JP6457596B2 - Activated sludge treatment method, sludge generation amount control method, and microorganism - Google Patents
Activated sludge treatment method, sludge generation amount control method, and microorganism Download PDFInfo
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- JP6457596B2 JP6457596B2 JP2017153319A JP2017153319A JP6457596B2 JP 6457596 B2 JP6457596 B2 JP 6457596B2 JP 2017153319 A JP2017153319 A JP 2017153319A JP 2017153319 A JP2017153319 A JP 2017153319A JP 6457596 B2 JP6457596 B2 JP 6457596B2
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Description
本発明は、活性汚泥の処理方法、汚泥発生量の抑制方法、並びに、微生物に関する。本発明は、活性汚泥法による廃水処理で発生する余剰汚泥の削減に有用なものである。 The present invention relates to a method for treating activated sludge, a method for suppressing sludge generation, and a microorganism. The present invention is useful for reducing excess sludge generated in wastewater treatment by the activated sludge method.
日本における産業廃水処理及び下水処理の多くは活性汚泥法により行われている。活性汚泥法による廃水処理においては、処理過程で大量に発生する余剰汚泥の処分が問題となっている。余剰汚泥は産業廃棄物であり、通常、焼却した後に埋め立てて処理されている。しかし、余剰汚泥の焼却と埋め立てには環境負荷や処理コストの問題があるため、余剰汚泥を減らすための技術開発が必要である。例えば、汚泥発生量がより低い廃水処理技術の開発や、発生した汚泥を減容化する技術の開発が求められる。 Most of industrial wastewater treatment and sewage treatment in Japan are carried out by the activated sludge method. In the wastewater treatment by the activated sludge method, disposal of excess sludge generated in a large amount during the treatment process is a problem. Excess sludge is industrial waste and is usually disposed of after being incinerated. However, incineration and landfilling of surplus sludge has problems of environmental load and treatment cost, so technology development to reduce surplus sludge is necessary. For example, development of wastewater treatment technology with a lower sludge generation amount and development of technology for reducing the volume of generated sludge are required.
特許文献１には、光合成緑藻類の乾燥物を活性汚泥に添加して余剰汚泥を削減する技術が開示されている。この技術では、光合成緑藻類の乾燥物によって活性汚泥中の微生物活性を高めることにより、活性汚泥の発生量を低減している。 Patent Document 1 discloses a technique for reducing excess sludge by adding a dry product of photosynthetic green algae to activated sludge. In this technique, the amount of activated sludge generated is reduced by increasing the microbial activity in the activated sludge with a dried product of photosynthetic green algae.
特許文献２には、余剰汚泥に廃水原水の一部を混合することにより、廃水原水の水処理と余剰汚泥を分解処理の両方を同時に行う技術が開示されている。この技術によれば、余剰汚泥の処理効率が向上するとされている。また、その背景技術として、余剰汚泥の細胞壁を破壊して溶菌することで余剰汚泥をＢＯＤ成分とし、これを活性汚泥槽に返送して好気的に分解し、余剰汚泥を減容化する技術について記載されている。 Patent Document 2 discloses a technique for simultaneously performing both water treatment of wastewater raw water and decomposition treatment of excess sludge by mixing a part of the raw wastewater with excess sludge. According to this technology, it is said that the processing efficiency of excess sludge is improved. In addition, as a background technology, the surplus sludge is broken down and lysed to make the surplus sludge into a BOD component, which is returned to the activated sludge tank and decomposed aerobically to reduce the excess sludge. Is described.
上記のように、活性汚泥法による廃水処理において、余剰汚泥を削減するための方策が種々検討されている。しかし、下水道普及率の増加等に伴って余剰汚泥の発生量は増加傾向にあり、余剰汚泥を削減するための更なる技術開発が望まれる。
そこで本発明は、新たな原理に基づいた、活性汚泥を削減するための一連の技術を提供することを目的とする。
As described above, various methods for reducing excess sludge have been studied in wastewater treatment by the activated sludge method. However, with the increase in the sewerage penetration rate, the amount of surplus sludge generated is increasing, and further technological development for reducing surplus sludge is desired.
Then, an object of this invention is to provide a series of techniques for reducing activated sludge based on a new principle.
本発明の１つの様相は、アセチルトランスフェラーゼを生産する第１の微生物を活性汚泥に添加及び接触させて、第１の微生物が生産したアセチルトランスフェラーゼを前記活性汚泥に作用させ、前記活性汚泥中に含まれるアミノ酸又はペプチドのアミノ基をアセチル化する第１の工程を包含することを特徴とする活性汚泥の処理方法である。 One aspect of the present invention is that a first microorganism that produces acetyltransferase is added to and brought into contact with activated sludge, and the acetyltransferase produced by the first microorganism is allowed to act on the activated sludge and is contained in the activated sludge. It is a processing method of activated sludge characterized by including the 1st process of acetylating the amino group of an amino acid or a peptide.
本様相は活性汚泥の処理方法に係るものである。本様相では、アセチルトランスフェラーゼを生産する第１の微生物（アセチルトランスフェラーゼ生産微生物）を活性汚泥に添加及び接触させることにより、第１の微生物が生産したアセチルトランスフェラーゼを活性汚泥に作用させる。これにより、活性汚泥中に含まれるアミノ酸又はペプチドのアミノ基をアセチル化する（第１の工程）。アミノ基がアセチル化されたアミノ酸やペプチドは微生物に資化され難いので、本様相によれば、活性汚泥中の微生物の無用な増殖が抑えられ、結果として余剰汚泥の発生が抑制される。 This aspect relates to a method for treating activated sludge. In this aspect, the first microorganism that produces acetyltransferase (acetyltransferase-producing microorganism) is added and brought into contact with activated sludge so that the acetyltransferase produced by the first microorganism acts on the activated sludge. Thereby, the amino group of the amino acid or peptide contained in activated sludge is acetylated (1st process). Since amino acids and peptides whose amino groups are acetylated are hardly assimilated by microorganisms, according to this aspect, unnecessary growth of microorganisms in activated sludge is suppressed, and as a result, generation of excess sludge is suppressed.
好ましくは、プロテアーゼを生産する第２の微生物を前記活性汚泥に添加及び接触させて、第２の微生物が生産したプロテアーゼを前記活性汚泥に作用させる第２の工程をさらに包含する。 Preferably, the method further includes a second step of adding a second microorganism producing protease to the activated sludge and bringing it into contact with the activated sludge so that the protease produced by the second microorganism acts on the activated sludge.
