JP5849297B2 - Method for producing biodegradable plastic degrading enzyme and Pseudozyma antarctica used therefor - Google Patents
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Description
本発明は、特に、生分解性マルチフィルム等の農業資材を、環境に大きな負荷を与えずに分解するために使用され、遺伝子組み換え技術を用いずに行われる、生分解性プラスチック分解酵素の製造方法、及びこの製造方法に使用されるPseudozyma antarcticaに関する。 The present invention is particularly used for degrading agricultural materials such as biodegradable multi-films without degrading the environment, and producing biodegradable plastic degrading enzymes without using genetic recombination technology. The present invention relates to a method and Pseudozyma antarctica used in this production method.
マルチ栽培技術は、地中の温度の上昇や雑草の防除、地面の保湿などにより作物の育成を促進する効果が高く、日本の畑作に欠かせない栽培技術である。マルチ栽培に使用されるマルチフィルムの国内使用量は現在、年間約４万トン程度となっており、近年では、生分解性のマルチフィルムも開発されている。ここで、マルチフィルムは、圃場に薄く且つ広く敷設されるものであるため、マルチフィルムの圃場からの回収には多くの労力と費用が必要であった。生分解性マルチフィルムを使用することにより、マルチフィルムの圃場からの回収と処分に要する労力とコストを削減し、省力的な農法が実現可能となる。このため、国内の農業就業者の老齢化と働き手の不足が深刻な日本では、生分解性マルチフィルムが他の国に先駆けて導入されており、従来の生分解性を有さないマルチフィルム（例えば、ポリエチレン製マルチフィルム）と、生分解性マルチフィルムの価格差が小さくなるにつれて、流通量も年々増加している。
２００６年の全世界における生分解性マルチフィルムの流通量は１１５０トン程度で、その後も徐々に増加して２００９年度には約１．５倍の１７００トン程度となったと推計されており、今後も流通量は更に増加することが予測される。また、マルチフィルムメーカーでは、国内で使用されている、生分解性を有さないポリエチレン製マルチフィルムの約７割程度が、今後、生分解性マルチフィルムに置き換えられていくと見込んでいる。更に、マルチフィルムの使用量が、全世界の８割を超えている中国でもポリエチレン製マルチフィルム残渣の問題が顕著化しており、生分解性マルチフィルムの分解を制御する技術の開発が急がれている。
Multi-cultivation technology is an indispensable cultivation technique for Japanese field farming because it is highly effective in promoting the cultivation of crops by increasing the temperature in the ground, controlling weeds, and moisturizing the ground. The amount of domestic use of multi-film used for multi-cultivation is currently about 40,000 tons per year. In recent years, biodegradable multi-film has been developed. Here, since the multi-film is thinly and widely laid in the field, much labor and cost are required for collecting the multi-film from the field. By using a biodegradable multifilm, labor and cost required for collecting and disposing of the multifilm from the field can be reduced, and a labor-saving farming method can be realized. For this reason, biodegradable multifilms have been introduced in Japan ahead of other countries in Japan, where the aging of agricultural workers in Japan and the shortage of workers are serious. For example, as the price difference between a polyethylene multi-film and a biodegradable multi-film becomes smaller, the distribution amount is also increasing year by year.
The global distribution of biodegradable multi-films in 2006 was around 1150 tons, and after that it was estimated to increase gradually to about 1700 tons in FY2009, about 1.5 times. The distribution volume is expected to increase further. In addition, multifilm manufacturers expect that about 70% of non-biodegradable polyethylene multifilms used in Japan will be replaced with biodegradable multifilms in the future. Furthermore, even in China, where the amount of multifilm used exceeds 80% of the world, the problem of polyethylene multifilm residue has become more prominent, and the development of technology to control the degradation of biodegradable multifilms has been urgently needed. ing.
ここで、生分解性マルチフィルムの普及を促進するにあたっては、農業資材に必要とされる十分な強度を有する生分解性マルチフィルムを、使用後において効率的に分解可能な技術を開発することが求められている。
生分解性プラスチックを分解するための技術については、これまで、土壌、汚泥、又は空気中に浮遊する埃などから採取された微生物や、Pseudozyma属の葉面酵母（Pseudozyma antarctica）や無胞子不完全菌目に属する糸状菌が、生分解性プラスチック分解酵素を分泌することを見出している（特許文献１から５）。これらの菌類を使用して圃場に薄く且つ広く敷設される生分解性マルチフィルムを効率的に分解するため、生分解性プラスチックに対する分解活性がより高い酵素液を用いることや、大量の生分解性プラスチック分解酵素を生産することが検討されている。
Here, in promoting the spread of biodegradable multi-film, it is necessary to develop a technology that can efficiently decompose biodegradable multi-film having sufficient strength required for agricultural materials after use. It has been demanded.
As for technologies for degrading biodegradable plastics, microorganisms collected from soil, sludge or dust floating in the air, Pseudozyma antarctica and incomplete spore It has been found that filamentous fungi belonging to Mycelia secrete biodegradable plastic-degrading enzymes (Patent Documents 1 to 5). In order to efficiently decompose biodegradable multi-films that are thin and widely laid in the field using these fungi, use an enzyme solution with higher degrading activity for biodegradable plastics, or a large amount of biodegradable The production of plastic degrading enzymes is under consideration.
生分解性プラスチック分解酵素の大量生産は、遺伝子組み換え技術を使用した手法により行われることがある。しかしながら、遺伝子組み換え技術は、遺伝子組み換え体の流出防止のための、生産や安全性評価のコストが必要であるため、実際の農業現場において採用することは難しい。従って、自然界から単離された、安全性の確認されている微生物を用い、遺伝子組み換え技術を用いずに生分解性プラスチック分解酵素を大量生産する技術を開発することが求められている。 Mass production of biodegradable plastic degrading enzymes may be performed by means of genetic recombination techniques. However, genetic recombination techniques are difficult to adopt in actual agricultural sites because they require production and safety evaluation costs to prevent the outflow of genetically modified organisms. Therefore, it is required to develop a technique for mass-producing biodegradable plastic degrading enzymes using microorganisms isolated from nature and confirmed to be safe without using genetic recombination techniques.
