JP5344836B2 - New protease - Google Patents
New protease Download PDFInfo
- Publication number
- JP5344836B2 JP5344836B2 JP2008079526A JP2008079526A JP5344836B2 JP 5344836 B2 JP5344836 B2 JP 5344836B2 JP 2008079526 A JP2008079526 A JP 2008079526A JP 2008079526 A JP2008079526 A JP 2008079526A JP 5344836 B2 JP5344836 B2 JP 5344836B2
- Authority
- JP
- Japan
- Prior art keywords
- protease
- ala
- activity
- buffer
- fraction
- 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.)
- Expired - Fee Related
Links
- 108091005804 Peptidases Proteins 0.000 title claims description 62
- 239000004365 Protease Substances 0.000 title claims description 62
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims description 57
- 230000000694 effects Effects 0.000 claims description 56
- 101710180012 Protease 7 Proteins 0.000 claims description 37
- 239000000758 substrate Substances 0.000 claims description 21
- 230000009471 action Effects 0.000 claims description 18
- LKDMKWNDBAVNQZ-WJNSRDFLSA-N 4-[[(2s)-1-[[(2s)-1-[(2s)-2-[[(2s)-1-(4-nitroanilino)-1-oxo-3-phenylpropan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound OC(=O)CCC(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)CC1=CC=CC=C1 LKDMKWNDBAVNQZ-WJNSRDFLSA-N 0.000 claims description 15
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 claims description 11
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims description 11
- 239000003905 agrochemical Substances 0.000 claims description 8
- MRXDGVXSWIXTQL-HYHFHBMOSA-N (2s)-2-[[(1s)-1-(2-amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2-[[(2s)-4-methyl-1-oxo-1-[[(2s)-1-oxo-3-phenylpropan-2-yl]amino]pentan-2-yl]amino]-2-oxoethyl]carbamoylamino]-3-phenylpropanoic acid Chemical compound C([C@H](NC(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C=O)C1NC(N)=NCC1)C(O)=O)C1=CC=CC=C1 MRXDGVXSWIXTQL-HYHFHBMOSA-N 0.000 claims description 6
- OLVPQBGMUGIKIW-UHFFFAOYSA-N Chymostatin Natural products C=1C=CC=CC=1CC(C=O)NC(=O)C(C(C)CC)NC(=O)C(C1NC(N)=NCC1)NC(=O)NC(C(O)=O)CC1=CC=CC=C1 OLVPQBGMUGIKIW-UHFFFAOYSA-N 0.000 claims description 6
- 239000003443 antiviral agent Substances 0.000 claims description 6
- 108010086192 chymostatin Proteins 0.000 claims description 6
- PTHRPHGMGFMCSS-USNOLKROSA-N 4-[[(2s)-1-[[(2s)-1-[(2s)-2-[[(2s)-4-methyl-1-(4-nitroanilino)-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound N([C@@H](CC(C)C)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CCC(O)=O PTHRPHGMGFMCSS-USNOLKROSA-N 0.000 claims description 5
- 239000004480 active ingredient Substances 0.000 claims description 5
- 230000000717 retained effect Effects 0.000 claims description 3
- 239000000872 buffer Substances 0.000 description 53
- 241000724252 Cucumber mosaic virus Species 0.000 description 33
- 239000000523 sample Substances 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 31
- 208000015181 infectious disease Diseases 0.000 description 30
- 102000004169 proteins and genes Human genes 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 28
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 239000000243 solution Substances 0.000 description 24
- 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 22
- 238000000034 method Methods 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 241000723873 Tobacco mosaic virus Species 0.000 description 21
- 229940088598 enzyme Drugs 0.000 description 21
- 230000002401 inhibitory effect Effects 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 241000243686 Eisenia fetida Species 0.000 description 14
- 241000700605 Viruses Species 0.000 description 14
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 12
- 239000012149 elution buffer Substances 0.000 description 12
- 241000361919 Metaphire sieboldi Species 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000007986 glycine-NaOH buffer Substances 0.000 description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 244000061176 Nicotiana tabacum Species 0.000 description 9
- 241000219977 Vigna Species 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 8
- 235000010726 Vigna sinensis Nutrition 0.000 description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 8
- 239000000287 crude extract Substances 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 238000005571 anion exchange chromatography Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000003902 lesion Effects 0.000 description 7
- 230000000593 degrading effect Effects 0.000 description 6
- 230000009385 viral infection Effects 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 5
- 239000012506 Sephacryl® Substances 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 241001233061 earthworms Species 0.000 description 5
- 238000001641 gel filtration chromatography Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 244000068988 Glycine max Species 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 4
- 101710162629 Trypsin inhibitor Proteins 0.000 description 4
- 229940122618 Trypsin inhibitor Drugs 0.000 description 4
- 208000036142 Viral infection Diseases 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000002753 trypsin inhibitor Substances 0.000 description 4
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 3
- 231100000674 Phytotoxicity Toxicity 0.000 description 3
- 108090000787 Subtilisin Proteins 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- -1 for example Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000005185 salting out Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 102000003846 Carbonic anhydrases Human genes 0.000 description 2
- 108090000209 Carbonic anhydrases Proteins 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 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 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 101710196208 Fibrinolytic enzyme Proteins 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 102000004407 Lactalbumin Human genes 0.000 description 2
- 108090000942 Lactalbumin Proteins 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 108010065081 Phosphorylase b Proteins 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 241001429095 Tomarus Species 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 2
- 238000003277 amino acid sequence analysis Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 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 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 229910010271 silicon carbide Inorganic materials 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 239000010455 vermiculite Substances 0.000 description 2
- 229910052902 vermiculite Inorganic materials 0.000 description 2
- 235000019354 vermiculite Nutrition 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 235000021241 α-lactalbumin Nutrition 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 241001533462 Bromoviridae Species 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 102100025566 Chymotrypsin-like protease CTRL-1 Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000724253 Cucumovirus Species 0.000 description 1
- 240000004244 Cucurbita moschata Species 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 241000230129 Eisenia <Phaeophyceae> Species 0.000 description 1
- IBHSRCBKJMEBQB-VMPREFPWSA-N Fetidine Chemical compound CN1CCC2=CC(OC)=C(O)C3=C2[C@@H]1CC1=C3C=C(OC)C(OC2=C(OC)C(OC)=CC=C2C[C@@H]2N(C)CCC=3C=C(C(=CC=32)OC)OC)=C1 IBHSRCBKJMEBQB-VMPREFPWSA-N 0.000 description 1
- IBHSRCBKJMEBQB-UHFFFAOYSA-N Fetidine Natural products CN1CCC2=CC(OC)=C(O)C3=C2C1CC1=C3C=C(OC)C(OC2=C(OC)C(OC)=CC=C2CC2N(C)CCC=3C=C(C(=CC=32)OC)OC)=C1 IBHSRCBKJMEBQB-UHFFFAOYSA-N 0.000 description 1
- 101000856199 Homo sapiens Chymotrypsin-like protease CTRL-1 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 241001075689 Lagunaria Species 0.000 description 1
- 241001495098 Lumbricus rubellus Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 101710174798 Lysenin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108020005543 Satellite RNA Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 108700018667 Streptomyces subtilisin inhibitor Proteins 0.000 description 1
- 101710151905 Subtilisin inhibitor Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 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 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000007973 glycine-HCl buffer Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 108010070324 lumbrokinase Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 230000001047 pyretic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は新規なプロテアーゼに関する。より詳しくは、ミミズ由来の成分から分離された新規プロテアーゼであって、ウイルス増殖阻害作用を有するプロテアーゼに関するものである。 The present invention relates to a novel protease. More specifically, the present invention relates to a novel protease isolated from earthworm-derived components and having a virus growth inhibitory action.
従来、ミミズからいくつかの生理活性タンパク質が単離され、生理学的及び生化学的研究が行われている。例えば、フェチジン（分子量40,000：溶血活性、抗細菌活性、血液凝集活性）、アイゼニアポア（分子量38,000：溶血活性）、CCF-1（細胞溶解因子１、42,000：β-1,3グルカンとリポ多糖に結合）、ライセニン（分子量41,000：スフィンゴミエリンに特異的に結合）などのほか、ルンブロフェブリン（解熱作用）及びルンブロキナーゼ（血栓溶解作用）などが知られている（非特許文献１及び２）。 Conventionally, several physiologically active proteins have been isolated from earthworms, and physiological and biochemical studies have been conducted. For example, Fetidine (molecular weight 40,000: hemolytic activity, antibacterial activity, blood aggregation activity), Eisenia pore (molecular weight 38,000: hemolytic activity), CCF-1 (cytolytic factor 1, 42,000: β-1,3 glucan and lipopolysaccharide) ), Lysenin (molecular weight 41,000: specifically binds to sphingomyelin), lumbrofebrin (pyretic action), lumbrokinase (thrombolytic action), etc. are known (Non-patent Documents 1 and 2) .
最近、本発明者らは、ミミズの破砕物の懸濁液の上清からゲル濾過により分離された抗ウイルス感染作用及び増殖阻害作用を有する医薬を報告した（国際公開WO2006/93164）。この刊行物には、ミミズから2種類の抗ウイルス活性を有するタンパク質のフラクション〔フラクションNo.54〜58（フラクションI）、分子量40,000；及びフラクションNo.59〜64（フラクションII）、分子量20,000〕が得られたことが開示されており、後者のフラクションについてN-末端アミノ酸はSVGGSNAXPX-であることが明らかにされている。これらのフラクションはウイルス被覆タンパクに対する分解活性を有しており、特に後者のフラクションについては強いウイルス感染阻害活性が認められたことが開示されている。
本発明の課題は、ウイルス増殖阻害作用を有する新規なプロテアーゼを提供することにある。 An object of the present invention is to provide a novel protease having a virus growth inhibitory action.
本発明者らは、上記の国際公開（WO2006/93164）に記載された手法により、シマミミズの破砕物の懸濁液の上清からさらに新規タンパク質を分離すべく鋭意研究を行った。その結果、上記国際公開において報告したフラクションII（最大活性フラクションはNo.60）に若干遅れて溶出されるフラクションNo.63に上記プロテアーゼとは異なる新規なプロテアーゼが含まれることを見出した。また、そのプロテアーゼを単離及び精製し、該プロテアーゼがキモトリプシン様の作用を有しており、特に低温においても酵素活性を維持し、耐熱性も備えた有用なプロテアーゼであることを見出した。本発明は上記の知見を基にして完成された。 The inventors of the present invention conducted intensive research to further isolate a novel protein from the supernatant of a suspension of crushed earthworms by the method described in the above International Publication (WO2006 / 93164). As a result, it was found that a novel protease different from the above protease was contained in fraction No. 63, which was eluted with a slight delay from fraction II (maximum active fraction No. 60) reported in the above international publication. Moreover, the protease was isolated and purified, and the protease was found to be a useful protease having a chymotrypsin-like action, maintaining enzyme activity even at low temperatures, and having heat resistance. The present invention has been completed based on the above findings.
