JPS6339898A - Novel peptide mr-33-a substance and production thereof - Google Patents
Novel peptide mr-33-a substance and production thereofInfo
- Publication number
- JPS6339898A JPS6339898A JP61184754A JP18475486A JPS6339898A JP S6339898 A JPS6339898 A JP S6339898A JP 61184754 A JP61184754 A JP 61184754A JP 18475486 A JP18475486 A JP 18475486A JP S6339898 A JPS6339898 A JP S6339898A
- Authority
- JP
- Japan
- Prior art keywords
- substance
- reaction
- streptomyces
- formula
- ether
- 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
- 239000000126 substance Substances 0.000 title claims description 37
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 4
- 241000187747 Streptomyces Species 0.000 claims abstract description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 8
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 6
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 4
- -1 Isovaleryl Chemical group 0.000 claims description 3
- 241000187180 Streptomyces sp. Species 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 abstract description 6
- 239000004365 Protease Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 abstract description 5
- 108091005804 Peptidases Proteins 0.000 abstract description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000000354 decomposition reaction Methods 0.000 abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 abstract description 2
- 238000002844 melting Methods 0.000 abstract description 2
- 230000008018 melting Effects 0.000 abstract description 2
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 229920001817 Agar Polymers 0.000 description 5
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 239000000049 pigment Substances 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- 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 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
- 102000035195 Peptidases Human genes 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 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
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 208000016057 CHAND syndrome Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 244000201986 Cassia tora Species 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
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 238000010266 Sephadex chromatography Methods 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 231100000504 carcinogenesis Toxicity 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
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 235000019646 color tone Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- LMBMDLOSPKIWAP-UHFFFAOYSA-N embutramide Chemical compound OCCCC(=O)NCC(CC)(CC)C1=CC=CC(OC)=C1 LMBMDLOSPKIWAP-UHFFFAOYSA-N 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000004817 gas chromatography Methods 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
- 239000003292 glue Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- KSSNXJHPEFVKHY-UHFFFAOYSA-N phenol;hydrate Chemical compound O.OC1=CC=CC=C1 KSSNXJHPEFVKHY-UHFFFAOYSA-N 0.000 description 1
- NCAIGTHBQTXTLR-UHFFFAOYSA-N phentermine hydrochloride Chemical compound [Cl-].CC(C)([NH3+])CC1=CC=CC=C1 NCAIGTHBQTXTLR-UHFFFAOYSA-N 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose 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[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- QXADHXQCAQTNGW-UHFFFAOYSA-M sodium;boric acid;hydroxide Chemical compound [OH-].[Na+].OB(O)O QXADHXQCAQTNGW-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 229940124597 therapeutic agent Drugs 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は新規なペプチド及びその製造法に関する。さら
に詳しく述べ九ば、式(1)
(但し、上式中のI 5oval tr)lはインバレ
リル基を表わし、Phe及びIltばそれぞれフェニル
アラニンおよびインロイシンの残基を示す。)で示され
る化合物(MR−33−A物質)及び本発明者らによっ
て分離されたストレプトミセス属に属する微生物を培養
し、その培養物からMR−33−A物質を採取するMR
−35−A物質の製造法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide and a method for producing the same. More specifically, formula (1) (wherein I5ovaltr)l in the above formula represents an invaleryl group, and Phe and Ilt represent phenylalanine and inleucine residues, respectively. ) (MR-33-A substance) and a microorganism belonging to the genus Streptomyces isolated by the present inventors are cultured, and MR-33-A substance is collected from the culture.
-35-Relating to a method for producing a substance.
