JPH02255081A - Protease-producing bacterium, protease produced from the same bacterium and purification thereof - Google Patents
Protease-producing bacterium, protease produced from the same bacterium and purification thereofInfo
- Publication number
- JPH02255081A JPH02255081A JP1076421A JP7642189A JPH02255081A JP H02255081 A JPH02255081 A JP H02255081A JP 1076421 A JP1076421 A JP 1076421A JP 7642189 A JP7642189 A JP 7642189A JP H02255081 A JPH02255081 A JP H02255081A
- Authority
- JP
- Japan
- Prior art keywords
- protease
- strain
- opc
- bacterium
- producing
- 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.)
- Pending
Links
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 17
- 239000004365 Protease Substances 0.000 title claims abstract description 17
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 title claims abstract description 13
- 238000000746 purification Methods 0.000 title claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000004440 column chromatography Methods 0.000 claims abstract description 7
- 239000003929 acidic solution Substances 0.000 claims abstract description 6
- 239000000843 powder Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims abstract description 6
- GMKMEZVLHJARHF-UHFFFAOYSA-N 2,6-diaminopimelic acid Chemical compound OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 claims abstract description 5
- 241000203622 Nocardiopsis Species 0.000 claims abstract description 5
- 210000002421 cell wall Anatomy 0.000 claims abstract description 5
- 150000003904 phospholipids Chemical class 0.000 claims abstract description 4
- 241001221335 Nocardiopsis sp. Species 0.000 claims abstract 2
- 238000004458 analytical method Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 13
- 241000186361 Actinobacteria <class> Species 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 6
- 229920005654 Sephadex Polymers 0.000 claims description 4
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 229920002684 Sepharose Polymers 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 239000002689 soil Substances 0.000 abstract description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 108091005658 Basic proteases Proteins 0.000 description 4
- 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 3
- 101710127332 Protease I Proteins 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 101710137710 Thioesterase 1/protease 1/lysophospholipase L1 Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 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 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
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 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
- 239000012138 yeast extract Substances 0.000 description 2
- 241001156739 Actinobacteria <phylum> Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101100186606 Caenorhabditis elegans ncx-7 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-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
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-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
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 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
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000576755 Sclerotia Species 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 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
- 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
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- YFZOUMNUDGGHIW-UHFFFAOYSA-M p-chloromercuribenzoic acid Chemical compound OC(=O)C1=CC=C([Hg]Cl)C=C1 YFZOUMNUDGGHIW-UHFFFAOYSA-M 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 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
- 238000007670 refining Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、好アルカリ放線菌に関し、特に、プロテアー
ゼ生産菌、この菌体から産生したプロテアーゼ及びその
精製方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to alkaliphilic actinomycetes, and in particular to protease-producing bacteria, protease produced from the bacteria, and a method for purifying the same.
（従来の技術と発明が解決しようとする課題）放線菌は
多種多様の二次代謝産物を生産することから、実用的に
も重要な菌群である。しかしながら、好アルカリ放線菌
の分離およびその利用に関する研究は、他の異常環境下
に生育する微生物と比較して、報告は限られている。(Prior art and problems to be solved by the invention) Actinomycetes are a group of bacteria that are practically important because they produce a wide variety of secondary metabolites. However, there are limited reports on the isolation and utilization of alkaliphilic actinomycetes compared to other microorganisms that grow in abnormal environments.
本発明は異常環境下に生育する微生物の探索および利用
を目的として、好アルカリ放線菌の分離を行い、好アル
カリ放線菌の産生ずるアルカリプロテアーゼの精製とそ
の性質を解明することを目的とする。The purpose of the present invention is to isolate alkaliphilic actinomycetes, purify alkaline protease produced by alkaliphilic actinomycetes, and elucidate its properties, with the aim of searching for and utilizing microorganisms that grow in abnormal environments.
