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVOXLKUUVCCCSU-ZPFDUUQYSA-N 0.000 description 1
- 241001620960 Pseudomonas aeruginosa PAK Species 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-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
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 1
- PPNPDKGQRFSCAC-CIUDSAMLSA-N Ser-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPNPDKGQRFSCAC-CIUDSAMLSA-N 0.000 description 1
- XVWDJUROVRQKAE-KKUMJFAQSA-N Ser-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=CC=C1 XVWDJUROVRQKAE-KKUMJFAQSA-N 0.000 description 1
- LLSLRQOEAFCZLW-NRPADANISA-N Ser-Val-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LLSLRQOEAFCZLW-NRPADANISA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- GKWNLDNXMMLRMC-GLLZPBPUSA-N Thr-Glu-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O GKWNLDNXMMLRMC-GLLZPBPUSA-N 0.000 description 1
- DXPURPNJDFCKKO-RHYQMDGZSA-N Thr-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DXPURPNJDFCKKO-RHYQMDGZSA-N 0.000 description 1
- MQVGIFJSFFVGFW-XEGUGMAKSA-N Trp-Ala-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MQVGIFJSFFVGFW-XEGUGMAKSA-N 0.000 description 1
- NXJZCPKZIKTYLX-XEGUGMAKSA-N Trp-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N NXJZCPKZIKTYLX-XEGUGMAKSA-N 0.000 description 1
- DDJHCLVUUBEIIA-BVSLBCMMSA-N Trp-Met-Phe Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)CCSC)C(O)=O)C1=CC=CC=C1 DDJHCLVUUBEIIA-BVSLBCMMSA-N 0.000 description 1
- UABYBEBXFFNCIR-YDHLFZDLSA-N Tyr-Asp-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UABYBEBXFFNCIR-YDHLFZDLSA-N 0.000 description 1
- DWAMXBFJNZIHMC-KBPBESRZSA-N Tyr-Leu-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O DWAMXBFJNZIHMC-KBPBESRZSA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- PFMAFMPJJSHNDW-ZKWXMUAHSA-N Val-Cys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N PFMAFMPJJSHNDW-ZKWXMUAHSA-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
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229940051921 muramidase Drugs 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000001066 phage therapy Methods 0.000 description 1
- 108010024607 phenylalanylalanine Proteins 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010072644 valyl-alanyl-prolyl-glycine Proteins 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 230000001018 virulence Effects 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种铜绿假单胞菌噬菌体裂解酶及应用，人工铜绿假单胞菌噬菌体裂解酶，其核酸序列1和2，氨基酸序列3和4，利用基因工程技术，在原有革兰氏阴性菌噬菌体裂解酶的N端添加聚阳离子九肽、疏水五肽开发能够高效杀灭铜绿假单胞菌、恶臭假单胞菌、地衣芽孢杆菌、枯草芽孢杆菌的人工裂解酶。该裂解酶及其衍生物可单独使用或与表面活性剂协同作用，能够特异型灭活铜绿假单胞菌、芽孢杆菌，该裂解酶可去除以及抑制由上述微生物形成的生物被膜，为目前防治铜绿假单胞菌、地衣芽孢杆菌、枯草芽孢杆菌的感染和控制食品中的地衣芽孢杆菌、枯草芽孢杆菌等细菌造成的生物被膜污染提供一种安全、无毒副作用的酶制剂来源。