本発明者らは生体の制御機構におけるプロテアーゼの重
要な役割に着目し、新規なプロテアーゼ阻害物質を探索
した結果、ストレプトミセス・寅に寓する微生物がプロ
テアーゼ活性を強く阻害する作用を示す物質を産生ずる
事実を発見し、その培養液より有効成分を単離したとこ
ろ、この物質が上記(1)式で示される新規なペプチド
であることを確認し、本発明を完成させたつ
本発明に使用されるMR−33−A物質生産菌の一例と
しては、鳥取系の土壌より新たに分離されたAK−33
株がある。The present inventors focused on the important role of proteases in biological control mechanisms, and as a result of searching for new protease inhibitors, a microorganism similar to Streptomyces tora produces a substance that strongly inhibits protease activity. After discovering the fact that this occurs and isolating the active ingredient from the culture solution, it was confirmed that this substance is a novel peptide represented by the above formula (1). An example of MR-33-A substance-producing bacteria is AK-33, which was newly isolated from Tottori soil.
There are stocks.
この菌株の菌学的性状は下記の通りである。The mycological properties of this strain are as follows.
1、 形態
顕微鏡下では良く分岐した基土菌糸より気菌糸が良く伸
長し、その先端はらせん状となる。輪生分岐は認められ
ない。1. Morphology Under a microscope, aerial hyphae elongate better than well-branched substratum hyphae, and their tips become spiral-shaped. Whorl branching is not recognized.
電子顕微鏡での観察による胞子表面の形態は平滑である
。胞子は円筒型で大きさは0.5〜G、8×08〜1,
6ミクロンであり、通常10個以上の胞子の連鎖が認め
られる。The morphology of the spore surface observed with an electron microscope is smooth. The spores are cylindrical and the size is 0.5~G, 8x08~1,
The diameter is 6 microns, and chains of 10 or more spores are usually observed.
2、各種培地上での生育状態 各種寒天培地上の性状は以下の通シである。2. Growth status on various media The properties on various agar media are as follows.
培養温度は28°Cで観察は21日間培養後に行った。The culture temperature was 28°C, and observations were made after 21 days of culture.
色調の記1哉は、標阜色彩図表A(日本色彩研究所)に
よった。The color tones are based on Shibefu Color Chart A (Japan Color Research Institute).
(注)G;生育 A;気菌糸
几;裏面 S;可溶性色素
3、生理的性質
(1)生育温度範囲:15°C−37°C(2)ゼラチ
ンの液化:#性
(3)スターチの加水分解:陽性(弱い)(4)脱脂牛
乳の凝固:陰性
ペプトン化:陽性
(5)メラニン様色素
チロシン寒天:陽性
ペプトン・イースト・鉄寒天:陽性
4 炭素源の利用性(プリドハム・ゴトリーブ寒天培地
、25°C培養)
(1)利用する
D−グルコース、D−キシロース、D−フラクトース、
D−ガラクトース、イノ/トール、D−マンニトール
(2)利用しない
L−アラビノース、L−ラムノース、ラフィノース、ン
ユクロース、すIJシン5 全細胞加水分解物中のジア
ミノピ2ノリン酸LL型
以上の性状を要約すると、AK−33株はストレプトミ
セス属に属し、気菌糸の先端はらせん状で10個以上の
胞子連鎖を形成し、胞子表面は平滑である。気菌糸の色
調は初期は白〜灰色で、成熟するにつれ黄味〜緑味を帯
びる。種々の培地で黄ないし褐色系の可溶性色素を産生
じ、チロシン寒天、ペプトン・イースト・鉄基天上での
メラニン様色素の産生能は強い。(Note) G: Growth A: Aerial mycelium; Back side S: Soluble pigment 3, physiological properties (1) Growth temperature range: 15°C-37°C (2) Liquefaction of gelatin: # quality (3) Starch Hydrolysis: Positive (weak) (4) Coagulation of skim milk: Negative Peptonization: Positive (5) Melanin-like pigment tyrosine agar: Positive Peptone/yeast/iron agar: Positive 4 Carbon source availability (Pridham-Gotlieb agar medium , 25°C culture) (1) D-glucose, D-xylose, D-fructose to be used,
D-galactose, ino/tol, D-mannitol (2) Unused L-arabinose, L-rhamnose, raffinose, nucrose, SuIJshin 5 Summary of properties of diaminopininophosphate LL type and above in whole cell hydrolyzate Then, the AK-33 strain belongs to the genus Streptomyces, the tips of the aerial hyphae are spiral and form chains of 10 or more spores, and the spore surface is smooth. The color of the aerial mycelium is white to gray at the beginning, and becomes yellowish to greenish as it matures. It produces yellow to brown soluble pigments on various media, and has a strong ability to produce melanin-like pigments on tyrosine agar, peptone, yeast, and iron base.