すなわち、本発明により、下記の特性：
(a)分子量が約27,000であり、
(b)N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成し、その作用はキモスタチンにより阻害され、
(c)N-サクシニル-Ala-Ala-Pro-Leu-p-ニトロアニリドに対しては実質的に分解作用を有せず、
(d)至適pHが9.5であり、pH5.0〜10.0の範囲でpH9.5における活性に対して約80％以上の活性が維持され、
(e)至適温度が40℃であり、60℃において40℃における活性に対して約80%の活性が維持され、
(f)4℃において40℃における活性の約60%が保持され、
(g)N末端のアミノ酸配列がVal-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Glyである
を有するプロテアーゼAが提供される。
That is, according to the present invention, the following characteristics:
(a) the molecular weight is about 27,000,
(b) N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is decomposed as a substrate to produce p-nitroaniline, the action of which is inhibited by chymostatin,
(c) N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide has substantially no decomposing action,
(d) The optimum pH is 9.5, and an activity of about 80% or more is maintained with respect to the activity at pH 9.5 in the range of pH 5.0 to 10.0,
(e) the optimum temperature is 40 ° C., and the activity at about 80% is maintained at 60 ° C. relative to the activity at 40 ° C .;
(f) about 60% of the activity at 40 ° C is retained at 4 ° C;
(g) Protease A having an N-terminal amino acid sequence of Val-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Gly is provided.
また、本発明により、下記の特性：
(a)分子量が約26,000であり、
(b)N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成し、その作用はフェニルメチルスフホニルフルオリドにより実質的に完全に阻害され、
(c)N-グルタリル-L-Phe-p-ニトロアニリド及びN-α-ベンゾイル-L-Arg-p-ニトロアニリドに対しては実質的に分解作用を有せず、
(d)至適pHが9.5であり、pH6.0〜11.5の範囲でpH9.5における活性に対して約80％以上の活性が維持され、
(e)至適温度が60℃であり、60℃以下で活性が維持され、
(f)N末端のアミノ酸配列がIle-Ile-Gly-Gly-Ser-Asn-Ala-Ser-Pro-Gly-Gluである
を有するプロテアーゼBが提供される。
In addition, according to the present invention, the following characteristics:
(a) the molecular weight is about 26,000,
(b) Decomposing N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate to produce p-nitroaniline, the action of which is substantially completely inhibited by phenylmethylsulfonyl fluoride And
(c) N-glutaryl-L-Phe-p-nitroanilide and N-α-benzoyl-L-Arg-p-nitroanilide have substantially no decomposition action,
(d) The optimum pH is 9.5, and an activity of about 80% or more is maintained with respect to the activity at pH 9.5 in the range of pH 6.0 to 11.5,
(e) the optimum temperature is 60 ° C, the activity is maintained at 60 ° C or less,
(f) Protease B having an N-terminal amino acid sequence of Ile-Ile-Gly-Gly-Ser-Asn-Ala-Ser-Pro-Gly-Glu is provided.
別の観点からは、上記プロテアーゼA又はBを有効成分として含む抗ウイルス剤が提供される。この抗ウイルス剤はヒトを含む哺乳類動物におけるウイルス感染症の予防及び／又は治療のための医薬、並びに植物におけるウイルス感染症の予防及び／又は治療のための農薬として有用である。上記プロテアーゼA又はBは特に低温環境下において活性を維持する特性を有していることから、上記の抗ウイルス剤は低温環境下で用いるための農薬として特に有用である。 From another viewpoint, an antiviral agent containing protease A or B as an active ingredient is provided. This antiviral agent is useful as a medicament for preventing and / or treating viral infections in mammals including humans, and as an agrochemical for preventing and / or treating viral infections in plants. Since the protease A or B has the property of maintaining activity particularly in a low temperature environment, the antiviral agent is particularly useful as an agrochemical for use in a low temperature environment.
本発明により提供される新規プロテアーゼA又はBは、耐熱性にも優れており、特に低温において活性を失わないという従来公知のプロテアーゼには認められなかった優れた性質を有している。また、優れた抗ウイルス作用を有するという特徴もある。従って、本発明のプロテアーゼA又はBは、低温環境や高温環境において発生した植物ウイルス感染症に対する予防及び／又は治療などに極めて有用である。 The novel protease A or B provided by the present invention is excellent in heat resistance, and has an excellent property that has not been recognized by conventionally known proteases, especially that it does not lose its activity at low temperatures. In addition, it has a feature of having an excellent antiviral effect. Therefore, the protease A or B of the present invention is extremely useful for prevention and / or treatment of plant virus infections occurring in a low temperature environment or a high temperature environment.
本発明のプロテアーゼA又はBは、例えば、シマミミズの破砕物懸濁液から分離・精製することができる。シマミミズの破砕物懸濁液からタンパク質画分を調製する方法についてはWO2006/93164に記載された方法を参照することができる。例えば、シマミミズ(50g)を凍結乾燥し、細かく破砕し（ミミズは凍結乾燥前は1日絶食）；この破砕物に50mMトリス塩酸（Tris-HCl）pH7.0の緩衝液を加えよく懸濁させ、次いで遠心分離（分離条件；回転数・10,000rpm、時間・20min、4℃）を行って上清を回収し、この上清に80%飽和になるように硫酸アンモニウムを加え、塩析させるために一夜低温室に放置し、さらに遠心分離（分離条件；10,000rpm、20min、4℃）を行い、沈殿物を回収して20mMトリス塩酸（Tris-HCl）pH7.0の緩衝液に溶解することができる。原料として用いるミミズは特に限定されず、本発明のプロテアーゼを有するものであれば、シマミミズのほかツリミミズ科やフトミミズ科に属するミミズなどを用いることもできる。 Protease A or B of the present invention can be separated and purified from a crushed suspension of earthworm, for example. The method described in WO2006 / 93164 can be referred to for the method for preparing the protein fraction from the crushed suspension of earthworm. For example, the earthworm (50 g) is lyophilized and crushed finely (the earthworm is fasted for 1 day before lyophilization); a buffer solution of 50 mM Tris-HCl (pH 7.0) is added to this crushed material and suspended well. Then, centrifugation (separation conditions: rotation speed 10,000 rpm, time 20 min, 4 ° C) is performed to recover the supernatant, and ammonium sulfate is added to the supernatant to 80% saturation for salting out. It can be left overnight in a low temperature room and further centrifuged (separation conditions; 10,000 rpm, 20 min, 4 ° C.) to collect the precipitate and dissolve it in a buffer solution of 20 mM Tris-HCl pH 7.0. it can. The earthworm used as a raw material is not particularly limited, and if it has the protease of the present invention, it is possible to use earthworms, worms belonging to the earthworm family or the earthworm family, and the like.
このタンパク質溶液を、DEAE-Toyopearl650Mによる陰イオン交換クロマトグラフィー、Sephacryl S-200 ゲルろ過クロマトグラフィー、及びResource Qによるイオンクロマトグラフィーを行うことにより、本発明のプロテアーゼA又はBを含む精製された活性画分を回収することができる。本明細書の実施例に本発明のプロテアーゼの分離及び精製方法の詳細を示したので、当業者は本発明のプロテアーゼを容易に入手することができる。なお、本発明のプロテアーゼはSDS-PAGEにより単一のタンパク質と認められる程度まで精製することができる。本発明のプロテアーゼA又はBには、本明細書の実施例に具体的に記載したシマミミズより分離された天然由来のプロテアーゼのほか、該シマミミズ由来のプロテアーゼのアミノ酸配列において1又は数個のアミノ酸が置換、挿入、及び／又は欠失しており、かつ上記(a)ないし(g)の特徴を有するプロテアーゼが包含される。このようなプロテアーゼは一般的には遺伝子工学的な手法により入手可能であり、あるいはシマミミズ以外の他の動物から分離・精製することができる。 The protein solution is subjected to anion exchange chromatography using DEAE-Toyopearl650M, Sephacryl S-200 gel filtration chromatography, and ion chromatography using Resource Q, thereby purifying the purified active fraction containing protease A or B of the present invention. Minutes can be recovered. Since the details of the method for separating and purifying the protease of the present invention are shown in the Examples of the present specification, those skilled in the art can easily obtain the protease of the present invention. The protease of the present invention can be purified to the extent that it is recognized as a single protein by SDS-PAGE. The protease A or B of the present invention includes one or several amino acids in the amino acid sequence of the protease derived from the earthworm, in addition to the naturally occurring protease isolated from the earthworm specifically described in the examples of the present specification. Proteases that are substituted, inserted, and / or deleted and that have the characteristics (a) to (g) above are included. Such proteases are generally available by genetic engineering techniques, or can be separated and purified from animals other than earthworms.
本発明のプロテアーゼは、分子量が約27,000であり、WO2006/93164に記載されたフラクションIに含まれる活性成分（分子量40,000）、及びフラクションIIに含まれる活性成分（分子量20,000）とは異なる分子量を有する。上記のように精製されたプロテアーゼのN末端のアミノ酸配列を決定したところVal-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Gly（VIAGGXAAIIG）であり、WO2006/93164に記載された部分精製タンパク質（N末端のアミノ酸配列がSVGGSNAXPX）とは異なることが確認された。以上の結果から、本発明のプロテアーゼはWO2006/93164に記載された活性物質とは異なるタンパク質であると結論された。 The protease of the present invention has a molecular weight of about 27,000 and has a different molecular weight from the active ingredient (molecular weight 40,000) contained in fraction I described in WO2006 / 93164 and the active ingredient (molecular weight 20,000) contained in fraction II. . The N-terminal amino acid sequence of the protease purified as described above was determined to be Val-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Gly (VIAGGXAAIIG), described in WO2006 / 93164 It was confirmed that the partially purified protein (N-terminal amino acid sequence was SVGGSNAXPX) was different. From the above results, it was concluded that the protease of the present invention is a protein different from the active substance described in WO2006 / 93164.
本発明のプロテアーゼは、N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成するが、その作用はキモスタチンにより阻害される。また、本発明のプロテアーゼはN-グルタリル-Phe-p-ニトロアニリドも基質として分解してp-ニトロアニリンを生成するが、基質としての反応性は、N-グルタリル-Phe-p-ニトロアニリドよりもN-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドのほうが高い。また、本発明のプロテアーゼは、N-サクシニル-Ala-Ala-Pro-Leu-p-ニトロアニリドに対しては実質的に分解作用を有しない。 The protease of the present invention decomposes using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate to produce p-nitroaniline, but its action is inhibited by chymostatin. In addition, the protease of the present invention also decomposes N-glutaryl-Phe-p-nitroanilide as a substrate to produce p-nitroaniline, but the reactivity as a substrate is higher than that of N-glutaryl-Phe-p-nitroanilide. N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is also higher. Further, the protease of the present invention has substantially no degrading action on N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide.
