JPH04287685A - Collagenase producing bacterium - Google Patents
Collagenase producing bacteriumInfo
- Publication number
- JPH04287685A JPH04287685A JP12888491A JP12888491A JPH04287685A JP H04287685 A JPH04287685 A JP H04287685A JP 12888491 A JP12888491 A JP 12888491A JP 12888491 A JP12888491 A JP 12888491A JP H04287685 A JPH04287685 A JP H04287685A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- strains
- negative
- positive
- production
- 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
- 102000029816 Collagenase Human genes 0.000 title claims abstract description 11
- 108060005980 Collagenase Proteins 0.000 title claims abstract description 11
- 229960002424 collagenase Drugs 0.000 title claims abstract description 11
- 241000894006 Bacteria Species 0.000 title claims abstract description 9
- 241000589564 Flavobacterium sp. Species 0.000 claims abstract description 7
- 238000004519 manufacturing process Methods 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 235000013372 meat Nutrition 0.000 claims description 4
- 235000000346 sugar Nutrition 0.000 claims description 4
- 150000008163 sugars Chemical class 0.000 claims description 4
- 239000001052 yellow pigment Substances 0.000 claims description 4
- 102000016938 Catalase Human genes 0.000 claims description 2
- 108010053835 Catalase Proteins 0.000 claims description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- 238000003794 Gram staining Methods 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- 102000004316 Oxidoreductases Human genes 0.000 claims description 2
- 108090000854 Oxidoreductases Proteins 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 108010046334 Urease Proteins 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 244000052616 bacterial pathogen Species 0.000 claims description 2
- 239000007789 gas Substances 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 235000013336 milk Nutrition 0.000 claims description 2
- 239000008267 milk Substances 0.000 claims description 2
- 210000004080 milk Anatomy 0.000 claims description 2
- 230000000877 morphologic effect Effects 0.000 claims description 2
- 150000002823 nitrates Chemical class 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000000049 pigment Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 230000019086 sulfide ion homeostasis Effects 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 3
- 239000000512 collagen gel Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 201000000628 Gas Gangrene Diseases 0.000 description 1
- 241000193159 Hathewaya histolytica Species 0.000 description 1
- 208000003618 Intervertebral Disc Displacement Diseases 0.000 description 1
- 206010050296 Intervertebral disc protrusion Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【0001】0001
【産業上の利用分野】本発明は、土壌より分離して得ら
れたフラホバクテリウム属に属する新規微生物に関し、
更に詳しくは、コラゲナーゼを産生する新規微生物の提
供に関する。[Industrial Application Field] The present invention relates to a new microorganism belonging to the genus Flaphobacterium isolated from soil.
More specifically, the present invention relates to the provision of a novel microorganism that produces collagenase.
【0002】0002
【従来の技術】コラゲナーゼは、従来、クロストリジウ
ム・ヒストリティカム(Clostridium h
istolyticum)の生産する酵素が最も良く知
られている。かかるコラゼナーゼは、動物組織の細胞分
散などの生化学試薬として幅広く利甲されるだけなく、
最近では椎間板ヘルニアの治療など臨床領域においても
、需要が増大しつつある。しかし、本菌はガス壊疽菌と
して古くから知られる嫌気性の病原菌であり、酵素の大
量生産には適さない。[Prior Art] Collagenase has conventionally been used in Clostridium histolyticum (Clostridium h.
istolyticum) is the best known. Collasenase is not only useful as a biochemical reagent for dispersing animal tissue cells, but also has a wide range of uses.
Recently, demand has been increasing in clinical fields such as the treatment of intervertebral disc herniation. However, this bacterium is an anaerobic pathogen known for a long time as gas gangrene, and is not suitable for mass production of enzymes.
【0003】0003
【発明が解決しようとする課題】本発明は、大量生産か
容易で経済性のあるコラゲナーゼ生産菌を自然界より検
索し、フラボバクテリウム属に属する259−1株およ
び489−1株という新規な微生物が培地中にコラゲナ
ーゼを産生することを見いだし、本発明を完成したもの
である。[Problems to be Solved by the Invention] The present invention searches nature for collagenase-producing bacteria that can be easily mass-produced and is economical. The present invention was completed based on the discovery that collagenase was produced in the culture medium.
【0004】0004
【課題を解決するための手段】しかるに、本発明は以下
の菌学的性質を有するフラボバクテリウム・エスピー(
Flavobacterium sp.)259−1
株(微工研菌寄第12005号)、およびフラボバクテ
リウム・エスピー489−1株(微工研菌寄第1200
6号)からなる群から選択されたコラゲナーゼ産生微生
物の提供に関するものである[Means for Solving the Problems] However, the present invention relates to Flavobacterium sp. having the following mycological properties.
Flavobacterium sp. )259-1
strain (Feikoken Bibori No. 12005), and Flavobacterium sp.