Description
技术领域
本发明设计属于生物工程领域，特别涉及人工铜绿假单胞菌噬菌体裂解酶(Endolysin或称为溶壁酶)及其作为杀菌活性成分在食品生物被膜中的应用。
背景技术
生物被膜(Bacterial biofilm)是指菌体为了适应环境，粘附于不同材质界面，分泌大量的胞外多糖、蛋白质和核酸等不均一的胞外基质，将菌体自身包裹在其中而形成的大量菌体聚集膜样物。生物被膜为细菌提供了一种保护性生活方式，其形成有利于微生物持续定植、抵抗宿主免疫系统清除、提高对抗生素的耐受性以及遗传物质的交换。生物被膜黏附于接触表面，菌体嵌入聚合物保护层导致生物被膜一旦形成就难于清除，并且被膜菌的形态结构、生理生化特性、致病性以及对环境因子的敏感性等都与浮游细菌有显著的不同，其对抗生素的耐药性比浮游菌增加10～1000倍，成为一种潜在的交叉污染源，因此，食品行业中微生物生物膜形成机制以及生物被膜的抑制和去除成为近几年来研究的热点问题之一。
生物被膜主要存在于以下食品行业：乳制品、肉类、家禽、海鲜和蔬菜加工行业，根据食品加工行业的不同，细菌的种类和污染途径有所不同。在海鲜产品行业中，最容易形成生物膜的病原菌是弧菌属、嗜水气单胞菌、沙门氏菌、单核细胞增生李斯特氏菌。在新鲜农产品行业，存在于产品处理设备上的沙门氏菌、大肠杆菌O157：H7、李斯特菌、志贺氏菌、蜡状芽孢杆菌、产气荚膜梭菌和鼠疫等细菌能吸附到植物组织上形成生物膜。在乳品行业中，大多数污染来自清洗设备的不足和致病细菌的存在；在肉类工业中，大肠杆菌O157：H7能引起病原性污染。在家禽行业中，沙门氏菌和空肠弯曲菌是最常见的病原体。总的来说，食品行业中的生物被膜具有屏障作用，能抵抗外界抗菌剂对细菌的杀菌作用，对食品行业具有极其严重的危害。
目前，针对于生物被膜的危害，市场上已经出现具有一定的去除生物被膜作用的化学清洗剂。化学清洁剂可高效去除生物被膜，但其给人体带来一定的危害及潜在危害，给设备带来一定的腐蚀性，同时，还对环境造成一定的环保问题。例如，清洁剂中含有的氯，会改变水的滋味，对人体及环境造成一定的负面影响；噬菌体被认为是控制生物被膜的新型有效方法，但噬菌体使用的过程中存在很多劣势，例如，针对不同的生物被膜需要不同的噬菌体，噬菌体宿主范围窄，无法消除对噬菌体不敏感的菌体；裂解能力不强；对菌体有筛选作用；且噬菌体的使用过程中存在一定危险性。
噬菌体裂解酶是噬菌体病毒复制晚期合成的一类细胞壁水解酶，又称内溶素、溶胞壁酶，可以水解宿主菌的肽聚糖结构，属于一种自然酶。与抗生素治疗、噬菌体疗法相比，裂解酶具有很多噬菌体与抗生素所不具备的优势：菌体难以对裂解酶产生耐受性、裂解酶受抗体的裂解效率影响小、裂解酶的裂解谱比噬菌体的裂解谱广、裂解酶与抗生素具有协同作用、裂解酶对细菌的裂解能力更强、裂解酶与裂解酶之间存在协同作用、裂解酶对人体正常细胞无毒性作用、裂解酶不会转移毒力基因等。
发明内容
本发明的一个目的是提供两种改造后的核苷酸序列，该两种核苷酸序列编码人工铜绿假单胞菌噬菌体裂解酶。
本发明的目的是通过以下技术方案实现的：
一种铜绿假单胞菌噬菌体裂解酶，其特征在于：其核酸序列如序列1或2所示。
一种铜绿假单胞菌噬菌体裂解酶基因，其特征在于：其基因序列如序列3或4所示。
一种编码有铜绿假单胞菌噬菌体人工裂解酶的质粒。
一种含有编码有铜绿假单胞菌噬菌体人工裂解酶的质粒的重组菌。
一种铜绿假单胞菌噬菌体裂解酶的制备方法，采用如序列5、序列6所示的引物和如序列7、序列8所示的引物，从铜绿假单胞菌噬菌体基因组DNA中扩增铜绿假单胞菌噬菌体裂解酶基因；
具体步骤如下
⑴从铜绿假单胞菌噬菌体基因组中扩增人工铜绿假单胞菌噬菌体裂解酶基因；
⑵构建表达人工铜绿假单胞菌噬菌体裂解酶的重组表达载体；
⑶将重组表达载体转化大肠杆菌感受态细胞，筛选得到表达人工铜绿假单胞菌噬菌体裂解酶的工程菌；
⑷异丙基-β-D-硫代吡喃半乳糖苷诱导表达，获得重组基因表达产物；
⑸将重组基因表达产物，经镍柱亲和层析纯化分离，得到重组的人工铜绿假单胞菌噬菌体裂解酶。
铜绿假单胞菌噬菌体裂解酶用于抑制铜绿假单胞菌、芽孢杆菌生长的应用。
一种菌体裂解剂，包括铜绿假单胞菌噬菌体裂解酶与表面活性剂组合。
而且，所述表面活性剂为聚赖氨酸。
本发明的优点和积极效果是：
1、本专利采用基因工程菌株发酵生产的人工噬菌体裂解酶作为杀菌剂，该酶通过水解细菌细胞壁肽聚糖上糖与肽间的酰胺键或肽内氨基酸残基间的连键最终使宿主细胞裂解，该人工裂解酶相比于野生型裂解酶其具有安全无毒、水溶性好、抑菌谱广优点外其裂解酶的热稳定性以及杀菌活性有一定的提高。
2、本发明提供的铜绿假单胞菌噬菌体的人工裂解酶可以用于抑制铜绿假单胞菌、芽孢杆菌的生长，防治污染，本人工铜绿假单胞菌噬菌体裂解酶与表面活性剂LAB-35以及聚赖氨酸具有协同作用，提高杀菌活性，作为杀菌剂中的优良混合，制备新型杀菌剂。
附图说明
图1为裂解酶HPP-GP57基因、PCNP-GP57的表达质粒的构建与鉴定。
图1A为HPP-GP57基因扩增，图1B为HPP-GP57表达质粒的酶切验证。
图1C为PCNP-GP57基因扩增，图1D为PCNP-GP57表达质粒的酶切验证。
图2为人工裂解酶HPP-GP57、PCNP-GP57表达产物的鉴定分析。
泳道1：纯化后的野生型裂解酶GP57。
泳道2：纯化后的改造裂解酶HPP-GP57。
泳道3：纯化后的改造裂解酶PCNP-GP57。
图3为裂解酶GP57及其人工裂解酶HPP-GP57、PCNP-GP57的热稳定性分析。
图3A为裂解酶GP57的热稳定性分析，图3B为裂解酶HPP-GP57的热稳定性分析。
图3C为裂解酶PCNP-GP57的热稳定性分析图。
图4为裂解酶GP57及其人工裂解酶HPP-GP57、PCNP-GP57的杀菌能力分析。
图5为裂解酶与表面活性剂的协同作用。
图5A为铜绿假单胞菌作为底物时裂解酶与表面活性剂的协同作用，图5B为芽孢杆菌作为底物时裂解酶与表面活性剂的协同作用。
图6为裂解酶对聚苯乙烯材质上单一生物被膜的影响。
图6A为裂解酶对聚苯乙烯材质上单一阳性菌生物被膜的影响，图6B为裂解酶对聚苯乙烯材质上单一阴性菌生物被膜的影响。
图7为裂解酶以及衍生裂解酶对聚苯乙烯材质上成熟细菌生物被膜的裂解作用图