本発明のプロテアーゼがN-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成する場合の至適pHは約9.5であり、その作用はpH5.0〜10.0の範囲でpH9.5の活性に対して約80％以上に維持される。pH3.0及びpH11.0における活性はpH9.5における活性に対してそれぞれ約40％程度である。上記作用に関して、本発明のプロテアーゼの至適温度は約40℃であり、60℃において40℃における活性の約80%程度が保持され、かつ4℃においては40℃における活性の約60%程度が保持される。40℃〜50℃の間でほぼ最高の活性が維持される。また、熱安定性に関しては、50℃まではほとんど活性を失わず、60℃でも40℃における活性の80％以上が残存する。70℃では20％以上の活性が残存し、80℃ではほとんど失活する。 When the protease of the present invention decomposes using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate to produce p-nitroaniline, the optimum pH is about 9.5, and its action is pH 5. It is maintained above about 80% for pH 9.5 activity in the range of 0 to 10.0. The activity at pH 3.0 and pH 11.0 is about 40% of the activity at pH 9.5. Regarding the above action, the optimum temperature of the protease of the present invention is about 40 ° C., about 80% of the activity at 40 ° C. is maintained at 60 ° C., and about 60% of the activity at 40 ° C. is maintained at 4 ° C. Retained. The highest activity is maintained between 40 ° C and 50 ° C. Regarding thermal stability, almost no activity is lost up to 50 ° C., and even at 60 ° C., 80% or more of the activity at 40 ° C. remains. At 70 ° C, 20% or more of the activity remains, and at 80 ° C it is almost inactivated.
本発明のプロテアーゼBは、分子量が約26,000であり、上記のプロテアーゼBとは異なる分子量を有する。精製されたプロテアーゼBのN末端のアミノ酸配列を決定したところIle-Ile-Gly-Gly-Ser-Asn-Ala-Ser-Pro-Gly-Glu（IIGGSNASPGE）であり、WO2006/93164に記載された部分精製タンパク質（N末端のアミノ酸配列がSVGGSNAXPX）及び上記のプロテアーゼAとは異なることが確認された。一方、このN末端のアミノ酸配列はE. fetida由来のフィブリン分解酵素（EFE-ｄ及びEFE-e：Biotechnology Letters, 25, pp.1105-1109, 2003）とN-末端から11残基で100％の相同性を有していた。以上の結果から、本発明のプロテアーゼBはWO2006/93164に記載された活性物質及び上記のプロテアーゼAとは異なるタンパク質であると結論された。 Protease B of the present invention has a molecular weight of about 26,000 and has a molecular weight different from that of protease B described above. The N-terminal amino acid sequence of the purified protease B was determined to be Ile-Ile-Gly-Gly-Ser-Asn-Ala-Ser-Pro-Gly-Glu (IIGGSNASPGE), a part described in WO2006 / 93164 It was confirmed that it was different from the purified protein (N-terminal amino acid sequence SVGGSNAXPX) and the above-mentioned protease A. On the other hand, this N-terminal amino acid sequence consists of E. fetida-derived fibrin degrading enzymes (EFE-d and EFE-e: Biotechnology Letters, 25, pp. 1105-1109, 2003) and 100% of 11 residues from the N-terminal. It had the homology of. From the above results, it was concluded that protease B of the present invention is a protein different from the active substance described in WO2006 / 93164 and the above-mentioned protease A.
本発明のプロテアーゼBは、N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成するが、その作用はフェニルメチルスフホニルフルオリドにより実質的に完全に阻害される。また、本発明のプロテアーゼBはN-グルタリル-L-Phe-p-ニトロアニリド及びN-α-ベンゾイル-L-Arg-p-ニトロアニリドに対しては実質的に分解作用を有しない。本発明のプロテアーゼBの至適pHは9.5であり、pH6.0〜11.5の範囲でpH9.5における活性に対して約80％以上の活性が維持され、至適温度は60℃である。また、60℃以下でその活性が実質的に維持される。 Protease B of the present invention decomposes using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate to produce p-nitroaniline, but its action is substantially reduced by phenylmethylsulfonyl fluoride. Completely inhibited. In addition, the protease B of the present invention has substantially no degrading action on N-glutaryl-L-Phe-p-nitroanilide and N-α-benzoyl-L-Arg-p-nitroanilide. The optimum pH of protease B of the present invention is 9.5, the activity of about 80% or more is maintained with respect to the activity at pH 9.5 in the range of pH 6.0 to 11.5, and the optimum temperature is 60 ° C. Moreover, the activity is substantially maintained at 60 ° C. or lower.
本発明のプロテアーゼA又はBの抗ウイルス作用は、例えば、WO2006/93164に記載された方法に従って、例えばトマトモザイクウイルス（ToMV）、タバコモザイクウイルス（TMV）、キュウリモザイクウイルス（CMV）などの植物ウイルス、又はヒトインフルエンザA型ウイルスなどの哺乳類動物感染性ウイルスを用いて容易に検定することができる。 The antiviral action of protease A or B of the present invention can be achieved, for example, according to the method described in WO2006 / 93164, for example, plant viruses such as tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV). Or can be easily assayed using a mammalian infectious virus such as human influenza A virus.
本発明のプロテアーゼA又はBの用途は特に限定されないが、抗ウイルス作用を有していることから、例えば植物のほか人畜や魚介類を含めた広い生物種においてウイルスの感染及び増殖を阻害する目的で使用することができ、抗ウイルス剤として医薬又は農薬の形態で用いることができる。 Although the use of protease A or B of the present invention is not particularly limited, it has an antiviral action, so that it is intended to inhibit viral infection and growth in a wide variety of species including plants, humans and fishery products, for example. And can be used in the form of a pharmaceutical or agrochemical as an antiviral agent.
医薬として用いる場合には、1種又は2種以上の製剤用添加物を用いて経口又は非経口投与に適する医薬組成物の形態として調製することができ、例えば、錠剤、丸剤、散剤、顆粒剤、シロップ剤、液剤、懸濁剤、乳剤、顆粒剤、又はカプセル剤などの経口投与に適する医薬組成物、あるいは注射剤、点滴剤、坐剤などの非経口投与に適する医薬組成物として調製することができる。用いられる製剤用添加物としては、賦形剤、崩壊補助剤、安定化剤、防腐剤などを挙げることができるが、これらに限定されることはない。医薬の投与量は特に限定されず、患者の体重、年齢、症状、ウイルス疾患の種類などに応じて適宜決定することができるが、例えば、一日あたり0.001 mg〜1,000 mg程度の範囲で選択することができる。 When used as a medicament, it can be prepared in the form of a pharmaceutical composition suitable for oral or parenteral administration using one or more pharmaceutical additives, for example, tablets, pills, powders, granules Prepared as a pharmaceutical composition suitable for oral administration such as an injection, syrup, solution, suspension, emulsion, granule or capsule, or as a pharmaceutical composition suitable for parenteral administration such as an injection, infusion, suppository, etc. can do. Examples of the additive for formulation used include, but are not limited to, excipients, disintegration aids, stabilizers, preservatives and the like. The dose of the drug is not particularly limited and can be appropriately determined according to the patient's weight, age, symptoms, type of viral disease, etc., for example, selected within the range of about 0.001 mg to 1,000 mg per day be able to.
農薬として用いる場合には、1種又は2種以上の補助剤を用いて乳剤、粉剤、水和剤、液剤、粒剤、懸濁製剤などの種々の形態の農薬用組成物を製剤することができる。これらの農薬用組成物はそのまま使用するか、又は水などの希釈剤で所定濃度に希釈して使用することができる。用いられる補助剤としては、担体、乳化剤、懸濁剤、分散剤、展着剤、浸透剤、湿潤剤、増粘剤、安定剤などが挙げられるが、これらに限定されることはない。農薬の使用濃度又は使用量は特に限定されず、対象作物、使用方法、製剤形態、対象ウイルスなどによって異なるが、茎葉処理の場合には有効成分濃度として0.1〜10,000 ppm程度であり、土壌処理の場合には10〜100,000 g/ha程度であり、適宜増減することができる。 When used as agricultural chemicals, various forms of agricultural chemical compositions such as emulsions, powders, wettable powders, liquids, granules and suspensions can be formulated using one or more adjuvants. it can. These agrochemical compositions can be used as they are, or diluted to a predetermined concentration with a diluent such as water. Adjuvants used include, but are not limited to, carriers, emulsifiers, suspending agents, dispersing agents, spreading agents, penetrating agents, wetting agents, thickening agents, stabilizers and the like. The concentration or amount of pesticide used is not particularly limited and varies depending on the target crop, method of use, formulation, target virus, etc., but in the case of foliage treatment, the active ingredient concentration is about 0.1 to 10,000 ppm, In some cases, it is about 10-100,000 g / ha and can be increased or decreased as appropriate.
以下、本発明を実施例によりさらに具体的に説明するが、本発明の範囲は下記の実施例により限定されることはない。
例１：プロテアーゼA
(a)硫安画分の調製
1日絶食させ凍結乾燥したシマミミズ（E. fetida） 15 gを破砕し、50 mM Tris-HCl 緩衝液(pH 7.0)で懸濁した。懸濁液を遠心分離（27,720 g，20 min，４℃）し、上清をE. fetida粗抽出物として得た。E. fetida粗抽出物に飽和80%になるよう硫安を添加し、よく攪拌した後、一晩冷蔵庫で静置した。遠心分離（27,720 g, 20 min, 4℃）を行い、上清と沈殿に分画し、沈殿画分に少量の50 mM Tris-HCl buffer, pH 7.0を加え、沈殿物を完全に溶解した。
(b)DEAE-TOYOPEARL 650Mによる陰イオン交換クロマトグラフィー
担体を初期バッファー 1 リットルで洗浄、溶出バッファー600 mlで活性化し、スタートバッファー 1リットルで平衡化した。試料5 mlを負荷し、スタートバッファー600 mlで洗浄後、分速1.5 mlで初期バッファー300 mlと溶出バッファー300 mlとを用いて塩のグラジエントによりタンパク質の溶出を行った。溶出した試料は、試験管1本当たり7 mlとして100画分に分画し、各画分のA280における吸光度を測定した。
担体：DEAE-TOYOPEARL 650M（TOSOH CORPORATION）
カラム：直径2.5 cm×25 cm
初期バッファー：20 mM Tris-HCl buffer, pH 7.0
溶出バッファー：1 M NaClを含む20 mM Tris-HCl buffer, pH 7.0
試料：E. fetida粗抽出物の80%飽和飽和硫安塩析後の沈殿物を少量のスタートバッファーに溶解し、20 mM Tris-HCl buffer, pH 7.0で一晩透析した。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by the following examples.
Example 1: Protease A
(a) Preparation of ammonium sulfate fraction
15 g of E. fetida fasted and lyophilized for 1 day was crushed and suspended in 50 mM Tris-HCl buffer (pH 7.0). The suspension was centrifuged (27,720 g, 20 min, 4 ° C.), and the supernatant was obtained as an E. fetida crude extract. Ammonium sulfate was added to the E. fetida crude extract so as to reach 80% saturation, and after stirring well, the mixture was allowed to stand overnight in a refrigerator. Centrifugation (27,720 g, 20 min, 4 ° C.) was performed to fractionate the supernatant and the precipitate, and a small amount of 50 mM Tris-HCl buffer, pH 7.0 was added to the precipitate fraction to completely dissolve the precipitate.
