CN101586086A - 一种耐有机溶剂蛋白酶及其产生菌株 - Google Patents
一种耐有机溶剂蛋白酶及其产生菌株 Download PDFInfo
- Publication number
- CN101586086A CN101586086A CNA2009100278092A CN200910027809A CN101586086A CN 101586086 A CN101586086 A CN 101586086A CN A2009100278092 A CNA2009100278092 A CN A2009100278092A CN 200910027809 A CN200910027809 A CN 200910027809A CN 101586086 A CN101586086 A CN 101586086A
- Authority
- CN
- China
- Prior art keywords
- organic solvent
- gly
- ala
- protease
- asn
- 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
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 131
- 239000003960 organic solvent Substances 0.000 title claims abstract description 117
- 239000004365 Protease Substances 0.000 title claims abstract description 76
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 241000607720 Serratia Species 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 230000001580 bacterial effect Effects 0.000 claims description 46
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 102000035195 Peptidases Human genes 0.000 abstract description 120
- 241000607715 Serratia marcescens Species 0.000 abstract description 35
- 244000005700 microbiome Species 0.000 abstract description 12
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 6
- 239000008346 aqueous phase Substances 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 229920001184 polypeptide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 229940098362 serratia marcescens Drugs 0.000 abstract description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 235000019419 proteases Nutrition 0.000 description 64
- 102000004190 Enzymes Human genes 0.000 description 51
- 108090000790 Enzymes Proteins 0.000 description 51
- 230000000694 effects Effects 0.000 description 44
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 26
- 241000894006 Bacteria Species 0.000 description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000007788 liquid Substances 0.000 description 16
- 235000019833 protease Nutrition 0.000 description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 10
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 8
- 229910021645 metal ion Inorganic materials 0.000 description 8
- 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 7
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 7
- 241000282326 Felis catus Species 0.000 description 7
- 108010006035 Metalloproteases Proteins 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 6
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000013543 active substance Substances 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 238000013016 damping Methods 0.000 description 6
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 4
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 4
- PDAWDNVHMUKWJR-ZETCQYMHSA-N Gly-Gly-His Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CNC=N1 PDAWDNVHMUKWJR-ZETCQYMHSA-N 0.000 description 4
- WXLYNEHOGRYNFU-URLPEUOOSA-N Ile-Thr-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N WXLYNEHOGRYNFU-URLPEUOOSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 102000005741 Metalloproteases Human genes 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 108010047495 alanylglycine Proteins 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 108010009297 diglycyl-histidine Proteins 0.000 description 4
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 238000007747 plating Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- BGHCVCJVXZWKCC-UHFFFAOYSA-N tetradecane Chemical compound CCCCCCCCCCCCCC BGHCVCJVXZWKCC-UHFFFAOYSA-N 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 3
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000009413 insulation Methods 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 230000000452 restraining effect Effects 0.000 description 3
- 238000012882 sequential analysis Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 108010046845 tryptones Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 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 2
- BGNLUHXLSAQYRQ-FXQIFTODSA-N Ala-Glu-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O BGNLUHXLSAQYRQ-FXQIFTODSA-N 0.000 description 2
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 2
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 2
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 2
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 2
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 2
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 2
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 2
- AENHOIXXHKNIQL-AUTRQRHGSA-N Ala-Tyr-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H]([NH3+])C)CC1=CC=C(O)C=C1 AENHOIXXHKNIQL-AUTRQRHGSA-N 0.000 description 2
- TTXYKSADPSNOIF-IHRRRGAJSA-N Arg-Asp-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O TTXYKSADPSNOIF-IHRRRGAJSA-N 0.000 description 2
- RKRSYHCNPFGMTA-CIUDSAMLSA-N Arg-Glu-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O RKRSYHCNPFGMTA-CIUDSAMLSA-N 0.000 description 2
- HGKHPCFTRQDHCU-IUCAKERBSA-N Arg-Pro-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O HGKHPCFTRQDHCU-IUCAKERBSA-N 0.000 description 2
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 2
- 241000186063 Arthrobacter Species 0.000 description 2
- XWGJDUSDTRPQRK-ZLUOBGJFSA-N Asn-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O XWGJDUSDTRPQRK-ZLUOBGJFSA-N 0.000 description 2
- 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 2