本発明の発明者らは、上記課題を解決するために鋭意研究を行った。その結果、キシロースを含有する培地中でPseudozyma antarcticaを培養することにより、遺伝子組み換え技術を使用せずに生分解性プラスチック分解酵素を大量に生産できることを見出し、本発明を完成するに至った。具体的には、本発明は以下のものを提供する。 The inventors of the present invention have intensively studied to solve the above problems. As a result, it was found that by culturing Pseudozyma antarctica in a medium containing xylose, a large amount of biodegradable plastic degrading enzyme can be produced without using genetic recombination technology, and the present invention has been completed. Specifically, the present invention provides the following.
本発明の第一の態様は、キシロースを含有する培地中でPseudozyma antarcticaを培養することを特徴とする生分解性プラスチック分解酵素の製造方法である。
本発明の第二の態様は、受託番号FERM P-22155のPseudozyma antarcticaである。
A first aspect of the present invention is a method for producing a biodegradable plastic degrading enzyme, wherein Pseudozyma antarctica is cultured in a medium containing xylose.
The second aspect of the present invention is Pseudozyma antarctica with accession number FERM P-22155.
本発明によれば、キシロースを含有する培地中でPseudozyma antarcticaを培養するため、遺伝子組み換え技術を用いずに生分解性プラスチック分解酵素を大量に生産することができる。このように大量に生産された生分解性プラスチック分解酵素は、圃場に敷設された生分解性マルチフィルムを分解するために使用でき、安全且つ効率的に生分解性マルチフィルムを分解することができる。 According to the present invention, since Pseudozyma antarctica is cultured in a medium containing xylose, a large amount of biodegradable plastic degrading enzyme can be produced without using a genetic recombination technique. The biodegradable plastic-degrading enzyme produced in large quantities in this way can be used for degrading the biodegradable multifilm laid in the field, and can safely and efficiently degrade the biodegradable multifilm. .
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
＜生分解性プラスチック分解酵素の製造方法＞
本発明の生分解性プラスチック分解酵素の製造方法は、遺伝子組み換え技術を用いず、キシロースを含有する培地中でPseudozyma antarcticaを培養することを特徴とする。
<Method for producing biodegradable plastic degrading enzyme>
The method for producing a biodegradable plastic degrading enzyme of the present invention is characterized in that Pseudozyma antarctica is cultured in a medium containing xylose without using a genetic recombination technique.
［Pseudozyma antarctica］
本発明の生分解性プラスチック分解酵素の製造方法においては、葉面酵母であるPseudozyma antarcticaを使用する。Pseudozyma antarcticaとしては、研究施設等において提供されている株や、自然界から単離された株等、任意の株を使用することができる。具体的には、独立行政法人理化学研究所バイオリソースセンターにおいて提供されているPseudozyma antarctica JCM10317株や、茨城県において採取された稲籾から単離された受託番号FERM P-22155のPseudozyma antarcticaを使用することができるが、特に、グルコース存在下においても生分解性プラスチック分解酵素を大量に発現可能な受託番号FERM P-22155のPseudozyma antarcticaを使用することが好ましい。
［培地］
本発明の生分解性プラスチック分解酵素の製造方法においては、キシロースを含有する培地を使用する。キシロースを含有する培地としては、葉面酵母の培養に適し、炭素源としてキシロースを含有する限りにおいて特に限定されるものではなく、合成培地、半合成培地、及び天然培地のいずれをも使用することができる。これらの培地に添加する成分としては、動植物組織の抽出物／加水分解物を用いてもよいし、化学的に合成された、窒素、リン、ミネラル、及び／又は炭素を含む化合物を用いてもよい。
本発明の生分解性プラスチック分解酵素の製造方法に使用する培地としては、Pseudozyma antarcticaの培養に特に適しているという点で、FMM培地(Fungal Minimum Medium)を用いることが好ましく、特に、炭素源として、グルコース及びキシロース、好ましくはキシロースのみを含有するFMM培地を用いることが特に好ましい。
本発明の生分解性プラスチック分解酵素の製造方法に使用する培地に含まれるキシロースの含有量は、２質量％以上１０質量％以下であることが好ましく、２質量％以上８質量％以下であることが更に好ましい。キシロースの含有量を上記の範囲内のものとすることにより、生分解性プラスチック分解酵素を効率的に生産誘導し、Pseudozyma antarcticaの生育状態を良好なものとすることができる。
また、本発明の生分解性プラスチック分解酵素の製造方法に使用する培地が、キシロース及びグルコースを含む場合には、グルコースの含有量は、１質量％以上４質量％以下であることが好ましく、１質量％以上２質量％であることが更に好ましい。また、キシロース及びグルコースの合計量は、２質量％以上１０質量％以下であることが好ましく、２質量％以上８質量％以下であることが更に好ましい。
本発明の生分解性プラスチック分解酵素の製造方法に使用する培地は、酸又は塩基で、ｐＨを5.0以上8.0以下、好ましくは5.5以上7.0以下に調整することが好ましい。ｐＨの調整に使用される酸又は塩基は、特に限定されるものではないが、培地への窒素源の供給も兼ねて、アンモニア水を用いてｐＨを調整することが好ましい。
[Pseudozyma antarctica]
In the method for producing a biodegradable plastic degrading enzyme of the present invention, Pseudozyma antarctica which is a foliar yeast is used. As Pseudozyma antarctica, any strain such as a strain provided in a research facility or a strain isolated from the natural world can be used. Specifically, use Pseudozyma antarctica JCM10317 strain provided by RIKEN BioResource Center, or Pseudozyma antarctica with accession number FERM P-22155 isolated from rice straw collected in Ibaraki Prefecture. However, it is particularly preferable to use Pseudozyma antarctica with accession number FERM P-22155 that can express a large amount of biodegradable plastic-degrading enzyme even in the presence of glucose.