(b) Anion exchange chromatography with DEAE-TOYOPEARL 650M The support was washed with 1 liter of initial buffer, activated with 600 ml of elution buffer, and equilibrated with 1 liter of start buffer. After loading 5 ml of the sample and washing with 600 ml of the start buffer, the protein was eluted with a salt gradient using 300 ml of the initial buffer and 300 ml of the elution buffer at a speed of 1.5 ml per minute. The eluted sample was fractionated into 100 fractions of 7 ml per test tube, and the absorbance at A 280 of each fraction was measured.
Carrier: DEAE-TOYOPEARL 650M (TOSOH CORPORATION)
Column: Diameter 2.5 cm x 25 cm
Initial buffer: 20 mM Tris-HCl buffer, pH 7.0
Elution buffer: 20 mM Tris-HCl buffer with 1 M NaCl, pH 7.0
Sample: The precipitate after 80% saturated saturated ammonium sulfate salting out of E. fetida crude extract was dissolved in a small amount of start buffer and dialyzed overnight against 20 mM Tris-HCl buffer, pH 7.0.
(b)Sephacyl S-200によるゲルろ過クロマトグラフィー
バッファー 2 リットルで平衡化したカラムに試料を注入し、分速0.5 mlでバッファーを流して溶出を行った。溶出した試料は、試験管1本当たり5 mlとして100画分に分画し、各画分のA280における吸光度を測定した。
担体：Sephacryl(登録商標) S-200 High Resolution（GEヘルスケアバイオサイエンス）
カラム：直径2.5 cm×100 cm
バッファー：20 mM Tris-HCl buffer＋0.2 M NaCl, pH 7.0
サンプル：DEAE-TOYOPEARL 650Mによる画分のうち、CMV pepo阻害活性のあったfractionI-IIIを80％飽和硫安塩析し、少量のバッファーに溶解した。
(b) Gel filtration chromatography using Sephacyl S-200 The sample was injected into a column equilibrated with 2 liters of buffer, and elution was performed by flowing the buffer at a rate of 0.5 ml per minute. The eluted sample was fractionated into 100 fractions as 5 ml per test tube, and the absorbance at A 280 of each fraction was measured.
Carrier: Sephacryl (registered trademark) S-200 High Resolution (GE Healthcare Bioscience)
Column: Diameter 2.5 cm x 100 cm
Buffer: 20 mM Tris-HCl buffer + 0.2 M NaCl, pH 7.0
Sample: Among fractions obtained by DEAE-TOYOPEARL 650M, fraction I-III having CMV pepo inhibitory activity was precipitated with 80% saturated ammonium sulfate and dissolved in a small amount of buffer.
CMV pepo阻害作用検定は以下の方法により行なった。検定ウイルスとしてキュウリモザイクウイルス（Cucumber mosaic virus：CMV）pepo系統を用いた。Bromoviridae科 Cucumovirus属に属し、直径28〜30nmの粒子形態をとる。CMVは40科191種以上の植物に感染し、植物ウイルスの中でもっとも広い宿主範囲を持つウイルスの一つである（Palukaitis and Arenal, 2003）。CMV pepo系統は1973年に尾崎らが和歌山県で採種した、モザイク症状を示すペポカボチャより分離したサテライトRNAを含まない病原性の強い分離株である（関西病中研報, 15, pp.135-136, 1973）。この株はラグナリア系統群のサブグループIに属する。 The CMV pepo inhibitory effect assay was performed by the following method. A cucumber mosaic virus (CMV) pepo strain was used as the assay virus. Bromoviridae belongs to the genus Cucumovirus and takes the form of particles with a diameter of 28-30 nm. CMV infects more than 191 plants in 40 families and is one of the broadest host range of plant viruses (Palukaitis and Arenal, 2003). The CMV pepo strain is a highly virulent isolate that does not contain satellite RNA isolated from pepo pumpkin with mosaic symptoms, which was cultivated in Wakayama Prefecture by Ozaki et al. In 1973 (Kansai Disease Research Institute, 15, pp.135-136). , 1973). This strain belongs to subgroup I of the Lagunaria family.
検定植物としてはジュウロクササゲ（Vigna unguiculate cv. Kurodane-sanjaku）を用いた。バーミキュライトを満たした鉢にササゲの種子を播種し温室内に置き、発芽した苗をセルトレイに移植し育苗した。実験には、子葉が最大に展開したものを直径9 cmのプラスチックポットに移植し用いた。
検定方法は、都丸の方法によった（都丸, 農薬実験法 2 殺菌剤編、深見順一ほか編, ソフトサイエンス社, pp.83-115, 1981）。検定には、CMV pepoと局部病斑宿主であるササゲを用いた。試料は、ウイルス液と等量になるよう混合した。対照のウイルス液も試料の溶媒のみで同様に処理した。最大に展開したササゲの子葉に、研磨剤としてカーボランダムを均一にふりかけ、子葉片方に試料とウイルス液の混合液を、他方には試料の溶媒とウイルス液の混合液を塗布した。接種より２日後、試料葉及び対照葉に形成された壊死斑の数を測定し、CMV pepo感染阻害性を検定した。感染阻害率を次のように算出した。感染阻害率(%)=(1−処理区病斑数／対照区病斑数)×100
As a test plant, Vigna unguiculate cv. Kurodane-sanjaku was used. Cowpea seeds were sown in a pot filled with vermiculite, placed in a greenhouse, and the germinated seedlings were transplanted to cell trays to grow seedlings. In the experiment, the cotyledon with the largest expansion was transplanted into a plastic pot with a diameter of 9 cm.
The test method was based on the method of Tomaru (Tomaru, Agricultural Chemical Experiment 2 Disinfectant, Junichi Fukami et al., Soft Science, pp.83-115, 1981). The test used CMV pepo and cowpea, a local lesion host. The sample was mixed with the virus solution in an equal volume. The control virus solution was similarly treated with the sample solvent alone. Carborundum was uniformly sprinkled as a polishing agent on the cotyledon of the cowpea that had been developed to the maximum, and a mixed solution of the sample and virus solution was applied to one of the cotyledons, and a mixed solution of the sample solvent and virus solution was applied to the other. Two days after the inoculation, the number of necrotic spots formed in the sample leaves and the control leaves was measured, and CMV pepo infection inhibitory activity was assayed. The infection inhibition rate was calculated as follows. Infection inhibition rate (%) = (1-number of lesions in treatment group / number of lesions in control group) x 100
(c)Resource Qによるイオン交換クロマトグラフィー
カラムをスタートバッファー30 mlで洗浄後、溶出バッファー30 mlで活性化し、スタートバッファー60 mlで平衡化した。試料を負荷し、スタートバッファー60 mlで洗浄した後、分速2.5 mlで1 M NaClの直線勾配がかかるように、初期バッファー 15 mlと溶出バッファー 15 mlを流した。溶出した試料は、試験管1本当たり0.3 mlとして60画分に分画し、各画分のA280における吸光度を測定した。
カラム：Resouce Q, 6 ml （GEヘルスケアバイオサイエンス）
初期バッファー：20 mM Tris-HCl buffer, pH 7.0
溶出バッファー：1 M NaClを含む20 mM Tris-HCl buffer, pH 7.0
サンプル：Sephaceyl S-200 fraction による画分のうち、CMV pepo阻害活性を示したfractionIIIを、20 mM Tris-HCl buffer, pH 7.0で一晩透析した。
(c) Ion exchange chromatography using Resource Q The column was washed with 30 ml of start buffer, then activated with 30 ml of elution buffer, and equilibrated with 60 ml of start buffer. After loading the sample and washing with 60 ml of start buffer, 15 ml of initial buffer and 15 ml of elution buffer were flowed so that a linear gradient of 1 M NaCl was applied at a speed of 2.5 ml per minute. The eluted sample was fractionated into 60 fractions as 0.3 ml per test tube, and the absorbance at A 280 of each fraction was measured.
Column: Resouce Q, 6 ml (GE Healthcare Bioscience)
Initial buffer: 20 mM Tris-HCl buffer, pH 7.0
Elution buffer: 20 mM Tris-HCl buffer with 1 M NaCl, pH 7.0
Sample: Among the fractions by Sephaceyl S-200 fraction, fraction III showing CMV pepo inhibitory activity was dialyzed overnight against 20 mM Tris-HCl buffer, pH 7.0.
(d)阻害活性画分のタンパク質定量
タンパク質定量をBradford法（Bradford, Anal. Biochem., 72, pp.248-254, 1976）により行った。スタンダードとして牛血清アルブミン（和光純薬工業株式会社）を用いた。結果を表１に示す。
Resource Qにより得られたフラクションII(図１C)をドデシル硫酸ナトリウム(SDS)-ポリアクリルアミドゲル電気泳動法により分析した。結果を図２に示す。電気泳動的に単一のバンドを得られ、分子量は約27,000であると推定された。
分子量マーカーには、低分子量マーカーである以下のものを使用した。
ホスホリラーゼb（分子量：94,000）
牛血清アルブミン（分子量：67,000）
卵白アルブミン（分子量：43,000）
カルボニックアンヒドラーゼ（分子量：30,000）
大豆トリプシンインヒビター（分子量：20,100）
α-ラクトアルブミン（分子量：14,400）
(d) Protein quantification of the inhibitory activity fraction Protein quantification was performed by the Bradford method (Bradford, Anal. Biochem., 72, pp. 248-254, 1976). Bovine serum albumin (Wako Pure Chemical Industries, Ltd.) was used as a standard. The results are shown in Table 1.
Fraction II (FIG. 1C) obtained by Resource Q was analyzed by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. The results are shown in FIG. A single band was obtained electrophoretically and the molecular weight was estimated to be about 27,000.
As the molecular weight marker, the following low molecular weight markers were used.
Phosphorylase b (molecular weight: 94,000)
Bovine serum albumin (molecular weight: 67,000)
Ovalbumin (molecular weight: 43,000)
Carbonic anhydrase (molecular weight: 30,000)
Soybean trypsin inhibitor (molecular weight: 20,100)
α-Lactalbumin (Molecular weight: 14,400)
(f)各画分のCMV pepo感染阻害作用検定
各クロマトグラフィーによる分画後、A280において吸収ピークが見られた画分において、CMV pepo感染阻害作用検定を行った。検定の際の各画分は、最終濃度0.05 mg/mlとなるよう調製した。結果を表２に示す。
(g)N末端アミノ酸配列解析
精製したタンパク質をSDS-PAGEに供し、PVDF膜に転写した。膜を水洗し、0.25% Coomassie Brilliant Blue R-250, 5%メタノール溶液で染色し、その後、５分間50% methanol溶液で洗浄した。目的のタンパク質を含むバンドを切り出し、アミノ酸シークエンサーにてアミノ末端からのアミノ酸配列を決定した（Applied Biosystems Model 491 Prociseシリーズ）。その結果、このタンパク質のN末端からのアミノ酸配列はVal-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Glyであった。その結果を基にして、National Center for Biotechnology Information（NCBI）のBLAST（http://www.ncbi.nih.gov）を用いて既にアミノ酸配列が報告されているタンパク質との相同性の比較を行ったところ、Lumbricus rubellus由来のフィブリン分解酵素と相同性が認められた（表３）。
(g) N-terminal amino acid sequence analysis The purified protein was subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was washed with water, stained with 0.25% Coomassie Brilliant Blue R-250, 5% methanol solution, and then washed with 50% methanol solution for 5 minutes. A band containing the target protein was cut out, and the amino acid sequence from the amino terminus was determined with an amino acid sequencer (Applied Biosystems Model 491 Procise series). As a result, the amino acid sequence from the N-terminus of this protein was Val-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Gly. Based on the results, homology was compared with proteins whose amino acid sequences had already been reported using BLAST (http://www.ncbi.nih.gov) of the National Center for Biotechnology Information (NCBI). As a result, homology with the fibrinolytic enzyme derived from Lumbricus rubellus was recognized (Table 3).