- XWFPGQVLOVGSLU-CIUDSAMLSA-N Asn-Gln-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XWFPGQVLOVGSLU-CIUDSAMLSA-N 0.000 description 2
- QYXNFROWLZPWPC-FXQIFTODSA-N Asn-Glu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QYXNFROWLZPWPC-FXQIFTODSA-N 0.000 description 2
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 2
- ZMUQQMGITUJQTI-CIUDSAMLSA-N Asn-Leu-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZMUQQMGITUJQTI-CIUDSAMLSA-N 0.000 description 2
- REQUGIWGOGSOEZ-ZLUOBGJFSA-N Asn-Ser-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N REQUGIWGOGSOEZ-ZLUOBGJFSA-N 0.000 description 2
- JWQWPRCDYWNVNM-ACZMJKKPSA-N Asn-Ser-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N JWQWPRCDYWNVNM-ACZMJKKPSA-N 0.000 description 2
- SNYCNNPOFYBCEK-ZLUOBGJFSA-N Asn-Ser-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O SNYCNNPOFYBCEK-ZLUOBGJFSA-N 0.000 description 2
- HCZQKHSRYHCPSD-IUKAMOBKSA-N Asn-Thr-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HCZQKHSRYHCPSD-IUKAMOBKSA-N 0.000 description 2
- LMIWYCWRJVMAIQ-NHCYSSNCSA-N Asn-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N LMIWYCWRJVMAIQ-NHCYSSNCSA-N 0.000 description 2
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 2
- CYCKJEFVFNRWEZ-UGYAYLCHSA-N Asp-Ile-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O CYCKJEFVFNRWEZ-UGYAYLCHSA-N 0.000 description 2
- PYXXJFRXIYAESU-PCBIJLKTSA-N Asp-Ile-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PYXXJFRXIYAESU-PCBIJLKTSA-N 0.000 description 2
- AYFVRYXNDHBECD-YUMQZZPRSA-N Asp-Leu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O AYFVRYXNDHBECD-YUMQZZPRSA-N 0.000 description 2
- UMHUHHJMEXNSIV-CIUDSAMLSA-N Asp-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UMHUHHJMEXNSIV-CIUDSAMLSA-N 0.000 description 2
- HJCGDIGVVWETRO-ZPFDUUQYSA-N Asp-Lys-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(O)=O)C(O)=O HJCGDIGVVWETRO-ZPFDUUQYSA-N 0.000 description 2
- ZQFRDAZBTSFGGW-SRVKXCTJSA-N Asp-Ser-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZQFRDAZBTSFGGW-SRVKXCTJSA-N 0.000 description 2
- ITGFVUYOLWBPQW-KKHAAJSZSA-N Asp-Thr-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ITGFVUYOLWBPQW-KKHAAJSZSA-N 0.000 description 2
- KACWACLNYLSVCA-VHWLVUOQSA-N Asp-Trp-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KACWACLNYLSVCA-VHWLVUOQSA-N 0.000 description 2
- 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 2
- RKXVTTIQNKPCHU-KKHAAJSZSA-N Asp-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O RKXVTTIQNKPCHU-KKHAAJSZSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- VPQZSNQICFCCSO-BJDJZHNGSA-N Cys-Leu-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VPQZSNQICFCCSO-BJDJZHNGSA-N 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 2
- PKVWNYGXMNWJSI-CIUDSAMLSA-N Gln-Gln-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O PKVWNYGXMNWJSI-CIUDSAMLSA-N 0.000 description 2
- CLPQUWHBWXFJOX-BQBZGAKWSA-N Gln-Gly-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O CLPQUWHBWXFJOX-BQBZGAKWSA-N 0.000 description 2
- SXGMGNZEHFORAV-IUCAKERBSA-N Gln-Lys-Gly Chemical compound C(CCN)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N SXGMGNZEHFORAV-IUCAKERBSA-N 0.000 description 2
- YPFFHGRJCUBXPX-NHCYSSNCSA-N Gln-Pro-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCC(N)=O)C(O)=O YPFFHGRJCUBXPX-NHCYSSNCSA-N 0.000 description 2
- RONJIBWTGKVKFY-HTUGSXCWSA-N Gln-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O RONJIBWTGKVKFY-HTUGSXCWSA-N 0.000 description 2
- XMWNHGKDDIFXQJ-NWLDYVSISA-N Gln-Thr-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O XMWNHGKDDIFXQJ-NWLDYVSISA-N 0.000 description 2
- GJLXZITZLUUXMJ-NHCYSSNCSA-N Gln-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N GJLXZITZLUUXMJ-NHCYSSNCSA-N 0.000 description 2
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 2
- ZSWGJYOZWBHROQ-RWRJDSDZSA-N Glu-Ile-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSWGJYOZWBHROQ-RWRJDSDZSA-N 0.000 description 2
- RBXSZQRSEGYDFG-GUBZILKMSA-N Glu-Lys-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O RBXSZQRSEGYDFG-GUBZILKMSA-N 0.000 description 2
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 description 2
- BRFJMRSRMOMIMU-WHFBIAKZSA-N Gly-Ala-Asn Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O BRFJMRSRMOMIMU-WHFBIAKZSA-N 0.000 description 2
- 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 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
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 2
- AIJAPFVDBFYNKN-WHFBIAKZSA-N Gly-Asn-Asp Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN)C(=O)N AIJAPFVDBFYNKN-WHFBIAKZSA-N 0.000 description 2
- 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 2
- XRTDOIOIBMAXCT-NKWVEPMBSA-N Gly-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)CN)C(=O)O XRTDOIOIBMAXCT-NKWVEPMBSA-N 0.000 description 2
- LURCIJSJAKFCRO-QWRGUYRKSA-N Gly-Asn-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LURCIJSJAKFCRO-QWRGUYRKSA-N 0.000 description 2
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 2
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 2
- NPSWCZIRBAYNSB-JHEQGTHGSA-N Gly-Gln-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NPSWCZIRBAYNSB-JHEQGTHGSA-N 0.000 description 2
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 2
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 2
- FQKKPCWTZZEDIC-XPUUQOCRSA-N Gly-His-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 FQKKPCWTZZEDIC-XPUUQOCRSA-N 0.000 description 2
