JP3798336B2 - Enzyme production method using rush - Google Patents

Description

注：財団法人日本食品分析センター調べ
【００１５】
更に、イグサはタンパク分をあまり含まないことも培養基質としての利点の一つである。イグサのタンパク分は、１００ｇあたり約４ｇ程度であり、このようにタンパク分の少ないものを培養基質として用いた場合に、比活性の高い酵素が生産できると考えられる。つまり、含有されているタンパクのほとんどが生産された目的酵素になるということである。
【００１６】
このように、イグサは、他の植物、例えば、小麦、またはミカンと比較して、タンパク分の含有量が少ないため、生産された酵素の比活性が高くなり精製が容易になると共に、混在する不純タンパク質によって、培養させる目的である酵素を切断したり、または培養の働きを低下させたりすることがなくなるのである。
【００１７】
培養前の糸状菌株に含有されるペクチナーゼの一種である活性したポリガラクツロナーゼのそれぞれの含有量を表２に示す。
【表２】

注：ＩＦＯ３０１０２、ＩＦＯ３１１２５、ＩＦＯ５２３８はAspergillusであり、その他についてはRhizopusである。
【００１８】
ペクチナーゼは、果汁、果実酒の清澄化、植物性繊維の精錬等に広く一般的に使用され、植物の組織中に含有されるペクチンに作用する酵素の一つである。ポリガラクツロナーゼは、ペクチナーゼの一種であり、ポリガラクツロン酸をガラクツロン酸に分解する酵素である。
【００１９】
（実験例１）
この実験例１においては、糸状菌の一つであるRhizopus oryzae ＩＦＯ４６９７株をポリガラクツロン酸生産菌として使用した。使用菌株をポテトデキストロース寒天培地を用い、略３０℃程度の温度で７日間培養したものを実験に使用した。培養した斜面に滅菌水を３ｍｌ加えたものを胞子懸濁液にし、接種液とした。
【００２０】
国産（熊本県八代地方産）の泥染めイグサを水洗し乾燥したものを略８ｍｍの長さに切断し、ミキサー（Hamilton Beach：Model ９１１：１５７００ｒｐｍ）で粉末にしたものを培養の基質として使用した。
【００２１】
酵素の培養については、培地には、前記粉末にしたイグサ５ｇと、純水１７ｍｌと、ペクチン（Nacalai tesque社製）０．５ｇとを三角フラスコに入れて綿栓をし、１２１℃の温度で略２０分間程度オートクレーブ（殺菌）したものを使用した。
【００２２】
殺菌後のイグサ培地、即ち、三角フラスコに前記接種液を滴下し、直射日光を避けた室内において、略２５〜３０℃の温度に維持し３日間に渡って培養した。
【００２３】
このように培養した後、前記三角フラスコに純水を略４０ｍｌ入れて、室温にて一昼夜放置した後に濾過して、酵素抽出液を得た。
【００２４】
（比較例１）
この比較例１においては、実験例１の粉末にしたイグサに換えて、固体を基質にする場合に一般的に用いられている小麦ふすまを培養の基質として使用し、その他については、実験例１と同様にして酵素抽出液を得た。
【００２５】
実験例１と比較例１とのポリガラクツロナーゼの含有量における比較については、夫々１ｇあたりのポリガラクツロナーゼ活性と比活性の含有量を比較した。
【００２６】
酵素抽出液に含有される活性した酵素を測定する方法としては、ペクチン酸溶液（Nacalai tesque社製）の１％溶液（０．１Ｍ酢酸緩衝液、ｐＨ４．５）０．５ｍｌに、前記酵素抽出液を０．５ｍｌ加えて、４０℃の温度で略１５分間反応させた。
【００２７】
次に、前記反応させた液体を沸騰した水浴中で略５分間加熱して反応を停止させて、遊離したガラクツロン酸量をＤＮＳ試薬によって測定した。
【００２８】
このＤＮＳ試薬においては、４．５％の水酸化ナトリウム水溶液３００ｍｌ中に、１％のＤＮＳ試薬８８０ｍｌと、ロッセル塩２５５ｇとを加え、該ロッセル塩が溶解されるまで撹拌して、溶液Ａを得る。また、これとは別に、１０％の水酸化ナトリウム水溶液２２ｍｌ中に、結晶フェノール１０ｇと、水とを加えて、１００ｍｌにし、この１００ｍｌから６９ｍｌを取って、その６９ｍｌ中に炭酸水素ナトリウム６．９ｇを加えて、溶液Ｂを得る。これらの溶液Ａ、Ｂを混合して、２日間放置して濾過したものを前記ＤＮＳ試薬として用いた。
【００２９】
前記反応液に、前記ＤＮＳ試薬１ｍｌを入れて撹拌した後、沸騰した水浴中で略１０分間加熱し、その後、１０分間氷冷させ、これを遠心分離機（３０００回転、５分間）にかけて、その上清を水で１０倍に希釈し、分光光度計（HITACHI：Ｕ−１１００）によって、５５０ｎｍにおける吸光度を測定した。
【００３０】
このようにして得られた吸光度と、あらかじめ作成しておいた検量線とを用いて還元糖濃度を算出した。その際に、酵素単位（Ｕ）は、１分間に酵素溶液１ｍｌがガラクツロン酸１ｍｇを遊離する量（ｍｇ−ガラクツロン酸／ｍｉｎ）を１（Ｕ）として規定した。
【００３１】
実験例１と比較例１とに含有されるポリガラクツロナーゼの含有量を表３に示す。
【表３】