本様相では、さらに、プロテアーゼを生産する第２の微生物（プロテアーゼ生産微生物）を活性汚泥に添加及び接触させて、第２の微生物が生産したプロテアーゼを前記活性汚泥に作用させる（第２の工程）。これにより、活性汚泥中に存在する微生物を溶菌させることができる。本様相では、第２の微生物が生産したプロテアーゼによって活性汚泥中の微生物を積極的に溶菌させ、当該微生物に由来するアミノ酸やペプチドを積極的に活性汚泥中に放出させる。そして、これらのアミノ酸やペプチドのアミノ基をアセチル化し（第１の工程）、微生物に資化され難くする。本様相によれば、活性汚泥中の微生物の無用な増殖が抑えられるとともに、活性汚泥中の微生物を積極的に溶菌させることで、余剰汚泥をさらに削減することができる。 In this aspect, a second microorganism that produces protease (protease-producing microorganism) is added to and contacted with activated sludge, and the protease produced by the second microorganism is allowed to act on the activated sludge (second step). . Thereby, the microorganisms which exist in activated sludge can be lysed. In this aspect, the microorganisms in the activated sludge are actively lysed by the protease produced by the second microorganism, and amino acids and peptides derived from the microorganisms are actively released into the activated sludge. Then, the amino group of these amino acids and peptides is acetylated (first step) to make it difficult to be assimilated by microorganisms. According to this aspect, unnecessary proliferation of microorganisms in the activated sludge can be suppressed, and surplus sludge can be further reduced by positively lysing the microorganisms in the activated sludge.
好ましくは、第１の微生物と第２の微生物とを同時に活性汚泥に接触させる。 Preferably, the first microorganism and the second microorganism are simultaneously contacted with activated sludge.
かかる構成により、溶菌した微生物から放出されるアミノ酸やペプチドに対してアセチルトランスフェラーゼを直ちに作用させることができる。 With this configuration, acetyltransferase can immediately act on amino acids and peptides released from lysed microorganisms.
好ましくは、第２の微生物が、Bacillus属に属する微生物である。 Preferably, the second microorganism is a microorganism belonging to the genus Bacillus.
好ましくは、第２の微生物が、SAS-1946502 (NITE P-02327)である。 Preferably, the second microorganism is SAS-1946502 (NITE P-02327).
好ましくは、第１の微生物が、Curtobacterium属に属する微生物である。 Preferably, the first microorganism is a microorganism belonging to the genus Curtobacterium.
好ましくは、第１の微生物が、SAS-1946503 (NITE P-02328)である。 Preferably, the first microorganism is SAS-1946503 (NITE P-02328).
本発明の他の様相は、上記の活性汚泥の処理方法によって活性汚泥を処理することを特徴とする汚泥発生量の抑制方法である。 Another aspect of the present invention is a method for suppressing sludge generation, characterized in that activated sludge is treated by the above-described activated sludge treatment method.
本様相は、上記の活性汚泥の処理方法によって活性汚泥を処理し、余剰汚泥の発生量を抑制するものである。 In this aspect, the activated sludge is treated by the above-described activated sludge treatment method, and the generated amount of excess sludge is suppressed.
本発明の他の様相は、SAS-1946503 (NITE P-02328)である微生物である。 Another aspect of the present invention is a microorganism which is SAS-1946503 (NITE P-02328).
本様相の微生物は、活性汚泥中で安定的に保持され、かつアセチルトランスフェラーゼを高生産することができる。本様相の微生物によれば、上記した方法における第１の工程を効率的に行うことができる。 The microorganism of this aspect is stably maintained in activated sludge and can produce acetyltransferase at a high level. According to the microorganism of this aspect, the first step in the above-described method can be efficiently performed.
本発明の他の様相は、SAS-1946502 (NITE P-02327)である微生物である。 Another aspect of the invention is a microorganism which is SAS-1946502 (NITE P-02327).
本様相の微生物は、活性汚泥中で安定的に保持され、かつプロテアーゼを高生産することができる。本様相の微生物によれば、上記した方法における第２の工程を効率的に行うことができる。 The microorganism of this aspect can be stably retained in activated sludge and can produce a high amount of protease. According to the microorganism of this aspect, the second step in the above-described method can be efficiently performed.
本発明によれば、活性汚泥法による廃水処理において、余剰汚泥の発生量を効率的に削減することができる。 According to the present invention, it is possible to efficiently reduce the amount of excess sludge generated in wastewater treatment by the activated sludge method.
まず本発明の微生物、すなわち、アセチルトランスフェラーゼ生産微生物であるSAS-1946503 (NITE P-02328)と、プロテアーゼ生産微生物であるSAS-1946502 (NITE P-02327)について説明する。これらの微生物は、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）に寄託されている。寄託の詳細を以下に示す。 First, the microorganism of the present invention, that is, SAS-1946503 (NITE P-02328) which is an acetyltransferase-producing microorganism and SAS-1946502 (NITE P-02327) which is a protease-producing microorganism will be described. These microorganisms are deposited with the Patent Microorganism Deposit Center (NPMD) of the National Institute of Technology and Evaluation. Details of the deposit are shown below.
＜アセチルトランスフェラーゼ生産微生物＞
表示：SAS-1946503
受託番号：NITE P-02328
受領日：２０１６年８月９日
<Acetyltransferase-producing microorganism>
Display: SAS-1946503
Accession Number: NITE P-02328
Date of receipt: August 9, 2016
＜プロテアーゼ生産微生物＞
表示：SAS-1946502
受託番号：NITE P-02327
受領日：２０１６年８月９日
<Protease-producing microorganism>
Display: SAS-1946502
Accession Number: NITE P-02327
Date of receipt: August 9, 2016
SAS-1946503 (NITE P-02328)の菌学的性質は以下のとおりである。「＋」は陽性、「−」は陰性を示す。 The mycological properties of SAS-1946503 (NITE P-02328) are as follows. “+” Indicates positive and “−” indicates negative.
〔形態等〕
・細胞形態：桿菌（０．７〜０．８μｍ×１．５〜２．０μｍ）
・グラム染色性：＋
・胞子の有無：−
・運動性：＋
・コロニー形態（培地：Nutrient agar、培養時間：４８時間、培養温度：３０℃）
直径：１．０ｍｍ
色調：黄色
形：円形
***状態：レンズ状
周縁：全縁
表面の形状など：スムーズ
透明度：不透明
粘稠度：バター様
[Form etc.]