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 2
- CQMFNTVQVLQRLT-JHEQGTHGSA-N Gly-Thr-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CQMFNTVQVLQRLT-JHEQGTHGSA-N 0.000 description 2
- PNUFMLXHOLFRLD-KBPBESRZSA-N Gly-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 PNUFMLXHOLFRLD-KBPBESRZSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- IMCHNUANCIGUKS-SRVKXCTJSA-N His-Glu-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IMCHNUANCIGUKS-SRVKXCTJSA-N 0.000 description 2
- HQKADFMLECZIQJ-HVTMNAMFSA-N His-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N HQKADFMLECZIQJ-HVTMNAMFSA-N 0.000 description 2
- KHUFDBQXGLEIHC-BZSNNMDCSA-N His-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 KHUFDBQXGLEIHC-BZSNNMDCSA-N 0.000 description 2
- HYWZHNUGAYVEEW-KKUMJFAQSA-N His-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N HYWZHNUGAYVEEW-KKUMJFAQSA-N 0.000 description 2
- ZVKDCQVQTGYBQT-LSJOCFKGSA-N His-Pro-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O ZVKDCQVQTGYBQT-LSJOCFKGSA-N 0.000 description 2
- DMZOUKXXHJQPTL-GRLWGSQLSA-N Ile-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N DMZOUKXXHJQPTL-GRLWGSQLSA-N 0.000 description 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 2
- DLFAACQHIRSQGG-CIUDSAMLSA-N Leu-Asp-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O DLFAACQHIRSQGG-CIUDSAMLSA-N 0.000 description 2
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 2
- LVTJJOJKDCVZGP-QWRGUYRKSA-N Leu-Lys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LVTJJOJKDCVZGP-QWRGUYRKSA-N 0.000 description 2
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 2
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 2
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 2
- MPOHDJKRBLVGCT-CIUDSAMLSA-N Lys-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N MPOHDJKRBLVGCT-CIUDSAMLSA-N 0.000 description 2
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 2
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 2
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 2
- LUAJJLPHUXPQLH-KKUMJFAQSA-N Lys-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCCN)N LUAJJLPHUXPQLH-KKUMJFAQSA-N 0.000 description 2
- RPWQJSBMXJSCPD-XUXIUFHCSA-N Lys-Val-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(O)=O RPWQJSBMXJSCPD-XUXIUFHCSA-N 0.000 description 2
- SMVTWPOATVIXTN-NAKRPEOUSA-N Met-Ser-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SMVTWPOATVIXTN-NAKRPEOUSA-N 0.000 description 2
- SOAYQFDWEIWPPR-IHRRRGAJSA-N Met-Ser-Tyr Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O SOAYQFDWEIWPPR-IHRRRGAJSA-N 0.000 description 2
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 2
- BJEYSVHMGIJORT-NHCYSSNCSA-N Phe-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BJEYSVHMGIJORT-NHCYSSNCSA-N 0.000 description 2
- HTXVATDVCRFORF-MGHWNKPDSA-N Phe-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N HTXVATDVCRFORF-MGHWNKPDSA-N 0.000 description 2
- BYAIIACBWBOJCU-URLPEUOOSA-N Phe-Ile-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BYAIIACBWBOJCU-URLPEUOOSA-N 0.000 description 2
- OSBADCBXAMSPQD-YESZJQIVSA-N Phe-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N OSBADCBXAMSPQD-YESZJQIVSA-N 0.000 description 2
- OWSLLRKCHLTUND-BZSNNMDCSA-N Phe-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OWSLLRKCHLTUND-BZSNNMDCSA-N 0.000 description 2
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 2
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 2
- UNBFGVQVQGXXCK-KKUMJFAQSA-N Phe-Ser-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O UNBFGVQVQGXXCK-KKUMJFAQSA-N 0.000 description 2
- GLJZDMZJHFXJQG-BZSNNMDCSA-N Phe-Ser-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GLJZDMZJHFXJQG-BZSNNMDCSA-N 0.000 description 2
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 2
- JSGWNFKWZNPDAV-YDHLFZDLSA-N Phe-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JSGWNFKWZNPDAV-YDHLFZDLSA-N 0.000 description 2
- KLOQCCRTPHPIFN-DCAQKATOSA-N Pro-Met-Gln Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@@H]1CCCN1 KLOQCCRTPHPIFN-DCAQKATOSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- BRKHVZNDAOMAHX-BIIVOSGPSA-N Ser-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N BRKHVZNDAOMAHX-BIIVOSGPSA-N 0.000 description 2
- TYYBJUYSTWJHGO-ZKWXMUAHSA-N Ser-Asn-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O TYYBJUYSTWJHGO-ZKWXMUAHSA-N 0.000 description 2
- XWCYBVBLJRWOFR-WDSKDSINSA-N Ser-Gln-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O XWCYBVBLJRWOFR-WDSKDSINSA-N 0.000 description 2
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 2
- MLSQXWSRHURDMF-GARJFASQSA-N Ser-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CO)N)C(=O)O MLSQXWSRHURDMF-GARJFASQSA-N 0.000 description 2
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 2
- PCJLFYBAQZQOFE-KATARQTJSA-N Ser-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N)O PCJLFYBAQZQOFE-KATARQTJSA-N 0.000 description 2
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 2
- 108050000761 Serpin Proteins 0.000 description 2
- 102000008847 Serpin Human genes 0.000 description 2
- LHUBVKCLOVALIA-HJGDQZAQSA-N Thr-Arg-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O LHUBVKCLOVALIA-HJGDQZAQSA-N 0.000 description 2
- PQLXHSACXPGWPD-GSSVUCPTSA-N Thr-Asn-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PQLXHSACXPGWPD-GSSVUCPTSA-N 0.000 description 2