Item 6) relates to the provision of collagenase-producing microorganisms selected from the group consisting of
【0005】A.形態学的性質
(1)形および大きさ
いずれの株も桿菌、大きさは、
259−1株:0.8×4.0〜4.5μm489−1
株:0.8×2.0〜2.5μm(2)多形性
259−1株:0.8×22.0μmに伸長する。
489−1株:なし
(3)運動性
いずれの株もなし。
(4)胞子
いずれの株もなし。
(5)グラム染色
いずれの株も陰性。
(6)抗酸性
いずれの株も陰性。[0005]A. Morphological properties (1) Shape and size All strains are bacilli, and the size is: 259-1 strain: 0.8 x 4.0 to 4.5 μm 489-1
Strain: 0.8 x 2.0 to 2.5 μm (2) Polymorphism 259-1 strain: Elongates to 0.8 x 22.0 μm. 489-1 strain: None (3) None of the motile strains. (4) No spores of either strain. (5) Gram staining Negative for all strains. (6) Acid-fast All strains were negative.
【0006】B.生育状態
(1)肉汁寒天平板培養
259−1株:コロニーは円形、光沢あり、不透明な黄
白色、半球状。
489−1株:コロニーは円形、光沢あり、不透明な黄
色、半球状。
(2)肉汁寒天斜面培養
259−1株:帯状に生育し、光沢のある不透明な黄白
色のコロニーを形成する。
489−1株:帯状に生育し、光沢のある不透明な黄色
のコロニーを形成する。
(3)肉汁液体培養
いずれの株も全体に濁る。
(4)肉汁ゼラチン穿刺培養。
いずれの株も液化する。
(5)リトマス・ミルク
いずれの株も凝固する。B. Growth status (1) Strain 259-1 cultured on broth agar plate: Colonies are round, glossy, opaque yellowish white, and hemispherical. Strain 489-1: Colonies are round, shiny, opaque yellow, and hemispherical. (2) Broth agar slant culture 259-1 strain: Grows in a band shape and forms glossy, opaque yellow-white colonies. Strain 489-1: Grows in a band shape and forms shiny, opaque yellow colonies. (3) Meat juice liquid culture All strains become cloudy throughout. (4) Meat juice gelatin puncture culture. Both strains liquefy. (5) Both litmus and milk strains coagulate.
【0007】C.生理学的性質
(1)硝酸塩の還元
いずれの株も陰性。
(2)脱窒反応
いずれの株も陰性。
(3)MRテスト
いずれの株も陰性。
(4)VP反応
いずれの株も陰性。
(5)インドールの生成
259−1株:陰性。
489−1株:陽性。
(6)硫化水素の生成
259−1株:弱い陽性。
489−1株:陽性。
(7)デンプンの加水分解
259−1株:陰性。
489−1株:陽性。
(8)クエン酸の利用
259−1株:コーサーの培地、クリステンセンの培地
で利用する。
489−1株:コーサーの培地では利用しない。クリス
テンセンの培地では利用する
(9)無機窒素源の利用
いずれの株も硝酸塩、アンモニウム塩を利用しない。
(10)色素の生成
259−1株:黄色の色素をわずかに生成する。
489−1株:黄色の色素を生成する。
(11)ウレアーゼ
いずれの株も陽性。
(12)オキシダーゼ
いずれの株も陽性。
(13)カタラーゼ
いずれの株も陽性。
(14)生育の温度範囲
いずれの株も10〜37℃
(15)生育のpH範囲
259−1株:4.8〜9.6
489−1株:5.7〜8.1
(16)酸素に対する態度
いずれの株も好気性。
(17)O−Fテスト
259−1株:陰性
489−1株:酸化型
(18)糖類からのガスの生成
いずれの菌もなし。
(19)糖類からの酸の生成
表1の通り。C. Physiological properties (1) Nitrate reduction All strains are negative. (2) Denitrification reaction Negative for all strains. (3) MR test All strains were negative. (4) VP reaction All strains were negative. (5) Indole production strain 259-1: Negative. 489-1 strain: Positive. (6) Hydrogen sulfide production strain 259-1: Weak positive. 489-1 strain: Positive. (7) Hydrolysis of starch 259-1 strain: Negative. 489-1 strain: Positive. (8) Utilization of citric acid 259-1 strain: Used in Kosar's medium and Christensen's medium. 489-1 strain: Not used in Kosar's medium. (9) Utilization of inorganic nitrogen sources None of the strains utilizes nitrates and ammonium salts, which are used in Christensen's medium. (10) Production of pigment 259-1 strain: Produces a slight amount of yellow pigment. 489-1 strain: produces yellow pigment. (11) Urease All strains were positive. (12) Oxidase All strains were positive. (13) Catalase All strains were positive. (14) Temperature range for growth 10-37℃ for all strains (15) pH range for growth 259-1 strain: 4.8-9.6 489-1 strain: 5.7-8.1 (16) Oxygen Attitude towards Both strains are aerobic. (17) O-F test 259-1 strain: Negative 489-1 strain: Oxidized type (18) No production of gas from sugars. (19) Production of acid from sugars As shown in Table 1.