-Cell morphology: Neisseria gonorrhoeae (0.7-0.8 μm × 1.5-2.0 μm)
-Gram stainability: +
・ Spore presence:
・ Mobility: +
-Colony morphology (medium: Nutrient agar, culture time: 48 hours, culture temperature: 30 ° C)
Diameter: 1.0mm
Color: Yellow Shape: Circular Raised: Lens-like Edge: All edges Surface shape, etc .: Smooth Transparency: Opaque Consistency: Butter-like
〔生育温度〕
・３７℃：＋（弱い）
・４５℃：−
[Growth temperature]
・ 37 ℃: + (weak)
・ 45 ℃:-
・カタラーゼ反応：＋
・オキシダーゼ反応：−
・グルコースからの酸／ガス産生（酸産生／ガス産生）：−／−
・グルコースのＯ／Ｆテスト（酸化／発酵）：−／−
Catalase reaction: +
・ Oxidase reaction:-
Acid / gas production from glucose (acid production / gas production):-/-
Glucose O / F test (oxidation / fermentation):-/-
〔生化学試験〕
・硝酸塩還元：−
・ピラジンアミダーゼ：＋
・ピロリドニルアリルアミダーゼ：−
・アルカリフォスファターゼ：−
・β−グルクロニダーゼ：−
・β−ガラクトシダーゼ：＋
・α−グルコシダーゼ：＋
・Ｎ−アセチル−β−グルコサミニダーゼ：−
・エスクリン（β−グルコシダーゼ）：＋
・ウレアーゼ：−
・ゼラチン加水分解：＋
・カタラーゼ：＋
[Biochemical test]
・ Nitrate reduction:-
・ Pyrazineamidase: +
・ Pyrrolidonyl allylamidase:-
・ Alkaline phosphatase:-
・ Β-glucuronidase:-
Β-galactosidase: +
・ Α-Glucosidase: +
N-acetyl-β-glucosaminidase:-
・ Esculin (β-glucosidase): +
・ Urease:-
・ Gelatin hydrolysis: +
-Catalase: +
〔発酵性試験〕
・ブドウ糖：＋
・リボース：−
・キシロース：＋
・マンニトール：−
・マルトース：＋
・乳糖：−
・白糖：−
・グリコーゲン：−
[Fermentation test]
・ Glucose: +
・ Ribose:-
・ Xylose: +
・ Mannitol:-
-Maltose: +
・ Lactose:-
・ Sucrose:-
・ Glycogen:-
・カゼイン加水分解：＋
・リパーゼ活性（Tween 80）：＋
・Acetate資化性：＋（弱い）
・Fumarate資化性：＋（弱い）
Casein hydrolysis: +
・ Lipase activity (Tween 80): +
・ Acetate assimilation: + (weak)
・ Fumarate utilization: + (weak)
・１６ｒＤＮＡ塩基配列の相同性
Curtobacterium flaccumfaciens：９９．９％
・ Homology of 16rDNA base sequence
Curtobacterium flaccumfaciens: 99.9%
以上の結果より、SAS-1946503 (NITE P-02328)はCurtobacterium flaccumfaciensと同定された。 Based on the above results, SAS-1946503 (NITE P-02328) was identified as Curtobacterium flaccumfaciens.
SAS-1946502 (NITE P-02327)の菌学的性質は以下のとおりである。「＋」は陽性、「−」は陰性を示す。 The mycological properties of SAS-1946502 (NITE P-02327) are as follows. “+” Indicates positive and “−” indicates negative.
〔形態等〕
・細胞形態：桿菌（０．８〜０．９μｍ×１．５〜２．５μｍ）
・グラム染色性：＋
・胞子の有無：＋
・運動性：＋
・コロニー形態（培地：Nutrient agar、培養時間：４８時間、培養温度：３０℃）
直径：２．０〜３．０ｍｍ
色調：クリーム色
形：円形
***状態：レンズ状
周縁：糸状
表面の形状など：スムーズ
透明度：不透明
粘稠度：バター様
[Form etc.]
-Cell morphology: Neisseria gonorrhoeae (0.8-0.9 μm × 1.5-2.5 μm)
-Gram stainability: +
・ Spore presence: +
・ Mobility: +
-Colony morphology (medium: Nutrient agar, culture time: 48 hours, culture temperature: 30 ° C)
Diameter: 2.0-3.0mm
Color tone: Cream color Shape: Circular Raised state: Lens shape Perimeter: Thread shape Surface shape, etc .: Smooth Transparency: Opaque Consistency: Butter-like
〔生育温度〕
・３７℃：＋
・４５℃：＋
[Growth temperature]
・ 37 ℃: +
・ 45 ℃: +
・カタラーゼ反応：＋
・オキシダーゼ反応：＋
・グルコースからの酸／ガス産生（酸産生／ガス産生）：−／−
・グルコースのＯ／Ｆテスト（酸化／発酵）：−／−
Catalase reaction: +
・ Oxidase reaction: +
Acid / gas production from glucose (acid production / gas production):-/-
Glucose O / F test (oxidation / fermentation):-/-
〔発酵性試験〕
・グリセロール：＋
・エリスリトール：−
・Ｄ−アラビノース：−
・Ｌ−アラビノース：＋
・リボース：＋
・Ｄ−キシロース：＋
・Ｌ−キシロース：−
・アドニトール：−
・β−メチル−Ｄ−キシロース：−
・ガラクトース：＋
・グルコース：＋
・フラクトース：＋
・マンノース：＋
・ソルボース：−
・ラムノース：−
・ズルシトール：−
・イノシトール：−
・マンニトール：＋
・ソルビトール：＋
・α−メチル−Ｄ−マンノシド：−
・α−メチル−Ｄ−グルコシド：＋
・Ｎ−アセチルグルコサミン：−
・アミグダリン：＋
・アルブチン：＋
・エスクリン：＋
・サリシン：＋
・セロビオース：＋
・マルトース：＋
・ラクトース：＋
・メリビオース：−
・サッカロース：＋
・トレハロース：＋
・イヌリン：＋
・メレチトース：−
・ラフィノース：＋
・でんぷん：−
・グリコーゲン：−
・キシリトール：−
・ゲンチオビオース：＋
・Ｄ−ツラノース：−
・Ｄ−リキソース：−
・Ｄ−タガトース：−
・Ｄ−フコース：−
・Ｌ−フコース：−
・Ｄ−アラビトール：−
・Ｌ−アラビトール：−
・グルコネート：−
・２−ケトグルコネート：−
・５−ケトグルコネート：−
[Fermentation test]
・ Glycerol: +
・ Erythritol:-
・ D-arabinose:-
・ L-arabinose: +
・ Ribose: +
D-xylose: +
・ L-xylose:-
・ Adonitol:-
.Beta.-methyl-D-xylose:-
・ Galactose: +
・ Glucose: +
・ Fructose: +
・ Mannose: +
・ Sorbose:-
・ Rhamnose:-
・ Zulcitol:-
・ Inositol:-
・ Mannitol: +
・ Sorbitol: +
Α-methyl-D-mannoside: −
Α-methyl-D-glucoside: +
・ N-acetylglucosamine:-
・ Amygdalin: +
・ Arbutin: +
・ Esculin: +
-Salicin: +
・ Cellobiose: +
-Maltose: +
・ Lactose: +
・ Meribiose:-
・ Sucrose: +
・ Trehalose: +
・ Inulin: +
・ Meletitol:-
・ Raffinose: +
・ Starch:-
・ Glycogen:-
・ Xylitol:-
・ Gentiobiose: +
・ D-Turanorose:-
・ D-lyxose:-
・ D-Tagatose:-
・ D-Fucose:-
・ L-Fucose:-
・ D-arabitol:-
・ L-arabitol:-
・ Gluconate:-
2-ketogluconate:-
・ 5-ketogluconate:-
〔生化学試験〕
・β−ガラクトシダーゼ：−
・アルギニンジヒドロラーゼ：−
・リシンデカルボキシラーゼ：−
・オルニチンデカルボキシラーゼ：−
・クエン酸の利用性：−
・Ｈ2Ｓ産生：−
・ウレアーゼ：−
・トリプトファンデアミナーゼ：−
・インドール産生：−
・アセトイン産生（ＶＰ）：−
・ゼラチナーゼ：＋
・硝酸塩還元：−
[Biochemical test]
.Beta.-galactosidase:-
Arginine dihydrolase:-
-Lysine decarboxylase:-
Ornithine decarboxylase:-
・ Utility of citric acid:-
・ H 2 S production: −
・ Urease:-
Tryptophan deaminase:-
・ Indole production:-
Acetoin production (VP):-
・ Gelatinase: +
・ Nitrate reduction:-
・嫌気条件下での生育：＋（弱い）
・５０℃での生育：＋
・１０％ ＮａＣｌでの生育：−
・カゼインの加水分解：＋
・でんぷんの加水分解：−
・ Growth under anaerobic conditions: + (weak)
・ Growth at 50 ℃: +
・ Growth with 10% NaCl:-
Casein hydrolysis: +
・ Starch hydrolysis:-
・１６ｒＤＮＡ塩基配列の相同性
Bacillus amyloliquefaciens：９９．８％
・ Homology of 16rDNA base sequence
Bacillus amyloliquefaciens: 99.8%
以上の結果より、SAS-1946502 (NITE P-02327)はBacillus amyloliquefaciensに近縁なBacillus sp.と同定された。 From the above results, SAS-1946502 (NITE P-02327) was identified as a Bacillus sp. Closely related to Bacillus amyloliquefaciens.