(h)基質特異性
2.5 mMの合成基質溶液（各合成基質を、4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）0.06 mlを、37℃で５分間保温した後、上記精製タンパク質（以下、プロテアーゼAと呼ぶ）を0.06 ml加え、37℃、30分間反応を行い、分光光度計を用いて405 nmにおける吸光度を測定して、遊離したp-ニトロアニリン量を定量した。酵素1単位（U）は、上記の反応条件において1分間に1 μmolのp-ニトロアニリンを遊離させるのに必要な酵素量とした。結果を表４に示す。この結果、本発明のプロテアーゼAはN-サクシニル-Ala-Ala-Pro-Phe p-ニトロアニリド及びN-グルタリル-L-Phe-p-ニトロアニリドに対して高い分解活性を有していたが、N-サクシニル-Ala-Ala-Pro-Leu-p-ニトロアニリドに対しては全く分解活性を示さなかった。
(h) Substrate specificity
After incubating 0.06 ml of 2.5 mM synthetic substrate solution (each synthetic substrate dissolved in 0.1 M glycine-NaOH buffer containing 4% DMSO, pH 9.5) at 37 ° C. for 5 minutes, the purified protein (hereinafter referred to as the purified protein) 0.06 ml) was added, and the mixture was reacted at 37 ° C. for 30 minutes. The absorbance at 405 nm was measured using a spectrophotometer, and the amount of p-nitroaniline released was quantified. One unit (U) of enzyme was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline per minute under the above reaction conditions. The results are shown in Table 4. As a result, protease A of the present invention had a high degrading activity against N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and N-glutaryl-L-Phe-p-nitroanilide. N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide showed no degradation activity.
本発明のプロテアーゼA 0.06 mlと各種阻害剤を最終濃度が以下に記す濃度になるように0.06 ml加えて混合し、37℃、30分間保温した後、基質として5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH9.5に溶解したもの）を0.06 ml加え、37℃、30分間反応を行った後に酵素活性を測定した。結果を表５に示す。下記に使用した阻害剤と各濃度を示した。この結果、本発明のプロテアーゼA の活性はキモスタチンにより強く阻害されたが、それ以外の阻害剤ではほとんど活性の低下は認められなかった。
1 mM ε-Aminocapronic acid(e-ACA)
1 mM Phenylmethylsulfonyl fluoride(PMSF)
1 mM 2,2'-Bipyridyl
0.01 mg/ml Chymostatin
1 mM Phenantorolin
1 mM EDTA
0.1 mM E-64
1 mM ε-Aminocapronic acid (e-ACA)
1 mM Phenylmethylsulfonyl fluoride (PMSF)
1 mM 2,2'-Bipyridyl
0.01 mg / ml Chymostatin
1 mM Phenantorolin
1 mM EDTA
0.1 mM E-64
(j)最適pHおよびpH安定性
基質として2.5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液と4% DMSOとを含むpH 2.5〜11.5の種々の緩衝液0.06 mlを37℃で5分間保温した後、本発明のプロテアーゼAを0.06 ml加え、37℃で30分間反応を行った。反応後、酵素活性を測定した。使用した各種緩衝液及びそのpH範囲は次のとおりである。
pH 2.5〜3.5 0.1 M グリシン-HCl 緩衝液
pH 3.5〜6.0 0.1 M 酢酸緩衝液
pH 6.0〜8.0 0.1 M リン酸緩衝液
pH 8.0〜9.0 0.1 M Tris-HCl 緩衝液
pH 9.0〜11.5 0.1 M グリシン-NaOH 緩衝液
(j) Optimum pH and pH stability 37 0.06 ml of various buffers at pH 2.5-11.5 containing 2.5 mM N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide solution and 4% DMSO as substrates After incubating at 5 ° C. for 5 minutes, 0.06 ml of protease A of the present invention was added, and the reaction was carried out at 37 ° C. for 30 minutes. After the reaction, enzyme activity was measured. The various buffers used and their pH ranges are as follows.
pH 2.5-3.5 0.1 M glycine-HCl buffer
pH 3.5-6.0 0.1 M acetate buffer
pH 6.0-8.0 0.1 M phosphate buffer
pH 8.0-9.0 0.1 M Tris-HCl buffer
pH 9.0-11.5 0.1 M glycine-NaOH buffer
pH 3.0〜11.5の種々の緩衝液0.03 mlと本発明のプロテアーゼA 0.03 mlとを混合し、37℃で60分間反応させた後、2.5 mMのN-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）を加え、37℃で30分反応を行い、酵素活性に及ぼすpHの影響を調べた。使用した各種緩衝液及びそのpH範囲は次のとおりである。
pH 3.0〜6.0 0.1 M酢酸緩衝液
pH 6.0〜8.0 0.1 M リン酸緩衝液
pH 8.0〜9.0 0.1 M Tris-HCl 緩衝液
pH 9.0〜11.5 0.1 M グリシン-NaOH 緩衝液
After mixing 0.03 ml of various buffers of pH 3.0 to 11.5 and 0.03 ml of protease A of the present invention and reacting at 37 ° C. for 60 minutes, 2.5 mM N-succinyl-Ala-Ala-Pro-Phe-p -Nitroanilide solution (1 M glycine-NaOH buffer containing 4% DMSO, dissolved in pH 9.5) was added and reacted at 37 ° C for 30 minutes to examine the effect of pH on enzyme activity. The various buffers used and their pH ranges are as follows.
pH 3.0-6.0 0.1 M acetate buffer
pH 6.0-8.0 0.1 M phosphate buffer
pH 8.0-9.0 0.1 M Tris-HCl buffer
pH 9.0-11.5 0.1 M glycine-NaOH buffer
(k)最適温度および熱安定性
基質として2.5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）0.06 mlを、0〜80℃で5分間保温した後、本発明のプロテアーゼA液を0.06 ml加え、各種温度で30分間反応を行った後に酵素活性を測定した。また、本発明のプロテアーゼA 0.06 mlを20〜80℃の各種温度で60分間反応させた後、基質として2.5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）0.06 mlを加え、37℃、30分間反応を行った後、酵素活性を測定した。本発明のプロテアーゼAの最適温度は40℃であり、pH 9.5で60℃までの加熱による活性の低下は認められなかった。
(k) Optimal temperature and thermal stability 2.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide solution as substrate (0.1 M glycine-NaOH buffer containing 4% DMSO, pH 9.5) ) After incubating 0.06 ml at 0 to 80 ° C. for 5 minutes, 0.06 ml of the protease A solution of the present invention was added and reacted at various temperatures for 30 minutes, and then enzyme activity was measured. Further, after reacting 0.06 ml of the protease A of the present invention at various temperatures of 20 to 80 ° C. for 60 minutes, a 2.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide solution (4% DMSO) was used as a substrate. 0.06 ml dissolved in 0.1 M glycine-NaOH buffer solution, pH 9.5) was added and reacted at 37 ° C. for 30 minutes, and then the enzyme activity was measured. The optimum temperature of protease A of the present invention was 40 ° C., and no decrease in activity due to heating up to 60 ° C. at pH 9.5 was observed.
(l)プロテアーゼAの活性測定
基質として2.5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）0.06 mlを、37℃、5分間保温した後、各分画液を0.06 ml加え、各種温度で30分間反応を行った後、酵素活性を測定した。本発明のプロテアーゼAの収量は0.4 %であり、乾燥重量 15 g（全タンパク質量：2300 mg）のE. fetidaから、最終0.5 mgの目的タンパク質を回収した。
(l) Activity measurement of protease A 2.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide solution as a substrate (dissolved in 0.1 M glycine-NaOH buffer containing 4% DMSO, pH 9.5) After incubating 0.06 ml at 37 ° C. for 5 minutes, 0.06 ml of each fraction was added, reacted at various temperatures for 30 minutes, and then enzyme activity was measured. The yield of protease A of the present invention was 0.4%, and a final protein of 0.5 mg was recovered from E. fetida having a dry weight of 15 g (total protein amount: 2300 mg).
(m)植物ウイルス感染阻害作用
本発明のプロテアーゼAのCMV pepoに対する有効阻止濃度を、ササゲにおける混合接種法により調べた。CMV pepo 10 μg/mlに対し本発明のプロテアーゼAを1 〜100 μg/mlに調製した。本発明のプロテアーゼAが10 μg/mlのCMV pepoに対し80％以上の感染阻害率を示す濃度は30 μg/ml以上であった（図４）。
(m) Plant virus infection inhibitory action The effective inhibitory concentration of protease A of the present invention against CMV pepo was examined by the mixed inoculation method in cowpea. Protease A of the present invention was prepared at 1 to 100 μg / ml with respect to 10 μg / ml of CMV pepo. The concentration at which the protease A of the present invention showed an infection inhibition rate of 80% or more for 10 μg / ml CMV pepo was 30 μg / ml or more (FIG. 4).
種々のプロテアーゼのCMV pepo感染抑制作用をササゲを用いた混合接種法により調べて、本発明のプロテアーゼAの作用と比較した。各合成酵素の検定濃度は、CMV pepo 10 μg/mlに対し、0.1 mg/mlで行った。結果を表７に示す（表中、Enzymeは本発明のプロテアーゼAを示す）。
(n)ToMV阻害作用の検定
本発明のプロテアーゼAのTMVに対する影響をタバコを用いた半葉法により調べた。検定ウイルスとしてトマトモザイクウイルス（Tomato mosaic virus ：ToMV）を用いた。このウイルスは、従前はTobacco mosaic virus（TMV）のトマト系統と呼ばれていたウイルスであり、TMVとほぼ同様の性質をもつ。検定植物としてはタバコ（Nicotiana tabacum cv. Xanthi nc）にN遺伝子を導入したものを用いた。このタバコはTMV（ToMV含む）の感染に対して過敏感反応を示し、感染部位に局部壊死斑を示す。
(n) Assay for ToMV Inhibitory Effect The effect of protease A of the present invention on TMV was examined by the half-leaf method using tobacco. Tomato mosaic virus (ToMV) was used as a test virus. This virus used to be a tomato line of Tobacco mosaic virus (TMV) and has almost the same properties as TMV. As the test plant, tobacco (Nicotiana tabacum cv. Xanthi nc) into which the N gene was introduced was used. This cigarette shows a hypersensitive response to TMV (including ToMV) infection and shows local necrotic spots at the site of infection.