图7A为对十株革兰氏阳性菌有不同程度的裂解作用，图7B为对十株革兰氏阴性菌中的SK98的生物被膜具有裂解作用
表1为裂解酶GP57的底物特异性的分析。
具体实施方式
下面结合实施例，对本发明进一步说明，下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
本发明提供一种铜绿假单胞菌噬菌体裂解酶，其核酸序列为Seq ID NO.1所示的全部或部分核酸序列；或其核酸序列为Seq ID NO.2所示的全部或部分核酸序列。
本发明提供一种制备铜绿假单胞菌噬菌体裂解酶的制备方法，采用引物5’-GAGCTCATGGGATCCTTCTTCGTAGCACCGGGCTCCTCCGCTCTAACTGAGCAAG-3’和引物5’-CTGCAGTTACTTGAAGGATTGATAGG-3’；5’-GCGGGATCCATGAAACGCAAGAAACGTAAGAACGCAAAGCTCTAACTGAGCAAG-3’和引物5’-CGGGGTACCTTACTTGAAGGATTGATAGG-3’；从铜绿假单胞菌噬菌体基因组DNA中扩增人工铜绿假单胞菌噬菌体裂解酶基因；具体步骤如下
⑴从铜绿假单胞菌噬菌体基因组中扩增人工铜绿假单胞菌噬菌体裂解酶基因；
⑵构建表达人工铜绿假单胞菌噬菌体裂解酶的重组表达载体；
⑶将重组表达载体转化大肠杆菌感受态细胞，筛选得到表达人工铜绿假单胞菌噬菌体裂解酶的工程菌；
⑷异丙基-β-D-硫代吡喃半乳糖苷诱导表达，获得重组基因表达产物；
⑸将重组基因表达产物，经镍柱亲和层析纯化分离，得到重组的人工铜绿假单胞菌噬菌体裂解酶。
上述过程具体操作通过以下几个实施例来具体说明。
实施例1噬菌体基因组的提取
(一)粗制噬菌体颗粒
⑴宿主菌的制备：从固体培养基上挑取铜绿假单胞菌单个菌落，接种于5mL LB液体培养基中，
振荡培养6-8h。
⑵噬菌体纯培养液的制备：挑取单个噬菌斑，接种于5mL对数期宿主菌培养液中，
振荡培养4-6h，而后将裂解液于10000×g离心10min，上清液即为噬菌体纯培养液。
⑶噬菌体粗制颗粒的制备：将过夜培养物转接到100mL液体LB培养基中，接种量为1％，扩增培养至对数期(OD600约0.4)，加入5mL噬菌体纯培养液，
振荡培养6-8h后得到噬菌体裂解液。向裂解液中加入DNase I和RNase A至终浓度为5μg/mL，混匀后
静置1h。而后加入NaCl至终浓度为0.1mol/L，混匀溶解后冰浴1h，12000×g离心20min。将上清液转到另一离心管中后，加入PEG6000至终浓度为10％(w/v)，充分振荡溶解后于
静置过夜，12000×g离心20min，弃上清。用500μLTM(0.05mol/L Tris-HCl pH 7.5，0.2％MgSO4·7H2O)缓冲液将沉淀重悬，并用等体积的氯仿反复抽提一次，12000×g离心10min，以除去重悬液中的PEG6000，最终得到噬菌体颗粒的粗提物。
(二)噬菌体基因组DNA的制备
⑴在纯化的噬菌体颗粒中加入DNase I和RNase A，终浓度为1μg/mL，
静置1h，以降解残留的宿主菌的DNA或RNA；
⑵然后加入1mol/L EDTA(pH 8.0)至终浓度50mmol/L，终止DNase I及RNase A活性；
⑶加蛋白酶K至终浓度为50μg/mL，加SDS至终浓度为0.5％，混匀，
l h，消化蛋白质；
⑷加入等体积苯酚：氯仿：异戊醇(25∶24∶1)混匀，12000×g离心10min，收集上清；
⑸步骤4重复3次；收集上清，等体积氯仿，混匀，12000×g，10min，收集上清；
⑹加入1/10体积3mol/L NaAC及2倍体积预冷的95％乙醇，混匀，12000×g，10min，沉淀DNA；
⑺加70％乙醇(500μL)于沉淀中，并将盖紧的离心管颠倒数次，12000×g离心5min，回收DNA。
⑻去掉上清液，除去管壁上的酒精液滴，将开口的离心管室温干燥10min，而后用双蒸水重悬DNA。
实施例2重组质粒的构建
1、目的片段的获得
⑴引物设计
根据裂解酶基因序列(Seq ID NO.2)，设计引物：
HPP-Lysgp57-F：5’-GAGCTCATGGGATCCTTCTTCGTAGCACCGGGCTCCTCCGCTCTAACTGAGCAAG-3’
HPP-Lysgp57-R：5’-CTGCAGTTACTTGAAGGATTGATAGG-3’
PCNP-Lysgp57-F：5’-GCGGGATCCATGAAACGCAAGAAACGTAAGAACGCAAAGCTCTAACTGAGCAAG-3’
PCNP-Lysgp57-R：5’-CGGGGTACCTTACTTGAAGGATTGATAGG-3’；
⑵50μL反应体系：
⑶PCR反应条件：
2、化学转化实验(T-easy重组载体的构建)
⑴将gp57基因PCR产物与T-easy载体进行连接，连接体系为：
10μL反应体系：
将反应体系混匀，
过夜连接。
⑵大肠杆菌DH5a化学感受态细胞制备：
a、细菌培养：挑取大肠杆菌DH5a单个菌落于5mL液体LB培养基中，
过夜培养。转接过夜培养物至200mL LB培养基中，接种量为3％，继续培养对数期中期(OD600约0.35-0.4)，将培养液转入离心管中冰浴，至完全冷却；
b、0.1mol/L氯化镁处理溶液：将培养液于
3500×g离心10min。菌体沉淀用20mL氯化镁溶液重悬后冰浴5min，然后于
3500×g离心10min，弃上清；
c、0.1mol/L氯化钙处理溶液：10mL氯化钙溶液重悬沉淀后冰浴20min，而后
3500×g离心10min，弃上清；
d、0.1mol/L氯化钙/15％甘油保存：用1mL混合溶液重悬沉淀，混匀后以100μL/管(离心管预先冰浴10min)分装，最终于
保存；
⑶取3μL PCR产物与质粒载体T-easy的连接产物和100μL大肠杆菌DH5a感受态细胞混匀。冰浴30min后于
热激30s，而后于冰上静置2min。加入800μL预冷的LB液体培养基，于
振荡培养30min后，取100μL涂于(含100μg/mL氨苄青霉素，50μg/mL IPTG)的LB平板上，
过夜培养，通过蓝白菌落筛选，选取白色菌落。
挑取白色菌落至含有100μg/mL的氨苄青霉素和25μg/mL的LB液体培养基中过夜震荡培养，碱裂解法提取质粒，并用限制性内切酶进行酶切验证，如图1所示，大小与预期相符，表明构建正确。