[Culture medium]
In the method for producing a biodegradable plastic degrading enzyme of the present invention, a medium containing xylose is used. The medium containing xylose is not particularly limited as long as it is suitable for culturing foliar yeast and contains xylose as a carbon source, and any of a synthetic medium, a semi-synthetic medium, and a natural medium should be used. Can do. As an ingredient added to these media, an extract / hydrolyzate of animal or plant tissue may be used, or a chemically synthesized compound containing nitrogen, phosphorus, mineral, and / or carbon may be used. Good.
As the medium used in the method for producing the biodegradable plastic degrading enzyme of the present invention, FMM medium (Fungal Minimum Medium) is preferably used in terms of being particularly suitable for cultivation of Pseudozyma antarctica. It is particularly preferred to use an FMM medium containing only glucose and xylose, preferably xylose.
The content of xylose contained in the medium used in the method for producing the biodegradable plastic degrading enzyme of the present invention is preferably 2% by mass or more and 10% by mass or less, and preferably 2% by mass or more and 8% by mass or less. Is more preferable. By setting the xylose content within the above range, the biodegradable plastic-degrading enzyme can be efficiently produced and induced, and the growth state of Pseudozyma antarctica can be improved.
Moreover, when the culture medium used for the manufacturing method of the biodegradable plastic degrading enzyme of this invention contains xylose and glucose, it is preferable that content of glucose is 1 to 4 mass%. More preferably, it is at least 2% by mass. The total amount of xylose and glucose is preferably 2% by mass or more and 10% by mass or less, and more preferably 2% by mass or more and 8% by mass or less.
The medium used in the method for producing a biodegradable plastic degrading enzyme of the present invention is an acid or a base, and the pH is preferably adjusted to 5.0 or more and 8.0 or less, preferably 5.5 or more and 7.0 or less. The acid or base used for adjusting the pH is not particularly limited, but it is also preferable to adjust the pH using aqueous ammonia in combination with the supply of a nitrogen source to the medium.
ここで、本発明の生分解性プラスチック分解酵素の製造方法に使用できる培地において使用できるキシロースとしては、試薬として市販されているキシロースのみではなく、バイオエタノールを製造する過程で、草本系又は木質系バイオマス原料中のヘミセルロースに由来するキシロース溶液等、キシロース以外の化学物質の製造過程において副産物として生成されるキシロースを含むキシロース溶液も用いることができる。
このようなバイオマス原料の分解産物に由来するキシロース溶液を用いることにより、間伐材や廃材、農産物の非可食部（例えば、小麦ふすま、米ぬか、稲わら、麦わら、トウモロコシ茎部、サトウキビバガス、ソルガムバガス等）等の資材を有効に活用して生分解性プラスチック分解酵素の生産を行うことができる。特に、現在のバイオエタノール生産技術においては、バイオマスの糖化液中のグルコースを利用してエタノールを生産することができるものの、ヘミセルロースに由来するキシロースは、バイオエタノールの生産に用いることができないため、キシロースを多く含む蒸留廃液が多量に排出されていた。本発明の生分解性プラスチック分解酵素の製造方法によれば、これら、キシロースを多く含む蒸留廃液も有効活用することができるため、経済的に生分解性プラスチック分解酵素を生産することができる。
バイオマス原料の分解産物に由来するキシロース溶液には、キシロース以外にも、微量のグルコースを含んでいるが、特に、本発明の生分解性プラスチック分解酵素の製造方法においては、キシロース以外の炭素源が含まれていたとしても、Pseudozyma antarcticaが生分解性プラスチック分解酵素を生産することができるため、このようなキシロース溶液であっても、生分解性プラスチック分解酵素の製造方法に好適に用いることができる。
なお、本発明の生分解性プラスチック分解酵素の製造方法において、培地にバイオマス原料の分解産物に由来するキシロース溶液を用いる場合、グルコース存在下における生分解性プラスチック分解酵素の生産能に特に優れる、受託番号FERM P-22155のPseudozyma antarcticaを用いることが好ましい。
Here, the xylose that can be used in the medium that can be used in the method for producing the biodegradable plastic degrading enzyme of the present invention is not limited to xylose that is commercially available as a reagent, but in the process of producing bioethanol, herbaceous or woody A xylose solution containing xylose produced as a by-product in the production process of chemical substances other than xylose, such as a xylose solution derived from hemicellulose in a biomass raw material, can also be used.
By using a xylose solution derived from such biomass raw material decomposition products, thinned and waste wood, non-edible parts of agricultural products (eg wheat bran, rice bran, rice straw, straw, corn stalk, sugar cane bagasse, sorghum) It is possible to produce biodegradable plastic degrading enzymes by effectively using materials such as bagasse. In particular, in the current bioethanol production technology, ethanol can be produced using glucose in the saccharified biomass, but xylose derived from hemicellulose cannot be used for bioethanol production. A large amount of distillation waste liquid containing a large amount of was discharged. According to the method for producing a biodegradable plastic degrading enzyme of the present invention, these waste liquids containing a lot of xylose can also be used effectively, so that the biodegradable plastic degrading enzyme can be produced economically.
In addition to xylose, the xylose solution derived from the biomass raw material degradation product contains a small amount of glucose. In particular, in the method for producing a biodegradable plastic degrading enzyme of the present invention, a carbon source other than xylose is present. Even if it is contained, Pseudozyma antarctica can produce biodegradable plastic degrading enzymes, so even such a xylose solution can be suitably used in a method for producing biodegradable plastic degrading enzymes. .