（課題を解決するための手段）
本発明は、細胞壁が２．６−ジアミノピメリン酸分析に
よりメソ型で細胞壁タイプＩｎ／Ｃ型であり、リン脂質
はＰＩ型であるノカルジオプシス属に属する微生物（好
アルカリ放線菌、Ｎｏｃａｒｄ １ｏｐｓ ｉ ｓｓｐ
．ＯＰＣ−２１０株、微工研菌寄第１０５０８号（ＦＥ
ＲＭ Ｐ−１０５０８））であるプロテアーゼ生産菌及
びこの菌体から産生したプロテアーゼである。(Means for Solving the Problems) The present invention provides a microorganism belonging to the genus Nocardiopsis (alkaliphilic Actinobacteria, Nocard 1ops i ssp
．． OPC-210 strain, Microtechnical Research Institute No. 10508 (FE
RM P-10508)) and the protease produced from this bacterial cell.
また、本発明のプロテアーゼ生産菌から産生したプロテ
アーゼの精製方法は、前記のＯＰＣ７２１０株からアセ
トンパウダーを得、これを弱酸性溶液（好ましくは酢酸
アンモニウム水溶液）に熔解し、透析後、カラムクロマ
トグラフィーにより精製することを特徴とする。この際
、カラムクロマトグラフィーによる精製を、前記弱酸性
溶液で平衡化したＤＥＡＲ−セファデックスＡ−５０カ
ラムに素通りさせ、次いでＣＭ−セファロースＣＬ −
６Ｂカラムクロマトグラフィーにより行うことが好まし
い。In addition, the method for purifying protease produced from protease-producing bacteria of the present invention involves obtaining acetone powder from the above-mentioned OPC7210 strain, dissolving it in a weakly acidic solution (preferably an aqueous ammonium acetate solution), dialysis, and then using column chromatography. Characterized by refining. At this time, purification by column chromatography was carried out by passing it through a DEAR-Sephadex A-50 column equilibrated with the weakly acidic solution, and then CM-Sepharose CL-
Preferably, this is carried out by 6B column chromatography.
本発明で利用する微生物は、昭和６２年１１月に京都府
の土壌より新たに分離したものである。その分離株（以
下．ＯＰＣ−２１０株と称する）の菌学的性質について
説明すると下記の通りである。The microorganisms used in the present invention were newly isolated from soil in Kyoto Prefecture in November 1985. The mycological properties of the isolated strain (hereinafter referred to as OPC-210 strain) are as follows.
因みに．ＯＰＣ−２１０株は、工業技術院微生物工業技
術研究所に、微工研菌寄第１０５０８号（ＦＥＲＭＰ−
１０５０８）として寄託されている。By the way. OPC-210 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 10508 (FERMP-
10508).
１）形態上の性質
基中菌糸は、適度に分岐している、通常の条件下では分
断しない。気菌糸はイースト・麦芽寒天培地（ＩＳＰ培
地培地階上で豊富に着生し、胞子形成は良好である。気
菌糸の分岐は単純分岐である。胞子のう、菌核、便毛胞
子は、観察されない。1) Morphological properties Medium hyphae are moderately branched and do not divide under normal conditions. Aerial hyphae grow abundantly on yeast/malt agar medium (ISP medium), and sporulation is good. Aerial hyphae are simply branched. Sporangium, sclerotia, and fecal hair spores are Not observed.
胞子形成の初期には、気菌子が多少ジグザグ状に見え、
その後長い断片に分断し、それがさらに不均一な大きさ
の、表面平滑な胞子に分断する。走査型電子顕微鏡によ
る観察では、胞子はレモン型であり、径約ＩＸ１．７
ミクロンの大きさを有し、胞子表面は平滑状である。At the beginning of sporulation, the aerial mycelium appears somewhat zigzag-shaped;
It then fragments into long fragments, which in turn fragment into unevenly sized, smooth-surfaced spores. When observed using a scanning electron microscope, the spores are lemon-shaped and approximately IX1.7 in diameter.
It has a micron size and the spore surface is smooth.