3、表达质粒的构建
⑴大肠杆菌M15化学感受态细胞制备：
a、细菌培养：挑取大肠杆菌M15单个菌落于5mL液体LB培养基(含卡那霉素25μg/mL)中，
过夜培养。转接过夜培养物至200mL LB培养基(含卡那霉素25μg/mL)中，接种量为1％，继续培养对数期(OD600约0.35-0.4)，将培养液转入离心管中冰浴，至完全冷却；
b、0.1mol/L氯化镁溶液处理：将培养液于
3500×g离心10min。菌体沉淀用20mL氯化镁溶液重悬后冰浴5min，然后于
3500×g离心10min；
c、0.1mol/L氯化钙溶液处理：10mL氯化钙溶液重悬沉淀后冰浴20min，而后
3500×g离心10min；
d、0.1mol/L氯化钙/15％甘油保存：用2mL混合溶液重悬沉淀，混匀后以200μL/管(离心管预先冰浴10min)分装，最终于
保存；
⑵用BamHI对质粒pQE30以及克隆有裂解酶gp57基因的T载体质粒进行酶切，酶切体系如下：
gp57基因的100μL酶切反应体系：
将反应体系混匀，
水浴4h。
⑶使用胶回收试剂盒对pQE30质粒以及裂解酶gp57基因片段进行回收.
⑷将pQE30质粒去磷酸化，并将质粒进行回收
⑸将gp57酶切回收产物与去磷酸化后的pQE-30质粒载体进行连接，连接体系如下：
gp57回收产物 4μL
载体pQE-30 1μL
DNA连接酶Solution I 5μL
将反应体系混匀，
过夜连接。
⑹取5μL连接产物和100μL大肠杆菌M15感受态细胞混匀。冰浴30min后于
热激30s，而后于冰上静置2min。加入800μL预冷的LB液体培养基，于
110rpm振荡培养15min后，培养物涂布于含有100μg/mL氨苄青霉素、25μg/mL卡那霉素的LB平板上，
倒置培养过夜。
⑺挑取菌落至5ml LB液体培养基中过夜震荡培养，碱裂解法提取质粒进行双酶切验证，结果表明质粒载体中具有目的核苷酸序列。将鉴定正确的重组质粒载体命名为PCNP-gp57-pQE30、HPP-gp57-pQE30，并将载有重组质粒的工程菌命名为JZY16-01、JZY16-02。
挑取白色菌落至含有100μg/mL的氨苄青霉素和25μg/mL的LB液体培养基中过夜震荡培养，碱裂解法提取质粒，并用限制性内切酶进行酶切验证，如图1所示，大小与预期相符，初步表明构建正确，对重组质粒进行测序，测序结果表明重组质粒PCNP-gp57-pQE30、HPP-gp57-pQE30与设计序列一致，表明构建成功。
实施例3裂解酶在大肠杆菌中的高效表达
将冻存的重组菌JZY16-01、JZY16-02菌液接种单菌落于20mL LB液体培养基(含氨苄青霉素100μg/mL、卡那霉素25μg/mL)中，
220r/min，过夜振荡培养，次日，按照1％的接种量将菌液加入到新鲜的1L LB液体培养基(含氨苄青霉素100μg/mL、卡那霉素25μg/mL)中，
180rpm培养至OD600＝0.6，加入诱导剂IPTG(至终浓度为1mmol/L)。诱导培养4h后，离心，4000r/min、15min收集菌体。每1g菌体(湿重)重悬于3mL的破菌缓冲液(Tris-HCl20mmol/L，NaCl 250mmol/L、PMSF 1mmol/L，pH 7.4)中，充分混匀后，超声波破碎细胞，离心10000×g，
离心15min，去除不溶性细胞碎片，上清液用0.22μm无菌滤膜过滤，获得粗酶液。
将粗酶液用His亲和层析镍柱(GE Healthcare，Sweden)纯化，具体按试剂盒说明步聚进行。获得蛋白命名为裂解酶HPP-GP57、PCNP-GP57。
SDS-PAGE分析结果如图2所示，纯化后的裂解酶GP57、HPP-GP57、PCNP-GP57泳道中分别出现22.73KDa、23.67KDa、23.97KDa的融合蛋白条带，说明裂解酶均为可溶表达并且可通过Ni2+-NTA亲和层析纯化。通过Quantity One软件(Bio-Rad)分析SDS-PAGE电泳图，结果发现，纯化的裂解酶蛋白的纯度均能够达到90％以上。
实施例4裂解酶GP57的底物特异性
1、将过夜培养物按3％接种量转接到20mL LB培养基中，培养至OD600＝0.6-0.8；将菌液置于离心管中，4000g，15min，4℃离心。
2、革兰氏阴性菌去除细胞外膜后同革兰氏阳性菌一样用10mmol/L磷酸盐缓冲液(pH＝7)洗2次；
3、取等体积的裂解酶加入96孔板至裂解酶终浓度为50μg/mL，用混匀的菌悬液170μL补至总反应体积为200μL；
4、以等体积缓冲液为阴性对照，设置3个平行，每隔3min测定吸光度OD600，反应温度为室温。
结果如表1所示，表明裂解酶对铜绿假单胞菌、霍乱弧菌、恶臭假单胞菌、枯草芽孢杆菌、地衣芽孢杆菌有极其明显的裂解作用，这表明该裂解酶GP57不仅能裂解革兰氏阴性菌还能裂解革兰氏阳性菌，具有一定的广谱性。
实施例5裂解酶GP57、HPP-GP57、PCNP-GP57热稳定性的测定
1、铜绿假单胞菌去除细胞外膜。
2、取9份体积为100μL的裂解酶，分别置于20-100℃的水浴锅中处理15min，置于室温复性20min。
3、分别取经不同温度处理的裂解酶30μL加入96孔板，用混匀的菌悬液170μL补至总反应体积为200μL，裂解酶终浓度为0.05μg/mL；以等体积缓冲液为阴性对照，设置3个平行，每隔3min测定吸光度OD600，反应温度为室温。
结果如图3所示，经不同温度处理后，裂解酶GP57、HPP-GP57、PCNP-GP57均仍能保持一定的裂解能力，40℃处理15min，GP57的酶活只是原来的10％，裂解酶HPP-GP57、PCNP-GP57分别还剩60％、80％以上，这表明衍生裂解酶HPP-GP57、PCNP-GP57的热稳定性较好。
实施例6裂解酶GP57以及衍生裂解酶HPP-GP57、PCNP-GP57的杀菌能力分析
1、将过夜培养物按3％接种量转接到100mL LB培养基中，培养至OD600＝0.6-0.8
2、将菌液置于离心管中，4000×g，4℃，离心10min富集细胞，用0.9％的生理盐水重悬细胞，4000×g，4℃，离心15min，洗一次，再用适量0.9％的生理盐水将M15大肠杆菌、铜绿假单胞菌PAK、枯草芽孢杆菌、地衣芽孢杆菌重悬至OD600＝0.8-0.9左右，备用。