試料区用の試料として、最終濃度が本発明のプロテアーゼA 0.1 mg/ml、ToMV 50 μg/mlとなるように本発明のプロテアーゼA溶液とToMV液とを等量混合した。一方、対照区には溶媒（20 mM Tri-HC buffer, pH 7.0）とToMV液（最終濃度50 μg/ml）を等量混合したものを用いた。これらの試料をボルテックスでよく懸濁した後、4℃で30分間静置した。5〜6葉期に生長したN. tabacum cv. Xanthi ncの最大展開葉にカーボランダムをふりかけ、最大展開葉の半葉を処理区、他方を対照区としてそれぞれに本酵素とToMVとの混合液、溶媒とToMVとの混合液を接種した。2〜3日後に形成された壊死斑数を計測し、感染阻害率を以下のとおりに算出した。感染阻害率（%）=(1−処理区病斑数／対照区病斑数)×100 本発明のプロテアーゼAのToMVに対する感染阻害率は85%であった。
例２：プロテアーゼB
(a)硫安画分の調製
1日絶食させ凍結乾燥したシマミミズ（E. fetida） 15 gを破砕し、50 mM Tris-HCl 緩衝液(pH 7.0)で懸濁した。懸濁液を遠心分離（27,720 g，20 min，４℃）し、上清をE. fetida粗抽出物として得た。E. fetida粗抽出物に飽和80%になるよう硫安を添加し、よく攪拌した後、一晩冷蔵庫で静置した。遠心分離（27,720 g, 20 min, 4℃）を行い、上清と沈殿に分画し、沈殿画分に少量の50 mM Tris-HCl buffer, pH 7.0を加え、沈殿物を完全に溶解した。
(b)DEAE-TOYOPEARL 650Mによる陰イオン交換クロマトグラフィー
担体を初期バッファー 1 リットルで洗浄、溶出バッファー600 mlで活性化し、スタートバッファー 1リットルで平衡化した。試料5 mlを負荷し、スタートバッファー600 mlで洗浄後、分速1.5 mlで初期バッファー300 mlと溶出バッファー300 mlとを用いて塩のグラジエントによりタンパク質の溶出を行った。溶出した試料は、試験管1本当たり7 mlとして100画分に分画し、各画分のA280における吸光度を測定した。
担体：DEAE-TOYOPEARL 650M（TOSOH CORPORATION）
カラム：直径2.5 cm×25 cm
初期バッファー：20 mM Tris-HCl buffer, pH 7.0
溶出バッファー：1 M NaClを含む20 mM Tris-HCl buffer, pH 7.0
試料：E. fetida粗抽出物の80%飽和飽和硫安塩析後の沈殿物を少量のスタートバッファーに溶解し、20 mM Tris-HCl buffer, pH 7.0で一晩透析した。
As samples for the sample section, equal amounts of the protease A solution of the present invention and the ToMV solution were mixed so that the final concentrations were 0.1 mg / ml of protease A of the present invention and 50 μg / ml of ToMV. On the other hand, the control group was a mixture of an equal amount of a solvent (20 mM Tri-HC buffer, pH 7.0) and a ToMV solution (final concentration 50 μg / ml). These samples were well suspended by vortexing and then allowed to stand at 4 ° C. for 30 minutes. Sprinkle carborundum on the largest unfolded leaves of N. tabacum cv. Xanthi nc grown in the 5th to 6th leaf stages. Inoculated with a mixture of solvent and ToMV. The number of necrotic plaques formed after 2 to 3 days was counted, and the infection inhibition rate was calculated as follows. Infection inhibition rate (%) = (1−number of lesions in the treatment group / number of lesions in the control group) × 100 The infection inhibition rate of the protease A of the present invention against ToMV was 85%.
Example 2: Protease B
(a) Preparation of ammonium sulfate fraction
15 g of E. fetida fasted and lyophilized for 1 day was crushed and suspended in 50 mM Tris-HCl buffer (pH 7.0). The suspension was centrifuged (27,720 g, 20 min, 4 ° C.), and the supernatant was obtained as an E. fetida crude extract. Ammonium sulfate was added to the E. fetida crude extract so as to reach 80% saturation, and after stirring well, the mixture was allowed to stand overnight in a refrigerator. Centrifugation (27,720 g, 20 min, 4 ° C.) was performed to fractionate the supernatant and the precipitate, and a small amount of 50 mM Tris-HCl buffer, pH 7.0 was added to the precipitate fraction to completely dissolve the precipitate.
(b) Anion exchange chromatography with DEAE-TOYOPEARL 650M The support was washed with 1 liter of initial buffer, activated with 600 ml of elution buffer, and equilibrated with 1 liter of start buffer. After loading 5 ml of the sample and washing with 600 ml of the start buffer, the protein was eluted with a salt gradient using 300 ml of the initial buffer and 300 ml of the elution buffer at a speed of 1.5 ml per minute. The eluted sample was fractionated into 100 fractions of 7 ml per test tube, and the absorbance at A 280 of each fraction was measured.
Carrier: DEAE-TOYOPEARL 650M (TOSOH CORPORATION)
Column: Diameter 2.5 cm x 25 cm
Initial buffer: 20 mM Tris-HCl buffer, pH 7.0
Elution buffer: 20 mM Tris-HCl buffer with 1 M NaCl, pH 7.0
Sample: The precipitate after 80% saturated saturated ammonium sulfate salting out of E. fetida crude extract was dissolved in a small amount of start buffer and dialyzed overnight against 20 mM Tris-HCl buffer, pH 7.0.
(b)Sephacyl S-200によるゲルろ過クロマトグラフィー
蒸留水1リットルで洗浄後、溶出バッファー 1 リットルで平衡化したカラムに試料25mlを負荷し、分速0.5 mlでバッファーを流して溶出を行った。溶出した試料は、試験管1本当たり5 mlとして100画分に分画し、各画分のA280における吸光度を測定した。A280のピーク部分とその周辺における感染阻害率を混合接種法により測定し、効果のあった画分についてプロテアーゼ活性を測定した。
担体：Sephacryl(登録商標) S-200 High Resolution（GEヘルスケアバイオサイエンス）
カラム：直径2.5 cm×91cm
バッファー：20 mM Tris-HCl buffer＋0.2 M NaCl, pH 7.0
サンプル：DEAE-TOYOPEARL 650Mによる画分のうち、fraction No.32〜46
(b) Gel filtration chromatography using Sephacyl S-200 After washing with 1 liter of distilled water, 25 ml of the sample was loaded onto a column equilibrated with 1 liter of elution buffer, and the buffer was run at 0.5 ml / min for elution. The eluted sample was fractionated into 100 fractions as 5 ml per test tube, and the absorbance at A 280 of each fraction was measured. The inhibition rate of infection at the peak portion of A 280 and the vicinity thereof was measured by the mixed inoculation method, and the protease activity was measured for the effective fraction.
Carrier: Sephacryl (registered trademark) S-200 High Resolution (GE Healthcare Bioscience)
Column: Diameter 2.5 cm x 91 cm
Buffer: 20 mM Tris-HCl buffer + 0.2 M NaCl, pH 7.0
Sample: Fraction No. 32-46 out of fractions by DEAE-TOYOPEARL 650M
プロテアーゼ活性測定は以下の方法で行った。サンプル50 μlと、基質であるハマルステンカゼインの終濃度が1%となるように50 mM Borate-NaOH buffer(pH 10.0)を加えて調整を行った(表８)。37℃の恒温槽で10分間反応させた後、0.44 Mのトリクロロ酢酸を1.0 ml加えて反応を停止させた。コントロールには反応停止後サンプルを50 μl添加した。その後、再び37℃の恒温槽で20分間静置して、それぞれの濾液1.0 mlに0.44 M の炭酸ナトリウム2.5 mlと1.0 Nのフェノール試薬0.5 mlを加えて発色させた。37℃の恒温槽で20分間反応させた後、660 nmでの吸光度をBECKMAN DU Series 7400を用いて測定した。1 Uは1分間に1 μmolのTyrosineを遊離する酵素量と定義し、20〜100 μg/mlのTyrosine標準溶液から作成した標準曲線から遊離量を求めた。
(c)Resource Qによるイオン交換クロマトグラフィー
カラムを30 mlのmilliQで洗浄後、初期バッファー30 mlで平衡化し、試料1 mlを負荷して初期バッファー 15 mlと溶出バッファー 15 mlの間でNaClグラジエントを形成してタンパク質を溶出した。溶出した試料は、試験管1本当たり0.5 mlとして60画分に分画し、各画分のA280における吸光度を測定した。A280のピーク部分とその周辺における感染阻害率を混合接種法により検定し、効果のあった画分についてプロテアーゼ活性を測定した。
カラム：Resouce Q, 6 ml （GEヘルスケアバイオサイエンス）
初期バッファー：20 mM Tris-HCl buffer, pH 7.0
溶出バッファー：0.5 M NaClを含む20 mM Tris-HCl buffer, pH 7.0
サンプル：Sephaceyl S-200 fraction によるFraction No. 55〜57を初期バッファーで一晩透析したもの
(c) Ion exchange chromatography with Resource Q After washing the column with 30 ml of milliQ, equilibrate with 30 ml of initial buffer, load 1 ml of sample and change the NaCl gradient between 15 ml of initial buffer and 15 ml of elution buffer. Formed and eluted the protein. The eluted sample was fractionated into 60 fractions as 0.5 ml per test tube, and the absorbance at A 280 of each fraction was measured. The inhibition rate of infection in the peak portion of A 280 and the vicinity thereof was tested by the mixed inoculation method, and the protease activity was measured for the effective fraction.
Column: Resouce Q, 6 ml (GE Healthcare Bioscience)
Initial buffer: 20 mM Tris-HCl buffer, pH 7.0
Elution buffer: 20 mM Tris-HCl buffer with 0.5 M NaCl, pH 7.0
Sample: Fraction No. 55-57 with Sephaceyl S-200 fraction dialyzed overnight against initial buffer
(d)阻害活性画分のタンパク質定量
タンパク質定量をBradford法（Bradford, Anal. Biochem., 72, pp.248-254, 1976）により行った。スタンダードとして牛血清アルブミン（和光純薬工業株式会社）を用いた。結果を表９に示す。
Resource Qにより得られたフラクションNo.27をSDS-ポリアクリルアミドゲル電気泳動法により分析した。アクリルアミド濃度は分離ゲルを15%、濃縮ゲルを3%として、20mAを負荷して電気泳動を行った。結果を図６に示す。電気泳動的に単一のバンドを得られ、分子量は約26,000であると推定された。この物質の分子量は例１で得られたプロテアーゼAに類似しているが、ResourceQにおける溶出パターンが若干異なるものであった。
分子量マーカーには、低分子量マーカーである以下のものを使用した。
ホスホリラーゼb（分子量：94,000）
牛血清アルブミン（分子量：67,000）
卵白アルブミン（分子量：43,000）
カルボニックアンヒドラーゼ（分子量：30,000）
大豆トリプシンインヒビター（分子量：20,100）
α-ラクトアルブミン（分子量：14,400）
(d) Protein quantification of the inhibitory activity fraction Protein quantification was performed by the Bradford method (Bradford, Anal. Biochem., 72, pp. 248-254, 1976). Bovine serum albumin (Wako Pure Chemical Industries, Ltd.) was used as a standard. The results are shown in Table 9.