本発明者らはAK−33株をストレプトミセス・エスピ
ー−AK−33(Slreptampces S/
IAK−33)と命名した。The present inventors used Streptomyces sp. AK-33 (Slreptampces S/
IAK-33).
本菌株は工業技術院微生物工業技術研究所に受託番号微
工研菌寄第8851号として寄託されている。This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 8851.
本発明物質の生産はストレプトミセス・エスピー・AK
−33株を培養し、その培養物より採取することにより
行われる。培養法は一般微生物の培養方法に準じて行わ
れるが、通常は液体培地による深部培養法が有利である
。培養は好気的条件下で行われ、温度はストレプ+−ミ
セス・エスピー・AK−33株が生育し本発明物質を産
生ずる範囲で適宜に変更しうるが、一般に28°C前後
で行われる。The substance of the present invention is produced by Streptomyces sp. AK.
-33 strain is cultured and collected from the culture. The culture method is carried out according to the culture method of general microorganisms, but deep culture method using a liquid medium is usually advantageous. Cultivation is carried out under aerobic conditions, and the temperature can be changed as appropriate as long as the Strep+-Mrs. sp. AK-33 strain grows and produces the substance of the present invention, but it is generally carried out at around 28°C. .
培地としては放線菌の培養に用いられる通常の原料が使
用されるが、hiR−33A 物質の産生には、L−
インロインンを適当量添加すると良い。As the medium, ordinary raw materials used for culturing actinomycetes are used, but for the production of hiR-33A substance, L-
It is good to add an appropriate amount of yin loin.
培養時間は培地の組成、温度条件により異なるが、多く
の場合好気的条件下に2〜4日の培養で本発明物質の産
生蓄積量は最大となる。Although the culture time varies depending on the composition of the medium and temperature conditions, in most cases, the amount of production and accumulation of the substance of the present invention is maximized after 2 to 4 days of culture under aerobic conditions.
本発明物質は主として培養ろ夜中に蓄積される。培養物
からの目的物質の単離・精製には目的物質の化学的特性
に基く種々の方法が使用できる。The substance of the present invention is mainly accumulated during the night of culture. Various methods can be used to isolate and purify a target substance from a culture, depending on the chemical properties of the target substance.
例えば、n−ブタノールなどの溶媒による抽出、イオン
交換樹脂、イオン交換セファデックスなどのイオン交換
体によるイオン交換クロマトグラフィー、アルキル基を
結合させたシリカゲルを用いた逆相クロマトグラフィー
などを適当に組み合わせて使用することにより、本発明
物質は粉末状に単離される。For example, an appropriate combination of extraction with a solvent such as n-butanol, ion exchange chromatography using an ion exchanger such as ion exchange resin or ion exchange Sephadex, and reversed phase chromatography using silica gel bonded with an alkyl group, etc. When used, the substance of the invention is isolated in powder form.