【表1】
尚、図1には259−1株の、図2には489−1株の
電子顕微鏡による菌体の拡大写真を示した。以下、本発
明菌株のスクリーニング方法について例示する。[Table 1] FIG. 1 shows enlarged photographs of the bacterial cells of the 259-1 strain, and FIG. 2 shows enlarged photographs of the bacterial cells of the 489-1 strain, taken with an electron microscope. The method for screening the strain of the present invention will be exemplified below.
【0008】[0008]
【実施例】(1)1次スクリーニング
培地1を3ml分注した試験管に土壌懸濁水25ulを
加え、30℃で2日間培養した。この培養上澄をペーパ
ーディスクに加え、コラーゲンゲルプレート上に供試し
た。30℃で18時間インキュベートした後、ハローを
形成した試料についてその培養液を培地2で希釈培養し
、土壌単離菌を得た。[Example] (1) 25 ul of soil suspension water was added to a test tube into which 3 ml of primary screening medium 1 was dispensed, and cultured at 30°C for 2 days. This culture supernatant was added to a paper disk and sampled on a collagen gel plate. After incubating at 30° C. for 18 hours, the culture solution of the sample in which a halo was formed was diluted and cultured in medium 2 to obtain soil isolated bacteria.
【0009】(2)2次スクリーニング(1)の方法で
得た土壌単離菌を培地1で30℃、2日間培養した。こ
の培養上澄をペーパーディスクに加え、コラーゲンゲン
ゲルプレート上に供試した。30℃で18時間インキュ
ベートした後、ハローを形成した微生物をコラゲナーゼ
生産菌として採取した。(2) Secondary screening The soil isolate obtained by the method of (1) was cultured in medium 1 at 30°C for 2 days. This culture supernatant was added to a paper disk and placed on a collagen gel plate. After incubation at 30°C for 18 hours, microorganisms that formed a halo were collected as collagenase-producing bacteria.
【0010】0010
【0011】[0011]
【発明の効果】本発明は、大量生産が容易で経済性のあ
るコラゲナーゼ生産菌を自然界より得たものである。[Effects of the Invention] The present invention provides a collagenase-producing bacterium obtained from nature that is easy and economical to mass-produce.
【図1】259−1株の菌体の電子顕微鏡写真を示す。FIG. 1 shows an electron micrograph of bacterial cells of strain 259-1.
【図2】489−1株の菌体の電子顕微鏡写真を示す。FIG. 2 shows an electron micrograph of bacterial cells of strain 489-1.
Claims (1)
テリウム・エスピー(Flavobacterium
sp.)259―1株(微工研菌寄第12005号)
、およびフラボバクテリウム・エスピー489−1株(
微工研菌寄第12006号)からなる群から選択された
コラゲナーゼ産生微生物。 A.形態学的性質 (1)形および大きさ いずれの株も桿菌、大きさは、 259−1株:0.8×4.0〜4.5μm489−1
株:0.8×2.0〜2.5μm(2)多形性 259−1株:0.8×22.0μmに伸長する。 489−1株:なし (3)運動性 いずれの株もなし。 (4)胞子 いずれの株もなし。 (5)グラム染色 いずれの株も陰性。 (6)抗酸性 いずれの株も陰性。 B.生育状態 (1)肉汁寒天平板培養 259−1株:コロニーは円形、光沢あり、不透明な黄
白色、半球状。 489−1株:コロニーは円形、光沢あり、不透明な黄
色、半球状。 (2)肉汁寒天斜面培養 259−1株:帯状に生育し、光沢のある不透明な黄白
色のコロニーを形成する。 489−1株:帯状に生育し、光沢のある不透明な黄色
のコロニーを形成する。 (3)肉汁液体培養 いずれの株も全体に濁る。 (4)肉汁ゼラチン穿刺培養。 いずれの株も液化する。 (5)リトマス・ミルク いずれの株も凝固する。 C.生理学的注質 (1)硝酸塩の還元 いずれの株も陰性。 (2)脱窒反応 いずれの株も陰性。 (3)MRテスト いずれの株も陰性。 (4)VP反応 いずれの株も陰性。 (5)インドールの生成 259−1株:陰性。 489−1株:陽性。 (6)硫化水素の生成 259−1株:弱い陽性。 489−1株:陽性。 (7)デンプンの加水分解 259−1株:陰性。 489−1株:陽性。 (8)クエン酸の利用 259−1株:コーサーの培地、クリステンセンの培地
で利用する。 489−1株:コーサーの培地では利用しない。クリス
テンセンの培地では利用する。 (9)無機窒素源の利用 いずれの株も硝酸塩、アンモニウム塩を利用しない。 (10)色素の生成 259−1株:黄色の色素をわずかに生成する。 489−1株:黄色の色素を生成する。 (11)ウレアーゼ いずれの株も陽性。 (12)オキシダーゼ いずれの株も陽性。 (13)カタラーゼ いずれの株も陽性。 (14)生育の温度範囲 いずれの株も10〜37℃ (15)生育のpH範囲 259−1株:4.8〜9.6 489−1株:5.7〜8.1 (16)酸素に対する態度 いずれの株も好気性。 (17)O−Fテスト 259−1株:陰性 489−1株:酸化型 (18)糖類からのガスの生成 いずれの菌もなし。 (19)糖類からの酸の生成 表1の通り。 【表1】Claim 1: Flavobacterium sp. having the following mycological properties:
sp. ) 259-1 strain (Feikoken Bacterium No. 12005)
, and Flavobacterium sp. 489-1 strain (
Collagenase-producing microorganisms selected from the group consisting of (Feikoken Bibori No. 12006). A. Morphological properties (1) Shape and size All strains are bacilli, and the size is: 259-1 strain: 0.8 x 4.0 to 4.5 μm 489-1