In addition, in the method for producing a biodegradable plastic degrading enzyme of the present invention, when a xylose solution derived from a biomass raw material degradation product is used for the culture medium, the production performance of the biodegradable plastic degrading enzyme in the presence of glucose is particularly excellent. Pseudozyma antarctica number FERM P-22155 is preferably used.
［培養条件］
本発明の生分解性プラスチック分解酵素の製造方法において、Pseudozyma antarcticaを培養する条件は、葉面酵母の培養条件として従来公知の培養条件を採用することができる。具体的には、２５℃以上３２℃以下の好気的条件で、２４時間以上の前培養を行った後、同様の温度で７２時間以上培養する条件を挙げることができる。Pseudozyma antarcticaを前培養する際の培地は、従来公知の培地を使用することができるが、通常は、YM培地(Yeast extract Malt extract medium)等の天然培地を使用することが好ましい。
Pseudozyma antarcticaの培養に当たっては、生分解性プラスチック分解酵素を含む培養液をより効率的に得るために、半回分培養又は連続培養を行うことが好ましい。即ち、本発明においては、キシロースを含む培地中でPseudozyma antarcticaを一定時間培養した後、Pseudozyma antarcticaの培養液を一定割合で回収し、培養装置に残った培養液に、回収した培養液の量と同量のキシロースを含む培地を添加して更にPseudozyma antarcticaを培養する方法、及びキシロースを含む培地中でPseudozyma antarcticaを培養しながら、一定の速度でPseudozyma antarcticaの培養液を回収し、培養液を回収する速度と同じ速度でキシロースを含む培地を追加する方法もまた提供される。
Pseudozyma antarcticaの培養に当たって、半回分培養を行う場合、培養液へ新たな培地を追加した後、２４時間以上４８時間以下の間、培養することが好ましい。培養時間を上記の通り設定することにより、Pseudozyma antarcticaの増殖速度や得られる培養液中の酵素濃度を高く維持することができる。また、半回分培養の１回の培養後に抜き取る培養液の量は、全培養液の５０％以上８０％以下であることが好ましい。
Pseudozyma antarcticaの培養に当たって、連続培養を行う場合、培養液への培地の追加と培養液の回収は、0.25v/(v/d)以上0.75V/(v/d)以下の速度で行うことが好ましい。斯かる速度で新たな培地を追加し、培養液を回収することにより、培養液中の酵素濃度を高く維持しつつ、Pseudozyma antarcticaの増殖速度を高く維持することができる。
Pseudozyma antarcticaの培養に用いる培養装置は、特に限定されないが、大量培養が可能であり、温度、通気量、ｐＨ等の培養条件を一定に保つことができるという点から、ジャーファーメンターを用いることが好ましい。
[Culture conditions]
In the method for producing a biodegradable plastic-degrading enzyme of the present invention, the conditions for culturing Pseudozyma antarctica can employ conventionally known culture conditions as culturing conditions for foliar yeast. Specifically, it is possible to list conditions in which pre-culture is performed for 24 hours or more under aerobic conditions of 25 ° C. or more and 32 ° C. or less, and then cultured at the same temperature for 72 hours or more. A conventionally known medium can be used as a medium for pre-culturing Pseudozyma antarctica, but it is usually preferable to use a natural medium such as a YM medium (Yeast extract Malt extract medium).
In culturing Pseudozyma antarctica, it is preferable to perform semi-batch culture or continuous culture in order to obtain a culture solution containing a biodegradable plastic-degrading enzyme more efficiently. That is, in the present invention, after culturing Pseudozyma antarctica in a medium containing xylose for a certain period of time, a culture solution of Pseudozyma antarctica is collected at a certain ratio, and the amount of the collected culture solution is added to the culture solution remaining in the culture apparatus. A method of culturing Pseudozyma antarctica by adding a medium containing the same amount of xylose, and recovering the culture solution of Pseudozyma antarctica at a constant rate while culturing Pseudozyma antarctica in a medium containing xylose Also provided is a method of adding a medium containing xylose at the same rate as it does.
In culturing Pseudozyma antarctica, when performing semi-batch culture, it is preferable to culture for 24 hours to 48 hours after adding a new medium to the culture solution. By setting the culture time as described above, the growth rate of Pseudozyma antarctica and the enzyme concentration in the obtained culture solution can be maintained high. Moreover, it is preferable that the quantity of the culture solution extracted after one culture of a half-batch culture is 50% or more and 80% or less of the whole culture solution.
When culturing Pseudozyma antarctica, when performing continuous culture, the medium should be added to the culture medium and the culture medium should be collected at a rate of 0.25 v / (v / d) to 0.75 V / (v / d). preferable. By adding a new medium at such a rate and collecting the culture solution, the growth rate of Pseudozyma antarctica can be kept high while maintaining the enzyme concentration in the culture solution high.
The culture apparatus used for culturing Pseudozyma antarctica is not particularly limited, but a jar fermenter is used from the viewpoint that mass culture is possible and culture conditions such as temperature, aeration volume, and pH can be kept constant. preferable.