1、 元素分析値:
CUN
実測値 59,55 8.32 15.90理論値 5
9.97 8.52 16.14(C26H4204N
6・L(20として)2 分子量:502.65
(計算値、C26H4204N6として)6 融点:
163−166°C(分解)4、 比旋光度: Ca
”JD22−30. c; + 1.5(C= 1.1
05 in CH30H)5、紫外線吸収スペクトル
:250,257及び262M!に7エニルアラニンに
特異的な吸収が昭められる。1. Elemental analysis value: CUN Actual value 59,55 8.32 15.90 Theoretical value 5
9.97 8.52 16.14 (C26H4204N
6.L (as 20)2 Molecular weight: 502.65 (calculated value, as C26H4204N6)6 Melting point:
163-166°C (decomposition) 4, specific rotation: Ca
"JD22-30. c; + 1.5 (C = 1.1
05 in CH30H) 5, UV absorption spectrum: 250, 257 and 262M! The absorption specific to 7-enylalanine is improved.
6、赤外吸収スペクトル;
KJBr−1゜
ν m 、 3300−3250 (NH)ax
2960 (CHand CH2)
1640 (amid/l 、 yuanidium)
154 Q (amide l )
1450 C1)htn&! )
700 (phlntl )
7 各種溶媒に対する溶解性:
メタノール、エタノール、ジメチルスルホキシドに可溶
、水、アセトン、酢酸エチル、エーテル、クロロホルム
、石油エーテルには難溶である。6. Infrared absorption spectrum; KJBr-1゜ν m, 3300-3250 (NH)ax 2960 (CHand CH2) 1640 (amide/l, yuanidium)
154 Q (amide l) 1450 C1)htn&! ) 700 (phlntl) 7 Solubility in various solvents: Soluble in methanol, ethanol, and dimethyl sulfoxide, slightly soluble in water, acetone, ethyl acetate, ether, chloroform, and petroleum ether.
8 呈色反応:
坂口反応、Rpdon −Sm1th反応 及び2、5
.5−トリフェニルテトラゾリウムクロライドに陽性、
ニンヒドリン反応には陰性である。8 Color reaction: Sakaguchi reaction, Rpdon-Sm1th reaction, and 2, 5
.. positive for 5-triphenyltetrazolium chloride;
The ninhydrin reaction is negative.
9、 構成成分:
本物質に関して酸分解(6N HCjl、 110°C
124時間)を行い、その分解物を7リ力ゲル薄層クロ
マトグラフィーによシ分析したところ、フェニルアラニ
ン、インロインンが検出された。まだ、酸分解物に関し
てエーテル抽出ヲ行い、そのエーテル層をガスクロマト
グラフィーで分析したところ、1sovalerit:
acidが検出された。9. Components: Acid decomposition (6N HCjl, 110°C) of this substance.
124 hours), and the decomposed product was analyzed by 7L gel thin layer chromatography, and phenylalanine and inroinine were detected. However, when the acid decomposition product was extracted with ether and the ether layer was analyzed by gas chromatography, it was found that 1 sovalerit:
Acid was detected.
さらに、本物質をK Mn 04を用いて酸化し、その
酸化物に関しても同様にアミノ酸の分析ヲ行ったところ
、フェニルアラニン、イソロイシン及びアルギニンが検
出された。Furthermore, when this substance was oxidized using K Mn 04 and the oxide was similarly analyzed for amino acids, phenylalanine, isoleucine, and arginine were detected.
本物質 酸化物
フェニルアラニン 0.95 0.91イソロイシ
ン 1、DO1,00
アルギニン 1.04アミノ酸配
列:
本物質の酸化物を0.5 yytlのトリス−塩酸緩衝
液(pH8,0)に溶かし、tarbozp lye/
ytidaseA0.5μmi’及びcarboscp
/ye戸tidase B 5 pfを加えて25°
Cで反応させ、一定時間間隔で反応液を採取し、ノリ力
ゲル薄層クロマトグラフィーで分析を行った。展開溶媒
にはフェノール水(3:1)を用いニンヒドリン試薬で
発色させたところ、初めにアルギニンが検出され、次い
でイソロイシンの遊離が認められた。なお、フェニルア
ラニンは検出されなかった。This substance Oxide Phenylalanine 0.95 0.91 Isoleucine 1, DO 1,00 Arginine 1.04 Amino acid sequence: Dissolve the oxide of this substance in 0.5 yytl of Tris-HCl buffer (pH 8,0), and add it to the tarbozp lye /
ytidaseA0.5μmi' and carboscp
/ye door tidase B 5 pf added and 25°
The reaction solution was collected at fixed time intervals and analyzed by glue gel thin layer chromatography. When phenol water (3:1) was used as a developing solvent and color was developed with a ninhydrin reagent, arginine was detected first, followed by the release of isoleucine. Note that phenylalanine was not detected.