- PZVGOVRNGKEFCB-KKHAAJSZSA-N Thr-Asn-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N)O PZVGOVRNGKEFCB-KKHAAJSZSA-N 0.000 description 2
- NLSNVZAREYQMGR-HJGDQZAQSA-N Thr-Asp-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NLSNVZAREYQMGR-HJGDQZAQSA-N 0.000 description 2
- LGNBRHZANHMZHK-NUMRIWBASA-N Thr-Glu-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O LGNBRHZANHMZHK-NUMRIWBASA-N 0.000 description 2
- BNGDYRRHRGOPHX-IFFSRLJSSA-N Thr-Glu-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O BNGDYRRHRGOPHX-IFFSRLJSSA-N 0.000 description 2
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 2
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 2
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 2
- WYLAVUAWOUVUCA-XVSYOHENSA-N Thr-Phe-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O WYLAVUAWOUVUCA-XVSYOHENSA-N 0.000 description 2
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 2
- NJGMALCNYAMYCB-JRQIVUDYSA-N Thr-Tyr-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O NJGMALCNYAMYCB-JRQIVUDYSA-N 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- OETOOJXFNSEYHQ-WFBYXXMGSA-N Trp-Ala-Asp Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O)=CNC2=C1 OETOOJXFNSEYHQ-WFBYXXMGSA-N 0.000 description 2
- PXQPYPMSLBQHJJ-WFBYXXMGSA-N Trp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N PXQPYPMSLBQHJJ-WFBYXXMGSA-N 0.000 description 2
- JVTHMUDOKPQBOT-NSHDSACASA-N Trp-Gly-Gly Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O)=CNC2=C1 JVTHMUDOKPQBOT-NSHDSACASA-N 0.000 description 2
- UIRPULWLRODAEQ-QEJZJMRPSA-N Trp-Ser-Glu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 UIRPULWLRODAEQ-QEJZJMRPSA-N 0.000 description 2
- SGQSAIFDESQBRA-IHPCNDPISA-N Trp-Tyr-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SGQSAIFDESQBRA-IHPCNDPISA-N 0.000 description 2
- VCXWRWYFJLXITF-AUTRQRHGSA-N Tyr-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VCXWRWYFJLXITF-AUTRQRHGSA-N 0.000 description 2
- IELISNUVHBKYBX-XDTLVQLUSA-N Tyr-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 IELISNUVHBKYBX-XDTLVQLUSA-N 0.000 description 2
- TZXFLDNBYYGLKA-BZSNNMDCSA-N Tyr-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 TZXFLDNBYYGLKA-BZSNNMDCSA-N 0.000 description 2
- PMDWYLVWHRTJIW-STQMWFEESA-N Tyr-Gly-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PMDWYLVWHRTJIW-STQMWFEESA-N 0.000 description 2
- NOOMDULIORCDNF-IRXDYDNUSA-N Tyr-Gly-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NOOMDULIORCDNF-IRXDYDNUSA-N 0.000 description 2
- XUIOBCQESNDTDE-FQPOAREZSA-N Tyr-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O XUIOBCQESNDTDE-FQPOAREZSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- UEOOXDLMQZBPFR-ZKWXMUAHSA-N Val-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N UEOOXDLMQZBPFR-ZKWXMUAHSA-N 0.000 description 2
- GXAZTLJYINLMJL-LAEOZQHASA-N Val-Asn-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N GXAZTLJYINLMJL-LAEOZQHASA-N 0.000 description 2
- BEGDZYNDCNEGJZ-XVKPBYJWSA-N Val-Gly-Gln Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O BEGDZYNDCNEGJZ-XVKPBYJWSA-N 0.000 description 2
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 2
- QRVPEKJBBRYISE-XUXIUFHCSA-N Val-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N QRVPEKJBBRYISE-XUXIUFHCSA-N 0.000 description 2
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 2
- KISFXYYRKKNLOP-IHRRRGAJSA-N Val-Phe-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N KISFXYYRKKNLOP-IHRRRGAJSA-N 0.000 description 2
- PGQUDQYHWICSAB-NAKRPEOUSA-N Val-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N PGQUDQYHWICSAB-NAKRPEOUSA-N 0.000 description 2
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 2
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 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
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 2
- 108010059898 glycyl-tyrosyl-lysine Proteins 0.000 description 2
- 108010010147 glycylglutamine Proteins 0.000 description 2
- 108010020688 glycylhistidine Proteins 0.000 description 2
- 108010087823 glycyltyrosine Proteins 0.000 description 2
- 108010092114 histidylphenylalanine Proteins 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 108010027338 isoleucylcysteine Proteins 0.000 description 2
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 2
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 2
- 108010003700 lysyl aspartic acid Proteins 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 108010038320 lysylphenylalanine Proteins 0.000 description 2
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 2
- 108010084572 phenylalanyl-valine Proteins 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 239000003001 serine protease inhibitor Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 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 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 101710089384 Extracellular protease Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 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 1
- 102000010750 Metalloproteins Human genes 0.000 description 1
- 108010063312 Metalloproteins Proteins 0.000 description 1
- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- ZPIILPIOQFBBCP-UHFFFAOYSA-N SC(C)O.SC(C)O Chemical compound SC(C)O.SC(C)O ZPIILPIOQFBBCP-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101100242191 Tetraodon nigroviridis rho gene Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical group C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 241001156497 bacterium MH6 Species 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- DIOQZVSQGTUSAI-NJFSPNSNSA-N decane Chemical compound CCCCCCCCC[14CH3] DIOQZVSQGTUSAI-NJFSPNSNSA-N 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical group OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- DIOQZVSQGTUSAI-UHFFFAOYSA-N n-butylhexane Natural products CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- -1 phenylbenzene hexafluorophosphoric acid Chemical compound 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000003946 protein process Effects 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- ZJHHPAUQMCHPRB-UHFFFAOYSA-N urea urea Chemical compound NC(N)=O.NC(N)=O ZJHHPAUQMCHPRB-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供一种具有有机溶剂耐受性的沙雷氏菌属菌株,命名为Serratiamarcescens MH6,其保藏登记号为:CCTCC M 208205。本发明还提供该菌株产生的耐有机溶剂蛋白酶,该耐有机溶剂蛋白酶具有SEQ ID NO:2所示的氨基酸序列,其编码基因具有SEQ ID NO:1所示的核苷酸序列。本发明提供的菌株Serratia marcescens MH6是能够耐受有机溶剂的极端微生物,能够产生耐有机溶剂蛋白酶,丰富了极端微生物的种类和耐有机溶剂蛋白酶的来源。该蛋白酶较其它的天然蛋白酶具有良好的有机溶剂耐受性,有望应用于非水相中多肽的合成及关于其有机溶剂耐受机理的理论研究。
Description
技术领域
本发明涉及一种耐有机溶剂蛋白酶产生菌株以及该耐有机溶剂蛋白酶,属于微生物学与酶学领域。
背景技术
蛋白酶广泛应用于洗涤剂、食品、制药、制革、诊断试剂、污水处理等领域。1984年,Klibanov等人发现蛋白酶在一些有机溶剂中保存较长时间仍然具有活性,而且可以催化合成肽键的反应。这一发现促进了非水酶学的兴起。大量研究表明,有机溶剂中进行酶促反应有许多优点,如有机底物的溶解性大大增强、酶的稳定性提高、防止微生物污染、产物易纯化等。更重要的是,在有机溶剂中利用蛋白酶催化合成肽,可以避免氨基酸的外消旋作用,不需要对氨基酸侧链进行保护及脱保护,因此降低成本。研究表明,只有当酶分子具有一定的构象时,才能表现出催化活力。水直接或间接地参与维持酶分子构象的非共价作用力,维持酶的活性构象变化所需的“柔性”。因此,有机溶剂尤其是亲水性有机溶剂对天然来源的蛋白酶分子的催化活性影响很大。为了改善酶在有机溶剂中的稳定性,人们采用各种方法如酶的定向进化或化学修饰、固定化等策略。但是效果并不能满足实际催化的需要。
耐有机溶剂极端微生物能在富含有机溶剂的环境中生存,因此其所产胞外蛋白酶很有可能具有一定的有机溶剂耐受性。在已报道的有机溶剂耐受性微生物中以革兰氏阴性菌为主,仅有少量文献报道了具有有机溶剂耐受性的革兰氏阳性菌如Bacillus(芽孢杆菌属),Rhodococcus(红球菌属)和Arthrobacter(节杆菌属)等。目前已报道的产溶剂稳定性蛋白酶的微生物多为Pseudomonas(假单胞菌属)菌株。国内关于耐有机溶剂微生物产蛋白酶的相关报道并不多。其中也未见Serratia marcescens产耐有机溶剂蛋白酶的报道。
发明内容
本发明的目的是提供一种具有优良有机溶剂耐受性能的蛋白酶及其产生菌株。
本发明提供一种具有有机溶剂耐受性的沙雷氏菌属菌株,命名为Serratiamarcescens MH6,保藏单位:中国典型培养物保藏中心(湖北省武汉市武汉大学),保藏日期2008年11月7日,其保藏登记号为:CCTCCNO:M208205。
菌株Serratia marcescens MH6为耐有机溶剂蛋白酶产生菌。筛选过程如下:
初筛:采用不同浓度环己烷、DMF、DMSO等有机溶剂为筛选压力从油污土样中筛选获得耐有机溶剂极端微生物。然后,采用牛奶琼脂平板培养基,从上述耐有机溶剂极端微生物中筛选蛋白酶产生菌株,共获得6株蛋白酶高产菌。
复筛:通过检测各菌株产蛋白酶能力及所产蛋白酶的有机溶剂耐受性,从初筛菌中选取具有优良有机溶剂耐受性能的蛋白酶的产生菌。菌株MH6产蛋白酶能力最强,活力高达1204U/mL;该菌株的粗酶液具有优良有机溶剂耐受性能因此,最终选择菌株MH6作为耐有机溶剂蛋白酶产生菌。
菌株MH6经过BIOLOG自动细菌鉴定仪鉴定和16S rDNA序列分析,表明该菌株属于沙雷氏菌属,为Serratia marcescens,并命名Serratia marcescens MH6。
菌株Serratia marcescens MH6为革兰氏阴性短杆菌株,无芽孢,周生鞭毛,菌落为红色,大小为1μm×1.5~0.7×1.0μm。在LB培养基中生长24小时后,菌落大小为1.5~2.5mm。该菌株生长温度范围为20~45℃,最适生长温度为35℃,生长pH为5~9,最适pH为8.0,在有氧条件下生长。
本发明对菌株Serratia marcescens MH6产生的耐有机溶剂蛋白酶进行了纯化:(1)硫酸铵沉淀法初步纯化耐有机溶剂蛋白酶。将粗酶液置于冰浴中,加入50%饱和度的(NH4)2SO4沉淀杂蛋白,然后向上清液中加入(NH4)2SO4至饱和度60%,获得沉淀。经检测,沉淀中的蛋白酶活力回收达到69.1%。(2)离子交换层析法进一步纯化耐有机溶剂蛋白酶。通过SDS-PAGE电泳分析,表明:经过两步纯化,该蛋白酶已经达到电泳纯,其亚基分子量约为52~54kDa;最终,蛋白酶纯化倍数(表3)为5.98,回收率为19.87%,比活达到42388.57U/mg。该耐有机溶剂蛋白酶,基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的分子量为52-54KDa。
菌株Serratia marcescens MH6产生的耐有机溶剂蛋白酶具有良好的有机溶剂耐受性,在考察的15种有机溶剂中,多数有机溶剂对蛋白酶的酶活力几乎没有影响;DMSO、DMF对蛋白酶酶活力有一定影响,但处理24h后相对酶活力仍然保持在60%以上;异丙醇、丙酮、乙醇、乙腈对酶活影响较大,在24h以后,相对酶活力小于10%以下。
菌株Serratia marcescens MH6产生的耐有机溶剂蛋白酶在50%(v/v)的长链烷烃如十二烷、环己烷、乙酸乙酯中比水相中稳定。
菌株Serratia marcescens MH6产生的耐有机溶剂蛋白酶的最适反应pH为8.0-8.5,在pH6~8.5,能保留最大活力的50%以上;该酶在pH6~8的范围具有很好的稳定性。该酶的最适反应温度为40-45℃,在30、40和45℃放置一小时后,仍然能保留初始活力的70%以上,较稳定。当温度大于等于50℃时,该酶活力随着放置时间的变化迅速失活。
Ca2+、Mg2+和Ni2+离子对该耐有机溶剂蛋白酶有激活作用,而高浓度的离子溶液对酶活有抑制作用。
丝氨酸蛋白酶抑制剂(PMSF)对该酶活力有轻微抑制,表面活性剂Tween80、TritonX-100等对蛋白酶活有激活作用,而SDS对该酶活有显著抑制;巯基乙醇对该蛋白酶活有明显的抑制作用,表明该蛋白酶结构中有二硫键存在。
本发明分离克隆了菌株Serratia marcescens MH6产生的耐有机溶剂蛋白酶,所述的耐有机溶剂蛋白酶具有SEQ ID NO:2所示的氨基酸序列。该耐有机溶剂蛋白酶的编码基因具有SEQ ID NO:1所示的核苷酸序列。LC/MS/MS(HPLC-mass spectrometry analysis)分析结果显示,该耐有机溶剂蛋白酶属于Serratia marcescens金属蛋白酶家族。在NCBI上进行比对,发现该蛋白酶编码基因与Serratia marcescens metalloprotease gene(Serratia marcescens SM6金属蛋白酶)同源性为98%。CDS区域编码基因对应的氨基酸比对结果发现相似度为99%,有四个氨基酸不同。
本发明提供的菌株Serratia marcescens MH6是能够耐受有机溶剂的极端微生物,能够产生耐有机溶剂蛋白酶,丰富了极端微生物的种类和耐有机溶剂蛋白酶的来源。该蛋白酶与其它的天然蛋白酶相比,具有良好的有机溶剂耐受性,有望应用于非水相中多肽的合成及关于其有机溶剂耐受机理的理论研究。
附图说明
图1是牛奶琼脂平板筛选照片;
图2是耐有机溶剂蛋白酶的SDS-PAGE电泳分析,其中1-标准分子量蛋白,2-硫酸铵沉淀后蛋白酶液,3-DEAE-Sepharose FF离子交换后蛋白酶液;
图3表示不同有机溶剂对蛋白酶的影响;
图4表示蛋白酶在有机溶剂中的稳定性;
图5表示耐有机溶剂蛋白酶的最适反应pH和pH稳定性,其中图5(1)表示耐有机溶剂蛋白酶的最适反应pH,图5(1)表示耐有机溶剂蛋白酶的pH稳定性;
图6表示耐有机溶剂蛋白酶的最适反应温度和热稳定性,其中图6(1)表示耐有机溶剂蛋白酶的最适反应温度,图6(2)表示耐有机溶剂蛋白酶的温度稳定性。
具体实施方式
实施例一
本实验说明产耐有机溶剂蛋白酶的天然菌株的筛选程序。
初筛:获得能够耐受有机溶剂并产蛋白酶的菌株。采用不同浓度环己烷、DMF、DMSO等有机溶剂为筛选压力从油污土样中筛选获得耐有机溶剂极端微生物。然后,采用牛奶琼脂平板培养基从上述耐有机溶剂极端微生物中筛选蛋白酶产生菌株。根据各菌株在牛奶琼脂平板培养基上形成的菌落与透明圈直径之比值,初步筛选到6株蛋白酶高产菌(图1)。由于此6株菌能够耐受有机溶剂,其产生的蛋白酶也很有可能具有有机溶剂耐受性。
复筛:通过检测各菌株产蛋白酶能力及所产蛋白酶的有机溶剂耐受性,从初筛菌中选取具有优良有机溶剂耐受性能的蛋白酶的产生菌。将初筛获得的菌株接种到产酶发酵培养基,于35℃,摇床转速为180r/min,培养24h。将发酵液离心(4℃,9000r/min离心15min),上清液为粗酶液。检测各粗酶液的蛋白酶活性,其中菌株MH6产蛋白酶能力最强,活力高达1204U/mL。取菌株MH6粗酶液1mL,分别加入等体积的十六烷、十四烷、十二烷、癸烷、乙酸乙酯、正辛烷、丙酮、乙醇、二甲基甲酰胺(DMF)和二甲基亚砜(DMSO)等,于30℃、180rpm震荡处理24h后,以酪蛋白为底物检测蛋白酶剩余酶活;实验表明该酶具有优良有机溶剂耐受性能。在DMF中处理后仍能保持60%以上酶活,尤其在疏水有机溶剂中处理24h后保持80%以上酶活。因此,最终选择菌株MH6作为耐有机溶剂蛋白酶产生菌,进行后续研究。
牛奶琼脂平板培养基(MLB):胰蛋白胨5g/L,酵母粉3g/L,脱脂奶粉12g/L,琼脂18g/L。
产酶发酵培养基:胰蛋白胨10g/L,(NH4)2SO41.8g/L,KH2PO40.5g/L,MgSO40.3g/L,CaCl21.0g/L,NaCl 1.0g/L,甘油5ml/L。
蛋白酶活力检测方法为:配制酪蛋白含量为2%(W/V)的pH为8.0的Tris-HCl缓冲液作为反应底物。取1mL稀释适当倍数的粗酶液,加入1mL反应底物,40℃保温10min后加入4mL TCA反应终止液(含有0.11M三氯乙酸,0.22M醋酸钠,0.33M醋酸)。将该反应混合物于4℃静置15min,12000rpm离心25min,取上清液于280nm处检测紫外吸光值。每一个单位(U)蛋白酶活力定义为,在40℃,pH为8.0的反应条件下,每毫升粗酶液每分钟催化产生1μg酪氨酸所需的酶量。
实施例二
本实验说明耐有机溶剂蛋白酶产生菌MH6的生理生化性质及其鉴定
菌株Serratia marcescens MH6为革兰氏阴性短杆菌株,无芽孢,周生鞭毛,大小为1μm×1.5~0.7×1.0μm。在LB培养基中生长24小时后,菌落为红色,菌落大小为1.5~2.5mm。该菌株生长温度范围为20~45℃,最适生长温度为35℃,生长pH为5~9,最适pH为8.0,在有氧条件下生长。其生理生化性质见(表1)。
LB培养基配方:胰蛋白胨10g/L,酵母粉5g/L,NaCl 1.0g/L。