Fraction No. 27 obtained by Resource Q was analyzed by SDS-polyacrylamide gel electrophoresis. The acrylamide concentration was 15% for the separation gel and 3% for the concentration gel, and electrophoresis was performed with 20 mA applied. The results are shown in FIG. A single band was obtained electrophoretically and the molecular weight was estimated to be about 26,000. The molecular weight of this substance is similar to protease A obtained in Example 1, but the elution pattern in ResourceQ is slightly different.
As the molecular weight marker, the following low molecular weight markers were used.
Phosphorylase b (molecular weight: 94,000)
Bovine serum albumin (molecular weight: 67,000)
Ovalbumin (molecular weight: 43,000)
Carbonic anhydrase (molecular weight: 30,000)
Soybean trypsin inhibitor (molecular weight: 20,100)
α-Lactalbumin (Molecular weight: 14,400)
(f)各画分のCMV pepo感染阻害作用検定
各クロマトグラフィーによる分画後、A280において吸収ピークが見られた画分において、CMV pepo感染阻害作用検定を行った。検定の際の各画分は、最終濃度0.05 mg/mlとなるよう調製した。結果を表２に示す。
(g)N末端アミノ酸配列解析
精製した上記タンパク質（以下、プロテアーゼBと呼ぶ）をSDS-PAGEに供した後、分離ゲルをPVDF膜に転写した。膜をmilliQで洗浄し、0.25% Coomassie Brilliant Blue R-250で染色した後 50%メタノール溶液で脱色して目的のバンドを確認した。その後再びmilliQで洗浄し、目的のタンパク質を含むバンドを切り出し、アミノ酸シークエンサーにてアミノ末端からのアミノ酸配列を決定した（Applied Biosystems Model 491 Prociseシリーズ）。その結果を元に既にアミノ酸配列が報告されているタンパク質との相同性の比較を、National Center for Biotechnology Information（NCBI）のBLAST（http://www.ncbi.nih.gov）を用いて行った。この結果、プロテアーゼBは既知のE. fetida由来のフィブリン分解酵素（EFE-ｄ及びEFE-e：Biotechnology Letters, 25, pp.1105-1109, 2003）とN-末端から11残基で100％の相同性を有していたが、例1で得られたプロテアーゼAとはほとんど相同性が認められず、別のタンパク質であると結論された。結果を表11に示す。表中、EFE-a〜EFE-eはEisenia fetida由来のフィブリン分解酵素である。
(g) N-terminal amino acid sequence analysis After the purified protein (hereinafter referred to as protease B) was subjected to SDS-PAGE, the separation gel was transferred to a PVDF membrane. The membrane was washed with milliQ, stained with 0.25% Coomassie Brilliant Blue R-250, and decolorized with a 50% methanol solution to confirm the target band. Thereafter, it was washed again with milliQ, a band containing the target protein was cut out, and the amino acid sequence from the amino terminus was determined with an amino acid sequencer (Applied Biosystems Model 491 Procise series). Based on the results, comparison of homology with proteins whose amino acid sequences have already been reported was performed using BLAST ( http://www.ncbi.nih.gov ) of the National Center for Biotechnology Information (NCBI). . As a result, protease B is a known fibrinolytic enzyme derived from E. fetida (EFE-d and EFE-e: Biotechnology Letters, 25, pp. 1105-1109, 2003) and 100% of 11 residues from the N-terminus. Although it had homology, it was concluded that it was a different protein with little homology with protease A obtained in Example 1. The results are shown in Table 11. In the table, EFE-a to EFE-e are fibrin degrading enzymes derived from Eisenia fetida.
(h)基質特異性
2.5 mMの合成基質溶液（各合成基質を、4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH 9.5に溶解したもの）0.06 mlを、37℃で５分間保温した後、本発明のプロテアーゼBを0.06 ml加え、37℃、30分間反応を行い、分光光度計を用いて405 nmにおける吸光度を測定して、遊離したp-ニトロアニリン量を定量した。酵素1単位（U）は、上記の反応条件において1分間に1 μmolのp-ニトロアニリンを遊離させるのに必要な酵素量とした。結果を表１２に示す。この結果、本発明のプロテアーゼBはN-サクシニル-Ala-Ala-Pro-Phe p-ニトロアニリド及びN-サクシニル-Ala-Ala-Pro-Leu-p-ニトロアニリドに対して高い分解活性を有していたがN-グルタリル-L-Phe-p-ニトロアニリド及びN-α-ベンゾイル-L-Arg-p-ニトロアニリドに対してに対してはほとんど分解活性を示さなかった。
(h) Substrate specificity
After incubating 0.06 ml of a 2.5 mM synthetic substrate solution (each synthetic substrate dissolved in 0.1 M glycine-NaOH buffer containing 4% DMSO, pH 9.5) at 37 ° C. for 5 minutes, protease B of the present invention 0.06 ml was added, reacted at 37 ° C. for 30 minutes, and the absorbance at 405 nm was measured using a spectrophotometer to quantify the amount of p-nitroaniline released. One unit (U) of enzyme was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline per minute under the above reaction conditions. The results are shown in Table 12. As a result, the protease B of the present invention has high degradation activity against N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide. However, it showed little degradation activity against N-glutaryl-L-Phe-p-nitroanilide and N-α-benzoyl-L-Arg-p-nitroanilide.
(i)阻害剤による影響
本発明のプロテアーゼ0.06 mlと各種阻害剤を最終濃度が以下に記す濃度になるように0.06 ml加えて混合し、37℃、30分間保温した後、基質として5 mM N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリド溶液（4% DMSOを含む0.1 M グリシン-NaOH 緩衝液, pH9.5に溶解したもの）を0.06 ml加え、37℃、30分間反応を行った後に酵素活性を測定した。結果を表１３に示す。下記に使用した阻害剤と各濃度を示した。この結果、本発明のプロテアーゼの活性はPMSFにより強く阻害され、SBTI、キモトリプシン、TPCKによっても阻害されたが、それ以外の阻害剤ではほとんど活性の低下は認められなかった。
(i) Influence of inhibitors 0.06 ml of the protease of the present invention and various inhibitors are added and mixed at 0.06 ml so that the final concentration is as described below, and incubated at 37 ° C. for 30 minutes, and then 5 mM N as a substrate -Add 0.06 ml of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide solution (dissolved in 0.1 M glycine-NaOH buffer, pH 9.5 containing 4% DMSO) and react at 37 ° C for 30 minutes Enzyme activity was measured after doing. The results are shown in Table 13. The inhibitor used and each concentration are shown below. As a result, the activity of the protease of the present invention was strongly inhibited by PMSF and was also inhibited by SBTI, chymotrypsin, and TPCK, but almost no decrease in activity was observed with other inhibitors.
1 mM ε-Aminocapronic acid(e-ACA)
1 mM Phenylmethylsulfonyl fluoride(PMSF)
0.01mg/ml Streptomyces サブチリシン Inhibitor (SSI)
1 mM 2,2'-Bipyridyl
0.01(0.1) mg/ml Chymostatin
1 mM Phenantorolin
1 mM EDTA
0.1 mM E-64
1 mM N-tosyl-L-phenylalanine chloromethyl ketone(TPCK)
1 mM ε-Aminocapronic acid (e-ACA)
1 mM Phenylmethylsulfonyl fluoride (PMSF)
0.01mg / ml Streptomyces Subtilisin Inhibitor (SSI)
1 mM 2,2'-Bipyridyl
0.01 (0.1) mg / ml Chymostatin
1 mM Phenantorolin
1 mM EDTA
0.1 mM E-64
1 mM N-tosyl- L -phenylalanine chloromethyl ketone (TPCK)
(j)最適pH、pH安定性、最適温度、及び熱安定性
例１の(j)及び(k)に記載された方法に従って本発明のプロテアーゼBの最適pH、pH安定性、最適温度、及び熱安定性を調べた。この結果、最適pHは9.5付近、pH安定性は6.0〜11.5であり、最適温度は60℃であった。また、pH9.5において60℃までは活性の低下が認められなかった。
(j) Optimal pH, pH stability, optimal temperature, and thermal stability According to the method described in Example 1 (j) and (k), the optimal pH, pH stability, optimal temperature, and The thermal stability was investigated. As a result, the optimum pH was around 9.5, the pH stability was 6.0 to 11.5, and the optimum temperature was 60 ° C. Also, no decrease in activity was observed up to 60 ° C at pH 9.5.
(h)プロテアーゼBの作用
CMV に対する作用を調べるために、本発明のプロテアーゼBとCMVをそれぞれ下記の濃度で20μlずつ等量混合して、37℃で30分間反応させた後、SDS-PAGEを行った。アクリルアミド濃度は、分離ゲルを15%、濃縮ゲルを3%として、電気泳動は20 mAで行った。分子量マーカーには上記と同様の低分子量マーカーを使用した。
(i)酵素(100μg/ml)
(ii)酵素(100μg/ml)＋ CMV(100μg/ml)
(iii)酵素(10μg/ml) ＋ CMV(100μg/ml)
(iv)酵素(1μg/ml) ＋ CMV(100μg/ml)
(v)CMV(100μg/ml)
結果を図８に示す。レーン(ii)及びレーン(iii)において分子量15,000のバンドが認められ、このバンドはCMVの外被タンパク質であるCMV CPの分解物であると考えられた。従って、本発明のプロテアーゼBはCMV CPに作用して外被タンパク質を分解し、CMVの粒子構造を破壊するものと考えられる。
(h) Action of protease B
In order to examine the action on CMV, protease B of the present invention and CMV were mixed in equal amounts of 20 μl each at the following concentrations, reacted at 37 ° C. for 30 minutes, and then subjected to SDS-PAGE. The acrylamide concentration was 15% for the separation gel, 3% for the concentrated gel, and electrophoresis was performed at 20 mA. As the molecular weight marker, the same low molecular weight marker as described above was used.
(i) Enzyme (100 μg / ml)
(ii) Enzyme (100 μg / ml) + CMV (100 μg / ml)
(iii) Enzyme (10 μg / ml) + CMV (100 μg / ml)
(iv) Enzyme (1 μg / ml) + CMV (100 μg / ml)
(v) CMV (100 μg / ml)
The results are shown in FIG. A band having a molecular weight of 15,000 was observed in lane (ii) and lane (iii), and this band was considered to be a degradation product of CMV CP, which is a coat protein of CMV. Therefore, it is considered that the protease B of the present invention acts on CMV CP to degrade the coat protein and destroy the particle structure of CMV.