表１
２）各種培地上の成育状態
表１に示す各種培地にて２７℃の温度で７〜１４日間培
養した結果、ＯＰＣ，−２１０株は酵母エキス・麦芽エ
キス寒天培地（ＩＳＰ培地隘２）、スターチ・無機塩寒
天培地（ＩＳＰ培地培地階上での生育は極めて良好であ
る。チロシン寒天培地（ＩＳＰ培地ＮｃＸ７） 、グル
コース・アスパラギン培地での成育は適度であり、スタ
ーチ合成寒天培地では殆ど生育しない。観察した生育状
態は表１に示す通りである。Table 1 2) Growth status on various media As a result of culturing on the various media shown in Table 1 at a temperature of 27°C for 7 to 14 days, OPC, -210 strain was grown on yeast extract/malt extract agar medium (ISP medium 2). , Growth on starch/inorganic salt agar medium (ISP medium) is extremely good.Growth on tyrosine agar medium (ISP medium NcX7), glucose/asparagine medium is moderate, and almost no growth on starch synthetic agar medium. No. The observed growth conditions are shown in Table 1.
３）生理的性質
ａ）成育温度及びｐＨ
２７〜３２℃で良好な成育を示し、またその至適ｐ）１
は、８〜１０．３であった。3) Physiological properties a) Growth temperature and pH Shows good growth at 27-32°C, and its optimum p) 1
was 8 to 10.3.
ｂ）スターチの分解 陽性
Ｃ）ミルクの凝固 陰性
ｄ）メラニン色素の生成 陰性
ｅ）炭素源の利用および分解性
プリドハム・ゴツトリーブ寒天培地（ＴＳＰ培地陥９）
に各種炭素源をそれぞれ１％となるよう＊ＪＩＳ Ｚ
８７２１準拠
標準色索表（財団法人
日本規格協会）に従った。b) Decomposition of starch Positive C) Coagulation of milk Negative d) Production of melanin pigment Negative e) Utilization and degradability of carbon sources Prudham-Gottlieb agar (TSP medium 9)
*JIS Z
8721 compliant standard color table (Japanese Standards Association) was followed.
に添加して同化性について観察した。The assimilation properties were observed.
Ｄ−グルコース 強く利用する
イノシトール 利用しない
ラクトース 利用する
Ｄ−マンニトール 利用する
Ｄ−マンノース 僅かに利用するも不明確Ｄ−ラフィ
ノース 利用しない
Ｌ−ラムノース 利用しない
シュクロース 利用しない
Ｄ−キシロース 強く利用する
４）細胞壁組成
ルシバリエ等の方法（Ｔｈｅ ｃｈｅｍｏｔａｘｏｎｏ
ｍｙ ｏｆａｃｔｉｎｏｍｙｃｅｔｅ、 Ｉｎ ： ＾
ｃｔｉｎｏｍｙｃｅｔｅ ｔａｘｎｏｍｙ（Ｄｉｅｉｚ
、 Ａ ＆ Ｄ、 Ｈ，Ｔｈａｔｅｒ ｅｄ、’、 ５
ｏｃｉｅｔｙ ｆｏｒＩｎｄｕｓｔｒｉａｌ Ｍｉｃｒ
ｏｂｉｏｌｏｇｙ＋ Ｖｉｒｇｉｎｉａ、 ｐｐ ２２
５（１９８０） ）によりＯＰＣ−２１０株の菌体中の
２，６ジアミノビメリン酸を分析した結果、メソ型であ
り細胞壁タイプＩＩＩ／Ｃ型であり、特徴的な還元糖お
よびミコール酸は認められなかった。D-glucose Inositol is strongly utilized Lactose is not utilized D-mannitol is utilized D-mannose is utilized Slightly utilized but unclear D-raffinose L-rhamnose is not utilized Sucrose is not utilized D-xylose is not utilized strongly 4) Cell wall Composition The method of Lucivalier et al.
my ofactinomycete, In : ^
ctinomycete taxnomy (Dieiz
, A & D, H, Thatter ed,', 5
ociety forIndustrial Micro
obiology+ Virginia, pp 22
(5 (1980)) analyzed 2,6-diaminobimelic acid in the cells of OPC-210 strain, and found that it was meso-type and cell wall type III/C, and characteristic reducing sugars and mycolic acids were not observed. Ta.