Strain: 0.8 x 2.0 to 2.5 μm (2) Polymorphism 259-1 strain: Elongates to 0.8 x 22.0 μm. 489-1 strain: None (3) None of the motile strains. (4) No spores of either strain. (5) Gram staining Negative for all strains. (6) Acid-fast All strains were negative. B. Growth status (1) Strain 259-1 cultured on broth agar plate: Colonies are round, glossy, opaque yellowish white, and hemispherical. Strain 489-1: Colonies are round, shiny, opaque yellow, and hemispherical. (2) Broth agar slant culture 259-1 strain: Grows in a band shape and forms glossy, opaque yellow-white colonies. Strain 489-1: Grows in a band shape and forms shiny, opaque yellow colonies. (3) Meat juice liquid culture All strains become cloudy throughout. (4) Meat juice gelatin puncture culture. Both strains liquefy. (5) Both litmus and milk strains coagulate. C. Physiological injection (1) Reduction of nitrate All strains were negative. (2) Denitrification reaction Negative for all strains. (3) MR test All strains were negative. (4) VP reaction All strains were negative. (5) Indole production strain 259-1: Negative. 489-1 strain: Positive. (6) Hydrogen sulfide production strain 259-1: Weak positive. 489-1 strain: Positive. (7) Hydrolysis of starch 259-1 strain: Negative. 489-1 strain: Positive. (8) Utilization of citric acid 259-1 strain: Used in Kosar's medium and Christensen's medium. 489-1 strain: Not used in Kosar's medium. Used in Christensen's medium. (9) Use of inorganic nitrogen sources Neither strain uses nitrates or ammonium salts. (10) Production of pigment 259-1 strain: Produces a slight amount of yellow pigment. 489-1 strain: produces yellow pigment. (11) Urease All strains were positive. (12) Oxidase All strains were positive. (13) Catalase All strains were positive. (14) Temperature range for growth 10-37℃ for all strains (15) pH range for growth 259-1 strain: 4.8-9.6 489-1 strain: 5.7-8.1 (16) Oxygen Attitude towards Both strains are aerobic. (17) O-F test 259-1 strain: Negative 489-1 strain: Oxidized type (18) No production of gas from sugars. (19) Production of acid from sugars As shown in Table 1. [Table 1]
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP12888491A JPH04287685A (en) | 1991-03-15 | 1991-03-15 | Collagenase producing bacterium |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP12888491A JPH04287685A (en) | 1991-03-15 | 1991-03-15 | Collagenase producing bacterium |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH04287685A true JPH04287685A (en) | 1992-10-13 |
Family
ID=14995740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP12888491A Pending JPH04287685A (en) | 1991-03-15 | 1991-03-15 | Collagenase producing bacterium |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH04287685A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58102108A (en) * | 1981-12-14 | 1983-06-17 | Mitsubishi Electric Corp | Laser range finder |
JP2015047134A (en) * | 2013-09-03 | 2015-03-16 | 和歌山県 | Sweetfish cold-water disease-bacteria derived toxin |
-
1991
- 1991-03-15 JP JP12888491A patent/JPH04287685A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58102108A (en) * | 1981-12-14 | 1983-06-17 | Mitsubishi Electric Corp | Laser range finder |
JP2015047134A (en) * | 2013-09-03 | 2015-03-16 | 和歌山県 | Sweetfish cold-water disease-bacteria derived toxin |
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