［生分解性プラスチック分解酵素の分離・精製］
本発明の生分解性プラスチック分解酵素の製造方法においては、Pseudozyma antarcticaを培養することにより得られた培養液を、そのまま、生分解性プラスチックの分解に使用してもよいが、生分解性プラスチック分解酵素を含む溶液の保存性を向上させるためには、Pseudozyma antarcticaを含む培養液から、Pseudozyma antarcticaの菌体を分離することが好ましい。Pseudozyma antarcticaの培養液から菌体を分離するには、遠心分離や加圧ろ過など、従来公知の方法を採用することができる。なお、遠心分離によりPseudozyma antarcticaの菌体を除去した場合、上清に微量の菌体が混入する可能性があるため、保存性を更に向上するためには、遠心分離の後、更にろ過滅菌等を行うことが好ましい。ろ過滅菌を行うに際して使用するメンブランフィルターの孔径は、０．４５μｍ以下であることが好ましい。また、菌体を分離した生分解性プラスチック分解酵素の溶液に対して、更に限外ろ過等の処理を行うことにより、生分解性プラスチック分解酵素を高濃度で含有する酵素液を得ることができる。
本発明の生分解性プラスチック分解酵素の製造方法においては、必要に応じて、生分解性プラスチック分解酵素を精製してもよい。その場合、陰イオン交換クロマトグラフィー、アフィニティークロマトグラフィー等、タンパク質の精製方法として従来公知の精製方法を採用することができる。
なお、本発明でいう、生分解性プラスチック分解酵素とは、生分解性プラスチックの分解能を有する限りにおいて、特に限定されるものではないが、クチナーゼであることが好ましい。
[Separation and purification of biodegradable plastic-degrading enzymes]
In the method for producing a biodegradable plastic degrading enzyme of the present invention, the culture solution obtained by culturing Pseudozyma antarctica may be used as it is for degrading the biodegradable plastic. In order to improve the preservability of the solution containing the enzyme, it is preferable to isolate Pseudozyma antarctica cells from the culture solution containing Pseudozyma antarctica. In order to separate the cells from the culture solution of Pseudozyma antarctica, conventionally known methods such as centrifugation and pressure filtration can be employed. In addition, when the cells of Pseudozyma antarctica are removed by centrifugation, a trace amount of cells may be mixed into the supernatant. To further improve the storage stability, further filtration sterilization, etc. after centrifugation It is preferable to carry out. The pore size of the membrane filter used when performing filter sterilization is preferably 0.45 μm or less. In addition, an enzyme solution containing the biodegradable plastic-degrading enzyme at a high concentration can be obtained by further performing a treatment such as ultrafiltration on the biodegradable plastic-degrading enzyme solution from which the cells have been separated. .
In the method for producing a biodegradable plastic degrading enzyme of the present invention, the biodegradable plastic degrading enzyme may be purified as necessary. In that case, conventionally known purification methods such as anion exchange chromatography and affinity chromatography can be employed.
The biodegradable plastic-degrading enzyme referred to in the present invention is not particularly limited as long as it has the resolution of biodegradable plastic, but is preferably cutinase.
以下、本発明について実施例を挙げて詳細に説明する。なお、本発明は、以下に挙げる実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. Note that the present invention is not limited to the following examples.
＜実施例１；生分解性プラスチック分解酵素の産生に好適な培養条件の選択１＞
三角フラスコを用い、Pseudozyma antarcticaが生分解性プラスチック分解酵素を産生する際の、好適な条件の選定を行った。培養条件の選定に使用した株は、独立行政法人理化学研究所バイオリソースセンターにおいて提供されているPseudozyma antarctica JCM10317株、及び茨城県において採取された稲籾から単離された受託番号FERM P-22155のPseudozyma antarcticaであった。
まず、生分解性プラスチック分解酵素の産生に好適な培地の条件を検討するため、FMM培地中の各種栄養素の濃度を変更した改変FMM培地を作成し、各培地中で、Pseudozyma antarctica JCM10317株の生分解性プラスチック分解酵素の産生能を検討した。具体的には、前培養として、Pseudozyma antarctica JCM10317株を表１に示すYM培地中、３０℃で２４時間培養した後、表２に示すFMM培地の各成分のうち、KH2PO4、MgSO4・7H2O、Yeast Extractの濃度を、表２に記載された濃度の範囲で変更した改変FMM培地に、炭素源として8質量%のグリセロールを添加した培地を、100mL容の三角フラスコ中に20mL用意し、これに前培養液200μLを接種して、３０℃、200rpmで９６時間まで培養した。
次いで、20mM Tris-HCl緩衝液（ｐＨ6.8）に660nmにおける吸光度が0.65となるようにＰＢＳＡエマルジョンを懸濁させた懸濁液1.9mLを内径10mmの試験管に入れ、上記の培養により得られた培養上清を100μL加えて、３０℃、180rpmで１５分間振とう処理して吸光度の減少を測定した。１分あたり吸光度を1.0減少させる酵素量を1Uとした。
KH2PO4、MgSO4・7H2O、Yeast Extractの濃度を、通常用いたれるFMM培地の濃度（KH2PO4について０．０２％、MgSO4・7H2Oについて０．０２％、Yeast Extractについて０．１％）に対して、それぞれ、１倍量、３倍量、又は５倍量に変更した培地を使用して得られた酵素液のPBSA分解活性を図１（Ａ）に、Pseudozyma antarctica JCM10317株の乾燥菌体重量を図１（Ｂ）に示す。
表１：YM培地の組成
表２：FMM培地の組成
<Example 1: Selection of culture conditions suitable for production of biodegradable plastic degrading enzyme 1>
Using an Erlenmeyer flask, suitable conditions were selected when Pseudozyma antarctica produced a biodegradable plastic-degrading enzyme. The strains used for selection of the culture conditions are Pseudozyma antarctica JCM10317 strain provided by RIKEN BioResource Center, and Pseudozyma with accession number FERM P-22155 isolated from rice straw collected in Ibaraki Prefecture. It was antarctica.
First, in order to examine the conditions of the medium suitable for the production of biodegradable plastic degrading enzymes, modified FMM mediums with various nutrient concentrations in the FMM medium were prepared, and the Pseudozyma antarctica JCM10317 strain was produced in each medium. The production ability of degradable plastic degrading enzyme was examined. Specifically, as a preculture, Pseudozyma antarctica JCM10317 strain was cultured at 30 ° C. for 24 hours in the YM medium shown in Table 1, and among the components of the FMM medium shown in Table 2, KH 2 PO 4 , MgSO 4 20 mL of a medium in which 8% by mass of glycerol was added as a carbon source to a modified FMM medium in which the concentration of 7H 2 O and Yeast Extract was changed within the concentration range shown in Table 2, was placed in a 100 mL Erlenmeyer flask. Prepared, 200 μL of the preculture solution was inoculated, and cultured at 30 ° C. and 200 rpm for 96 hours.