リン脂質は、ｐＨｌ型であった。The phospholipids were of pHl type.
上述した性状からみて、○Ｐ（、−２１０株はノカルジ
オプシス属に属する微生物く放線菌）であると判定され
、Ｎｏｃａｒｄｉｏｐｓｉｓ ｓｐ、 ＯＰ Ｃ−２１
０株と命名した。In view of the above-mentioned properties, it was determined that the strain ○P (, -210 strain is a microorganism actinomycete belonging to the genus Nocardiopsis), and Nocardiopsis sp, OP C-21
It was named 0 stock.
（発明の効果）
多種多様の二次代謝産物を生産する放線菌は、実用的に
も重要な菌群である。本発明によれば、好アルカリ放線
菌の産生ずるアルカリプロテアーゼの精製が可能である
。本好アルカリ放線菌の産生ずるアルカリプロテアーゼ
を高率的に分離することによって、その利用範囲を拡大
することができる。(Effects of the Invention) Actinomycetes, which produce a wide variety of secondary metabolites, are a group of bacteria that are practically important. According to the present invention, it is possible to purify alkaline protease produced by alkaliphilic actinomycetes. By efficiently isolating alkaline protease produced by alkaline-loving actinomycetes, the range of its use can be expanded.
また、これまでに極限状態で活性を有する酵素は主とし
て、好熱性細菌から得られたものである。Furthermore, enzymes that are active under extreme conditions have so far been mainly obtained from thermophilic bacteria.
しかしながら、本発明で得られたプロテアーゼは耐熱性
で至適ｐｎがアルカリ性側にある。この点から、通常生
物の酵素と比較すること、あるいは突然変異株を用いて
アミノ酸残基の置換と活性との相関性を調べることによ
り、酵素の構造と機能、また酵素の有効利用について多
（の知見を得ることが可能である。However, the protease obtained in the present invention is heat resistant and has an optimum pn on the alkaline side. From this point of view, we can learn more about the structure and function of enzymes, as well as the effective use of enzymes, by comparing them with enzymes from normal organisms or by investigating the correlation between amino acid residue substitutions and activity using mutant strains. It is possible to obtain knowledge of
（実施例） 以下、実施例に基づき本発明を具体的に説明する。(Example) Hereinafter, the present invention will be specifically explained based on Examples.
（実施例１）
ＯＰＣ−２１０株の分離：昭和６２年１１月に京都府の
土壌より得られた好アルカリ放線菌の菌株を堀越らの方
法に準じて分離した（Ｋ、 Ｈｏｒｉｋｏｓｂｉ＋八ｇ
ｒｉｃ、 Ｂへｏｌ、Ｃｈｅｍ、、３５． １４０７
（１９７１））。(Example 1) Isolation of strain OPC-210: A strain of alkaliphilic actinomycetes obtained from soil in Kyoto Prefecture in November 1985 was isolated according to the method of Horikoshi et al.
ric, B to ol, Chem,, 35. 1407
(1971)).
培養条件：培地組成が１重量％のグルコース、１重量％
のカゼイン、０．５重量％のイースト抽出物、０．１重
量％のに２ＨＰＯ４，０，０２重量％ノＭｇＳＯ４，−
７Ｈ２０，１重量％のＮａ、ＣＯ３からなるｐＨ１０，
０の培地にて、２７°Ｃの培養温度で、種培養２日間、
副培養５日間、１２０ｒｐｍ、振幅７ ｃｍの振盪培養
を行った。Culture conditions: medium composition: 1% glucose, 1% by weight
casein, 0.5 wt% yeast extract, 0.1 wt% 2HPO4, 0.02 wt% MgSO4,-
7H20, pH 10, consisting of 1% by weight Na, CO3,
Seed culture for 2 days at a culture temperature of 27°C in a medium of
Subculturing was performed for 5 days with shaking at 120 rpm and an amplitude of 7 cm.