【００３２】
表３から明らかなように、比較例１の小麦ふすまを基質として使用した場合よりも、実験例１のイグサを粉末にしたものを基質にした方が、比活性の高い酵素を大量に得ることができることが解る。
【００３３】
（実験例２）
この実験例２においては、実験例１の培地に使用した量をそれぞれ２０倍、即ち、イグサ１００ｇと、純水３４０ｍｌと、ペクチン（Nacalai tesque社製）１０ｇとを使用し、その他については、実験例１と同様にして培養した。
【００３４】
このようにして培養した実験例２に含有されるポリガラクツロナーゼの含有量を測定したところ、イグサ１ｇあたりのポリガラクツロナーゼ活性は４７２０Ｕ／ｇ−igusaであり、ポリガラクツロナーゼ比活性は５３００Ｕ／ｍｇ−protein・g-igusaであった。
【００３５】
この結果より明らかなように、実験例１のポリガラクツロナーゼの活性または比活性の含有量よりも、実験例２の方がそれぞれ増加していることから、培養させる効率が高くなっていることが解る。
【００３６】
一般的な固体の基質を使用した糸状菌の培養においては、培地の量を増加させることによって、その培養できる量が減少することもあるが、本発明の実験例２から明らかなように、イグサを基質にして糸状菌を培養した場合には、培地を増加させることによって、その培養できる量を増加させることができるため、ポリガラクツロナーゼを大量に培養する際にその効果が大きくなることが解る。
【００３７】
なお、実験例においては、泥染めイグサを用いたが、本発明に係るイグサを用いた酵素の培養方法はこれに限定されるものではなく、例えば、無染イグサまたは新芽イグサ等を用いても良い。
【００３８】
更に、イグサは微生物の成育にとって好ましい環境であるため、その利用は酵素の培養に限定されるものではなく、例えば、医薬品工業等における微生物による代謝物の生産においても、利用することができるのである。
【００３９】
【発明の効果】
以上説明したように、本発明に係るイグサを用いた酵素の生産方法は、植物を基質とする酵素の培養方法であって、少なくとも水洗したイグサを粉末にし、該イグサ粉末を殺菌し培養基質として糸状菌を培養するようにしたことにより、イグサは食物繊維及びミネラル成分を多量に含有するため微生物が繁殖しやすく、且つイグサはタンパク質の含有量が少ないため、この粉末を培養の基質にすることによって、得られる酵素の比活性が高く、目的とする酵素を精製することが容易であると共に、共存するプロテアーゼによって目的とする酵素が切断されることがなく、さらに、酵素を大量に培養し生産する際に、培地を多くすることで効率よく培養量を多くできるという優れた効果を奏する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an enzyme used for processing, for example, a pharmaceutical material such as an enzyme preparation or a food product.
[0002]
[Prior art]
Conventionally, as a method for producing an enzyme such as bacteria or yeast, a production method by liquid culture using a liquid substance (including a jelly-like substance) as a substrate is generally used. For an enzyme such as a fungus, a production method by solid culture using a solid as a substrate is used. As a method for producing an enzyme using solid as a substrate by solid culture, a method using a natural plant or the like made of wheat bran or citrus juice as a substrate is known.
[0003]
[Problems to be solved by the invention]
However, natural plants consisting of wheat bran or citrus juice residue generally contain a large amount of protein, so that the specific activity of the obtained enzyme is low and it is difficult to purify the target enzyme. In some cases, the target enzyme may be cleaved by a coexisting protease, and the productivity of the enzyme is poor.
[0004]
Therefore, in the conventional production method by solid culture of an enzyme using a plant as a substrate, there is a problem to be solved by developing a culture substrate that is easy to propagate microorganisms and low in protein content and improving productivity. have.
[0005]
[Means for Solving the Problems]
As a specific means for solving the problems of the conventional examples described above, the present invention is a method for producing an enzyme using a plant as a substrate, wherein at least the washed rush is powdered, the rush powder is sterilized, and filamentous fungi are used as a culture substrate. The present invention provides a method for producing an enzyme using rush characterized by culturing.
[0006]