3、用0.9％的生理盐水作阴性对照，不同裂解酶作为实验组，反应1h，用0.9％的生理盐水10梯度稀释，取合适浓度涂板，37℃，倒置静置过夜培养，计菌落数。裂解酶GP57、HPP-GP57、PCNP-GP57终浓度为200μg/mL。由涂板获得的数据带入公式抗菌活性(％)＝(I0-Ii)/I0％(I0：0h未处理的菌落数；Ii：被处理1h后的菌落数)获得抗菌活性即杀菌率。
结果如图4A所示，以PAK为指示菌时，衍生裂解酶PCNP-GP57的杀菌率是裂解酶GP57的1.33倍，如图4B所示，以地衣芽孢杆菌1686为指示菌时，衍生裂解酶PCNP-GP57的杀菌率是裂解酶GP57的1.24倍，说明衍生裂解酶PCNP-GP57的杀菌能力高于裂解酶GP57。
实施例7裂解酶与表面活性剂的协同作用
1、将过夜培养物按3％接种量转接到100mL LB培养基中，培养至OD600＝0.6-0.8；
2、将菌液置于离心管中，4000×g，4℃，离心10min富集细胞，用0.9％的生理盐水重悬细胞，4000×g，4℃，离心15min，洗一次，再用适量0.9％的生理盐水将M15大肠杆菌重悬至OD600＝0.9左右，备用
3、用0.9％的生理盐水作阴性对照，裂解酶、LAB-35、聚赖氨酸作为实验组，反应1h，用0.9％的生理盐水10倍梯度稀释后，取合适浓度涂板，37℃，倒置静置过夜培养，计菌落数。LAB-35终浓度为0.004％，聚赖氨酸终浓度为10μg/mL，裂解酶终浓度为200μg/mL。由涂板获得的数据带入公式杀菌率＝Lg(I0/Ii)时(I0：0h未处理的菌落数；Ii：被处理1h后的菌落数)获得抗菌活性值，除去对照，与最高抗菌活性值相比获得相对酶活。
结果如图5所示，实验中，LAB-35终浓度为0.004％，聚赖氨酸终浓度为10μg/mL，裂解酶终浓度为200μg/mL，裂解酶GP57、HPP-GP57、PCNP-GP57分别与LAB-35、聚赖氨酸混合验证裂解酶与表面活性剂的协同作用时，发现以铜绿假单胞菌为底物时，阴性对照、GP57、HPP-GP57、PCNP-GP57、PL、LAB-35、GP57+PL、HPP-GP57+PL、PCNP-GP57+PL、GP57+LAB-35、HPP-GP57+LAB-35、PCNP-GP57+LAB-35的相对活细胞率(％)分别为100、37.5、56.4、16.9、96.8、94.2、11.2、40.2、6.9、51.6、53.4、25.6(图5A)，可明显看出裂解酶与聚赖氨酸具有协同作用，与LAB-35没有协同作用，以枯草芽孢杆菌为底物的情况下，阴性对照、GP57、HPP-GP57、PCNP-GP57、PL、LAB-35、GP57+PL、HPP-GP57+PL、PCNP-GP57+PL、GP57+LAB-35、HPP-GP57+LAB-35、PCNP-GP57+LAB-35的相对活细胞率(％)分别为100、13.8、44.4、12.7、11.6、69.2、5.6、5.6、5.2、11.4、11.4、11.2(图5B)，证明裂解酶与聚赖氨酸、LAB-35均具有协同作用，可进一步提高裂解酶对枯草芽孢杆菌的杀菌能力，为裂解酶的应用提供有效参考数据
实施例8
裂解酶以及衍生裂解酶对聚苯乙烯材质上细菌生物被膜形成的抑制作用
1、向96孔板中每孔加入200μL配制好的1/3液体培养基，分别按照3％、1％接种量将SK98、地衣芽孢杆菌接种到1/3液体培养基，以无菌生理盐水为阴性对照，将裂解酶加到96孔板中，至终浓度为500μg/mL，于37℃下孵育48h。
2、取出培养板，弃掉培养液与浮游菌，静态水洗3次去除生物被膜表面粘附的游离菌。每孔加200μL结晶紫染色15min后，静态水洗3次去结晶紫，每孔加200μL无水乙醇溶解15min，将含有生物被膜的无水乙醇移至新的96孔板中，测定OD595。
结果如图6所示，对九株阳性菌有不同程度的抑制作用，最高可使生物被膜的形成量降低94.9％；只对SK98、PAK、D1、E1、B12、B13、B15、TJ45、TJ54、6-5十株革兰氏阴性菌中的SK98的生物被膜具有明显抑制。
实施例9
裂解酶以及衍生裂解酶对聚苯乙烯材质上成熟细菌生物被膜的裂解作用
1、向96孔板中每孔加入200μL配制好的1/3液体培养基，按照1％接种量将菌接种到1/3液体培养基，以无菌生理盐水为阴性对照，于37℃下孵育48h。
2、取出培养板，弃掉培养液与浮游菌，静态水洗3次去除生物被膜表面粘附的游离菌。
3、将裂解酶加到96孔板中，至每孔的终浓度为500μg/mL，室温作用2h，弃掉裂解酶，静态水洗3次去除生物被膜表面被裂解的生物被膜，每孔加200μL结晶紫染色15min后，静态水洗3次去处结晶紫，每孔加200μL无水乙醇溶解15min，将含有生物被膜的无水乙醇移至新的96孔板中，测定OD595。
结果如图7所示，对十株革兰氏阳性菌有不同程度的裂解作用，最高可去除91.2％的生物被膜量；只对SK98、PAK、D1、E1、B12、B13、B15、TJ45、TJ54、6-5十株革兰氏阴性菌中的SK98的生物被膜具有裂解作用。
表1
SEQUENCE LISTING
<110> 天津科技大学
<120> 铜绿假单胞菌噬菌体裂解酶及应用
<130> 2017-04-07
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 196
<212> PRT
<213> 铜绿假单胞菌噬菌体裂解酶
<400> 1
Met Gly Ser Phe Phe Val Ala Pro Gly Ser Ser Ala Leu Thr Glu Gln
1 5 10 15
Asp Phe Gln Ser Ala Ala Asp Asp Leu Gly Val Asp Val Ala Ser Val
20 25 30
Lys Ala Val Thr Lys Val Glu Ser Arg Gly Ser Gly Phe Leu Leu Ser
35 40 45
Gly Val Pro Lys Ile Leu Phe Glu Arg His Trp Met Phe Lys Leu Leu
50 55 60
Lys Arg Lys Leu Gly Arg Asp Pro Glu Ile Asn Asp Val Cys Asn Pro