アルカリプロテアーゼの精製および諸性質二〇ｐｃ−２
１０株の培養濾液から常法により３倍容量の冷アセトン
を添加し、アセトンパウダーを得た。このパウダー３０
．０ｇを１０ｍＭ ＣＨ３ＣＯ０ＮＨｎ（ｐＨ６，６）
２０．０−に溶解し、上澄みを同じ緩衝液で一夜透析後
、同溶液で平衡化したＤＥＡＥ−セファデックスＡ５０
カラム（セファデックスはファーマシア社製商品名）に
よりクロマトグラフを行った。吸着しないで素通りした
分画をＦｒ−１とした。Ｆｒ−１は、さらに０．ＩＭＮ
ａＣｆを含む１０ｍＭｃ７）ＣＨ３ＣＯＯＮＨ４，（ｐ
Ｈ５，０）で平衡化したＣＭ−セファロースＣＬ −６
Ｂカラム（セファロースはファーマシア社製商品名、カ
ラム寸法は１．９ Ｘ ５０．０ｃｍ）により１回クロ
マトグラフを行い精製した（プロテアーゼＩ）。Purification and properties of alkaline protease 20pc-2
Acetone powder was obtained by adding 3 times the volume of cold acetone to the culture filtrate of the 10 strains in a conventional manner. This powder 30
．． 0g to 10mM CH3CO0NHn (pH 6,6)
DEAE-Sephadex A50 was dissolved in 20.0- and the supernatant was dialyzed overnight in the same buffer and then equilibrated in the same solution.
Chromatography was performed using a column (Sephadex is a trade name manufactured by Pharmacia). The fraction that passed through without being adsorbed was designated as Fr-1. Fr-1 is further 0. IMN
10mM Mc7) containing aCf CH3COONH4, (p
CM-Sepharose CL-6 equilibrated with H5,0)
Purification was performed by performing chromatography once using a B column (Sepharose is a trade name manufactured by Pharmacia, column dimensions: 1.9 x 50.0 cm) (protease I).
各精製フラクシヲンについて表２に示した。Table 2 shows each purified fraction.
表２ ａ）プロティンはローリ−らの方法によって決定した。Table 2 a) Protein was determined by the method of Lowry et al.
ｂ） １単位の酵素活性を酵素溶液１．０ｍｉ’、１
分間当りのチロシン１．０μｇとして定義した。b) 1 unit of enzyme activity in enzyme solution 1.0 mi', 1
Defined as 1.0 μg of tyrosine per minute.
このプロテアーゼＩについては電気泳動的に均一な標品
が得られた。また、本酵素の諸性質について検討を加え
、次の結果を得た。An electrophoretically homogeneous sample of this protease I was obtained. We also investigated various properties of this enzyme and obtained the following results.
）分子量：約２１，０００ （ＳＤＳ電気泳動法、Ｋ
。) Molecular weight: approximately 21,000 (SDS electrophoresis method, K
.
Ｗｅｂｅｒ＋ Ｍ、 ０ｓｂｏｒｎ、 Ｊ、 Ｂｉｏｌ
、 Ｃｈｅｍ、２４４＋ ４４０６ｉｉ）至適ｐＨ：
１０〜１２（基質カゼイン）ｉｉｉ ）至適温度＝６０
〜７０°Ｃ（基質カゼイン）ｉｖ）熱安定性： ｐＨ１
０で３０分間処理では５０℃まで安定であるが、７０℃
では完全に失活する。Weber+ M, 0sborn, J, Biol
, Chem, 244+ 4406ii) Optimal pH:
10-12 (substrate casein) iii) Optimal temperature = 60
~70°C (substrate casein) iv) Thermal stability: pH 1
When treated at 0 for 30 minutes, it is stable up to 50℃, but at 70℃
Then it becomes completely inactive.
ｖ）ｐＨ安定性：ｐ８４〜８では６０℃、３０分間処理
で８０％以上の残存活性があるが、ｐＨ１０ではほぼ失
活する。v) pH stability: At pH 84-8, there is a residual activity of 80% or more after treatment at 60° C. for 30 minutes, but at pH 10, the activity is almost lost.