In the present invention, the culture substrate includes pectin; and obtaining pectinase by the culture as additional requirements.
[0007]
The method for producing an enzyme using the rush according to the present invention uses a rush containing a large amount of dietary fiber and mineral components and a low protein content, and using the rush as a culture substrate. By using it, microorganisms can be easily propagated, and the target enzyme can be cultured and efficiently produced.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in detail based on specific embodiments.
In the present invention, rush is used as a substrate for culturing the enzyme. Rabbits are composed of dietary fiber, approximately 63% of its components, and are one of the plants that have a relatively high dietary fiber content even among plants belonging to nature. Presents.
[0009]
Thus, since the inside of a rush is a porous sponge shape, it contains a lot of air in the inside, and is a suitable environment for microorganisms that prefer oxygen, that is, aerobic fungi.
[0010]
This is a method for producing an enzyme using a rush having such characteristics as a so-called plant substrate. When using the rush, the rushed and dried rush is washed with water to remove deposits and further dried. The rush is pulverized into powder by appropriate means. The powder in this case does not have to be uniform. For example, the powder may be in a state where a coarse product and a fine product are mixed.
[0011]
The rush powder is sterilized by adding the required amount of pure water and pectin and heat-treating it, and is cooled to room temperature. A medium exhibiting a shape is inoculated, and an enzyme to be cultured, for example, a filamentous fungus (liquid), is inoculated on the medium and cultured for several days while maintaining a predetermined temperature. In this case, depending on the type of enzyme to be cultured, for example, the culture may be performed over 7 to 30 days or more.
[0012]
The medium using the rush is formed in a compartmented flat container with a layer thickness of a predetermined thickness, but it is preferable for the growth of fungi to form a thin layer with a relatively wide spread as a whole. Is. However, depending on the type of enzyme to be cultured, for example, the medium may be formed with a relatively thick layer in a deep bottle-like container.
[0013]
After culturing the target enzyme in this way, the container is charged with a required amount (more than twice the volume of the medium) of pure water or sterilized water, and is allowed to stand for about a day and night while maintaining the room temperature. The rush and moisture are filtered by a filtering means, and the filtrate becomes an enzyme extract, after which the enzyme extract is purified by a predetermined means.
[0014]
In addition, rush contains a large amount of minerals such as potassium, calcium, magnesium, sodium, and iron, and abundantly contains essential components for the growth of microorganisms. Therefore, Table 1 shows the content of mineral components contained in the rush.
[Table 1]

Note: Survey by Japan Food Analysis Center [0015]
Furthermore, rush is one of the advantages as a culture substrate that it does not contain much protein. The protein content of rush is about 4 g per 100 g, and it is considered that an enzyme with a high specific activity can be produced when such a low protein content is used as a culture substrate. That is, most of the contained protein becomes the target enzyme produced.
[0016]
Thus, the rush has a low protein content compared to other plants, such as wheat or mandarin, so that the specific activity of the produced enzyme is increased and purification is facilitated and mixed. The impure protein does not cleave the enzyme that is the object of culturing or reduce the function of culturing.
[0017]
Table 2 shows the respective contents of active polygalacturonase, which is a kind of pectinase contained in the filamentous strain before culture.
[Table 2]

Note: IFO 30102, IFO 31125, IFO 5238 are Aspergillus, others are Rhizopus.
[0018]
Pectinase is one of the enzymes that act widely on pectin contained in plant tissues, and is widely used for clarification of fruit juice, fruit wine, refining of vegetable fiber, and the like. Polygalacturonase is a kind of pectinase and is an enzyme that decomposes polygalacturonic acid into galacturonic acid.
[0019]
(Experimental example 1)
In Experimental Example 1, Rhizopus oryzae IFO4697 strain, which is one of filamentous fungi, was used as a polygalacturonic acid-producing bacterium. The strain used was a potato dextrose agar medium and cultured for 7 days at a temperature of about 30 ° C. for the experiment. A spore suspension obtained by adding 3 ml of sterilized water to the cultured slope was used as an inoculum.
[0020]
Washed and dried mud-dyed rush from Japan (Yatsushiro, Kumamoto Prefecture), cut into a length of about 8 mm, and powdered with a mixer (Hamilton Beach: Model 911: 15700 rpm) was used as the substrate for culture. .
[0021]
For the culture of the enzyme, the medium contains 5 g of rushed rush, 17 ml of pure water and 0.5 g of pectin (manufactured by Nacalai tesque) in an Erlenmeyer flask. What was autoclaved (sterilized) for about 20 minutes was used.
[0022]
The inoculum was dropped into a sterilized rush medium, that is, an Erlenmeyer flask, and the cells were cultured for 3 days while maintaining a temperature of about 25 to 30 ° C. in a room avoiding direct sunlight.
[0023]
After culturing in this way, about 40 ml of pure water was put into the Erlenmeyer flask, left standing at room temperature for a whole day and then filtered to obtain an enzyme extract.
[0024]
(Comparative Example 1)
In this comparative example 1, wheat bran generally used in the case of using a solid as a substrate is used as a substrate for culturing instead of the powdered rush of experimental example 1; In the same manner, an enzyme extract was obtained.
[0025]
About the comparison in the content of polygalacturonase between Experimental Example 1 and Comparative Example 1, the content of polygalacturonase activity per 1 g and the specific activity were respectively compared.
[0026]
As a method for measuring the active enzyme contained in the enzyme extract, the enzyme extract was added to 0.5 ml of a 1% solution (0.1 M acetate buffer, pH 4.5) of a pectate solution (manufactured by Nacalai Tesque). 0.5 ml of the solution was added and reacted at a temperature of 40 ° C. for approximately 15 minutes.
[0027]
Next, the reaction liquid was heated in a boiling water bath for about 5 minutes to stop the reaction, and the amount of liberated galacturonic acid was measured with a DNS reagent.
[0028]
In this DNS reagent, 880 ml of 1% DNS reagent and 255 g of Rossel salt are added to 300 ml of 4.5% sodium hydroxide aqueous solution, and stirred until the Rossel salt is dissolved to obtain a solution A. . Separately, 10 g of crystalline phenol and water are added to 22 ml of a 10% aqueous sodium hydroxide solution to make 100 ml, and from this 100 ml to 69 ml, 6.9 g of sodium bicarbonate is added to 69 ml. To obtain solution B. These solutions A and B were mixed, allowed to stand for 2 days and filtered, and used as the DNS reagent.
[0029]
The reaction solution was stirred with 1 ml of the DNS reagent, heated in a boiling water bath for about 10 minutes, then cooled on ice for 10 minutes, and this was subjected to a centrifuge (3000 rpm, 5 minutes). The supernatant was diluted 10-fold with water, and the absorbance at 550 nm was measured with a spectrophotometer (HITACHI: U-1100).
[0030]
The reducing sugar concentration was calculated using the absorbance thus obtained and a calibration curve prepared in advance. At that time, the enzyme unit (U) was defined as 1 (U), where 1 ml of enzyme solution liberates 1 mg of galacturonic acid per minute (mg-galacturonic acid / min).
[0031]
Table 3 shows the content of polygalacturonase contained in Experimental Example 1 and Comparative Example 1.
[Table 3]

[0032]
As is apparent from Table 3, a larger amount of enzyme having a higher specific activity can be obtained when the powdered rush of Experimental Example 1 is used as a substrate than when wheat bran of Comparative Example 1 is used as a substrate. I understand that
[0033]
(Experimental example 2)
In Experimental Example 2, the amount used in the medium of Experimental Example 1 was 20 times, that is, 100 g of rush, 340 ml of pure water, and 10 g of pectin (Nacalai tesque) were used. The culture was carried out in the same manner as in Example 1.
[0034]
When the content of polygalacturonase contained in Experimental Example 2 cultured as described above was measured, the polygalacturonase activity per gram of rush was 4720 U / g-igusa, and the specific activity of polygalacturonase was It was 5300 U / mg-protein · g-igusa.
[0035]
As is clear from this result, since the content of Experimental Example 2 is higher than the content of the activity or specific activity of Polygalacturonase of Experimental Example 1, the efficiency of culturing is higher. I understand.
[0036]
In culturing filamentous fungi using a general solid substrate, increasing the amount of the medium may decrease the amount that can be cultured. However, as apparent from Experimental Example 2 of the present invention, the rush When the filamentous fungus is cultured with the substrate as the substrate, the amount of the cultivatable can be increased by increasing the medium, so that the effect is increased when cultivating polygalacturonase in large quantities. I understand.
[0037]
In the experimental examples, mud-dyed rush was used. However, the enzyme culture method using the rush according to the present invention is not limited to this. For example, undyed rush or newly sprouted rush can be used. good.
[0038]
Furthermore, since rush is a favorable environment for the growth of microorganisms, its use is not limited to enzyme culture, and can be used, for example, in the production of metabolites by microorganisms in the pharmaceutical industry and the like. .
[0039]
【The invention's effect】
As described above, the method for producing an enzyme using the rush according to the present invention is a method for culturing an enzyme using a plant as a substrate, and at least the washed rush is made into powder, the rush powder is sterilized and used as a culture substrate. By cultivating filamentous fungi, rushes contain a large amount of dietary fiber and minerals, so microorganisms are easy to propagate, and rushes have low protein content, so this powder should be used as a substrate for culture. The enzyme has a high specific activity, and it is easy to purify the target enzyme. The target enzyme is not cleaved by the coexisting protease, and the enzyme is cultured and produced in large quantities. When this is done, an excellent effect is achieved in that the amount of culture can be increased efficiently by increasing the culture medium.

Claims (3)

A method for producing an enzyme using a plant as a substrate,
Powder at least washed rushes,
A method for producing an enzyme using rush characterized by sterilizing the rush powder and culturing filamentous fungi as a culture substrate. In the culture substrate,
The method for producing an enzyme using the rush as claimed in claim 1, wherein pectin is included. By the culture,
The method for producing an enzyme using the rush as claimed in claim 1, wherein pectinase is obtained.