65 70 75 80
Lys Ala Gly Gly Tyr Leu Gly Gly Gln Ala Glu His Glu Arg Leu Asp
85 90 95
Lys Ala Val Lys Met Asp Arg Asp Cys Ala Leu Gln Ser Ala Ser Trp
100 105 110
Gly Leu Phe Gln Ile Met Gly Phe His Trp Glu Ala Leu Gly Tyr Ala
115 120 125
Ser Val Gln Ala Phe Val Asn Ala Gln Tyr Ala Ser Glu Gly Ser Gln
130 135 140
Leu Asn Thr Phe Val Arg Phe Ile Lys Thr Asn Pro Ala Ile His Lys
145 150 155 160
Ala Leu Lys Ser Lys Asp Trp Ala Glu Phe Ala Arg Arg Tyr Asn Gly
165 170 175
Pro Asp Tyr Lys Lys Asn Asn Tyr Asp Val Lys Leu Ala Glu Ala Tyr
180 185 190
Gln Ser Phe Lys
195
<210> 2
<211> 195
<212> PRT
<213> 铜绿假单胞菌噬菌体裂解酶及应用
<400> 2
Met Lys Arg Lys Lys Arg Lys Lys Arg Lys Ala Leu Thr Glu Gln Asp
1 5 10 15
Phe Gln Ser Ala Ala Asp Asp Leu Gly Val Asp Val Ala Ser Val Lys
20 25 30
Ala Val Thr Lys Val Glu Ser Arg Gly Ser Gly Phe Leu Leu Ser Gly
35 40 45
Val Pro Lys Ile Leu Phe Glu Arg His Trp Met Phe Lys Leu Leu Lys
50 55 60
Arg Lys Leu Gly Arg Asp Pro Glu Ile Asn Asp Val Cys Asn Pro Lys
65 70 75 80
Ala Gly Gly Tyr Leu Gly Gly Gln Ala Glu His Glu Arg Leu Asp Lys
85 90 95
Ala Val Lys Met Asp Arg Asp Cys Ala Leu Gln Ser Ala Ser Trp Gly
100 105 110
Leu Phe Gln Ile Met Gly Phe His Trp Glu Ala Leu Gly Tyr Ala Ser
115 120 125
Val Gln Ala Phe Val Asn Ala Gln Tyr Ala Ser Glu Gly Ser Gln Leu
130 135 140
Asn Thr Phe Val Arg Phe Ile Lys Thr Asn Pro Ala Ile His Lys Ala
145 150 155 160
Leu Lys Ser Lys Asp Trp Ala Glu Phe Ala Arg Arg Tyr Asn Gly Pro
165 170 175
Asp Tyr Lys Lys Asn Asn Tyr Asp Val Lys Leu Ala Glu Ala Tyr Gln
180 185 190
Ser Phe Lys
195
<210> 3
<211> 591
<212> DNA
<213> 铜绿假单胞菌噬菌体裂解酶基因
<400> 3
atgggatcct tcttcgtagc accgggctcc tccgctctaa ctgagcaaga cttccaatcg 60
gctgccgatg atctcggagt cgatgttgcc agtgtaaagg ccgtcactaa ggtagagagt 120
cgtgggagcg gctttctact ttctggcgtc cctaagattc tattcgaaag gcactggatg 180
ttcaagcttc tcaaaaggaa gctaggtcgt gaccctgaaa taaacgacgt ttgcaaccct 240
aaagctggag gatacctcgg cggacaagcg gagcacgaac gtctagataa agcagtcaaa 300
atggatagag actgcgcact tcaaagtgcc tcttggggcc tattccagat tatgggattc 360
cattgggagg cactaggtta tgcgagtgtt caggcatttg tcaatgccca gtatgctagc 420
gagggatcgc aactaaacac ttttgtgcgc ttcatcaaga ccaacccggc aattcacaaa 480
gctttaaagt ctaaggactg ggcagaattc gcaagaaggt ataacgggcc ggattacaag 540
aaaaacaact acgatgttaa gctagcagaa gcctatcaat ccttcaagta a 591
<210> 4
<211> 588
<212> DNA
<213> 铜绿假单胞菌噬菌体裂解酶基因
<400> 4
atgaaacgca agaaacgtaa gaaacgcaaa gctctaactg agcaagactt ccaatcggct 60
gccgatgatc tcggagtcga tgttgccagt gtaaaggccg tcactaaggt agagagtcgt 120
gggagcggct ttctactttc tggcgtccct aagattctat tcgaaaggca ctggatgttc 180
aagcttctca aaaggaagct aggtcgtgac cctgaaataa acgacgtttg caaccctaaa 240
gctggaggat acctcggcgg acaagcggag cacgaacgtc tagataaagc agtcaaaatg 300