ｖｉ）安定化：ｐＨ２，５０℃、１５０分間処理で残存
活性は約２５％であるが、１ｍＭのＣａ ２ ＋存在下
では、５０％まで安定化される。vi) Stabilization: The residual activity is about 25% after treatment at pH 2, 50° C., for 150 minutes, but is stabilized to 50% in the presence of 1 mM Ca 2+.
ｖｉｉ ）阻害剤の影響：２ｍＭのＰＣＭＢおよび１０
ｍＭのＥＤＴＡでは阻害されなかった。vii) Effect of inhibitors: 2mM PCMB and 10
It was not inhibited by mM EDTA.
菌株同定：菌学的性状（培養性状ならびに生理生化学的
性状）はＩＳＰの方法（Ｅ、 Ｂ、Ｓｈｉｒｌｉｎｇａ
ｎｄ Ｄ、 Ｇｏｔｔｌｉｅｂ、 Ｉｎｔ、 Ｊ、 ５
ｙｓｔ、 Ｂａｃｔｅｒｉｏｌ、。Strain identification: Mycological properties (culture properties and physiological biochemical properties) were determined using the ISP method (E, B, Shirlinga
nd D, Gottlieb, Int, J, 5
yst, Bacteriol,.
川、 ３１３ （１９６６））に準じ、また化学的分類
法（ジアミノピメリン酸、還元糖、ミコール酸、ならび
にリン脂質）は、レシェヴアリエルらの方法（Ｍ。Kawa, 313 (1966)), and the chemical classification method (diaminopimelic acid, reducing sugars, mycolic acids, and phospholipids) was according to the method of Rechev Ariel et al. (M.
Ｐ、 Ｌｅｃｂｅｖａｌｉｅｒ ａｎｄ Ｈ，Ａ、 Ｌ
ｅｃｈｅｖａｌｉｅｒ Ｉｎｔ、 Ｊ。P, Lecbevalier and H, A, L
echevalier Int, J.
５ｙｓｔ、 Ｂａｃｔｅｒｉｏｌ、、 ２０．４３５
（１９７０））に従った。5yst, Bacteriol, 20.435
(1970)).
なお結果は、表３ならびに表４に示した。The results are shown in Tables 3 and 4.
表４
尚、プロテアーゼｌは５ｍＭのＰＭＳＦで完全に阻害さ
れることから、セリンプロテアーゼであることを認めた
。Table 4 In addition, since protease I was completely inhibited by 5mM PMSF, it was confirmed that it is a serine protease.
Claims (6)
メソ型で細胞壁タイプIII／Ｃ型であり、リン脂質はＰ
III型であるノカルジオプシス属に属する微生物（好ア
ルカリ放線菌、Ｎｏｃａｒｄｉｏｐｓｉｓ ｓｐ．ＯＰ
Ｃ−２１０株、微工研菌寄第１０５０８号（ＦＥＲＭ
Ｐ−１０５０８））であるプロテアーゼ生産菌。(1) The cell wall is meso type and cell wall type III/C type as determined by 2,6-diaminopimelic acid analysis, and the phospholipid is P
Type III microorganisms belonging to the genus Nocardiopsis (alkaliphilic actinomycetes, Nocardiopsis sp. OP
Strain C-210, Microtechnical Research Institute No. 10508 (FERM
P-10508)) is a protease producing bacterium.
ウダーを得、これを弱酸性溶液に溶解し、透析後、カラ
ムクロマトグラフィーにより精製することを特徴とする
プロテアーゼ生産菌から産生したプロテアーゼの精製方
法。(2) Purification of protease produced from protease-producing bacteria, characterized in that acetone powder is obtained from OPC-210 strain according to claim 1, dissolved in a weakly acidic solution, dialyzed, and purified by column chromatography. Method.
酸性溶液で平衡化したＤＥＡＥ−セファデックスＡ−５
０カラムに素通りさせ、次いで、ＣＭ−セファロースＣ
Ｌ−６Ｂカラムクロマトグラフィーにより行う請求項２
記載の方法。(3) DEAE-Sephadex A-5 purified by column chromatography and equilibrated with the weakly acidic solution
0 column, then CM-Sepharose C
Claim 2 carried out by L-6B column chromatography
Method described.