Next, 1.9 mL of a suspension in which PBSA emulsion is suspended in 20 mM Tris-HCl buffer (pH 6.8) so that the absorbance at 660 nm is 0.65 is placed in a test tube having an inner diameter of 10 mm, and obtained by the above culture. 100 μL of the obtained culture supernatant was added, and the mixture was shaken at 30 ° C. and 180 rpm for 15 minutes, and the decrease in absorbance was measured. The amount of enzyme that decreases the absorbance per minute by 1.0 was defined as 1U.
The concentrations of KH 2 PO 4 , MgSO 4 .7H 2 O, and Yeast Extract were adjusted to the concentrations of commonly used FMM media (0.02% for KH 2 PO 4 , 0.02% for MgSO 4 .7H 2 O, Yeast FIG. 1 (A) shows the PBSA degradation activity of an enzyme solution obtained using a medium that has been changed to a 1-fold, 3-fold, or 5-fold volume, respectively. The dry cell weight of Pseudozyma antarctica JCM10317 strain is shown in FIG.
Table 1: Composition of YM medium
Table 2: Composition of FMM medium
図１から明らかなように、KH2PO4、MgSO4・7H2O、Yeast Extractの濃度を、それぞれ、通常のFMM培地に用いられる量の３倍量にした改変FMM培地（Ａ）では、通常のFMM培地を用いた場合に比べ、培養後９６時間の酵素活性が、約２倍（通常のFMM培地における生分解性プラスチック分解酵素の酵素活性0.23U/mLに比べ、0.42U/mL）となった。一方、KH2PO4、MgSO4・7H2O、Yeast Extractの濃度を、それぞれ、通常のFMM培地に用いられる量の５倍量にした改変FMM培地（Ｂ）では、培養後９６時間後の生分解性プラスチック分解酵素の酵素活性は0.54U/mL程度で、改変FMM培地（Ａ）で培養した際の生分解性プラスチック分解酵素の酵素活性と大きく変わらなかった。 As is clear from FIG. 1, in the modified FMM medium (A) in which the concentrations of KH 2 PO 4 , MgSO 4 .7H 2 O, and Yeast Extract are each three times the amount used in a normal FMM medium, Compared with normal FMM medium, enzyme activity 96 hours after culture is approximately double (0.42 U / mL compared to 0.23 U / mL enzyme activity of biodegradable plastic-degrading enzyme in normal FMM medium) It became. On the other hand, in the modified FMM medium (B) in which the concentrations of KH 2 PO 4 , MgSO 4 .7H 2 O, and Yeast Extract were each 5 times the amount used for normal FMM medium, 96 hours after culturing The enzymatic activity of the biodegradable plastic degrading enzyme was about 0.54 U / mL, which was not significantly different from the enzymatic activity of the biodegradable plastic degrading enzyme when cultured in the modified FMM medium (A).
＜実施例２；生分解性プラスチック分解酵素の産生に好適な培養条件の選択２＞
Pseudozyma antarcticaにおいて、生分解性プラスチック分解酵素の生産誘導に関与する炭素源を特定するため、図２に示す１０種類の炭素源を添加した改変FMM培地（Ａ）を用いて、炭素源の種類が生分解性プラスチック分解酵素の産生能に与える影響を調べた。
Pseudozyma antarctica JCM10317株をYM培地中、３０℃で２４時間培養した後、図２に示した１０種類の炭素源を、濃度が８質量％となるようにそれぞれ添加した、この炭素源添加改変FMM培地（Ａ）60mLを300mLの三角フラスコに入れて、上記前培養液600μLを接種し、３０℃、撹拌速度200rpmで培養した。
培養液を遠心分離して得られた培養上清100μLについて、実施例１と同様に酵素活性を測定した。結果を図２に示す。
図２から明らかなように、炭素源としてキシロースを用いたときに、生分解性プラスチック分解酵素の産生が亢進し、高い酵素活性を有する酵素液を得られることが分かった。
<Example 2: Selection of culture conditions suitable for production of biodegradable plastic-degrading enzyme 2>
In Pseudozyma antarctica, in order to identify the carbon sources involved in the induction of biodegradable plastic-degrading enzyme production, the modified FMM medium (A) to which 10 types of carbon sources shown in FIG. The influence of biodegradable plastic degrading enzyme on the production ability was investigated.
After culturing Pseudozyma antarctica JCM10317 strain in YM medium at 30 ° C. for 24 hours, this carbon source-added modified FMM medium was added to each of the 10 carbon sources shown in FIG. (A) 60 mL was placed in a 300 mL Erlenmeyer flask, inoculated with 600 μL of the preculture solution, and cultured at 30 ° C. and a stirring speed of 200 rpm.
The enzyme activity was measured in the same manner as in Example 1 with respect to 100 μL of the culture supernatant obtained by centrifuging the culture solution. The results are shown in FIG.
As apparent from FIG. 2, it was found that when xylose was used as the carbon source, the production of biodegradable plastic-degrading enzyme was enhanced, and an enzyme solution having high enzyme activity could be obtained.
＜実施例３；生分解性プラスチック分解酵素の産生に特に好適な株の選定＞
８質量％のグルコース又はキシロースを添加した改変FMM培地（Ａ）を用い、実施例２と同様の条件下、Pseudozyma antarctica JCM10317株と、受託番号FERM P-22155のPseudozyma antarctica（ＧＢ４（１）Ｗと表示）を９６時間培養した。
各培養液を遠心分離して得られた培養上清100μLについて、実施例１と同様に生分解性プラスチック分解酵素の酵素活性を測定した。結果を図３に示す。
図３に見られるように、受託番号FERM P-22155のPseudozyma antarcticaの生分解性プラスチック分解酵素の産生量は、Pseudozyma antarctica JCM10317株の生分解性プラスチック分解酵素の産生量に比較して有意に高く、受託番号FERM P-22155のPseudozyma antarcticaが、生分解性プラスチック分解酵素の産生に特に適していることが分かった。
<Example 3; Selection of strain particularly suitable for production of biodegradable plastic degrading enzyme>
Pseudozyma antarctica JCM10317 strain and Pseudozyma antarctica (GB4 (1) W) with accession number FERM P-22155 were used under the same conditions as in Example 2 using a modified FMM medium (A) supplemented with 8% by weight of glucose or xylose. Indications) were cultured for 96 hours.