表1菌株MH6的部分生理生化特征
特征 | 结果 | 特征 | 结果 |
尿素 | - | 山梨醇 | + |
氧化酶反应 | - | D-果糖 | + |
蔗糖 | + | 乳糖 | - |
明胶反应 | + | 麦芽糖 | - |
葡萄糖 | + | 甘露糖 | + |
注:+(阳性):能利用;-(阴性):不能利用。
经过BIOLOG自动细菌鉴定仪和16S rDNA序列分析,菌株MH6属于沙雷氏菌属,为Serratia marcescens,并命名Serratia marcescens MH6。
考察Serratia marcescens MH6的溶剂耐受性。500mL三角瓶中MLB培养基的装液量为40mL,菌株Serratia marcescens MH6的接种量为2%(体积百分数)。在各培养体系中分别加入不同的有机溶剂溶剂(11种),添加量为20%(体积百分数),其中有机溶剂含量为20%,总体系为50ml,以橡胶塞封口,于37℃,摇床180rpm培养,培养时间24h取样检测OD660(表2)。实验结果表明,菌株MH6能在含有不同有机溶剂的培养基中生长(对照中不添加有机溶剂),具有一定的有机溶剂耐受性。Log Po/w大于2.0的有机溶剂如十二烷,庚烷,环己烷对MH6的生长影响较小;Log Po/w小于2.0的有机溶剂如异丙醇,乙醇能明显抑制菌株MH6的生长。
表2有机溶剂对菌株Serratia marcescensMH6生长的影响
溶剂 | LogP | 生长 |
空白 | - | ++ |
十二烷 | 5.6 | ++ |
正庚烷 | 4.0 | ++ |
环己烷 | 3.2 | ++ |
苯 | 2.5 | - |
甲苯 | 2.0 | + |
正丁醇 | 0.8 | - |
乙酸乙酯 | 0.68 | ++ |
异丙醇 | 0.05 | - |
乙醇 | -0.24 | - |
DMF | -1.0 | + |
DMSO | -1.35 | + |
注:++:生长良好;+:可以生长;-:不可以生长。
实施例三
本实验说明菌株Serratia marcescens MH6所产耐有机溶剂蛋白酶的纯化程序。
菌株Serratia marcescens MH6在产酶培养基中培养28h,将所得的发酵液离心(9000rmp,4℃)20min,取上清液作为粗酶液。
硫酸铵沉淀法初步纯化耐有机溶剂蛋白酶。将粗酶液置于冰浴中,一边搅拌一边缓慢加入(NH4)2SO4至50%饱和度,然后离心(9000rmp,4℃)20min,取上清液继续加入(NH4)2SO4至60%饱和度,再次离心(9000rmp,4℃)20min,将所得沉淀用Tris-HCl缓冲液(50mmol/L,pH8.0)溶解。经检测,沉淀中的蛋白酶活力回收率达到69.1%。
离子交换层析法进一步纯化耐有机溶剂蛋白酶。DEAE Fast Flow离子交换层析柱事先采用Tris-HCl缓冲液(20mmol/L,pH7.5)平衡,将硫酸铵沉淀法所得沉淀的Tris-HCl溶液作为进一步纯化的样品,上样至DEAE Fast Flow离子交换层析柱,然后用含盐(NaCl浓度为1mol/L)的Tris-HCl缓冲液(20mmol/L,pH7.5,)进行梯度洗脱,收集具有蛋白酶活性的洗脱液。通过SDS-PAGE电泳分析(图2),表明:经过两步纯化,该蛋白酶已经达到电泳纯,其亚基分子量约为52~54kDa;最终,蛋白酶纯化倍数(表3)为5.98,回收率为19.87%,比活达到42388.57U/mg。
表3耐有机溶蛋白酶MH6的纯化步骤及结果
总活力(U) | 总蛋白(mg) | 比活力(U/mg) | 回收率(%) | 纯化倍数 | |
粗酶液 | 74672 | 10.53 | 7091.35 | 100 | 1 |
(NH4)2SO4 | 51535 | 5.04 | 10221.14 | 69.1 | 1.44 |
(60%) | |||||
DEAE Fast Flow | 14836 | 0.35 | 42388.57 | 19.87 | 5.98 |
注:蛋白质浓度采用考马斯亮兰法测定
实施例四
本实验说明菌株Serratia marcescens MH6所产耐有机溶剂蛋白酶的酶学性质。
(1)溶剂耐受性
采用实施例三的方法,获得耐有机溶剂蛋白酶。将该蛋白酶稀释液分别加入等体积有机溶剂如十六烷、十四烷、环己烷、DMF、DMSO、丙酮、乙醇,在30℃、180rpm振荡24h后,检测残留蛋白酶活力(图3)。对照中加入等体积Tris-HCl缓冲液(0.05M,pH8.0)。实验结果表明,该蛋白酶具有良好的有机溶剂耐受性,在考察的15种有机溶剂中,多数有机溶剂对蛋白酶的酶活力几乎没有影响;DMSO、DMF对蛋白酶酶活力有一定影响,但处理24h后相对酶活力仍然保持在60%以上;异丙醇、丙酮、乙醇、乙腈对酶活影响较大,在24h以后,相对酶活力小于10%以下。
(2)溶剂稳定性
通常,疏水有机溶剂不直接与酶接触,所以抑制酶活力能力较亲水性有机溶剂小,本研究中考查了几种疏水有机溶剂对蛋白酶的稳定性的影响。如图4所示,此蛋白酶在50%(v/v)的长链烷烃如十二烷、环己烷、乙酸乙酯中比水相(control中添加的是缓冲液)中稳定。
(3)耐有机溶剂蛋白酶最适反应pH值和pH稳定性
以不同pH值的酪蛋白为底物,检测菌株Serratia marcescens MH6所产耐有机溶剂蛋白酶的最适反应pH。由图5(1)可见,该耐有机溶剂蛋白酶的最适反应pH为8.0-8.5,反应pH为6~8.5时,蛋白酶活力能保留最大活力的50%以上(以pH8.0条件下的蛋白酶活力为100%)。
向该耐有机溶剂蛋白酶中加入不同pH(pH介于6~9)的缓冲溶液,于40℃保温1h后,检测蛋白酶活力(以初始蛋白酶活力为对照),以考察pH对该耐有机溶剂蛋白酶稳定性的影响。由图5(2)可知,该蛋白酶在pH6~8的范围具有很好的稳定性。
(4)最适反应温度和热稳定性
在Tris-HCl缓冲体系(0.05M,pH8.0)下,检测菌株Serratia marcescens MH6所产耐有机溶剂蛋白酶的最适反应温度。从图6(1)可以看出,该酶最适反应温度为40-45℃。
将菌株Serratia marcescens MH6所产耐有机溶剂蛋白酶在30℃、40℃、45℃、50℃、55℃下分别放置1h以后,测定残留蛋白酶活力,以考察温度对该蛋白酶稳定性的影响(图6(2))。该蛋白酶在30、40和45℃水浴中处理后,仍然能保留初始活力的70%以上,较稳定。当温度大于等于50℃时,该酶活力随着放置时间的变化迅速失活。
(5)金属离子对蛋白酶活力的影响
向该耐有机溶剂蛋白酶中加入不同浓度的EDTA(乙二胺四乙酸二钠)和1,10-Phenathroline(邻菲罗林),4℃下放置30min后,检测残留蛋白酶活力(表4(1))。结果显示,随着EDTA浓度的增加,抑制作用逐渐加强,10mM的EDTA对酶活有明显的抑制作用;1mM的1,10-Phenathroline几乎完全抑制该酶活力。
向该耐有机溶剂蛋白酶中,加入各种金属离子,考察金属离子对其酶活力的影响。操作过程如下:向该耐有机溶剂蛋白酶中加入EDTA除去溶液中的微量离子,使其终浓度分别为为1mmol L-1和5mmol L-1,于4℃下放置30min后,分别添加Ca2+、Ni2+、Zn2+、Mg2+、Cu2+、Fe2+、Co2+、Mn2+等二价金属离子(对应于EDTA浓度,各金属离子终浓度为分别为1mmol L-1和5mmol L-1),于4℃下放置30min后,检测蛋白酶活力。对照中不加入任何金属离子。由表4(2)可见,1mmol L-1的Ca2+、Mg2+、Ni2+离子对该蛋白酶有激活作用,而高浓度的离子溶液对酶活有抑制作用。
表4(1)金属离子鳌合剂对蛋白酶活力的影响
表4(2)金属离子对蛋白酶活力的影响
(6)表面活性剂及抑制剂对蛋白酶活性的影响
向该耐有机溶剂蛋白酶中,分别加入不同浓度的表面活性剂和抑制剂,在25℃放置10min后,检测蛋白酶活力,考察其对该酶活力的影响。对照中不添加任何抑制剂和表面活性剂。由表5可见,丝氨酸蛋白酶抑制剂(PMSF)对该酶活力有轻微抑制,表面活性剂Tween80、TritonX-100等对蛋白酶活有激活作用,而SDS对该酶活有显著抑制;巯基乙醇对该蛋白酶活有明显的抑制作用,表明该蛋白酶结构中有二硫键存在。
表5变性剂和表面活性剂的影响
注:PMSF二苯基氟磷酸,glutathione还原型的谷胱甘肽,DTT二硫苏糖醇,Urea尿素,Mercaptoethanol巯基乙醇,SDS十二烷基磺酸钠,L-Cysteine L-半胱氨酸,CTAB十二烷基三甲基溴化铵。
实施例五
本实验说明粘质沙雷氏菌Serratia marcescens MH6所产耐有机溶剂蛋白酶的LC/MS/MS分析以及其编码基因的分离克隆程序。
纯化后的耐有机溶剂蛋白酶经SDS-PAGE电泳分析后,切出含有目的蛋白的单一条带。将该目的蛋白经过胰蛋白酶水解后获得中间肽段,(片段1)GNGIQINGK,(片段2)FSSTNVAGDTGLSK,(片段3)TGDTVYGFNSNTGR和(片段4)SFSDVGGLK,委托国家生物医学分析中心(NBCA)进行LC/MS/MS序列分析。将所得的片段信息通过NCBI蛋白酶数据库比对,结果显示四条肽段序列与粘质沙雷氏菌Serratia marcescens SM6金属蛋白酶氨基酸相应序列相同,因此根据该金属蛋白酶的序列设计引物来扩增目的蛋白的基因序列。
采用酚-氯仿法抽提菌体总DNA。根据粘质沙雷氏菌Serratia marcescensSM6金属蛋白酶基因序列,设计简并引物(PF:GTGGCTTACGGGGAGGTTAT;PR:TCCGTTGCTGTGTTACACGA),扩增该耐有机溶剂蛋白酶的编码序列。PCR反应参数为:94℃预变性5min;94℃变性1min;55℃退火2min;72℃延伸2min;循环30轮后,72℃保温10min。根据该反应条件,扩增到了约1.6kb的PCR片段。根据测定此蛋白的分子量,推断此酶蛋白的编码基因长度不超过1.5kb。将此片段连接到pMD18-T载体,进行序列测定。结果表明,本实验克隆的基因序列包括了之前LC/MS/MS分析得到的肽段序列。在NCBI上进行比对,发现该蛋白酶编码基因与Serratia marcescens metalloprotease gene(Serratia marcescens SM6金属蛋白酶)同源性为98%。CDS区域编码基因对应的氨基酸比对结果发现相似度为99%,有四个氨基酸不同。该蛋白酶片段含信号肽序列17个氨基酸,编码成熟蛋白酶的氨基酸个数为487。
序列表
<110>南京工业大学
<120>一种耐有机溶剂蛋白酶及其产生菌株
<130>njut2009
<160>4
<170>PatentIn version 3.3
<210>1
<211>1515
<212>DNA
<213>Serratia marcescens MH6
<220>
<221>CDS
<222>(1)..(1512)
<220>
<221>sig_peptide
<222>(1)..(51)
<220>
<221>mat_peptide
<222>(100)..()
<400>1
atg tct atc tgt ctg att gat atc aat cag gta atg agt gga atc gaa 48
Met Ser Ile Cys Leu Ile Asp Ile Asn Gln Val Met Ser Gly Ile Glu
-30 -25 -20
cca atg caa tct act aaa aag gca att gaa att act gaa tcc agc ctt 96
Pro Met Gln Ser Thr Lys Lys Ala Ile Glu Ile Thr Glu Ser Ser Leu
-15 -10 -5
gcg gcc gcg gca acc ggc tac gat gct gta gat gac ctg ctg cat tat 144
Ala Ala Ala Ala Thr Gly Tyr Asp Ala Val Asp Asp Leu Leu His Tyr
-1 1 5 10 15
cat gag cgg ggt aac ggg att cag att aat ggc aag gat tca ttt tct 192
His Glu Arg Gly Asn Gly Ile Gln Ile Asn Gly Lys Asp Ser Phe Ser
20 25 30
aac gag caa gct ggg ttg ttt att acc cgc gag aac caa acc tgg aac 240