gatagagact gcgcacttca aagtgcctct tggggcctat tccagattat gggattccat 360
tgggaggcac taggttatgc gagtgttcag gcatttgtca atgcccagta tgctagcgag 420
ggatcgcaac taaacacttt tgtgcgcttc atcaagacca acccggcaat tcacaaagct 480
ttaaagtcta aggactgggc agaattcgca agaaggtata acgggccgga ttacaagaaa 540
aacaactacg atgttaagct agcagaagcc tatcaatcct tcaagtaa 588
<210> 5
<211> 55
<212> DNA
<213> HPP-Lysgp57-F
<400> 5
gagctcatgg gatccttctt cgtagcaccg ggctcctccg ctctaactga gcaag 55
<210> 6
<211> 26
<212> DNA
<213> HPP-Lysgp57-R
<400> 6
ctgcagttac ttgaaggatt gatagg 26
<210> 7
<211> 55
<212> DNA
<213> PCNP-Lysgp57-F
<400> 7
cgcggatcca tgaaacgcaa gaaacgtaag aaacgcaaag ctctaactga gcaag 55
<210> 8
<211> 29
<212> DNA
<213> PCNP-Lysgp57-R
<400> 8
cggggtacct tacttgaagg attgatagg 29
Claims (6)
1.一种铜绿假单胞菌噬菌体裂解酶，其特征在于：其氨基酸序列如序列1或2所示。
2.一种铜绿假单胞菌噬菌体裂解酶基因，其特征在于：其基因序列如序列3或4所示。
3.一种含有权利要求2所述的铜绿假单胞菌噬菌体裂解酶基因的质粒。
4.一种含有权利要求3所述的质粒的重组菌。
5.权利要求1所述的铜绿假单胞菌噬菌体裂解酶作为制备抑制铜绿假单胞菌生长的制剂的应用。
6.一种菌体裂解剂，其特征在于：包括权利要求1所述的铜绿假单胞菌噬菌体裂解酶与表面活性剂组合；所述表面活性剂为聚赖氨酸。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710231999.4A CN107058265B (zh) | 2017-04-11 | 2017-04-11 | 铜绿假单胞菌噬菌体裂解酶及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710231999.4A CN107058265B (zh) | 2017-04-11 | 2017-04-11 | 铜绿假单胞菌噬菌体裂解酶及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107058265A CN107058265A (zh) | 2017-08-18 |
| CN107058265B true CN107058265B (zh) | 2020-08-28 |
Family
ID=59602309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710231999.4A Active CN107058265B (zh) | 2017-04-11 | 2017-04-11 | 铜绿假单胞菌噬菌体裂解酶及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058265B (zh) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108126190B (zh) * | 2017-11-02 | 2021-01-26 | 重庆医科大学附属第一医院 | 分枝杆菌噬菌体裂解酶Lysin-Guo1的制备与应用 |
| CN108103050A (zh) * | 2018-02-11 | 2018-06-01 | 中国医药集团总公司四川抗菌素工业研究所 | 一种铜绿假单胞菌噬菌体裂解酶、其编码基因、重组表达载体及其制备方法和应用 |
| CN108531469B (zh) * | 2018-04-08 | 2021-07-13 | 海南师范大学 | 一种蜡样芽孢杆菌噬菌体裂解酶及其制备方法和应用 |
| CN109371011A (zh) * | 2018-11-26 | 2019-02-22 | 天津科技大学 | 一种新的提取噬菌体基因组dna的方法 |
| CN111019876B (zh) * | 2019-12-30 | 2023-04-28 | 延安大学 | 一种铜绿假单胞菌工程菌的构建方法及应用 |
| CN112143747B (zh) * | 2020-09-09 | 2022-09-13 | 昆明理工大学 | 一种噬菌体裂解酶及其基因、基因重组表达载体与应用 |
| CN114288393B (zh) * | 2021-12-31 | 2023-08-22 | 安徽医科大学 | 两种生物酶联用在抑制铜绿假单胞菌生物膜形成上的应用 |
| CN114807104B (zh) * | 2022-04-11 | 2024-02-20 | 西南大学 | 肺炎克雷伯菌噬菌体裂解酶及其制备方法和应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2157100A1 (en) * | 2008-08-19 | 2010-02-24 | Profos AG | Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins |
| CN105543256A (zh) * | 2016-01-12 | 2016-05-04 | 天津科技大学 | 一种噬菌体的裂解酶及杀菌应用 |
-
2017