The enzyme activity of the biodegradable plastic degrading enzyme was measured in the same manner as in Example 1 for 100 μL of the culture supernatant obtained by centrifuging each culture solution. The results are shown in FIG.
As can be seen in FIG. 3, the production of biodegradable plastic degrading enzyme of Pseudozyma antarctica with accession number FERM P-22155 is significantly higher than that of Pseudozyma antarctica JCM10317. Pseudozyma antarctica with accession number FERM P-22155 has been found to be particularly suitable for the production of biodegradable plastic degrading enzymes.
＜実施例４；大容量での生分解性プラスチック分解酵素の生産＞
5L容のジャーファーメンターに、表３に示した組成の培地3Lを加え、受託番号FERM P-22155のPseudozyma antarcticaを実施例１記載の条件と同様の条件でYM培地で培養した前培養液30mLを接種して、３０℃、撹拌速度500rpm、通気量8LPMで培養を行った。高泡形成による培養液の流出を防止するため、消泡剤（商品名「信越シリコーンKM-72F」、信越化学工業株式会社製）を60mL添加した。
窒素源の追加とｐＨの調整のため、培地にアンモニア水を滴下してｐＨを６．０に調整した。培養開始２４時間後から、表４に示した組成の流加培地を0.5L/dの速度で流加した。
流加培地の添加の開始から２４時間毎に所定量の培養液を回収し、培養液を遠心分離して得られた培養上清100μLについて、実施例１と同様に酵素活性を測定すると共に、得られた培養液中の乾燥菌体重量を測定した。結果を図４に示す。
図４から明らかなように、流加培地の添加に伴い生分解性プラスチック分解酵素の酵素活性は増加していき、流加培地の添加開始から１２０時間後には約21U/mL（比活性からの推定濃度0.3g/L）の酵素を生産できた。
表３：ジャーファーメンター用培地
表４：生分解性プラスチック分解酵素生産誘導用流加培地
※ディフコ社製
Example 4 Production of Biodegradable Plastic Degrading Enzyme with Large Capacity
3 L of medium having the composition shown in Table 3 was added to a 5 L jar fermenter, and Pseudozyma antarctica with accession number FERM P-22155 was cultured in YM medium under the same conditions as described in Example 1 30 mL of preculture solution Was cultured at 30 ° C., a stirring speed of 500 rpm, and an aeration rate of 8 LPM. In order to prevent the culture medium from flowing out due to the formation of high bubbles, 60 mL of an antifoaming agent (trade name “Shin-Etsu Silicone KM-72F”, manufactured by Shin-Etsu Chemical Co., Ltd.) was added.
In order to add a nitrogen source and adjust the pH, aqueous ammonia was added dropwise to the medium to adjust the pH to 6.0. From 24 hours after the start of the culture, a fed-batch medium having the composition shown in Table 4 was fed at a rate of 0.5 L / d.
A predetermined amount of the culture solution is collected every 24 hours from the start of the addition of the fed-batch medium, and about 100 μL of the culture supernatant obtained by centrifuging the culture solution, the enzyme activity is measured in the same manner as in Example 1, The dry cell weight in the obtained culture broth was measured. The results are shown in FIG.
As is apparent from FIG. 4, the enzymatic activity of the biodegradable plastic-degrading enzyme increases with the addition of the fed-batch medium, and is about 21 U / mL (from the specific activity from 120 hours after the start of the fed-fed medium addition). An enzyme with an estimated concentration of 0.3 g / L) could be produced.
Table 3: Medium for jar fermenters
Table 4: Feeding medium for inducing biodegradable plastic degradation enzyme production
* Difco
＜実施例５；半回分培養による生分解性プラスチック分解酵素の生産＞
実施例４では、約21U/mL（約0.3g/L）の酵素を生産できたが、前培養で十分な量の菌体を確保するために1.5日程度の時間を要するため、前培養を含めた全過程を通じた酵素生産速度は0.06g/L/dである。この酵素生産速度を向上させるため、培養後の培養液の一部を利用して、更に生分解性プラスチック分解酵素の生産を行う半回分培養により生分解性プラスチック分解酵素の培養を行った。
5Lのジャーファーメンターに表３に示した組成の培地を3L加え、YM培地中、実施例１と同様の条件で前培養した受託番号FERM P-22155のPseudozyma antarcticaの前培養液30mLを添加し、３０℃、撹拌速度500rpm、通気量8LPMで培養を行った。高泡形成による培養液の流出を防止するため、消泡剤（商品名「信越シリコーンKM-72F」、信越化学工業株式会社製）を５０倍希釈で添加した。
培地には、窒素源の追加とｐＨの調整のため、アンモニア水を滴下してｐＨを６．０に調整した。培養開始２４時間後から、表４に示した組成の流加培地を0.5L/dで流加した。
流加培地の添加開始後７２時間の時点で2Lの培養液を回収し、残る1Lの培養液に対して、表３に記載の組成の培地を2L添加し、流加培地の流加を更に継続して培養を続けた。以後、24時間ごとに2Lの培養液の回収と2Lの培地の添加を計７回行った。
得られた培養液について、実施例１に示した方法と同様の方法により、生分解性プラスチック分解酵素の酵素活性を測定し、菌体の乾燥重量の測定も行った。結果を図４に示す。
各培養液から得られた培養上清中の酵素活性は、平均して14U/mL（約0.2g/L）となり、酵素生産速度は0.2g/L/dとなった。
<Example 5: Production of biodegradable plastic degrading enzyme by semi-batch culture>
In Example 4, an enzyme of about 21 U / mL (about 0.3 g / L) could be produced, but it takes about 1.5 days to secure a sufficient amount of cells in the preculture. The enzyme production rate throughout the entire process is 0.06 g / L / d. In order to improve the enzyme production rate, the biodegradable plastic degrading enzyme was cultured by semi-batch culture in which a part of the culture broth after the cultivation was further used to produce the biodegradable plastic degrading enzyme.