Asn Glu Gln Ala Gly Leu Phe Ile Thr Arg Glu Asn Gln Thr Trp Asn
35 40 45
ggt tac aag gta ttt ggc cag ccg gtc aaa tta acc ttc tcc ttc ccg 288
Gly Tyr Lys Val Phe Gly Gln Pro Val Lys Leu Thr Phe Ser Phe Pro
50 55 60
gac tat aag ttc tct tcc acc aac gtc gcc ggc gat acc ggg ctg agc 336
Asp Tyr Lys Phe Ser Ser Thr Asn Val Ala Gly Asp Thr Gly Leu Ser
65 70 75
aag ttc agc gcg gaa cag cag cag cag gct aag ctg tcg ctg cag tcc 384
Lys Phe Ser Ala Glu Gln Gln Gln Gln Ala Lys Leu Ser Leu Gln Ser
80 85 90 95
tgg gcc gac gtc gcc aat atc acc ttc acc gaa gtg gcg gcc ggt caa 432
Trp Ala Asp Val Ala Asn Ile Thr Phe Thr Glu Val Ala Ala Gly Gln
100 105 110
aag gcc aat atc acc ttc ggc aat tac agc cag ggt cgt ccc ggc cac 480
Lys Ala Asn Ile Thr Phe Gly Asn Tyr Ser Gln Gly Arg Pro Gly His
115 120 125
tat gat tat ggt acc cag gcc tac gcc ttc ctg ccg aac acc att tgg 528
Tyr Asp Tyr Gly Thr Gln Ala Tyr Ala Phe Leu Pro Asn Thr Ile Trp
130 135 140
cag ggc cag gat ttg ggc ggc cag acc tgg tac aac gtc aac caa tcc 576
Gln Gly Gln Asp Leu Gly Gly Gln Thr Trp Tyr Asn Val Asn Gln Ser
145 150 155
aac gtg aag cat ccg gcg acc gaa gac tac ggc cgc cag acg ttc acc 624
Asn Val Lys His Pro Ala Thr Glu Asp Tyr Gly Arg Gln Thr Phe Thr
160 165 170 175
cat gag att ggc cat gcg ctg ggc ctg agc cac ccg ggc gac tac aac 672
His Glu Ile Gly His Ala Leu Gly Leu Ser His Pro Gly Asp Tyr Asn
180 185 190
gcc ggt gag ggc aac ccg acc tat aac gac gtc acc tat gcg gaa gat 720
Ala Gly Glu Gly Asn Pro Thr Tyr Asn Asp Val Thr Tyr Ala Glu Asp
195 200 205
acc cgc cag ttc agc ctg atg agc tac tgg agt gaa acc aat acc ggt 768
Thr Arg Gln Phe Ser Leu Met Ser Tyr Trp Ser Glu Thr Asn Thr Gly
210 215 220
ggc gac aac ggc ggt cac tat gcc gcg gct ccg ctg ctg gat gac att 816
Gly Asp Asn Gly Gly His Tyr Ala Ala Ala Pro Leu Leu Asp Asp Ile
225 230 235
gcc gcc att cag cat ctg tat ggc gcc aac ctg tcg acc cgc acc ggc 864
Ala Ala Ile Gln His Leu Tyr Gly Ala Asn Leu Ser Thr Arg Thr Gly
240 245 250 255
gac acc gtg tac ggc ttt aac tcc aat acc ggt cgt gac ttc ctc agc 912
Asp Thr Val Tyr Gly Phe Asn Ser Asn Thr Gly Arg Asp Phe Leu Ser
260 265 270
acc acc agc aat tcg cag aaa gtg atc ttt gcg gcc tgg gat gcg ggt 960
Thr Thr Ser Asn Ser Gln Lys Val Ile Phe Ala Ala Trp Asp Ala Gly
275 280 285
ggc aac gat acc ttc gac ttc tcc ggt tat acc gct aac cag cgc atc 1008
Gly Asn Asp Thr Phe Asp Phe Ser Gly Tyr Thr Ala Asn Gln Arg Ile
290 295 300
aac ctg aat gag aaa tcg ttc tcc gac gtg ggc ggc ctg aag ggc aac 1056
Asn Leu Asn Glu Lys Ser Phe Ser Asp Val Gly Gly Leu Lys Gly Asn
305 310 315
gtc tcg atc gcc gcc ggt gtg acc att gag aac gcc act ggc ggt tcc 1104
Val Ser Ile Ala Ala Gly Val Thr Ile Glu Asn Ala Thr Gly Gly Ser
320 325 330 335
ggc aac gac gtg atc gtc ggc aac gcg gcc aac aac gtg ctg aaa ggc 1152
Gly Asn Asp Val Ile Val Gly Asn Ala Ala Asn Asn Val Leu Lys Gly
340 345 350
ggc gcg ggt aac gac gtg ctg ttc ggc ggc ggc ggg gcg gat gaa ctg 1200
Gly Ala Gly Asn Asp Val Leu Phe Gly Gly Gly Gly Ala Asp Glu Leu
355 360 365
tgg ggc ggt gcc ggc aaa gac atc ttt gtg ttc tct gcc gcc agc gat 1248
Trp Gly Gly Ala Gly Lys Asp Ile Phe Val Phe Ser Ala Ala Ser Asp
370 375 380
tct gca ccg ggt gct tcc gac tgg atc cgc gac ttc caa aaa ggg atc 1296
Ser Ala Pro Gly Ala Ser Asp Trp Ile Arg Asp Phe Gln Lys Gly Ile
385 390 395
gac aag atc gac ctg tcg ttc ttc aat aaa gaa gcg aat agc agt gat 1344
Asp Lys Ile Asp Leu Ser Phe Phe Asn Lys Glu Ala Asn Ser Ser Asp
400 405 410 415
ttc atc cac ttc gtc gat cac ttc agc ggc acg gcc ggt gag gcg ctg 1392
Phe Ile His Phe Val Asp His Phe Ser Gly Thr Ala Gly Glu Ala Leu
420 425 430
ctg agc tac aac gcg tcc agc aat gtg acc gat ttg tcg gtg aac atc 1440
Leu Ser Tyr Asn Ala Ser Ser Asn Val Thr Asp Leu Ser Val Asn Ile
435 440 445
ggc ggg cat cag gcg ccg gac ttc ctg gtg aaa atc gtc ggc cag gta 1488
Gly Gly His Gln Ala Pro Asp Phe Leu Val Lys Ile Val Gly Gln Val
450 455 460
gac gtc gcc acg gac ttt atc gtg taa 1515
Asp Val Ala Thr Asp Phe Ile Val
465 470
<210>2
<211>504
<212>PRT
<213>Serratia marcescens MH6
<400>2
Met Ser Ile Cys Leu Ile Asp Ile Asn Gln Val Met Ser Gly Ile Glu
-30 -25 -20
Pro Met Gln Ser Thr Lys Lys Ala Ile Glu Ile Thr Glu Ser Ser Leu
-15 -10 -5
Ala Ala Ala Ala Thr Gly Tyr Asp Ala Val Asp Asp Leu Leu His Tyr
-1 1 5 10 15
His Glu Arg Gly Asn Gly Ile Gln Ile Asn Gly Lys Asp Ser Phe Ser
20 25 30
Asn Glu Gln Ala Gly Leu Phe Ile Thr Arg Glu Asn Gln Thr Trp Asn
35 40 45
Gly Tyr Lys Val Phe Gly Gln Pro Val Lys Leu Thr Phe Ser Phe Pro
50 55 60
Asp Tyr Lys Phe Ser Ser Thr Asn Val Ala Gly Asp Thr Gly Leu Ser
65 70 75
Lys Phe Ser Ala Glu Gln Gln Gln Gln Ala Lys Leu Ser Leu Gln Ser
80 85 90 95
Trp Ala Asp Val Ala Asn Ile Thr Phe Thr Glu Val Ala Ala Gly Gln
100 105 110
Lys Ala Asn Ile Thr Phe Gly Asn Tyr Ser Gln Gly Arg Pro Gly His
115 120 125
Tyr Asp Tyr Gly Thr Gln Ala Tyr Ala Phe Leu Pro Asn Thr Ile Trp
130 135 140
Gln Gly Gln Asp Leu Gly Gly Gln Thr Trp Tyr Asn Val Asn Gln Ser
145 150 155
Asn Val Lys His Pro Ala Thr Glu Asp Tyr Gly Arg Gln Thr Phe Thr
160 165 170 175
His Glu Ile Gly His Ala Leu Gly Leu Ser His Pro Gly Asp Tyr Asn
180 185 190
Ala Gly Glu Gly Asn Pro Thr Tyr Asn Asp Val Thr Tyr Ala Glu Asp
195 200 205
Thr Arg Gln Phe Ser Leu Met Ser Tyr Trp Ser Glu Thr Asn Thr Gly
210 215 220
Gly Asp Asn Gly Gly His Tyr Ala Ala Ala Pro Leu Leu Asp Asp Ile
225 230 235
Ala Ala Ile Gln His Leu Tyr Gly Ala Asn Leu Ser Thr Arg Thr Gly
240 245 250 255
Asp Thr Val Tyr Gly Phe Asn Ser Asn Thr Gly Arg Asp Phe Leu Ser
260 265 270
Thr Thr Ser Asn Ser Gln Lys Val Ile Phe Ala Ala Trp Asp Ala Gly