本発明の微生物を培養する方法としては、好気性微生物の培養方法として一般的な方法をそのまま採用することができる。例えば、適当な炭素源等を含有する液体培地を用いて、通気及び撹拌して培養することができる。培養温度としては、例えば５℃〜４０℃、好ましくは１５℃〜４０℃、より好ましくは２５℃〜３７℃の範囲を選択することができる。 As a method for culturing the microorganism of the present invention, a general method for culturing aerobic microorganisms can be employed as it is. For example, using a liquid medium containing an appropriate carbon source or the like, it can be cultured with aeration and agitation. As culture | cultivation temperature, the range of 5 to 40 degreeC, for example, Preferably 15 to 40 degreeC, More preferably, 25 to 37 degreeC can be selected.
本発明の活性汚泥の処理方法は、アセチルトランスフェラーゼを生産する第１の微生物を活性汚泥に添加及び接触させて、第１の微生物が生産したアセチルトランスフェラーゼを前記活性汚泥に作用させ、前記活性汚泥中に含まれるアミノ酸又はペプチドのアミノ基をアセチル化する第１の工程を包含する。
また本発明の汚泥発生量の抑制方法は、上記の活性汚泥の処理方法によって活性汚泥を処理し、汚泥発生量を抑制するものである。
In the activated sludge treatment method of the present invention, the first microorganism producing acetyltransferase is added to and contacted with activated sludge, and the activated sludge produced by the first microorganism is allowed to act on the activated sludge. The 1st process of acetylating the amino group of the amino acid or peptide contained in is included.
Moreover, the sludge generation amount suppression method of the present invention treats activated sludge by the above-described activated sludge processing method, and suppresses the sludge generation amount.
第１の微生物を活性汚泥に添加及び接触させる方法としては、第１の微生物を活性汚泥に添加して組み込み、他微生物と共存（共生）させることが挙げられる。すなわち、第１の微生物を活性汚泥に添加し、活性汚泥内で安定的に維持されるよう調整する。
第１の微生物の活性汚泥への添加は、１回だけ行ってもよいし、複数回行ってもよい。
As a method for adding and contacting the first microorganism to the activated sludge, the first microorganism can be added to and incorporated in the activated sludge and coexist with other microorganisms (symbiosis). That is, the first microorganism is added to the activated sludge and adjusted so as to be stably maintained in the activated sludge.
The addition of the first microorganism to the activated sludge may be performed only once or a plurality of times.
第１の微生物としては、アセチルトランスフェラーゼを生産する微生物であれば特に限定はない。第１の微生物は、アセチルトランスフェラーゼを細胞外に分泌する微生物であることが好ましい。ただし、アセチルトランスフェラーゼが細胞内に保持されている場合でも、第１の工程が有効に行われることが十分に期待できる。なぜならば、アミノ酸レベルの物質は、微生物の細胞内外を自由に透過できることがアミノ酸発酵により明確化されているからである。
また第１の微生物は、Curtobacterium属に属する微生物であることが好ましく、SAS-1946503 (NITE P-02328)であることが特に好ましい。
The first microorganism is not particularly limited as long as it is a microorganism that produces acetyltransferase. The first microorganism is preferably a microorganism that secretes acetyltransferase outside the cell. However, even when acetyltransferase is retained in the cell, it can be sufficiently expected that the first step is effectively performed. This is because amino acid fermentation has clarified that substances at the amino acid level can freely penetrate inside and outside the cells of microorganisms.
The first microorganism is preferably a microorganism belonging to the genus Curtobacterium, and particularly preferably SAS-1946503 (NITE P-02328).
第１の工程では、第１の微生物が生産したアセチルトランスフェラーゼの作用により、活性汚泥中のアミノ酸やペプチドのアミノ基をアセチル化する。これにより、アミノ酸とペプチドが微生物に資化され難くなり、結果的に活性汚泥の発生量が抑えられる。 In the first step, the amino group of the activated sludge and the amino group of the peptide are acetylated by the action of the acetyltransferase produced by the first microorganism. Thereby, amino acids and peptides are hardly assimilated by microorganisms, and as a result, the generation amount of activated sludge is suppressed.
好ましい実施形態では、プロテアーゼを生産する第２の微生物を活性汚泥に添加及び接触させて、第２の微生物が生産したプロテアーゼを活性汚泥に作用させる第２の工程をさらに行う。 In preferable embodiment, the 2nd microorganisms which produce protease are added and made to contact with activated sludge, and the 2nd process of making protease produced by the 2nd microorganisms act on activated sludge is further performed.
第２の微生物を活性汚泥に添加及び接触させる方法としては、第２の微生物を活性汚泥に添加して組み込み、他微生物と共存（共生）させることが挙げられる。例えば、第１の微生物とともに第２の微生物を活性汚泥に添加し、活性汚泥内で安定的に維持されるよう調整する。
第２の微生物の添加は、１回だけ行ってもよいし、複数回行ってもよい。また第２の微生物の添加は、第１の微生物の添加と同時に行ってもよいし、別々に行ってもよい。
As a method for adding and contacting the second microorganism to the activated sludge, it is possible to add the second microorganism to the activated sludge and incorporate it so as to coexist with other microorganisms (symbiosis). For example, the second microorganism is added to the activated sludge together with the first microorganism so as to be stably maintained in the activated sludge.
The addition of the second microorganism may be performed only once or a plurality of times. The addition of the second microorganism may be performed simultaneously with the addition of the first microorganism, or may be performed separately.
第２の微生物としては、プロテアーゼを生産する微生物であれば特に限定はない。第２の微生物は、プロテアーゼを細胞外に分泌する微生物であることが好ましい。第２の微生物は、Bacillus属に属する微生物であることが好ましく、SAS-1946502 (NITE P-02327)であることが特に好ましい。 The second microorganism is not particularly limited as long as it is a microorganism that produces protease. The second microorganism is preferably a microorganism that secretes a protease outside the cell. The second microorganism is preferably a microorganism belonging to the genus Bacillus, and particularly preferably SAS-1946502 (NITE P-02327).
第２の工程では、第２の微生物が生産したプロテアーゼの作用により、活性汚泥中に存在する微生物を溶菌させる。溶菌させることにより、微生物内のアミノ酸とペプチドを活性汚泥中に積極的に放出させる。そして活性汚泥中のアミノ酸とペプチドは、第１の工程によってアミノ基がアセチル化される。 In the second step, the microorganisms present in the activated sludge are lysed by the action of the protease produced by the second microorganism. By lysis, amino acids and peptides in the microorganism are actively released into the activated sludge. In the activated sludge, amino groups and peptides are acetylated in the first step.
本発明の活性汚泥の処理方法は、回分法、連続法のいずれの手法にも用いることができる。例えば、活性汚泥に第１の微生物と第２の微生物を添加して活性汚泥に組み込む。そして、第１の微生物と第２の微生物が活性汚泥中で安定的に維持されることにより、廃水の連続処理に対応することができる。 The activated sludge treatment method of the present invention can be used for both batch and continuous methods. For example, the first microorganism and the second microorganism are added to the activated sludge and incorporated into the activated sludge. And it can respond to a continuous treatment of wastewater by the 1st microorganisms and the 2nd microorganisms being stably maintained in activated sludge.
以下、実施例をもって本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
（１）新規微生物のスクリーニング
余剰汚泥発生量が少なく（余剰汚泥引き抜き量が少なく）かつ汚泥沈降性が良好（ＳＶ６０≒５５〜６５）な活性汚泥実処理施設における硝化槽、返送汚泥槽、調整槽の汚泥を分離源とした。特にプロテアーゼを生産する第２の微生物の選抜については、各汚泥に８０℃、３０分間のヒートショックを与え、各汚泥の上清を含有する平板培地にて複数の微生物を分離した。アセチルトランスフェラ―ゼを生産する第１の微生物の選抜については、ヒートショックを行わず室温にて複数の微生物を分離した。これらの分離により、強力にアセチルトランスフェラーゼ生産能を有する微生物と強力にプロテアーゼ生産能を有する微生物を各々１つ選抜した。
(1) Screening for new microorganisms Nitrification tanks, return sludge tanks, and adjustment tanks in an activated sludge actual treatment facility with little excess sludge generation (small excess sludge extraction amount) and good sludge settling (SV60 ≒ 55-65) The sludge was used as the separation source. In particular, for the selection of the second microorganism that produces protease, each sludge was subjected to heat shock at 80 ° C. for 30 minutes, and a plurality of microorganisms were separated on a plate medium containing the supernatant of each sludge. For the selection of the first microorganism producing acetyltransferase, a plurality of microorganisms were separated at room temperature without heat shock. By the separation, one microorganism having a strong acetyltransferase-producing ability and one microorganism having a strong protease-producing ability were selected.
選抜されたアセチルトランスフェラーゼ生産微生物は、上述した形態と生理・生化学的性状を有していた。さらに、ｒＤＮＡの塩基配列を解析したところ、配列番号１に示す塩基配列を有しており、Curtobacterium flaccumfaciensの当該配列と９９．９％の相同性を有していた。以上の結果より、アセチルトランスフェラーゼ生産微生物はCurtobacterium flaccumfaciensと同定された。当該微生物を「SAS-1946503」と命名し、独立行政法人 製品評価技術基盤機構 特許微生物寄託センターに寄託した（NITE P-02328）。 The selected acetyltransferase-producing microorganism had the above-described form and physiological / biochemical properties. Furthermore, when the base sequence of rDNA was analyzed, it had the base sequence shown in SEQ ID NO: 1 and had 99.9% homology with the sequence of Curtobacterium flaccumfaciens. From the above results, the acetyltransferase-producing microorganism was identified as Curtobacterium flaccumfaciens. The microorganism was named “SAS-1946503” and deposited with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation (NITE P-02328).
選抜されたプロテアーゼ生産微生物は、上述した形態と生理・生化学的性状を有していた。さらに、ｒＤＮＡの塩基配列を解析したところ、配列番号２に示す塩基配列を有しており、Bacillus amyloliquefaciensの当該配列と９９．８％の相同性を有していた。以上の結果より、プロテアーゼ生産微生物はBacillus amyloliquefaciensに近縁なBacillus sp.と同定された。当該微生物を「SAS-1946502」と命名し、独立行政法人 製品評価技術基盤機構 特許微生物寄託センターに寄託した（NITE P-02327）。 The selected protease-producing microorganism had the above-described form and physiological / biochemical properties. Furthermore, when the base sequence of rDNA was analyzed, it had the base sequence shown in SEQ ID NO: 2 and had 99.8% homology with the relevant sequence of Bacillus amyloliquefaciens. From the above results, the protease-producing microorganism was identified as Bacillus sp. Closely related to Bacillus amyloliquefaciens. The microorganism was named “SAS-1946502” and deposited with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation (NITE P-02327).
（２）ハロー形成試験
上記の実処理の活性汚泥（返送汚泥槽）を遠心分離（10,000rpm、10分間）した後、フィルター（ADVANTEC No.1 保留粒子径:6.0μm）でろ過した溶液（返送汚泥上清）を調製した。
表１に示す組成の基本培地に返送汚泥上清を最終濃度１０％となるよう添加した液体培地（以下、「返送汚泥上清含有培地」を称する）に、SAS-1946502（目的菌）と上記の活性汚泥より分離同定した大腸菌（宿主菌）を１：１の割合で植菌し、数日間振とう培養した（目的菌の前培養）。
(2) Halo formation test The activated sludge (return sludge tank) of the above actual treatment was centrifuged (10,000 rpm, 10 minutes), and then filtered through a filter (ADVANTEC No.1 retained particle size: 6.0 μm) (return) Sludge supernatant) was prepared.
SAS-1946502 (target fungus) and the above were added to a liquid medium (hereinafter referred to as “returned sludge supernatant-containing medium”) in which the return sludge supernatant was added to the basic medium having the composition shown in Table 1 to a final concentration of 10%. Escherichia coli (host fungus) separated and identified from the activated sludge was inoculated at a ratio of 1: 1 and cultured with shaking for several days (preculture of the target fungus).
１％ジェランガムを含む返送汚泥上清含有培地の平板培地に、上記の大腸菌（宿主菌）の培養液２０μＬをアプライし、スプレッダーで均一に塗り広げた。この操作を２回繰り返した（培養液４０μＬ分）。平板培地を３０℃で１日間静置培養した（宿主菌の前培養）。 20 μL of the above E. coli (host fungus) culture solution was applied to a plate medium of a return sludge supernatant-containing medium containing 1% gellan gum and spread evenly with a spreader. This operation was repeated twice (for 40 μL of culture solution). The plate medium was cultivated at 30 ° C. for 1 day (host culture).
静置培養後の平板培地に、目的菌の前培養液を１プレートあたり１０μＬ×３スポットとしてアプライした。クリーンベンチ内でスポット面が垂れない程度に乾燥させた後、３０℃で静置し、ハロー形成を経時観察した。結果を図１に示す。図１（ａ）〜図１（ｄ）はそれぞれ静置後０時間（試験開始時）、２４時間、４８時間、７２時間経過時の平板培地の写真である。
すなわち、静置後２４時間で宿主菌の溶菌に起因するハロー形成が認められ（図１（ｂ））、４８時間経過時にはシャーレの広範囲まで行き渡る強いハロー形成が認められた（図１（ｃ））。
The preculture solution of the target bacteria was applied as 10 μL × 3 spots per plate to the plate medium after stationary culture. After drying to such an extent that the spot surface does not sag in the clean bench, the plate was allowed to stand at 30 ° C., and halo formation was observed over time. The results are shown in FIG. FIG. 1 (a) to FIG. 1 (d) are photographs of the plate medium after standing for 0 hour (at the start of the test), 24 hours, 48 hours, and 72 hours, respectively.
That is, halo formation due to lysis of the host bacteria was observed 24 hours after standing (FIG. 1 (b)), and strong halo formation reaching a wide range of the petri dish was observed after 48 hours (FIG. 1 (c)). ).
（３）プロテアーゼ活性測定
返送汚泥上清含有培地にSAS-1946502を植菌し、３０℃で１日間振とう培養した。培養液を遠心分離し、上清を回収した。Ａｍｐｌｉｔｅプロテアーゼ測定キット（コスモバイオ社）を用い、下記手順で上清のプロテアーゼ活性を測定した。基質としてカゼインの蛍光接合体を用いた。陽性コントロールとしてトリプシンを用いた。
(3) Protease activity measurement SAS-1946502 was inoculated into the medium containing the returned sludge supernatant and cultured with shaking at 30 ° C for 1 day. The culture solution was centrifuged and the supernatant was collected. The protease activity of the supernatant was measured by the following procedure using Amplite protease measurement kit (Cosmo Bio). A fluorescent conjugate of casein was used as a substrate. Trypsin was used as a positive control.
９６穴ブラックプレートの各穴に試料５０μＬと基質希釈液５０μＬを入れ、混合した。蛍光プレートリーダー（SynergyTM H1 (BIOTeK) Gen5 2.01）を用いて、励起波長４９０ｎｍ、蛍光波長５２５ｎｍの条件で蛍光増加量を検出し、５分毎に３０分間、蛍光強度を測定した。この間、暗室で３０℃の条件で酵素反応させた。
その結果、SAS-1946502の上清に強いプロテアーゼ活性が認められた。陽性コントロールのトリプシンのプロテアーゼ活性を１００Ｕ／ｍＬとした場合、SAS-1946502の上清のプロテアーゼ活性は４７Ｕ／ｍＬであった。なお、トリプシンは高純度の精製品であるのに対し、SAS-1946502の上清は未精製の培養液である。
50 μL of the sample and 50 μL of the substrate diluent were placed in each hole of the 96-well black plate and mixed. Using a fluorescence plate reader (Synergy ™ H1 (BIOTeK) Gen5 2.01), an increase in fluorescence was detected under the conditions of an excitation wavelength of 490 nm and a fluorescence wavelength of 525 nm, and the fluorescence intensity was measured every 5 minutes for 30 minutes. During this time, the enzyme reaction was carried out in the dark at 30 ° C.
As a result, strong protease activity was observed in the supernatant of SAS-1946502. When the protease activity of the positive control trypsin was 100 U / mL, the protease activity of the supernatant of SAS-1946502 was 47 U / mL. Note that trypsin is a highly purified product, whereas the supernatant of SAS-1946502 is an unpurified culture solution.
（４）薄層クロマトグラフィーによるアセチルトランスフェラーゼ活性確認＜１＞
返送汚泥上清含有培地にSAS-1946503を１白金耳植菌し、３０℃で１日間振とう培養した（前培養）。３００ｍＬ容の三角フラスコ（バッフル付）に返送汚泥上清含有培地１００ｍＬを仕込み、前培養液１００μＬを添加して、３０℃で１日間振とう培養した（本培養）。
本培養液を遠心分離して上清を廃棄し、菌体を得た。菌体に０．１Ｍ リン酸緩衝液（ｐＨ７．０）と３，４−ジクロロアニリン（３，４−ＤＣＡ）とアセチル−ＣｏＡリチウム塩を添加して、３０℃で１日振とうした（酵素反応）。１Ｎ ＮａＯＨを添加してｐＨを１０〜１１にすることにより酵素反応を停止させた。
同量のジエチルエーテルを添加し、ボルテックスミキサーで３０分間攪拌した後、室温で１日静置した。自然分離した上澄み（有機相）を回収し、TLC Silica gel 60 F254 (Millipore)にスポットした。展開液（クロロホルム：アセトン＝９１：９）で展開した後に、紫外線でスポットを検出した。結果を図２に示す。図２中、（ａ）は３，４−ＤＣＡ（コントロール）、（ｂ）は測定試料をスポットして展開したものである。
すなわち測定試料から、３，４−ＤＣＡのアミノ基がアセチル化されて生成した３，４−ジクロロアセトアニリン（３，４−ＤＣＡＡ）のスポットが検出された。これにより、SAS-1946503がアミノ基をアセチル化するアセチルトランスフェラーゼを生産することが示された。
(4) Confirmation of acetyltransferase activity by thin layer chromatography <1>
One platinum ear of SAS-1946503 was inoculated into the return sludge supernatant-containing medium and cultured with shaking at 30 ° C. for 1 day (pre-culture). A 300 mL Erlenmeyer flask (with a baffle) was charged with 100 mL of the returned sludge supernatant-containing medium, 100 μL of the preculture was added, and cultured with shaking at 30 ° C. for 1 day (main culture).
The culture broth was centrifuged and the supernatant was discarded to obtain bacterial cells. 0.1 M phosphate buffer (pH 7.0), 3,4-dichloroaniline (3,4-DCA), and acetyl-CoA lithium salt were added to the cells and shaken at 30 ° C. for 1 day (enzyme). reaction). The enzyme reaction was stopped by adding 1N NaOH to bring the pH to 10-11.
The same amount of diethyl ether was added, and the mixture was stirred for 30 minutes with a vortex mixer and then allowed to stand at room temperature for 1 day. The supernatant (organic phase) separated naturally was collected and spotted on TLC Silica gel 60 F254 (Millipore). After developing with a developing solution (chloroform: acetone = 91: 9), spots were detected with ultraviolet rays. The results are shown in FIG. In FIG. 2, (a) shows 3,4-DCA (control), and (b) shows a sample developed by spotting.
That is, a spot of 3,4-dichloroacetoaniline (3,4-DCAA) generated by acetylation of the amino group of 3,4-DCA was detected from the measurement sample. This showed that SAS-1946503 produces an acetyltransferase that acetylates the amino group.
（５）薄層クロマトグラフィーによるアセチルトランスフェラーゼ活性確認＜２＞
返送汚泥上清含有培地にSAS-1946503を１白金耳植菌し、３０℃で１日間振とう培養した（前培養）。３００ｍＬ容の三角フラスコ（バッフル付）に返送汚泥上清含有培地１００ｍＬを仕込み、前培養液１００μＬを添加して、３０℃で３日間振とう培養した（本培養）。
本培養液を遠心分離して沈殿以外の上清を得た。上清に３，４−ＤＣＡとアセチル−ＣｏＡリチウム塩を添加して、３０℃で１日振とうした（酵素反応）。１Ｎ ＮａＯＨを添加してｐＨを１０〜１１にすることにより酵素反応を停止させた。別途、１Ｎ ＮａＯＨを添加しない（酵素反応を停止させない）試料も作製した。
ジエチルエーテルを１０ｍＬ添加し、ボルテックスミキサーで３０分間攪拌した後、上相（ジエチルエーテル層）を別の３０ｍＬ褐色ビンに移した。下相（水層）を再びジエチルエーテルで抽出し、上相（ジエチルエーテル層）を３０ｍＬ褐色ビンに移した。ジエチルエーテルで２回抽出した溶液を熱水（１００℃程度）で加熱濃縮し、TLC Silica gel 60 F254 (Millipore)にスポットした。展開液（クロロホルム：アセトン＝９１：９）で展開した後に、紫外線でスポットを検出した。結果を図３に示す。図３中、（ａ）は３，４−ＤＣＡと３，４−ＤＣＡＡの混合物（コントロール）、（ｂ）は３，４−ＤＣＡ（コントロール）、（ｃ）は１Ｎ ＮａＯＨを添加しなかった測定試料、（ｄ）は１Ｎ ＮａＯＨを添加した測定試料をスポットして展開したものである。
すなわち（ｃ）と（ｄ）の測定試料から、３，４−ＤＣＡのアミノ基がアセチル化されて生成した３，４−ＤＣＡＡのスポットが検出された。これにより、SAS-1946503がアミノ基をアセチル化するアセチルトランスフェラーゼを生産し、細胞外に分泌することが示された。
(5) Confirmation of acetyltransferase activity by thin layer chromatography <2>
One platinum ear of SAS-1946503 was inoculated into the return sludge supernatant-containing medium and cultured with shaking at 30 ° C. for 1 day (pre-culture). A 300 mL Erlenmeyer flask (with a baffle) was charged with 100 mL of the returned sludge supernatant-containing medium, 100 μL of the preculture was added, and cultured with shaking at 30 ° C. for 3 days (main culture).
The culture broth was centrifuged to obtain a supernatant other than the precipitate. 3,4-DCA and acetyl-CoA lithium salt were added to the supernatant and shaken at 30 ° C. for 1 day (enzyme reaction). The enzyme reaction was stopped by adding 1N NaOH to bring the pH to 10-11. Separately, a sample in which 1N NaOH was not added (enzymatic reaction was not stopped) was also prepared.
After adding 10 mL of diethyl ether and stirring with a vortex mixer for 30 minutes, the upper phase (diethyl ether layer) was transferred to another 30 mL brown bottle. The lower phase (aqueous layer) was extracted again with diethyl ether, and the upper phase (diethyl ether layer) was transferred to a 30 mL brown bottle. The solution extracted twice with diethyl ether was concentrated by heating with hot water (about 100 ° C.) and spotted on TLC Silica gel 60 F254 (Millipore). After developing with a developing solution (chloroform: acetone = 91: 9), spots were detected with ultraviolet rays. The results are shown in FIG. In FIG. 3, (a) is a mixture of 3,4-DCA and 3,4-DCAA (control), (b) is 3,4-DCA (control), and (c) is a measurement in which 1N NaOH was not added. Sample (d) is developed by spotting a measurement sample to which 1N NaOH has been added.
That is, a spot of 3,4-DCAA produced by acetylation of the amino group of 3,4-DCA was detected from the measurement samples of (c) and (d). Thus, it was shown that SAS-1946503 produces acetyltransferase that acetylates the amino group and secretes it outside the cell.
（６）活性汚泥の処理実験
SAS-1946503（NITE P-02328）とSAS-1946502（NITE P-02327）を、それぞれ表１に示す基本培地を用いて３０℃で４８時間振とう培養した。得られた各培養液を遠心分離して菌体を集め、生理食塩水で２回洗浄した。洗浄した各菌体を生理食塩水で懸濁し（濁度：SAS-1946503：OD＝13.6、SAS-1946502：OD＝0.87）、SAS-1946503とSAS-1946502の各菌体懸濁液を調製した。表２の組成からなる原水１Ｌに、各菌体懸濁液を１０ｍＬずつ添加した。表２において、活性汚泥は、活性汚泥浮遊物質（ＭＬＳＳ）が９０００ｍｇ／Ｌの濃縮汚泥を終濃度２００ｍｇ／Ｌとなるように添加した。
(6) Treatment experiment of activated sludge
SAS-1946503 (NITE P-02328) and SAS-1946502 (NITE P-02327) were each cultured with shaking at 30 ° C. for 48 hours using the basic media shown in Table 1. Each culture solution obtained was centrifuged to collect the cells and washed twice with physiological saline. Each washed cell was suspended in physiological saline (turbidity: SAS-1946503: OD = 13.6, SAS-1946502: OD = 0.87), and each cell suspension of SAS-1946503 and SAS-1946502 was prepared. . 10 mL of each bacterial cell suspension was added to 1 L of raw water having the composition shown in Table 2. In Table 2, the activated sludge was added so that the activated sludge suspended solids (MLSS) was 9000 mg / L and the concentrated sludge had a final concentration of 200 mg / L.
菌体懸濁液を添加した原水について、スターラーを用いて通気攪拌処理（２５℃、２５０ｒｐｍ）を行った。処理開始から０時間、２時間、１６時間、２４時間、４０時間経過時における汚泥濃度を、ガラス繊維ろ紙法によって測定し、各処理時間における汚泥減容率を算出した。コントロールとして、菌体懸濁液を添加しない原水を用いて同様の操作を行った。結果を表３に示す。 The raw water to which the bacterial cell suspension was added was subjected to aeration and stirring treatment (25 ° C., 250 rpm) using a stirrer. The sludge concentration at the time of 0 hours, 2 hours, 16 hours, 24 hours, and 40 hours from the start of the treatment was measured by the glass fiber filter paper method, and the sludge volume reduction rate at each treatment time was calculated. As a control, the same operation was performed using raw water to which no cell suspension was added. The results are shown in Table 3.
すなわち、菌体懸濁液を添加した場合（微生物添加）には、時間の経過とともに汚泥が減容し、２４時間経過時には２１％の減容率を示した。４０時間経過時でも１５％の減容率を示しており、微生物溶菌後の再増殖が抑制されていた。
一方、コントロールでは２４時間経過時に減容率が０％となり、溶菌後に微生物が再増殖していた。
以上より、SAS-1946503（NITE P-02328）とSAS-1946502（NITE P-02327）を活性汚泥に添加することにより、汚泥発生量を抑制できることが明らかとなった。
That is, when the bacterial cell suspension was added (microorganism addition), the volume of sludge decreased with the passage of time, and a volume reduction rate of 21% was exhibited after 24 hours. Even after 40 hours, the volume reduction rate was 15%, and regrowth after microbial lysis was suppressed.
On the other hand, in the control, the volume reduction rate was 0% after 24 hours, and the microorganisms re-growth after lysis.
From the above, it was revealed that the amount of sludge generated can be suppressed by adding SAS-1946503 (NITE P-02328) and SAS-1946502 (NITE P-02327) to the activated sludge.
（７）アセチルトランスフェラーゼの基質特異性
SAS-1946503（NITE P-02328）を、表１に示す基本培地を用いて３０℃で２４時間振とう培養した。培養液を遠心分離し、得られた上清を粗酵素液とした。遠心式限外ろ過フィルター(30K, Merck Millipore Ltd.)を用いて、粗酵素液１５ｍＬを５００μＬ程度まで濃縮した。得られた濃縮液を脱イオン水で５倍希釈したものを濃縮粗酵素液とした。
コントロールとして、粗酵素液に代えて基本培地を用いて同様の操作を行い、ブランク用濃縮液を得た。
(7) Substrate specificity of acetyltransferase
SAS-1946503 (NITE P-02328) was cultured with shaking in the basic medium shown in Table 1 at 30 ° C. for 24 hours. The culture solution was centrifuged, and the resulting supernatant was used as a crude enzyme solution. Using a centrifugal ultrafiltration filter (30K, Merck Millipore Ltd.), 15 mL of the crude enzyme solution was concentrated to about 500 μL. The obtained concentrated solution was diluted 5-fold with deionized water to obtain a concentrated crude enzyme solution.
As a control, the same operation was performed using a basic medium in place of the crude enzyme solution to obtain a blank concentrate.
濃縮粗酵素液１ｍＬに各種Ｌ−アミノ酸を２０〜２５ｍｇ、アセチル基供与体としてアセチルコエンザイムＡ三リチウム塩約０．５ｍｇを添加して、インキュベーターシェーカーにて３０℃で２４時間反応させた。ブランク用濃縮液を用いて同様の反応を行ったものをブランク溶液とした。反応終了後、反応液を脱イオン水で４倍に希釈し、遠心式限外ろ過フィルター(3K, Merck Millipore Ltd.)で２回ろ過した。得られたろ過液を、高速液体クロマトグラフィー／質量分析（ＬＣ／ＭＳ）を行うまで４℃で保存した。 To 1 mL of the concentrated crude enzyme solution, 20 to 25 mg of various L-amino acids and about 0.5 mg of acetyl coenzyme A trilithium salt as an acetyl group donor were added and reacted at 30 ° C. for 24 hours in an incubator shaker. What performed the same reaction using the concentrate for blanks was made into the blank solution. After completion of the reaction, the reaction solution was diluted 4 times with deionized water and filtered twice with a centrifugal ultrafiltration filter (3K, Merck Millipore Ltd.). The obtained filtrate was stored at 4 ° C. until high performance liquid chromatography / mass spectrometry (LC / MS) was performed.
ろ過液を３０％（ｖ／ｖ）アセトニトリルで希釈し、遠心分離した。上清を孔径０．２０μｍのフィルターでろ過し、ＬＣ／ＭＳで分析した。得られたデータを解析ソフト(Bruker Daltonics Data Analysis)でデータ処理を行い、粗酵素液ピークと標準試料ピークを比較することで、アセチルアミノ酸のピークを判別した。そして、粗酵素液ピークとブランク溶液ピークを比較し、アセチルトランスフェラーゼの影響を検討した。ブランク溶液ピークに対する粗酵素液ピークのピーク面積比を算出し、ピーク面積比２．０以上を「○：アセチル化あり」、ピーク面積比１．０超２．０未満を「△：アセチル化あり（弱い）」、１．０以下を「×：アセチル化なし」と評価した。 The filtrate was diluted with 30% (v / v) acetonitrile and centrifuged. The supernatant was filtered through a 0.20 μm pore size filter and analyzed by LC / MS. The obtained data was processed with analysis software (Bruker Daltonics Data Analysis), and the peak of the acetylamino acid was determined by comparing the crude enzyme solution peak with the standard sample peak. And the crude enzyme liquid peak and the blank solution peak were compared, and the influence of acetyltransferase was examined. The peak area ratio of the crude enzyme solution peak to the blank solution peak is calculated. A peak area ratio of 2.0 or more is “◯: with acetylation”, and a peak area ratio of more than 1.0 and less than 2.0 is “Δ: with acetylation” (Weak) ”and 1.0 or less were evaluated as“ ×: no acetylation ”.
＜ＬＣ／ＭＳの条件＞
・ＬＣ機器：Agilent 1100 Series
・ＭＳ機器：Bruker Esquire 3000 Plus
・ＬＣ条件：移動相：０．１％ギ酸アセトニトリル溶液／超純水＝３０／７０
試薬注入量：２μＬ
流速：０．２５ｍＬ／ｍｉｎ
温度：３０℃
・カラム：ジーエルサイエンスInertsil ODS-3 (３μｍ、３．０×１５０ｍｍ）
・ＭＳ条件：ＥＳＩ（エレクトロスプレーイオン化法）、Ion Polarity Positive
・解析ソフト：Bruker Daltonics Data Analysis
<Conditions of LC / MS>
・ LC equipment: Agilent 1100 Series
・ MS equipment: Bruker Esquire 3000 Plus
LC condition: mobile phase: 0.1% formic acid acetonitrile solution / ultra pure water = 30/70
Reagent injection volume: 2 μL
Flow rate: 0.25 mL / min
Temperature: 30 ° C
・ Column: GL Sciences Inertsil ODS-3 (3μm, 3.0 × 150mm)
MS conditions: ESI (electrospray ionization method), Ion Polarity Positive
・ Analysis software: Bruker Daltonics Data Analysis
結果を表４に示す。表４ではアミノ酸を酸性、塩基性、親水性、疎水性に分類している。すなわち、セリン、アスパラギン、ロイシン、イソロイシン、メチオニン、フェニルアラニン、及びトリプトファンについて、アミノ基のアセチル化が確認された。リジン、グルタミン、及びバリンについても、活性は弱かったがアミノ基のアセチル化が確認された。他のアミノ酸については、アミノ基のアセチル化は確認されなかった。 The results are shown in Table 4. Table 4 classifies amino acids into acidic, basic, hydrophilic, and hydrophobic properties. That is, acetylation of amino groups was confirmed for serine, asparagine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. Lysine, glutamine, and valine were also less active, but acetylation of the amino group was confirmed. For other amino acids, acetylation of the amino group was not confirmed.
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