Add 3 L of medium with the composition shown in Table 3 to a 5 L jar fermenter, and add 30 mL of precultured Pseudozyma antarctica with accession number FERM P-22155 pre-cultured under the same conditions as in Example 1 in YM medium. The culture was performed at 30 ° C., a stirring speed of 500 rpm, and an aeration rate of 8 LPM. An antifoaming agent (trade name “Shin-Etsu Silicone KM-72F”, manufactured by Shin-Etsu Chemical Co., Ltd.) was added at a 50-fold dilution to prevent the culture solution from flowing out due to the formation of high bubbles.
To the medium, ammonia water was added dropwise to adjust the pH to 6.0 in order to add a nitrogen source and adjust the pH. From 24 hours after the start of the culture, a fed-batch medium having the composition shown in Table 4 was fed at 0.5 L / d.
At 72 hours after the start of addition of the fed-batch medium, 2 L of the culture solution is collected, and 2 L of the medium having the composition shown in Table 3 is added to the remaining 1 L of the culture solution, and the fed-batch medium is further fed. The culture was continued. Thereafter, every 24 hours, 2 L of the culture solution was collected and 2 L of medium was added a total of 7 times.
With respect to the obtained culture broth, the enzyme activity of the biodegradable plastic degrading enzyme was measured by the same method as shown in Example 1, and the dry weight of the cells was also measured. The results are shown in FIG.
The enzyme activity in the culture supernatant obtained from each culture medium was 14 U / mL (about 0.2 g / L) on average, and the enzyme production rate was 0.2 g / L / d.
＜実施例６；草本系バイオマスを利用した生分解性プラスチック分解酵素の生産＞
実施例１から５においては、キシロースを単独の炭素源として添加した培地において生分解性プラスチック分解酵素の生産を行ったが、本実施例では、草本系バイオマスを添加して培養中にこれを分解し、キシロース以外にもグルコース等の炭素源が存在するキシロース溶液を提供して生分解性プラスチック分解酵素の生産を行った。
まず、300mL容の三角フラスコに小麦ふすまを2.5g、水を50mL加え、１２１℃で１５分間加温加圧滅菌した。これに、硝酸ナトリウム0.01g、硝酸アンモニウム0.01g、おから0.15g、米ぬか0.1gを加えた培地をそれぞれ用意し、必要に応じて、小麦ふすま中のセルロースの加水分解を補助する機能を有するアクレモニウムセルラーゼを加えた。
実施例１と同様の条件で得られたYM培地による前培養液600μLを各培地に加え、３０℃、撹拌速度200rpmで培養した。
得られたそれぞれの培養上清100μLについて、実施例１と同様に酵素活性を測定した。結果を図６に示す。
図６から明らかなように、小麦ふすまのみを加えた培地においても生分解性プラスチック分解酵素の酵素活性は９６時間で0.95U/mLとなったものの、硝酸ナトリウムの添加により、この酵素活性が1.05U/mLまで増加し、更にセルラーゼを加えることにより1.25U/mLまで増加した。
<Example 6: Production of biodegradable plastic degrading enzyme using herbaceous biomass>
In Examples 1 to 5, biodegradable plastic-degrading enzymes were produced in a medium supplemented with xylose as the sole carbon source. In this example, herbaceous biomass was added and decomposed during cultivation. In addition, a biodegradable plastic degrading enzyme was produced by providing a xylose solution containing a carbon source such as glucose in addition to xylose.
First, 2.5 g of wheat bran and 50 mL of water were added to a 300 mL Erlenmeyer flask, and heated and autoclaved at 121 ° C. for 15 minutes. A medium containing 0.01g sodium nitrate, 0.01g ammonium nitrate, 0.15g okara and 0.1g rice bran is prepared, and if necessary, Acremonium has a function to assist the hydrolysis of cellulose in wheat bran. Cellulase was added.
600 μL of a pre-culture solution with a YM medium obtained under the same conditions as in Example 1 was added to each medium and cultured at 30 ° C. and a stirring speed of 200 rpm.
The enzyme activity was measured in the same manner as in Example 1 for each of the obtained culture supernatants (100 μL). The results are shown in FIG.
As is apparent from FIG. 6, the enzyme activity of the biodegradable plastic degrading enzyme was 0.95 U / mL in 96 hours even in the medium containing only wheat bran, but this enzyme activity was 1.05 by adding sodium nitrate. It increased to U / mL, and further increased to 1.25 U / mL by adding cellulase.
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| WO2021157618A1 (en) | 2020-02-03 | 2021-08-12 | 日本甜菜製糖株式会社 | Seedling-growing pot body with long-term stability, and decomposition promotion method therefor |
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| JP7181542B2 (en) | 2017-03-21 | 2022-12-01 | 国立研究開発法人農業・食品産業技術総合研究機構 | A novel strain of Pseudozyma antactica |
| JP7473137B2 (en) | 2017-03-21 | 2024-04-23 | 国立研究開発法人農業・食品産業技術総合研究機構 | A novel strain of Pseudozyma antarctica |
| WO2021157618A1 (en) | 2020-02-03 | 2021-08-12 | 日本甜菜製糖株式会社 | Seedling-growing pot body with long-term stability, and decomposition promotion method therefor |
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