275 280 285
Gly Asn Asp Thr Phe Asp Phe Ser Gly Tyr Thr Ala Asn Gln Arg Ile
290 295 300
Asn Leu Asn Glu Lys Ser Phe Ser Asp Val Gly Gly Leu Lys Gly Asn
305 310 315
Val Ser Ile Ala Ala Gly Val Thr Ile Glu Asn Ala Thr Gly Gly Ser
320 325 330 335
Gly Asn Asp Val Ile Val Gly Asn Ala Ala Asn Asn Val Leu Lys Gly
340 345 350
Gly Ala Gly Asn Asp Val Leu Phe Gly Gly Gly Gly Ala Asp Glu Leu
355 360 365
Trp Gly Gly Ala Gly Lys Asp Ile Phe Val Phe Ser Ala Ala Ser Asp
370 375 380
Ser Ala Pro Gly Ala Ser Asp Trp Ile Arg Asp Phe Gln Lys Gly Ile
385 390 395
Asp Lys Ile Asp Leu Ser Phe Phe Asn Lys Glu Ala Asn Ser Ser Asp
400 405 410 415
Phe Ile His Phe Val Asp His Phe Ser Gly Thr Ala Gly Glu Ala Leu
420 425 430
Leu Ser Tyr Asn Ala Ser Ser Asn Val Thr Asp Leu Ser Val Asn Ile
435 440 445
Gly Gly His Gln Ala Pro Asp Phe Leu Val Lys Ile Val Gly Gln Val
450 455 460
Asp Val Ala Thr Asp Phe Ile Val
465 470
<210>3
<211>20
<212>DNA
<213>Artificial
<220>
<223>PF
<400>3
gtggcttacg gggaggttat 20
<210>4
<211>20
<212>DNA
<213>Artificial
<220>
<223>PR
<400>4
tccgttgctg tgttacacga 20
Claims (3)
1、一种具有有机溶剂耐受性的沙雷氏菌属菌株,命名为Serratia marcescensMH6,其保藏登记号为:CCTCC NO:M208205。
2、一种权利要求1所述菌株产生的耐有机溶剂蛋白酶,其特征在于所述耐有机溶剂蛋白酶具有SEQ ID NO:2所示的氨基酸序列。
3、根据权利要求2所述的耐有机溶剂蛋白酶,其特征在于所述耐有机溶剂蛋白酶的编码基因具有SEQ ID NO:1所示的核苷酸序列。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009100278092A CN101586086B (zh) | 2009-05-15 | 2009-05-15 | 一种耐有机溶剂蛋白酶及其产生菌株 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009100278092A CN101586086B (zh) | 2009-05-15 | 2009-05-15 | 一种耐有机溶剂蛋白酶及其产生菌株 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101586086A true CN101586086A (zh) | 2009-11-25 |
CN101586086B CN101586086B (zh) | 2011-05-04 |
Family
ID=41370517
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2009100278092A Expired - Fee Related CN101586086B (zh) | 2009-05-15 | 2009-05-15 | 一种耐有机溶剂蛋白酶及其产生菌株 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101586086B (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174422A (zh) * | 2010-11-22 | 2011-09-07 | 南京工业大学 | 一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用 |
CN102329745A (zh) * | 2011-07-27 | 2012-01-25 | 南京工业大学 | 高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用 |
CN107884351A (zh) * | 2017-10-30 | 2018-04-06 | 广东百味佳味业科技股份有限公司 | 松肉粉中木瓜蛋白酶活力的检测方法 |
CN112105730A (zh) * | 2018-03-07 | 2020-12-18 | 北极酶 As 公司 | 热不稳定蛋白酶 |
-
2009
- 2009-05-15 CN CN2009100278092A patent/CN101586086B/zh not_active Expired - Fee Related
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174422A (zh) * | 2010-11-22 | 2011-09-07 | 南京工业大学 | 一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用 |
CN102329745A (zh) * | 2011-07-27 | 2012-01-25 | 南京工业大学 | 高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用 |
CN102329745B (zh) * | 2011-07-27 | 2014-06-25 | 南京工业大学 | 高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用 |
CN107884351A (zh) * | 2017-10-30 | 2018-04-06 | 广东百味佳味业科技股份有限公司 | 松肉粉中木瓜蛋白酶活力的检测方法 |
CN112105730A (zh) * | 2018-03-07 | 2020-12-18 | 北极酶 As 公司 | 热不稳定蛋白酶 |
Also Published As
Publication number | Publication date |
---|---|
CN101586086B (zh) | 2011-05-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wang et al. | Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium | |
CN100422207C (zh) | 二肽生产方法,所用的l-氨基酸酰胺水解酶,以及l-氨基酸酰胺水解酶生产方法 | |
Liang et al. | Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation | |
Elbanna et al. | Purification and characterization of halo-alkali-thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt | |
Annamalai et al. | Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis | |
CN110777136B (zh) | 一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用 | |
Abou-Elela et al. | Alkaline protease production by alkaliphilic marine bacteria isolated from Marsa-Matrouh (Egypt) with special emphasis on Bacillus cereus purified protease | |
CN110656063B (zh) | 一种产耐高温蛋白酶菌株 | |
CN105176951A (zh) | 一种新型碱性蛋白酶突变体 | |
CN101586086B (zh) | 一种耐有机溶剂蛋白酶及其产生菌株 | |
CN110511897B (zh) | 一种沙雷氏菌及其在蛋白酶生产中的应用 | |
CN101240254B (zh) | 一种耐有机溶剂蛋白酶高产菌以及该耐有机溶剂蛋白酶的基因和应用 | |
CN107964557A (zh) | 一种提高芽孢杆菌抗菌脂肽产量的发酵方法 | |
Kumar et al. | Purification and characterization of a small size protease from Bacillus sp. APR-4 | |
Du et al. | Isolation and purification of Bacillus amyloliquefaciens D1 protease and its application in the fermentation of soybean milk to produce large amounts of free amino acids | |
CN107557314B (zh) | 产蛋白酶菌株ls20-2-2及其产低温蛋白酶的方法 | |
CN116426509B (zh) | 一种碱性蛋白酶组合突变体及其应用 | |
Bich Thuy et al. | Characterization of multiple extracellular proteases produced by a Bacillus subtilis strain and identification of the strain | |
CN102021125B (zh) | 一种耐有机溶剂蛋白酶产生菌及其所产耐有机溶剂蛋白酶的基因和应用 | |
CN104212739A (zh) | 一种蜡样芽孢杆菌及其在制备亚硝酸盐还原酶中的应用 | |
CN103667201B (zh) | 海洋微生物菌株ys0810产的碱性过氧化氢酶及其分离纯化方法 | |
Shahbazi et al. | A novel low molecular weight extracellular protease from a moderately halophilic bacterium Salinivibrio sp. strain MS-7: production and biochemical properties | |
US8802416B2 (en) | Thermostable organic solvent tolerant protease from gram-positive bacteria | |
Bhuyar | Isolation, partial purification and characterization of protease enzyme from proteolytic bacteria from dairy soil | |
Yuan et al. | Identification of an alkaline protease producing bacterium isolated from panjin red beach, China |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110504 Termination date: 20170515 |
|
CF01 | Termination of patent right due to non-payment of annual fee |