以上の結果から、本発明物質の化学構造式は下記と決定
された。From the above results, the chemical structural formula of the substance of the present invention was determined to be as follows.
T
〔本発明物質の生理活性〕
本発明物質と基質溶液とを混合し、67°Cで5分間プ
レインキュベイトしてから、酵素溶液を混合して反応を
開始させる。基質としては0.2%カゼインを用い、そ
の他に4 mMンステイン及び50薊ホウ酸−水酸ナト
リウム緩衝液を加え、37°Cで30分反応させる。次
いで反応液に0.17 M過塩素酸を加えて反応を停止
させ遠心分離後、上滑の28071?ffにおける吸光
度を測定し、対照液との対比から阻害率を求める。T [Physiological activity of the substance of the present invention] The substance of the present invention and the substrate solution are mixed and pre-incubated at 67°C for 5 minutes, and then the enzyme solution is mixed to start the reaction. 0.2% casein is used as a substrate, and 4 mM protein and 50% boric acid-sodium hydroxide buffer are added, and the reaction is allowed to proceed at 37°C for 30 minutes. Next, 0.17 M perchloric acid was added to the reaction solution to stop the reaction, and after centrifugation, 28071? The absorbance at ff is measured and the inhibition rate is determined from comparison with the control solution.
結果
本物質0.3μグは5μ2のパパイン活性を50%阻害
し、同様に本物質4.2μ2は511?のトリブ/ン活
性を50チ阻害した。Results: 0.3μg of this substance inhibited the papain activity of 5μ2 by 50%, and similarly, 4.2μ2 of this substance inhibited 511? It inhibited the tributane activity of 50%.
麦芽エキス、酵母エキス、ブドウ糖及びL−インロイシ
ンからなる合成培地2500ysl (PI(7,4)
にAK−33株の純培養物を接種し、30°Cで72時
間培養した後培養物をろ過し、ろ液を水酸化ナトリウム
でpH9,0に調整した後、ダウエックス1×4カラム
(CI型)(75X120朋)を通過させ、不純物をカ
ラムに吸着させて除いた。非吸着画分を減圧下に500
肩lまで濃縮し、1−ブタノール(50C1+tX3)
を用いて抽出を行った。Synthetic medium 2500ysl consisting of malt extract, yeast extract, glucose and L-inleucine (PI(7,4)
was inoculated with a pure culture of AK-33 strain and incubated at 30°C for 72 hours. The culture was filtered. The filtrate was adjusted to pH 9.0 with sodium hydroxide, and then transferred to a DOWEX 1×4 column ( CI type) (75 x 120 mm) was passed through, and impurities were removed by adsorption onto the column. The non-adsorbed fraction was dried under reduced pressure for 500 min.