(i)感染阻害率
本発明のプロテアーゼBのCMV に対する感染阻害効果の有効濃度を、ササゲにおける混合接種法により調査した。本酵素を1.00 〜100μg/mlに調製し、各濃度での感染阻害率を求めた。また、既知のプロテアーゼであるα-キモトリプシンとサブチリシンについても濃度を0.05 〜1.00 mg/mlに調整し、各濃度での感染阻害率を求めた。本発明のプロテアーゼBは20μg/ml以上の濃度では、ほぼ100%の感染阻害率を維持していた。感染阻害率が50%となる濃度であるIC50を求めたところ3.84μg/mlであり、α-キモトリプシンと サブチリシンのIC50はそれぞれ396μg/ml及び74.3μg/mlであった。この結果、プロテアーゼBは類似のプロテアーゼに比べて約20倍の低濃度で感染阻害作用を有することが示された。
(i) Infection inhibition rate The effective concentration of the inhibitory effect of protease B of the present invention on CMV was investigated by the mixed inoculation method in cowpea. This enzyme was prepared at 1.00-100 μg / ml, and the infection inhibition rate at each concentration was determined. In addition, α-chymotrypsin and subtilisin, which are known proteases, were adjusted to a concentration of 0.05 to 1.00 mg / ml, and the infection inhibition rate at each concentration was determined. Protease B of the present invention maintained an infection inhibition rate of almost 100% at a concentration of 20 μg / ml or more. The IC 50 at which the infection inhibition rate was 50% was determined to be 3.84 μg / ml, and the IC 50 of α-chymotrypsin and subtilisin were 396 μg / ml and 74.3 μg / ml, respectively. As a result, it was shown that protease B has an infection inhibitory action at a concentration about 20 times lower than that of a similar protease.
(j)葉表塗布法による感染阻害作用の検定
プロテアーゼBを用いて葉表塗布法による検定を行った。プロテアーゼの濃度は100 μg/mlに調整した。また、プロテアーゼBが葉の表面で直接作用しているのかを調べているために、葉が乾燥した30分後にサンプルを水で完全に洗い流し、その後再び乾燥させてから葉の両面に10μg/mlのCMV溶液を綿棒で接種した対照区を準備した。この結果、葉表塗布法では97.7%の感染阻害率を示したが、一旦サンプルを洗い流している対照区では感染阻害効果が認められなかった。
(j) Inhibition of Infection Inhibitory Effect by Leaf Surface Application Method An assay by the leaf surface application method was performed using protease B. The protease concentration was adjusted to 100 μg / ml. Also, since we are investigating whether protease B is acting directly on the leaf surface, wash the sample thoroughly with water 30 minutes after the leaf has dried, then dry it again and then 10 μg / ml on both sides of the leaf. A control group inoculated with a cotton swab was prepared. As a result, the leaf surface application method showed an infection inhibition rate of 97.7%, but no infection inhibition effect was observed in the control group once the sample was washed away.
(k)ToMVに対する感染阻害作用の検定
タバコモザイクウイルスを用い、0.1 M リン酸 buffer（pH 7.0）で100μg/mlの濃度に調整したToMVと試料を1：１となるようにエッペンチューブに加え、接種濃度が50μg/mlになるよう混合液を作成した。つぎに、対照用として試料の代わりに20mM Tris-HCl buffer（pH7.0）を用いたものを作成した。そして、それぞれをボルテックスで十分攪拌後、氷中で30分間静置した。検定植物としてタバコ(ナス科、Nicotiana tabacum cv. Xanthi nc) を用い、バーミキュライトを満たした鉢にタバコの種子を播種し温室内に置き、発芽した苗をセルトレイに移植して育苗した。2〜3週間後に、鉢に１個体ずつ定植し、7〜8葉期のものを実験に用いた。タバコの葉の中から、同じ程の大きさの葉を２枚選び、混合接種法を用いて検定した。対照葉では多数の病斑が見られるのに対して試料葉ではほとんど見られず、本発明のプロテアーゼBが感染阻害作用を有することが確認できた。
(k) Inhibition of infection inhibition against ToMV Using tobacco mosaic virus, add ToMV adjusted to a concentration of 100 μg / ml with 0.1 M phosphate buffer (pH 7.0) and the sample to 1: 1 to an Eppendorf tube, A mixed solution was prepared so that the inoculum concentration was 50 μg / ml. Next, a sample using 20 mM Tris-HCl buffer (pH 7.0) instead of the sample was prepared as a control. Each was sufficiently stirred by vortexing and allowed to stand in ice for 30 minutes. Tobacco seeds were used as a test plant (Nitriciana tabacum cv. Xanthi nc). Tobacco seeds were sown in a pot filled with vermiculite, placed in a greenhouse, and the germinated seedlings were transplanted to cell trays to grow seedlings. Two to three weeks later, one plant was planted one by one in a pot, and the 7-8 leaf stage was used for the experiment. Two leaves of the same size were selected from the tobacco leaves and tested using the mixed inoculation method. A large number of lesions were observed in the control leaves, but hardly any in the sample leaves, confirming that the protease B of the present invention has an infection inhibitory action.
(l)植物の葉表面における薬害の有無
本発明のプロテアーゼBのササゲへの薬害の有無を調べるために、100μg/mlの試料溶液を用いて混合接種法を行い、2日後と7日後の試料溶液塗布群とコントロール群の葉の様子をそれぞれ観察した。タバコに関しても同様の方法で薬害の有無を調べた。試料溶液塗布群では特に目立った外傷はなく、コントロール群においても病斑以外の目立った外傷はなかった。無処理の葉と成長の度合いも同程度であった。タバコの葉においても同様の結果が得られた。
(l) Presence / absence of phytotoxicity on leaf surface of plant In order to examine the presence / absence of phytotoxicity to cowpea of protease B of the present invention, mixed inoculation was performed using a sample solution of 100 μg / ml, and samples after 2 and 7 days The state of the leaves of the solution application group and the control group was observed. For tobacco, the same method was used to investigate the presence of phytotoxicity. There was no particularly noticeable trauma in the sample solution application group, and there was no noticeable trauma other than lesions in the control group. The degree of growth was similar to that of untreated leaves. Similar results were obtained with tobacco leaves.
Claims (3)
(a)分子量が約27,000であり、
(b)N-サクシニル-Ala-Ala-Pro-Phe-p-ニトロアニリドを基質として分解してp-ニトロアニリンを生成し、その作用はキモスタチンにより阻害され、
(c)N-サクシニル-Ala-Ala-Pro-Leu-p-ニトロアニリドに対しては実質的に分解作用を有せず、
(d)至適pHが9.5であり、pH5.0〜10.0の範囲でpH9.5における活性に対して約80％以上の活性が維持され、
(e)至適温度が40℃であり、60℃において40℃における活性に対して約80%の活性が維持され、
(f)4℃において40℃における活性の約60%が保持され、
(g)N末端のアミノ酸配列がVal-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Glyである
を有するプロテアーゼA。 The following characteristics:
(a) the molecular weight is about 27,000,
(b) N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is decomposed as a substrate to produce p-nitroaniline, the action of which is inhibited by chymostatin,
(c) N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide has substantially no decomposing action,
(d) The optimum pH is 9.5, and an activity of about 80% or more is maintained with respect to the activity at pH 9.5 in the range of pH 5.0 to 10.0,
(e) the optimum temperature is 40 ° C., and the activity at about 80% is maintained at 60 ° C. relative to the activity at 40 ° C .;
(f) about 60% of the activity at 40 ° C is retained at 4 ° C;
(g) Protease A having an N-terminal amino acid sequence of Val-Ile-Ala-Gly-Gly-X-Ala-Ala-Ile-Ile-Gly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008079526A JP5344836B2 (en) | 2007-04-12 | 2008-03-26 | New protease |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007104429 | 2007-04-12 | ||
| JP2007104429 | 2007-04-12 | ||
| JP2008079526A JP5344836B2 (en) | 2007-04-12 | 2008-03-26 | New protease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2008278881A JP2008278881A (en) | 2008-11-20 |
| JP5344836B2 true JP5344836B2 (en) | 2013-11-20 |
Family
ID=40140238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2008079526A Expired - Fee Related JP5344836B2 (en) | 2007-04-12 | 2008-03-26 | New protease |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5344836B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011177105A (en) * | 2010-03-01 | 2011-09-15 | Osaka Prefecture Univ | Protease derived from earthworm |
| JP6196406B1 (en) * | 2017-04-06 | 2017-09-13 | ワキ製薬株式会社 | Method for producing enzyme activity promoter and content of enzyme activity promoter |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2006093164A1 (en) * | 2005-02-28 | 2008-08-07 | 大阪府 | Viral infection and growth inhibitor containing earthworm-derived components |
-
2008
- 2008-03-26 JP JP2008079526A patent/JP5344836B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2008278881A (en) | 2008-11-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization | |
| Brown et al. | A role for pectinase and protease inhibitors in fungal rot development in tomato fruits | |
| Völkel et al. | Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia: Purification, cloning, sequencing and specific inhibition by its expressed propeptide | |
| JP5344836B2 (en) | New protease | |
| Healy et al. | The lysine-specific proteinase from Armillaria mellea is a member of a novel class of metalloendopeptidases located in Basidiomycetes | |
| Wang et al. | Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis | |
| CN116675748A (en) | A plant immune activation protein FpCDP1 secreted by Fusarium graminearum and its application | |
| KR100346928B1 (en) | Antimicrobial protein | |
| JPH09509842A (en) | Novel thiol protease inhibitor | |
| JP2011177105A (en) | Protease derived from earthworm | |
| Khuraishi et al. | Isolation and purification of anticoagulant enzymes from plant latex. | |
| US20170233712A1 (en) | Process for the prevention and suppression of bacterial diseases in plants | |
| CN115975981B (en) | Xanthomonas disease-resistant related protein, and encoding gene and application thereof | |
| KR101016688B1 (en) | Proteins involved in the transmission of peptidoglycan recognition signals, genes encoding them, and bacterial infection detection kits including the same | |
| CN116157018B (en) | Biostimulants and biosafety peptides and their use in agriculture | |
| JPH0223158B2 (en) | ||
| US20110200541A1 (en) | Recombinant preparation of bromelain inhibitors and bromelain inhibitor precursor | |
| Izrael-Živković et al. | Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853 | |
| KR100453574B1 (en) | Serine protease derived from insects, method for extracting said serin protease and composition for drug and food using the same | |
| El-Hadedy et al. | Streptomyces lavendulae Protease Inhibitor: Purification, Gene Overexpression, and 3‐Dimensional Structure | |
| JP4245804B2 (en) | Novel hemolytically active protein and gene encoding the protein | |
| KR102751632B1 (en) | A composition for preventing or controlling diseases caused by plant viruses comprising azurin protein derived from Pseudomonas chlororaphis O6 | |
| CN102899304A (en) | Protein having phytopathogen killing activity, and coding gene and application thereof | |
| KR102751628B1 (en) | A composition for preventing or controlling diseases caused by plant viruses comprising antiviral protein derived from Pseudomonas chlororaphis O6 | |
| McManus et al. | Wounding induces a series of closely related trypsin/chymotrypsin inhibitory peptides in leaves of tobacco |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20110325 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20110325 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20110328 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130528 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130712 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20130806 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20130813 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |