JP5022044B2 - Method for producing new uricase - Google Patents
Method for producing new uricase Download PDFInfo
- Publication number
- JP5022044B2 JP5022044B2 JP2007013003A JP2007013003A JP5022044B2 JP 5022044 B2 JP5022044 B2 JP 5022044B2 JP 2007013003 A JP2007013003 A JP 2007013003A JP 2007013003 A JP2007013003 A JP 2007013003A JP 5022044 B2 JP5022044 B2 JP 5022044B2
- Authority
- JP
- Japan
- Prior art keywords
- uricase
- cellulosimicrobium
- strain
- culture
- genus
- 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.)
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Description
本発明は、新規なウリカーゼの製造方法に関する。より詳細には、本発明は、耐熱性に優れるセルロシミクロビウム属（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）に属する微生物由来の新規なウリカーゼの製造方法に関する。 The present invention relates to a novel method for producing uricase. More specifically, the present invention relates to a method for producing a novel uricase derived from a microorganism belonging to the genus Cellulosimicrobium having excellent heat resistance.
ウリカーゼ（ＥＣ１．７．３．３）は、尿酸をアラントイン、過酸化水素、および炭酸ガスに分解する、即ち、尿酸＋Ｏ２＋２Ｈ２Ｏ→アラントイン＋ＣＯ２＋Ｈ２Ｏ２の反応を触媒する、酸化的分解活性を有する酵素である。このウリカーゼは、種々の動物組織（例えば、肝臓、腎臓）中や微生物組織中に広く存在する。現在、ウリカーゼは、人体内の尿酸蓄積に起因する種々の疾患の臨床診断を目的とした、血液または尿中に存在する尿酸の測定用酵素として使用されている。 Uricase (EC 1.7.3.3) decomposes uric acid into allantoin, hydrogen peroxide, and carbon dioxide, ie, catalyzes the reaction of uric acid + O 2 + 2H 2 O → allantoin + CO 2 + H 2 O 2. It has an enzymatic degradation activity. This uricase is widely present in various animal tissues (eg, liver, kidney) and microbial tissues. Currently, uricase is used as an enzyme for measuring uric acid present in blood or urine for the purpose of clinical diagnosis of various diseases caused by uric acid accumulation in the human body.
ウリカーゼの微生物による生産は広く行われている（例えば、特許文献１〜３参照）。なかでも、特許文献１では、天然界から分離したトルロプシス属に属する酵母がウリカーゼを著量に生産することが提案されている。また、特許文献２では、多量のウリカーゼを生産するミクロコッカス・ロゼウスが開示され、該微生物が生産するウリカーゼの活性の最適条件は、他の細菌から製造されたウリカーゼと異なり、生理液のｐＨ値に近いことが見出されている。さらに、特許文献３では、バチルス・エスピー ＴＢ−９０菌株が、広い作用ｐＨ域を有する好熱性微生物由来のウリカーゼを生産することを開示している。
しかしながら、上記特許文献１〜３で得られるウリカーゼは、いずれも高温（６０℃以上）では不安定である。これは、臨床診断において保存している酵素製品の品質低下の原因となり得る。例えば、特許文献３で得られるウリカーゼは好熱性微生物由来であるが、５０℃を超えると失活が起こり、６０℃付近では、活性は約５０％程度維持されるのみである（第４図参照）。これは、臨床診断において保存している酵素製品の品質低下の原因となり得る。 However, all of the uricases obtained in Patent Documents 1 to 3 are unstable at high temperatures (60 ° C. or higher). This can cause a reduction in the quality of the stored enzyme product in clinical diagnosis. For example, the uricase obtained in Patent Document 3 is derived from a thermophilic microorganism, but inactivation occurs when the temperature exceeds 50 ° C., and the activity is only maintained at about 50% around 60 ° C. (see FIG. 4). ). This can cause a reduction in the quality of the stored enzyme product in clinical diagnosis.
したがって、本発明は、上記事情を鑑みてなされたものであり、耐熱性に優れた新規なウリカーゼの製造方法を提供することを目的とする。 Therefore, the present invention has been made in view of the above circumstances, and an object thereof is to provide a novel method for producing uricase having excellent heat resistance.
本発明者らは、上記課題を達成するために鋭意検討を行なった結果、耐熱性に優れたウリカーゼ生産能を有する微生物について、広範囲にわたり検索を行なった。その結果、自然界（土壌）から分離したセルロシミクロビウムに属する細菌（Ｕ−６８９株）が、既知のウリカーゼよりも耐熱性に優れていることを見出し、本発明を完成するに至った。 As a result of intensive studies in order to achieve the above-mentioned problems, the present inventors have extensively searched for microorganisms having a uricase-producing ability excellent in heat resistance. As a result, it was found that a bacterium (U-689 strain) belonging to cellulosimicrobium isolated from the natural world (soil) is superior in heat resistance to known uricase, and the present invention has been completed.
すなわち、上記目的は、セルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）属に属し、ウリカーゼの生産能を有する微生物を培養する工程（１）と、前記工程（１）で得られた培養物からウリカーゼを採取する工程（２）と、を含む、ウリカーゼの製造方法によって達成される。 That is, the above object is to collect a uricase from the step (1) of culturing a microorganism belonging to the genus Cellulosimicrobium and having the ability to produce uricase, and the culture obtained in the step (1). It is achieved by a method for producing uricase, comprising step (2).
本発明のウリカーゼは、６０℃で２０分間熱処理しても失活せず、耐熱性に優れるため、酵素製品の保存安定性が良好となり、臨床診断における酵素製品の長期保存が可能である。 The uricase of the present invention does not inactivate even when heat-treated at 60 ° C. for 20 minutes and is excellent in heat resistance. Therefore, the storage stability of the enzyme product is improved, and the enzyme product can be stored for a long time in clinical diagnosis.
以下本発明を詳細に説明する。 The present invention will be described in detail below.
本発明に用いられるウリカーゼ産生菌は、上記諸性質を有するウリカーゼを産生するものであれば、特に制限されないが、好ましくはセルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）属（以下、括弧内を省略する）に属し、より好ましくはセルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｓｐ．）Ｕ−６８９ ＦＥＲＭ−Ｐ２１０９９菌株（以下、該菌株を「Ｕ−６８９株」と称する）である。上記細菌は、ウリカーゼ生産能を有する限り、自然界に存在するものであっても、または人為的な遺伝子操作などによって変異を受けたものであってもよい。上記細菌によって産生されたウリカーゼは、菌体内に蓄積されてもよく、あるいは菌体内または菌体外に分泌されてもよいが、酵素の精製工程の簡略化を考慮すると、好ましくは菌体外（培養液中）に生産・蓄積する。 The uricase-producing bacterium used in the present invention is not particularly limited as long as it produces uricase having the above-mentioned properties, but preferably it belongs to the genus Cellulosimicrobium (hereinafter omitted in parentheses). More preferably, Cellulosimicrobium sp. U-689 FERM-P21099 strain (hereinafter referred to as “U-689 strain”). As long as the bacterium has the ability to produce uricase, the bacterium may exist in nature or may have been mutated by artificial genetic manipulation. The uricase produced by the bacterium may be accumulated in the microbial cell or secreted into or out of the microbial cell. However, considering simplification of the enzyme purification process, the uricase is preferably extracellular ( Produced and accumulated in the culture medium).
本発明に係るＵ−６８９株の性質について以下詳細に述べる。 The properties of the U-689 strain according to the present invention will be described in detail below.
Ｕ−６８９株は、本発明者らによって、岐阜県内の土壌から、尿酸を唯一の炭素源とした培地の集積培養により分離された菌種であり、平成１８年年１１月１７日付で独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ−２１０９９として寄託されている。 The U-689 strain is a bacterial species isolated from the soil in Gifu Prefecture by the present inventors by enrichment culture of a medium using uric acid as the sole carbon source. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number FERM P-21099.
また、上記Ｕ−６８９株は、以下の（a）形態的性質、（ｂ）培養的性質、（ｃ）生理学的性質、（ｄ）その他の性質を有する。下記表において、「＋」は、陽性を、「−」は、陰性を、および「ｗ」は、反応が弱いことを、それぞれ示す。 The U-689 strain has the following (a) morphological properties, (b) culture properties, (c) physiological properties, and (d) other properties. In the following table, “+” indicates positive, “−” indicates negative, and “w” indicates weak reaction.
ＢＬＡＳＴを用いた細菌基準株データベースに対する相同性検索の結果、Ｕ−６８９株の１６Ｓ ｒＤＮＡ塩基配列はセルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）由来の１６Ｓ ｒＤＮＡに対し高い相同性を示し、相同率９９．５％でセルロシミクロビウム・セルランス（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｃｅｌｌｕｌａｎｓ）ＤＳＭ４３８７９株の１６Ｓ ｒＤＮＡに対し最も高い相同性を示した。ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬに対する相同性検索の結果においても、本菌株の１６Ｓ ｒＤＮＡはセルロシミクロビウム由来の１６Ｓ ｒＤＮＡに対し高い相同性を示し、基準株ではセルロシミクロビウム・フンケイ（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｆｕｎｋｅｉ） ＡＴＣＣ ＢＡＡ−８８６株の１６Ｓ ｒＤＮＡに対し相同率９９．７％の相同性を示した。また、Ｕ−６８９株の１６Ｓ ｒＤＮＡと細菌基準株データベースに対する相同性検索上位３０株の１６Ｓ ｒＤＮＡにセルロシミクロビウム・フンケイの１６Ｓ ｒＤＮＡの加えて行なった簡易分子系統解析の結果、本菌株はセルロシミクロビウム・フンケイの１６Ｓ ｒＤＮＡと系統枝を形成し、近縁であることが示された。しかし両者の１６Ｓ ｒＤＮＡは完全には一致しておらず、本菌株がセルロシミクロビウム・フンケイに近縁であるものの、種としては同種になる可能性は低い。 As a result of homology search against a bacterial reference strain database using BLAST, the 16S rDNA base sequence of U-689 strain showed high homology to 16S rDNA derived from Cellulosimicrobium, and the homology rate was 99.5. % Showed the highest homology to the 16S rDNA of the Cellulosimicrobium cellulans DSM 43879 strain. Also in the results of the homology search for GenBank / DDBJ / EMBL, the 16S rDNA of this strain shows high homology to the 16S rDNA derived from cellulosimicrobium, and the reference strain is Cellulosimicrobium funkkei. ) A homology of 99.7% with the 16S rDNA of ATCC BAA-886 strain was shown. In addition, as a result of a simple molecular phylogenetic analysis performed by adding 16S rDNA of Cellulosimicrobium funkei to 16S rDNA of U-689 strain and 16S rDNA of the top 30 homology search against the bacterial reference strain database, It formed a phylogenetic branch with 16S rDNA of Cellulosimicrobium funky and was shown to be closely related. However, both 16S rDNAs do not completely match, and although this strain is closely related to Cellulosimicrobium funkei, it is unlikely to be the same species.
本菌株は運動性を有さないグラム陽性桿菌で、培養時間の経過に伴い形態が変化する「桿菌−球菌生活環［ロッド コッカス サイクル（ｒｏｄ−ｃｏｃｃｕｓ ｃｙｃｌｅ）］」を示した。Ｎｕｔｒｉｅｎｔ ａｇａｒ平板培地上でのコロニー色は黄色を呈し、培養一週間後には基底菌糸を形成した。カタラーゼ反応は陽性、オキシダーゼ反応は陰性を示し、硝酸塩を還元し、ゼラチンを加水分解し、グルコースおよびキシロースを発酵し、マンニトールを発酵しなかった。また、カゼインを加水分解し、Ｌ−アラビノース、グリセロールおよびマンノースを酸化し、Ｄ−ソルビトールを酸化しなかった。これらの性状は、１６Ｓ ｒＤＮＡ塩基配列解析において近縁性が示唆されたセルロシミクロビウム・フンケイやセルロシミクロビウム・セルランスの性状と一致すると考えられるが、運動性を示さない点においてはセルロシミクロビウム・フンケイよりもセルロシミクロビウム・セルランスを支持した。 This strain is a Gram-positive rod that has no motility, and exhibited a “rod-coccus cycle” whose morphology changes with the passage of time. The colony color on the Nutrient agar plate medium was yellow, and basal mycelium was formed after one week of culture. Catalase reaction was positive, oxidase reaction was negative, nitrate was reduced, gelatin was hydrolyzed, glucose and xylose were fermented, and mannitol was not fermented. In addition, casein was hydrolyzed, L-arabinose, glycerol and mannose were oxidized, and D-sorbitol was not oxidized. These properties are thought to be consistent with the properties of Cellulosimicrobium funky and Cellulosimicrobium cerance, which were suggested to be related in the 16S rDNA nucleotide sequence analysis, but they do not exhibit motility. He supported Cellulosimicrobium cerance than Cellulosimicrobium funkei.
以上の１６Ｓ ｒＤＮＡ塩基配列解析の結果および諸性質から、本菌株は属レベルではセルロシミクロビウムに含まれると考えられる。しかし、１６Ｓ ｒＤＮＡ塩基配列解析の結果はセルロシミクロビウム・フンケイ（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｆｕｎｋｅｉ）、生理・生化学性状においてはセルロシミクロビウム・セルランス（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｃｅｌｌｕｌａｎｓ）を支持することから、本菌株をセルロシミクロビウム属（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｓｐ．）Ｕ−６８９と命名した。 From the results and various properties of the 16S rDNA base sequence analysis described above, this strain is considered to be contained in cellulosimicrobium at the genus level. However, since the results of 16S rDNA nucleotide sequence analysis support Cellulosimicrobium funkei and Cellulosimicrobium cellulans in physiological and biochemical properties, this strain is considered to be a cell. It was named Cellulosimicrobium sp. U-689.
このＵ−６８９株は、平成１８年１１月１７日付で、日本国茨城県つくば市東１−１−１ つくばセンター（郵便番号３０５−８５６６） 中央第６独立行政法人 産業技術総合研究所 特許生物寄託センターにＦＥＲＭ−Ｐ２１０９９として寄託されている。 This U-689 strain was dated November 17, 2006, Tsukuba Center, 1-1-1 Tsukuba City, Ibaraki, Japan (Postal Code: 305-866), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Deposited at the center as FERM-P21099.
以上のことから、本発明の第二は、ウリカーゼ生産能を有するセルロシミクロビウム属（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｓｐ．）Ｕ−６８９ ＦＥＲＭ−Ｐ２１０９９菌株を提供する。 In view of the above, the second aspect of the present invention provides a Cellulosimicrobium sp. U-689 FERM-P21099 strain having uricase-producing ability.
（セルロシミクロビウム属由来のウリカーゼの製造方法）
本発明のウリカーゼの製造方法は、セルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）属に属し、ウリカーゼの生産能を有する微生物を培養する工程（１）と、前記工程（１）で得られた培養物からウリカーゼを採取する工程（２）と、を含むものである。
(Method for producing uricase derived from the genus Cellulosimicrobium)
The method for producing uricase of the present invention includes a step (1) of culturing a microorganism belonging to the genus Cellulosimicrobium and having the ability to produce uricase, and a uricase from the culture obtained in the step (1). A step (2) of collecting
本発明の一実施態様として、セルロシミクロビウム属に属する細菌を用いたウリカーゼの製造方法の各工程について以下に述べる。 As one embodiment of the present invention, each step of the method for producing uricase using bacteria belonging to the genus Cellulosimicrobium will be described below.
（１）セルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）属に属し、ウリカーゼの生産能を有する微生物を培養する工程。 (1) A step of culturing a microorganism belonging to the genus Cellulosimicrobium and capable of producing uricase.
上記製造方法に使用される培地は、セルロシミクロビウム属に属する細菌が生育する限り特に制限されず、炭素源、窒素源、無機物、その他使用菌株の必要とする微量栄養素を程よく含有するものであれば、合成培地、天然培地のいずれも使用可能である。また、上記培地は、固体培地、液体培地または半流動培地のいずれであってもよい。 The medium used in the above production method is not particularly limited as long as bacteria belonging to the genus Cellulosimicrobium grow, and contains moderate amounts of micronutrients required by the carbon source, nitrogen source, inorganic substances, and other strains used. If so, either a synthetic medium or a natural medium can be used. The medium may be a solid medium, a liquid medium, or a semi-fluid medium.
炭素源としては、セルロシミクロビウム属に属する細菌が良好に生育でき、所望のウリカーゼを生産できるものであればいずれの炭素源であってもよい。具体的には、グルコース、シュークロース、ガラクトースなどの糖類；エタノール、グリセロール、ソルビトールなどのアルコール類；クエン酸、リンゴ酸、コハク酸などの有機酸；デンプンまたはその組成画分、焙焼デキストリン、加工デンプン、デンプン誘導体、物理処理デンプン及びα−デンプン等の炭水化物などが挙げられる。また、尿酸などを炭素源として使用してもよい。好ましくは、グルコース、シュークロース、ガラクトースなどの糖類、エタノール、グリセロール、ソルビトールなどのアルコール類、クエン酸、リンゴ酸、コハク酸などの有機酸、尿酸が使用される。これらの炭素源は、単独あるいは２種以上の混合物の形態で使用できる。 As a carbon source, any carbon source may be used as long as bacteria belonging to the genus Cellulosimicrobium can grow well and produce a desired uricase. Specifically, sugars such as glucose, sucrose and galactose; alcohols such as ethanol, glycerol and sorbitol; organic acids such as citric acid, malic acid and succinic acid; starch or a fraction thereof, roasted dextrin, and processing Examples thereof include carbohydrates such as starch, starch derivatives, physically treated starch, and α-starch. Uric acid or the like may be used as a carbon source. Preferably, sugars such as glucose, sucrose and galactose, alcohols such as ethanol, glycerol and sorbitol, organic acids such as citric acid, malic acid and succinic acid, and uric acid are used. These carbon sources can be used alone or in the form of a mixture of two or more.
窒素源としては、一般的にセルロシミクロビウム属に属する細菌の培養に使用するのと同様のものが使用でき、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウムなどのアンモニウム塩、尿素、Ｌ−グルタミン酸などのアミノ酸類、あるいは尿酸などの無機あるいは有機の窒素化合物が使用できる。さらに、窒素源としては、ペプトン、ポリペプトン、肉エキス、酵母エキス、大豆加水分解物、大豆粉末、ミルクカゼイン、カザミノ酸、コーンスティープリカー等の窒素含有天然物を使用してもよい。これらのうち、塩化アンモニウム、硫酸アンモニウム、尿素、Ｌ−グルタミン酸などのアミノ酸類、尿酸などの無機あるいは有機窒素化合物、ペプトン、肉エキス、酵母エキスなの窒素含有天然物が好ましい。これらの窒素源は、単独あるいは２種以上の混合物の形態で使用できる。 As the nitrogen source, those generally used for culturing bacteria belonging to the genus Cellulosimicrobium can be used. For example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, L- Amino acids such as glutamic acid or inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, corn steep liquor and the like may be used as the nitrogen source. Among these, amino acids such as ammonium chloride, ammonium sulfate, urea, and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid, nitrogen-containing natural products such as peptone, meat extract, and yeast extract are preferable. These nitrogen sources can be used alone or in the form of a mixture of two or more.
無機物としても、一般的にセルロシミクロビウム属に属する細菌の培養に使用するのと同様のものが使用でき、例えば、例えば、塩化ナトリウム、塩化カリウム、塩化カルシウム、リン酸カリウム、リン酸ナトリウム、硫酸マグネシウム、硫酸アンモニウム等の、マグネシウム、マンガン、カルシウム、ナトリウム、カリウム、銅、鉄及び亜鉛などのリン酸塩、塩酸塩、硫酸塩及び酢酸塩などが用いられる。そのほか、チアミン、ビオチンなどのビタミン類、さらに必要に応じて、アデニン、ウラシルなどの核酸関連物質が使用されてもよい。これらの無機物は、単独あるいは２種以上の混合物の形態で使用できる。 As the inorganic substance, those generally used for culturing bacteria belonging to the genus Cellulosimicrobium can be used. For example, sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate And phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, hydrochlorides, sulfates and acetates, such as magnesium sulfate and ammonium sulfate. In addition, vitamins such as thiamine and biotin, and if necessary, nucleic acid-related substances such as adenine and uracil may be used. These inorganic substances can be used alone or in the form of a mixture of two or more.
セルロシミクロビウム属に属する細菌の培養は、好気的条件下、通常振盪培養あるいは通常撹拌培養で行なわれる。その際の培養条件は、培地の組成や培養法によって適宜選択され、本菌株が増殖し目的とする酵素であるウリカーゼが失活せずに効率よく産生できる条件であれば特に制限されない。通常は、培養温度は２５〜４５℃で行う。培地のｐＨは５．０〜９．０、好ましくは６．０〜８．０の範囲である。培養時間は、通常２０〜７２時間、好ましくは２４〜４８時間培養する。本発明では、ウリカーゼは、上記したようにして得られたセルロシミクロビウム属に属する細菌の培養液中および／または菌体中に生成・蓄積されるが、特に本発明の特に好ましい実施形態であるＵ−６８９株を使用する場合には、以下の実施例で詳述するように、Ｕ−６８９株の菌体内に生産されたウリカーゼが、耐熱性に優れるため、好ましい。 Cultivation of bacteria belonging to the genus Cellulosimicrobium is carried out under aerobic conditions, usually by shaking culture or normally stirring culture. The culture conditions at that time are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the strain can grow and the uricase, which is the target enzyme, can be efficiently produced without being inactivated. Usually, the culture temperature is 25 to 45 ° C. The pH of the medium is in the range of 5.0 to 9.0, preferably 6.0 to 8.0. The culture time is usually 20 to 72 hours, preferably 24 to 48 hours. In the present invention, uricase is produced and accumulated in the culture solution and / or in the bacterial body of the bacterium belonging to the genus Cellulosimicrobium obtained as described above. Particularly preferred embodiments of the present invention When the U-689 strain is used, uricase produced in the cells of the U-689 strain is preferable because it is excellent in heat resistance, as described in detail in the following Examples.
（２）前記工程（１）で得られた培養物からウリカーゼを採取する工程。 (2) A step of collecting uricase from the culture obtained in the step (1).
本発明のウリカーゼの単離方法を以下に記載する。 The method for isolating the uricase of the present invention is described below.
産生したウリカーゼが菌体内に蓄積または分泌される場合には、培養終了後、培養液から菌体を遠心分離などにより回収し、ついでこの菌体から適当な手段でウリカーゼを適当な溶媒（例えば、トリトン Ｘ−１００など）で抽出する。遠心分離などによってこの抽出液を処理して不溶性成分を除去した後、酸沈殿、有機溶媒沈殿（例えば、エタノール、アセトンなどによる溶媒沈澱）、塩析（例えば、硫安による塩析）、透析、各種クロマトグラフィー（例えば、イオン交換クロマトグラフィー、セファデックスクロマトグラフィー、アフィニティクロマトグラフィー、ゲル濾過クロマトグラフィー、疎水クロマトグラフィーなど）、限外濾過、凍結乾燥、電気泳動などの、当業者が通常用いる酵素精製方法によって精製することにより、純度の高いウリカーゼが得られる。なお、ウリカーゼの精製度合いは、得られたウリカーゼを電気泳動にかけることにより確認でき、電気泳動的に単一になるまで精製することにより、純度の高いウリカーゼが得られる。または、培養終了後、濾過または遠心分離などにより菌体を培養液から回収し、ついでこの菌体を適当な手段（例えば、超音波、自己消化法、ガラスビーズ等を用いた物理的破砕など）で破砕して、細胞破砕物を得、当該破砕物から遠心分離などにより上清液を得る。この上清液を、上記と同様の精製方法を使用することによって、純度の高いウリカーゼを得てもよい。 When the produced uricase is accumulated or secreted in the microbial cells, the microbial cells are recovered from the culture solution by centrifugation after completion of the culture, and then uricase is removed from the microbial cells by an appropriate means (for example, Triton X-100 etc.). After processing this extract by centrifugation, etc. to remove insoluble components, acid precipitation, organic solvent precipitation (for example, solvent precipitation with ethanol, acetone, etc.), salting out (for example, salting out with ammonium sulfate), dialysis, various types Enzyme purification methods commonly used by those skilled in the art, such as chromatography (eg, ion exchange chromatography, Sephadex chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, etc.), ultrafiltration, lyophilization, electrophoresis, etc. The uricase with high purity can be obtained by purifying by the above. The degree of purification of the uricase can be confirmed by subjecting the obtained uricase to electrophoresis, and a highly purified uricase can be obtained by purifying the uricase until it becomes single electrophoretically. Alternatively, after completion of the culture, the cells are collected from the culture solution by filtration or centrifugation, and then the cells are collected by appropriate means (for example, ultrasonic, self-digestion, physical disruption using glass beads, etc.) To obtain a cell lysate, and a supernatant is obtained from the crushed material by centrifugation or the like. A highly purified uricase may be obtained from this supernatant by using the same purification method as described above.
また、産生したウリカーゼが菌体外（培養物中）に分泌される場合は、培養液から濾過または遠心分離により菌体を除去し、濾液または上清を得る。こうして得られた濾液または上清に、直接上記と同様の精製方法を適用することによって、純度の高いウリカーゼが得られる。 When the produced uricase is secreted outside the cells (in the culture), the cells are removed from the culture solution by filtration or centrifugation to obtain a filtrate or supernatant. A highly purified uricase can be obtained by directly applying the same purification method to the filtrate or supernatant thus obtained.
本発明の製造方法により得られるウリカーゼは、上述したように、人体内の尿酸蓄積に起因する種々の疾患の臨床診断を目的として、血液または尿中に存在する尿酸の測定用酵素として使用できる。この際、ウリカーゼを使用する尿酸の定量法としては下記の方法がある。 As described above, the uricase obtained by the production method of the present invention can be used as an enzyme for measuring uric acid present in blood or urine for the purpose of clinical diagnosis of various diseases caused by uric acid accumulation in the human body. At this time, the uric acid quantification method using uricase includes the following methods.
（ｉ）尿酸とウリカーゼとを反応させることにより生成する過酸化水素量を測定する方法
この際、過酸化水素量の測定方法としては、例えば下記（ア）〜（ウ）の方法がある：
（ア）過酸化水素を４−アミノアンチピリンとフエノールあるいはその誘導体とパーオキシダーゼの存在下で反応させて、過酸化水素量に比例して生成する色素を吸光度から定量する（酵素比色法中ウリカーゼ・パーオシダーゼ法）方法；
（イ）酸化水素とアルコールとをカタラーゼの存在下で反応させ、生じたアルデヒドをアセチルアセトン及びアンモニアと縮合させて生成する色素を吸光度から定量する（酵素比色法中ウリカーゼ・カタラーゼ法）方法；および
（ウ）上記（イ）のカタラーゼによる反応生成物のアルデヒドと還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）とをアルコールデヒドロゲナーゼの存在下で反応させてＮＡＤを生成せしめ、その際、減少するＮＡＤＨを定量する（紫外部吸収法）方法。
(I) Method for measuring the amount of hydrogen peroxide produced by reacting uric acid and uricase At this time, examples of methods for measuring the amount of hydrogen peroxide include the following methods (a) to (c):
(A) Hydrogen peroxide is reacted with 4-aminoantipyrine and phenol or its derivative in the presence of peroxidase, and the dye produced in proportion to the amount of hydrogen peroxide is quantified from the absorbance (uricase in the enzyme colorimetric method).・ Perosidase method) method;
(I) a method in which hydrogen oxide and alcohol are reacted in the presence of catalase, and the resulting aldehyde is condensed with acetylacetone and ammonia to determine the amount of dye produced from the absorbance (uricase-catalase method in the enzyme colorimetric method); and (C) The reaction product aldehyde of the above (a) catalase is reacted with reduced nicotinamide adenine dinucleotide (NADH) in the presence of alcohol dehydrogenase to produce NAD, and the decreased NADH is quantified. (Ultraviolet absorption method)
（ｉｉ）尿酸をウリカーゼと反応させる際に消費される酸素あるいは生成する炭酸ガスを測定する方法（電極法）。 (Ii) A method of measuring oxygen consumed when reacting uric acid with uricase or generated carbon dioxide (electrode method).
（ｉｉｉ）反応の前後における尿酸由来の紫外部吸収の差を測定する方法（紫外部吸収法）。 (Iii) A method for measuring a difference in ultraviolet absorption derived from uric acid before and after the reaction (ultraviolet absorption method).
（ＩＶ）ウリカーゼ処理及び未処理の試料に化学的尿酸定量法（例えば、リンタングステン酸法）。 (IV) Chemical uric acid determination method (for example, phosphotungstic acid method) for uricase-treated and untreated samples.
本発明の製造方法により得られるウリカーゼは、上記方法のいずれも適用できるが、好ましくは（ｉ）（ア）の方法に使用できる。 Any of the above methods can be applied to the uricase obtained by the production method of the present invention, but it can be preferably used in the method (i) (a).
以下実施例により本発明を詳細に説明するが、本実施例により本発明の範囲は制限されるものではない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited by these examples.
（実施例１）
（１）セルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｓｐ．）Ｕ−６８９株の培養
以下挙げる組成；トリプトン（ＢＤ（Ｂｅｃｔｏｎ，Ｄｉｃｋｉｎｓｏｎ ａｎｄ Ｃｏｍｐａｎｙ）社製）１％（ｗ／ｖ）、酵母エキス（ＢＤ（Ｂｅｃｔｏｎ，Ｄｉｃｋｉｎｓｏｎ ａｎｄ Ｃｏｍｐａｎｙ）社製）０．１％（ｗ／ｖ）、塩化ナトリウム（関東化学製）０．５％（ｗ／ｖ）、リン酸一カリウム（関東化学製）０．２％（ｗ／ｖ）、尿酸（和光純薬工業社製）０．２％（ｗ／ｖ）からなる培養液を調製した後、ｐＨをＮａＯＨで７．０に調整し、１２１℃で２０分間滅菌した。
Example 1
(1) Culture of Cellulosimicrobium sp. U-689 strain The following composition: Tryptone (BD (Becton, Dickinson and Company)) 1% (w / v), yeast extract (BD (Becton) , Manufactured by Dickinson and Company) 0.1% (w / v), sodium chloride (manufactured by Kanto Chemical) 0.5% (w / v), monopotassium phosphate (manufactured by Kanto Chemical) 0.2% (w / V), a culture solution consisting of 0.2% (w / v) uric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was prepared, pH was adjusted to 7.0 with NaOH, and sterilized at 121 ° C. for 20 minutes.
次に、得られた培養液を、滅菌したＬ字試験管に５ｍｌずつ分注し、これに１．５％寒天を含有するブイヨン培地で予め培養しておいたＵ−６８９株を、上記Ｌ字試験管の培養液に接種し、３０℃で１２０ｒｐｍの速度で撹拌させながら、２４時間培養を行った。 Next, 5 ml of the obtained culture solution was dispensed into a sterilized L-shaped test tube, and U-689 strain previously cultured in a bouillon medium containing 1.5% agar was added to the above-mentioned L The inoculum was inoculated into a culture solution in a letter-shaped test tube, and cultured for 24 hours while stirring at 30 ° C. at a speed of 120 rpm.
（２）酵素の採取
上記（１）で得られた培養液を遠心分離し、得た菌体に、５ｍＬのリン酸緩衝液（１０ｍＭ、ｐＨ＝７）および３ｇのガラスビーズを加えて混合し、３分間振盪して、菌体を物理的に破砕した。この菌体破砕物を遠心分離により上清を回収し、この上清を酵素液（総ウリカーゼ活性 １．８Ｕ）とした。
(2) Collection of enzyme The culture solution obtained in (1) above is centrifuged, and 5 mL of phosphate buffer (10 mM, pH = 7) and 3 g of glass beads are added to and mixed with the obtained cells. The cells were shaken for 3 minutes to physically disrupt the cells. The supernatant of the crushed cells was collected by centrifugation, and this supernatant was used as an enzyme solution (total uricase activity 1.8 U).
なお、本実施例において、ウリカーゼの活性は、下記方法によって測定した。 In this example, the activity of uricase was measured by the following method.
（評価例１）ウリカーゼ活性の測定方法
ウリカーゼの活性の測定は、ウリカーゼ・ペルオキシダーゼ法を用い、５５０ｎｍにおける４−アミノアンチピリンとＴＯＤＢ（Ｎ，Ｎ−ビス（４−スルホブチル）−３−メチルアニリン）の酸化縮合体のキノン色素を分光光度計により行なった。すなわち、０．８３ｍＭ 尿酸溶液 １２０μｌ、７５ｍＭ ＴＯＤＢ ５μｌ、７５ｍＭ ４−アミノアンチピリン ５μｌ、８０Ｕ／ｍｌ ペルオシキダーゼ ５μｌ、酵素液 ６５μｌを混合し、室温（２５℃）で１時間反応させた後、５５０ｎｍの吸光度を測定した。なお、ウリカーゼの単位は、上記試験条件下において毎分１マイクロモルの尿酸を分解する酵素の量（力価）を、「１Ｕ（ユニット）」と定義した。
(Evaluation Example 1) Method for Measuring Uricase Activity The activity of uricase was measured using uricase peroxidase method, and 4-aminoantipyrine and TODB (N, N-bis (4-sulfobutyl) -3-methylaniline) at 550 nm. The oxidative condensate quinone dye was measured with a spectrophotometer. Specifically, 120 μl of 0.83 mM uric acid solution, 5 μl of 75 mM TODB, 5 μl of 75 mM 4-aminoantipyrine, 5 μl of 80 U / ml peroxidase, and 65 μl of enzyme solution were mixed and reacted at room temperature (25 ° C.) for 1 hour, and then the absorbance at 550 nm was obtained. It was measured. The unit of uricase was defined as “1 U (unit)” as the amount (titer) of an enzyme that decomposes 1 micromole of uric acid per minute under the above test conditions.
（評価例２）酵素の温度安定性
標品としては、上記実施例１（２）で得られた酵素液を使用した。
(Evaluation Example 2) Temperature Stability of Enzyme As a sample, the enzyme solution obtained in Example 1 (2) was used.
本発明のウリカーゼ標品を、種々の温度（２０℃、３０℃、４０℃、５０℃、６０℃）に２０分間保持した後、上記評価例１に記載の方法に従って、ウリカーゼ活性を測定した。２０℃で２０分保持した後の活性を１００として、各温度における相対残存活性を算出して、その結果を図１に示す。図１から、本発明の製造方法によって得られたウリカーゼは、６０℃で２０分間の熱処理に対しても、８５％を超える活性を維持しており、優れた耐熱製を有することが示される。 After maintaining the uricase preparation of the present invention at various temperatures (20 ° C., 30 ° C., 40 ° C., 50 ° C., 60 ° C.) for 20 minutes, the uricase activity was measured according to the method described in Evaluation Example 1 above. Relative residual activity at each temperature was calculated with the activity after holding at 20 ° C. for 20 minutes as 100, and the result is shown in FIG. FIG. 1 shows that the uricase obtained by the production method of the present invention maintains an activity exceeding 85% even after heat treatment at 60 ° C. for 20 minutes, and has excellent heat resistance.
Claims (3)
前記工程（１）で得られた培養物からウリカーゼを採取する工程（２）と、
を含む、ウリカーゼの製造方法であって、
前記セルロシミクロビウム（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ）属に属する微生物が、セルロシミクロビウム属（Ｃｅｌｌｕｌｏｓｉｍｉｃｒｏｂｉｕｍ ｓｐ．）Ｕ−６８９ ＦＥＲＭ−Ｐ２１０９９菌株である、ウリカーゼの製造方法。 A step (1) of culturing a microorganism belonging to the genus Cellulosimicrobium and capable of producing uricase;
Collecting uricase from the culture obtained in the step (1) (2);
A method for producing uricase , comprising :
A method for producing uricase, wherein the microorganism belonging to the genus Cellulosimicrobium is a Cellulosimicrobium sp. U-689 FERM-P21099 strain .
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| JP5010291B2 (en) * | 2007-01-15 | 2012-08-29 | シーシーアイ株式会社 | Method for producing new uricase |
| JP5113557B2 (en) * | 2008-03-04 | 2013-01-09 | 一般財団法人電力中央研究所 | Microorganism reducing hexavalent chromium and environmental purification method using the microorganism |
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| JP5010291B2 (en) * | 2007-01-15 | 2012-08-29 | シーシーアイ株式会社 | Method for producing new uricase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US11647746B2 (en) | 2012-02-20 | 2023-05-16 | Basf Se | Enhancing the antimicrobial activity of biocides with polymers |
| US11666050B2 (en) | 2012-02-20 | 2023-06-06 | Basf Se | Enhancing the antimicrobial activity of biocides with polymers |
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