Concentrate to shoulder level and add 1-butanol (50C1+tX3)
Extraction was performed using
この1−フリノール層を減圧下に濃縮・乾固させてから
これを0.01 M IJン酸塩緩衝液(pH8,0)
40C]+tに溶かし、CM−8ephadesc C
−25(Na型)のカラム(≠60x35C]朋)に吸
着させ、このカラムを同じ緩衝液1000屑lで洗浄後
、塩化ナトリウムの濃度グラジェント(0,0−0,4
M)で溶出させた。この溶出液を100m1まで減圧下
に濃縮し、ODSカラム($10X400朋)を用いて
Illした後、LiChro戸re戸RP−8カラム(
φ10x240m)に負荷し、水100m?で洗浄を行
い活性物質を80係メタノールで溶出分画した。この活
性分画を合併し減圧下に濃縮・乾固させ、メタノール−
エーテルから沈澱させて、はぼ純粋な本発明物質8〜が
白色の無定形物質として得られたO
〔発明の効果〕
本発明のΔ1R−33−A 物質はグロテアーゼ活性
を強く阻害する。一般にプロテアーゼは炎症、発癌、癌
転移、筋ジストロフィー等の様々な病態現象と密接な関
係を有し、本発明物質は、これらの病態現象の解析に有
効であると共に疾病の治療薬として期待されるものであ
る。This 1-furinol layer was concentrated and dried under reduced pressure, and then added to 0.01 M IJ phosphate buffer (pH 8,0).
40C]+t, CM-8ephadesc C
-25 (Na type) column (≠60
M) was eluted. This eluate was concentrated under reduced pressure to 100 ml, filtered using an ODS column ($10X400), and then refilled with a LiChro RP-8 column (
φ10x240m) and 100m of water? The active substance was eluted and fractionated with 80% methanol. The active fractions were combined, concentrated under reduced pressure and dried, and methanol-
By precipitation from ether, almost pure substances of the invention 8~ were obtained as white amorphous substances. Effects of the Invention The Δ1R-33-A substance of the invention strongly inhibits grotease activity. In general, proteases are closely related to various pathological phenomena such as inflammation, carcinogenesis, cancer metastasis, and muscular dystrophy, and the substances of the present invention are effective in analyzing these pathological phenomena and are expected to be therapeutic agents for diseases. It is.
また、アフィニティクロマトグラフィーの活性基として
プロテアーゼの精製に使用される等の発酵工業への応用
も考えられる。Further, applications in the fermentation industry, such as use as an active group in affinity chromatography to purify protease, are also considered.
(特許出願人 丸石製薬株式会社) (代理人 弁理士 糟谷 安)(Patent applicant Maruishi Pharmaceutical Co., Ltd.) (Agent: Patent attorney Yasu Kasuya)
Claims (1)
基を表わし、Phe及びIleはそれぞれフェニルアラ
ニン及びイソロイシンの残基を示す。) 2、MR−33−A物質生産能を有するストレプトミセ
ス属に属する菌株を培養し、培養液よりMR−33−A
物質を採取することを特徴とする新規ペプチドMR−3
3−A物質の製造法。 3、MR−33−A物質生産菌がストレプトミセス・エ
スピー・AK−33株である特許請求の範囲第2項記載
の製造法。[Claims] 1. MR-33-A substance represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. (However, Isovaleryl in the above formula represents an isovaleryl group, and Phe and Ile represent phenylalanine and isoleucine residues, respectively.) 2. Cultivate a strain belonging to the genus Streptomyces that has the ability to produce MR-33-A substance. , MR-33-A from the culture solution
Novel peptide MR-3 characterized by collecting substances
3-Method for producing substance A. 3. The production method according to claim 2, wherein the MR-33-A substance-producing bacterium is Streptomyces sp. AK-33 strain.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61184754A JPH085911B2 (en) | 1986-08-05 | 1986-08-05 | Novel peptide MR-33-A substance and method for producing the same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61184754A JPH085911B2 (en) | 1986-08-05 | 1986-08-05 | Novel peptide MR-33-A substance and method for producing the same |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6339898A true JPS6339898A (en) | 1988-02-20 |
JPH085911B2 JPH085911B2 (en) | 1996-01-24 |
Family
ID=16158758
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61184754A Expired - Fee Related JPH085911B2 (en) | 1986-08-05 | 1986-08-05 | Novel peptide MR-33-A substance and method for producing the same |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH085911B2 (en) |
-
1986
- 1986-08-05 JP JP61184754A patent/JPH085911B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JPH085911B2 (en) | 1996-01-24 |
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