項３記載の方法。(4) The method according to claim 3, wherein the weakly acidic solution is an aqueous ammonium acetate solution.
ロテアーゼ。(5) A protease produced from the OPC-210 strain according to claim 1.
ーゼ。(6) A protease obtained by the purification method according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1076421A JPH02255081A (en) | 1989-03-30 | 1989-03-30 | Protease-producing bacterium, protease produced from the same bacterium and purification thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1076421A JPH02255081A (en) | 1989-03-30 | 1989-03-30 | Protease-producing bacterium, protease produced from the same bacterium and purification thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02255081A true JPH02255081A (en) | 1990-10-15 |
Family
ID=13604723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1076421A Pending JPH02255081A (en) | 1989-03-30 | 1989-03-30 | Protease-producing bacterium, protease produced from the same bacterium and purification thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02255081A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004111221A1 (en) | 2003-06-19 | 2004-12-23 | Novozymes A/S | Proteases |
| WO2005123911A2 (en) | 2004-06-21 | 2005-12-29 | Novozymes A/S | Proteases |
| US7179630B2 (en) | 2003-06-19 | 2007-02-20 | Novozymes A/S | Thermostable proteases |
| US7208310B2 (en) | 2003-06-19 | 2007-04-24 | Novozymes A/S | Proteases |
| US7588926B2 (en) | 2003-02-07 | 2009-09-15 | Novozymes A/S | Proteases |
| US7658965B2 (en) | 2000-02-08 | 2010-02-09 | Dsm Assets B.V. | Use of acid stable protease in animal feed |
| EP2284259A2 (en) | 2003-10-10 | 2011-02-16 | Novozymes A/S | Protease variants |
-
1989
- 1989-03-30 JP JP1076421A patent/JPH02255081A/en active Pending
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7658965B2 (en) | 2000-02-08 | 2010-02-09 | Dsm Assets B.V. | Use of acid stable protease in animal feed |
| US8067238B2 (en) | 2000-02-08 | 2011-11-29 | Dsm Ip Assets Bv | Use of acid stable protease in animal feed |
| US7588926B2 (en) | 2003-02-07 | 2009-09-15 | Novozymes A/S | Proteases |
| US7906310B2 (en) | 2003-02-07 | 2011-03-15 | Novozymes A/S | Proteases |
| WO2004111221A1 (en) | 2003-06-19 | 2004-12-23 | Novozymes A/S | Proteases |
| US7179630B2 (en) | 2003-06-19 | 2007-02-20 | Novozymes A/S | Thermostable proteases |
| US7208310B2 (en) | 2003-06-19 | 2007-04-24 | Novozymes A/S | Proteases |
| US8772011B2 (en) | 2003-10-10 | 2014-07-08 | Novozymes A/S | Protease variants |
| EP2284259A2 (en) | 2003-10-10 | 2011-02-16 | Novozymes A/S | Protease variants |
| US7892808B2 (en) | 2003-10-10 | 2011-02-22 | Norozymes A/S | Protease variants |
| EP2308966A1 (en) | 2003-10-10 | 2011-04-13 | Novozymes A/S | Protease variants |
| US8377677B2 (en) | 2003-10-10 | 2013-02-19 | Novozymes A/S | Protease variants |
| US9131711B2 (en) | 2003-10-10 | 2015-09-15 | Novozymes A/S | Protease variants |
| EP2258838A1 (en) | 2004-06-21 | 2010-12-08 | Novozymes A/S | Nocardiopsis proteases |
| WO2005123911A2 (en) | 2004-06-21 | 2005-12-29 | Novozymes A/S | Proteases |
| US8357408B2 (en) | 2004-06-21 | 2013-01-22 | Novozymes A/S | Proteases |
| EP2258839A1 (en) | 2004-06-21 | 2010-12-08 | Novozymes A/S | Nocardiopsis proteases |
| US9279115B2 (en) | 2004-06-21 | 2016-03-08 | Novozymes A/S | Proteases |
| US9279114B2 (en) | 2004-06-21 | 2016-03-08 | Novozymes A/S | Proteases |
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