- 2017-04-11 CN CN201710231999.4A patent/CN107058265B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2157100A1 (en) * | 2008-08-19 | 2010-02-24 | Profos AG | Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins |
| CN105543256A (zh) * | 2016-01-12 | 2016-05-04 | 天津科技大学 | 一种噬菌体的裂解酶及杀菌应用 |
Non-Patent Citations (2)
| Title |
|---|
| endolysin [Pseudomonas phage JG004];Garbe,J.et al.;《NCBI Reference Sequence: YP_007002453.1》;20130215;全文 * |
| Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL;Yves Briers et al.;《Molecular Microbiology》;20071231;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107058265A (zh) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107058265B (zh) | 铜绿假单胞菌噬菌体裂解酶及应用 | |
| AU2022202944B2 (en) | Altering microbial populations & modifying microbiota | |
| Labreuche et al. | Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses | |
| US20230193241A1 (en) | Altering microbial populations & modifying microbiota | |
| Wang et al. | Resistance mechanisms adopted by a Salmonella Typhimurium mutant against bacteriophage | |
| KR20220019697A (ko) | 항박테리아제 및 방법 | |
| CN111235119B (zh) | 一种融合抗菌蛋白的制备及应用 | |
| CN117802127B (zh) | 一种沙门氏菌噬菌体裂解酶及其基因、基因重组表达载体与应用 | |
| CN112852794B (zh) | 一种产气荚膜梭菌噬菌体裂解酶及应用 | |
| Tetz et al. | Effect of nucleolytic, proteolytic, and lipolytic enzymes on transfer of antibiotic resistance genes in mixed bacterial communities | |
| Liu et al. | Expression and biological activity of lytic proteins HolST-3 and LysST-3 of Salmonella phage ST-3 | |
| CN110452895B (zh) | 一种来源于噬菌体的溶菌酶及其基因与应用 | |
| Le et al. | Characterization and safety evaluation of partially purified bacteriocin produced by Escherichia coli E isolated from fermented pineapple Ananas comosus (L.) Merr | |
| KR20130078092A (ko) | 신규 박테리오파아지 ΦＥｐ14 | |
| EP3372091A1 (en) | Method of reducing contamination of an object with clostridium | |
| CN111471670A (zh) | 一种具有体外裂解活性的沙门氏菌宽谱裂解酶及其应用 | |
| CN114807106B (zh) | 一种裂解酶pEf51和穿孔素蛋白pEf191的应用 | |
| Sheng et al. | Isolation and rapid genetic characterization of a novel T4-like bacteriophage | |
| KR20160082141A (ko) | 크로노박터 사카자키의 박테리오파아지 pbes01 및 그 용도 | |
| CN114107271B (zh) | 具有体外裂解活性的耐热耐富营养沙门氏菌宽谱裂解酶及其制备与应用 | |
| Balko et al. | BACTERIOCINS OF SOME GROUPS OF GRAM-NEGATIVE BACTERIA. | |
| KR101449417B1 (ko) | 살모넬라 엔테리카의 박테리오파아지 및 그 용도 | |
| Islam et al. | Determination of optimum survivability factors of highly pathogenic Vibrio cholerae 01 serogroup specific bacteriophage JSF4ϕ | |
| Al-Saedi et al. | Inhibition of V. parahaemolyticus infection using the commensal E. coli HS in a tissue culture infection model | |
| RU2775669C2 (ru) | Изменение популяций микроорганизмов и модификация микробиоты |
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 |